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Cell signalling in induction and anterior-posterior patterning of the vertebrate central nervous system
Cell signalling in induction
and anterior-posterior
patterning of the vertebrate
central nervous system
Andrew Roche Institute Recent
experimental
reappraisal patterned studies,
P. McMahon
of Molecular
studies
in
of the mechanisms by dorsal
addressing
the
mesoderm.
This
Opinion
USA
have
patterning
The classic view, which has held sway for several decades, is that dorsal mesoderm, principally the notochord, is responsible for initial neural induction. Thereafter, signals derived from the notochord, which differ qualitatively or quantitatively along the mesodermal axis, are responsible for induction of specific regions of the CNS (for an excellent review, see [ 1.1). Thus, as Mangold showed in 1933 [2], anterior mesoderm placed in ventral regions induces anterior neural structures, whilst more posterior mesoderm induces posterior neural structures.
on these, and related
in patterning
the vertebrate
1993,
3:4-7
To determine whether planar signals pattern the neural plate two approaches have been adopted. In the first [ 6*], explants from early Xenopusgastrulae are placed in cul-
Abbreviations
@
Current
a
and
Recent experiments have addressed the issue of whether induction of the neural plate, and subsequent patterning, requires signals that arise vertically from dorsal mesoderm, or whether neural cells can signal through the plane of the neural plate to establish A-P patterning. Two different approaches, one in which ectoderm is cultured in combination with neural plate tissue [4], and the other in which exogastrulae are generated by preventing mesodermal involution (by incubation in high salt concentrations) [ 51, have established that neural markers are induced by apparent planar signalling between ectodermal cells. But can planar signals pattern the neural plate? The answer appears to be that planar signalling can give rise to a substantial, but not complete, CNS pattern.
The evidence that CNS specification can be separated into two phases, an initial general neural induction followed by more specific regional induction, is largely based on rather weak morphological criteria. One recent experiment in Xenopus suggests that initial induction results in the widespread expression of A-P characteristics, with subsequent mesodermal interactions refining the pattern along the neural axis [3-l. This study examined the expression of retinal opsin and a homeobox gene
4
to
(XEIBOX-6). In the tailbud embryo, these mark relatively anterior and posterior regions of the CNS, respectively. As expected, neural induction does not occur until the gastrula stage, when mesodenn is forming. When either anterior or more posterior mesodermal tissue from mid gastrula stage embryos is assayed for the ability to induce regional expression of the CNS markers, both induce opsin and XEIBOX-6 expression. Moreover, induction of these markers is not restricted within the neural ectoderm, both genes are broadly activated along the A-P axis. Therefore, neither the regional inductive capacity of the mesoderm, nor the responsive capacity of the ectoderm, is restricted at early stages. In contrast, by early neurula stages, when the neural plate has formed, dorsal mesoderm is only capable of inducing those neural markers appropriate for the particular A-P axial level. Although it is not clear to what extent this phenomenon is mirrored by other regionally distributed neural markers, or by the intact embryo, the implication that the regional character of both the inducing mesoderm and the responding ectoderm is gradually acquired is an interesting observation.
on neural
A-l-anterior-posterior;
led
plate is induced
system.
in Neurobiology
The problem of how the central nervous system (CNS) is established and subdivided into the various anatomical and functional domains that make up the brain and spinal cord has occupied experimental embyologists for over 60 years. The recent discovery of early markers, which allow unambiguous identification of CNS regions, and the growing sophistication of embryological and genetic techniques, which allow the probing of cell states and gene function, have significantly changed our view of CNS patterning. This review will focus on some recent observations that address the role cell signalling plays in neural induction and regional specification along the anterior-posterior (A-P) axis of the vertebrate CNS.
and anterior-posterior
review focuses
nervous
Introduction
induction
embryo
the neural
the role of cell signalling
Current
of mesoderm
Xenopus
by which
central
The influence
Biology, Nutley,
CNS-central
Biology
nervous
Ltd ISSN 09594388
system.
