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Cellular and molecular biology of neural crest cell lineage determination
REVIEWS
T he
development of the neural crest presents the challenging problem of how a migratory population of multipotential cells gives rise to a diverse array of differentiated derivatives. Because these derivatives include the neurons and glia of the peripheral nervous system (PNS), the neural crest also poses the problem of how different types of neurons are generated in development. For many years, the problem of neural crest devetopment was addressed primarily at the cellular level, using avian embryos as an experimental system’. More recently, it has become possible to ask questions about genes that control neural crest development, using the mouse, the chick and the zebrafish.
Neural crest cells detach from the neuroepithelium of the dorsal neural tube and migrate along a number of defined routes to various tissues, where they stop moving and d&rent&e into various cell types (Fig. 1). These cell types exhibit diversity along the rostrocaudal extent of the neuraxis and also within a single axial level (Fig. 2). For example, crest cells that emigrate from the trunk region of the neural tube produce sensory and autonomic neurons of multiple subclasses, their associated glia, melanves in the skin and, in some regions, the endocrine cells (chromaffm cells) of the adrenal gland, while enteric neurons of the gut are generated from a specific position anterior to the trunk regionz. The study of neural crest development at the cellular level has focused on the qut.,lion of how cell lineage and environmental influences control the generation of these different cell types (reviewed in Ref. 3). At the initiation of migration, many individual neural crest cells are multipotent. The normal fate of these cells can be changed by transplanting them to different locations or by exposing them to different environments in uitro
1
Sympathetic neurons Smooth muscle? of the neural crest in the trunk. A cross-xction through the trunk region of a mouse embryo is shown. Neural
F#;uIIE 1. Derivatives
crest cells in this region are known to give rise to sensory and autonomic (sympathetic or parasympathetic)neurons, Schwann @ial) cells and melanocytes. At other axial levels the crest generates smooth muscle cells in certain blood vessels (Fig. 2). The contribution to the wall of the dorsal aorta is speculative and has not been observed in avian embryos. Nevertheless, in mudne embryos a crest contribution to the smooth muscle of this blood vessel has not been excluded. Abbreviations: DA, dorsal aorta; N, notochord; NT, neural tube.
Cellular andmolecular biology ofneural crestcell lineagedetermination DAMDJ. ANDEESDN
(reviewed in Ref. 4). The fact that neural crest cells are plastic and sensitive to their local environment raises several further questions. What is the identity of the environmental factors that influence crest ceil fate, what lineages do they effect, and what cells or tissues produce them? How do these factors affect the fate of multipotent cells? At one extreme, the choice of fate could be made by a cell-autonomous mechanism (either stochastic or deterministic) and the factors would support the survival or proliferation of lineage-restricted cells; at the other extreme, the factors could cause multipotent cells to choose one fate (or a subset of fates) at the expense of others. Which mechanism operates in the neural crest? The fact that neural crest cells are initially multipotent and sensitive to environmental influences should not imply that lineage restrictions do not have a role in the deveiopment of the crest. IF post-migratory neural crest cells are transplanted back into the neural crest of younger host embryos, they generate some derivatives but not others5. This implies that neural crest cells gradually undergo restrictions in their developmental potential. However, it is not clear when and how restricted sublineages emerge, and why some derivatives are only produced from certain positions along the neuraxis. Furthermore, restrictions in developmental potential might occur randomly, sequentially, or according to some kind of hierarchy or pattern (e.g, Ref. 6). These issues, among others, are yet to be resolved. The molecular gemtic problems of neural crest development From one perspective, the problems of neural crest development at the molecular genetic level can be stated rather simply: what genes are important for neural crest development, and what do their products do? As in any developing system, these genes are likely to encompass growth factors and their receptors, signal transduction molecules and transcription factors. Indeed, there are already examples in each of these categories of genes that are essential for neural crest development. Genetic and biochemical analyses can allow, in principle,
REVIEWS one to piece together pathways and networks that con-
trol the development of panicular cell types. What is more difficult is to understand how the action of these genes fits in with the lineage restrictions, positional differences and environmental influences on neural crest cell fate that have been revealed by cellular analysis_ In some cases, the relationships are straightfomfard: in others they are less so. Perhaps most challenging is the problem of how to explain changes in the developmental potential, or state of commitment, of neural crest cells, properties defined operationalIy by cellular experiments, in terms of the act!ons of specific genes. In what follows, I briefly discuss selected experiments that begin to address some of the outstanding issues outlined above, focusing on work performed in mammalian systems where it has been possible to integrate cellular and molecular genetic approaches. Space limitations preclude a comprehensive survey of the extensive literature on neural crest development. In particular, this review emphasizes the development of lineages that arise from trunk neural crest (Figs 1 and 2). These lineages exclude bone and cartilage, which appear to represent a developmental capacity unique to cranial neural crest*. The cranial neural crest is aIso intimatefy involved in pattern formation in the head and neck region’. While significant advances have recently been achieved in understanding thii patterning process (e.g. Reefs8,9), a discussion of thii work is beyond the scope of this review. The reader is referred to recent review@-‘3 for further details.
Emc~~i~thatreglllak~crestlin~ Isolated mammalian neural crest cells are multiporent, self--renewing stem-like cells Conditions for the growth of mouse and rat neural crest cells in clonal culture have been establishedl4*15. Such clonal assays have allowed several impomnt cell biological issues in neural crest development to be investigated: the developmental potential of these isolated mammalian neural crest cells; whether such cells can undergo self-renewal, like stem cells in other system&j; the environmental signals that influence the differentiation of these cells; and whether the signals act setectively or instructively. In vitro, rodent neural crest cells are multipotent like their avian counterpart#,*s. In the rat, the cells have been shown to produce at least three differentiated cell types: autonomic neurons, glia and smooth muscle“W In the mouse, neurons, glia and melanocytes have been demonstrated to arise from single founder celW. The question of whether such isolated cells can also generate other crest derivatives, such as sensory neurons or cartilage20, remains open (although neurons expressing some sensory properties have been shown to develop in mass cultures of mouse neural crest21). Clonal analysis of avian neural crest cells, however, has provided evidence of such pleuripotency20.22, although not of self-renewal. The self-renewal of multipotent rat neural crest cells has been demonstrated by subcloning experiments*4. The properties of multipotency and self-renewal capacity are a characteristic of stem cells (reviewed in Ref. 161. Whether self-renewal of mammalian neural crest cells occurs irz Vito is not yet established, although
Sansory rwrons Glia Melanocytes
Cranial
FacW bone/cartilage 7
Hindbrain
Sensory neurons Glia
Metanocytes
Bone/cartilage
Vagal
Trllnk
Smooti muscle Glia Melancqtes Enteric neurons Endocrine cells (thyroid)
Sensory neurons Glia Melanocytes Autonomic neurons Chromaffin cells Smooth muscle?
