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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
IntematioDal Bureau 




PCT 

INTERNATIONAL APPLICATION PUBUSHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification ^ : 

C12N 15/12, A61K 48/00, 38/18, C12N 
5/06, GOIN 33/50 



Al 



(11) International Publication Ninnber: WO 95/10611 

(43) International Publication Date: 20 i^ril 1995 (20.04.95) 



(21) International Application Number: PCTAJS94/1 1745 

(22) International Ffling Date: 14 October 1994 (14.10.94) 



(30) Priority Data: 

08/136,74S 



14 October 1993 (14.10.93) 



US 



(71) Applicant: PRESIDENT AND FELLOWS OF HARVARD 

COLLEGE [USAJS]; 124 Mt Auburn Sticet. Cambridge. 
MA 02138 (US). 

(72) Inventors: MELTON. Douglas, A.; 22 Flocum Road, Lexing- 

ton, MA 02173 (US). HEMMATI-BRIVANLOU, AH; 325 
East 84th Street, New Yoik, NY 10028 (US). 

(74) Agoits: VINCENT, Matthew, P. et al.; Lahivc & Cockfield, 60 
State Street, Boston, MA 02109 (US). 



(81) Designated States: AU, CA, JP, KR, European* patent (AT, 
BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU. MC; NL, 
PT, SE). 



Published 

Widi intemationai search report. 

Before the expiration of the dme limit for amending the 
claims and to he republished in the event of the receipt of 
amendments. 



(54) Title: METHOD OF INDUCING AND MAINTAINING NEURONAL CELLS 



(57) Abstract 



The present invention makes available a method for inducing neuronal differentiation and preventing the dca& ox degeneration of 
neuronal cells both in vitro and in Wvo. The subject method stems £rom the unexpected finding that, contrary to traditional unterstanding 
of neural irKhicticn, the default fate of ectodermal tissue is neuronal rather than mesodermal and/or epidermal In particular, it has been 
discoveml that preventing or antagonizing & signalinig pathway in a cell for a growth factor of the TGF-^ family can result in neuronal 
differentiation of that cell. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States party to the PCT on dte front pages of pamphlets publishing international 
applications under the PCT. 



AT 


Atutria 


GB 


United Kingdom 


MR 


Mauritania 


AO 


Ausmdia 


GE 


Georgia 


MW 


Malawi 


BB 


Btfbftdos 


GN 


Guinea 


NE 


Nigff 


BE 


Belgium 


GR 


Greece 


NL 


Netherlands 


BF 


Burkiiu Paao 


HO 


Hungary 


NO 


.Norway 


BG 


Bolgaria 


IE 


Ireland 


NZ 


New Zealand 


ai 


Benin 


rr 


Ualy 


PL 


Poland 


BR 


Brazil 


JP 


Japan 


PT 


Ponugnl 


BY 


Belarus 


KE 


Kenya 


RO 




CA 


Canada 


KG 


Kyrgystan 


RU 


Russian Fedoation 


CF 


Central African RqRiblic 


KP 


Oanocrabc People's Rep)d>Uc 


SD 


Sudan 


CG 


Congo 




of Korea 


SE 


Sweden 


CH 


Switzerland 


KR 


Republic of Korea 


51 


Slovenia 


CI 


Cdte dlvoire 


KZ 


KazaUisun 


SK 


Slovakia 


CM 


CamoDOn 


U 


Liecbtenstein 


SN 


Senegal 


CN 


Cbina 


LK 


Sri Lanka 


TD 


Chad 


CS 


Czechoslovakia 


U7 


Luxemiboing 


TG 


Togo 


CZ 


Ciecfa RqnJblic 


LV 


Latvia 


TI 


Tajikistan 


DE 


Gennany 


MC 


Monaco 


TT 


Trinidad and Tobago 


DK 


Denmark 


MD 


RqnAUc of Moldova 


UA 


Ukraine 


ES 


Spain 


MG 


Madagascar 


US 


United Slates of Amoka 


n 


Hnland 


ML 


Mali 


uz 


Uzbekistan 


FR 


Rtnoe 


MN 


Mongolia 


VN 


Viet Nam 


GA 


Gabon 











wo 95/1061 1 PCT/US94/1 1745 

-3- 

Method of Inducing and Maintaining Neuronal Cells 

Background of the Invention 

Understanding the processes that lead from a fertilized egg to the formation of germ 
5 layers and subsequently to a body plan is a central goal of embryology. Much of what is 
known about the development of a vertebrate body plan comes from studies of amphibia 
where, at the tadpole stage, the main body axis consists of the dorsal structures notochord, 
spinal cord and somites organized anterior to posterior as head, trunk and tail. All animal 
tissues derive from the three germ layers and the mesoderm plays a pivotal role in organizing 
10 the body axis (Keller, R. in Methods in Cell Biology, eds Kay and Peng, Academic Press: San 
Diego, 1991). Mesodermal cells lead the movements of gastrulation (Keller et al. (1988) 
Development 103:193-210; and Wilson et al. (1989) Deve/opmew/ 105:155-166), are required 
for the patterning of the nervous system (Mangold et al. (1933) Natyrwissenschaften 21:761- 
766; and Hemmati-Brivanlou et al. (1990) Science 250:800-802), and themselves give rise to 
15 the muscular, skeletal, circulatory and excretory systems. Moreover, a portion of the dorsal 
mesoderm from early gastrula, the Spemann organizer, can induce and organize a second 
body axis following transplantation to another site (Spemann et al. (1924) Arch mikr Anat 
£«nvMecA 100:599-638). 

The origin of the nervous system in all vertebrates can be traced to the end of 
20 gastrulation. At this time, the ectoderm in the dorsal side of the embryo changes its fate from 
epidenna to neural. The newly formed neuroectoderm thickens to form a flattened structure 
called the neural plate which is characterized, in some vertebrates, by a central groove (neural 
groove) and thickened lateral edges (neural folds). At its early stages of differentiation, the 
neural plate already exhibits signs of regional differentiation along its anterior posterior (A-P) 
25 and mediolateral axis (M-L). The neural folds eventually fuse at the dorsal midline to form 
the neural tube which will differentiate into brain at its anterior end and spinal cord at its 
posterior end. Closure of the neural tube creates dorsal/ventral differences by virtue of 
previoiis mediolateral differentiation. Thus, at the end of neurulation, the nem-al tube has a 
clear anterior-posterior (A-P), dorsal ventral (D-V) and mediolateral (M-L) polarities (see, for 
30 example. Principles in Neural Science (3rd), eds. Kandel, Schwartz and Jessell, Elsevier 
Science Publishing Company: NY, 1991; and Developmental Biology (3rd), ed. S.F. Gilbert, 
Sinauer Associates: Sunderland MA, 1991). 

Before gastrulation the three germ layers are simply arranged, top to bottom, in a frog 
blastula. Ectoderm arises from the top, or animal pole; mesoderm from the middle, or 
35 marginal zone, and endoderm from the bottom or vegetal pole. Mesoderm can be induced in 
animal pole cells (animal caps) by signals emanating from the vegetal pole. Several peptide 



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growth factors have been identified that can induce mesoderm in animal caps in vitro. When 
animal cap tissue is explanted from a blastula embryo and cultured in isolation it develops 
into a ball of epidermis. But in the presence of a mesoderm inducing factor, the animal cap 
will differentiate into mesodermal derivatives, including notochord, muscle and blood. 
5 Members of the fibroblast growth factor family, in particular basic fibroblast growth factor 
(bFGF), and the transforming growth factor-p (TGF-P) family, notably activins and Vg-1, are 
potent inducers in this assay. Xenopus homologues of the Wnt gene family may also have a 
role in mesoderm induction. Both Xwntl (McMahon et al. (1989) Cell 58, 1075-1084) and 
Xwnt8 messenger RNAs elicit dorsal mesoderm formation when injected into the ventral side 

10 of an early embryo, an activity shared by Vg-1, and to a lesser extent by activin RNA. bPGF 
and activin protein* can be detected in the early embryo and although there are no data on the 
localization of activin, there is evidence that bFGF is present in the marginal zone and vegetal 
pole of early blastula. Vg-1 is present at the appropriate time and in the right region known to 
be responsible for mesoderm induction in vivo. Although Xwntl and XwntS are not present at 

15 the proper time or place to effect dorsal mesoderm induction, there may be other Xwnts that 
fulfill this role. 

Many types of communication take place among animal cells. These vary from long- 
range effects, such as those of rather stable hormones circulating in the blood and acting on 
any cells in the body that possess the appropriate receptors, however distant they are, to the 

20 fleeting effects of very unstable neurotransmitters operating over distances of only a few 
microns. Of particular importance in development is the class of cell interactions called 
embryonic induction; this includes influences operating between adjacent cells or in some 
cases over greater than 10 cell diameters (Saxen et al. (1989) Jnt J Dev Biol 33:21-48; and 
Gurdon et al. (1987) Development 99:285-306). Embryonic induction is defined as in 

25 interaction between one (inducing) and another (responding) tissue or cell, as a result of 
which the responding cells undergo a change in the direction of differentiation. This 
interaction is often considered one of the most important mechanism in vertebrate 
development leading to differences between cells and to the organization of cells into tissues 
and organs. Adult organs in vertebrates, and probably in invertebrates, are formed through an 

30 interaction between epithelial and mesenchymal cells, that is, between ectoderm/endoderm 
and mesoderm, respectively. 

The effects of developmental cell interactions are varied. Typically, responding cells 
are diverted from one route of cell differentiation to another, by inducing c-ells that differ 
from both the uninduced and induced states of the responding cells (inductions). Sometimes 
35 cells induce their neighbors to differentiate like themselves (homoiogenetic induction); in 
other cases a cell inhibits its neighbors from differentiating like itself Cell interactions in 
early development may be sequential, such that an initial induction between two cell types 



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wo 95/10611 PCTAJS94/11745 

leads to a progressive amplification of diversity. Moreover, inductive interactions occur not 
only in embryos, but in adult cells as well, and can act to establish and maintain 
morphogenetic patterns as vi^ell as induce differentiation (J.B. Gurdon (1992) Cell 68:185- 
199). 

5 While there has been considerable progress in identifying molecules responsible for 

mesoderm induction, practically nothing is known about the molecular nature of neural 
induction. Candidate neural pattemers are growth factors that are involved in mesoderm 
patterning in earlier stages and become localized later in a subset of cells in the nervous 
system. These molecules include different members of the Wnt, TGF-P and FGF families. 

10 Three members of the Wnt family Wnt-1, Wnt-3 and Wnt-3A, are localized in the roof plate 
(dorsal spinal cord) and a subset of brain cells. Good evidence that Wnt products pattern the 
neural tube comes from homozygote mice lacking the Wnt-1 gene product; these mutant mice 
display a strong abnormality, in the anterior hindbrain and posterior midbrain (a region that 
coincides with engrailed-2 expressing cells)(McMahon et al. (1992) CelL 69:581-595). Vg-1, 

15 BMP-4 (Jones et al. (1991) Development, 111:532-542) and dorsalin-1 (Blumberg et al. 
(1991) Science 253:194-196) are examples or TGF-p family members that display restricted 
expression in the embryonic nervous system (see also, Lyons et al. (1991) Trends Genet 
7:408-412; and Massague et al. (1990) J Biol Chem 265:21393-21396). Dorsalin-l inhibits 
the differentiation of motor neurons and induces migration of neural crest cells and thus may 

20 be involved in dorsal ventral patterning of the neural tube (Blumberg et al. (1991) Science 
253:194-196). Finally acidic FGF (aFGF), basic FGF (bFGF) as well as the newly 
characterized FGF from Xenopus embryos, XeFGF, (Isaacs et al. (1992) Development, 
1 14:71 1-20) are all expressed in some cells of the developing neiu-al tube (Weise et al. (1992) 
Cell & Tissue Research, 276:125-130; and Tannahill et al. (1992) Development, 115:695- 

25 702). 

Since the natural embryonic neural inducer or pattemer has yet to be characterized, 
the analysis of the mechanisms of induction and patterning is difficult. However, studies have 
demonstrated that notochord can induce and pattern neural structures (Jones et al. (1989) 
Development, 107:785-791; and Sharpe et al. (1987) Cell 50:749-758) which implies that 

30 the signals can travel vertically from the axial mesoderm to the overlying ectoderm. The 
finding that neuralization can be induced by mesoderm suggests that neural induction 
involves a signal acting in a paracrine fashion, the transduction of which appears to involve 
protein kinase C (Otte et al. (1991) Science, 251:570-573). A recent series of experiments, 
exploring one of Spemaim^s original ideas, have demonstrated that signals involved in both 

35 induction and patterning of the nervous system can also fravel through the plane of the 
ectoderm (Dixon et al. (1989) Development 106:749-757; Doniach et al. (1992) Science 
257:542-545; and Ruiz i Altaba, A, (1992) Development, 1 15:67-80). It is now accepted that 



SUBSTITUTE SHEET (RULE 26) 



wo 95/10611 PCTAJS94/11745 
both types of mechanisms coexist in the embryo and play a role in neurogenesis. 



Summary of the Invention 

The present invention makes available a method for inducing neuronal differentiation 
5 and preventing the death and/or degeneration of neuronal cells both in vitro and in vivo. The 
subject method stems from the unexpected finding that, contraiy to traditional understanding 
of neural induction, the default fate of ectodermal tissue is neuronal rather than mesodermal 
and/or epidermal. In particular, it has been discovered that preventing or antagonizing a 
signaling pathv^^ay in a cell for a grov^ factor of the TGF-p family (hereinafter "TGF-p-type 

10 grov^ factor"), can result in neuronal differentiation of that cell. In the subject method, 
signaling by the TGF-p-type growth factor is disrupted by antagonizing the inhibitory 
activity of the TGF-P-type growth factor. For instance, this can be accomplished by 
sequestering the grov^ factor with a grov^rth factor binding protein (such as an activin- 
binding protein where the neural-inhibitory grov^rth factor is activin) or by treating with an 

15 antagonist which competes with the grov^ factor for binding to a growth factor receptor on 
the surface of the cell of interest. 

In one embodiment of the subject method, inducing cells to differentiate to a neuronal 
cell phenotype comprises contacting the cells with an agent which antagonizes the biological 
action of at least one polypeptide growth factor of the TGF-p family which normally induces 
20 the cells to differentiate to a non-neuronal phenotype. The antagonizing agent can inhibit the 
biological activity of the TGF-p-type growth factor, for example, by preventing the growth 
factor from binding its receptors on the surface of the treated cells. In another embodiment, 
the antagonizing agent binds to the growth factor and sequesters the growth factor such that it 
cannot bind its receptors. 

25 To further illustrate the invention, the antagonizing agent can be selected from a 

group consisting of a follistatin, an a2-macroglobulin, a protein containing at least one 
follistatin module, and a truncated receptor for a growth factor of the TGF-P family. In the 
instance of the truncated receptor, it can be a soluble growth factor-binding domain of a TGF- 
P receptor, or, in another embodiment, the truncated receptor can be a membrane boimd 

30 receptor and comprises an extracellular growth factor-binding domain of a TGF- P receptor, a 
transmembrane domain for anchoring the extracellular domain to a cell surface membrane, 
and a dysfunctional cytoplasmic domain. In the latter embodiment, the truncated receptor is 
recombinantly expressed in the treated cell. 

In certain embodiments of the present method, the TGF-p-type growth factor which 
35 inhibits neuronal differentiation is an activin. In such instances, the method comprises 



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contacting the cells with an agent which disrupts the activin signaling pathway in the cells, 
causing the cells to default to neuronal differentiation, rather than, for instance, mesodermal 
and/or epidermal fates. 

The present method can be used in vitro, for example, to induce cells in culture to 
5 differentiate to a neuronal phenotype. Moreover, the present method is amenable to 
therapeutic application, and as described below, can be xised to treat neurodegenerative 
disorders associated with, for example, the progressive and persistent loss of neuronal cells, 
such as which occurs with Alzheimer's disease, Parkinson's disease, amyotrophic lateral 
sclerosis. Pick's disease, Huntington's disease, multiple sclerosis, neuronal damage resulting 
10 from anoxia-ischemia, neuronal damage resulting from trauma, and neuronal degeneration 
associated with a natural aging process. 