Cell signalling
in induction
mre before involution of the dorsal mesoderm (Keller explams). When two such sheets are cultured under a cover&p with their inner surfaces in contact, the mesodermal and ectodermal components undergo the typical convergence and extension of gastrulation, but in opposite directions. Thus, although the mesoderm and etoderm remain in contact, the former never comes to underlie the latter. Despite this, the expression of several regional CNS markers are induced with a correctly ordered spatial distribution along the A-P axis, similar to that seen in the Xenopus neurula. No induction of either general or specific neural markers occurs in the absence of adjacent mesoderm, suggesting that mesoderm is the source of the signal(s) that travels through the plane of the neural plate. Similarly, region specific neural markers are also expressed in exogastrulae despite the absence of mesoderm invagination under the ectoderm of the gastrula [7*]. Together these experiments convincingly demonstrate that contact between mesoderm and ectoderm is essential for neural induction and A-P patteming. However, as several aspects of CNS pattern can be established by signals passing through the plane of the neuroectoderm, vertical signalling from mesoderm to ectoderm is not necessary. Although neither set of experiments excludes a role for vertical mesodermal signalling during normal gastrulation, one must still explain how planar signalling can induce and pattern the neural plate. As more early markers are examined, it is possible that not all aspects of A-P pattern can be established by planar signals. The most anterior part of the CNS, the forebrain, is not apparently induced in exogastrulae unless anterior mesodemr is placed directly under the presumptive anterior neural ectoderm [7=]. Thus, vertical signals may be necessary for forebrain development. HOW does planar signalling lead to patterning? Attention has focused on a midline population of ectodermal cells, the notoplate, whose derivatives give rise to ventral midline cells of the CNS, termed the floorplate. The notoplate undergoes convergence and extension during normal gastrulation in exogastrulae and in Keller sandwiches. In accordance with regional specification in the CNS, which is symmetric about the ventral midline, signals derived from the notoplate would be expected to have equal effects on either side of the midline. There are several lines of evidence that link the floorplate and notochord in terms of their inductive properties [s] and the expression of putative regulatory genes [9,10]. However, little is known of the properties of the notoplate itself. Grafts of midline neural plate will induce neural expression suggesting that, before floor plate differentiation, the notoplate may participate in neural signalling [7*]. The failure to observe normal expression of a notoplate marker in exogastrulae, calls into question the normality of the notoplate in embryos capable of planar signalling [lo]. Moreover, induction of regional CNS markers still occurs in the absence of convergence and extension of ectoderm, which presumably includes the notoplate (in open-faced Keller explants), and when ventral ectoderm, which normally never forms neural tissue, is placed next to dorsal mesoderm [ 6*]. In summary, although planar signalling can occur, the mechanism by which signals are passed through the plane of the ecto-
and patterning
of the central
nervous
system
McMahon
derm to induce and pattern the neural plate is currently a mystery. Signals What are the signals governing neural induction and patterning? Considerable circumstantial evidence indicates that members of the activin family of peptide growth factors participate in the induction of dorsal mesodemr, including notochord (for a review, see [ll]). In addition, one line of experimentation suggests that, directly or indirectly, activin can induce neural tissue in the absence of a preceding notochordal induction [ 121. In these experiments, animal caps from ultra-vi olet irradiated Xenopus embryos (so called ventralized embryos) were treated with high doses of activin. Normally, these caps form ciliated epidermis. In the presence of activin, however, several neural markers were induced. Moreover, two of these, engrailed and Krox20 (which in normal neurulae are expressed in tight bands restricted to the mid/hindbrain and hindbrain regions, respectively) were induced in localized strips in the ventralized ectodermal explants, suggesting that both induction and patterning of some CNS regions was occurring. The failure to identify appreciable amounts of notochord using a notochord-specific antibody suggests that activin directly, or through some other non-notochordal cell type, can induce and pattern neural tissue. Recent experiments appear to argue against a direct role for activin signalling in neural induction [ 13’1. Indeed, they indicate that suppression of activin signalling is normally required to induce neural tissue. The involvement of activin has been studied by injecting RNA encoding a Xenopus activin receptor, in which the serinethreonine kinase domain has been deleted, into a Xenopus embtyo. Animal caps taken from such embryos and treated with activin fail to induce mesoderm. Thus, expression of the truncated receptor apparently inhibits activin signalling, although the mechanism by which this occurs, and the degree to which inhibition is specific to activin receptor signalling alone, is unclear. Interestingly, animal cap cells expressing truncated receptor did not form epidemral tissue, their normal fate in the absence of mesoderm induction. Instead, expression of a general neural marker, N-CAM, was induced in the absence of any mesoderm. These results suggest that blocking a low level of activin signalling in the animal cap itself, may be required to convert ectodermal cells from an epidermal to a neural cell fate. This explanation is consistent with an earlier observation that dissociated animal caps from blastulae can give neural cell types directly, in the absence of any mesodermal induction [ 141. These experiments require rather sparse seeding of animal cap cells, possibly lowering, below some threshold value, the local concentration of activin required to inhibit neural induction. If this general view is correct, then neural inducing factors would be predicted to interfere, at some level, with endogenous activin signalling. This may occur by suppressing the ability of activin to bind to its own receptor, for which two peptide factors, follistatin and inhibin, are likely candidates. Alternatively, a second signalling pathway may lead to suppression of