FIGURE 2. Variationsin crest derivatives produced at different levels along the rostrocaudal extent of the neuraxis. Only major subdivisions of the neumxis and a simplified subset ofcrest derivatives are shown. The results are based primarily on fate-mapping experiments in avian embtyosl. (For a fuller description, see the monograph by k Douarinl.)
in avian embryos some multipotent cells can be recovered from peripheral tissues colonized by the neural crest (reviewed in Ref. 231, suggesting that self-renewal does occur. Neural crest cellfates can be determiued Ly instruct& environmental signaLs in vitm In the rat, three growth factors have been identified that bias the dserentiation of neural crest cells along dii tinct lineages: glial growth factor (GGF, a neuregulin2*~25), which promotes glial differentiatior@; transforming growth factor p CTGF-p), which promotes smooth musde differentiation; and bone morphogenetic proteins 2 and 4 (BMP2/4), which promote the diierentiation of autonomic neurons and, to a lesser extent, smooth muscle*g. Serial observation of individual clones strongly suggests that, in each case, the growth factor acts to promote the development of one lineage at the expense of otherO~26 (Fig. 3). Thus, the neural crest is one of the few systems in which growth factors have been demonstrated to influence instructively the fate of muftipotent stem cells. By contrast, in haematopoiesis there is clear evidence that at least some growth factors, such as erythropoietin, act selectivelyz7. This does not mean that growth factors do not exert selective effects on crest development as well: indeed there is evidence that GGF cm also act as a survival factor for Schwann cell
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affect other neura1 crest lineages that give rise to internal structures, such as the emetic nervous system. For exampie, endotheh 3 and its receptor are required for melanogenesis and enteric neurogenesis (reviewed in Ref. 37). However, this linkage is not always maintained: for e.xample, Steel factor, plateletderived growth factor, and their respective receptors are required for melanocyte, but not enteric, development (reviewed in Ref. 381, while glialderived neurotrophic factor and its receptor RET are required for enteric neurogenesis, but not melanogenesis (reviewed in Ref. 391. Interestingly, muFIGUIE 3. Growthfactors thar promore development of diierent tations in the human counterparts of some of these neural crest lineages from mammalianneural crejt stem cells genes are associated with various diis, such as in u&n Each of the factors indicatedis Iiely to work in an piebaldism, agangliogenesis of the bowel and multiple instructive, ratherthana selective, manneras determined by endocrine neoplasia, an inheritd cancer (reviewed in clonalanalysis. (Reproduced withpermission fromRef.19-I Refs 37, 40). Finally, endotheh 1 and its receptor are required for the development of craniofacial stmctures precursors~, and neurotrophins were or&ally identiand the major o&low vessels of the hear@42, which are populated by crest-derived cartilage and smooth fied by their survival promoting effects on crestderived peripheral neurons (reviewed in Ref. 29). muscle, respectively. These in z&a data leave Opt31 the The observation that growth factors can instructively questions of whether these environmental signals act as lineage determinants, survival factors, mitogens, or difinfluence the fate of neural crest cells raises additional interesting questions. Is the effect of a given factor pri- ferentiation signals, and whether they act singly or in marily positive (i.e. to promote a given fate), negative combination with other factors. In ujttvexperiments are (i.e. to inhibit alternative fates), or both? How does a beginning to address these issue&a. neural crest cell integrate the combined influence of opposing signals? Are neural crest ceils equally competent Transcriitkfactorsirnportantinneuralcrest to respond to all signals at the same time, or are there kagedek&lWon windows of opportunity for the cells to respond to difRole of the bHLH transcription fuctor mammalian ferent signals, which change with time? How do these achaete-scute homologlre 1 (MASHI) in autonomic growth-factor-signalling systems interface with other neunzgenesis celt-cell signalling systems, such as NOTCH and its liSeveral transcription factors that are important in gandsw, which are known to be expressed by developneural crest development have been identified, principally using reverse-genetic techniques. Two mammalian ing neural crest derivatives as welB*? homologues of the Dnxopbilu proneural genes achaeteSources and requirements for environmental signals scute, encoding basic-helix-loop-helix (bHLH) tranu&ting neural crest dewlopment in vivo scription factors, were isolated from a cefl line of neural Although factors such as GGF, TGF-8 and BMPU4 crest origin45. Of these two genes, the mammalian can influence neural crest cell fate in vitro, it is not cer- achaete-scute homologue I (Mu&I) is expressed in precursors of all autonomic neurons, but not in those of tain that they play this role in viva. The evidence is not yet ali in, but most of it is consistent with the in vitro sensory neurons4‘ (enigmatically, Mash2 is expressed results. All three growth factors are expressed in the only in the placenta‘?. A targeted mutation in MashI blocks the development of sympathetic, parasympathetic appropriate places at appropriate times: neuregulins and and a subset of enteric neuror&s#@. In vitro analysis of their receptors in developing peripheral nerves (where Schwanncells are located)32;TGF-p in the major vessels wild-type and Mu&l mutant neural crest ceils has sugof the heatt (whose smooth muscle ceils are crest- gested that the gene is required for neuronal differentiderivedP; and BMP2/4 in the do& aorta and gut ation, probably after cells have become committed to a (structures near or within which various classes of autoneuronal fate%. However, the gene is expressed initially nomic neurons develop) l9*3*.Targeted mutations in the in more primitive cell@, raising the possibility that it neuregulin gene reduce the number of glial cells associhas an earlier role not revealed by the null mutation. ated with peripheral nenes35, while a TGF-p knockout This idea is supported by recent gain-of-function experiinterferes with cardiac development (reviewed in Ref. 36). ments, which suggest that MASH1 maintains competence Unfortunately, embryos homozygous for mutations in for neurogenesis in uncommitted post-migratory neural BMP2 or BMP4 die too early to assess a requirement for crest cells74. Thus, MASH1 might play multiple roles at these factors in autonomic neurogenesis (A. Bradley, successive stages of autonomic neurogenesis, only one pers. cormnun.). Tissue-specificknockouts of these li- of which is revealed by conventional loss-of-function gands or their receptors will, therefore, be necessary to mutational analysis, address this issue. Recent data have shed light on the extracellular sigA number of other growth factors have been renals that induce the synthesis of MASH1 in neural crest vealed, by genetic experiments, to be important in cells. In vitro, MASH1is induced within six hours by mammalian crest development. Many of these factors BMP2, which, as mentioned earlier, also promotes the are required for proper melanogenesis, reflecting the fact differentiation of autonomic neurons (the cell type in that dominant coat colour mutations are easily detected which MASH1 function is required)l”. In vivo, MASHlin mice37. However, some of the same mutations also expressing cells, which are precursors of sympathetic TIG JULY1997 VOL. 13 No. 7
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REVIEWS neurons, first appear adjacent to the dorsal aorta, which expresses BMP2 (Refs 19, 461. Explants of dorsal aom can induce the synthesis of MASH1 in cultured neural crest cells, and thii induction can be blocked by noggin (A. Groves, N. Shah and DJ. Anderson, unpublished), which inhibits the action of BMP2 and BMP4 (Ref. 51). These results suggest that BMP2 and/or BMP4 can induce the production of MASH1 in &I, although, for reasons mentioned earlier, it has nor yet been possible to verify this genetically. Taken together, these data provide one of the first examples of a link between a growth factor and an essential transcription factor in controlling the development of a specific neural crest sublineage.
