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The link between extrinsic signaling, progenitor cell specification and neuronal subtype identity is central to the developmental organization of the vertebrate central nervous system. In the hindbrain and spinal cord, distinctions in the rostrocaudal identity of progenitor cells are associated with the generation of different motor neuron subtypes. Two fundamental classes of motor neurons, those with dorsal (dMN) and ventral (vMN) exit points, are generated over largely non-overlapping rostrocaudal domains of the caudal neural tube. Cdx and Hox genes are important determinants of the rostrocaudal identity of neural progenitor cells, but the link between early patterning signals, neural Cdx and Hox gene expression, and the generation of dMN and vMN subtypes, is unclear. Using an in vitro assay of neural differentiation, we provide evidence that an early Wnt-based program is required to interact with a later retinoic acid- and fibroblast growth factor-mediated mechanism to generate a pattern of Cdx and Hox profiles characteristic of hindbrain and spinal cord progenitor cells that prefigure the generation of vMNs and dMNs.

Specification of neuronal fate in the vertebrate central nervous system depends on the profile of transcription factor expression by neural progenitor cells, but the precise roles of such factors in neurogenesis remain poorly characterized. Two closely related transcriptional repressors, Nkx6.2 and Nkx6.1, are expressed by progenitors in overlapping domains of the ventral spinal cord. We provide genetic evidence that differences in the level of repressor activity of these homeodomain proteins underlies the diversification of interneuron subtypes, and provides a fail-safe mechanism during motor neuron generation. A reduction in Nkx6 activity further permits V0 neurons to be generated from progenitors that lack homeodomain proteins normally required for their generation, providing direct evidence for a model in which progenitor homeodomain proteins direct specific cell fates by actively suppressing the expression of transcription factors that direct alternative fates.

Within the developing vertebrate nervous system, the mechanisms that coordinate neuronal subtype identity with generic features of neuronal differentiation are poorly defined. We show here that a bHLH protein, Olig2, is expressed selectively by motor neuron progenitors and has a key role in specifying the subtype identity and pan-neuronal properties of developing motor neurons. The role of Olig2 in the specification of motor neuron subtype identity depends on regulatory interactions with progenitor homeodomain proteins, whereas its role in promoting pan-neuronal properties is associated with expression of another bHLH protein, Ngn2. Both aspects of Olig2 function appear to depend on its activity as a transcriptional repressor. Together, these studies show that Olig2 has a critical role in integrating diverse features of motor neuron differentiation in the developing spinal cord.

Distinct classes of neurons are generated at defined positions in the ventral neural tube in response to a gradient of Sonic Hedgehog (Shh) activity. A set of homeodomain transcription factors expressed by neural progenitors act as intermediaries in Shh-dependent neural patterning. These homeodomain factors fall into two classes: class I proteins are repressed by Shh and class II proteins require Shh signaling for their expression. The profile of class I and class II protein expression defines five progenitor domains, each of which generates a distinct class of postmitotic neurons. Cross-repressive interactions between class I and class II proteins appear to refine and maintain these progenitor domains. The combinatorial expression of three of these proteins--Nkx6.1, Nkx2.2, and Irx3--specifies the identity of three classes of neurons generated in the ventral third of the neural tube.

During neural development in vertebrates, a spatially ordered array of neurons is generated in response to inductive signals derived from localized organizing centres. One organizing centre that has been proposed to have a role in the control of neural patterning is the roof plate. To define the contribution of signals derived from the roof plate to the specification of neuronal cell types in the dorsal neural tube, we devised a genetic strategy to ablate the roof plate selectively in mouse embryos. Embryos without a roof plate lack all the interneuron subtypes that are normally generated in the dorsal third of the neural tube. Using a genetically based lineage analysis and in vitro assays, we show that the loss of these neurons results from the elimination of non-autonomous signals provided by the roof plate. These results reveal that the roof plate is essential for specifying multiple classes of neurons in the mammalian central nervous system.

The generation of distinct classes of neurons at defined positions within the developing vertebrate nervous system depends on inductive signals provided by local cell groups that act as organizing centers. Genetic and embryological studies have begun to elucidate the processes that control the pattern and identity of neuronal cell types. Here we discuss the cellular interactions and molecular mechanisms that direct neuronal cell fates in the dorsal half of the vertebrate central nervous system. The specification of dorsal neuronal cell fates appears to depend on a cascade of inductive signals initiated by cells of the epidermal ectoderm that flank the neural plate and propagated by roof plate cells within the neural tube. Members of the transforming growth factor-beta (TGF beta) family of secreted proteins have a prominent role in mediating these dorsalizing signals. Additional signals involving members of the Wnt and fibroblast growth factor (FGF) families may also contribute to the proliferation and differentiation of dorsal neuronal cell types.