.5
6
Development the activin signal transduction pathway within ectodermal cells, counteracting the effect of ectodermally-produced activin. Protein kinase C isozymes have been implicated in the intracellular neural induction pathway [ 151, and, therefore, are possible targets for such regulation. Thus, although it is clear that injection of a truncated activin receptor can induce neural cells, there is much to be learned about how this experimental manipulation relates to the in vivo situation. With respect to CNS patterning, it will be fascinating to determine whether such animal caps show a general expression of neural markers, or regional distributions consistent with prepatterning of the ectoderm. What is the nature of the signalling molecules involved in patterning the neural plate? One candidate is retinoic acid. Application of retinoic acid to gastrulating Xenopus [16,17], zebrafish 1181 and mouse embryos [Ip] leads to perturbation of midbrain and hindbrain patterning, possibly due to the misexpression of retinoic acid sensitive Hox genes [ 19.1. Indeed, by applying retinoic acid to neural explants, it is possible to mimic the normal induction of some regional markers along the neural axis, suggesting that retinoic acid may initiate their expression [ 201. As these explants contain mesodermal derivatives, it is not clear whether retinoic acid acts directly, or indirectly through midline mesoderm, to activate regional gene expression. If retinoic acid is involved in signalling in vivo, the most likely source is the posterior region of the embryo. Studies in the mouse have established that the node, which is thought to represent the mammalian counterpart of the Xenopus dorsal blastopore lip and the chick Hensen’s node, produces retinoic acid in keeping with this hypothesis [21]. Thus, retinoic acid may play a broad early role in patterning caudal regions of the CNS, perhaps through the regional activation of homeobox genes. As well as the long range signalling discussed above, chick grafting studies indicate that local cell signalling may regulate the expression of putative regional determinants in the vertebrate CNS. Grafting of midhindbrain regions from early somite stage chick embryos into the forebrain leads to ectopic midbrain development by the graft, but more surprisingly, host cells are also recruited into the midbrain pathway [22]. This can be visualized soon after grafting by the induction of ectopic engruiled expression in host cells at the site of the graft [23,24]. Engruiled is expressed in, and probably contributes to, the specification of the midbrain/hindbrain region. Thus, grafted cells are able to signal to, and change the fate of, neighboring forebrain cells, another example of planar signalling. A strong candidate for this interaction is the signalling molecule Wnt-1. Wnt-1 is essential for midbrain development and is expressed with striking overlap with the engraileddomain [ 251. Drosophila studies have established that the Wnt-1 orthologue, wingless, is required for the maintenance of engruiled expression in neighboring cells in the Drosophila segment. Is Wnt-1 the signal activating ectopic engrailed expression in the forebrain, and by inference, the signal regulating normal engrailed expression in the mid/hindbrain region? At present, this question remains unanswered. It seems unlikely that Wnt-1 is directly responsible for ectopic en-
grailed expression, as grafts of anterior hindbrain, which do not express Wnt-1, appear to induce ectopic engrailed expression in the forebrain [26]. Whatever the mechanism by which Wnt-1 regulates midbrain development, gene disruption studies in the mouse have clearly established that Wnt-1 signalling is essential for normal CNS patterning [25]. Signalling may regulate not only the establishment, but also the polarity, of engruiled expression in the midbrain. Normal engrailed expression is highest at the posterior end of the midbrain, decreasing anteriorly, apparently due to a repressive signal(s) emanating from the diencephalic-mesencephalic border [ 271. Thus, the cytoarchitectonic development of the chick midbrain (tectum), and the patterning of retinotectal projections, which show an A-P gradient, may depend on signals that attenuate normal engrailed expression. Conclusion
and perspective
The induction and subsequent early patterning of the vertebrate CNS is critically dependent upon mesodermallyderived signals. Intact ectodermal explants, in the absence of dorsal mesoderm, do not give rise to neural tissue. An exception occurs in explants in which activin signalling is inhibited, suggesting that neural induction results, at least in part, from preventing endogeneous activin signalling within the dorsal ectoderm. Vertical signalling from dorsal mesoderm may specify the most anterior regions of the CNS. However, mesodermdependent signalling within the plane of the ectoderm can, under experimental conditions, lead to substantial patterning of more posterior regions of the developing CNS. Finally, local cellular interactions within the neural plate modulate regional expression of putative determinants, and may be responsible for determining the underlying polarity of specific brain regions. Although many signals have been hypothesized to explain certain experimental observations, relatively little is known about the identity of the signals themselves, or their mechanism of action; the signalling pathways and target genes. With the identification of an increasing number of putative signalling molecules, the closer we come to martying together the observations of decades of classical experimental embryology with the actual molecules that regulate the developmental processes suggested by these manipulative procedures. References
and recommended
Papers review, . ..
of particular interest, published have been highlighted as: of special interest of outstanding interest
1. .
