Neuronal subtype diversification is essential for the establishment of functional neural circuits, and yet the molecular events underlying neuronal diversity remain largely to be defined. During spinal neurogenesis, the p2 progenitor domain, unlike others in the ventral spinal cord, gives rise to two intermingled but molecularly distinct subtypes of interneurons, termed V2a and V2b. We show here that the Foxn4 winged helix͞forkhead transcription factor is coexpressed with the bHLH factor Mash1 in a subset of p2 progenitors. Loss of Foxn4 function eliminates Mash1 expression and V2b neurons and causes a fate-switch to V2a neurons, whereas the absence of Mash1 displays a similar but less severe phenotype. Overexpression of Foxn4 alone in spinal neural progenitors promotes the V2a fate at the expense of the V2b fate, whereas Mash1 suppresses both the V2a and V2b fates. However, coexpression of both Foxn4 and Mash1 promotes the V2b fate while inhibiting the V2a fate, indicating that Foxn4 cooperates with Mash1 to specify the identity of V2b neurons from bipotential p2 progenitors.winged-helix͞forkhead ͉ transcription factor ͉ p2 progenitor ͉ spinal neurogenesis

SUMMARYIn caudal regions of the diencephalon, sonic hedgehog (Shh) is expressed in the ventral midline of prosomeres 1-3 (p1-p3), which underlie the pretectum, thalamus and prethalamus, respectively. Shh is also expressed in the zona limitans intrathalamica (zli), a dorsally projecting spike that forms at the p2-p3 boundary. The presence of two Shh signaling centers in the thalamus has made it difficult to determine the specific roles of either one in regional patterning and neuronal fate specification. To investigate the requirement of Shh from a focal source of expression in the ventral midline of the diencephalon, we used a newly generated mouse line carrying a targeted deletion of the 525 bp intronic sequence mediating Shh brain enhancer-1 (SBE1) activity. In SBE1 mutant mice, Shh transcription was initiated but not maintained in the ventral midline of the rostral midbrain and caudal diencephalon, yet expression in the zli was unaffected. In the absence of ventral midline Shh, rostral thalamic progenitors (pTH-R) adopted the molecular profile of a more caudal thalamic subtype (pTH-C). Surprisingly, despite their early mis-specification, neurons derived from the pTH-R domain continued to migrate to their proper thalamic nucleus, extended axons along their normal trajectory and expressed some, but not all, of their terminal differentiation markers. Our results, and those of others, suggest a model whereby Shh signaling from distinct spatial and temporal domains in the diencephalon exhibits unique and overlapping functions in the development of discrete classes of thalamic interneurons.

The three vertebrate Gli proteins play a central role in mediating Hedgehog(Hh)-dependent cell fate specification in the developing spinal cord; however,their individual contributions to this process have not been fully characterized. In this paper, we have addressed this issue by examining patterning in the spinal cord of Gli2;Gli3 double mutant embryos, and in chick embryos transfected with dominant activator forms of Gli2 and Gli3. In double homozygotes, Gli1 is also not expressed; thus, all Gli protein activities are absent in these mice. We show that Gli3 contributes activator functions to ventral neuronal patterning, and plays a redundant role with Gli2 in the generation of V3 interneurons. We also show that motoneurons and three classes of ventral neurons are generated in the ventral spinal cord in double mutants, but develop as intermingled rather than discrete populations. Finally, we provide evidence that Gli2 and Gli3 activators control ventral neuronal patterning by regulating progenitor segregation. Thus, multiple ventral neuronal types can develop in the absence of Gli function, but require balanced Gli protein activities for their correct patterning and differentiation.

Shh-Gli signaling controls cell fates in the developing ventral neural tube by regulating the patterned expression of transcription factors in neural progenitors. However, the molecular mechanisms that limit target gene responses to specific domains are unclear. Here, we show that Wnt pathway inhibitors regulate the threshold response of a ventral Shh target gene, Nkx2.2, to establish its restricted expression in the ventral spinal cord. Identification and characterization of an Nkx2.2 enhancer reveals that expression is directly regulated by positive Shh-Gli signaling and negative Tcf repressor activity. Our data indicate that the dorsal limit of Nkx2.2 is controlled by Tcf4-mediated transcriptional repression, and not by a direct requirement for high-level Shh-Gli signaling, arguing against a simple model based on differential Gli factor affinities in target genes. These results identify a transcriptional mechanism that integrates graded Shh and Wnt signaling to define progenitor gene expression domains and cell fates in the neural tube.

With the realization that many proto-oncogenes and tumor suppressor genes are expressed and have important functions during mammalian development, it is clear that cancer often involves the inappropriate activation of genetic pathways used during normal development. A signaling cascade that has been of considerable interest to both developmental and cancer biologists involves the Hedgehog (Hh) family of secreted proteins. To date, the only transcription factors shown to be directly downstream of Hh are the zinc-finger containing proteins Cubitus interruptus (Ci) and Gli, in flies and vertebrates, respectively. The identification of many of the genes and proteins involved in Hh signaling has come largely from genetic and biochemical studies in Drosophila. Ci mediates Hh signaling through a Hh-dependent set of protein modifications that alter the activity of Ci on Hh target genes. Recent evidence suggests vertebrate Gli proteins may be similarly regulated. The interest in this pathway has taken on added importance with the identification of mutations in Hh pathway genes, including Gli genes, in several human developmental disorders and cancers. We discuss models for how Gli proteins mediate Hh signaling in both vertebrate development and cancers.