0
Neural crest
_
Smooth muscle
Other sensory sublineages
Enteric
WUUE4. Basic-heiix-ioopheiii transcription factors associated with the developmentof differentneural crest sublineages.The sequentia: king of factors within a given lineage indicates sequential expression,not dependent function, although in Xenopus, neumgedn 1 OgnlJ hasbeen shown to activate expression of XenopusNerlmD(Ref. 52).
A subfumi@ ofbHLHtranscription Genes marked by an asterisk have been shown to be essential for the indicated iieages factors for seh?_wy neurogenesis ill uim by targetedmutagenesis in mouse. Because MASH1 is not synthesized in sensory ganglia, it seems likely that there are other bHLH transcription factors mutant animals sensory neurons appear to differentiate that play a role analogous to that of MASH1 in sensory normally but then die, suggesting that these factors might neurogenesis. Recently, two genes have been isolated that control, for example, the expression of neurotrophins appear to tillill thii expectation: ne~m&~in I (Ref. 52) or their receptors. ISLETI, a founding member of the WM family of homeoproteins, is required for the differentiand neurogenin 2 (also known as Matb4A)y-ji. Both ation or survival of sensory as well as motorneuron@. genes are expressed in sensory but not autonomic ganglia, complementary to Mash2 (Fig. 4). In Xenopus embryos, PAX3, a paired homeodornain transcription factor, is essentiai for the development of sensory ganglia as revealed nawcgenin I and a Xenopzrs homoiogue (X-#@r-Z) by the Splotch mutation (reviewed in Ref. 621, although function as neuronai determination genes, activating whether it is autonomously involved in the development expression of the bHLH factor NEUROD and of downof neurons, glia63, or both, remains to be determined. stream markers, such as N-tubuiinsz. In mouse embryos, NEUROD (Ref. 55), and the bHLH factors NSCLl and 2 Several transcription fdctors have been shown to be essential for different stages in the development of (Refs 56, 57) are also expressed following the neuroSchwann ceils in viva, including the POUdomain factor genins (Refs 52, 54, 581, suggesting that these bHLH SCfP/TSTl/OC%, and the zinc finger protein KROX20 proteins function in a cascade in the sensory lineage CRefs64-66; reviewed in Ref. 67). However, these genes (Fig. 4). Interestingly, nezlrogetzin 2 is expressed in are probably expressed too late to function in Schwann crest-, otic- and trigeminai-piacodederived cranial xncell lineage determination. The microphtbulmia gene sory ganglia, whereas newoge?zin 2 is expressed in those ganglia whose neurons derive from the epibranchiai encodes a bHLH-Zip protein that is specii%zaliyexpressed piacodesj3.5*. These data suggest that these two bHLH in meianocyte precursors (Fig. 4) and that is probably factors can control the development of different suba regulator of pigment cell-specific genes (reviewed in classes of sensory neurons. Targeted mutations in these Ref. 68). Ho3c genes and retinoic acid receptors are genes, which are in progress, should test this hypothesis. clearly important for the development of mesectodermai neural crest derivatives in the hindbrain region, including the branchial arches (reviewed in Ref. 69). Finally, the bHLH proteins eHAND (Refs 70, 71) and Literally dozens of different transcription factors in dHAND (Ref. 72) are expressed in neural crest-derived various gene families have been shown to be expressed smooth muscle ceils (Fig. 4) in the major outdow tracts in the neural crest or its derivatives i>zvir~. A full listing of the heart, and have been functionally implicated in of these is beyond the scope of this review, so those cardiac development by antisense experiments72. factors for which essential roles in neural crest development have been demonstrated by genetic experiments are highlighted, focusing on the lineages in which they The analysis of neural crest development has entered are required. a new era, as the combination of reverse-genetic experiSeveral transcription factors importanr for sensory ments and in tfitmstudies continues to identify imporneurogenesis have been identified. The POLldomain taut genes and to establish their cellular mechanisms homeoprotein Br&0/3a (renamed Pou4fll) is essential for of action. Furthermore, the recent completion of a iargethe differentiation of subsets of sensory neuron@@. In scale saturation mutagenesis experiment in zebrafish TIC JULY1997 VOL. 13 No. 7
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REVXEWS 32 Camway, K.L. and Burden, S.J. (19%) Cim. Opit2. Net&~ivl. 5.606-612 33 Millan. F.A. ef nl. (1991) &Iewio~~~~c?it111, 131-144 34 y;fyd M.J. and Mchlahon, A.P, (1995) &v. Biul. 172,
(reviewed in Ref. 73) promises a cornucopia of new genes that are important in crestdevetopment that could not have been identified by any other means. At the same time, ongoing cellular experiments will establish the partem of lineage restrictions that occur and should identify extracellular signals that influence the development of different crest sublineages. The ability to add or to delete genes from neural crest cells and then to chaIlenge them, by transplantation in c4:o or exposure to defined signals in vitro, should help to shed new light on the important issue of when and how these multipotent cells become committed to particular fates.
35 hkyer, R. and Birchmeier. C. (19951 ~lirrrrre378, 386-390 36 King&y. D.M. (1994) Gews &II. 8, 133-146 37 Bash. G.S. (1996) Trends Genei. 15, 299-305 38 Marusich, M.F. and Weston, J.A. (1991) Cwr. Opin. GeW. De2.L1,221-229 39 Robertson, K. and Mason, I. (1997) Trrr2rksGenet. 13, l-3 40 Mak. Y.F. and Ponder, B+A.J. (1%) C!tn: Opin. Genet+ Drv. 6,82-S 41 Kurihard. Y. efnl (lm) ,~am-e368,703-710 42 Kurihan. Y. el al. (199%J. Clin. huvst. 96, 293-300
Aclmowkdgeme~ts work described from the author-s laboratory was supported by the NIH, Muscular Dystrophy Association and the Howard Hughes Medical institute. I thank members of my laboratory for helpful discussions, and Nirao Shah for the preparation of Fig. 1.