SLACK JMW,
2.
MANGOLD0: Induktionsfihigkeit der Neurula van Urodelen. 43:761-766.
reading
within
the annual
period
of
TANNAHILLD: Mechanisms of Anterior-Posterior Axis Specification in Vertebrates. Development 1992, 114:285-302. This review provides a modem interpretation of 70 years worth of Xeqws embryology relating to A-P specification of the early CNS. der Verscheidenen Naturwissenschaften
Bezirke 1933,
Cell signalling in induction and patterning of the central nervous system McMahon 3.
.
SAHA MS, GRAINGER Rht A Labile Period in the Determination of the Anterior-Posterior Axis During Early Neural
Development in Xenopus Neuron 1992, 8:1003_1014. Expression of regional specific neural markers was examined in cultured explants of ectoderm and mesoderm taken from mid gastrula (stage 11.5) and early neurula (stage 14) embryos. Neural induction occurs at mid gastrula stages. At this time, mesodermal regions are not restricted in their inductive capacity along the A-P axis. By neurula stages, however, the inductive capacity of mesoderm is restricted. Anterior and posterior mesoderm only induce expression of anterior or posterior neural markers, respectively. 4.
SERVETNICK
M, GRAINGERRM: Homeogenetic Dev Biol 1991, 14773-82.
GODSAVE SF, SLACKJMW: Clonal Analysis of Mesoderm Induction in Xenopus laevis. Dev Biol 1989, 134:486490.
15.
OTIT AP, MOONRT: Protein Kinase C Isozymes Have Dii-
tlnct Roles in Neural Induction and Competence Cell 1992, 68:1021-1029.
16.
Rulv ALTABAA, JESSELLTM: Retinoic Acid Modiies the Pattern of Cell Differentiation in the Central Nervous System of Neurula Stage Xenopus Embryos. Development 1991, 112:945-958.
17.
PAPALOPULU N, CLARKEJDW, BRADLEYL, WI&lNSON D, KRUMLAUF R, HOLDERN: Retinoic Acid Causes Abnormal Development and Segmental Patterning of the Anterior Hindbrain in Xenopus Embryos. Development 1991, 113:1145-1158.
18.
HOLDERN, HILLJ: Retinoic Acid Modifies Development of the Midbrain-Hindbraln Border and Affects Cranial Ganglion Formation in Zebra&h Embryos. Development 1991, 113:1159-1170.
108:59%604. DONIACH T, PHILUPS CR, GERHARTJC: Planar Induction of Anteroposterior Pattern in the Developing Central Nervous System of Xenopus Laevis. Science 1992, 257542-545. Whole mount in situ hybridization or antibody analysis was performed on explants of early gastrulae. Although mesodermal and ectodermal extension occur in opposite directions, all neural markers examined were induced. Moreover, the induction of specific regional markers was localized to discrete regions of the neural plate, which corresponded to their normal position. Thus, neural induction and A-P patterning occur by a signal or signals travelling through the plane of the neural plate. .
7.
.
RUIZIALTABAA: Planar and Vertical Signals in the Induction and Patterning of the Xenopus Nervous System. Deuelw ment 1992, 116:67+30.
The normal induction of a number of neural markers demarcating spew cific regions of the neural plate occurs in exogastrulae. Thus, signals within the plane of the neural plate can result in A-P patterning. A midline ectodermal structure, the notoplate, is shown to have inductive properties. 8.
YANAL)A T, PIACZEKM, TANAKAH, DODD J, JESSELLTM: Control Nervous System: Polarof Cell Pattern in the Developing izing Activity of the Floor Plate and Notochord. Cell 1991, 64~635-647.
CONLON RA, ROSSANTJ: Exogenous Retinoic Acid Rapidly Induces Anterior Ectopic Expression of Murlne Hex-2 Genes In Vivo. Development 1992, 116:357-368. Intraperitoneal injection of retinoic acid into pregnant mice results in the rapid activation of ectopic expression of various numbers of the Hox-B family in the neural plate of early embryos. Thus, retinoic acid disturbs the normal A-P distribution of Hex-gene expression. 19. .
