Cell, Vol. 64, 471-473, February8, 1991,Copyright© 1991 by Cell Press
Motorizing the Spinal Cord Andrew Lumsden Division of Anatomy and Cell Biology United Medical and Dental Schools Guy's Hospital London SE1 9RT England
Possession of a notochord and possession of a dorsal hollow nerve cord are two of the principal characteristics of vertebrates. A flexible, compression-resisting rod along the midline of the body, the notochord is the primary axial skeleton of embryos and a template for the vertebrae of adults. The nerve cord, immediately overlying the notochord, is a bilateral structure (see figure, part A). Neurogenic lateral plates are linked across the midline by roof and floor plates of specialized but nonneurogenic epithelium. Neurons develop in a ventral to dorsal progression. Motor neurons develop first, project their axons out of the central nervous system, and are confined to the ventral region of the lateral plate. Sensory axons from the periphery enter the dorsal region of late-differentiating local circuit neurons. Here and in the intermediate midlateral region are also relay neurons that connect with targets either in the same side or in the opposite side of the central nervous system. The large majority of crossing axons pass through the floor plate, forming a ventral commissure that is continuous along the length of the hindbrain and spinal cord. Thus, the dorsoventral axis of the cord has a distinctly polarized pattern of neurogenesis, neuronal type, and axonal trajectory; how is this pattern formed? During early development, the floor plate of the spinal neural tube directly contacts the notochord and coextends with it along the body axis. This close association extends to abnormalities of development; in cases of notochord duplication in chick embryos, wherever a supernumerary notochord contacts the neural tube, a "floor plate reaction" occurs and the spinal cord develops two ventral poles (Watterson, 1965). Similarly, if the notochord is removed early in amphibian development, the resulting spinal cord has no floor plate (Holtfreter, 1934; Kitchen, 1949). The notochord develops from dorsal axial mesoderm cells (chordamesoderm), which are involved in the primary induction of the neural plate from dorsal ectoderm (Spemann, 1938). Could these midline cells continue to signal to the overlying neuroectoderm later in development, influencing patterning down the dorsoventral axis of the young neural tube? A systematic analysis of the effect of the notochord was undertaken by van Straaten and his colleagues. When a length of notochord was grafted within 80 p.m of the lateral surface of the closed neural tube of stage 12-15 (2 day) chick embryos, that side of the spinal cord was enlarged, and the area occupied by early-differentiating ventral neurons (histochemically stained for acetylcholinesterase) and their emergent roots increased (van Straaten et al., 1985a). That no additional floor plate developed was at-
Minireview
tributed to a loss of ability to respond at the late stage of the operation. However, a floor plate reaction did occur when the notochord was grafted at stages 9-11, provided that it lay within 25 I~m of the surface of the closing neural tube (van Straaten et al., 1985b, 1988; Smith and Schoenwolf, 1989). If the donor notochord was implanted beside the host notochord, the host floor plate broadened out above the donor tissue; if the notochord was implanted more dorsally, a distinct second floor plate formed. A dual influence of the grafted notochord was deduced. First, a briefly transient, proximity-dependent interaction at around stage 10 induces a floor plate. Second, at a greater distance from the tube or at developmental stages beyond 12, the grafted notochord elicits an increase in neuronal precursor proliferation and differentiation. Does the ventral region of the neural tube depend on the notochord in a similar way during normal development? It would seem so, for when the most recently formed region of the notochord is extirpated at stage 10, the spinal cord develops neither a floor plate nor ventral roots (van Straaten and Drukker, 1987); the absence of emergent axons, together with the lack of acetylcholinesterasestaining ventral neurons, suggests the failure of motor neuron development. These experiments raise the possibility that the notochord plays a crucial role in ventralizing the neural tube--creating a median divide that bilateralizes the neurogenic regions and then locally regulating their pattern of neurogenesis. In this case, primary (neural) induction by chordamesoderm would leave the neuroectoderm with a dorsal fate, perhaps the default state of the whole neural plate, awaiting further signals from the midline mesoderm to specify motor regions. van Straaten's experiments are less than conclusive, however, because the results are assessed largely by morphology. The ectopic