43 Lhav, K. et rrl. (1996) Proc. Nafl.Acad. Sci. U. S. A. 93, 3892-.3897 44 Reid. K. d al.(1996) De&k@zen? 122. 391l-3919 45 Johnson, J.E.. Birren, S.J. and Anderson, D.J. (1990) NatWe 346, 858-861 46 Lo, L. et al. (1991) Gerres L&L!. 5, 1524-1537 47 Guillemot. F. et (~1. WW ~!ntlrre 3771.333-336 48 Guillemot, F. et crl. (1993) Cell75 463-476 49 Bku.IglUnd. E. et al. (1996) &w’lop??Ienl122. 309-320 50 Sommer. L, e! al. (1995) Keilrotl Ii. 1245-1258 51 Zimmerman. L.B., Dejesus-Escobar. J.M. and Hadand. R.M. (1996) CeU86. 599-606 52 Ma. Q.. Kintner. C. and Anderson, D.J. Cef!(in press) 53 Grddwohl. G., Fade, C. and Guillemot. F. (1996) Deu. BiCJ/. 180. 227~%I
Ref2 me~ouarin. NM. (1382) ?%be A’t?~tr~tI crest, Cambridge
University Press L.eDouarin, NM. (1980) hkMure 286, 663-669 : Bronner-Fraser. M.E.(1993) Czrrr Opirr. Cnrr!. Del,. 3. 641-647 4 Le Douarin, NM., ZiUer,C. and Couly, G.F. (1993) Dev. Biol. t53, 24-49 5 Le Douarin, NM. (19861 Sciejrce231. 1515-1522 6 Weston, J.A. (1991) Corn: Top. Deu. Viol. 25. 133453 7 Noden. D.M. (1993)j. iVt~+~~bk~l. 2% 248-261 61 Graham. A., Heyman. 1. and Lumsden. A. ( 19%) DeveIopment 119,233-245 9 Birghauer, E. et al. (1995) Dewlopnw~l121. 935-945 IO Stemple, D.L. and Anderson. DJ. t 19931 &L? Bid. 159.
54 Sommer. L.. Ma, Q and Anderson. 0.J. (1%) Mol. Cell. h&wrwi. 8, 221-241
55 Lee,J.E. etnl.(1995) Science268. 836-844
12-23
11 Le Douarin. NM., Dupin, E. and Ziller. C. (1994) C~rw. 12 13 14 IS 16
Opin. Genet. Dev. 4, 6885-695 Bronner-Fraser, M. (1994) FASEBJ. 8.699-706 Lumsden. A. and Krumlauf, R. (1996) Sciwce274. 1109-1115 Stemple, D.L. and Anderson. D.J. (1992) Ce1171. 973-985 Ito, K.. Morita. T. and Sieber-Blum. M. (19931 i&%1.Biro!. 157,517-525 Morrison, S.J.. Shah. N. and Anderson. DJ. (1997) Cell88.
287-298 1Y Sieher-Blum, M. and Cohen. A. (I9801 &l: Hiol 80. 96-106
18 Baroffio. A., Dupin. E. and Le Doudrin. NM. (1988) ~%nz. ?iatI. Acad. SC!. U. S. A. 85. 5325-5329 19 Shah, N., Groves. A. and Anderson, D.J. ( 1996) Cell85, 331-343 20 Baroffio, A.+Dupin. E. and Le Douarin. N.M. (1991) Developnlerzt112. 301-305 21 Murphy, M. etal. (1991) Proc. Xatl, Acad. Sci. U. S. A. 88. 22 23 24 25
3498-3501 Sieber-Blum. Sieher-Blum, Lemke. G.E. Marchionni,
27 28 29 30
Wu, H. et al. (1995) Cell83,59-67 Dong, Z. etal. (1995) Nettrnjl Ii, 585-596 Thoenen, H. (1991) Trmtds Nerrrosci.14, 165-170 Artavanis-Tsakonas, S., Matsuno, K. and Fortini, M.E,
M. (1989) Scierzce 243, 1608-1610 M. er al. (1993jj. &wrobiol. 24, 173-184
and Brockes. J.P. (1984) i. Nelrrosci. 4,75-83 M.A.etd. (1993) rVot~r~362,312-318 26 Shah, N.M. et al. (1994) Ceil77.349~360
(19951Sc&ce268, 225-232 31 W&master, G., Roberts, V.J.and Lemke, G. (1991) Lkmlopment
113, 199-205
56 Begley. CC. et al. (1992) Prc,c. :V~tl. Acncl. Sci I! S. A. 89 +42 57 G&l. V. t’ lNI. (lc992) Cell Grcwfb D@r. 3. 143-148 58 Ma. );, d ~1.J, i’%WmCi. (in press) 59 McEvilly. K.J. d al.(199G~ X?rrrm38$ 574-577 60 Xiang. M.Q+ rl (ii, (1996) Proc. had Acad Sci. U. S. A. 93. 11950-l 1955 61 Pfaff. S.L. el al. C 19961 Ceil 84, 309320 62 Gruss, P. and Watcher. C. (1992) CelI69.719-722 63 Kioussi. C., Gross. hl K. and Grws. P. (199j) Nwnwr 15. j j3-562 64 Topilko. P. et nl. (1994) kr~wr371.796-799 65 Weinstein, DE. Burr&. P.G. and Lemke. G. (1995) ivfr~l.cell. Atiwosci. 6. 212-229 66 Bermingham. J.R. et (11.(1996) Gtws &II. 10, 1751-1762 67 Zorick, T.S, and Len&. G. (1996l Cwr. Opi”. Cell Biol. 8. 870-876 68 Moore. K.J. (l!Y$lj) 7ksds Geuet. 11. 442-448 69 Hunt. P. :md Krumlauf, H. (1992) Annu. Rev. Cell Biol. 8. 227-256 70 Cserjesi, P. ei al. (1995) L&&r.Biol. 170.664-678 71 Hollonherg. SM. ef a/. (1995) dlf~l. Cell. Eiol. 15. 3813-3822 72 StivdStaKi.D., Csejesi, P. and Olson. E.N. (19%) Scie#zcr no. 1995-1999 73 Eisen. J.S. (1996) CeI87, 969-977 Refermce added in proof 74 Lo. l., et NI. Cnrr, Biol. (in press)
D J. Att&rson is in the Divisiw ofBiology 2167G, Howard Hughes Medical Institute, Cali/omiu Institute Tecbmoiogl:Pasaderla, CA 91l.3, USA.