20.
SHARPE CR: Retinoic Acid can Mimic Endogenous Signals Involved in Transformation of the Xenopus Nervous System. Neuron 1991, 7:233-247.
21.
HOGANBLM, THALLER C, EICHELE G: Evidence that Hensen’s Node is a Site of Retinoic Acid Synthesis. Nature 1992, 359237-241.
22.
ALVARALZ~MAUART R-M,
23.
GARDNERCA, BARALDKF: The Cellular Environment Controls the Expression of Engrailed-Like Protein in the Cranial Neuroepithelium of Quail-Chick Chime& Embryos. Development 1991, 113:1037-1048.
24.
MARTINEZ S, WASSEFM, ALVARAD~MALUUIT RM: Induction of a Mesencephalic Phenotype in the 2-Day-Old Chick Prosencephalon is Preceded by the Early Expression of the Homeobox Gene En. Neuron 1991, 6:971-981.
9.
DIRKSENML, JAMRICHM: A Novel, Activin Inducible, Blastopore Lip-Speci6c Gene of Xenopus Laevfs Contains a Fork Head DNA Binding Domain. Genes Dev 1992: 6:59+608.
10.
TM: Pintallavis, Rum ATTABAA, JESSELL
Expressed in the Organizer and Midline Cells of Frog Embryos: Involvement in the Development of the Neural Axis. Development
11.
JESSELL TM, MELTON DA Diisible Factors in Vertebrate Embryonic Induction. Cell 1992, 68~257~270.
12.
BOLCEME, HEMMATI-BRIVANLOU A, KUSHNER PD, GARLAND RM: Ventral Ectoderm of Xenopus Forms Neural Tissue, Including Hlndbrain in Response to Activin. Development 1992, 115%&a.
26.
HEMMAX-BRIVANLOU A, MELTON DA A Truncated Activin Receptor Inhibits Mewderm Induction and Formation of Axial Structures in Xenopus Embryos. Nature 1992, 359-l.
27.
a Gene
1992, 116:81-93.
13. .
Animal caps and intact Xenopus embryos expressing a truncated Xeno pus activin receptor (XAK1) were examined. Activin has been implicated in induction of mescderm in the embryo. Expression of the
truncated receptor inhibited formation of mesoderm by animal caps treated with activin, and also suppressed mesoderm formation in the intact embryo. Surprisingly, animal caps switched on a neural marker,
in Xeno
pus
Rum ALTABAA Neural Expression of the Xenopus Homeobox Gene XHox3: Evidence for a Patterning Neural Sig nal that Spreads Through tbe Ectoderm. Development 1990,
6.
these
14.
Neural Induc-
tion in Xenopus 5.
suggesting that a normal low level of activin signalling between cells suppresses activation of neural development.
25.
MARTINEZ S, IANCE-JONES CC: Pluripo tentiality of the 2-Day-Old Avian Germinative Neuroepithelium. Dev Biol 1990, 139~75-88.
MCMAHON AP, JOYNER AL, BRADLEYA, MCMAHON JA The
Midbrain-Hindbrain Phenotype of Wnt-1-/ W&%1-Mice Results from Stepwise Deletion of Engrailed-Expressing Cells by 9.5 Days Postcoitum. Cell 1992, 69:581-595. BALLY-CUIF L, ALVARADC-MAUART R-M, DARNELLDK, WA~SEFM: Relationship Between Wnt-1 and En-2 Expression Domains During Early Development of Normal and Ectopic Met-Mesencephalon. Development 1992, 115:99+1w. ITA.SAKIN, ICHIJO H, HAMA c, MATSUNOT, NAKAMURA H: ES-
tablishment of Rostrocaudal Polarity in Tectal Primordium: EngraiZed Expression and Subsequent Tectal Polarity. De velopment 1991, 113:1133-1144. AP McMahon, Roche Institute of Molecular Street, Nutley, New Jersey 07110-1199, USA
Biology,
340 Kingsland
.7
and anterior-posterior
patterning of the vertebrate
central nervous system
Andrew Roche Institute Recent
experimental
reappraisal patterned studies,
P. McMahon
of Molecular
studies
in
of the mechanisms by dorsal
addressing
the
mesoderm.
