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Mesodermal control of neural cell identity in vertebrates
Mesodermal
control of neural cell identity in vertebrates Jane Dodd Columbia
It has long neural
been
appreciated
cells is controlled
Several recent act throughout
New York, New York, USA
University,
experiments early
the
differentiation
have revealed
neural
nature
Current
that
in part by inductive development
and
patterning
of
signals from the mesoderm.
that distinct
mesodermal
and have begun
signals
to address the
and sources of such signals.
Opinion
in Neurobiology
Introduction Vertebrate neural development begins with an inductive interaction between two of the primary gem3 layers of the embryo, the ectoderm and the mesoderm. Signals from dorsal mesoderm transform adjacent ectoderm into neuroectoderm, resulting in the formation of the neural plate. The neural plate becomes regionalized along anterior-posterior and mediolateral axes and neurons differentiate in specific locations. This review will focus on some recent developments that have contributed to our understanding of the role of mesodem-i in the induction and patterning of neural cell identity.
Neural induction: the origins and properties of the inducing signal in Xenopus Much of our current understanding of mesodermal and neural induction comes from studies of the frog, Xeno pus kzevk. Neural induction in Xenopus is controlled by signals from the organizer, a region of the embryo that differentiates into dorsal mesodermal structures such as notochord and somites. One approach that has been used to identify molecules involved in neural induction has been to test known growth factors for their ability to promote neural differentiation in isolated explants of blastula stage ectoderm (animal cap assay). Neural tissue can be induced in this assay by a number of polypeptide growth factors, the most potent of which is activin [ 141. Act&in-A, a growth factor identiiied as a regulator of follicle stimulating hormone release from the pituitary [5], is the active component of two factors, XTC/MIF and PIF, previously characterized as potent mesodermal inducers [ 2-41. In animal cap explants treated with high concentrations of activin-A large amounts of neural tissue form in addition to mesodermal tissue, raising the possibility that activin may have neural as well as mesodermal inducing capacity [I-4,6*].
1992, 2:3-8
I .’
Does activin induce neural tissue directly [4,6*,7] or does it induce it indirectly through the induction of dorsal mesoderm? Maternal activin-like activity can be detected in very early Xenopus embryos [ES]. Furthermore, the molecular cloning of Xenopus act&in-A and activin-B (the activity of which appears to be indistinguishable from activin-A) has revealed that act&in-B RNA is expressed in the stage 9 blastula [ 6.1, which may be sufficiently early for a role in neural induction. In contrast, activin-A RNAis expressed too late for it to function as a primary inducer of neural tissue. Two lines of evidence suggest that activins are not direct mediators of neural induction but act to induce mesoderm, which then induces neural tissue. First, XTC/MIF (activin A) appears to induce mesoderm but not neural tissue in single ectodermal cap cells [PI. In contrast, when cells exposed to XTC/MIF are reaggregated with naive ectodermal cells, NCAM RNA is expressed, presumably in the naive cells that had been induced to become neural. These results suggest that the neural response observed in ectodemral caps exposed to activin-A is mediated indirectly by signals from induced mesodemr. The second line of evidence comes from studies of the neural inducing properties of the chick organizer, Hensen’s node, whose actions differ from those of act&in-A in the animal cap assay [lo*]. Unlike activin, Hensen’s node induces large amounts of neural tissue in Xenopus ectoderm without inducing detectable mesoderm. In addition, the period of competence of the ectoderm to form neural tissue in response to activin is much shorter than it is for Hensen’s node, as might be expected if activin acts through intermediate steps. These results suggest that the neural inducing capacity of the organizer cannot be ascribed solely to an activin-like molecule. It is possible, however, that the neural inducing properties of organizer tissue are a result of the action of an activinlike molecule together with a second agent that alters the response properties of the ectoderm to this factor.
Abbreviation CSPG-chondroitin
_,-
sulphate proteoglycan.
@ Current Biology Ltd ISSN 0959488
4
Development
Mesoderm
control
patterning
of the neural
of antero-posterior tube
Distinct classes of neuron develop at different positions along the antero-posterior axis of the neural tube. Two proposals have been offered for the mechanisms underlying antero-posterior patterning in the neural tube (Fig. 1). First, positional information along the antero-posterior axis of the dorsal mesoderm may be transmitted to overlying neuroectoderm (Fig. la) [11,12*]. Second, signals from the organizer may travel through the plane of the ectoderm and impose pattern on the tissue independent of involuting mesoderm (Fig. lb) [ 131.
(a) Neur;~tod;~
vi
fb) Neur~c~~~
F>)
Organizer
(c) Neur;::;;
vr_
Organizer
Fig. 1. A diagram illustrating the mesodermal control of anteroposterior regionalization of the neural plate. The patterned expression of a homeobox gene such as XIHbox7 (a,b) or En-2 Cc) is illustrated by a black band. (a) Signals from underlying mesoderm impose pattern on neuroectoderm. fb) Propagation of signals within the planes of the ectoderm and the mesoderm imposes pattern on both tissues simultaneously. Cc)Signals from underlying mesoderm regulate or stabilize pattern imposed by signals propagated in the plane of the ectoderm.