floor plates certainly have the thin-walled look of a normal floor plate, are nonneurogenic, and have an appropriately low proliferation rate. But do they match the naturally formed structure, property for property? Similarly, the use of a marker, acetylcholinasterass, that appears in a broad range of young neuronal phenotypes compromises the ability to identify specific types of neurons. Undeniably, dorsal neurons differentiate precociously in the presence of a grafted notochord, but are they motor neurons? Finally, does the notochord affect neurogenesis directly, or is the floor plate also involved perhaps as an intermediary? These questions are substantially resolved by two recent studies in which the results of notochord addition and deletion operations have been assessed both by functional activity (Placzek et al., 1990) and by cell type-specific monoclonal antibodies (Yamada et al., 1991). One functional marker for floor plate differentiation is a diffusible activity that promotes and orients the growth of commissural axons in vitro and is thus presumed to be responsible for attracting their growth cones to the ventral midline (Tessier-Lavigne et al., 1988). This activity was assayed by Placzek et al. in explanted lateral regions of
Cell 472
A
c
Cross-Sections through the Developing Chick Spinal Cord at 1.5 Days (Left) and 4 Days (Right) (A) During normal development, the floor plate (stippled) develops above the notochord (n), and motor neurons subsequently differentiate in adjacent lateral plate. AC4 antigen is expressed in more dorsal regions (grey). (B) Grafting a donor notochord (n') alongside the folding neural plate results in an additional floor plate and a third column of motor neurons. AC4 expression retracts. (C) Removing the notochord from beneath the neural plate results in the absence of both floor plate and motor neurons. AC4 expression extends through the ventral region of the cord.
chick spinal cord that had previously either received a notochord graft or been contacted by notochord in vitro. When cocultured with explants of rat dorsal cord, these pretreated regions promote commissural axon outgrowth with a potency equivalent to that of normal floor plate. The activity is lacking, furthermore, in ventral cord from which the notochord had previously been resected. Together with the demonstration by Yamada et al. that floor platespecific antigens (FPll2) are expressed in notochord-influenced lateral plate and not in notochord-deprived spinal cord, these data show that the notochord is required and sufficient for floor plate development. Using a monoclonal antibody (SC1) that distinguishes motor neurons from other spinal cord neurons, Yamada et al. have also found that the notochord does influence the production of motor neurons. When implanted alongside the lateral plate, an additional notochord elicits the formation of motor neurons in the dorsal neural tube close to but not contiguous with the induced ectopic floor plate (see figure, part B). As in their normal ventral location, an unlabeled region (called X by Yamada et al.) separates the SCl-positive neurons from the FPl-positive floor plate
cells. When the notochord is implanted in the dorsal midline, disrupting the roof plate, bilaterally symmetrical columns of motor neurons form in the dorsal cord. The ectopic motor neurons in these animals apparently develop from precursors destined for another fate rather than by the rescue of a population of specific motor neuron progenitors: although there is a significant increase in the number of motor neurons compared with controls, there is no overall increase in the number of cells. That ventral cell types differentiate at the expense of dorsal ones is also suggested by reciprocal changes in the distribution of dorsal markers. Both AC4, a surface antigen of cells restricted to dorsal and intermediate regions of the spinal cord, and CRABP, an intracellular marker for relay neurons in these regions, are expressed over a reduced area after notochord grafts. Consistently, when Yamada et al. remove the notochord early in development, the spinal cord lacks SC1- or FPl-positive cells, and both the AC4 antigen and CRABP are expressed throughout the ventral region (see figure, part C). Lineage analysis in the chick (Leber et al., 1990) predicts that motor neurons do not arise from specific precursors but share a common precursor with other neuronal types; such cells might be expected to be distributed throughout the proliferative (in ner) layer of the neural tube. The findings of Yamada et al. suggest that signals from the notochord are required to drive these precursors toward a motor fate. Does the notochord directly induce motor neuron differentiation, or does its influence depend on the prior induction of floor plate and signaling within the neuroepithelium? Two lines of evidence suggest direct