This
Opinion
USA
have
patterning
The classic view, which has held sway for several decades, is that dorsal mesoderm, principally the notochord, is responsible for initial neural induction. Thereafter, signals derived from the notochord, which differ qualitatively or quantitatively along the mesodermal axis, are responsible for induction of specific regions of the CNS (for an excellent review, see [ 1.1). Thus, as Mangold showed in 1933 [2], anterior mesoderm placed in ventral regions induces anterior neural structures, whilst more posterior mesoderm induces posterior neural structures.
on these, and related
in patterning
the vertebrate
1993,
3:4-7
To determine whether planar signals pattern the neural plate two approaches have been adopted. In the first [ 6*], explants from early Xenopusgastrulae are placed in cul-
Abbreviations
@
Current
a
and
Recent experiments have addressed the issue of whether induction of the neural plate, and subsequent patterning, requires signals that arise vertically from dorsal mesoderm, or whether neural cells can signal through the plane of the neural plate to establish A-P patterning. Two different approaches, one in which ectoderm is cultured in combination with neural plate tissue [4], and the other in which exogastrulae are generated by preventing mesodermal involution (by incubation in high salt concentrations) [ 51, have established that neural markers are induced by apparent planar signalling between ectodermal cells. But can planar signals pattern the neural plate? The answer appears to be that planar signalling can give rise to a substantial, but not complete, CNS pattern.
The evidence that CNS specification can be separated into two phases, an initial general neural induction followed by more specific regional induction, is largely based on rather weak morphological criteria. One recent experiment in Xenopus suggests that initial induction results in the widespread expression of A-P characteristics, with subsequent mesodermal interactions refining the pattern along the neural axis [3-l. This study examined the expression of retinal opsin and a homeobox gene
4
to
(XEIBOX-6). In the tailbud embryo, these mark relatively anterior and posterior regions of the CNS, respectively. As expected, neural induction does not occur until the gastrula stage, when mesodenn is forming. When either anterior or more posterior mesodermal tissue from mid gastrula stage embryos is assayed for the ability to induce regional expression of the CNS markers, both induce opsin and XEIBOX-6 expression. Moreover, induction of these markers is not restricted within the neural ectoderm, both genes are broadly activated along the A-P axis. Therefore, neither the regional inductive capacity of the mesoderm, nor the responsive capacity of the ectoderm, is restricted at early stages. In contrast, by early neurula stages, when the neural plate has formed, dorsal mesoderm is only capable of inducing those neural markers appropriate for the particular A-P axial level. Although it is not clear to what extent this phenomenon is mirrored by other regionally distributed neural markers, or by the intact embryo, the implication that the regional character of both the inducing mesoderm and the responding ectoderm is gradually acquired is an interesting observation.
on neural
A-l-anterior-posterior;
led
plate is induced
system.
in Neurobiology
The problem of how the central nervous system (CNS) is established and subdivided into the various anatomical and functional domains that make up the brain and spinal cord has occupied experimental embyologists for over 60 years. The recent discovery of early markers, which allow unambiguous identification of CNS regions, and the growing sophistication of embryological and genetic techniques, which allow the probing of cell states and gene function, have significantly changed our view of CNS patterning. This review will focus on some recent observations that address the role cell signalling plays in neural induction and regional specification along the anterior-posterior (A-P) axis of the vertebrate CNS.
and anterior-posterior
review focuses
nervous
Introduction
induction
embryo
the neural
the role of cell signalling
Current
of mesoderm
Xenopus
by which
central
The influence
Biology, Nutley,
CNS-central
Biology
nervous
Ltd ISSN 09594388
system.