The expression pattern of a number of homeobox genes in early vertebrate embryos has been interpreted to support the first proposal. For example, the Xenopus gene, XlHbox 1, is expressed early in a narrow band of nuclei at exactly the same antero-posterior position in neural crest, neural tube and underlying axial and lateral mesoderm [ 141. Similarly, the mouse gene Hox-2.9 is expressed in a highly restricted fashion in the developing hindbrain region of the neural tube and underlying mesoderm. The anterior boundaries of neural and mesodermal expression are exactly in register [15*3. The distributions of XlHbox 1 and Hox-2.9 have been suggested to reflect the transmission of positional coordinates from the axial mesoderm to the overlying unpatterned ectoderm (Fig. la) [11,14,15*]. In fact, a signal that dill&es through the plane of the mesoderm, and also through the ectoderm as the two tissues elongate in parallel, could specify an antero-posterior pattern in both tissues (Fig. lb) [ 131. Indeed, signals that are propagated over quite large distances within the plane of the ectoderm have been demonstrated in Xenopus [ 13,16,17]. The possibility that antero-posterior patterning is controlled by position-specific signals from the notochord has been approached experimentally in Xenopus The
homeobox protein, En-2, is found in a discrete band of cells within anterior neural plate, but not in mesoderm. The expression of En-2 was measured in Xeno pus animal cap ectoderm in response to notochord explants taken from different axial levels [12*]. Anterior notochordal pieces induced four times as much En-2 as posterior notochord taken from a donor of the same age, providing some support for the idea that the anteroposterior pattern is regulated by the properties of axial mesoderm (Fig. la). Potential developmental differences between anterior and posterior notochord from embryos of the same stage, however, may account for apparent positional differences in the ability of the notochord to induce En-2 expression. In addition, the effect may be a consequence of the formation of different amounts of neural tissue. It therefore remains unclear which of the two schemes (Fig.la,b) predominate in the patterning of the anteroposterior axis. It seems likely that more than one type of signal will contribute. In fact, the finding that some En-2 protein is generated in response to posterior notochord in Xenopus suggests that additional mechanisms contribute to the antero-posterior regulation of En-2 expression observed in duo. The recent finding that transplants of mesencephalic neuroectoderm that express En-2 induce the expression of En-2 by prosencephalic neuroectoderm [ 18*,19’] has lent support to the idea that there is an En-2inducing signal that is propagated in the plane of the ectoderm in the chick. The normal absence of En-2 expression in neuroectoderm adjacent to the mesencephalon also suggests that the final pattern of neural expression of En-2 in the hindbrain is dependent on a second regulatory signal, perhaps acting to stabilize En-2 expression (Fig. lc) [18*, 19*]. Evidence for synergism in the actions of involuting mesoderm and a spreading neural signal in Xenopus [ 161 supports a role for involuting axial mesoderm in the regulation of neural phenotype and/or the patterning of the neural tube along the anteroposterior axis. Thus, a signal that alters the response of ectoderm to notochord-derived factors may provide a basis for antero-posterior specification (Fig. lc). Such a signal could arise from the organizer and spread through the pregastrula ectoderm, acting to prepattem the ectoderm and generate a differential response to subsequent, e.g. notochord-derived, signals. Indeed, the early gastrula ectoderm appears to be regionalized with respect to its response to activin-A [20*,21*] and also in the expression of distinct isoforms of protein kinase C [22*].
Acquisition
of cell type-specific
neural
properties Mesodermal signals also appear to be involved later in development in the specification of individual neural cell types. The characterization of markers that identify subsets of cells in the early neural tube [23*] has made it possible to test the idea [24-271 that dorsoventral patterning in the spinal cord and hindbrain is initiated by signals from the notochord.
Mesodermal
control of neural cell identity in vertebrates Dodd
Mesodermal
control
of neural
crest cell
migration (a)
fb)
Fig. 2. A diagram
illustrating mesodermal control of the specification of neural cell type. (a) An inductive signal from the notochord (NJ induces the differentiation of the floor plate (F) in overlying neuroectoderm. Signals (arrows) from the floor plate regulate the differentiation and patterning of neurons in the developing neural tube. fb) Signals from the notochord and the floor plate control the differentiation and patterning of neurons in the developing neural tube. X represents a class of neural cells found adjacent to the floor plate (in the hindbrain these express S-hydroxytryptamine). M, motor neuron [23*,30*1.
Grafting and ablation studies in the chick have demonstrated that one of the first neural cell groups to dif ferentiate within the neural tube, the floor plate, does so in response to a local signal from the notochord [28,29,30*]. In vitro experiments have further shown that a notochord signal alone is sufficient to induce many, and perhaps all, of the differentiated properties of the floor plate [30-l. These studies have also revealed that contact between the notochord and neural epithelium is required for floor plate induction. This may reflect the fact that the notochord-derived induction signal is membrane bound or that the diffusion distance of a secreted factor is lim ited, perhaps by binding to extracellular matrix. Such a role for the extracellular matrix is emerging as a common mechanism for limiting diffusion distances and for the presentation of signalling molecules in development D-341. Grafting experiments have also provided evidence that the fate of cells in the ventral neuroepithelium is defined by cell position with respect to the ventral midline. Signals from both the notochord and the floor plate can control the pattern of identified neuronal cell types that appear in specific positions along the dorsoventral axis of the neural tube [ 23.1. It is unclear which of the two ventral midline cell groups is more important in establishing neuronal cell fate in vivo (Fig. 2b). The induction of the floor plate by the notochord may represent a necessary first step in the dorsoventral patterning of the central nervous system. The acquisition of patterning properties by the floor plate may, however, be required to offset the ventral displacement of the notochord from the neural tube that occurs soon after floor plate induction, thus maintaining induction at the midline of the neural tube.
Mesoderm also appears to regulate the fate of neural crest cells that give rise to the peripheral nervous system. The differentiation of neural crest cells is determined by the environment through which they migrate. For instance, signals from the notochord appear to influence the migratory path of sympathetic ganglion precursor cells and their differentiation in the vicinity of the dorsal aorta (Fig.3) [35,36]. Recent notochord ablation and neural tube rotation experiments suggest that inhibitory signals from the notochord and/or the peri-notochordal matrix are essential for the appropriate migration of sympathetic ganglion precursors [37-j. A chondroitin sulphate proteoglycan (CSPG) or related molecule has been previously suggested to mediate’& influence of the notochord on neural crest cell migration [36]. To test the biochemical nature of the inhibitory activity in vivo, isolated notochords were treated in vitro with a panel of enzymes and the treated notochords grafted into chick embryos at sites in the neural crest pathway [38]. The size of the crest cell-free zone around the notochord was then examined. The results support the idea that a CSPG, potentially secreted into the pen-notochordal matrix by the notochord, inhibits neural crest cell migration.
(a)
(b)
0
A
0
A
Fig. 3 A diagram
illustrating mesodermal control of neural crest cell migration and differentiation. (a) Sympathetic ganglia 6) differentiate in the vicinity of the dorsal aorta (A) after migrating past the notochord (NJ. fb) In the absence of a notochord neural crest cells invade the midline region ventral to the neural tube and do not migrate to the dorsal aorta. Sympathetic ganglia do not form. Cc) When the neural tube and notochord are inverted neural crest cells migrate dorsally past the notochord, but do not come into the vicinity of the dorsal aorta. Sympathetic ganglia do not form F37.1.
The idea that multiple mesodermally-derived signals act in concert to achieve neural cell patterning is also illustrated in this system. Signals from both the notochord (or the perichordal matrix) and the aorta appear to be required for catecholamine synthetic enzyme expression by sympathetic ganglia. It remains to be determined whether the notochord affects neural crest cell fate by the action of secreted factors that are bound to the notochordal matrix, and whether it provides a differentiation signal
5
6
Development
or merely a physical barrier that influences the course of crest cells and their exposure to a signal from another cellular source.