induction. First, ectopic motor neurons can differentiate without, apparently, the formation of a nearby floor plate (van Straaten, 1985a; Yamada et al., 1991). This happens when the neural tube lies 25-80 p.m from an implanted notochord, roughly the same distance that separates the notochord and the earliest motor neurons in the normal embryo. The correlation of effect with distance suggests a graded inductive signal with different responses at two thresholds. Second, there is emerging evidence from zebrafish that the floor plate may not be required for motor neuron production. In the cyclops mutant (cyc-l), the floor plate is absent. The defect appears to be in the competence of midline cells to respond to the notochord signal, since the phenotype can be rescued by transplantatibn of wild-type gastrula ectoderm into mutants (Hatta et al., 1991). Although they have not yet been described, motor neurons are presumably present in cyc-1 despite the absence of a floor plate--the animal is motile. Fish and amphibian larvae, however, have a very early differentiating type of motor neuron not found in birds and mammals; these primary motor neurons appear to develop constitutively in the neuroectoderm without a requirement for either floor plate or notochord (Clarke et al., 1991). A relevant question then is whether cyc-1 develops secondary motor neurons, the homologs of those in higher vertebrates, in line with the expectations of a direct induction by notochord. There is compelling evidence, however, that the floor plate can regulate motor neurogenesis independently of
Minireview 473
the notochord. When floor plate is grafted next to the lateral surface of the neural tube, expression of both FP1 and SC1 antigens is induced in the adjacent neuroepithelium, the ectopic motor neurons being separated from the induced floor plate by a region of unlabeled cells (Yamada et al., 1991). The floor plate therefore has the capacity to suppress and promote motor neuron differentiation at a constant distance. With respect to these properties, the floor plate seems to mimic the notochord, raising the question of which is instrumental during normal development, or whether both midline structures are involved. The acquisition of signaling capacity by the floor plate suggests that the ventral pole of the neural tube is set up to go it alone and that the further signaling potential of the notochord is redundant. An organizing center within the neuroepithelium would perhaps allow greater precision of positional signaling than one lying outside that is subject to displacement. A further question is: if floor plate can induce floor plate, how is its width set? One possibility is that during normal floor plate induction, the notochord also inhibits the reaction in region X. Despite these remaining problems, the coupling of embryo manipulation with specific antibody markers has produced a clearer insight into the process of motor neuron differentiation. It has, furthermore, indicated a likely mechanism for patterning: the floor plate, itself dependent on the notochord for its induction, may constitute a polarizing region for the dorsoventral axis of the lateral plate. Motor neurons, in this case, would differentiate from multipotential precursors according to their position in a concentration gradient of morphogen diffusing from this source. Retinoic acid is a candidate molecule for signaling positional information within the neural tube. The floor plate produces retinoids, the biological activity of which has been assayed by the ability to respecify the pattern of digits in the chick wing (Wagner et al., 1990). Interestingly, both the notochord (Wagner et al., 1990) and its progenitor, Hensen's node (Hornbruch and Wolpert, 1986), also polarize in the chick wing assay, suggesting a way in which the notochord could signal motor neuron differentiation in the absence of a floor plate as, for example, in those cases where the grafted notochord lay outside the range for a floor plate reaction. The role of retinoids in dorsoventral signaling will be further elucidated by analyses of the distribution of nuclear retinoic acid receptors, the effects of retinoic acid on neural tube development in the chick, using the variety of position-marking antibodies now available, and identification of retinoic acid-inducible genes that could encode positional value. Genes have been isolated that have dorsoventrally restricted expression patterns in the young spinal cord, suggesting some involvement in dorsoventral patterning. These include genes in the Hox 2 cluster and Hox 3.1 (Graham et al., 1991), certain paired box-containing g e n e s (e.g., Pax2: Nornes et al., 1990; Pax8: Plachov et al., 1990), and Evx 1 (Bastian and Gruss, 1990). A useful approach would be to examine how the expression patterns of these genes change in response to grafting an additional notochord.