We have investigated the role of Bone Morphogenetic Protein 4 (BMP-4) and a BMP antagonist, chordin, in primitive streak formation and neural induction in amniote embryos. We show that both BMP-4 and chordin are expressed before primitive streak formation, and that BMP-4 expression is downregulated as the streak starts to form. When BMP-4 is misexpressed in the posterior area pellucida, primitive streak formation is inhibited. Misexpression of BMP-4 also arrests further development of Hensen's node and axial structures. In contrast, misexpression of chordin in the anterior area pellucida generates an ectopic primitive streak that expresses mesoderm and organizer markers. We also provide evidence that chordin is not sufficient to induce neural tissue in the chick. Misexpression of chordin in regions outside the future neural plate does not induce the early neural markers L5, Sox-3 or Sox-2. Furthermore, neither BMP-4 nor BMP-7 interfere with neural induction when misexpressed in the presumptive neural plate before or after primitive streak formation. However, chordin can stabilise the expression of early neural markers in cells that have already received neural inducing signals. These results suggest that the regulation of BMP signalling by chordin plays a role in primitive streak formation and that chordin is not sufficient to induce neural tissue.

Antibodies that block Sonic Hedgehog (SHH) signaling have been used to show that SHH activity is required for the induction of floor plate differentiation by the notochord and independently for the induction of motor neurons by both the notochord and midline neural cells. Motor neuron generation depends on two critical periods of SHH signaling: an early period during which naive neural plate cells are converted into ventralized progenitors and a late period that extends well into S phase of the final progenitor cell division, during which SHH drives the differentiation of ventralized progenitors into motor neurons. The ambient SHH concentration during the late period determines whether ventralized progenitors differentiate into motor neurons or interneurons, thus defining the pattern of neuronal cell types generated in the neural tube.

The vertebrate hedgehog-related gene, sonic hedgehog, is expressed in ventral domains along the entire rostrocaudal length of the neural tube, including the forebrain. Shh induces the differentiation of ventral neuronal cell types in explants derived from prospective forebrain regions of the neural plate. Neurons induced in explants derived from diencephalic and telencephalic levels of the neural plate express the LIM homeodomain protein Islet-1, but these neurons possess distinct identities that match those of the ventral neurons normally generated in these two subdivisions of the forebrain. These results, together with other studies of neuronal differentiation at caudal levels of the neural tube, suggest that a single inducing molecule, Shh, mediates the induction of distinct ventral neuronal cell types along the entire rostrocaudal extent of the embryonic central nervous system.

Background: The differentiation of floor plate cells and motor neurons in the vertebrate neural tube appears to be induced by signals from the notochord. The secreted protein encoded by the Sonic hedgehog (Shh) gene is expressed by axial midline cells and can induce floor plate cells in vivo and in vitro. Motor neurons can also be induced in vitro by cells that synthesize Sonic hedgehog protein (Shh). It remains unclear, however, if the motor-neuron-inducing activity of Shh depends on the synthesis of a distinct signaling molecule by floor plate cells. To resolve this issue, we have developed an in vitro assay which uncouples the notochord-mediated induction of motor neurons from floor plate differentiation, and have used this assay to examine whether Shh induces motor neurons in the absence of floor plate differentiation., Results: Floor plate cells and motor neurons were induced in neural plate explants grown in contact with the notochord, but only motor neurons were induced when explants were separated from the notochord. COS cells transfected with Shh induced both floor plate cells and motor neurons when grown in contact with neural plate explants, whereas only motor neurons were induced when the explants were grown at a distance from Shh-transfected COS cells. Direct transfection of neural plate cells with an Shh-expression construct induced both floor plate cells and motor neurons, with motor neuron differentiation occurring prior to, or coincidentally with, floor plate differentiation. The induction of motor neurons appears, therefore, not to depend on floor plate differentiation., Conclusions: The induction of motor neurons by Shh does not depend on distinct floor-plate-derived signaling molecules. Shh can, therefore, initiate the differentiation of two cell types that are generated in the ventral region of the neural tube. These results show that the early development of motor neurons involves the inductive action of Shh, whereas the survival of motor neurons at later stages of embryonic development requires neurotrophic factors.