Detailed Description Of The Invention 

As described herein, a collective group of experiments performed with either a 

15 truncated activin receptor conferring a dominant negative effect, or with a recombinant 
follistatin or inhibin, establish that a signaling pathway of a ^owth factor of the TGF-P 
family is involved in inhibiting neural induction in vivo. The present findings indicate, for 
the first time in vertebrates, that neuralization is a default state. As described in the Examples 
below, our results indicate that activin, or any other member of the TGF-P family that 

20 interacts with the truncated activin receptor, can inhibit neural induction, as these TGF-p 
signals instruct cells towards non-neuronal facts such as epidermal, mesodermal or 
endodermal fate. Inhibition of signal transduction by a TGF-p-type growth factor, by either 
the truncated activin receptor, follistatin, or inhibin, induced cells of the intact animal cap to 
switch to a neuronal fate in the absence of any detectable mesoderm. This data indicates that 

25 presumptive neural tissue in animal caps can respond to TGF-p-type growth factor and form 
mesodermal and/or epidermal tissues, but if this tissue specification by the factor is blocked, 
the cells become neural. As described below, activin is strongly implicated as the TGF-P- 
type growth factor that inhibits neuronal differentiation. Both activin and its receptor are 
present naturally in the animal cap, indicating that an endogenous ;:c:ivity that blocks activin 

30 signaling switches the tissue from an ectodermal to a neural fate, T.;us, endogenous activin 
may act as a neural inhibitor (acting to induce epidermal developmcn and/or mesoderm), and 
neuralization requires the inhibition of activin activity. 

The present invention makes available a method for inducing neuronal differentiation 
and/or preventing the death or degeneration of neuronal cells. In general, the method 
35 comprises contacting a cell, either in vivo or in vitro, with an agent capable of antagonizing 
the bioligical action of a protein from the family of transforming growth factor-Ps. The 



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mechanism of action of the antagonist can, for example, comprise: competitive or non- 
competitive binding to a cell-surface receptor for the growth factor; sequestration of the 
growth factor; or inhibition of signal transduction events mediated by the growth factor 
receptor. Representative embodiments are described in more detail below. 

5 The subject method stems from the unexpected finding that, contrary to traditional 

understanding of netiral induction, the default fate of ectodermal tissue is neuronal rather than 
epidermal. In particular, it has been discovered that preventing or antagonizing a TGF-p-type 
grov^ factor signaling pathway for a cell can result in neuronal differentiation of that cell. 
In the subject method, signaling by the TGF-P-type grov^ factor is disrupted by 
10 antagonizing the inhibitory activity of the TGF-p-type growth factor. For instance, this can 
be accomplished by sequestering the grov^ factor with a growth factor binding protein (such 
as an activin-binding protein) or by treating vnth an antagonist which competes vnth the 
grov^h factor for binding to a growth factor receptor on the surface of the cell of interest. 

As described herein, the present method can be used in vitro, for example, to induce 
15 cells in culture to differentiate to a neuronal phenotype. In one embodiment, the 
differentiated cells are subsequently continued in culture, and can be used to provide useful in 
vitro assay systems as well as valuable research tools for further understanding neural 
development. In another embodiment, the differentiated cells are used in vivo for 
transplantation. Moreover, the present method is amenable to therapeutic application, and as 
20 described below, can be used to treat neurodegenerative disorders associated with, for 
example, the progressive and persistent loss of neuronal cells, such as which occurs with 
Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and Himtington's 
disease. 

While the following description, for clarity, describes the use of agents which 
25 antagonize activin signaling, it is understood that many such agents can also bind or 
otherwise antagonize other TGF-p-type growth factors and thereby disrupt their inhibition, if 
any, of neuralization. As used herein, the terms "transforming growth factor-beta" and "TGF- 
P" denote a family of structurally related paracrine polypeptides found ubiquitously in 
vertebrates, and prototypic of a large family of metazoan growth, differentiation, and 
30 morphogenesis factors (see, for review, Massaque et al. (1990) Ann Rev Cell Biol 6:597-641; 
and Spom et al. (1992) J Cell Biol 119:1017-1021). Moreover, the present invention, namely 
the discovery that neuralization is a defauh state, will readily allow identification of other 
factors, including other TGF-p-like growth factors, which inhibit a cell from reaching this 
default (e.g. actively induce non-neuronal differentiation). In light of this understanding, 
35 agents which disrupt these factors are specifically contemplated by the present invention. 

An agent capable of antagonizing the signaling pathway of a TGF-P factor involved 



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in preventing neuronal differentiation, so as to cause a cell to default to neuronal 
differentiation, is herein referred to as a neuralizing agent, or "NA". 

In one embodiment, the NA is an activin-binding protein which can reduce the 
bioavailability of activin, e.g. by sequestering activin in the extracellular milieu, with 
exemplary activin-binduig agents including foUistatins, a2-macroglobulin, and activin 
receptors. Other activin-binding proteins of the present method can include agrin, agrin- 
related proteins^ and other proteins containing foUistatm modules. In a preferred 
embodiment, the NA has a binding affinity for activin on the order of, or greater than, that of 
either a foUistatin or an activin receptor. 

In an illustrative embodiment of the present method, the NA is a follistatin able to 
bind and sequester activin. FoUistatins are single chain, glycosylated polypeptides that were 
first isolated based on their ability to inhibit follicle-stimulating hormone release, FoUistatins 
from several species, including human, have been structurally characterized and cloned. 
(See, for example, Esch et al. (1987) Mol Endocrinology 11:849; Ling et al. International 
Publication No. WO 89/01945; Ling et al. U.S. Patent Serial No. 5,182,375; Ling et al. U.S. 
Patent Serial No. 5,041,538; and Inouye et al. (1991) Endocrinology 129:815-822). For 
instance, two forms of human follistatm have been cloned and expressed, one having 315 
amino acid residues, and one having 288 amino acid residues. (Inouye et al., aypGi). Human 
follistatin is available through the National Hormone and Pituitary Program of the NIH. In a 
one embodiment, the folUstatin of the present method is of the class of shorter foUistatins 
(e.g. the 288 a.a. human homolog), since, as described below, these forms appear to have a 
greater bmding affinity for activin, relative to the larger forms of follistatin. 

In another exemplary embodiment, the activin-binding protein can be an activm 
receptor, or portion thereof Activin receptors have also been cloned from several species. 
(Attisano et al. (1992) Cell 68:97-108; and Gerogi et al. (1990) Cell 61:635-645). In 
embodiments of the present invention in which it is desirable for the NA to be a diffrisible 
molecule, a soluble extracellular portion of an activin receptor can be used, provided the 
extracellular portion is chosen so as to retain activin-binding. For example, a soluble form of 
an activin receptor can be generated using the cloned activin receptor gene of Attisano et al., 
which includes an endogenous signal sequence for secretion (Attisano et al. (1992) Cell 
68:97-108). For mstance, a stop codon can be introduced at a site 5' of the gene encoding the 
transmembrane domain (e.g. the ACC encoding Thr-134 can be mutated to TAA). Moreover, 
as described below, the truncated activin receptor can be engineer-ed as fusion protein to 
include other polypeptide sequences. 

In yet another illustrative embodunent of the present invention, the cell can be 
contacted wath an activin antagonist which inhibits activin binding to its cognate receptor on 




wo 95/1061 1 PCT/US94/1 1 745 

-6- 

the treated cell by competitively, or non-competitively, binding to the receptor protein. Such 
neuralizing agents can be utilized to block activin signaling and thereby induce the treated 
cell to undergo neuronal differentiation or to maintain its existing neuronal differentiation. A 
number of potential activin antagonists of this type are known in the art, including the family 
5 of inhibins. Inhibins and activins were first isolated and purified from follicular fluid on the 
basis of their ability to inhibit (inhibin) or stimulate (activin) FSH release by pituitary cells. 
Mature inhibin is typically a heterodimeric glycoprotein composed of a common a-subunit 
and one of two P subunits, Pb- I" addition to inhibins, the subject invention can be 

carried out using activins that have been mutagenized to create activin variants which act 

10 antagonistically to activin in neuronal induction. Activins are homodimeric forms of inhibin 
P-subunits (e.g. p^p^ or PaPb)- Such antagonists can be generated, for example, by 
combinatorial mutagenesis techniques well known in the art (See, for example, Ladner et al. 
PCT publication WO 90/02909; Garrard et al., PCX publication WO 92/09690; Marks et al. 
(1992) J. Biol. Chem. 267:16007-16010; GriffUis et al. (1993) EMBO J 12:725-734; 

15 Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461). 
Furthermore, peptidomimetics (e.g. of activin or inhibin) or other small molecules, such as 
may be identified in the assays set out below, can be used to antagonize activin signalling by 
binding to the receptor and precluding functional binding (or receptor oligomerization) by 
activin. 

20 In still further embodiments, the neuralizing agent acts to block signal transduction by 

the activin receptor irrespective of activin binding. Such agents include dominant negative 
receptors which, unlike the soluble form of the receptor, are membrane boimd, e.g. which 
include a transmembrane domain and at least a portion of a cytoplasmic domain. Such 
receptors, rather than merely sequestering activin fi-om functional receptors, are incapable of 

25 activating appropriate intracellular second messenger pathways in response to activin 
binding. Expression of these dominant negative receptors in cells expressing wild-type 
receptor can render the cells substantially insensitive to activin, e.g. by formation of non- 
productive oligomers with the wild-type receptor. Likewise, other agents which inhibit the 
activin receptor second messenger pathways downstream of the activin receptor can be used 

30 to inhibit activin-mediated induction of cells. 

Yet another embodiment of the subject assay features the use of an isolated nucleic 
acid construct for inhibiting synthesis of an activin receptor in the targeted cell by "antisense" 
therapy. As used herein, "antisense" therapy refers to administration or in situ generation of 
oligonucleotide probes or their derivatives which specifically hybridizes (e.g. binds) under 
35 cellular conditions, with the cellular mRNA and/or genomic DNA encoding an activin 
receptor so as to inhibit expression of that receptor, e.g. by inhibiting transcription and/or 
translation. The binding may be by conventional base pair complementarity, or, for example. 



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in the case of binding to DNA duplexes, through specific interactions in the major groove of 
the double helix. In general, "antisense" therapy refers to the range of techniques generally 
employed in the art, and includes any therapy which relies on specific binding to 
oligonucleotide sequences. 

5 An antisense construct of the present invention can be delivered, for example, as an 

expression plasmid which, when transcribed in the cell, produces RNA which is 
complementary to at least a unique portion of the cellular mRNA which encodes an acitivin 
receptor. Alternatively, the antisense construct can be an oligonucleotide probe which is 
generated ex vivo and which, when introduced into the treated cell causes inhibition of 

10 expression by hybridizing with the mRNA and/or genomic sequences of an activin receptor 
gene. Such oligonucleotide probes are preferably modified oligonucleotide which are 
resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and is therefore 
stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are 
phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. 

15 Patents 5,176,996; 5^64,564; and 5,256,775). Additionally, general approaches to 
constructing oligomers usefiil in antisense therapy have been reviewed, for example, by van 
der Rrol et al. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659- 
2668. 

In certain embodiments, when appropriate, the neuralizing agent can be a chimeric 

20 protein comprising a moiety that binds a component of the extracellular matrix. Such a 
chimeric NA can be usefiil in circumstances wherein diffusion of the NA fi-om a treatment 
site is undesirable, and will fimction to such an end by virtue of localizing the chimeric NA at 
or proximate a treatment site. An NA of this embodiment can be .generated as the product of 
a fiision gene, or by chemical cross-linking. For instance, a number of proteins have been 

25 characterized fi-om the extracellular matrix (ECM) of tissues that will support the localization 
of a chimeric NA at a target site. One example of a well characterized protein is fibronectin. 
Fibronectin is a large adhesive glycoprotein with multiple functional domains. Several of 
these domains have matrix attachment activity. For example, one of these is a single "type- 
Ill repeat" which contains a tetrapeptide sequence R-G-D-S (Pierschbacher et al. (1984) 

30 Nature 309:30-3; and Komblihtt et al. (1985) EMBO 4:1755-9). Peptides as small as 
pentapeptides containing these amino acids are able to support attachment to a cell through 
binding ECM components (Ruoslahti et al. (1987) Science 238:^ ' 1-497; Pierschbacheret al. 
(1987) J BioL Chem. 262:17294-8.; Hynes (1987) Cell 48:549-54; and Hynes (1992) Cell 
69:11-25). In fact, several companies have commercialized products based on this cell 

35 attachment sequence for use as reagents in cell culture and various biomaterials applications. 
See for example recent catalogs from Telios Pharmaceutical, BRL, Stratagene, Protein 
Polymer Technologies etc., as well as U.S. Patent Nos. 4,517,686; 4,589,881; 4,578,079; 



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4,614,517; 4,661,111; and 4,792,525. Accordingly, fibronectin binding sequences can be 
added, for example, to the soluble activin receptor described herein. 

Another aspect of the present invention relates to a method of inducing and/or 
maintaining a differentiated state, and/or enhancing survival of a neural cell responsive to 
activin induction, by contacting the cell with a neuralizing agent (e.g. an activin antagonist). 
For instance, it is contemplated by the invention that, in light of the present finding of an 
apparently broad involvement of activin in the formation of ordered spatial arrangements of 
differentiated neural tissues in vertebrates, the subject method could be used to generate 
and/or maintain an array of different neural tissue ibo& in vitro and in vivo. The neuralizing 
agent can be, as appropriate, any of the preparations described above, including isolated 
polypeptides, gene therapy constructs, antisense molecules, peptidomimetics or agents 
identified in the drug assays provided herein. 

For example, the present method is applicable to cell culture technique. In vitro 
neuronal culture systems have proved to be fundamental and indispensable tools for the study 
of neural development, as well as the identification of neurotrophic factors such as nerve 
growth factor (NGF), ciliary trophic factors (CNTF), and brain derived neurotrophic factor 
(BDNF). Once a neuronal cell has become terminally-differentiated it typically will not 
change to another terminally differentiated cell-type. However, neuronal cells can 
nevertheless readily lose their differentiated state. This is commonly observed when they are 
grown in culture from adult tissue, and when they form a blastema during regeneration. The 
present method provides a means for ensuring an adequately restrictive environment in order 
to maintain neuronal cells at various stages of differentiation, and can be employed, for 
instance, in cell cultures designed to test the specific activities of other trophic factors. In 
such embodiments of the subject method, the cultured cells can be contacted with a 
neuralizing agent of the present invention in order to induce neuronal differentiation (e.g. of a 
stem cell), or to maintain the integrity of a culture of terminally-differentiated neuronal cells 
by preventing loss of differentiation. The source of the neuralizing agent in the culture can be 
derived from, for example, a purified or semi-purified protein composition added directly to 
the cell culture media, or alternatively, released from a polymeric device which supports the 
growth of various neuronal cells and which has been doped with the neuralizing agent. If 
appropriate, the source of the neuralizing agent can also be a cell that is co-cultured with the 
intended neuronal cell and which produces a recombinant neuralizing agent. Alternatively, 
the source can be the neuronal cell itself which as been engineered to produce a recombinant 
neuralizing agent. In an exemplary embodiment, a naive neuronal cell (e.g. a stem cell) is 
treated with an activin antagonist in order to induce differentiation of the cells into, for 
example, sensory neurons or, alternatively, motomeurons. Such neuronal cultures can be 
used as convenient assay systems as well as sources of implantable cells for therapeutic 



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To further illustrate potential uses, it is noted that intracerebral grafting has emerged 
as an additional approach to central nervous system therapies. For example, one approach to 
repairing damaged brain tissues involves the transplantation of cells from fetal or neonatal 
5 animals into the adult brain (Dunnett et al. (1987) J Exp Biol 123:265-289; and Freund et al. 
(1985) J Neurosci 5:603-616). Fetal neurons from a variety of brain regions can be 
successfully incorporated into the adult brain, and such grafts can alleviate behavioral 
defects. For example, movement disorder induced by lesions of dopaminergic projections to 
the basal ganglia can be prevented by grafts of embryonic dopaminergic neurons. Complex 

10 cognitive functions that are impaired after lesions of the neocortex can also be partially 
restored by grafts of embryonic cortical cells. Thus, use of activin antagonist for 
maintenance of neuronal cell cultures can help to provide a source of implantable neuronal 
tissue. The use of a neuralizing agent in the culture can be to prevent loss of differentiation, 
or where fetal tissue is used, especially neuronal stem cells, a neuralizing agent of the present 

15 invention can be used to induce differentiation. 