References
Bastian, H., and Gruss, P. (1990). EMBOJ. 9, 1839-1852. Clarke,J. D.W., Holder,N., Soffe,S. R., and Storm-Mathisen,J. (1991). Development,in press. Graham, A., Maden, M., and Krumlauf, R. (1991). Development,in press. Hatta, K., Kimmel, C. B., Ho, R. K., and Walker, C. (1991). Nature, in
press. Holffreter,J. (1934).Amh. Exp. Zellforsch. 15, 281-301. Hornbruch, A., and Wolpert, L. (1986).J. Embryol. Exp. Morphol.87, 163-174. Kitchen, I. C. (1949).J. Exp. Zool. 112, 393-415. Leber,S. M., Breedlove,S. M., and Sanes,J. R. (1990).J. Neurosci.10, 2451-2462. Nornes, H. O., Dressier,G. R., Knapik, E. W., Deutsch, U., and Gruss, P. (1990). Development109, 797-809. Plachov,D., Chowdhury,K., Walther,C., Simon,D., Guenet,J. L., and Gruss, P. (1990). Development110, 643-651. Placzek,M., Tessier-Lavigne,M., Yamada,T., Jesse,, T., and Dodd,J. (1990). Science250, 985-988. Smith, J. L., and Schoenwolf,G. C. (1989). J. Exp. Zool. 250, 49-62. Spemann,H. (1938).EmbryonicDevelopmentand Induction(NewHaven, Connecticut:YaleUniversityPress). Tessier-Lavigne,M., Placzek,M., Lumsden,A. G. S., Dodd, J., and Jesse,, T. M. (1988). Nature336, 775-778. van Straaten,H. W. M., and Drukker,J. (1987).In MesenchymaI-EpithelialInteractionsin NeuralDevelopment,J. R. Woolfet al., eds.(Berlin: Springer-Verlag),pp. 153-162. van Straaten,H. W. M., Thors, F., Hoessels,E. L., Hekking,J. W. M., and Drukker,J. (1985a).Dev. Biol. 110, 247-254. van Straaten,H. W. M., Hekking,J. W. M., Thors,E, Wiertz, E. L. J. M., and Drukkar,J. (1985b).Acta Morphol. Neerl.-Scand.23, 91-97. van Straaten,H. W. M, Hekking,J. W. M., Wiertz-Hoessels,E. J. L. M., Thors, F., and Drukker,J. (1988). Anat. Embryol. 177, 317-324. Wagner, M., Thaller,C., Jesse,, T., and Eichele,G. (1990).Nature345, 819-822. Watterson, R. L. (1965).In Organogenesis,R. L. de Haan and H. Ursprung, eds. (New York: Holt, Rinehartand Winston), pp. 129-159. Yamada,T., Placzek,M., Tanaka,H., Dodd,J., andJessell,T. M. (1991). Cell 64, this issue.
Motorizing the Spinal Cord Andrew Lumsden Division of Anatomy and Cell Biology United Medical and Dental Schools Guy's Hospital London SE1 9RT England
Possession of a notochord and possession of a dorsal hollow nerve cord are two of the principal characteristics of vertebrates. A flexible, compression-resisting rod along the midline of the body, the notochord is the primary axial skeleton of embryos and a template for the vertebrae of adults. The nerve cord, immediately overlying the notochord, is a bilateral structure (see figure, part A). Neurogenic lateral plates are linked across the midline by roof and floor plates of specialized but nonneurogenic epithelium. Neurons develop in a ventral to dorsal progression. Motor neurons develop first, project their axons out of the central nervous system, and are confined to the ventral region of the lateral plate. Sensory axons from the periphery enter the dorsal region of late-differentiating local circuit neurons. Here and in the intermediate midlateral region are also relay neurons that connect with targets either in the same side or in the opposite side of the central nervous system. The large majority of crossing axons pass through the floor plate, forming a ventral commissure that is continuous along the length of the hindbrain and spinal cord. Thus, the dorsoventral axis of the cord has a distinctly polarized pattern of neurogenesis, neuronal type, and axonal trajectory; how is this pattern formed? During early development, the floor plate of the spinal neural tube directly contacts the notochord and coextends with it along the body axis. This close association extends to abnormalities of development; in cases of notochord duplication in chick embryos, wherever a supernumerary notochord contacts the neural tube, a "floor plate reaction" occurs and the spinal cord develops two ventral poles (Watterson, 1965). Similarly, if the notochord is removed early in amphibian development, the resulting spinal cord has no floor plate (Holtfreter, 1934; Kitchen, 1949). The notochord develops from dorsal axial mesoderm cells (chordamesoderm), which are involved in the primary induction of the neural plate from dorsal ectoderm (Spemann, 1938). Could these midline cells continue to signal to the overlying neuroectoderm later in development, influencing patterning down the dorsoventral axis of the young neural tube? A systematic analysis of the effect of the notochord was undertaken by van Straaten and his colleagues. When a length of notochord was grafted within 80 p.m of the lateral surface of the closed neural tube of stage 12-15 (2 day) chick embryos, that side of the spinal cord was enlarged, and the area occupied by early-differentiating ventral neurons (histochemically stained for acetylcholinesterase) and their emergent roots increased (van Straaten et al., 1985a). That no additional floor plate developed was at-