Does mesoderm
impose pattern on adjacent
tissue by mechanical
means?
Mesoderm may also intluence the segmental organization of the developing spinal cord. The spinal cord displays a pattern of indentations, known as myelomeres, the origins of which have been proposed either to be a result of mechanical moukling by the adjacent mesoderm or to correspond, like rhombomeres, to segmental patterns of cell division and differentiation. The role of the somitic mesoderm in the generation of myelomeres has now been investigated in embryos in which the pattern of segmentation of somitic mesoderm is altered [ 391. The registration of the myelomeres corresponds exactly to the pattern of somite segmentation, while the patterns of differentiated neurons, examined at later stages, within the spinal cord do not. The myelomeres are therefore likely to reflect the physical properties of the adjacent mesoderm rather than an inherent pattern of neuronal organization (Fig. 4). Thus, in this situation the mesoden-n, rather than exerting its effect by an inductive mechanism, appears to confer patterning on the neural tube by a mechanical effect.
’
c
I I
’
m
I I
n
c:
’
Fig. 4. A diagram illustrating mesodermal control of the segmental organization of the spinal cord. Somites 6) impose a local anteroposterior pattern on the neural tube (n) such that myelomeres Cm) are found in register with somite boundaries, while the migration of clonally related neuronal groups (c) is restricted by boundaries that align with the middle of the somites. Neither myelomeres nor migratory boundaries exist in neural tube adjacent to nonsegmented somitic mesoderm (N) [39*,40*1.
In the spinal cord, it had been thought that lineage restrictions along the antero-posterior axis did not exist. A re-examination of this question, by single cell marking in the developing chick spinal cord has revealed segmental boundaries that restrict the migration of clones in an antero-posterior direction [40*]. The boundaries of clonally related neural cell groups form only after segmenta tion of adjacent somitic mesoderm and are aligned with the middle of each somite (Fig. 41, a Ending that has also been interpreted to result from mechanical compression of the neural tube as a consequence of somite formation. Lineage studies after disruption of somite boundaries
[39] could be used to test this idea further. In addition, the maintenance of periodic restrictions in cell migration requires the presence of adjacent somitic mesoderm, suggesting another example of a mesoderm-derived factor acting to stabilize rather than generate pattern. The lack of segmental organization in the spinal cord at later stages is presumably due to migration and rearrangement of cells [40*]. It cannot be ruled out, however, that early clonal relationships between spinal neurons may be important for later organization of appropriate spinal circuitry.
Conclusions The results described in these recent papers suggest that mesodermaliy derived signals have sequential roles in neural cell differentiation. Initially, dorsal mesoderm induces the formation of the neural plate. Mesodermal signals also appear to initiate or regulate antero-posterior regionalization of the neural plate. Subsequently, local signals from the notochord are essential for the induction and patterning of neurons along the dorsoventral axis of the neural tube. Induction of the floor plate by the notochord appears to be a critical first step in the subsequent cell differentiation of the developing central nervous system. Mesodermal signals also control neural cell identity in the peripheral nervous system. The migration and differentiation of neural crest cells appears to be influenced by signals from somites, from the notochord and from the dorsal aorta. Finally, the mesoderm appears to have mechanical as well as inductive roles in the development of a patterned vertebrate central nervous system. A major focus of current work is to identify the mesodermal signals and to describe their sites and mechanisms of action in the developing embryo. Such analyses should resolve how many distinct mesodermally-derived induction signals exist. A relatively small range of inducing factors produce different effects depending on the competence of the ectoderm to respond. How mesodermal signals interact with each other and whether they act sinply as triggers of cellular differentiation or are required to have a sustained action, should also be resolved with the characterization of the signalling properties of mesodermal cells.
References
and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: . of special interest .. of outstanding interest
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10. .
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11.
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12. .
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18. .
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20. .
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7
8
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NE?XGREEN DF, SCHEELM, KAXNERV: Morphogenesis of Scle-
rotome and Neural Crest in Avian Embryos: In Vfvo and In Vftro Studies on the Role of Notochordal Extracelhrlar Matrix. Cell TrYisRes 1986, 244:233_313.
STERNCD, ARTINGER KB, BRONNER-FRASER M: Tissue Interactions Affecting the Migration and Differentiation of Neural Crest Cells in the Chick Embryo. Devebpment 1991, 113:207-216. Neural tube rotations and notochord grafts and ablations were performed in chick embryos to examine the inlluence of the notochord and other local structures on the migration and ditferentiation of neural crest-derived sympathetic ganglion precursors. Migrating neural crest cells avoided the notochord and the matrix surrounding it. The diUerentiation of the sympathetic ganglia appeared to depend on proximity to both the notochord and the dorsal aorta. 37. .
38.
PETIWAY2, GUILL~RYG, BRONNFXFRASER M: Absence of Neural Crest Cells from the Region Surrounding Implanted Notochords In Situ. Dev Biol 1991, 142:335345.
LIM T-M, JAQUESKF, STERNCD, KEYNESRJ: An Evaluation of Myelomeres and Segmentation of the Chick Embryo Spinal Cord. Deuelopment 1991, 113:227-238. The possibility that myelomeres represent intrinsic segmentation within the spinal cord was examined by disrupting the patterns of cell proliferation in the spinal cord using colchicine. This did not at&t myelomere formation. The role of the somites in the formation of myelomeres was investigated by using a heat shock to disrupt the pattern of somite formation The pattern of myelomeres conformed exactly to the abnormal pattern of somites, suggesting mechanical moulding of the neuroepithelium by the neighbouring somites. 39. .
STERN CD, JAQUESKF, Lm T-M, FRASERSE, KF(NEs RJ: Seg mental Lineage Restrictions in the Chick Embryo Spinal Cord Depend on the Adjacent Somites. Development 1991, 113:23‘+244. The possibility that the spinal cord is intrinsically segmented in the antero-posterior axis was examined by mapping the migration of clones of cells derived from single labelfed cells in the chick neural tube. Cells labelled before overt segmentation of the adjacent mesodenn did not obey boundaries, whereas those labelled after mesodennal segrnentadon were restricted in their distribution by invisible boundaries aligned with the middle of each somite. Manipulation of the somite pattern caused alignment of the clonal boundaries with the new pattern of somites. The results are interpreted as reflecting a mechanical intluence of the somite on the adjacent neural tube. 40. .