Stem cells useful in the present invention are generally known. For example, several 
neural crest cells have been identified, some of which are multipotent and likely represent 
uncommitted neural crest cells, and others of which can generate only one type of cell, such 
as sensory neurons, and likely represent committed progenitor cells. ThcTole of an activin- 

20 disrupting agent employed in the present method to culture such stem cells can be to induce 
differentiation of the xmconmiitted progenitor and thereby give rise to a committed progenitor 
cell, or to cause further restriction of the developmental fate of a committed progenitor cell 
towards becoming a terminally-differentiated neuronal cell For example, the present method 
can be used in vitro to induce and/or maintain the differentiation of neural crest cells into 

25 glial cells, Schwann cells, chromaffin cells, cholinergic sympathetic or parasympathetic 
neurons, as well as peptidergic and serotonergic neurons. The neuralizing agent can be used 
alone, or can be used in combination with other neurotrophic factors which act to more 
particularly enhance a particular differentiation fate of the neuronal progenitor cell. In the 
later instance, the neuralizing agent might be viewed as ensuring that the treated cell has 

30 achieved a particular phenotypic state such that the cell is poised along a certain 
developmental pathway so as to be properly induced upon contact with a secondary 
neurotrophic factor. In similar fashion, even relatively xmdifferentiated stem cells or 
primative neuroblasts can be maintained in culture and caused to differentiate by treatment 
with the subject neuralizing agents. Exemplary primative cell cultures comprise cells 

35 harvested from the nueral plate or neural tube of an embryo even before much overt 
differentiation has occurred. 

The method of the present invention will also facilitate further determination of a 



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potential role of follistatin as a "morphogen", that is, a molecule whose tight threshold of 
concentration determines specific cell fate during development (Wolpert, L. (1969) 1 Theor 
Biol 25:1-47). One of the first molecules to qualify as a morphogen was bicoid, a DNA 
binding protein whose graded distribution in the syncytium of the Drosophila embryo leads to 
5 the generation of specific cell fates (Driever et al. (1988) Cell 54:95-1 04). More recently two 
factors, activin in Xenopus embryos (Green et al. (1992) Cell 71:731-739) and 
decapentaplegic (dpi), (Ferguson et al. (1992) Cell 71 :45M61)) in Drosophila embryos have 
been showed to act as morphogens in vitro. Both of these factors belong to the TGF-P 
superfamily of peptide growth factors and both can specify different cell fates at tight 

10 thresholds of concentration. Since follistatin is an inhibitor of activin and both activin ligand 
and receptor RNAs are expressed in the presumptive ectoderm, follistatin, like activin, may 
have morphogenic activity. Both the dominant negative activin receptor and follistatin, as 
described below, elicit neural tissue formation directly. Based on this data, it is asserted that 
neural tissue represents the default state vis-a-vis activin signaling in presumptive ectoderm. 

15 In this model, the amount of activin ligand and receptor present in the cap maintains the cells 
as epidermal, and additional activin changes the cells* fate to mesodermal. By inhibiting 
activin to varying degrees, follistatin could also act as a morphogen. 

In an illustrative embodiment of an in vitro assay system, to test if follistatin (or 
another NA) can act as a morphogen, dissociated animal cap cells can be cultured and dosed 

20 with activin at a concentration sufficient to turn on a general mesodermal marker such as 
brachyury upon reassociation. The activity of activin can then be challenged with small 
incremental changes in follistatin protein concentration. Indicators that follistatin might serve 
as a morphogen will include the observation of small concentration differences giving rise to 
different cell fates, distinguishable by histology or through the use of cell-type specific 

25 molecular markers. These studies will allow for the determination of the extent of the 
number of independent cellular fates that exist in the presumptive ectoderm as well as, what 
proportion of animal cap cells become neural in response to different concentrations of 
follistatin. 

Similar studies can be perfomned with stem cells, such as the neural crest cells 
30 described above, to determine if the concentration of follistatin (or other NA) is influential on 
the path of neuronal differentiation of xmcommitted and committed progenitor cells, and 
ultimately whether concentration effects the particular terminally-differentiated derivation of 
the progenitor cells which arise. 

In another embodiment, in vitro cell cultures can be used for the identification, 
35 isolation, and study of genes and gene products that are expressed in response to disruption of 
activin signaling, and therefore likely involved in neurogenesis. These genes would be 
"downstream" of the activin signal, and required for neuronal differentiation. For example, if 



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new transcription is required for the neuralization, a subtractive cDNA library prepared with 
control animal caps and animal caps treated with follistatin can be used to isolate genes that 
are turned on or turned off by this process. The powerful subtractive library methodology 
incorporating PCR technology described by Wang and Brown is an example of a 
5 methodology useful in conjunction with the present invention to isolate such genes (Wang et 
al. (1991) Proc.NatLAcadScL USA 88:11505-11509). For example, this approach has been 
used successfully to isolate more than sixteen genes involved in tail resorption vnih and 
without thyroid hormone treatment in Xenopus. Utilizing control and treated caps, the 
induced pool can be subtracted from the uninduced pool to isolate genes that are turned on, 

10 and then the iminduced pool from the induced pool for genes that are turned off. From this 
screen, it is expected that two classes of mRNAs can be identified. Class I RNAs would 
include those RN/V . pressed in untreated caps and reduced or eliminated in induced caps, 
that is ' ■ down-; -? -lated population of RNAs. Class II RNAs include RNAs that are 
upre*. in respoxise to induction and thus more abundant in treated than in untreated 

15 caps. ^ extracted from treated vs untreated caps can be used as a primary test for the 
classil :m of the clones isolated from the libraries. Clones of each class can be further 
charac: - d by sequencing and, their spatiotemporal distribution determined in the embryo 
by whole mount in situ and developmental northern blots analysis. 

For example, in one embodiment of this subtractive assay, special attention can be 

20 given to genes that prove to be an immediate early response to neural induction. To qualify as 
such, these genes should fulfill the following four criteria. First, the RNA should appear 
quickly (10 to 30 minutes) following application of the inducer. To test this requirement, 
RNA can be isolated at different times from induced caps and scored for gene expression by 
northern blots. Second, the induction of the gene should not require previous protein 

25 synthesis. Thus, caps can be incu ^vith eye' heximide (S^ig/ml) prior to and during short 
incubation with follistatin (30 m ' after -h the caps -can be allowed to remain in 
follistatin fc nger periods of tir - ) minuie^) and then analyzed by northern blotting. 
This strateg y ■ ^ been used in a S; l : situation when Mix. 1, a homeobox gene exhibiting 
an immediate :^ly response to boiii activin and bFGF was isolated from Xenopus animal 

30 caps (Rosa, F.M. (1989) Cell 57:965-974). These conditions are sufficient to inhibit ^5% to 
80% of the protein synthesis during f^e period of indu n and to abolish the indt on of 
muscle actin mRNA in response tc n in late explai >lce et al. (1993) Devel .mntal 
Biology 160). Thir ■ immediate isponse genei Id be expressed as a . jsult of 

contact with the : acer and no. a secondary Ji :ell induction. One nv^tbod to 

35 differentiate be- these two re •;>■: is to dissociate ^ cells of the animal cap in Ca/Mg 
free medium, ; ■ distatin and « on'jp e the amount of the induced transcript in dissociated 
cells versus inia<: :aps. If the levt' ^ ^e comparable in both types of caps, then it may be 
concluded that ceh-cell contact was not required for this induction and it is thus likely a direct 



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response of follistatin treatment. Finally, these genes would be expected to be present and 
activated in the nervous system during neurogenesis. 

Once isolated, the genes regulated by follistatin can be sequenced and their embryonic 
distribution can be determined by vi^holemount approaches. If their embryonic expression is 
5 in agreement with a possible neurogenic function, they can be tested for neuralizing activity 
in animal caps and in embryos as described herein for follistatin and other NAs. 

Moreover, the present invention provides assays for identifying novel neuralizing 
agents. For example, an assay can comprise animal cap cells, or equivalent cells thereof, 
cultured in the presence of a TGF-P-type factor which inhibits neuralizationm such as activin. 
10 A portion of the cells are contacted with a candidate agent, and neuronal differentiation of 
any of the cells, is scored for by the presence of a neuronal marker, such as NCAM, being 
expressed by the cells. 

Other embodiments of the assay can score simply for the ability of an added agent to 
inhibit protein-protein interaction between a TGF-P and its cognate receptor. For instance, 
15 in one embodiment, the assay evaluates the ability of a compoimd to modulate binding 
between an activin polypeptide and ah activin receptor. A variety of assay formats will 
suffice and, in light of the present inventions, will be comprehended by skilled artisan. 

In many drug screening programs which test libraries of compotmds and natural 
extracts, high throughput assays are desirable in order to maximize the number of compounds 

20 surveyed in a given period of time. Assays which are performed in cell-free systems, such as 
may be derived with purified or semi-purified proteins, are often preferred as "primary" 
screens in that they can be generated to permit rapid development and relatively easy 
detection of an alteration in a molecular target which is mediated by a test compound. 
Moreover, the effects of cellular toxicity and/or bioavailability of the test compound can be 

25 generally ignored in the in vitro system, the assay instead being focused primarily on the 
effect of the drug on the molecular target as may be manifest in an alteration of binding 
affinity v^th receptor proteins. Accordingly, in an exemplary screening assay of the present 
invention, the compound of interest is contacted with an activin receptor polypeptide which is 
ordinarily capable of binding an activin protein. To the mixture of the compound and 

30 receptor is then added a composition containing an activin polypeptide. Detection and 
quantification of receptor/activin complexes provides a means for determining the 
compoimd's efficacy at inhibiting complex formation between the receptor protein and the 
activin polypeptide. The efficacy of the compotmd can be assessed by generating dose 
response curves from data obtained using various concentrations of the test compound. 

35 Moreover, a control assay can also be performed to provide a baseline for comparison. In the 
control assay, isolated and purified activin polypeptide is added to a composition containing 



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the receptor protein, and the formation of receptor/activin complex is quantitated in the 
absence of the test compoimd. 

Complex formation between the activin polypeptide and an activin receptor may be 
detected by a variety of techniques. For instance, modulation of the formation of complexes 
5 can be quantitated using, for example, detectably labelled proteins such as radiolabelled, 
fluorescently labelled, or enzymatically labelled activin polypeptides, by immunoassay, or by 
chromatographic detection. 

Accordingly, a wide range of agents can be tested, such as proteins and polypeptide: 
as well as peptidomimetics and other small molecules (including natural products). For 

10 instance, a drug screening assay described above can be used in the reduction of the activin of 
inhibin proteins to generate mimetics, e.g. peptide or non-pepti'?e agents, which are able to 
disrupt binding of an activin polypeptide of the present invention with an activin receptor. 
By employing, for example, scanning mutagenesis to map the critical amino acid residues of 
the activin protein involved in binding the activin receptor, peptidomimetic compounds can 

15 be generated which mimic those residues in bindin£: lo the receptor and which consequently 
can inhibit binding of activin to its receptor, as may be detected in a screeing assay as 
described herein. For instance, non-hydrolyzable peptide analogs of such residues can be 
generated using benzodiazepine (e.g., see Freidinger et al. in Peptides: Chemistry and 
Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see 

20 Huffman et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: 
■ Jen, Netherlands, 1988), substituted gama lactam rings (Garvey et al. in Peptides: 
i emistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), 
keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295; and Ewenson et 
al. in Peptides: Structure and Function (Proceedings of the 9th American Peptide 

25 Symposium) Pierce Chemical Co. Rockland, IL, 1985), P-tum dipeptide cores (Nagai -et al. 
(1985) Tetrahedron Lett 26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231), 
and p-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun\26'A\9\ and Dann 
et al. ( 1 986) Biochem Biophys Res Commun 1 34:7 1 ). 

In addition to the implantation of cells cultured in the presence of an NA and other in 
30 vitro uses described above, yet another objective of the present invention concerns the 
therapeutic application of activin-disrupting agents to enhance survival of neurons and other 
neuronal cells in both the central nervous system and the peripheral nervous system. The 
ability of folHstatin to regulate neuronal differentiation not only during development of the 
nervous system but also presumably in the adult state indicates that NAs can be reasonably 
35 expected to facilitate control of adult neurons with regard to maintenance, functional 
performance, and aging of normal cells; repair and regeneration processes in chemically or 
mechanically lesioned cells; and prevention of degeneration and death which result from loss 



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of differentiation in certain pathological conditions. In light of this understanding, the 
present invention specifically contemplates applications of the subject method to the 
treatment of (prevention and/or reduction of the severity of) neiirological conditions deriving 
from: (i) acute, subacute, or chronic injury to the nervous system, including traumatic injury, 
5 chemical injury, vasal injury and deficits (such as the ischemia resulting from stroke), 
together with infectious/inflammatory and tumor-induced injury; (ii) aging of the nervous 
system including Alzheimer's disease; (iii) chronic neurodegenerative diseases of the 
nervous system, including Parkinson's disease, Huntington's chorea, amylotrophic lateral 
sclerosis and the like, as well as spinocerebellar degenerations; (iv) chronic immimological 

10 diseases of the nervous system or affecting the nervous system, including multiple sclerosis; 
and (v) degenerative diseases of the retina- 
Many neurological disorders are associated with degeneration of discrete populations 
of neuronal elements and may be treated with a therapeutic regimen which includes a 
neuralizing agent of the present invention. For example, Alzheimer's disease is associated 

15 vwth deficits in several neurotransmitter systems, both those that project to the neocortex and 
those that reside with the cortex. For instance, the nucleus basalis in patients with 
Alzheimer's disease were observed to have a profound (75%) loss of neurons compared to 
age-matched controls. Although Alzheimer's disease is by far the most common form of 
dementia, several other disorders can produce dementia. Many are age-related, occurring in 

20 far greater incidence in older people than in younger. Several of there are degenerative 
diseases characterized by the death of neiu-ons in various parts of the central nervous system, 
especially the cerebral cortex. However, some forms of dementia are associated with 
degeneration of the thalmus or the white matter underlying the cerebral cortex. Here, the 
cognitive dysfunction results from the isolation of cortical areas by the degeneration of 

25 efferents and afferents. Himtington's disease involves the degeneration of intrastraital and 
cortical cholinergic neurons and GABAergic neurons. Pick's disease is a severe neuronal 
degeneration in the neocortex of the frontal and anterior temporal lobes, sometimes 
accompanied by death of neurons in the striatum. Treatment of patients suffering from such 
degenerative conditions can include the application of neuralizing agent polypeptides, or 

30 agents which mimic their effects, in order to manipulate, for example, the de-differentiation 
and apoptosis of neurons which give rise to loss of neurons. In preferred embodiments, a 
source of a neuralizing agent agent is stereotactically provided within or proximate the area 
of degeneration. 

In addition to degenerative-induced dementias, a pharmaceutical preparation of a 
35 neuralizing agent can be applied opportunely in the treatment of neurodegenerative disorders 
which have manifestations of tremors and involxmtary movements. Parkinson's disease, for 
example, primarily affects subcortical structures and is characterized by degeneration of the 



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nigrostriatal pathway, raphe nuclei, locus cereleus, and the motor nucleus of vagus. Ballism 
is typically associated with damage to the subthalmic nucleus, often due to acute vascular 
accident. Also included are neurogenic and myopathic diseases which ultimately affect the 
somatic division of the peripheral nervous system and are manifest as neuromuscular 
5 disorders. Examples include chronic atrophies such as amyotrophic lateral sclerosis, 
Guillain-Barre syndrome and chronic peripheral neuropathy, as well as other diseases which 
can be manifest as progressive bulbar palsies or spinal muscular atrophies. The present 
method is ammenable to the treatment of disorders of the cerebellum which result in 
hypotonia or ataxia, such as those lesions in the cerebellum which produce disorders in the 
10 limbs ipsilateral to the lesion. For instance, a preparation of a neuralizing agent of the 
present invention can be used to treat a restricted form of cerebellar corical degeneration 
involving the anterior lobes (vermis and leg areas) such as is common in alcoholic patients. 