Minireview
tributed to a loss of ability to respond at the late stage of the operation. However, a floor plate reaction did occur when the notochord was grafted at stages 9-11, provided that it lay within 25 I~m of the surface of the closing neural tube (van Straaten et al., 1985b, 1988; Smith and Schoenwolf, 1989). If the donor notochord was implanted beside the host notochord, the host floor plate broadened out above the donor tissue; if the notochord was implanted more dorsally, a distinct second floor plate formed. A dual influence of the grafted notochord was deduced. First, a briefly transient, proximity-dependent interaction at around stage 10 induces a floor plate. Second, at a greater distance from the tube or at developmental stages beyond 12, the grafted notochord elicits an increase in neuronal precursor proliferation and differentiation. Does the ventral region of the neural tube depend on the notochord in a similar way during normal development? It would seem so, for when the most recently formed region of the notochord is extirpated at stage 10, the spinal cord develops neither a floor plate nor ventral roots (van Straaten and Drukker, 1987); the absence of emergent axons, together with the lack of acetylcholinesterasestaining ventral neurons, suggests the failure of motor neuron development. These experiments raise the possibility that the notochord plays a crucial role in ventralizing the neural tube--creating a median divide that bilateralizes the neurogenic regions and then locally regulating their pattern of neurogenesis. In this case, primary (neural) induction by chordamesoderm would leave the neuroectoderm with a dorsal fate, perhaps the default state of the whole neural plate, awaiting further signals from the midline mesoderm to specify motor regions. van Straaten's experiments are less than conclusive, however, because the results are assessed largely by morphology. The ectopic floor plates certainly have the thin-walled look of a normal floor plate, are nonneurogenic, and have an appropriately low proliferation rate. But do they match the naturally formed structure, property for property? Similarly, the use of a marker, acetylcholinasterass, that appears in a broad range of young neuronal phenotypes compromises the ability to identify specific types of neurons. Undeniably, dorsal neurons differentiate precociously in the presence of a grafted notochord, but are they motor neurons? Finally, does the notochord affect neurogenesis directly, or is the floor plate also involved perhaps as an intermediary? These questions are substantially resolved by two recent studies in which the results of notochord addition and deletion operations have been assessed both by functional activity (Placzek et al., 1990) and by cell type-specific monoclonal antibodies (Yamada et al., 1991). One functional marker for floor plate differentiation is a diffusible activity that promotes and orients the growth of commissural axons in vitro and is thus presumed to be responsible for attracting their growth cones to the ventral midline (Tessier-Lavigne et al., 1988). This activity was assayed by Placzek et al. in explanted lateral regions of
Cell 472
A
c
Cross-Sections through the Developing Chick Spinal Cord at 1.5 Days (Left) and 4 Days (Right) (A) During normal development, the floor plate (stippled) develops above the notochord (n), and motor neurons subsequently differentiate in adjacent lateral plate. AC4 antigen is expressed in more dorsal regions (grey). (B) Grafting a donor notochord (n') alongside the folding neural plate results in an additional floor plate and a third column of motor neurons. AC4 expression retracts. (C) Removing the notochord from beneath the neural plate results in the absence of both floor plate and motor neurons. AC4 expression extends through the ventral region of the cord.