J Dodd, Department of Physiology and Cellular Biophysics,
Center for Neurobiology and Behavior, Columbia University, 630 West 168th Street, New York, New York 10025, USA
control of neural cell identity in vertebrates Jane Dodd Columbia
It has long neural
been
appreciated
cells is controlled
Several recent act throughout
New York, New York, USA
University,
experiments early
the
differentiation
have revealed
neural
nature
Current
that
in part by inductive development
and
patterning
of
signals from the mesoderm.
that distinct
mesodermal
and have begun
signals
to address the
and sources of such signals.
Opinion
in Neurobiology
Introduction Vertebrate neural development begins with an inductive interaction between two of the primary gem3 layers of the embryo, the ectoderm and the mesoderm. Signals from dorsal mesoderm transform adjacent ectoderm into neuroectoderm, resulting in the formation of the neural plate. The neural plate becomes regionalized along anterior-posterior and mediolateral axes and neurons differentiate in specific locations. This review will focus on some recent developments that have contributed to our understanding of the role of mesodem-i in the induction and patterning of neural cell identity.
Neural induction: the origins and properties of the inducing signal in Xenopus Much of our current understanding of mesodermal and neural induction comes from studies of the frog, Xeno pus kzevk. Neural induction in Xenopus is controlled by signals from the organizer, a region of the embryo that differentiates into dorsal mesodermal structures such as notochord and somites. One approach that has been used to identify molecules involved in neural induction has been to test known growth factors for their ability to promote neural differentiation in isolated explants of blastula stage ectoderm (animal cap assay). Neural tissue can be induced in this assay by a number of polypeptide growth factors, the most potent of which is activin [ 141. Act&in-A, a growth factor identiiied as a regulator of follicle stimulating hormone release from the pituitary [5], is the active component of two factors, XTC/MIF and PIF, previously characterized as potent mesodermal inducers [ 2-41. In animal cap explants treated with high concentrations of activin-A large amounts of neural tissue form in addition to mesodermal tissue, raising the possibility that activin may have neural as well as mesodermal inducing capacity [I-4,6*].
1992, 2:3-8
I .’
Does activin induce neural tissue directly [4,6*,7] or does it induce it indirectly through the induction of dorsal mesoderm? Maternal activin-like activity can be detected in very early Xenopus embryos [ES]. Furthermore, the molecular cloning of Xenopus act&in-A and activin-B (the activity of which appears to be indistinguishable from activin-A) has revealed that act&in-B RNA is expressed in the stage 9 blastula [ 6.1, which may be sufficiently early for a role in neural induction. In contrast, activin-A RNAis expressed too late for it to function as a primary inducer of neural tissue. Two lines of evidence suggest that activins are not direct mediators of neural induction but act to induce mesoderm, which then induces neural tissue. First, XTC/MIF (activin A) appears to induce mesoderm but not neural tissue in single ectodermal cap cells [PI. In contrast, when cells exposed to XTC/MIF are reaggregated with naive ectodermal cells, NCAM RNA is expressed, presumably in the naive cells that had been induced to become neural. These results suggest that the neural response observed in ectodemral caps exposed to activin-A is mediated indirectly by signals from induced mesodemr. The second line of evidence comes from studies of the neural inducing properties of the chick organizer, Hensen’s node, whose actions differ from those of act&in-A in the animal cap assay [lo*]. Unlike activin, Hensen’s node induces large amounts of neural tissue in Xenopus ectoderm without inducing detectable mesoderm. In addition, the period of competence of the ectoderm to form neural tissue in response to activin is much shorter than it is for Hensen’s node, as might be expected if activin acts through intermediate steps. These results suggest that the neural inducing capacity of the organizer cannot be ascribed solely to an activin-like molecule. It is possible, however, that the neural inducing properties of organizer tissue are a result of the action of an activinlike molecule together with a second agent that alters the response properties of the ectoderm to this factor.
Abbreviation CSPG-chondroitin
_,-
sulphate proteoglycan.
@ Current Biology Ltd ISSN 0959488
4
Development
Mesoderm
control
patterning
of the neural
of antero-posterior tube
Distinct classes of neuron develop at different positions along the antero-posterior axis of the neural tube. Two proposals have been offered for the mechanisms underlying antero-posterior patterning in the neural tube (Fig. 1). First, positional information along the antero-posterior axis of the dorsal mesoderm may be transmitted to overlying neuroectoderm (Fig. la) [11,12*]. Second, signals from the organizer may travel through the plane of the ectoderm and impose pattern on the tissue independent of involuting mesoderm (Fig. lb) [ 131.
(a) Neur;~tod;~
vi
fb) Neur~c~~~
F>)
Organizer
(c) Neur;::;;
vr_
Organizer
Fig. 1. A diagram illustrating the mesodermal control of anteroposterior regionalization of the neural plate. The patterned expression of a homeobox gene such as XIHbox7 (a,b) or En-2 Cc) is illustrated by a black band. (a) Signals from underlying mesoderm impose pattern on neuroectoderm. fb) Propagation of signals within the planes of the ectoderm and the mesoderm imposes pattern on both tissues simultaneously. Cc)Signals from underlying mesoderm regulate or stabilize pattern imposed by signals propagated in the plane of the ectoderm.