In yet another embodiment, the subject method is used to treat amyotrophic lateral 
sclerosis. ALS is a name given to a complex of disorders that comprise upper and lower 

15 motor neurons. Patients may present with progressive spinal muscular atrophy, progressive 
bulbar palsy, primary lateral sclerosis, or a combination of these conditions. The major 
pathological adnomality is characterized by a selective and progressive degeneration of the 
lower motor neurons in the spinal cord and the upper motor neurons in the cerebral cortex. 
The therapeutic application of an activin antagonist can be used alone or in conjunction with 

20 other neurotrophic factors such as CNTF, BDNF, or NGF to prevent and/or reverse motor 
neuron degeneration in ALS patients. 

The neuralizing agents of the present invention can also be used in the treatment of 
autonomic disorders of the peripheral nervous system, which include disorders affecting the 
innervation of smooth muscle endocrine tissue (such as glandular tissue). For instance, 
25 neuralizing agent compositions may be useful to treat tachycardia or atrial cardiac arrythmias 
which may arise from a degenerative condition of the nerves innervating the striated muscle 
of the heart. 

In yet another embodiment, the subject neuralizing agents can be used in Ae 
treatment of neoplastic or hyj^rplastic transformations, involving neural tissue. For instance, 

30 an activin antagonist likely to be capable of inducing differentiation of transformed neuronal 
cells to become post-mitotic or possibly apoptotic. Inhibition of activin-mediated inductive 
events may also involve disruption of autocrine loops, such as PDGF autostimulatory loops, 
believed to be involved in the neoplastic transformation of several neuronal tumors. The 
subject method may, therefore, be of use in the treatment of, for example, malignant gliomas, 

35 meduUoblastomas, neuroectodermal tumors, and ependymonas. 

The NA, or a pharmaceutically acceptable salt thereof, may be conveniently 



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formulated for administration with a biologically acceptable medium, such as water, buffered 
saline, poJyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the 
like) or suitable mixtures thereof. The optimum concentration of the active ingredient(s) in 
the chosen medium can be determined emperically, according to procedures well known to 
medicinal chemists. As used herein, "biologically acceptable medium" includes any and all 
solvents, dispersion media, and the like which may be appropriate for the desired route of 
administration of the pharmaceutical preparation. The use of such media for 
pharmaceutically active substances is known in the art. Except insofar as any conventional 
media or agent is incompatible with the activivity of the NA, its use in the pharamceutical 
preparation of the invention is contemplated. Suitable vehicles and their formulation 
inclusive of other proteins are described, for example, in the book Reminpon's 
Pharmaceytical Science^ (Remington*s Pharmaceutical Sciences. Mack Publishing Company, 
Easton, Pa., USA 1985). These vehicles include injectable "deposit formulations". Based on 
the above, the pharmaceutical formulation includes, although not exclusively, NA solutions 
or a freeze-dried powder of an NA (such as a follistatin) in association with one or more 
pharmaceutically acceptable vehicles or diluents, and contained in buffered media at a 
suitable pH and isosmotic with physiological fluids. For illustrative purposes only and 
without being limited by the same, possible composition of formulations which may be 
prepared in the form of solutions for the treatment of nervous sytem disorders with an NA are 
given in the della Valle U.S. Patent No. 5,218,094. In the case of freeze-dried preparations, 
supporting excipients such as, but not exclusively, mannitol or glycine may be used and 
appropriate buffered solutions of the desired volume will be provided so as to obtain adequate 
isotonic buffered solutions of the desired pH. Similar solutions may also be used for the 
pharmaceutical compositions of the NA in isotonic solutions of the desired volume and 
include, but not exclusively, the use of buffered saline solutions with phosphate or citrate at 
suitable concentrations so as to obtain at all times isotonic pharmaceutical preparations of the 
desired pH, for example, neutral pH. 

Methods of introduction of the NA at the site of treatment include, but are not limited 
to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, and 
intranasal. In addition, it may be desirable to introduce the pharmaceutical compositions of 
the invention into the central nervous system by any suitable route, including intraventricular 
and intrathecal injection; intraventricular injection may be facilitated by an intraventricular 
catheter, for example, attached to a reservoir, such as an Ommaya reservoir. 

Methods of introduction may also be provided by rechargable or biodegradable 
devices. Various slow release polymeric devices have been developed and tested in vivo in 
recent years for the controlled delivery of drugs, including proteinacious biopharmaceuticals. 
A variety of biocompatible polymers (including hydrogels), including both biodegradable and 



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non-degradable polymers, can be used to form an implant for the sustained release of an NA 
at a particular target site. Such embodiments of the present invention can be used for the 
delivery of an exogenously purified NA, which has been incorporated in the polymeric 
device, or for the delivery of an NA produced by a cell encapsulated in the polymeric device. 

5 An essential feature of certain embodiments of the implant is the linear release of the 

NA, which can be achieved through the manipulation of the polmer composition and form. 
By choice of monomer composition or polymerization technique, the amount of water, 
porosity and consequent penneability characteristics can be controlled. The selection of the 
shape, size, polymer, and method for implantation can be determined on an individual basis 

10 according to the disorder to be treated and the individual patient response. The generation of 
such implants is generally known in the art. See, for example, Concise Encvlopedia of 
Medical & Dental Materials , ed. by David Williams (MIT Press: Cambridge, MA, 1990); 
and the Sabel et al. U.S. Patent No. 4,883,666. In another embodiment of an implant, a 
source of cells producing the NA, or a solution of hydogel matrix containing purifed NA, is 

15 encapsulated in implantable hollow fibers. Such fibers can be pre-spvm and subsequently 
loaded with the NA source (Aebischer et al. U.S. Patent No. 4,892,538; Aebischer et al. U.S. 
Patent No. 5,106,627; Hof&nan et al. (1990) Expt. Neurobiol 1 10:39-44; Jaeger et al. (1990) 
Prog. Brain Res, 82:41-46; and Aebischer et al. (1991) J. Biomech. Eng, 113:178-183), or 
can be co-extruded with a polymer which acts to form a polymeric coat about the NA sotirce 

20 (Lim U.S. Patent No. 4,391,909; Sefton U.S. Patent No. 4,353,888; Sugamori et al. (1989) 
Trans, Am, Artif Intern. Organs 35:791-799; Sefton et al. (1987) Biotehnol Bioeng. 
29:1 135-1 143; and Aebischer et al. <1991) Biomaterials 12:50-55). 

In yet another embodiment of the present invention, the neuralizing agent ^:an be 
administered as part of a combinatorial therapy with other agents. For example, the 

25 combinatorial therapy can include an neuralizing agent such as follistatin with at least one 
trophic factor. Exemplary trophic factors include nerve growth factor, cilliary neurotrophic 
growth factor, schwanoma-derived growth factor, glial growth factor, stiatal-derived 
neuronotrophic factor, platelet-derived growth factor, and scatter factor (HGF-SF). Other 
neural inductive proteins, such as hedgehogAik^ proteins, noggin, and ligands of the Notch 

30 receptor, may also be used in conjunction with the subject neuralizing agent. Antimitogenic 
agents can also be used, as for example, when proliferation of surrounding glial cells or 
astrocytes is undesirable in the regeneration of nerve cells. Examples of such antimitotic 
agents include cytosine, arabinoside, 5-fluorouracil, hydrozyurea, and methotrexate. 

Moreover, certain of the neuralizing agents, such as the dominant negative activin 
35 receptors (either soluble of membrane bound), may be ammenable to delivery by gene 
therapy. For instance, expression constructs of the subject dominant negative receptors may 
be administered in any biologically effective carrier, e.g. any formulation or composition 



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capable of effectively delivering the dominant negative receptor gene to cells in vivo. 
Approaches include insertion of the mutant receptor gene in viral vectors including 
recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus- 1, or 
recombinant bacterial or eukaiyotic plasmids. While viral vectors transfect cells directly, 
plasmid DNA can also be delivered with the help of, for example, cationic liposomes 
(lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates, gramacidin S, 
artificial viral envelopes or other such intracellular carriers, as well as direct injection of the 
gene construct or CaP04 precipitation carried out in vivo. It will be appreciated that because 
transduction of appropriate target cells represents the critical first step in gene therapy, choice 
of the particular gene delivery system will depend on such factors as the phenotype of the 
intended target and the route of administration, e.g. locally or systemically. Furthermore, it 
will be recognized that the particular gene construct provided for in vivo transduction of 
activin expression are also useful for in vitro transduction of cells, such as for use in the ex 
vivo tissue culture systems described above. 



The fact that neuralization was closely linked to mesoderm induction has hampered 
most of the previous effort invested in the molecular characterization of neural inducers and 
pattemers. Thus, attempts to isolate factors involved in neural induction and patterning have 
ended in the identification and characterization of mesoderm inducers and modifiers. The 
data described in the Examples below demonstrate that two activin antagonists, the dominant 
negative form of the activin receptor (AlXARl) and follistatin both elicit direct neuralization 
in embryonic explants without a prerequirement for mesoderm induction. In addition, a full 
length cDNA for Xenopus follistatin has been isolated and its embryonic localization shown 
to be in perfect agreement with a role in neural development in vivo. The observation that 
antagonizing the activin signal results in neuralization suggests, for the first time, that neural 
induction in vertebrates represents a default state. 

Additionally, as described in the Examples below, the truncated activin receptor does 
not block mesoderm induction by exogenous FGF in animal caps, and yet endogenous FGF 
does not induce mesoderm in a significant fi*action of embryos injected with AlXARl. 
Furthermore, when half the embryo is injected with AlXARl, that half lacks Xbra expression 
in all embryos tested (n=25) even though FGF is a potent inducer of brachyury in the animal 
cap assay. These data indicates that endogenous FGF signaling is not sufficient to rescue 
brachyury expression or mesoderm induction in the marginal zone of embryos injected with 
AlXARl . At the same time, it is clear fi*om other experiments with a dominant negative FGF 
receptor that FGF plays an important role in axial patterning, particularly for posterior 



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Structures (Amaya et al. (1991) Cell. 66:257-270). Taken together, these findings raise the 
possibility that FGF signaling at the time of mesoderm induction requires a functional activin 
pathway. 

The invention now being generally described, it will be more readily understood by 
5 reference to the following examples which are included merely for purposes of illustration of 
certain aspects and embodiments of the present invention, and are not intended to limit the 



To demonstrate the ertion that neuralization represents a default state requiring the 
inhibition of endogenous activin molecules, the ability of a dominant negative activin 
receptor to induce neuralization ectopically in embryos was assigned. A truncated version of 

15 XARl was constructed to contain the entire extracellular and transmembrane domains but 
which lacks nearly all of the cytoplasmic domain, including the serine/threonine kinase. To 
construct AlXARl, a fragment of DNA from XARl (Hemmati-Brivanlou et al. (1992) Dev 
Dyn 194:1-1 1) encoding the entire extracelltilar domain (including the signal sequence), the 
transmembrane domain and 10 amino acid residues of the cytoplasmic domain and entirely 

20 free of 5' and 3* untranslated sequences was subcloned into pSP64T (Kreig et al. (1984) Nuc 
Acid Res 12:7057-7070). The linearized plasmid was transcribed in vitro with SP6 to 
generated capped sense RNA. The resulting RNA encoding the truncated activin receptor was 
injected into cells of either wild type or ventralized embryos. In wild type embryos two types 
of experiments were performed. In the first set of experiments, RNA encoding AIXARI was 

25 injected in the animal pole of the early embryo (e.g. two cell stage). The animal pole is the 
region of the embryos that gives rise to the fiiture ectoderm which in turn becomes epidermal 
or neural in fate. Targeting AlXARl to the site of prospective ectoderm leads to the 
generation of embryos with grossly exaggerated neiiral structures mostly of anterior 
character. For instance, embryos form up to eight eyes and five cement glands, and whole 

30 mount immxmohistochemistry with an anti-NCAM antibody reveals that most of the cells of 
the embryo are positively stained with a general neural marker. Since the animal pole of the 
embryo participates in neurogenesis, this experiment demonstrates that neural induction has 
been amplified. 



35 were injected with AlXARl RNA. These cells normally give rise to endoderm and never 
form neural tissues. Surprisingly, injection of AlXARl in vegetal pole cells leads to the 
formation of ectopic neural structures in the embryo. Lineage tracing experiments in which a 



invention. 



10 



Example 1 

Inhibition of activin signaling by a truncated activin receptor 
induces neural structures in vivo 



* 



In the second set, a few cells of the vegetal pole of the early cleavage stage embryo 




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single vegetal pole cell is coinjected with AlXARl and P-Gal RNAs shows that the fate of 
the injected vegetal cell is changed when compared to the controls injected with globin and P 
-Gal RNAs. 

Moreover, while injected control cells (e.g. globin injected) populate mostly 
5 endodermal tissues, the cells that have received the AlXARl mostly occupy dorso-anterior 
positions in the embryo. Whole mount immunohistochemistry of such injected embryo 
depicts neural tissue that has expanded, and that this expansion correlates with the presence 
of the lineage tracer (P-Gal). These experiments illustrate that AlXARl can neuralize 
embryonic ceils in vivo by recruiting cells and changing their original fate, thereby increasing 
10 the amount of neural tissue dramatically. 

The ability of AlXARl to induce neural structures in ventralized embryos completely 
lacking axial structures was also explored. Embryos were UV irradiated during the first cell 

cycle and co-injected with RNAs encoding AlXARl and P-Gal in a single blastomere of the 

noun 

early blastula stage. The embryos were then allowed to develop imtil sibling non UV 
15 irradiated embryos reached tailbud stages and were then stained for P-Gal and for the 
presence of the neural marker NCAM. Comparison of the staining of the neuroaxis of a 
normal embryo (which is substantially lacking in.UV irradiated embryos) with ventralized 
embryos expressing AlXARl and P-Gal demonstrating that (i) AlXARl can induce neural 
tissue in embryos otherwise lacking dorsal structures, (ii) the neural tissue actually forms a 
20 structure that resembles a neural tube, and (iii) the cells that have received the RNA as 
marked by the lineage tracer are part of the neural tissue. These observations further 
establish that AlXARl can neuralize tissues in vivo even in embryos where no axial 
mesodeim is present. 

An other observation made during these experiments was that while the truncated 
25 receptor completely and specifically blocked the early morphogenetic response of animal 
caps exposed to activin, it did not affect the response to bFGF. An \inexpected observation is 
that animal caps derived fi-om AlXARl injected embryos, including those incubated in buffer 
alone, formed cement glands. Control and iminjected animal caps did not form cement gland 
or mesodermal tissues. 

30 Early and late molecular markers for mesoderm induction were also specifically 

blocked by the truncated activin receptor. In animal caps treated with activin, Xenopus 
brachyury (Xbra) and Xhox-4, a homeobox protein closely related to Mix-I (Rosa, F. M. 
(1989) Cell 57:965-974), were used as immediate early markers for mesoderm induction. 
When animal caps injected with the AlXARl RNA were incubated with activin, the 

35 expression of both Xbra and Xhox-4 was blocked. The specificity of this block was 
demonstrated by the fact that induction of Xbra by bFGF was not inhibited. In fact, Xbra 



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induction by bFGF was enhanced in the presence of AIXARl . In midgastrulae, brachyury is 
normally present as a ring in the lower part of the marginal zone (Smith et al. (1991) Cell 
67:79-87) and is a marker for prospective dorsal, lateral, as well as ventral mesoderm, Xhox- 
4, like Mix-1, marks the early vegetal cells as well as the prospective dorsal and ventral 

5 mesoderm. Therefore, the inhibition of Xbra and Xhox-4 expression indicated that all types 
of mesoderm were blocked by the truncated receptor. Further support for this assertion came 
from the fact that goosecoid, a marker for head mesoderm at the midgastrula stage, was 
absent in all injected caps, and the induction of Xwnt-8 expression, a marker for ventral 
mesoderm, in response to activin, was blocked by injection of the truncated activin receptor. 

10 Expression of muscle actin, a mesoderm-specific gene that is expressed at the end of 
gastrulation and increases during neurulation, was also selectively inhibited in animal caps 
injected with the truncated receptor. The block to induction of muscle actin was found to be 
dependent on the dose of AIXARl . It is interesting to note that bFGF induced muscle actin to 
roughly 10-fold higher levels in caps injected with the truncated activin receptor compared 

15 with control caps. This data, and the enhancement of Xbra expression in response to bFGF in 
the presence of AIXARl, is interpreted to indicate that activin antagonizes the mesoderm- 
inducing capacity of bFGF and that the mutant receptor, by blocking the effect of activin, 
amplifies or unmasks additional inducing activities of bFGF. This observation demonstrates a 
functional redundancy in embryonic cells whereby a block in one signaling pathway can lead 

20 to the enhancement of a parallel pathway to compensate for the effect. 