chick spinal cord that had previously either received a notochord graft or been contacted by notochord in vitro. When cocultured with explants of rat dorsal cord, these pretreated regions promote commissural axon outgrowth with a potency equivalent to that of normal floor plate. The activity is lacking, furthermore, in ventral cord from which the notochord had previously been resected. Together with the demonstration by Yamada et al. that floor platespecific antigens (FPll2) are expressed in notochord-influenced lateral plate and not in notochord-deprived spinal cord, these data show that the notochord is required and sufficient for floor plate development. Using a monoclonal antibody (SC1) that distinguishes motor neurons from other spinal cord neurons, Yamada et al. have also found that the notochord does influence the production of motor neurons. When implanted alongside the lateral plate, an additional notochord elicits the formation of motor neurons in the dorsal neural tube close to but not contiguous with the induced ectopic floor plate (see figure, part B). As in their normal ventral location, an unlabeled region (called X by Yamada et al.) separates the SCl-positive neurons from the FPl-positive floor plate
cells. When the notochord is implanted in the dorsal midline, disrupting the roof plate, bilaterally symmetrical columns of motor neurons form in the dorsal cord. The ectopic motor neurons in these animals apparently develop from precursors destined for another fate rather than by the rescue of a population of specific motor neuron progenitors: although there is a significant increase in the number of motor neurons compared with controls, there is no overall increase in the number of cells. That ventral cell types differentiate at the expense of dorsal ones is also suggested by reciprocal changes in the distribution of dorsal markers. Both AC4, a surface antigen of cells restricted to dorsal and intermediate regions of the spinal cord, and CRABP, an intracellular marker for relay neurons in these regions, are expressed over a reduced area after notochord grafts. Consistently, when Yamada et al. remove the notochord early in development, the spinal cord lacks SC1- or FPl-positive cells, and both the AC4 antigen and CRABP are expressed throughout the ventral region (see figure, part C). Lineage analysis in the chick (Leber et al., 1990) predicts that motor neurons do not arise from specific precursors but share a common precursor with other neuronal types; such cells might be expected to be distributed throughout the proliferative (in ner) layer of the neural tube. The findings of Yamada et al. suggest that signals from the notochord are required to drive these precursors toward a motor fate. Does the notochord directly induce motor neuron differentiation, or does its influence depend on the prior induction of floor plate and signaling within the neuroepithelium? Two lines of evidence suggest direct induction. First, ectopic motor neurons can differentiate without, apparently, the formation of a nearby floor plate (van Straaten, 1985a; Yamada et al., 1991). This happens when the neural tube lies 25-80 p.m from an implanted notochord, roughly the same distance that separates the notochord and the earliest motor neurons in the normal embryo. The correlation of effect with distance suggests a graded inductive signal with different responses at two thresholds. Second, there is emerging evidence from zebrafish that the floor plate may not be required for motor neuron production. In the cyclops mutant (cyc-l), the floor plate is absent. The defect appears to be in the competence of midline cells to respond to the notochord signal, since the phenotype can be rescued by transplantatibn of wild-type gastrula ectoderm into mutants (Hatta et al., 1991). Although they have not yet been described, motor neurons are presumably present in cyc-1 despite the absence of a floor plate--the animal is motile. Fish and amphibian larvae, however, have a very early differentiating type of motor neuron not found in birds and mammals; these primary motor neurons appear to develop constitutively in the neuroectoderm without a requirement for either floor plate or notochord (Clarke et al., 1991). A relevant question then is whether cyc-1 develops secondary motor neurons, the homologs of those in higher vertebrates, in line with the expectations of a direct induction by notochord. There is compelling evidence, however, that the floor plate can regulate motor neurogenesis independently of
Minireview 473