The expression pattern of a number of homeobox genes in early vertebrate embryos has been interpreted to support the first proposal. For example, the Xenopus gene, XlHbox 1, is expressed early in a narrow band of nuclei at exactly the same antero-posterior position in neural crest, neural tube and underlying axial and lateral mesoderm [ 141. Similarly, the mouse gene Hox-2.9 is expressed in a highly restricted fashion in the developing hindbrain region of the neural tube and underlying mesoderm. The anterior boundaries of neural and mesodermal expression are exactly in register [15*3. The distributions of XlHbox 1 and Hox-2.9 have been suggested to reflect the transmission of positional coordinates from the axial mesoderm to the overlying unpatterned ectoderm (Fig. la) [11,14,15*]. In fact, a signal that dill&es through the plane of the mesoderm, and also through the ectoderm as the two tissues elongate in parallel, could specify an antero-posterior pattern in both tissues (Fig. lb) [ 131. Indeed, signals that are propagated over quite large distances within the plane of the ectoderm have been demonstrated in Xenopus [ 13,16,17]. The possibility that antero-posterior patterning is controlled by position-specific signals from the notochord has been approached experimentally in Xenopus The
homeobox protein, En-2, is found in a discrete band of cells within anterior neural plate, but not in mesoderm. The expression of En-2 was measured in Xeno pus animal cap ectoderm in response to notochord explants taken from different axial levels [12*]. Anterior notochordal pieces induced four times as much En-2 as posterior notochord taken from a donor of the same age, providing some support for the idea that the anteroposterior pattern is regulated by the properties of axial mesoderm (Fig. la). Potential developmental differences between anterior and posterior notochord from embryos of the same stage, however, may account for apparent positional differences in the ability of the notochord to induce En-2 expression. In addition, the effect may be a consequence of the formation of different amounts of neural tissue. It therefore remains unclear which of the two schemes (Fig.la,b) predominate in the patterning of the anteroposterior axis. It seems likely that more than one type of signal will contribute. In fact, the finding that some En-2 protein is generated in response to posterior notochord in Xenopus suggests that additional mechanisms contribute to the antero-posterior regulation of En-2 expression observed in duo. The recent finding that transplants of mesencephalic neuroectoderm that express En-2 induce the expression of En-2 by prosencephalic neuroectoderm [ 18*,19’] has lent support to the idea that there is an En-2inducing signal that is propagated in the plane of the ectoderm in the chick. The normal absence of En-2 expression in neuroectoderm adjacent to the mesencephalon also suggests that the final pattern of neural expression of En-2 in the hindbrain is dependent on a second regulatory signal, perhaps acting to stabilize En-2 expression (Fig. lc) [18*, 19*]. Evidence for synergism in the actions of involuting mesoderm and a spreading neural signal in Xenopus [ 161 supports a role for involuting axial mesoderm in the regulation of neural phenotype and/or the patterning of the neural tube along the anteroposterior axis. Thus, a signal that alters the response of ectoderm to notochord-derived factors may provide a basis for antero-posterior specification (Fig. lc). Such a signal could arise from the organizer and spread through the pregastrula ectoderm, acting to prepattem the ectoderm and generate a differential response to subsequent, e.g. notochord-derived, signals. Indeed, the early gastrula ectoderm appears to be regionalized with respect to its response to activin-A [20*,21*] and also in the expression of distinct isoforms of protein kinase C [22*].
Acquisition
of cell type-specific
neural
properties Mesodermal signals also appear to be involved later in development in the specification of individual neural cell types. The characterization of markers that identify subsets of cells in the early neural tube [23*] has made it possible to test the idea [24-271 that dorsoventral patterning in the spinal cord and hindbrain is initiated by signals from the notochord.
Mesodermal
control of neural cell identity in vertebrates Dodd
Mesodermal
control
of neural
crest cell
migration (a)
fb)
Fig. 2. A diagram
illustrating mesodermal control of the specification of neural cell type. (a) An inductive signal from the notochord (NJ induces the differentiation of the floor plate (F) in overlying neuroectoderm. Signals (arrows) from the floor plate regulate the differentiation and patterning of neurons in the developing neural tube. fb) Signals from the notochord and the floor plate control the differentiation and patterning of neurons in the developing neural tube. X represents a class of neural cells found adjacent to the floor plate (in the hindbrain these express S-hydroxytryptamine). M, motor neuron [23*,30*1.
Grafting and ablation studies in the chick have demonstrated that one of the first neural cell groups to dif ferentiate within the neural tube, the floor plate, does so in response to a local signal from the notochord [28,29,30*]. In vitro experiments have further shown that a notochord signal alone is sufficient to induce many, and perhaps all, of the differentiated properties of the floor plate [30-l. These studies have also revealed that contact between the notochord and neural epithelium is required for floor plate induction. This may reflect the fact that the notochord-derived induction signal is membrane bound or that the diffusion distance of a secreted factor is lim ited, perhaps by binding to extracellular matrix. Such a role for the extracellular matrix is emerging as a common mechanism for limiting diffusion distances and for the presentation of signalling molecules in development D-341. Grafting experiments have also provided evidence that the fate of cells in the ventral neuroepithelium is defined by cell position with respect to the ventral midline. Signals from both the notochord and the floor plate can control the pattern of identified neuronal cell types that appear in specific positions along the dorsoventral axis of the neural tube [ 23.1. It is unclear which of the two ventral midline cell groups is more important in establishing neuronal cell fate in vivo (Fig. 2b). The induction of the floor plate by the notochord may represent a necessary first step in the dorsoventral patterning of the central nervous system. The acquisition of patterning properties by the floor plate may, however, be required to offset the ventral displacement of the notochord from the neural tube that occurs soon after floor plate induction, thus maintaining induction at the midline of the neural tube.
Mesoderm also appears to regulate the fate of neural crest cells that give rise to the peripheral nervous system. The differentiation of neural crest cells is determined by the environment through which they migrate. For instance, signals from the notochord appear to influence the migratory path of sympathetic ganglion precursor cells and their differentiation in the vicinity of the dorsal aorta (Fig.3) [35,36]. Recent notochord ablation and neural tube rotation experiments suggest that inhibitory signals from the notochord and/or the peri-notochordal matrix are essential for the appropriate migration of sympathetic ganglion precursors [37-j. A chondroitin sulphate proteoglycan (CSPG) or related molecule has been previously suggested to mediate’& influence of the notochord on neural crest cell migration [36]. To test the biochemical nature of the inhibitory activity in vivo, isolated notochords were treated in vitro with a panel of enzymes and the treated notochords grafted into chick embryos at sites in the neural crest pathway [38]. The size of the crest cell-free zone around the notochord was then examined. The results support the idea that a CSPG, potentially secreted into the pen-notochordal matrix by the notochord, inhibits neural crest cell migration.
(a)
(b)
0
A
0
A
Fig. 3 A diagram
illustrating mesodermal control of neural crest cell migration and differentiation. (a) Sympathetic ganglia 6) differentiate in the vicinity of the dorsal aorta (A) after migrating past the notochord (NJ. fb) In the absence of a notochord neural crest cells invade the midline region ventral to the neural tube and do not migrate to the dorsal aorta. Sympathetic ganglia do not form. Cc) When the neural tube and notochord are inverted neural crest cells migrate dorsally past the notochord, but do not come into the vicinity of the dorsal aorta. Sympathetic ganglia do not form F37.1.