The experiments reported here show that disruption of activin signaling can, in the 
extreme phenotype, prevent mesoderm induction and dorsal body axis formation. 
Histological differentiation of mesodermal tissue is missing, early and late molecular markers 
are blocked and gastrulation does not proceed. These results establish activin as a principal 
25 determinant of mesoderm induction in vivo and not merely a molecule that mimics some 
other inducer. 

Example J 

Endogenous Mesoderm Inducing Signal(s) Inhibited 

30 The experiments illustrated in Example 1 above demonstrate that a truncated activin 

receptor can block the induction of mesoderm in explanted animal cap cells. In whole 
embryos, mesoderm is derived from the marginal zone (the equatorial region of the blastula) 
rather than from animal cap cells, which normally follow an ectodermal fate. To test whether 
the truncated receptor can block induction of mesodermal markers in the marginal ?one of 

35 intact embryos, the truncated receptor was injected into one cell of two-cell embryos. In each 
case the uninjected half of the embryo served as a control The expression of brachyury, 
which is expressed as a complete ring in control embryos, was reduced to a half rmg in 



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injected embryos. This indicated that the truncated activin receptor blocked the induction of 
Xbra RNA in cells that form mesoderm in vivo. 

The experiments described herein further suggest that it would be possible to 
determine the phenotype of whole embryos lacking mesoderm and its accompanying 
5 morphogenetic movements. Embryos injected with the truncated activin receptor in both cells 
at the two-cell stage display a range of phenotypes, all of which are markedly deficient in 
mesodermal and axial development. 

10 TABLE] 

Axial defect produced in embryos injected with AIXARl 



RNA Injected 




AIXAR 




p-Globin 


Phenotype 


n 


Percent 


n 


Percent 


No axil structures 


40 


53 


0 


0 


Partial axial defects 


18 


24 


2 


2 


Normal embryos 


17 


23 


92 


98 


Nimiber of embryos scored 


75 


100 


94 


100 



Embryos were injected whh 4 ng of either AIXARl or p-globin RNA in the equatorial region of both 
15 blastomeres of the two-cell embryo. Embryos were allowed to develop until sibling uninjected controls reached 
the tail-bud stage, at which time the survivors were scored for their phenotypes. Of the embryos without axial 
structures, roughly half showed minimal evidence of gastrulation (scant bottle-cell formation only) and had no 
detectable muscle or notochord either by histology or by molecular marker assay. The others showed only 
partial gastrulation and contained less than 20% of the nonnal amount of these markers. Partial axial defects 
20 are animals without heads or tail and with less than 50% of the normal amount of notochord and muscle. This 
division into classes, extreme and partial defects is arbitrary in that a range of defect is observed, n ^Number of 
embryos. 

By the time controls reach the tail-bud stage, injected embryos showing the extreme 
25 phenotype (about 50% of those injected) were ^ossly deficient in their development. In the 
most extreme cases there was no sign of body axis formation, and although the embryos 
appear to have retained an animal-vegelal axis, there was little indication of an anterior- 
posterior or dorsal-ventral body plan. Occasionally, some bottle cells formed and 
gastrulation began, but invagination rarely proceeded beyond a small lip. As observed in 
30 injected animal cap explants, cement gland differentiation did occur. Histological sections 
revealed no evidence of mesodermal differentiation or gastrulation in the extreme cases. The 
blastocoel remained intact and there was virtually no rearrangement of the presumptive germ 
layers. Assays for molecular markers of differentiation showed that these embryos did not ♦ 
form notochord, express muscle actin or Xbra RNA. The absence of mesodermal 
35 differentiation, as assayed by muscle actin and Xbra RNA expression, was dependent on the 



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dose of truncated activin receptor RNA injected. At 4ng of injected RNA, muscle actin and 
brachyury expression were both drastically reduced. 

The rest of the enabryos showed, to varying degrees, rudimentary signs of mesodermal 
development, gastrulation and axis formation, but normal embryos were not produced. There 
5 was always a marked reduction in notochord and muscle formation, with most of the embryos 
in this class forming less than half the normal ? ■ ant of notochord or muscle. None of t : :e 
mesodermal or axial defects were observed to ai. v significant degree in embryos injected with 
control RNAs. 

10 Examples 



If disruption of activin signaling by the truncated activin receptor is the cause of the 
mesodermal and axial deficiencies observed, then injection of wild-type activin receptor 
should rescue the phenotype. Indeed, injecting increasing amounts of wild-type activin 
15 receptor RNA with a constant amount of RNA encoding the truncated receptor can rescue 
embryos, as judged by gross morphology and molecular assays for mesodermal markers. 
Interestingly, the rescue requires a relatively low amoxint of wild-type activin receptor and 
that larger concentrations of the wild-type receptor generate multiple and bent axes, as is 
observed by ectopic expression of the wild-type receptor alone. 



The studies set out in Examples 1-3 demonstrate that a truncated form of the activin 
receptor, such as AlXARl, inhibits activin signaling and alters the fate of animal cap cells 
25 from epidermal to neural. This neuralization of the cap, assayed by N-CAM expression, was 
direct in that no cells of mesodermal fate were detected. Fiirther characterization of die 
neuralizing activity of AlXARl was performed using animal cap explants. 

Xenopus embryos were injected at the 2-cell stage with different concentrations of 
AlXARl or control RNA. Animal caps were removed at blastula stages and cultured in saline 

30 solution until sibling controls reached the early tailbud stages. RNA was extracted and 
analyzed by Northern blot techniques for the presence of neural-specific markers. Animal 
caps injected v^dth AlXARl expressed two general neural markers: N-CAM, which is 
exclusively and ubiquitously expressed in the central nervous system (CNS) of Xenopus 
embryos and a transcript encoding the p-tubulin isotype II, which is also exclusively 

35 expressed in the CNS. Injection of globin RNA did not promote expression of these neural 



Rescue by wild-type activin receptor 



20 



pxampk 4 

Neuralization of Embryonic Ectoderm with AJXARI 



markers. 



In the preceding examples, it is shown that animal cap cells expressing the truncated 




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activin receptor do not express any mesodermal markers. Immediate early and late as well as 
dorsal and ventral mesodermal markers were all assayed and found not to be expressed in 
cells injected with AIXARl mRNA. In the present experiment, both neural markers, as well 
as the cement gland pCAGl) marker (Sive et al., (1989) Cell, 58:171-180), are expressed in 
5 the absence of axial mesoderm as tested by the muscle marker (cardiac actin; Gurdon et al., 
(1985) Cell 41:913-922. The cement gland is an ectodermal tissue located anterior to the 
forebrain. Thus, neuralization of prospective ectoderm by expression of AlXARl is neither 
preceded by, nor dependent upon, mesoderm induction, further confirming that the neural 
induction is direct. 



During early neurogenesis, the neural plate, although comprised of morphologically 
indistinguishable cells, displays an anteroposterior and mediolateral polarity. For example, 

15 engrailed-2 (En-2), a marker for midbrain-hindbrain structures (Hemmati-Brivanlou and 
Harland, (1989) Development 106:61 1-617), and Krox-20, a marker of rhombomeres 3 and 5 
in the hindbrain (Bradley et al., (1993) Mech Dev 40:73-84), are both expressed as stripes 
with sharp anteroposterior boundaries. Since AlXARl changes the fate of ectodermal cells 
from epidermal to neural, whether this neuralized tissue was patterned was tested by using 

20 Northern blots, immunohistochemistry, or reverse transcription polymerase chain reaction 
(RT-PCR) to score for a series of regionally expressed neural markers. 

The monoclonal antibody (MAb) 3C3 stains the entire CNS of Xenopus embryos, and 
MAb 25.4 stains mostly the sensory nervous system. Both MAbs were used to assay animal 
caps derived from embryos injected with either AlXARl or globin control mRNA. It was 

25 observed that animal caps injected with AlXARl (7 of 10), but not those injected with globin 
(0 of 10), stained positively v^th MAb 3C3. The cells expressing this neural-specific antigen 
were present in a cluster and not dispersed throughout the explant. Since lineage tracers 
coinjected with AlXARl mRNA showed that nearly all cells of the explanted animal caps 
contain AlXARl mRNA, these results indicate that only a subset of cells expressing 

30 AlXARl express the CNS antigen recognized by MAb 3C3. It was observed that animal cap 
cells expressing AlXARl stain with MAb 25.4 (8 of 10 animal caps), while those expressing 
globin do not. This suggests that some of the neuralized cells have adopted the fate of the 
sensory neurons. 

Expression of neural markers for different positions along the anteroposterior axis in 
35 animal caps was scored using RT-PCR, when sibling controls reached the tailbud stage. 
Opsin, an eye marker (Saha and Grainger, (1 993) Mol Brain Res 1 7:307-3 1 8), En-2, Krox-20, 
and tanabin were assayed. The eye is derived from the forebrain; thus, opsin expression 



10 



Example 5 

The Neural Tissue Induced by AJXARl Is Patterned 




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indicates the existence of forebrain cells. Expression of En-2 suggests the presence of 
midbrain cells. Tanabin is a neural-specific intermediate neurofilament that demarcates 
rhombomeres 2, 4, 6, and 8 of the hindbrain, the trigeminal ganglia, and a few cells in the 
eye, forebrain, and spinal cord. The presence of hindbrain cells is less certain, since Krox-20 

5 expression is very weak, and tanabin expression might suggest the presence of more anterior 
CMS cells, such as photoreceptors or forebrain. All these markers were found to be expressed 
in caps previously injected with AlXARl, but not in uninjected or globin-injected caps. The 
spinal cord-specific marker Xlhbox-6 (Wright et al., (1990) Development 109:225-234) was 
absent or expressed at low levels in the AlXARl explants. The presence of N-CAM and the 

10 absence of muscle actin expression in these injected explants demonstrate that these markers 
are expressed in the absence of mesoderm induction. These results are summarized below in 
Table 2. 

Table 2 

1 5 Neural Markers Scored in A nimal Caps Injected with the 

Dominant Negative Activin Receptor (DlXARl) 



Marker 


Expression 


General Neural Matters 




N-CAM 




(J-Tubulin Isotype II 


+ 


3C3 


+ 


Anteroposterior Markers 




Ospin 


+ 


Tanabin 


+ 


En'2 


+ 


Krox'20 


+ 


Xlhbox-6 




Dorsoventral Marker 




Tor 25.4 


+ 


Other Ectodemal Marker 




XAGJ 


+ 



For Table 2: markers in all cases have been scored when sibling controls have reached 
35 the tailbud stage. NCAM, p-tubulin isotype 11, and 3C3 are general neural maricers and are 
all expressed. The anteroposterior markers include opsin, a marker of the photoreceptors of 
the eye, En-2, which demarcates posterior midbrain and anterior hindbrain KroX'20, which 
demarcates rhombomeres 3 and 5 of the hindbrain, and tanabin, a marker of rhombomeres 2, 
4, 6, and 8, the trigeminal ganglia, and a few cells in the eye and spinal cord. Xlhbox-6 is a 
40 marker of the spinal cord. Tor 25.4 is a marker of sensory neurons. XAGl is cement gland 
marker. The data presented in this table are a combination of results obtained by Northern 
blots, RT-PCR, and whole-mount immunohistochemistry. 



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Example 6 

AIXARJ Diverts Prospective Ectodermal and Endodermal Blastomeres to a 
Neural Fate in Whole Embryos 

5 

The results described above show that inhibition of activin type II receptor signaling 
leads to neuralization and patterning of presumptive embryonic ectoderm in explants. To 
confirm that this nexiralization could occur in whole embryos, synthetic RNA encoding 
AlXARl was injected into prospective ectodermal cells of wild-type embryos. In the first set 

10 of experiments, RNA encoding AlXARl was injected into the ammal pole of the early 
embryo, the region that gives rise to the future ectoderm, which in turn becomes epidermal or 
neural. Targeting AlXARl to the site of prospective ectoderm was foxmd to lead to embryos 
with grossly exaggerated neural structures. Injected embryos form up to eight eyes and five 
cement glands. Histological examination revealed that AlXARl-injected embryos display a 

15 hypertrophy of the normal CNS as well as ectopic neural tissue. Thus, expression of 
AlXARl in the prospective ectoderm amplified the formation of neural tissue. 

In a second set of experiments, a single cell at the vegetal pole of 8-cell albino 
embryos was coinjected with AlXARl and p-galactosidase (P-gal) RNA (as a lineage tracer). 
Albinos were used because they are better suited for whole-mount analysis of lineage tracers 

20 and molecular markers. Of the four vegetal blastomeres, the two ventral blastomeres 
contribute primarily to posterior gut and posterior somites (Moody and Kline, (1990) Anat 
Embryol 182:347-362). The dorsal pair contribmes primarily to prechordal head mesoderm 
and pharyngeal endoderm. Neither dorsal nor ventral vegetal cells normally give rise to 
anterior neural tissue (Moody and Kline, Supra), Since albino embryos were injected, no 

25 distinction could be made between prospective dorsal or ventral vegetal blastomeres. The 
results demonstrated, in confirmation of the observations made above, that the fate of 
prospective dorsal and ventral vegetal blastomeres is changed to anterior neural tissue as a 
consequence of AlXARl expression. The progeny of vegetal blastomeres injected with 
control globin and p-gal RNA do not contribute to neural tissue (49 of 5D). However, 

30 coinjection of AlXARl and p-gal RNA mto vegetal cells did result in the relocation of most 
of the progeny of the injected cells into the dorsoanterior region of the embryo (50 of 50); 
This relocation of cells is similar to what has been observed in blastomeres expressing the 
homeobox gene goosecoid (Niehrs et al, (1993) Cell 72:491-503). In agreement with the fact 
that AlXARl induces anterior neural tissue in animal cap explants, most of these injected 

35 cells participate in neural tissue such as forebrain, midbrain, and eyes. In addition, some -cells 
have incorporated into axial mesodermal derivatives such as somites. 

In this same injection experiment, double staining with the neural-specific antibody 
3C3 confirmed that, in addition to populating the normal CNS, the progeny of injected cells 



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contributed to ectopic neural tissue detected. A dose of AlXARl mRNA (500 pg per 
embryo) that did not completely block mesoderm induction in animal cap explants was 
injected in these experiments. Thus, vegetal cells (prospective endoderm) expressing the 
truncated aetivin receptor change their fate to neural, whereas these cells would normally not 
5 contribute significantly to anterior neural structures. 

B^ampk 7 

AIXARI Neuralizes UV- Ventralized Embryos without Axial Rescue 

To investigate further the neuralizing properties of the truncated aetivin receptor, its 
10 effect on embryos ventralized by UV irradiation was determined. Irradiation of the vegetal 
pole of a fertilized egg during the first cell cycle leads to ventralization, the formation of 
embryos without the dorsal axial structures, somitic muscle, notochord, or neural tube 
(Malacinski et al., (1975) Dev Biol 56:24-39), Scharf and Gerhart, (1983) Dev Biol 99:15- 
87). UV-irradiated embryos were coinjected with RNAs encoding either a AlXARl and P- 
15 gal or globin and p-gal as a negative control. A single vegetal blastomere was injected at the 
8- to 16-cell stage, the embryos were allowed to develop until sibling non-UV-irradiated 
controls reached tailbud stages, and the embryos were stained for, p-gal and for the presence 
of the general neural marker 3C3. 

The results demonstrated that AlXARl can induce neural tissue in embryos that 
20 would ot; -rwise lack dorsal structures and that the induced neural tissue forms a tube-like 
structure resembling a neural tube. The cells that received the AlXARl RNA, as marked by 
the expression of P-gal, were part of the neural tissue. However, not all neural cells contain 
lineage tracer. Some neural cells could be formed secondarily by signals spreading from 
neural cells injected with AlXARl. The induction of neural structures, even though 
25 patterned as a rudimentary tube, does not apparently lead to the complete axial rescue of the 
ventralized phenotype of the UV embryos. These results further establish that a AlXARl can 
neuralize tissues in vivo, 

A complicating fact was the observation of some muscle-specific antigen (12/101 
staining) in UV embryos injected with AlXARl. The cells stained with 12/101 do not stain 
30 for p-gal and are therefore not derived from the injected blastomere. This result raises the 
interesting possibility that the neural tube could induce or pattern surrounding muscle. 

