Sonic hedgehog regulates Gli activator and repressor functions with spatial and temporal precision in the mid/hindbrain region

The midbrain and anterior hindbrain offer an ideal system in which to study the coordination of tissue growth and patterning in three dimensions. Two organizers that control anteroposterior (AP) and dorsoventral (DV) development are known, and the regulation of AP patterning by Fgf8 has been studied in detail. Much less is known about the mechanisms that control mid/hindbrain development along the DV axis. Using a conditional mutagenesis approach, we have determined how the ventrally expressed morphogen sonic hedgehog (Shh) directs mid/hindbrain development over time and space through positive regulation of the Gli activators (GliA) and inhibition of the Gli3 repressor (Gli3R). We have discovered that Gli2A-mediated Shh signaling sequentially induces ventral neurons along the medial to lateral axis, and only before midgestation. Unlike in the spinal cord, Shh signaling plays a major role in patterning of dorsal structures (tectum and cerebellum). This function of Shh signaling involves inhibition of Gli3R and continues after midgestation. Gli3R levels also regulate overall growth of the mid/hindbrain region, and this largely involves the suppression of cell death. Furthermore, inhibition of Gli3R by Shh signaling is required to sustain expression of the AP organizer gene Fgf8. Thus, the precise spatial and temporal regulation of Gli2A and Gli3R by Shh is instrumental in coordinating mid/hindbrain development in three dimensions.     

Impact of node ablation on the morphogenesis of the body axis and the lateral asymmetry of the mouse embryo during early organogenesis.

The node of the mouse gastrula is the major source of the progenitor cells of the notochord, the floor plate, and the gut endoderm. The node may also play a morphogenetic role since it can induce a partial body axis following heterotopic transplantation. The impact of losing these progenitor cells and the morphogenetic activity on the development of the body axes was studied by the ablation of the node at late gastrulation. In the ablated embryo, an apparently intact anterior-posterior body axis with morphologically normal head folds, neural tube, and primitive streak developed during early organogenesis. Cell fate analysis revealed that the loss of the node elicits de novo recruitment of neural ectoderm and somitic mesoderm from the surrounding germ-layer tissues. This leads to the restoration of the neural tube and the paraxial mesoderm. However, the body axis of the embryo was foreshortened and somite formation was retarded. Histological and gene expression studies reveal that in most of the node-ablated embryos, the notochord in the trunk was either absent or interrupted, and the floor plate was absent in the ventral region of the reconstituted neural tube. The loss of the node did not affect the differentiation of the gut endoderm or the formation of the mid- and hindgut. In the node-ablated embryo, expression of the Pitx2 gene in the lateral plate mesoderm was no longer restricted to the left side but was found on both sides of the body or was completely absent from the lateral plate mesoderm. Therefore, the loss of the node results in the failure to delineate the laterality of the body axis. The node and its derivatives therefore play a critical role in the patterning of the ventral neural tube and lateral body axis but not of the anterior-posterior axis during early organogenesis.     

Hedgehog signaling in gastrointestinal development and disease

The development of the gastrointestinal (GI) tract and its associated parenchymal organs depends on Hedgehog signals from the endoderm to the surrounding mesoderm. During development, Hedgehog signaling is essential for patterning the GI tract along anterior-posterior (A-P), dorsal-ventral (D-V), and radial axes, as well as in maintenance of stem cells. Our knowledge about these roles for Hedgehog signaling is derived from studies of developmental defects that result from disrupted or activated Hedgehog signaling in model organisms including mouse, chick, and frog. These studies provide evidence for distinct roles of specific Hedgehog ligands in GI development. Studies in model organisms have also elucidated how Hedgehog signaling may function in development and function of the GI tract in humans. Several diseases and congenital syndromes are known to result from genetic defects in Hedgehog signaling components, and this pathway may ultimately prove to be an important target for future diagnostic and therapeutic tools.     

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.     

