Spatial and temporal requirements for sonic hedgehog in the regulation of thalamic interneuron identity.

In 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.

FoxF1 and FoxL1 link hedgehog signaling and the control of epithelial proliferation in the developing stomach and intestine.

The hedgehog (Hh) signaling pathway is a key component of cross-talk during vertebrate gut development, involving endodermally secreted Sonic (Shh) and Indian hedgehog (Ihh) proteins that directly signal to adjacent mesoderm. Here we show that the closely linked mesenchymal forkhead transcription factors Foxf1 and Foxl1 are part of this signaling cascade. Analysis of conserved non-coding sequences surrounding Foxf1 and Foxl1 identified seven Gli binding sites, with two sites near Foxl1 being identical among mammalian, bird, fish, and amphibian species. In vitro experiments indicate that Gli2 binds to these Gli sites, several of which are critical for Gli2-mediated activation of a luciferase reporter in 293 cells. In addition, we demonstrate occupancy of one of these elements by Gli proteins in the intestine in vivo using chromatin immunoprecipitation. Furthermore, expression of both Foxf1 and Foxl1 is reduced in the Gli2/Gli3 mutant gut. These results provide compelling evidence that Foxf1 and Foxl1 are mediators of the Hh (endoderm) to mesoderm signaling pathway.

Opposing gradients of Gli repressor and activators mediate Shh signaling along the dorsoventral axis of the inner ear.

Organization of the vertebrate inner ear is mainly dependent on localized signals from surrounding tissues. Previous studies demonstrated that sonic hedgehog (Shh) secreted from the floor plate and notochord is required for specification of ventral (auditory) and dorsal (vestibular) inner ear structures, yet it was not clear how this signaling activity is propagated. To elucidate the molecular mechanisms by which Shh regulates inner ear development, we examined embryos with various combinations of mutant alleles for Shh, Gli2 and Gli3. Our study shows that Gli3 repressor (R) is required for patterning dorsal inner ear structures, whereas Gli activator (A) proteins are essential for ventral inner ear structures. A proper balance of Gli3R and Gli2/3A is required along the length of the dorsoventral axis of the inner ear to mediate graded levels of Shh signaling, emanating from ventral midline tissues. Formation of the ventral-most otic region, the distal cochlear duct, requires robust Gli2/3A function. By contrast, the formation of the proximal cochlear duct and saccule, which requires less Shh signaling, is achieved by antagonizing Gli3R. The dorsal vestibular region requires the least amount of Shh signaling in order to generate the correct dose of Gli3R required for the development of this otic region. Taken together, our data suggest that reciprocal gradients of GliA and GliR mediate the responses to Shh signaling along the dorsoventral axis of the inner ear.

Activation of Class I transcription factors by low level Sonic hedgehog signaling is mediated by Gli2-dependent and independent mechanisms.

The partitioning of the ventral neural tube into five distinct neuronal progenitor domains is dependent on the morphogenic action of the secreted protein Sonic hedgehog (Shh). The prevailing model stipulates that Class I genes are repressed and Class II genes are activated by high levels of Shh signaling and that sharp progenitor domain boundaries are established by the mutual repression of complementary pairs of Class I and Class II transcription factors. While core elements of this model are supported by experimental evidence, a number of issues remain unresolved. Foremost of these is a more thorough understanding of the mechanism by which Class I genes are regulated. In this study, we describe the consequences of Shh misexpression on Class I and Class II gene expression in the hindbrain of ShhP1 embryos. We observed that an ectopic source of Shh in the otic vesicle of ShhP1 embryos ventralized the adjacent hindbrain by inducing, rather than repressing, the expression of several Class I genes (Pax6, Dbx1, Dbx2). The Shh dependent activation of Class I genes was mediated, in part, by Gli2. These results bear significance on the model of ventral neural tube patterning as they suggest a dual role for Shh in the regulation of Class I genes, whereby low levels of Shh signaling initiate Class I gene transcription, while higher levels restrict the domains of Class I gene expression to intermediate positions of the neural tube through the activation of Class II transcriptional regulators.