Cloning of the Xenopus follistatin 

35 To further address whether aetivin is the endogenous inhibitor of neural 

differentiation, the activity of two known specific inhibitors of aetivin follistatin and inhibin, 
were assayed. Since no Xenopus fiiU length cDNAs for these two proteins were available. 



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the possible neuralizing activity of the corresponding rat genes were originally tested in vitro 
by the animal cap assay. Neuralization of Xenopus embryonic tissue was observed with both 
rat follistatin and inhibin. 

The same experiments were also performed with the Xenopus homologs. To perform 
5 this experiment, a fiiU length follistatin clone from a Xenopus cDNA library was isolated by 
standard protocols. The sequence of this clone contains a signal peptide, which is typically 
indicative of a secreted factor, and three closely related domains, previously reported as 
"follistatin modules" (Patthy et al. (1993) Trends in Neurol Science 16:76-81), that 
encompass about 75% of the mature protein. It is noted that follistatin modules have been 
10 recently recognized in at least four other proteins such as osteonectin, agrin, a protein SCI 
from rat brain, and human testican. While SCI, testican and osteonectin have one follistatin 
module, agrin has nine tandemly repeated modules at its N-terminus (Patthy et al. (1993) 
Trends in Neurol Science 16:76-81). 

In all species from which the follistatin cDNAs have been isolated, including humans, 
15 pigs, rats and Xenopus, two type of transcripts have been detected. Sequence analysis, SI 
mapping and RNase protection using the human, porcine and rat genomic clones have 
revealed that these two transcripts are generated by differential splicing. This differential 
splicing leads to the translation of two monomeric glycosylated proteins. The smaller 
molecular weight form in all species studied so far represents a carboxy-truncated form of the 
20 larger precursor and is generated by the removal of a highly acidic stretch of acidic amino 
acids from the larger precursor. The number of amino acids in each protein is used 
conventionally to name each subtype. Thus the hmnan follistatin proteins are called FS 315 
and FS 288; the rat, FS 344 and FS 317; and, the pig, FS 300 and FS 288. Using the same 
convention, the Xenopus follistatin is termed XFS 319. This protein is the homolog of the 
25 smaller subtype of follistatin cloned from other species and lacks the acidic carboxy terminus. 
Beside the two different lengths of the FS amino acid chains, the native proteins show 
variations in their degree of glycosylation, which also contributes to the heterogeneity in 
molecular weight of follistatin (Inouye et al. (1 991) Endocrinology, 129:8 1 5-822). 

30 Examph 9 

Generation of follistatin protein, synthetic RNA and expression vectors 

A plasmid allowing the production of large amount of active synthetic DNA for 
follistatin was created. This plasmid pSP64TXFS-319 was generated by subcloning the open 
reading frame of XFS-319 (SEQ ID No. 1) into the plasmid pSP64T. pSP64T is a derivative 
35 of the publicly available pSP64 vector which has been altered to include 5' and 3* untranslated 
regions of the xenopus P-globin gene to enhance mRNA stability and translation. This 
construct allows the production of large quantities of capped RNAs that is stable and 



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translationaly enhanced. An epitope tagged version of XFS-319, that is as active as the wild 
type construct in blocking activin, was generated. This was constructed by adding 12 amino 
acids originally derived from the human myc protein to the C-terminal end of XFS-319. This 
molecular tag is recognized by a monoclonal antibody (Mal9E10) and allows the injected 
5 folHstatin protein to be followed in whole embryos. 

Cell culture lines which overexpress a Xenopus follistatin pCFS-319) can be 
constructed using the pSP64TXFS-319 construct. Follovwng a protocol that has been proven 
successful in overexpressing human foUistatins in Chinese Hamster Ovary cell line (CHO 
cells) (Inouye et al. {\99\) Endocrinology, 129:815-822), the open reading frame of XFS-319 

10 can be subcloned into a plasmid containing an SV40 promoter and polyadenylation sequence 
(pSV2). This vector along with the same vector expressing dhfr (pSV2dhfr) is co-transfected 
by the calcium phosphate precipitation method into a dhfr-deficient CHO cell line (CHO- 
DG44). The Xenopus gene is amplified usmg methotrexate (MTX). Conditioned medium 
from these transfected cells, along with the untransfected controls, can be used directly, or, 

15 where purified protein is required, an activin affinity column can be employed to purify XFS- 
319 from the conditioned medium as previously described (Inouye et al. (1991) 
Endocrinology, 129:81 5-822). 

Several types of expression vectors can be used for the expression of XFS-319 in 
embryos. For example, XFS-319 can be placed under the control of the cytoskeletal actin 

20 promoter; a powerful, constitutively active promoter. Similarly, XFS-3 19 can be placed under 
the control of the Xenopus heat shock promoter. This promoter is only active when the 
temperature of the recipient embryos, usually kept at 20^C, is raised to 24*^C- 25®C (Harland 
et al. (1988) Development 102:837-852; and Vize et al. in Xenopus /flgv/^: Practical uses in 
Cell and Molecular Biologv .ed. Kay and Peng, Academic Press Inc:Floriv:.u, 1991). As 

25 zygotic transcription in Xenopus does not begin until mid-blastula stages (MBT) such 
constructs can be useful in determining the later function of follistatin in embryos. 



the case in mammals, two distinct RNAs of 2.4 and 3.6 kb are transcribed from the follistatin 
gene, and that the 2.4 kb message is present maternally in the fertilized egg. A partial cDNA 
clone encoding part of the larger Xenopus follistatin subtype was also isolated recently from 
Xenopus embryos (Tashiro et al. (1991) Biochem Biophys Res Comm, 174:1022-1027). The 
35 amino acid identity among species is extremely high, among mammals there is about 98% 
identity (Inouye et al. (1991) Endocrinology, 129:815-822) and between Xenopus and 
humans there is 86% identity. 



Example 10 

Expression of follistatin during Xenopus development 



30 



Northern blot analysis of RNA from different embryonic stages revealed that, as it is 




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The differential splicing leading to the generation of the two protein types does not 
seem to be under tissue specific regulation since both transcripts are present in all tissues 
expressing follistatin. The shorter of the human follistatin (HFS 288) has been shown to be 
8-10 times more potent in inhibiting activm both in vitro and in vivo (Inouye et al. (1991) 
5 Endocrinology. 129:815-822). In fact, the shorter form of the human follistatin is the most 
potent inhibitor of activin, even better than inhibin, the other specific inhibitor of activin 
(Inouye et al. (1991) Endocrinology. 129:815-822). Kinetic analyses of a mixture of human 
FS binding to activin iising a solid phase assay revealed that the FS-activin interaction is of 
high affinity similar to that estimated for activin binding to its receptor. Inhibin is 
10 approximately 500-1000-fold lower in relative potency as compared to activin in the FS 
binding assay. 

Example 11 

Follistatin is localized in the Spemann organizer 

15 Whole mount in situ hybridization (Henmiati-Brivanlou et al, (1990) Development 

1 10:325-330) with an anti-sense XFS-319 RNA was used to detect the spatial distribution of 
follistatin during different stages of Xenopus embryogenesis. The probe used in these 
experiments is that used on the northern blot and thus can recognize both follistatin 
transcripts. By this approach, follistatin RNA was first detected at the onset of gastrulation 

20 where a few cells of the organizer express XFS transcripts. In the gastrula, follistatin RNA is 
localized to the dorsal side. The localization is confirmed by RT-PCR on RNAs extracted 
fi^ora disected embryos at the onset of gastrulation, using the dorsally localized markers 
noggin (Smith et al. (1992) Cell 70:829-840) and Goosecoid (Blumberg et al. (1991) Science 
253:194-196) as controls. 

25 This region of the embryo has previously been characterized as a potent neural 

inducer in the animal cap assay. Expressions continues in the anterior two-thirds of the 
notochord in late gastrula and early neurula stage embryos. Transverse sections of these 
embryonic stages show that folUstatin RNA is exiwessed in a subset of cells of the prechordal 
and chordal mesoderm. This expression is strongest in the anterior involuting mesoderm and 

30 fades in intensity toward the posterior end. In early neurulae, when the neural plate is still 
open, the expression of follistatin is confined to head mesoderm and anterior notochord. A 
traverse section of these early neurula show that, in addition to the expression in the dorsal 
mesoderm, follistatin RNA can also be detected in a few cells of the hypochord, just ventral 
to the notochord. At these stages, the portion of the anterior notochord expressing follistatin 

35 touches the floor of the diencephalon (forebrain) and underlies the midbrain, hindbrain, and 
about half of the anterior spinal chord. The expression m the notochord extends posterioriy 
as development procedes and includes the entire notochord by stage 2 1 . The organizer region 
of the embryo has previously been characterized as a potent neural inducer in the animal cap 



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assay. Thus, the temporal and spatial localization of follistatin is in perfect agreement with 
the site generally predicted to contain the neural inducing activity. 

After the neural tube is formed, follistatin RNA is also detected in the forebrain, 
presumptive midbrain, midbrain-hindbrain jimction, and hindbrain. Furthermore, there is a 
transient follistatin expression in the pronephrous and ventrolateral mesoderm near the blood 
islands. There is also expression in photoreceptor of the retina at the tailbud stage. At the 
swimming tadpole stage, most of the expression is localized to the notochord and the head. 
In the forebrain, the expression of follistatin is confined to three stripes distributed 
dorsoventrally in the cells of the ventricxdar zone. There is no apparent staining in the floor 
or roofplate of the neural tube. 

Example 12 

Xenopus Follistatin RNA and Protein Block Mesoderm Formation and Morphogenetic 

Movements Induced by Activin 

The ability of follistatin to inhibit activin activity was tested by two independent 
approaches. In the first set of experiments, embryos were injected at the 2-ceIl stage in the 
animal pole of both blastomeres with 1 ng of either XFS-319 RNA or globin control RNA. 
The embryos were allowed to develop until they reached blastula stage 8, at which point the 
animal caps were dissected and incubated in either buffer alone or activin. While injection of 
control globin RNA apparently had no effect on activin induced morphogenetic movements, 
injection of XFS-319 completely blocked the morphogenetic movements associated with 
induction by activin treatment. In addition, animal caps in* ^cted with XFS-319 and incubated 
in buffer or activin were found to show clear cement glands, which parallels the effect of the 
dominant negative activin receptor described above. 

In the second set of experiments, either 50 ng of RNA encoding XFS-319 or 12 ng of 
RNA encoding Xenopus activin was injected into mature (stage 6) Xenopus oocytes. After 
48 hr, medium conditioned by these oocytes or by iminjected oocytes was collected and 
applied to animal cap explants of stage 8 blastula embryos. It was observed that when the 
explants were incubated in either buffer alone or conditioned medium from uninjected 
oocytes, they remained spherical. In contrast, explants incubated in conditioned mediimi fi-om 
activin-injected oocytes elongate drastically. Animal caps incubated in mediimi conditioned 
by oocytes injected with XFS-319 remained spherical. Finally, when conditioned media from 
oocytes injected with activin and XFS-319 were mixed at 1:1 ratio, the morphogenetic 
movement induced by activin is completely blocked. The results of these two experiments 
demonstrate that XFS-319, like its homologs in other species, is a potent inhibitor of activin. 

To assess the potency of this inhibition, a dose response study was carried out in 
which the animal poles of 2-cell stage embryos were injected with either control globin RNA 




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or different concentrations of XFS-3 1 9. As in the first set of experiments, animal caps from 
injected embryos were explanted at stage 8 and incubated with the potent mesoderm inducer 
activin. These caps were cultured until sibling uninjected embryos ^reached tailbud stage, at 
which point total RNA was isolated from the explants and analyzed; by Northern blotting. 
While 4 ng of the control globin RNA did not seem to interfere with the mesoderm inducing 
activity of activin, as assayed by the expression of the axial mesodermal marker muscle actin, 
250 pg of XFS-3 19 RNA was foimd to be enough to block activin action completely. In 
addition, none of the animal caps injected with 4.00, 1.00, or 0.25 ng of XFS-3 19 RNA and 
treated with activin (20 explants for each concentration) showed any sign of elongation. 



We tested for direct neural inducing activity of XFS-3 19 was also tested in animal cap 
explants. An indication that XFS-3 19 might possess this type of activity came from the 
observation that cement glands were induced in explants injected with XFS-3 19. Embryos 
were injected at the 2-cell stage in the animal pole v^th 2 ng of XFS-3 19 RNA, and it was 
found that the injection of this RNA elicits induction of neural tissue in the animal cap, as 
demonstrated by the expression of the general neural markers N-CAM and P-tubulin isotype 
11. These markers were expressed in the absence of muscle actin, suggesting that this 
induction is direct, i.e., without concomitant mesoderm induction. It is further noted that this 
represents the first evidence of a single endogenous embryonic molecule with neural inducing 
folllistation also provides evidence for activin as the inhibitor of neuralization. 

Induction of the general neural marker in these explants clearly demonstrates the 
neiiralizing activity of XFS-3 19. However, although this induction happens in the absence of 
the muscle actin marker, other mesodermal tissues could still be present. To address this 
question, XFS-3 19 or a control RNA was injected under the same conditions as described 
above. A portion of the animal cap explants were processed when sibling controls reached 
midgastrula stage to analyze the expression of immediate early mesodermal markers, and the 
rest were allowed to develop xmtil tailbud stage and then assayed for muscle actin and neural 
markers. Uninjected animal caps, or explants injected with either XFS-3 19 or control RNA 
incubated in buffer alone, failed to express any of the five mesodermal markers assayed. The 
early dorsal-specific mesodermal markers goosecoid and noggin, as well as the ventral 
marker Xwnt-8(Christian et al. (1991) Development 111:1045-1056 and the general «arly 
mesodermal markers X-bra (Smith et al., (1991) Cell 67:79-87) and Mix-1 are not expressed 
in caps that were injected with 2 ng of XFS-3 19 RNA. Uninjected animal caps, in contrast, 
did express all of these markers in reponse to activin. In addition, the explants that were left 
to develop until tailbud stage also were found to express the neural marker N-CAM, but not 
muscle actin, demonstrating that XFS-3 19 was active in inducing neural tissue in these 



pxample 1$ 
Follistatin Is a Direct Neural Inducer 




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experiments. These experiments fiuther support the notion that the neuralizing activity of 
XFS-319 happens in the absence of scorable mesoderm and is, by this definition, direct. In 
addition, the factitfiat follistatin does not induce noggin expression suggests that either the 
mechanism of neural iduction by follistatin is independent of noggin action or that follistatin 
5 acts downstream of noggin in this regard. 

Example 14 

Dose ofFolliststin RNA Required for Neural Induction 

We next aimed to characterize the direct neural inducing activity of follistatin by 
10 measuring the doses required for induction of a general neural marker and cement glands in 
animal cap explants. Embryos were injected at the 2-cel] stage in the animal pole with 
different concentrations of XFS-319 RNA. Animal caps from injected embryos were 
explanted at the blastula stage and allowed to develop until sibling uninjected controls 
reached the early tailbud stage. RNA extracted from explants injected vrith different 
15 concentrations of XFS-319 along udth RNA fi-om uninjected explants and control embryos 
were analyzed by Northern blots. It was also observed that the general neural marker P- 
tubxilin isotype II can be induced in these caps by injection of as little as 50 pg of XFS-319 
RNA, while the cement gland markerXAG-l (Sive et al., (1989) Cell 58:171-180) requires a 
minimum of 250 pg. The muscle actin panel again demonstrated that neuralization was direct 
20 and that the explants did not contain mesoderm. 



Example 15 
Follistatin Induces Anterior Neural Markers 

Examples 1-7 above demonstrate that interference v«th signaling through the type II 
25 activin receptor in embryonic explants results in the induction of anterior neural markers. 
Since follistatin interferes with activin signaling by a different mechanism, it is unportant to 
ask what type of neural tissue is induced by XFS-319 expression in animal cap explants. To 
this end, the same experiment described in Example 13 was performed, and RT-PCR was 
used to score for the range of anteroposterior neural markers. As was the case for the 
30 truncated activin receptor, the anterior neural markers opsin, which demarcates the 
photoreceptors of the retina derived from the forebrain, En-2, a marker of the midbrain- 
hindbrain junction, and tanabin, principally a marker of hindbrain, trigeminal ganglia, and a 
few cells of the eye, forebrain, and spinal cord were all induced by follistatin. Interestingly, 
Krox-20, another hindbrain marker, and Xlhbox-6, the spinal cord marker, are not detected in 
35 these caps. These results are summarized in Table 3. 