Early mouse endoderm is patterned by soluble factors from adjacent germ layers

Endoderm that forms the respiratory and digestive tracts is a sheet of approximately 500-1000 cells around the distal cup of an E7.5 mouse embryo. Within 2 days, endoderm folds into a primitive gut tube from which numerous organs will bud. To characterize the signals involved in the developmental specification of this early endoderm, we have employed an in vitro assay using germ layer explants and show that adjacent germ layers provide soluble, temporally specific signals that induce organ-specific gene expression in endoderm. Furthermore, we show that FGF4 expressed in primitive streak-mesoderm can induce the differentiation of endoderm in a concentration-dependent manner. We conclude that the differentiation of gastrulation-stage endoderm is directed by adjacent mesoderm and ectoderm, one of the earliest reported patterning events in formation of the vertebrate gut tube.     

Direct and indirect requirements of Shh/Gli signaling in early pituitary development

Induction of early pituitary progenitors is achieved through combined activities of signals from adjacent embryonic tissues. Previous studies have identified a requirement for oral ectoderm derived Sonic Hedgehog (Shh) in specification and/or proliferation of early pituitary progenitors, however how different Gli genes mediate Shh signaling to control pituitary progenitor development has not yet been determined. Here we show that Gli2, which encodes a major Gli activator, is required for proliferation of specific groups of pituitary progenitors but not for initial dorsoventral patterning. We further show that the action of Gli2 occurs prior to the closure of Rathke' pouch. Lastly, we show that Shh/Gli2 signaling controls the diencephalic expression of Bone morphogenetic protein 4 (Bmp4) and Fibroblast growth factor 8 (Fgf8), two genes that are known to play critical roles in patterning and growth of Rathke's pouch. Our results therefore suggest both cell-autonomous and non-cell-autonomous requirements for Gli2 in regulation of pituitary progenitor specification, proliferation and differentiation.     

Tail gut endoderm and gut/genitourinary/tail development: a new tissue-specific role for Hoxa13

Hoxa13 is expressed early in the caudal mesoderm and endoderm of the developing hindgut. The tissue-specific roles of Hoxa13 function have not been described. Hand-foot-genital syndrome, a rare dominantly inherited human malformation syndrome characterized by distal extremity and genitourinary anomalies, is caused by mutations in the HOXA13 gene. We show evidence that one specific HOXA13 mutation likely acts as a dominant negative in vivo. When chick HFGa13 is overexpressed in the chick caudal endoderm early in development, caudal structural malformations occur. The phenotype is specific to HFGa13 expression in the posterior endoderm, and includes taillessness and severe gut/genitourinary (GGU) malformations. Finally, we show that chick HFGa13 negatively regulates expression of Hoxd13 and antagonizes functions of both endogenous Hoxa13 and Hoxd13 proteins. We suggest a fundamental role for epithelial specific expression of Hoxa13 in the epithelial-mesenchymal interaction necessary for tail growth and posterior GGU patterning.     

Sonic hedgehog in the pharyngeal endoderm controls arch pattern via regulation of Fgf8 in head ectoderm

Fgf8 signalling is known to play an important role during patterning of the first pharyngeal arch, setting up the oral region of the head and then defining the rostral and proximal domains of the arch. The mechanisms that regulate the restricted expression of Fgf8 in the ectoderm of the developing first arch, however, are not well understood. It has become apparent that pharyngeal endoderm plays an important role in regulating craniofacial morphogenesis. Endoderm ablation in the developing chick embryo results in a loss of Fgf8 expression in presumptive first pharyngeal arch ectoderm. Shh is locally expressed in pharyngeal endoderm, adjacent to the Fgf8-expressing ectoderm, and is thus a candidate signal regulating ectodermal Fgf8 expression. We show that in cultured explants of presumptive first pharyngeal arch, loss of Shh signalling results in loss of Fgf8 expression, both at early stages before formation of the first arch, and during arch formation. Moreover, following removal of the endoderm, Shh protein can replace this tissue and restore Fgf8 expression. Overexpression of Shh in the non-oral ectoderm leads to an expansion of Fgf8, affecting the rostral-caudal axis of the developing first arch, and resulting in the formation of ectopic cartilage. Shh from the pharyngeal endoderm thus regulates Fgf8 in the ectoderm and the role of the endoderm in pharyngeal arch patterning may thus be indirectly mediated by the ectoderm.