the notochord. When floor plate is grafted next to the lateral surface of the neural tube, expression of both FP1 and SC1 antigens is induced in the adjacent neuroepithelium, the ectopic motor neurons being separated from the induced floor plate by a region of unlabeled cells (Yamada et al., 1991). The floor plate therefore has the capacity to suppress and promote motor neuron differentiation at a constant distance. With respect to these properties, the floor plate seems to mimic the notochord, raising the question of which is instrumental during normal development, or whether both midline structures are involved. The acquisition of signaling capacity by the floor plate suggests that the ventral pole of the neural tube is set up to go it alone and that the further signaling potential of the notochord is redundant. An organizing center within the neuroepithelium would perhaps allow greater precision of positional signaling than one lying outside that is subject to displacement. A further question is: if floor plate can induce floor plate, how is its width set? One possibility is that during normal floor plate induction, the notochord also inhibits the reaction in region X. Despite these remaining problems, the coupling of embryo manipulation with specific antibody markers has produced a clearer insight into the process of motor neuron differentiation. It has, furthermore, indicated a likely mechanism for patterning: the floor plate, itself dependent on the notochord for its induction, may constitute a polarizing region for the dorsoventral axis of the lateral plate. Motor neurons, in this case, would differentiate from multipotential precursors according to their position in a concentration gradient of morphogen diffusing from this source. Retinoic acid is a candidate molecule for signaling positional information within the neural tube. The floor plate produces retinoids, the biological activity of which has been assayed by the ability to respecify the pattern of digits in the chick wing (Wagner et al., 1990). Interestingly, both the notochord (Wagner et al., 1990) and its progenitor, Hensen's node (Hornbruch and Wolpert, 1986), also polarize in the chick wing assay, suggesting a way in which the notochord could signal motor neuron differentiation in the absence of a floor plate as, for example, in those cases where the grafted notochord lay outside the range for a floor plate reaction. The role of retinoids in dorsoventral signaling will be further elucidated by analyses of the distribution of nuclear retinoic acid receptors, the effects of retinoic acid on neural tube development in the chick, using the variety of position-marking antibodies now available, and identification of retinoic acid-inducible genes that could encode positional value. Genes have been isolated that have dorsoventrally restricted expression patterns in the young spinal cord, suggesting some involvement in dorsoventral patterning. These include genes in the Hox 2 cluster and Hox 3.1 (Graham et al., 1991), certain paired box-containing g e n e s (e.g., Pax2: Nornes et al., 1990; Pax8: Plachov et al., 1990), and Evx 1 (Bastian and Gruss, 1990). A useful approach would be to examine how the expression patterns of these genes change in response to grafting an additional notochord.
References
Bastian, H., and Gruss, P. (1990). EMBOJ. 9, 1839-1852. Clarke,J. D.W., Holder,N., Soffe,S. R., and Storm-Mathisen,J. (1991). Development,in press. Graham, A., Maden, M., and Krumlauf, R. (1991). Development,in press. Hatta, K., Kimmel, C. B., Ho, R. K., and Walker, C. (1991). Nature, in
press. Holffreter,J. (1934).Amh. Exp. Zellforsch. 15, 281-301. Hornbruch, A., and Wolpert, L. (1986).J. Embryol. Exp. Morphol.87, 163-174. Kitchen, I. C. (1949).J. Exp. Zool. 112, 393-415. Leber,S. M., Breedlove,S. M., and Sanes,J. R. (1990).J. Neurosci.10, 2451-2462. Nornes, H. O., Dressier,G. R., Knapik, E. W., Deutsch, U., and Gruss, P. (1990). Development109, 797-809. Plachov,D., Chowdhury,K., Walther,C., Simon,D., Guenet,J. L., and Gruss, P. (1990). Development110, 643-651. Placzek,M., Tessier-Lavigne,M., Yamada,T., Jesse,, T., and Dodd,J. (1990). Science250, 985-988. Smith, J. L., and Schoenwolf,G. C. (1989). J. Exp. Zool. 250, 49-62. Spemann,H. (1938).EmbryonicDevelopmentand Induction(NewHaven, Connecticut:YaleUniversityPress). Tessier-Lavigne,M., Placzek,M., Lumsden,A. G. S., Dodd, J., and Jesse,, T. M. (1988). Nature336, 775-778. van Straaten,H. W. M., and Drukker,J. (1987).In MesenchymaI-EpithelialInteractionsin NeuralDevelopment,J. R. Woolfet al., eds.(Berlin: Springer-Verlag),pp. 153-162. van Straaten,H. W. M., Thors, F., Hoessels,E. L., Hekking,J. W. M., and Drukker,J. (1985a).Dev. Biol. 110, 247-254. van Straaten,H. W. M., Hekking,J. W. M., Thors,E, Wiertz, E. L. J. M., and Drukkar,J. (1985b).Acta Morphol. Neerl.-Scand.23, 91-97. van Straaten,H. W. M, Hekking,J. W. M., Wiertz-Hoessels,E. J. L. M., Thors, F., and Drukker,J. (1988). Anat. Embryol. 177, 317-324. Wagner, M., Thaller,C., Jesse,, T., and Eichele,G. (1990).Nature345, 819-822. Watterson, R. L. (1965).In Organogenesis,R. L. de Haan and H. Ursprung, eds. (New York: Holt, Rinehartand Winston), pp. 129-159. Yamada,T., Placzek,M., Tanaka,H., Dodd,J., andJessell,T. M. (1991). Cell 64, this issue.