HNF-3 beta, a transcription factor of the winged-helix family, is expressed in embryonic and adult endoderm and also in midline cells of the node, notochord, and floor plate in mouse embryos. To define the function of HNF-3 beta, a targeted mutation in the HNF-3 beta locus was generated by homologous recombination in embryonic stem cells. Mice lacking HNF-3 beta die by embryonic day (E) 10-11. Mutant embryos examined from E6.5 to E9.5 do not form a distinct node and lack a notochord. In addition, mutant embryos show marked defects in the organization of somites and neural tube that may result from the absence of the notochord. The neural tube of mutant embryos exhibits overt anteroposterior polarity but lacks a floor plate and motor neurons. Endodermal cells are present but fail to form a gut tube in mutant embryos. These studies indicate that HNF-3 beta has an essential role in the development of axial mesoderm in mouse embryos.

The floor plate is located at the ventral midline of the neural tube and has been implicated in neural cell patterning and axon guidance. To address the cellular mechanisms involved in floor plate differentiation, we have used an assay that monitors the expression of floor-plate-specific antigens in neural plate explants cultured in the presence of inducing tissues. Contact-mediated signals from both the notochord and the floor plate act directly on neural plate cells to induce floor plate differentiation. Floor plate induction is initiated medially by a signal from the notochord, but appears to be propagated to more lateral cells by homeogenetic signals that derive from medial floor plate cells. The response of neural plate cells to inductive signals declines with embryonic age, suggesting that the mediolateral extent of the floor plate is limited by a loss of competence of neural cells. The rostral boundary of the floor plate at the midbrain-forebrain junction appears to result from the lack of inducing activity in prechordal mesoderm and the inability of rostral neural plate cells to respond to inductive signals.

Neural cell markers have been used to examine the effect of retinoic acid (RA) on the development of the central nervous system (CNS) of Xenopus embryos. RA treatment of neurula stage embryos resulted in a concentration-dependent perturbation of anterior CNS development leading to a reduction in the size of the forebrain, midbrain and hindbrain. In addition the overt segmental organization of the hindbrain was abolished by high concentrations of RA. The regional expression of two cell-specific markers, the homeobox protein Xhox3 and the neurotransmitter serotonin was also examined in embryos exposed to RA. Treatment with RA caused a concentration-dependent change in the pattern of expression of Xhox3 and serotonin and resulted in the ectopic appearance of immunoreactive neurons in anterior regions of the CNS, including the forebrain. Collectively, our results extend previous studies by showing that RA treatment of embryos at the neurula stage inhibits the development of anterior regions of the CNS while promoting the differentiation of more posterior cell types. The relevance of these findings to the possible role of endogenous retinoids in the determination of neural cell fate and axial patterning is discussed.

Cellular interactions in a variety of vertebrate non-neural tissues are thought to be mediated by cell surface carbohydrate structures. The detection of cell-specific surface carbohydrates and carbohydrate-binding proteins within the embryonic nervous system has raised the possibility that carbohydrate recognition may also contribute to the interactions of developing neurons. Soluble lactose-binding lectins constitute one class of carbohydrate-binding proteins expressed in the vertebrate nervous system. We describe here the isolation of cDNAs from rat brain libraries encoding one of these lectins, RL-14.5, and demonstrate that this protein is not only homologous to other soluble lectins, but also identical in primary sequence to a lectin present in at least one non-neural tissue. RNA blot analysis and in situ hybridization reveal a restricted pattern of expression of RL-14.5 mRNA within the rat nervous system. High levels of RL-14.5 mRNA are present in primary sensory neurons and motoneurons in the spinal cord and brain stem. Moreover, expression of RL-14.5 mRNA in sensory and motoneurons is detectable soon after neuronal differentiation. These findings, together with previous studies demonstrating the selective expression of oligosaccharide ligands for RL-14.5 on the same neurons, are consistent with the idea that carbohydrate-mediated interactions contribute to the development of this subset of mammalian neurons.

Little is known about the cellular and molecular mechanisms that determine neuronal cell fate and the patterning of neuronal connections in the vertebrate central nervous system. In this paper we summarize evidence which indicates that some aspects of neuronal differentiation and axon guidance are regulated by specialized epithelial cells that occupy the medial region of the neural plate and, later, the ventral midline of the spinal cord. This cell group, termed the notoplate/floor plate appears to constitute a distinct compartment within the neural plate that is more closely related in lineage and perhaps also in function to axial mesodermal cells of the underlying notochord than to other neural plate cells. Cells of the notoplate exhibit specialized mechanical and adhesive properties that may contribute to neurulation. At later stages of development, the floor plate appears to guide developing axons in the embryonic spinal cord by releasing a diffusible chemoattractant factor and by virtue of its specialized cell surface properties. The floor plate may also play a role in the determination of cell identity and patterning at earlier stages of neural tube development.