Cell signalling
in induction
mre before involution of the dorsal mesoderm (Keller explams). When two such sheets are cultured under a cover&p with their inner surfaces in contact, the mesodermal and ectodermal components undergo the typical convergence and extension of gastrulation, but in opposite directions. Thus, although the mesoderm and etoderm remain in contact, the former never comes to underlie the latter. Despite this, the expression of several regional CNS markers are induced with a correctly ordered spatial distribution along the A-P axis, similar to that seen in the Xenopus neurula. No induction of either general or specific neural markers occurs in the absence of adjacent mesoderm, suggesting that mesoderm is the source of the signal(s) that travels through the plane of the neural plate. Similarly, region specific neural markers are also expressed in exogastrulae despite the absence of mesoderm invagination under the ectoderm of the gastrula [7*]. Together these experiments convincingly demonstrate that contact between mesoderm and ectoderm is essential for neural induction and A-P patteming. However, as several aspects of CNS pattern can be established by signals passing through the plane of the neuroectoderm, vertical signalling from mesoderm to ectoderm is not necessary. Although neither set of experiments excludes a role for vertical mesodermal signalling during normal gastrulation, one must still explain how planar signalling can induce and pattern the neural plate. As more early markers are examined, it is possible that not all aspects of A-P pattern can be established by planar signals. The most anterior part of the CNS, the forebrain, is not apparently induced in exogastrulae unless anterior mesodemr is placed directly under the presumptive anterior neural ectoderm [7=]. Thus, vertical signals may be necessary for forebrain development. HOW does planar signalling lead to patterning? Attention has focused on a midline population of ectodermal cells, the notoplate, whose derivatives give rise to ventral midline cells of the CNS, termed the floorplate. The notoplate undergoes convergence and extension during normal gastrulation in exogastrulae and in Keller sandwiches. In accordance with regional specification in the CNS, which is symmetric about the ventral midline, signals derived from the notoplate would be expected to have equal effects on either side of the midline. There are several lines of evidence that link the floorplate and notochord in terms of their inductive properties [s] and the expression of putative regulatory genes [9,10]. However, little is known of the properties of the notoplate itself. Grafts of midline neural plate will induce neural expression suggesting that, before floor plate differentiation, the notoplate may participate in neural signalling [7*]. The failure to observe normal expression of a notoplate marker in exogastrulae, calls into question the normality of the notoplate in embryos capable of planar signalling [lo]. Moreover, induction of regional CNS markers still occurs in the absence of convergence and extension of ectoderm, which presumably includes the notoplate (in open-faced Keller explants), and when ventral ectoderm, which normally never forms neural tissue, is placed next to dorsal mesoderm [ 6*]. In summary, although planar signalling can occur, the mechanism by which signals are passed through the plane of the ecto-
and patterning
of the central
nervous
system
McMahon
derm to induce and pattern the neural plate is currently a mystery. Signals What are the signals governing neural induction and patterning? Considerable circumstantial evidence indicates that members of the activin family of peptide growth factors participate in the induction of dorsal mesodemr, including notochord (for a review, see [ll]). In addition, one line of experimentation suggests that, directly or indirectly, activin can induce neural tissue in the absence of a preceding notochordal induction [ 121. In these experiments, animal caps from ultra-vi olet irradiated Xenopus embryos (so called ventralized embryos) were treated with high doses of activin. Normally, these caps form ciliated epidermis. In the presence of activin, however, several neural markers were induced. Moreover, two of these, engrailed and Krox20 (which in normal neurulae are expressed in tight bands restricted to the mid/hindbrain and hindbrain regions, respectively) were induced in localized strips in the ventralized ectodermal explants, suggesting that both induction and patterning of some CNS regions was occurring. The failure to identify appreciable amounts of notochord using a notochord-specific antibody suggests that activin directly, or through some other non-notochordal cell type, can induce and pattern neural tissue. Recent experiments appear to argue against a direct role for activin signalling in neural induction [ 13’1. Indeed, they indicate that suppression of activin signalling is normally required to induce neural tissue. The involvement of activin has been studied by injecting RNA encoding a Xenopus activin receptor, in which the serinethreonine kinase domain has been deleted, into a Xenopus embtyo. Animal caps taken from such embryos and treated with activin fail to induce mesoderm. Thus, expression of the truncated receptor apparently inhibits activin signalling, although the mechanism by which this occurs, and the degree to which inhibition is specific to activin receptor signalling alone, is unclear. Interestingly, animal cap cells expressing truncated receptor did not form epidemral tissue, their normal fate in the absence of mesoderm induction. Instead, expression of a general neural marker, N-CAM, was induced in the absence of any mesoderm. These results suggest that blocking a low level of activin signalling in the animal cap itself, may be required to convert ectodermal cells from an epidermal to a neural cell fate. This explanation is consistent with an earlier observation that dissociated animal caps from blastulae can give neural cell types directly, in the absence of any mesodermal induction [ 141. These experiments require rather sparse seeding of animal cap cells, possibly lowering, below some threshold value, the local concentration of activin required to inhibit neural induction. If this general view is correct, then neural inducing factors would be predicted to interfere, at some level, with endogenous activin signalling. This may occur by suppressing the ability of activin to bind to its own receptor, for which two peptide factors, follistatin and inhibin, are likely candidates. Alternatively, a second signalling pathway may lead to suppression of