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T he
development of the neural crest presents the challenging problem of how a migratory population of multipotential cells gives rise to a diverse array of differentiated derivatives. Because these derivatives include the neurons and glia of the peripheral nervous system (PNS), the neural crest also poses the problem of how different types of neurons are generated in development. For many years, the problem of neural crest devetopment was addressed primarily at the cellular level, using avian embryos as an experimental system’. More recently, it has become possible to ask questions about genes that control neural crest development, using the mouse, the chick and the zebrafish.
Neural crest cells detach from the neuroepithelium of the dorsal neural tube and migrate along a number of defined routes to various tissues, where they stop moving and d&rent&e into various cell types (Fig. 1). These cell types exhibit diversity along the rostrocaudal extent of the neuraxis and also within a single axial level (Fig. 2). For example, crest cells that emigrate from the trunk region of the neural tube produce sensory and autonomic neurons of multiple subclasses, their associated glia, melanves in the skin and, in some regions, the endocrine cells (chromaffm cells) of the adrenal gland, while enteric neurons of the gut are generated from a specific position anterior to the trunk regionz. The study of neural crest development at the cellular level has focused on the qut.,lion of how cell lineage and environmental influences control the generation of these different cell types (reviewed in Ref. 3). At the initiation of migration, many individual neural crest cells are multipotent. The normal fate of these cells can be changed by transplanting them to different locations or by exposing them to different environments in uitro
1
Sympathetic neurons Smooth muscle? of the neural crest in the trunk. A cross-xction through the trunk region of a mouse embryo is shown. Neural
F#;uIIE 1. Derivatives
crest cells in this region are known to give rise to sensory and autonomic (sympathetic or parasympathetic)neurons, Schwann @ial) cells and melanocytes. At other axial levels the crest generates smooth muscle cells in certain blood vessels (Fig. 2). The contribution to the wall of the dorsal aorta is speculative and has not been observed in avian embryos. Nevertheless, in mudne embryos a crest contribution to the smooth muscle of this blood vessel has not been excluded. Abbreviations: DA, dorsal aorta; N, notochord; NT, neural tube.
Cellular andmolecular biology ofneural crestcell lineagedetermination DAMDJ. ANDEESDN
(reviewed in Ref. 4). The fact that neural crest cells are plastic and sensitive to their local environment raises several further questions. What is the identity of the environmental factors that influence crest ceil fate, what lineages do they effect, and what cells or tissues produce them? How do these factors affect the fate of multipotent cells? At one extreme, the choice of fate could be made by a cell-autonomous mechanism (either stochastic or deterministic) and the factors would support the survival or proliferation of lineage-restricted cells; at the other extreme, the factors could cause multipotent cells to choose one fate (or a subset of fates) at the expense of others. Which mechanism operates in the neural crest? The fact that neural crest cells are initially multipotent and sensitive to environmental influences should not imply that lineage restrictions do not have a role in the deveiopment of the crest. IF post-migratory neural crest cells are transplanted back into the neural crest of younger host embryos, they generate some derivatives but not others5. This implies that neural crest cells gradually undergo restrictions in their developmental potential. However, it is not clear when and how restricted sublineages emerge, and why some derivatives are only produced from certain positions along the neuraxis. Furthermore, restrictions in developmental potential might occur randomly, sequentially, or according to some kind of hierarchy or pattern (e.g, Ref. 6). These issues, among others, are yet to be resolved. The molecular gemtic problems of neural crest development From one perspective, the problems of neural crest development at the molecular genetic level can be stated rather simply: what genes are important for neural crest development, and what do their products do? As in any developing system, these genes are likely to encompass growth factors and their receptors, signal transduction molecules and transcription factors. Indeed, there are already examples in each of these categories of genes that are essential for neural crest development. Genetic and biochemical analyses can allow, in principle,
REVIEWS one to piece together pathways and networks that con-
trol the development of panicular cell types. What is more difficult is to understand how the action of these genes fits in with the lineage restrictions, positional differences and environmental influences on neural crest cell fate that have been revealed by cellular analysis_ In some cases, the relationships are straightfomfard: in others they are less so. Perhaps most challenging is the problem of how to explain changes in the developmental potential, or state of commitment, of neural crest cells, properties defined operationalIy by cellular experiments, in terms of the act!ons of specific genes. In what follows, I briefly discuss selected experiments that begin to address some of the outstanding issues outlined above, focusing on work performed in mammalian systems where it has been possible to integrate cellular and molecular genetic approaches. Space limitations preclude a comprehensive survey of the extensive literature on neural crest development. In particular, this review emphasizes the development of lineages that arise from trunk neural crest (Figs 1 and 2). These lineages exclude bone and cartilage, which appear to represent a developmental capacity unique to cranial neural crest*. The cranial neural crest is aIso intimatefy involved in pattern formation in the head and neck region’. While significant advances have recently been achieved in understanding thii patterning process (e.g. Reefs8,9), a discussion of thii work is beyond the scope of this review. The reader is referred to recent review@-‘3 for further details.
Emc~~i~thatreglllak~crestlin~ Isolated mammalian neural crest cells are multiporent, self--renewing stem-like cells Conditions for the growth of mouse and rat neural crest cells in clonal culture have been establishedl4*15. Such clonal assays have allowed several impomnt cell biological issues in neural crest development to be investigated: the developmental potential of these isolated mammalian neural crest cells; whether such cells can undergo self-renewal, like stem cells in other system&j; the environmental signals that influence the differentiation of these cells; and whether the signals act setectively or instructively. In vitro, rodent neural crest cells are multipotent like their avian counterpart#,*s. In the rat, the cells have been shown to produce at least three differentiated cell types: autonomic neurons, glia and smooth muscle“W In the mouse, neurons, glia and melanocytes have been demonstrated to arise from single founder celW. The question of whether such isolated cells can also generate other crest derivatives, such as sensory neurons or cartilage20, remains open (although neurons expressing some sensory properties have been shown to develop in mass cultures of mouse neural crest21). Clonal analysis of avian neural crest cells, however, has provided evidence of such pleuripotency20.22, although not of self-renewal. The self-renewal of multipotent rat neural crest cells has been demonstrated by subcloning experiments*4. The properties of multipotency and self-renewal capacity are a characteristic of stem cells (reviewed in Ref. 161. Whether self-renewal of mammalian neural crest cells occurs irz Vito is not yet established, although
Sansory rwrons Glia Melanocytes
Cranial
FacW bone/cartilage 7
Hindbrain
Sensory neurons Glia
Metanocytes
Bone/cartilage
Vagal
Trllnk
Smooti muscle Glia Melancqtes Enteric neurons Endocrine cells (thyroid)
Sensory neurons Glia Melanocytes Autonomic neurons Chromaffin cells Smooth muscle?