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TABLES 

Neural Markers Scored in Animal Caps Injected with XFS-3 19 RNA 



Marker 


Expression 


General Neural Matters 

v^wjAwi**! x^Vifumi JvxoiiwiD 




N-CAM 


T 


P-Tubulin Isotype II 


+ 


Anteroposterior Markers 




Opsin 




Tanabin 




En-2 




Krox'20 




mbox'6 




Dorsoventral Marker 




Other Ectodermal Marker 




XAGl 


-J- 



For Table 3: markers in all cases have been scored when sibling controls have reached 
the tailbud stage. N-CAM and p-tubulin isotype II are general neural markers and are botii 
expressed. The anteroposterior markers include opsin, which is a marker of the 
photoreceptors of the eye, En-2, which demarcates posterior midbrain and anterior hindlrain, 
KroX'20, which demarcates rhombomeres 3 and 5 of the hindbrain, and tanabm, a marker of a 
few cells of the forebrain and retina but mostly rhombomeres 2, 4, 6, and 8 and the trigeminal 
ganglia. Xlhbox-6 is a marker of the spinal cord. XAGl is a cement gland marker. The data 
presented in this table are a combination of results obtained by Northern blot and RT-PCR. 

From these results, we conclude that anterior neural tissue such as forebrain and 
midbrain is present in animal caps expressing XFS-3 19. The presence of hindbrain, however, 
is not well established by this type of assay. The absence of Ktox-20 expression suggests that 
rhombomeres 3 and 5 are not present in these explants. Tanabin expression -can be interpreted 
either as the presence of even-numbered rhombomeres or simply as confirmation of the 
presence of midbrain or forebrain. The absence of Xlhbox-6 signal suggests that posterior 
neural tissue such as spinal cord is absent. The staining patterns for N-CAM and muscle actin 
confirmed that this neuralization is direct. 

Example 16 

Actinin, but Not Noggin, Induces the Expression of Follistatin 

The protein noggin has been previously demonstrated to induce neural tissue in 
animal cap explants (Lamb et al., (1993) Science 262:713-718). However, while neural 
induction by noggin is direct, activin, in contrast, is not apparently a direct neural inducer, 
and the neural tissue in explants treated with activin likely results from a secondary induction 



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by mesoderm. It has been previously shown (Thomsen and Mehon, (1993) Cell 74:433- 
441)), and we have confirmed, that activin can induce the expression of noggin in animal cap 
explants (see Example 13). Thus, it could be argued that the indirect neural inducing activity 
of activin is mediated by noggin. FoIIistatin displays dh-ect neural inducing activity in 
5 explants; however, Example 13 demonstrates that this neural induction is not mediated by 
noggin. To investigate further a possible relationship among the neural inducing activity of 
noggin, follistatin, and activin, we decided to test the following: first, whether the neural 
inducing activity of noggin resulted in follistatin expression, and second, whether the ne\iral 
induction in explants and embryos, in response to activin, could be correlated with follistatin 
10 expression. 

Embryos at the 2-cell stage were injected vwth 1 ng of noggin RNA in the animal 
pole. At blastula stage (stage 8), the animal poles of these embryos along with those of the 
control injected and uninjected embryos were explanted. Half of the explants were allowed to 
develop xmtil mid-gastrula stage (stage 10.5), when neural induction has begun, and then 

15 were assayed by RT-PCR for follistatin expression. The other half were allowed to develop 
until early tailbud stage to provide a positive control for the function of noggin. Under this 
experimental condition, the expression of noggin in animal cap explants was not observed to 
induce follistatin. Thus, the neural inducing activity of noggin in animal cap explants is not 
mediated, at least at the transcriptional level, by follistatin. Noggin in these experiments has 

20 apparently been active in inducing the neural marker N-CAM directly in the explants. 

To determine if addition of activin to animal cap explants induces the expression of 
follistatin,? uninjected animal caps dissected at stage 8 were incubated in buffer alone or in 
the presence of activin. We found that addition of activin to animal caps induces the 
expression of XFS-319 RNA. Since activin can also induce the expression of noggin in these 
25 explants, we conclude that the neural inducing activity of activin may be mediated through 
either noggin or follistatin, or both. 

Example 17 

Follistatin Expression in Secondary Axes Induced by Activin 

30 When injected into the ventral side of the embryo, activin RNA can also induce a 

partial secondary axis that includes spinal cord and hindbrain, but not more anterior structures 
(Thomsen et al., (1990) Cell 63:485-493). Lineage-tracing experiments have shown that the 
cells that have received activin RNA do not participate in the induced ectopic neural tissue. 
Thus, we were prompted to ask whether follistatin could be involved in the induction of the 

35 ectopic neuraxis. Xenopus activin RNA or control RNA was injected into a single 
blastomere on the ventral side of embryos at the 8- to 16-cell stage. Embryos injected with 
Xenopus activin transcript, but not control RNA, displayed a partial secondary dorsal axis, 



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as previously reported. These embryos were analyzed at neurula stages (stage 21) for the 
expression and distribution of follistatin RNA by whole-mount in situ hybridization. It was 
noted that while follistatin RNA is present in the single notochord of the control, most 
embryos with double axes show the presence of follistatin in both the primary and the 
5 secondary axis (n = 18 of 25). Thus, the presence of follistatin in the secondary axis may 
account for the induction of the secondary neuraxis. These experiments demonstrate that 
activin can induce the expression of follistatin both in embryonic explants and in the context 
of the whole embryo. Thus, the neural inducing activity of activin may be mediated by either 
noggin or follistatin, or both. 



respond to a neural inductive stimulus and the size of the neural plate. To assay follistatin*s 

15 effect on the competence of the ectoderm, follistatin RNA can be injected in the animal pole 
of either one or both cells of a two cell stage embryo. For example, experimental embryos 
can be coinjected with different concentrations of myc-tagged follistatin and a single 
concentration of P-Gal. Control embryos will be injected with P-Gal alone. The embryos are 
allowed to develop until they reach the midneurula stage when the neural plate is still open, 

20 and then stained as wholemounts for P-Gal and NCAM simultaneously. Since the animal 
pole of the embryo contributes mostly to epidermal and neural derivatives, the control 
embryos are expected to display normal size neural plates with p-Gal staining in both the 
neuroectoderm and the epidermis. Several possible outcomes exist for the experimental 
embryos. In one scenario, no changes will be detected in the size of the neural plate 

25 regardless of the P-Gal distribution, indicating that follistatin did not modify the competence 
of the animal cap. In another scenario, the embryos still have an expanded neural plate and 
ectopic neural tissue, with most of the P-Gal and myc positive cells localized within the 
expanded neural tissue. This latter result would imply that follistatin is capable of changing 
the competence of the ectodermal cells for neural induction and has led to recruitment of 

30 epidermal-fated cells to adopt a neural fate. 

The ability of follistatin to induce neural structures on the ventral side can also be 
tested in similar fashion in the context of the whole embryo. To this end, follistatin and P-Gal 
RNA, or p-Gal RNA alone, is injected in the ventral side of an 8 cell stage embryo. Control 
and experimental embryos are harvested when sibling controls reach the tailbud stage, and 
35 examined by moiphology, histology and wholemoimt staining. Staining for NCAM can also 
be used to detect any ectopic neural tissue. The formation of a secondary neuroaxis or neural 
tissue would clearly indicate that follistatin can neuralize in vivo. The "P-Gal staining would 
again be informative as to the fate of the cells making the follistatin protein. In Spemann's 



10 



Example 18 

Ectopic injection of follistatin in wild type embryos 



There is a direct correlation between the competence of the ectodermal cells to 




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original experiment, most of the neural tissue in the secondary axis was derived from the 
ventral side of the host embryo and did not originate from the organizer. If ,P-Gal expressing 
cells do not participate in the induced ectopic neuroaxis, then experiment have demonstrated 
that foUistatin neural inducing ability parallels the neural inducer of the organizer. If no 
5 ectopic neural tissue is generated, then it might be concluded that other factors from the 
organizer are also required for the ectopic neural induction. Finally if all cells in the 
secondary neuroaxis have p-Gal, it would be consistent with a conclusion that even though 
follistatin can neuralize, this neuralization is not the same as the pathway used by the embryo. 

10 Example 19 

Injection of Follistatin in UV embryo 

A more strmgent assay of the neuralizing activity of follistatin in vivo is to test the 
ability of this factor to induce a neuroaxis in embryos completely lacking dorsal structures. 
UV irradiation of the Xenopus embryo during the first cell-cycle leads to embryos lacking a 

15 dorsal axis. These embryos have previously been used in an assay system to isolate factors 
involved in induction and patterning of the axial mesoderm (Smith et al. (1992) Cell 70:829- 
840). In addition most growth factors with organizer activity can rescue a complete or partial 
dorsal axis in these ventralized embryo. In one embodiment of an assay for other neuralizing 
activities, p-Gal and different concentrations of follistatin RNA are co-injected into a single 

20 blastomere of UV irradiated embryos. The embryos are then allowed to develop until early 
tailbud stages at which pomt they are examined by histology and stained as whole mount for 
P-Gal and NCAM protein. The control embryos can comprise p-Gal injected alone and 
uninjected embryos. The two latter controls allow assessment of the quality of UV 
ventralization. A given concentration of follistatin either will or will not induce an ectopic 

25 neural tissue. If it does, the level of P-Gal positive cells populating the induced neuroaxis can 
be determined. The character of the mesoderm surrounding the neural tissue can also be 
assessed to see if it maintains its ventral nature or whether it has been dorsalized. If the 
mesoderm is still ventral in nature, int might be concluded that follistatin has recruited ventral 
ectodermal cells for the formation of the ectopic neural tissue. If the mesoderm has been 

30 dorsalized (i.e. if markers of dorsal mesoderm such as muscle actin or notochord are present) 
then whether the mesodermal or the neural tissue was induced first will need to be 
determined. 



35 



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All of the above-cited references and publications are hereby incorporated by 
reference. 

Equivalents 

Those skilled in the art will recognize, or be able to ascertain using no more than 
routine experimentation, nxmierous equivalents to the specific methods and reagents 
described herein. Such equivalents are considered to be within the scope of this invention 
and are covered by the following claims. 



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245 250 255 

AGT AGA GTG GGT AGA GGT CGC TGT GCG CTG TGC GAT GAT CTG TGC GGA 932 
Ser Arg Val Gly Arg Gly. Arg Cys Ala Leu Cys Asp Asp Leu Cys Gly 
260 265 270 275 

GAG AGC AAG TCA GAC GAT ACA GTG TGC GCC AGC GAC AAC ACG ACT TAC 980 
Glu Ser Lys Ser Asp Asp Thr Val Cys Ala Ser Asp Asn Thr Thr Tyr 
280 285 290 

CCG AGC GAG TGC GCC ATG AAA CAG GCA GCC TGC TCC ACG <3GG ATT CTT 1028 
Pro Ser Glu Cys Ala Met Lys Gin Ala Ala Cys Ser Thr Gly lie Leu 
295 300 305 

15 TTG GAA GTG AAA CAC AGT GGA TCT TGC AAC TGT AAG TGAATTACCG 1074 
Leu Glu Val Lys His Ser Gly Ser Cys Asn Cys Lys 

310 315 320 



10 



CAACGCAGAG TAAGATTTCT AAAGGCAACC CCTCGGTAAT GAAGACTTTA AAGCAGCAAA 1134 
ATACTTTTTT TTTTTTTTTT TCCTTTTTTT CTAAGGGAAT TCAG 1178 

(2) INFORMATION FOR SEQ ID NO:2: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 319 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 

(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2: 

35 Met Leu Asn Glu Arg He Gin Pro Gly Met He Phe Leu Leu Thr Val 
15 10 15 

Ser Leu Cys His Phe Met Glu Tyr Arg Ala Val Gin Ala Gly Asn Cys 
20 25 30 

Trp Leu Gin Gin Ser Lys Asn Gly Arg Cys Gin Val Leu Tyr Arg Thr 
35 40 45 



20 



25 



30 



40 



Glu Leu Ser Lys Glu Glu Cys Cys Lys Thr Gly Arg Leu Gly Thr Ser 
45 50 55 60 

Trp Thr Glu Glu Asp Val Pro Asn Ser Thr Leu Phe Lys Trp Met He 
65 70 75 80 

50 Phe His Gly Gly Ala Pro His Cys He Pro Cys Lys <31u Thr Cys Glu 

85 90 95 

Asn Val Asp Cys Gly Pro Gly Lys Lys Cys Lys Met Asn Lys Lys Asn 
100 105 110 



55 



Lys Pro Arg Cys Val Cys Ala Pro Asp Cys Ser Asn He Thr Trp Lys 
115 120 125 



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Gly Ser Val Cys Gly lie Asp Gly Lys Thr Tyr Lys Asp Glu Cys Ala 
130 135 140 

Leu Leu Lys Ala Lys Cys Lys Gly Val Pro Glu Leu Asp Val Gin Tyr 
145 150 155 160 

Gin Gly Lys Cys Lys Lys Thr Cys Arg Asp Val Leu Cys Pro Gly Ser 
165 170 175 

Ser Ser Cys Val Val Asp Gin Thr Asn Asn Ala Tyr Cys Val Thr Cys 
180 185 190 

Asn Arg lie Cys Pro Glu Pro Thr Ser Pro Asp Gin Tyr Leu Cys Gly 
195 200 205 

Asn Asp Gly lie Thr Tyr Gly Ser Ala Cys His Leu Arg Lys Ala Thr 
210 215 220 

Cys Leu Leu Gly Arg Ser lie Gly Leu Ala Tyr Glu Gly Lys Cys lie 
225 230 235 240 

Lys Ala Lys Ser Cys Glu Asp He Gin Cys Ser Ala Gly Lys Lys Cys 
245 250 255 

Leu Trp Asp Ser Arg Val Gly Arg Gly Arg Cys Ala Leu Cys Asp Asp 
260 265 270 

Leu Cys Gly Glu Ser Lys Ser Asp Asp Thr Val Cys Ala Ser Asp Asn 
275 280 285 

Thr Thr Tyr Pro Ser Glu Cys Ala Met Lys Gin Ala Ala Cys Ser Thr 
290 295 300 

Gly He Leu Leu Glu Val Lys His Ser Gly Ser Cys Asn Cys Lys 
305 310 315 



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AGT AGA GTG GGT AGA GGT CGC TGT GCG CTG TGC GAT GAT CTG TGC GGA 932 
Ser Arg Val Gly Arg Gly Arg Cys Ala Leu Cys Asp Asp Leu Cys Gly 
260 265 270 275 

GAG AGC AAG TCA GAC GAT ACA GTG TGC GCC AGC GAC AAC ACG ACT TAC 980 
Glu Ser Lys Ser Asp Asp Thr Val Cys Ala Ser Asp Asn Thr Thr Tyr 
2B0 285 290 

CCG AGC GAG TGC GCC ATG AAA CA6 GCA GCC TGC TCC ACG GGG ATT CTT 1028 
Pro Ser Glu Cys Ala Met Lys Gin Tlla Ala Cys Ser Thr Gly lie Leu 
295 300 305 

15 TTG GAA GTG AAA CAC AGT GGA TCT TGC AAC TGT AAG TGAATTACCG 1074 
Leu Glu Val Lys His Ser Gly Ser Cys Asn Cys Lys 

310 315 320 



10 



20 



25 



30 



40 



55 



CAACGCAGAG TAAGATTTCT AAAGGCAACC CCTCGGTAAT GAAGACTTTA AAGCAGCAAA 1134 
ATACTTTTTT tTTTTTTTTT TCCTTTTTTT CTAAGGGAAT TCAG 1178 

(2) INFORMATION FOR SEQ ID NO: 2: 

(i) SEQUENCE CHARACTERISTICS: 