.5
6
Development the activin signal transduction pathway within ectodermal cells, counteracting the effect of ectodermally-produced activin. Protein kinase C isozymes have been implicated in the intracellular neural induction pathway [ 151, and, therefore, are possible targets for such regulation. Thus, although it is clear that injection of a truncated activin receptor can induce neural cells, there is much to be learned about how this experimental manipulation relates to the in vivo situation. With respect to CNS patterning, it will be fascinating to determine whether such animal caps show a general expression of neural markers, or regional distributions consistent with prepatterning of the ectoderm. What is the nature of the signalling molecules involved in patterning the neural plate? One candidate is retinoic acid. Application of retinoic acid to gastrulating Xenopus [16,17], zebrafish 1181 and mouse embryos [Ip] leads to perturbation of midbrain and hindbrain patterning, possibly due to the misexpression of retinoic acid sensitive Hox genes [ 19.1. Indeed, by applying retinoic acid to neural explants, it is possible to mimic the normal induction of some regional markers along the neural axis, suggesting that retinoic acid may initiate their expression [ 201. As these explants contain mesodermal derivatives, it is not clear whether retinoic acid acts directly, or indirectly through midline mesoderm, to activate regional gene expression. If retinoic acid is involved in signalling in vivo, the most likely source is the posterior region of the embryo. Studies in the mouse have established that the node, which is thought to represent the mammalian counterpart of the Xenopus dorsal blastopore lip and the chick Hensen’s node, produces retinoic acid in keeping with this hypothesis [21]. Thus, retinoic acid may play a broad early role in patterning caudal regions of the CNS, perhaps through the regional activation of homeobox genes. As well as the long range signalling discussed above, chick grafting studies indicate that local cell signalling may regulate the expression of putative regional determinants in the vertebrate CNS. Grafting of midhindbrain regions from early somite stage chick embryos into the forebrain leads to ectopic midbrain development by the graft, but more surprisingly, host cells are also recruited into the midbrain pathway [22]. This can be visualized soon after grafting by the induction of ectopic engruiled expression in host cells at the site of the graft [23,24]. Engruiled is expressed in, and probably contributes to, the specification of the midbrain/hindbrain region. Thus, grafted cells are able to signal to, and change the fate of, neighboring forebrain cells, another example of planar signalling. A strong candidate for this interaction is the signalling molecule Wnt-1. Wnt-1 is essential for midbrain development and is expressed with striking overlap with the engraileddomain [ 251. Drosophila studies have established that the Wnt-1 orthologue, wingless, is required for the maintenance of engruiled expression in neighboring cells in the Drosophila segment. Is Wnt-1 the signal activating ectopic engrailed expression in the forebrain, and by inference, the signal regulating normal engrailed expression in the mid/hindbrain region? At present, this question remains unanswered. It seems unlikely that Wnt-1 is directly responsible for ectopic en-
grailed expression, as grafts of anterior hindbrain, which do not express Wnt-1, appear to induce ectopic engrailed expression in the forebrain [26]. Whatever the mechanism by which Wnt-1 regulates midbrain development, gene disruption studies in the mouse have clearly established that Wnt-1 signalling is essential for normal CNS patterning [25]. Signalling may regulate not only the establishment, but also the polarity, of engruiled expression in the midbrain. Normal engrailed expression is highest at the posterior end of the midbrain, decreasing anteriorly, apparently due to a repressive signal(s) emanating from the diencephalic-mesencephalic border [ 271. Thus, the cytoarchitectonic development of the chick midbrain (tectum), and the patterning of retinotectal projections, which show an A-P gradient, may depend on signals that attenuate normal engrailed expression. Conclusion
and perspective
The induction and subsequent early patterning of the vertebrate CNS is critically dependent upon mesodermallyderived signals. Intact ectodermal explants, in the absence of dorsal mesoderm, do not give rise to neural tissue. An exception occurs in explants in which activin signalling is inhibited, suggesting that neural induction results, at least in part, from preventing endogeneous activin signalling within the dorsal ectoderm. Vertical signalling from dorsal mesoderm may specify the most anterior regions of the CNS. However, mesodermdependent signalling within the plane of the ectoderm can, under experimental conditions, lead to substantial patterning of more posterior regions of the developing CNS. Finally, local cellular interactions within the neural plate modulate regional expression of putative determinants, and may be responsible for determining the underlying polarity of specific brain regions. Although many signals have been hypothesized to explain certain experimental observations, relatively little is known about the identity of the signals themselves, or their mechanism of action; the signalling pathways and target genes. With the identification of an increasing number of putative signalling molecules, the closer we come to martying together the observations of decades of classical experimental embryology with the actual molecules that regulate the developmental processes suggested by these manipulative procedures. References
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