Essential Role forSonic hedgehogduring Hair Follicle Morphogenesis

AbstractThe hair follicle is a source of epithelial stem cells and site of origin for several types of skin tumors. Although it is clear that follicles arise by way of a series of inductive tissue interactions, identification of the signaling molecules driving this process remains a major challenge in skin biology. In this study we report an obligatory role for the secreted morphogen Sonic hedgehog (Shh) during hair follicle development. Hair germs comprising epidermal placodes and associated dermal condensates were detected in both control andShh−/− embryos, but progression through subsequent stages of follicle development was blocked in mutant skin. The expression ofGli1andPtc1was reduced inShh−/− dermal condensates and they failed to evolve into hair follicle papillae, suggesting that the adjacent mesenchyme is a critical target for placode-derived Shh. Despite the profound inhibition of hair follicle morphogenesis, late-stage follicle differentiation markers were detected inShh−/− skin grafts, as well as cultured vibrissa explants treated with cyclopamine to block Shh signaling. Our findings reveal an essential role for Shh during hair follicle morphogenesis, where it is required for normal advancement beyond the hair germ stage of development

Widespread Contribution of Gdf7 Lineage to Cerebellar Cell Types and Implications for Hedgehog-Driven Medulloblastoma Formation

The roof plate is a specialized embryonic midline tissue of the central nervous system that functions as a signaling center regulating dorsal neural patterning. In the developing hindbrain, roof plate cells express Gdf7 and previous genetic fate mapping studies showed that these cells contribute mostly to non-neural choroid plexus epithelium. We demonstrate here that constitutive activation of the Sonic hedgehog signaling pathway in the Gdf7 lineage invariably leads to medulloblastoma. Lineage tracing analysis reveals that Gdf7-lineage cells not only are a source of choroid plexus epithelial cells, but are also present in the cerebellar rhombic lip and contribute to a subset of cerebellar granule neuron precursors, the presumed cell-of-origin for Sonic hedgehog-driven medulloblastoma. We further show that Gdf7-lineage cells also contribute to multiple neuronal and glial cell types in the cerebellum, including glutamatergic granule neurons, unipolar brush cells, Purkinje neurons, GABAergic interneurons, Bergmann glial cells, and white matter astrocytes. These findings establish hindbrain roof plate as a novel source of diverse neural cell types in the cerebellum that is also susceptible to oncogenic transformation by deregulated Sonic hedgehog signaling

Shh and Gli3 activities are required for timely generation of motor neuron progenitors

AbstractGeneration of distinct ventral neuronal subtypes in the developing spinal cord requires Shh signaling mediated by the Gli family of transcription factors. Genetic studies of Shh−/−;Gli3−/− double mutants indicated that the inhibition of Gli3 repressor activity by Shh is sufficient for the generation of different neurons including motor neurons. In this study, we show that although ventral neural progenitors are initiated in normal numbers in Shh−/−;Gli3−/− mutants, the subsequent appearance of motor neuron progenitors shows a ∼20-hour lag, concomitant with a delay in the activation of a pan-neuronal differentiation program and cell cycle exit of ventral neural progenitors. Accordingly, the Shh−/−;Gli3−/− mutant spinal cord exhibits a delay in motor neuron differentiation and an accumulation of a ventral neural progenitor pool. The requirement of Shh and Gli3 activities to promote the timely appearance of motor neuron progenitors is further supported by the analysis of Ptch1−/− mutants, in which constitutive Shh pathway activity is sufficient to elicit ectopic and premature differentiation of motor neurons at the expense of ventral neural progenitors. Taken together, our analysis suggests that, beyond its well established dorso-ventral patterning function through a Gli3-derepression mechanism, Shh signaling is additionally required to promote the timely appearance of motor neuron progenitors in the developing spinal cord

Sonic hedgehog signaling directly targets Hyaluronic Acid Synthase 2, an essential regulator of phalangeal joint patterning

AbstractSonic hedgehog (Shh) signal, mediated by the Gli family of transcription factors, plays an essential role in the growth and patterning of the limb. Through analysis of the early limb bud transcriptome, we identified a posteriorly-enriched gene, Hyaluronic Acid Synthase 2 (Has2), which encodes a key enzyme for the synthesis of hyaluronan (HA), as a direct target of Gli transcriptional regulation during early mouse limb development. Has2 expression in the limb bud is lost in Shh null and expanded anteriorly in Gli3 mutants. We identified an ∼3kb Has2 promoter fragment that contains two strong Gli-binding consensus sequences, and mutation of either site abrogated the ability of Gli1 to activate Has2 promoter in a cell-based assay. Additionally, this promoter fragment is sufficient to direct expression of a reporter gene in the posterior limb mesenchyme. Chromatin immunoprecipitation of DNA-Gli3 protein complexes from limb buds indicated that Gli3 strongly binds to the Has2 promoter region, suggesting that Has2 is a direct transcriptional target of the Shh signaling pathway. We also showed that Has2 conditional mutant (Has2cko) hindlimbs display digit-specific patterning defects with longitudinally shifted phalangeal joints and impaired chondrogenesis. Has2cko limbs show less capacity for mesenchymal condensation with mislocalized distributions of chondroitin sulfate proteoglycans (CSPGs), aggrecan and link protein. Has2cko limb phenotype displays striking resemblance to mutants with defective chondroitin sulfation suggesting tight developmental control of HA on CSPG function. Together, our study identifies Has2 as a novel downstream target of Shh signaling required for joint patterning and chondrogenesis