FIGURE 2. Variationsin crest derivatives produced at different levels along the rostrocaudal extent of the neuraxis. Only major subdivisions of the neumxis and a simplified subset ofcrest derivatives are shown. The results are based primarily on fate-mapping experiments in avian embtyosl. (For a fuller description, see the monograph by k Douarinl.)
in avian embryos some multipotent cells can be recovered from peripheral tissues colonized by the neural crest (reviewed in Ref. 231, suggesting that self-renewal does occur. Neural crest cellfates can be determiued Ly instruct& environmental signaLs in vitm In the rat, three growth factors have been identified that bias the dserentiation of neural crest cells along dii tinct lineages: glial growth factor (GGF, a neuregulin2*~25), which promotes glial differentiatior@; transforming growth factor p CTGF-p), which promotes smooth musde differentiation; and bone morphogenetic proteins 2 and 4 (BMP2/4), which promote the diierentiation of autonomic neurons and, to a lesser extent, smooth muscle*g. Serial observation of individual clones strongly suggests that, in each case, the growth factor acts to promote the development of one lineage at the expense of otherO~26 (Fig. 3). Thus, the neural crest is one of the few systems in which growth factors have been demonstrated to influence instructively the fate of muftipotent stem cells. By contrast, in haematopoiesis there is clear evidence that at least some growth factors, such as erythropoietin, act selectivelyz7. This does not mean that growth factors do not exert selective effects on crest development as well: indeed there is evidence that GGF cm also act as a survival factor for Schwann cell
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affect other neura1 crest lineages that give rise to internal structures, such as the emetic nervous system. For exampie, endotheh 3 and its receptor are required for melanogenesis and enteric neurogenesis (reviewed in Ref. 37). However, this linkage is not always maintained: for e.xample, Steel factor, plateletderived growth factor, and their respective receptors are required for melanocyte, but not enteric, development (reviewed in Ref. 381, while glialderived neurotrophic factor and its receptor RET are required for enteric neurogenesis, but not melanogenesis (reviewed in Ref. 391. Interestingly, muFIGUIE 3. Growthfactors thar promore development of diierent tations in the human counterparts of some of these neural crest lineages from mammalianneural crejt stem cells genes are associated with various diis, such as in u&n Each of the factors indicatedis Iiely to work in an piebaldism, agangliogenesis of the bowel and multiple instructive, ratherthana selective, manneras determined by endocrine neoplasia, an inheritd cancer (reviewed in clonalanalysis. (Reproduced withpermission fromRef.19-I Refs 37, 40). Finally, endotheh 1 and its receptor are required for the development of craniofacial stmctures precursors~, and neurotrophins were or&ally identiand the major o&low vessels of the hear@42, which are populated by crest-derived cartilage and smooth fied by their survival promoting effects on crestderived peripheral neurons (reviewed in Ref. 29). muscle, respectively. These in z&a data leave Opt31 the The observation that growth factors can instructively questions of whether these environmental signals act as lineage determinants, survival factors, mitogens, or difinfluence the fate of neural crest cells raises additional interesting questions. Is the effect of a given factor pri- ferentiation signals, and whether they act singly or in marily positive (i.e. to promote a given fate), negative combination with other factors. In ujttvexperiments are (i.e. to inhibit alternative fates), or both? How does a beginning to address these issue&a. neural crest cell integrate the combined influence of opposing signals? Are neural crest ceils equally competent Transcriitkfactorsirnportantinneuralcrest to respond to all signals at the same time, or are there kagedek&lWon windows of opportunity for the cells to respond to difRole of the bHLH transcription fuctor mammalian ferent signals, which change with time? How do these achaete-scute homologlre 1 (MASHI) in autonomic growth-factor-signalling systems interface with other neunzgenesis celt-cell signalling systems, such as NOTCH and its liSeveral transcription factors that are important in gandsw, which are known to be expressed by developneural crest development have been identified, principally using reverse-genetic techniques. Two mammalian ing neural crest derivatives as welB*? homologues of the Dnxopbilu proneural genes achaeteSources and requirements for environmental signals scute, encoding basic-helix-loop-helix (bHLH) tranu&ting neural crest dewlopment in vivo scription factors, were isolated from a cefl line of neural Although factors such as GGF, TGF-8 and BMPU4 crest origin45. Of these two genes, the mammalian can influence neural crest cell fate in vitro, it is not cer- achaete-scute homologue I (Mu&I) is expressed in precursors of all autonomic neurons, but not in those of tain that they play this role in viva. The evidence is not yet ali in, but most of it is consistent with the in vitro sensory neurons4‘ (enigmatically, Mash2 is expressed results. All three growth factors are expressed in the only in the placenta‘?. A targeted mutation in MashI blocks the development of sympathetic, parasympathetic appropriate places at appropriate times: neuregulins and and a subset of enteric neuror&s#@. In vitro analysis of their receptors in developing peripheral nerves (where Schwanncells are located)32;TGF-p in the major vessels wild-type and Mu&l mutant neural crest ceils has sugof the heatt (whose smooth muscle ceils are crest- gested that the gene is required for neuronal differentiderivedP; and BMP2/4 in the do& aorta and gut ation, probably after cells have become committed to a (structures near or within which various classes of autoneuronal fate%. However, the gene is expressed initially nomic neurons develop) l9*3*.Targeted mutations in the in more primitive cell@, raising the possibility that it neuregulin gene reduce the number of glial cells associhas an earlier role not revealed by the null mutation. ated with peripheral nenes35, while a TGF-p knockout This idea is supported by recent gain-of-function experiinterferes with cardiac development (reviewed in Ref. 36). ments, which suggest that MASH1 maintains competence Unfortunately, embryos homozygous for mutations in for neurogenesis in uncommitted post-migratory neural BMP2 or BMP4 die too early to assess a requirement for crest cells74. Thus, MASH1 might play multiple roles at these factors in autonomic neurogenesis (A. Bradley, successive stages of autonomic neurogenesis, only one pers. cormnun.). Tissue-specificknockouts of these li- of which is revealed by conventional loss-of-function gands or their receptors will, therefore, be necessary to mutational analysis, address this issue. Recent data have shed light on the extracellular sigA number of other growth factors have been renals that induce the synthesis of MASH1 in neural crest vealed, by genetic experiments, to be important in cells. In vitro, MASH1is induced within six hours by mammalian crest development. Many of these factors BMP2, which, as mentioned earlier, also promotes the are required for proper melanogenesis, reflecting the fact differentiation of autonomic neurons (the cell type in that dominant coat colour mutations are easily detected which MASH1 function is required)l”. In vivo, MASHlin mice37. However, some of the same mutations also expressing cells, which are precursors of sympathetic TIG JULY1997 VOL. 13 No. 7
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REVIEWS neurons, first appear adjacent to the dorsal aorta, which expresses BMP2 (Refs 19, 461. Explants of dorsal aom can induce the synthesis of MASH1 in cultured neural crest cells, and thii induction can be blocked by noggin (A. Groves, N. Shah and DJ. Anderson, unpublished), which inhibits the action of BMP2 and BMP4 (Ref. 51). These results suggest that BMP2 and/or BMP4 can induce the production of MASH1 in &I, although, for reasons mentioned earlier, it has nor yet been possible to verify this genetically. Taken together, these data provide one of the first examples of a link between a growth factor and an essential transcription factor in controlling the development of a specific neural crest sublineage.