The idea that multiple mesodermally-derived signals act in concert to achieve neural cell patterning is also illustrated in this system. Signals from both the notochord (or the perichordal matrix) and the aorta appear to be required for catecholamine synthetic enzyme expression by sympathetic ganglia. It remains to be determined whether the notochord affects neural crest cell fate by the action of secreted factors that are bound to the notochordal matrix, and whether it provides a differentiation signal
5
6
Development
or merely a physical barrier that influences the course of crest cells and their exposure to a signal from another cellular source.
Does mesoderm
impose pattern on adjacent
tissue by mechanical
means?
Mesoderm may also intluence the segmental organization of the developing spinal cord. The spinal cord displays a pattern of indentations, known as myelomeres, the origins of which have been proposed either to be a result of mechanical moukling by the adjacent mesoderm or to correspond, like rhombomeres, to segmental patterns of cell division and differentiation. The role of the somitic mesoderm in the generation of myelomeres has now been investigated in embryos in which the pattern of segmentation of somitic mesoderm is altered [ 391. The registration of the myelomeres corresponds exactly to the pattern of somite segmentation, while the patterns of differentiated neurons, examined at later stages, within the spinal cord do not. The myelomeres are therefore likely to reflect the physical properties of the adjacent mesoderm rather than an inherent pattern of neuronal organization (Fig. 4). Thus, in this situation the mesoden-n, rather than exerting its effect by an inductive mechanism, appears to confer patterning on the neural tube by a mechanical effect.
’
c
I I
’
m
I I
n
c:
’
Fig. 4. A diagram illustrating mesodermal control of the segmental organization of the spinal cord. Somites 6) impose a local anteroposterior pattern on the neural tube (n) such that myelomeres Cm) are found in register with somite boundaries, while the migration of clonally related neuronal groups (c) is restricted by boundaries that align with the middle of the somites. Neither myelomeres nor migratory boundaries exist in neural tube adjacent to nonsegmented somitic mesoderm (N) [39*,40*1.
In the spinal cord, it had been thought that lineage restrictions along the antero-posterior axis did not exist. A re-examination of this question, by single cell marking in the developing chick spinal cord has revealed segmental boundaries that restrict the migration of clones in an antero-posterior direction [40*]. The boundaries of clonally related neural cell groups form only after segmenta tion of adjacent somitic mesoderm and are aligned with the middle of each somite (Fig. 41, a Ending that has also been interpreted to result from mechanical compression of the neural tube as a consequence of somite formation. Lineage studies after disruption of somite boundaries
[39] could be used to test this idea further. In addition, the maintenance of periodic restrictions in cell migration requires the presence of adjacent somitic mesoderm, suggesting another example of a mesoderm-derived factor acting to stabilize rather than generate pattern. The lack of segmental organization in the spinal cord at later stages is presumably due to migration and rearrangement of cells [40*]. It cannot be ruled out, however, that early clonal relationships between spinal neurons may be important for later organization of appropriate spinal circuitry.
Conclusions The results described in these recent papers suggest that mesodermaliy derived signals have sequential roles in neural cell differentiation. Initially, dorsal mesoderm induces the formation of the neural plate. Mesodermal signals also appear to initiate or regulate antero-posterior regionalization of the neural plate. Subsequently, local signals from the notochord are essential for the induction and patterning of neurons along the dorsoventral axis of the neural tube. Induction of the floor plate by the notochord appears to be a critical first step in the subsequent cell differentiation of the developing central nervous system. Mesodermal signals also control neural cell identity in the peripheral nervous system. The migration and differentiation of neural crest cells appears to be influenced by signals from somites, from the notochord and from the dorsal aorta. Finally, the mesoderm appears to have mechanical as well as inductive roles in the development of a patterned vertebrate central nervous system. A major focus of current work is to identify the mesodermal signals and to describe their sites and mechanisms of action in the developing embryo. Such analyses should resolve how many distinct mesodermally-derived induction signals exist. A relatively small range of inducing factors produce different effects depending on the competence of the ectoderm to respond. How mesodermal signals interact with each other and whether they act sinply as triggers of cellular differentiation or are required to have a sustained action, should also be resolved with the characterization of the signalling properties of mesodermal cells.
References
and recommended
reading
Papers of particular interest, published within the annual period of review, have been highlighted as: . of special interest .. of outstanding interest
1.
ASASHIMA M, NAKANOH, SHIMADA K, KINOSHITA K, ISHIK, SHIBAI H, UENO N: Mesodemal Induction in Early Amphibian Em-
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SMITHJC, PRICEBMJ, VAN NIMMENK, HLJYIEBROECK D: Identification of a Potent Xenopus Mescderm Inducing Factor as a Homologue of Activin-A Nature 1990, 3453729731.
Mesodermal control of neural cell identity in vertebrates Dodd 3.
EIJNDEN-VAN w AJM, VAN ZOELENT EJJ, VAN NIMMENK, KO~TERCH, SNOEKGT, DURSTONAJ, HUYLEBROECK D: ActivinLike Factor from a Xenopus lueuis CeII Line Responsible for Mesoderm Induction. Nature 1990, 347:732-734.
4.
SOKOLS, WONG GG, MELTONDA A Mouse Macrophage
Factor Induces Head Structures and Organizes a Body Axis in Xenopus Science 1990, 249~561-564.
5.
IING N, UENO N, YING SY, ESCH F, SHIMA~AKI S, HOITA M,
CUEVASP, GUILLEMIN R Inhibins and Activins. VitNorm 1988, 44:1-46.
6. .
THOMSENG, WOOLFT, WHITMAN M, SOKOLS, VAUGHAN J. VALE W, MELTONDA: Activins are Expressed Early in Xenopus Embryogenesis and can Induce Axial Mesoderm and Anterior Structures. CeN 1990, 63:485-493. Activin was tested in the animal cap assay and shown to induce the formation of notochord, muscle, neural tissue, and eyes arranged in a rudimentary axial pattern. Xenopus activins A and B were cloned and their developmental expression examined. Actiti-A was not detectable before the late gastruIa stage, while a&&-B was first detected in the blastula. Ectopic expression of activin-B produced a second body axis in embryos injected with synthetic mRNA. It is suggested that endogenous activinB is responsible for early induction and axial patterning in Xen@us and also that activin could induce neural tissue by a direct effect on the ectoderm.
7.
NEW HV, HOWES G, SMITH JC: Inductive Interactions in Early Embryonic Development. Curr @in Genet Dev 1991, 1:19&203.
8.