Manifestation of the Limb Prepattern: Limb Development in the Absence of Sonic Hedgehog Function

AbstractThe secreted protein encoded by the Sonic hedgehog (Shh) gene is localized to the posterior margin of vertebrate limb buds and is thought to be a key signal in establishing anterior–posterior limb polarity. In the Shh−/− mutant mouse, the development of many embryonic structures, including the limb, is severely compromised. In this study, we report the analysis of Shh−/− mutant limbs in detail. Each mutant embryo has four limbs with recognizable humerus/femur bones that have anterior–posterior polarity. Distal to the elbow/knee joints, skeletal elements representing the zeugopod form but lack identifiable anterior–posterior polarity. Therefore, Shh specifically becomes necessary for normal limb development at or just distal to the stylopod/zeugopod junction (elbow/knee joints) during mouse limb development. The forelimb autopod is represented by a single distal cartilage element, while the hindlimb autopod is invariably composed of a single digit with well-formed interphalangeal joints and a dorsal nail bed at the terminal phalanx. Analysis of GDF5 and Hoxd11–13 expression in the hindlimb autopod suggests that the forming digit has a digit-one identity. This finding is corroborated by the formation of only two phalangeal elements which are unique to digit one on the foot. The apical ectodermal ridge (AER) is induced in the Shh−/− mutant buds with relatively normal morphology. We report that the architecture of the Shh−/− AER is gradually disrupted over developmental time in parallel with a reduction of Fgf8 expression in the ridge. Concomitantly, abnormal cell death in the Shh−/− limb bud occurs in the anterior mesenchyme of both fore- and hindlimb. It is notable that the AER changes and mesodermal cell death occur earlier in the Shh−/− forelimb than the hindlimb bud. This provides an explanation for the hindlimb-specific competence to form autopodial structures in the mutant. Finally, unlike the wild-type mouse limb bud, the Shh−/− mutant posterior limb bud mesoderm does not cause digit duplications when grafted to the anterior border of chick limb buds, and therefore lacks polarizing activity. We propose that a prepattern exists in the limb field for the three axes of the emerging limb bud as well as specific limb skeletal elements. According to this model, the limb bud signaling centers, including the zone of polarizing activity (ZPA) acting through Shh, are required to elaborate upon the axial information provided by the native limb field prepattern

Shh pathway activation in Gdf7-lineage cells leads to cerebellar hyperplasia.

<p><i>Gdf7^Cre/+;SmoM2</i> gain-of-function mutant mice exhibit cerebellar defects. (A–B′) Hematoxylin-eosin staining of wild-type and <i>Gdf7^Cre/+;SmoM2</i> mutants. Prior to P10 histological sections of mutants are similar to control. (C–E′) <i>Gdf7^Cre/+;SmoM2</i> mutants over 14 days old develop ectopic foci of densely packed cells within the molecular layer of their cerebella. Higher magnification view of these foci reveals no discernible layer organization and resemblance to neoplastic lesions. Arrows in (D) indicate regions of hypercellularity. Boxed regions E and E″ are magnified and shown in the right adjacent panels. Arrows in D′ indicate nuclear molding. Arrowheads in D′ indicate apoptotic nuclei. (F-F″) The ectopic foci consist of cells of the Gdf7-lineage as indicated by their expression of SmoM2-YFP. (G-G″) Ectopic foci do not express differentiated neuronal marker NeuN. Abbreviations: EGL, external granular layer. ML, molecular layer. IGL, internal granular layer. T, tumor region. N, normal cerebellum.</p

A subset of Gdf7-lineage cells express neural stem cell markers.

<p>(A–B″′) Many cells within the tumor tissue of <i>Gdf7^Cre/+;SmoM2</i> mice coexpress neural stem cell marker Nestin (red) and glial marker GFAP (green). Arrows indicate co-localization. (C–E) Tumor cell lines can be invariably established from <i>Gdf7^Cre/+;SmoM2</i> cerebella. (F–G″) These cells highly express multiple neural stem cell markers Nestin and Sox2 and can undergo serial passages. (H–J) Here, one representative colony is shown when cultured for 3 and 6 days. (K–N) <i>Gdf7^Cre/+;SmoM2</i> cultured cells can differentiate into mature cerebellar cell types upon switching to culture media containing 10% FBS.</p