0
Neural crest
_
Smooth muscle
Other sensory sublineages
Enteric
WUUE4. Basic-heiix-ioopheiii transcription factors associated with the developmentof differentneural crest sublineages.The sequentia: king of factors within a given lineage indicates sequential expression,not dependent function, although in Xenopus, neumgedn 1 OgnlJ hasbeen shown to activate expression of XenopusNerlmD(Ref. 52).
A subfumi@ ofbHLHtranscription Genes marked by an asterisk have been shown to be essential for the indicated iieages factors for seh?_wy neurogenesis ill uim by targetedmutagenesis in mouse. Because MASH1 is not synthesized in sensory ganglia, it seems likely that there are other bHLH transcription factors mutant animals sensory neurons appear to differentiate that play a role analogous to that of MASH1 in sensory normally but then die, suggesting that these factors might neurogenesis. Recently, two genes have been isolated that control, for example, the expression of neurotrophins appear to tillill thii expectation: ne~m&~in I (Ref. 52) or their receptors. ISLETI, a founding member of the WM family of homeoproteins, is required for the differentiand neurogenin 2 (also known as Matb4A)y-ji. Both ation or survival of sensory as well as motorneuron@. genes are expressed in sensory but not autonomic ganglia, complementary to Mash2 (Fig. 4). In Xenopus embryos, PAX3, a paired homeodornain transcription factor, is essentiai for the development of sensory ganglia as revealed nawcgenin I and a Xenopzrs homoiogue (X-#@r-Z) by the Splotch mutation (reviewed in Ref. 621, although function as neuronai determination genes, activating whether it is autonomously involved in the development expression of the bHLH factor NEUROD and of downof neurons, glia63, or both, remains to be determined. stream markers, such as N-tubuiinsz. In mouse embryos, NEUROD (Ref. 55), and the bHLH factors NSCLl and 2 Several transcription fdctors have been shown to be essential for different stages in the development of (Refs 56, 57) are also expressed following the neuroSchwann ceils in viva, including the POUdomain factor genins (Refs 52, 54, 581, suggesting that these bHLH SCfP/TSTl/OC%, and the zinc finger protein KROX20 proteins function in a cascade in the sensory lineage CRefs64-66; reviewed in Ref. 67). However, these genes (Fig. 4). Interestingly, nezlrogetzin 2 is expressed in are probably expressed too late to function in Schwann crest-, otic- and trigeminai-piacodederived cranial xncell lineage determination. The microphtbulmia gene sory ganglia, whereas newoge?zin 2 is expressed in those ganglia whose neurons derive from the epibranchiai encodes a bHLH-Zip protein that is specii%zaliyexpressed piacodesj3.5*. These data suggest that these two bHLH in meianocyte precursors (Fig. 4) and that is probably factors can control the development of different suba regulator of pigment cell-specific genes (reviewed in classes of sensory neurons. Targeted mutations in these Ref. 68). Ho3c genes and retinoic acid receptors are genes, which are in progress, should test this hypothesis. clearly important for the development of mesectodermai neural crest derivatives in the hindbrain region, including the branchial arches (reviewed in Ref. 69). Finally, the bHLH proteins eHAND (Refs 70, 71) and Literally dozens of different transcription factors in dHAND (Ref. 72) are expressed in neural crest-derived various gene families have been shown to be expressed smooth muscle ceils (Fig. 4) in the major outdow tracts in the neural crest or its derivatives i>zvir~. A full listing of the heart, and have been functionally implicated in of these is beyond the scope of this review, so those cardiac development by antisense experiments72. factors for which essential roles in neural crest development have been demonstrated by genetic experiments are highlighted, focusing on the lineages in which they The analysis of neural crest development has entered are required. a new era, as the combination of reverse-genetic experiSeveral transcription factors importanr for sensory ments and in tfitmstudies continues to identify imporneurogenesis have been identified. The POLldomain taut genes and to establish their cellular mechanisms homeoprotein Br&0/3a (renamed Pou4fll) is essential for of action. Furthermore, the recent completion of a iargethe differentiation of subsets of sensory neuron@@. In scale saturation mutagenesis experiment in zebrafish TIC JULY1997 VOL. 13 No. 7
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REVXEWS 32 Camway, K.L. and Burden, S.J. (19%) Cim. Opit2. Net&~ivl. 5.606-612 33 Millan. F.A. ef nl. (1991) &Iewio~~~~c?it111, 131-144 34 y;fyd M.J. and Mchlahon, A.P, (1995) &v. Biul. 172,
(reviewed in Ref. 73) promises a cornucopia of new genes that are important in crestdevetopment that could not have been identified by any other means. At the same time, ongoing cellular experiments will establish the partem of lineage restrictions that occur and should identify extracellular signals that influence the development of different crest sublineages. The ability to add or to delete genes from neural crest cells and then to chaIlenge them, by transplantation in c4:o or exposure to defined signals in vitro, should help to shed new light on the important issue of when and how these multipotent cells become committed to particular fates.
35 hkyer, R. and Birchmeier. C. (19951 ~lirrrrre378, 386-390 36 King&y. D.M. (1994) Gews &II. 8, 133-146 37 Bash. G.S. (1996) Trends Genei. 15, 299-305 38 Marusich, M.F. and Weston, J.A. (1991) Cwr. Opin. GeW. De2.L1,221-229 39 Robertson, K. and Mason, I. (1997) Trrr2rksGenet. 13, l-3 40 Mak. Y.F. and Ponder, B+A.J. (1%) C!tn: Opin. Genet+ Drv. 6,82-S 41 Kurihard. Y. efnl (lm) ,~am-e368,703-710 42 Kurihan. Y. el al. (199%J. Clin. huvst. 96, 293-300
Aclmowkdgeme~ts work described from the author-s laboratory was supported by the NIH, Muscular Dystrophy Association and the Howard Hughes Medical institute. I thank members of my laboratory for helpful discussions, and Nirao Shah for the preparation of Fig. 1.