A%SHIMAM, NAKANOH, UCHIYAMA H, SUCINO H, NAKAMIJRA T, ETO Y, EJIMA D, NISHIMATSU S-I, UENO N, KINOSHITAK: Presence of Activin (Erythroid DiITerentiation Factor) in Unfertilized Eggs and BlastuIac of Xenopus Zaeuis. Proc Nat1Acad Sci USA 1991, 88:6511-6514.
GREENJBA, Sm JC: Graded Changes in Dose of a Xenopus Activin-A Homologue Elicit Stepwise Transitions in Embryonic CeU Fate. Nature 1990, 347:391-394. Dispersed Xenopusblastomeres were treatedwith XTC/MIF (activin-A), in a range of concentrations, reaggregated, and subsequently screened for the expression of mesodermal and neural properties. Notochord and muscle, but not neural tissue, formed within a narrow range of concentration. Neural transcripts were induced only when treated and untreated blastomercs were mixed, suggesting that activin induces neural tissue indirectly through the mescderm. 9. .
10. .
KI~VIXER C, DODD J: Hensen’s Node Induces Neural Tissue in Xenopus Ectoderm. Implications for the Action of the Organizer in Neural Induction. Development 1991, 113: in press. ‘Ihe actions of the chick organizer, Hensen’s node, and activin-A were compared in the Xen@us animal cap assay. Neural tissue induced by acdvir-A was ahvays accompanied by mesoderm induction. In contrast, Hensen’s node induced neural tissue in the absence of detectable mesoderm. In addition, several factors that altered the response of ectoderm to activin did not affect the response to Hensen’s node. The results suggest that activin alone cannot mediate the neural inducing effect of the organizer but do not rule out the possibility that acdvin acts in concert with another factor in Hensen’s node that changes the response of the ectoderm to activin.
11.
DEROBERTI~EM, OUVER G, WRIGHT CVE: Determination of AxiaI Polarity in the Vertebrate Embryo: Homeodomain Proteins and Homeogenetic Induction. Cell 1989, 57318!+191.
12. .
HEMMATI-Bruvmu A, S’IIZWART RM, v RM: Region-Specilic Neural Induction of an EngraiIed Protein by Anterior Notochord in Xenopus Science 1990, 250:80&802. Anterior and posterior regions of stage 12.5 Xenopus notochord were compared for their ability to induce En-2 in Xenopus animal caps. En2 expression, measured with an antibody, was four-fold more frequent in caps combined with anterior notochord than in caps combined with posterior notochord. 13.
RUIZALTABAAI: Neural Expression of the Xenopus Homeobox Gene XhoJc3: Evidence for a Patterning Neural Sig-
nal that Spreads Through the Ectoderm. Deuelqment 1990, 108:595&04. 14.
OLIVERG, WRIGHT CVE, HARDWICKE J, DEROBERTISEM: Differential Antero-Posterior Expression of Two Proteins Encoded by a Homeobox Gene in Xenopus and Mouse Embryos. EMBO J 1988, 7:319+3209.
FROHMAN MA, BOYLE M, m GR: Isolation of the Mouse Hox-2.9 Gene: Analysis of Embryonic Expression Sug gests that Positional Information Along the Anterior-Posterior Axis is Specified by Mesoderm. Devekgmerzt 1990, 110:589-&7. The mudne homeobox-containing gene, Hex-2.9, was cloned and its distribution in early embryos described Hex-2.9 is expressed in mesoderm that will participate in hindbrain formation, and the anterior boundary of expression coincides with the’anterior boundary in the overlying neural plate. 15. .
16.
DIXONJE, KINTNERCR Cellular Contacts Rtiquired for Neural Induction in Xenopus Embryos: Evigqce for Two Signals. Develqnnenf 1989, 106:74!+757., ,. -,
17.
SAVAGER, PHILLIPSCR: Signals from the’ Dorsal BIastopore Lip Region During GastruIation Bias the Ectoderm Toward a Nonepidermal Pathway of Differentiation in Xenopus lut+ vis Dev Biol 1989, 133157-168.
18. .
MARTWEZS, WA%EF M, ALVARD@MALL%T R-M: Induction of a Mesencephalic Phenotype in the Z-Day-Old Chick Prosencephalon is Preceded by the EarIy Expression of the Homeobox Gene en. Neuron 191, 6:971-981. Quail-chick chimeras were used to examine the effect of the environment on the fate of prospective cerebellar cells. Metencephalic cells that express high levels of En-2 were transplanted to prosencephalic sites and the expression pattern and histology of the chimeras examined. The metencephalic cells retained expression of En-2 and appeared to induce En-2 expression in prosencephalic cells that are normally En-2negative.
GARDNERCA, BARAU)KF: The Cellular Environment ControIs the Expression of engruiled-Iike in the Central Neuroepithelium of Quail-Chick Chhneric Embryos. Development 1991, 113:1037-1048. The expression pattern of En-2 is described in chick-quail chimeras in which the environment of En-2.expressing neural cells is altered. En2+ caudal mescncephalic cells (described as metencephalic in [18*]) maintain expression of En-2 when they are transplanted into prosencephalon (normaIIy En-2-). Furthermore, the mesencephalic cells ap pear to induce En-2 expression in the prosencephalic cells. Similarly, grafts of prosencephalon transplanted to mesencephalon expressed En-2. Ceils from medial mesencephalon, that express lower levels of En-2, tend to lose expression when transplanted to prosencephalon and do not appear to induce prosencephalic expression of En-Z. Mesoderm transplanted from the hindbrain to the prosencephalon did not induce En-2 expression. 19. .
20. .
RULZALTABA AI, JESSEU TM: Retinoic Acid Modifies MesodermaI Patterning in Early Xenopus Embryos. Genes Deu 1991, 5:175-187. The possibility that retinoic acid exerts its effects on the development of anterior axial structures through an action on the antero-posterior patterning mesoderm was examined in Xen0pu.s Retinoic acid was found to modify the tierentiation of anterior mesoderm in ectodermal caps treated with mesodern-inducing factors. In experiments to examine the physiological role of retinoic acid in uiuo,both dorsal and ventral ectcdenn were found to differ in their responses to mesoderm inducing factors in an animal cap assay. It is suggested that retinoic acid could act as a localized inhibitor in the embryo, modifying the effects of mesodermal inducing factors and thus contributing to axial patterning. 21. .