(A) LENGTH: 319 amino acids 

(B) TYPE: amino acid 
(D) TOPOLOGY: linear 



(ii) MOLECULE TYPE: protein 

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 

35 Met Leu Asn Glu Arg He Gin Pro Gly Met He Phe Leu Leu Thr Val 
1 5 XO 15 

Ser Leu Cys His Phe Met Glu Tyr Arg Ala Val Gin Ala Gly Asn Cys 
20 25 30 



Trp Leu Gin Gin Ser Lys Asn Gly Arg Cys Gin Val Leu Tyr Arg Thr 
35 40 45 



Glu Leu Ser Lys Glu Glu Cys Cys Lys Thr Gly Arg Leu Gly Thr. Ser 
45 50 55 60 

Trp Thr Glu Glu Asp Val Pro Asn Ser Thr Leu Phe Lys Trp Met He 
65 70 75 80 

50 Phe His Gly Gly Ala Pro His Cys He Pro Cys Lys Glu Thr Cys Glu 

85 90 95 



Asn Val Asp Cys Gly Pro Gly Lys Lys Cys Lys Met Asn Lys Lys Asn 
100 105 110 

Lys Pro Arg Cys Val Cys Ala Pro Asp Cys Ser Asn He Thr Trp Lys 
115 120 125 



SUBSTITUTE SHEET (RULE 26) 



wo 95/10611 



PCT/US94/11745 



Gly Ser Val Cys Gly lie Asp Gly Lys Thr Tyr Lys Asp Glu Cys Ala 
130 135 140 

Leu Leu Lys Ala Lys Cys Lys Gly Val Pro Glu Leu Asp Val Gin Tyr 
5 145 150 155 160 

Gin Gly Lys Cys Lys Lys Thr Cys Arg Asp Val Leu Cys Pro Gly Ser 
165 170 175 

10 Ser Ser Cys Val Val Asp Gin Thr Asn Asn Ala Tyr Cys Val Thr Cys 
180 185 190 

Asn Arg lie Cys Pro Glu Pro Thr Ser Pro Asp Gin Tyr Leu Cys Gly 
195 200 205 

15 

Asn Asp Gly lie Thr Tyr Gly Ser Ala Cys His Leu Arg Lys Ala Thr 
210 215 220 

Cys Leu Leu Gly Arg Ser He Gly Leu Ala Tyr Glu Gly Lys Cys He 
20 225 230 235 240 

Lys Ala Lys Ser Cys Glu Asp He Gin Cys Ser Ala Gly Lys Lys Cys 
245 250 255 

25 Leu Trp Asp Ser Arg Val Gly Arg Gly Arg Cys Ala Leu Cys Asp Asp 
260 265 270 

Leu Cys Gly Glu Ser Lys Ser Asp Asp Thr Val Cys Ala Ser Asp Asn 
275 280 285 

30 

Thr Thr Tyr Pro Ser Glu Cys Ala Met Lys Gin Ala Ala Cys Ser Thr 
290 295 300 

Gly He Leu Leu Glu Val Lys His Ser Gly Ser Cys Asn Cys Lys 
35 305 310 315 



SUBSTITUTE SHEET (RULE 26) 



wo 95/10611 



PCTAJS94/11745 



CLAIMS: 



5 

2. 

10 

3. 
4. 

15 

5. 

20 

6. 
7. 

25 

8. 

30 9. 
35 

10. 



A method for inducing a cell to differentiate to a neuronal cell phenotype, comprising 
contacting said cell with an agent which antagonizes the biological action of at least one 
polypeptide growth factor of the Transforming Growth Factor-p (TGF-P) family, said 
growth factor normally inducing said cell to differentiate to a non-neuronal phenotype. 

The method of claim 1, wherein said antagonizing agent inhibits the biological activity 
of said growth factor by preventing said grov^ factor from binding growth factor 
receptors on the surface of said cell. 

The method of claim 2, wherein said antagonizing agent binds said growth factor and 
sequesters said growth factor such that it cr: : '=ot bind said growth factor receptors. 

The method of claim 3, wherein said antagonizing agent is selected from a group 
consisting of a follistatin, an a2-macroglobulin, a protein containing at least one 
follistatin module, and a trunc ated receptor for a growth factor of the TGF-p family. 

The method of claim 4, wherein said truncated receptor comprises a soluble growth 
factor-binding domain of a TGF-P receptor. 

The method of claim 5, wherein said truncated receptor comprises a truncated activin 
receptor. 

The method of claim 2, wherein said antagonizing agent inhibits binding of said growth 
factor with said growth factor receptors via its own binding to said ^owth factor 
receptor. 

The method of claim 7, wherein said antagonizing agent is an inhibin. 

The method of claim 7, wherein said antagonizing agent is a polypeptide of said TGF-P 
family and which has one or more sites of amino acid mutation, said mutation 
diminishing an ability of said TGF-P polypeptide to induce said cell to differentiate to a 
non-neuronal phenotype, yet not substantially diminishing the binding of said activin to 
said growth factor receptor. 

The method of claim 9, wherein said TGF-P polypeptide is a mutated activin. 




wo 95/10611 



PCT/US94/11745 



11. 

12. 

5 

13. 

10 

14. 

15 

15. 

20 

16. 

25 17. 
18. 

30 

19. 

35 20. 



The method of claim 7, wherein said antagonizing agent is peptidyl fragment, or a 
peptidomimetic thereof, of a receptor-binding portion of an activin or inhibin protein. 

The method of claim 1, wherein said antagonizing agent is an antisense nucleic acid 
construct which inhibits expression of a receptor for said TGF-P polypeptide. 

The method of claim 1, wherein said antagonizing agent is dominant negative TGF-p 
receptor comprising an extracellular growth factor-binding domain of a TGF-P receptor, 
a transmembrane domain for anchoring said extracellular domain to a cell surface 
membrane, and a dysfunctional cytoplasmic domain, said dominant negative receptor 
being recombinantly expressed in said cell and inhibits the biological activity of said 
growth factor by inhibiting signal transduction by a naturally-occurring TGF-P receptor. 

The method of claim 1, wherein said growth factor is activin. 

The method of claim 1, wherein said cell is further contacted with a second growth 
factor having neurotrophic or neural inductive activity, such as a nerve growth factor, 
cilliary neurotrophic growth factor, schwanoma-derived growth factor, glial growth 
factor, stiatal-derived neuronotrophic factor, platelet-derived growth factor, scatter 
factor, a vertebrate hedgehog protein, noggin, and a ligand for a Notch receptor. 

The method of claim 1, wherein said cell is part of a host organism, and said 
antagonistic agent is delivered in the form of an in vivo therapeutic formulation. 

The method of claim 1 , wherein said neuronal cell comprises a neural progenitor cell. 

The method of claim 1, wherein said neuronal cell is selected from a group consisting 
of a melanocyte progenitor cell, a glial progenitor cell, a sensory neuron progenitor cell, 
a sympatho-adrenal progenitor cell, a parasympathetic progenitor cell, and an enteric 
progenitor cell. 

The method of claim 1, wherein said neuronal cell is a tOTninally-differentiated 
neuronal cell. 

The method of claim 19, wherein said terminally-diffCTentiated neuronal cell is selected 
from a group consisting of a microglial cell, a macroglia! cell, a Schwann cell, a 
cholinergic cell, a peptidergic cell, and a serotenergic cell. 




wo 95/10611 



PCT/US94/11745 



^7- 

2L The method of claim 1, wherein said cell is selected from a group consisting of an 
embryonic cell, a fetal cell, and a neonatal cell. 

22. A method for preventing death of a neuronal cell comprising contacting said cell with 
an agent which antagonizes the biological action of at least one polypeptide growth 
factor of the Transforming Growth Factor-P (TGF-P) family, said growth factor 
normally inducing said cell to differentiate to a non-neuronal phenotype. 

23. The method of claim 22, wherein said antagonizing agent is selected from a group 
consisting of a foUistatin, a truncated activin receptor, an a2-macroglobulin, an inhibin, 
and an antagonistic mutant of a polypeptide growth factor of the TGF-p family. 

24. The method of claim 22, wherein said cell is further contacted with a second growth 
factor having neurotrophic activity, such as a nerve growth factor, cilliary neurotrophic 
grov^rth factor, schwanoma-derived growth factor, glial growth factor, stiatal-derived 
neuronotrophic factor, platelet-derived growth factor, scatter factor, a vertebrate 
hedgehog protein, noggin, and a ligand for a Notch receptor. 

25. A method for inducing a cell to differentiate along a detennined neuronal pathway 
comprising, contacting said cell with an agent which disrupts a signaling pathway in 
said said cell of a growth factor of the TGF-p family, said signaling pathway normally 
inducing said cell to differentiate to a non-neuronal cell-type. 

26. The method of claim 25, wherein said signaling pathway is an activin-signaling 
pathway. 

27. A method for treating a degenerative disorder of the nervous system characterized by 
neuronal cell death, comprismg administering to a patient a therapeutically effective 
amount of an agent which antagonizes the biological action of at least one polypeptide 
growth factor of the Transforming Growth Factor-p (TGF-P) family, said growth factor 
normally inducing cells in said patient to differentiate to a non-neuronal phenotype. 

28. The method of claim 27, wherein said antagonizing agent is selected from a ^oup 
consisting of a foUistatin, a truncated activin receptor, an a2-macroglobulin, an inhibin, 
and an antagonistic mutant of a polypeptide growth factor of the TGF-P family. 

29. The method of claim 27, wherein said therapeutically effective amount of said 
antagonizing agent inhibits the de-differentiation of neuronal cells of said patient. 




wo 95/10611 



PCTAJS94/11745 



30. 

5 

31. 
32. 
10 33. 
34. 
35. 

15 

36. 

20 

37. 

25 

38. 

30 

39. 
35 40. 



The method of claim 27, wherein said therapeutically effective amount of said 
antagonizing agent induces the terminal differentiation of cells of said patient to a 
neural cell phenotype. 

The method of claim 30, wherein said neural cell phenotype is a glial cell. 

The method of claim 30 wherein said neural cell phenotype is a nerve cell. 

The method of claim 27 wherein said degenerative disorder is a neuromuscular disorder. 

The method of claim 27, wherein said degenerative disorder is a autonomic disorder. 

The method of claim 27, wherein said degenerative disorder is a central nervous system 
disorder. 

The method of claim 27, wherein said degenerative disorder is selected from a group 
consisting of Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis. 
Pick's disease, Huntington's disease, multiple sclerosis, neuronal damage resulting from 
anoxia-ischemia, neuronal damage resulting from trauma, and neuronal degeneration 
associated with a natural aging process. 

The method of claim 27, wherein a therapeutically effective amoimt of a second growth 
factor having neurotrophic activity is administered to said patient. 



The method of claim 37, wherein said second growth factor is selected from a group 
consisting of a nerve growth factor, cilliary neurotrophic growth factor, schwanoma- 
derived growth factor, glial growth factor, stiatal-derived neuronotrophic factor, 
platelet-derived grovrth factor. 

The method of claim 27, wherein a therapeutically effective amount of an antimitotic 
agent is administered to said patient in order to reduce the rate of growth of glial cells 
and favor the growth of nerve cells. 

The method of claim 39, wherein said antimitotic agent is selected from a group 
consisting of cytosine, arabinoside, 5-fluorouracil, hydrozyurea, and methotrexate. 




wo 95/10611 PCT/US94/11745 

-49- 

41. A method for identifying a neuralizing activity, comprising 

(i) culturing animal cap cells derived from an embryo, or equivalent cells thereof, in 
the presence of a polypeptide growth factor of the TGF-P family, said growth 
factor normally inducing said cells to differentiate to a non-neuronal phenotype, 
* 5 (ii) contacting said cells with a candidate agent, and 

(iii) detecting the neuronal differentiation of any of said cells, 
wherein neuronal differentiation of said cells in the presence of said candidate agent is 
indicative of a neuralizing activity. 



10 42. The method of claim 4 1 , wherein said growth factor is activin. 



43. The method of claim 41, wherein said neuronal differentiation is detected by scoring for 
the presence of a neural-specific marker on the surface of said cells. 

15 44. The method of claim 43, wherein said neural specific marker is NCAM, and the 
presence of NCAM is scored using a detectably labeled anti-NCAM antibody. 



SUBSTITUTE SHEET (RULE 26) 



Inter tnaX Application No 

PC I /us 94/11745 



A. CLASSinCATION OF SUBJECT MATTER 

IPC 6 C12N15/12 A61K48/00 A61K38/18 C12N5/06 G01N33/50 


According to IntemationAl Patent Oassificatton (IPQ or to both national dasstflcation and IPC 




B. FIELDS SEARCHED 


Minimum documentation scaixhcd (dassification system followed by classification symbols) 

IPC 6 C07K A61K 


DocurocntaticQ searched other than minimum documentation to the extent that such documents are inchuled in the fields searched 


Electronic data base consulted during the international search (name of data base and, where practical, search terms used) 


C. DOCUMENTS CONSIDERED TO BE RELEVANT 




Otation of document, with indication, where appropriate, of the relevant passages 


Relevant to daim No. 


X 


NATURE, 
vol.359, 1992 
pages 609 - 614 

A. HEMMATI-BRIVANLOU AND D.A- MELTON 'A 

truncated activin receptor inhibits 

mesoderm induction and formatijyi of axial 

structures in Xenopus embryos'^" 

see the whole document, especially the 

discussion. 


1-8, 

13-15, 

17,18, 

25,26, 

41-44 


X 


J. BIOL. CHEM., 
vol.267, 1992 
pages 7203 - 7206 

M. HASHIMOTO ET AL. 'Follistatin is a 
developmental 1y regulated cytokine in 
neural differentiation' 
see the whole document. 


1-4,17, 

18,25, 

26,41,42 






/- 




j )(| Further documents are listed in the continuation of box C 


1 1 Patent fiamily membos are listed in annex. 


Spcaalcatcgonet of ated documents: ^ later document published after the international filing date 

.^i- -. .-^^-.^ * or priority dau and not in conflict with the aprfication but 
A document dcfinmg the general state of the art which is not dtfi to inuierstand Ae priwaple or theory underiying the 

considered to be of particular relevance invention 
•E' Mrtier document bmputiished on or after the tnternatkjnal -x* document of particular relevance; the daimed invention 

nli^ cannot be considered novd or cannot be considered to 
"L* document which may throw doubts on priority daim(s) or involve an inventive step n4ien the document is taken alone 

which is dted to estaUials the publicaaon date of another -y- document of particular relevance; the daimed invention 

atation ox other spedal reason (as ^edfied) cannot be conadered to involve an inventive Oep when the 
'0* document referring to an oral disdosure, use, cxhibtticn or document is combined wiUi one or more other such docu- 

other means ments, such oombittation being obvious to a poson dolled 
'P* document published prior to ttie imemationr .^g date but in the ait 

later than the priority date claimed docuntent member of the same patent family 


Date of the actual completion of tite international scardt 


Date of mailing of the intematiooal search report 


8 February 1995 


23. 02. 95 




Name and mailing address of (he ISA 

European Patent Office, P.B. S81 8 Patendaan 3 
NL-2280 HVRijswijk 
Td.(-K31-70) 340-2040, TV. 31 651 eponl. 
Fax: (i- 31-70) 340-3016 


Authorized officer 

Yeats, S 



Fonn PCT/ISA/aiO (scamd ihcct) (July 1992) 



page 1 of 2 





Inter ' mal Application No 
PCi/US 94/11745 


1 QConOnuation) DOCUMEhJTS CONSIDERED TO BE RELEVANT 




Ca«gory uiauon oi flocument. with indication, where appropriate, of the lelevanl passages 


Relevant to cJaim No. 


1" DiULntn. DiUKHYb. Rh5. COMMUN., 
vol.173, 1990 
pages 193 - 200 

M. HASHIMOTO ET AL. 'Activin/EDF as an 
inhibitor of neural differentiation' 


1 



P,X I CELL, 

vol.77, 1994 
pages 273 - 281 

A. HEMMATI-BRIVANLOU AND D.A. MELTON 
'Inhibition of activin receptor signaling 
promotes neural ization in Xenopus' 
see the whole document. 

P.X I CELL, 

vol.77, 1994 
pages 283 - 295 

A. HEMMATI-BRIVANLOU ET AL. 'Follistatin, 
an antagonist of activin, is expressed in 
the Spemann organizer and displays direct 
neural izing activity' 
see the whole document. 



1-6, 

13-15, 

17,18, 

25,26, 

41-44 



1-4,14, 
15,17, 
18,25, 
26,41-44 



Foim PCT/lSA/aiO (amtinuttion of teoond theet) <July 1992) 



page 2 of 2 



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