43 Lhav, K. et rrl. (1996) Proc. Nafl.Acad. Sci. U. S. A. 93, 3892-.3897 44 Reid. K. d al.(1996) De&k@zen? 122. 391l-3919 45 Johnson, J.E.. Birren, S.J. and Anderson, D.J. (1990) NatWe 346, 858-861 46 Lo, L. et al. (1991) Gerres L&L!. 5, 1524-1537 47 Guillemot. F. et (~1. WW ~!ntlrre 3771.333-336 48 Guillemot, F. et crl. (1993) Cell75 463-476 49 Bku.IglUnd. E. et al. (1996) &w’lop??Ienl122. 309-320 50 Sommer. L, e! al. (1995) Keilrotl Ii. 1245-1258 51 Zimmerman. L.B., Dejesus-Escobar. J.M. and Hadand. R.M. (1996) CeU86. 599-606 52 Ma. Q.. Kintner. C. and Anderson, D.J. Cef!(in press) 53 Grddwohl. G., Fade, C. and Guillemot. F. (1996) Deu. BiCJ/. 180. 227~%I
Ref2 me~ouarin. NM. (1382) ?%be A’t?~tr~tI crest, Cambridge
University Press L.eDouarin, NM. (1980) hkMure 286, 663-669 : Bronner-Fraser. M.E.(1993) Czrrr Opirr. Cnrr!. Del,. 3. 641-647 4 Le Douarin, NM., ZiUer,C. and Couly, G.F. (1993) Dev. Biol. t53, 24-49 5 Le Douarin, NM. (19861 Sciejrce231. 1515-1522 6 Weston, J.A. (1991) Corn: Top. Deu. Viol. 25. 133453 7 Noden. D.M. (1993)j. iVt~+~~bk~l. 2% 248-261 61 Graham. A., Heyman. 1. and Lumsden. A. ( 19%) DeveIopment 119,233-245 9 Birghauer, E. et al. (1995) Dewlopnw~l121. 935-945 IO Stemple, D.L. and Anderson. DJ. t 19931 &L? Bid. 159.
54 Sommer. L.. Ma, Q and Anderson. 0.J. (1%) Mol. Cell. h&wrwi. 8, 221-241
55 Lee,J.E. etnl.(1995) Science268. 836-844
12-23
11 Le Douarin. NM., Dupin, E. and Ziller. C. (1994) C~rw. 12 13 14 IS 16
Opin. Genet. Dev. 4, 6885-695 Bronner-Fraser, M. (1994) FASEBJ. 8.699-706 Lumsden. A. and Krumlauf, R. (1996) Sciwce274. 1109-1115 Stemple, D.L. and Anderson. D.J. (1992) Ce1171. 973-985 Ito, K.. Morita. T. and Sieber-Blum. M. (19931 i&%1.Biro!. 157,517-525 Morrison, S.J.. Shah. N. and Anderson. DJ. (1997) Cell88.
287-298 1Y Sieher-Blum, M. and Cohen. A. (I9801 &l: Hiol 80. 96-106
18 Baroffio. A., Dupin. E. and Le Doudrin. NM. (1988) ~%nz. ?iatI. Acad. SC!. U. S. A. 85. 5325-5329 19 Shah, N., Groves. A. and Anderson, D.J. ( 1996) Cell85, 331-343 20 Baroffio, A.+Dupin. E. and Le Douarin. N.M. (1991) Developnlerzt112. 301-305 21 Murphy, M. etal. (1991) Proc. Xatl, Acad. Sci. U. S. A. 88. 22 23 24 25
3498-3501 Sieber-Blum. Sieher-Blum, Lemke. G.E. Marchionni,
27 28 29 30
Wu, H. et al. (1995) Cell83,59-67 Dong, Z. etal. (1995) Nettrnjl Ii, 585-596 Thoenen, H. (1991) Trmtds Nerrrosci.14, 165-170 Artavanis-Tsakonas, S., Matsuno, K. and Fortini, M.E,
M. (1989) Scierzce 243, 1608-1610 M. er al. (1993jj. &wrobiol. 24, 173-184
and Brockes. J.P. (1984) i. Nelrrosci. 4,75-83 M.A.etd. (1993) rVot~r~362,312-318 26 Shah, N.M. et al. (1994) Ceil77.349~360
(19951Sc&ce268, 225-232 31 W&master, G., Roberts, V.J.and Lemke, G. (1991) Lkmlopment
113, 199-205
56 Begley. CC. et al. (1992) Prc,c. :V~tl. Acncl. Sci I! S. A. 89 +42 57 G&l. V. t’ lNI. (lc992) Cell Grcwfb D@r. 3. 143-148 58 Ma. );, d ~1.J, i’%WmCi. (in press) 59 McEvilly. K.J. d al.(199G~ X?rrrm38$ 574-577 60 Xiang. M.Q+ rl (ii, (1996) Proc. had Acad Sci. U. S. A. 93. 11950-l 1955 61 Pfaff. S.L. el al. C 19961 Ceil 84, 309320 62 Gruss, P. and Watcher. C. (1992) CelI69.719-722 63 Kioussi. C., Gross. hl K. and Grws. P. (199j) Nwnwr 15. j j3-562 64 Topilko. P. et nl. (1994) kr~wr371.796-799 65 Weinstein, DE. Burr&. P.G. and Lemke. G. (1995) ivfr~l.cell. Atiwosci. 6. 212-229 66 Bermingham. J.R. et (11.(1996) Gtws &II. 10, 1751-1762 67 Zorick, T.S, and Len&. G. (1996l Cwr. Opi”. Cell Biol. 8. 870-876 68 Moore. K.J. (l!Y$lj) 7ksds Geuet. 11. 442-448 69 Hunt. P. :md Krumlauf, H. (1992) Annu. Rev. Cell Biol. 8. 227-256 70 Cserjesi, P. ei al. (1995) L&&r.Biol. 170.664-678 71 Hollonherg. SM. ef a/. (1995) dlf~l. Cell. Eiol. 15. 3813-3822 72 StivdStaKi.D., Csejesi, P. and Olson. E.N. (19%) Scie#zcr no. 1995-1999 73 Eisen. J.S. (1996) CeI87, 969-977 Refermce added in proof 74 Lo. l., et NI. Cnrr, Biol. (in press)
D J. Att&rson is in the Divisiw ofBiology 2167G, Howard Hughes Medical Institute, Cali/omiu Institute Tecbmoiogl:Pasaderla, CA 91l.3, USA.
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