%KOL S, MELTONDA: Pre-existent Pattern in Xenopus AnimaI Pole Revealed by Induction with Activin. Nature 1991, 351:409-411. The responses of prospective dorsal and ventral regions of Xen@use~toderm to activin were compared in an animal cap assay. In response to the same concentrations of acti-, dorsal blastula caps developed into head-like embtyoids containing notochord, muscle, neural tissue and eyes. Ventral caps, in contrast, developed predominantly into epi-
7
8
Development dermis, muscle and mesenchyme. Dorsoanterior structures were rarely obtained. These results reveal pre-patterning within the blastula ectoderm. 22. .
m AP, KRAMERlh%, DURSTONAJ: Protein Rinase C and Regulation of the Local Competence of Xenopus Ectoderm. Science 1991, 251~570-573. The distributions of protein kinase C isoqmes in Xenopus ectoderm were found to differ dramatically in ventral and dorsal regions. It remains to be determined whether the expression of protein kinase C reflects a role for this enzyme in regulating the response properties of the ectoderm. YAMADA T, PLKZEK M, TANAKA H, DODDJ, JESSELL TM: Control of Cell Pattern in the Developing Nervous System: Polarizing Activity of the Floor Plate and Notochord. Cell 1991, 64:63%48. Transplantation and ablation experiments suggest that the pattern of cell differentiation along the dorsoventral axis of the spinal cord and hind brain of the chick neural tube is regulated by signals from the notcchord and the Boor plate. Grafting of a notochord or floor plate to ectopic positions, or deleting notochord and Boor plate, resulted in changes in the fate and position of neural ceU types, defined by expression of specific antigens, 23. .
24.
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chord on Proliferation and Differentiation in the Neural Tube of the Chick Embryo. Devekpment 1989, 107:793803. 28.
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29.
SMITHJ, SCHOENWOLF GC: Notochordal Induction of Cell Wedging in the Chick Neural Plate and its Role in Neural Tube Formation. J Exp Zool 1989, 250:4962.
PUCZEK M, TESSIER-LAMGNE M, YAMADAT, JESSELL TM, DODD J: Mesodermal Control of Neural Cell Identity: Floor Plate Induction by the Notochord. Science 1990, 250:981-9&I The expression of a Hoor plate-specitic chemoattractant was used as a marker to examine the role of the notochord in chick floor plate development. Expression of the chemoattractant in lateral cells of the neural tube was induced by an ectopic notochord, and midline neural cells did not express the chemoattmctant after removal of the notochord early in development, Notochord explants also induce floor plate specific properties in explants of lateral neuroepithelium. Cell contact is required for this induction. The results indicate that the notochord is required for the expression of chemotropic activity by ventral midline ceils and suggest that signals from the notochord alone are sufficient to induce floor plate differentiation.
30. .
31.
RUOS~AHTI E, YAMAGUCHI Y: Proteoglycans as Modulators of Growth Factor Activities. Cell 1991, 64867-869.
32.
PARAKARVM, VUKICEV~C S, REDDIAH: Transforming Growth Factor B Type 1 Binds to Collagen Iv of Basement Membrane Mattix: Implications for Development. Deo Biof 1991, 143303308.
33.
LUEN LE, SENDTNERM, RAFF MC: Extracellukic Matrix-Associated Molecules Collaborate with Ciliary Neurotrophic Factor to Induce Type-2 Astrocyte Development. J CefI Bid 1990, 111:63%644.
34.
SCHUBERT D, KIMURAH: Substratum-Growth Factor Collaborations are Required for the Mitogenic Activities of Activin and FGF on Embryonal Carcinoma Cells. J Cell Biol 1991, 114841846.
35.
LEDOUARIN NM, TEILLET M-A The Migration of Neural Crest Cells to tbe Wall of the Digestive Tract in Avian Embryo. J Embryo1 E.Q Morphor 1973, 30:31-48.
36.
NE?XGREEN DF, SCHEELM, KAXNERV: Morphogenesis of Scle-
rotome and Neural Crest in Avian Embryos: In Vfvo and In Vftro Studies on the Role of Notochordal Extracelhrlar Matrix. Cell TrYisRes 1986, 244:233_313.
STERNCD, ARTINGER KB, BRONNER-FRASER M: Tissue Interactions Affecting the Migration and Differentiation of Neural Crest Cells in the Chick Embryo. Devebpment 1991, 113:207-216. Neural tube rotations and notochord grafts and ablations were performed in chick embryos to examine the inlluence of the notochord and other local structures on the migration and ditferentiation of neural crest-derived sympathetic ganglion precursors. Migrating neural crest cells avoided the notochord and the matrix surrounding it. The diUerentiation of the sympathetic ganglia appeared to depend on proximity to both the notochord and the dorsal aorta. 37. .
38.
PETIWAY2, GUILL~RYG, BRONNFXFRASER M: Absence of Neural Crest Cells from the Region Surrounding Implanted Notochords In Situ. Dev Biol 1991, 142:335345.
LIM T-M, JAQUESKF, STERNCD, KEYNESRJ: An Evaluation of Myelomeres and Segmentation of the Chick Embryo Spinal Cord. Deuelopment 1991, 113:227-238. The possibility that myelomeres represent intrinsic segmentation within the spinal cord was examined by disrupting the patterns of cell proliferation in the spinal cord using colchicine. This did not at&t myelomere formation. The role of the somites in the formation of myelomeres was investigated by using a heat shock to disrupt the pattern of somite formation The pattern of myelomeres conformed exactly to the abnormal pattern of somites, suggesting mechanical moulding of the neuroepithelium by the neighbouring somites. 39. .
STERN CD, JAQUESKF, Lm T-M, FRASERSE, KF(NEs RJ: Seg mental Lineage Restrictions in the Chick Embryo Spinal Cord Depend on the Adjacent Somites. Development 1991, 113:23‘+244. The possibility that the spinal cord is intrinsically segmented in the antero-posterior axis was examined by mapping the migration of clones of cells derived from single labelfed cells in the chick neural tube. Cells labelled before overt segmentation of the adjacent mesodenn did not obey boundaries, whereas those labelled after mesodennal segrnentadon were restricted in their distribution by invisible boundaries aligned with the middle of each somite. Manipulation of the somite pattern caused alignment of the clonal boundaries with the new pattern of somites. The results are interpreted as reflecting a mechanical intluence of the somite on the adjacent neural tube. 40. .
J Dodd, Department of Physiology and Cellular Biophysics,
Center for Neurobiology and Behavior, Columbia University, 630 West 168th Street, New York, New York 10025, USA