Gli2 and Gli3 play distinct roles in the dorsoventral patterning of the mouse hindbrain

Sonic Hedgehog (Shh) signaling plays a critical role during dorsoventral (DV) patterning of the developing neural tube by modulating the expression of neural patterning genes. Overlapping activator functions of Gli2 and Gli3 have been shown to be required for motoneuron development and correct neural patterning in the ventral spinal cord. However, the role of Gli2 and Gli3 in ventral hindbrain development is unclear. In this paper, we have examined DV patterning of the hindbrain of Shh(-/-), Gli2(-/-) and Gli3(-/-) embryos, and found that the respective role of Gli2 and Gli3 is not only different between the hindbrain and spinal cord, but also at distinct rostrocaudal levels of the hindbrain. Remarkably, the anterior hindbrain of Gli2(-/-) embryos displays ventral patterning defects as severe as those observed in Shh(-/-) embryos suggesting that, unlike in the spinal cord and posterior hindbrain, Gli3 cannot compensate for the loss of Gli2 activator function in Shh-dependent ventral patterning of the anterior hindbrain. Loss of Gli3 also results in a distinct patterning defect in the anterior hindbrain, including dorsal expansion of Nkx6.1 expression. Furthermore, we demonstrate that ventral patterning of rhombomere 4 is less affected by loss of Gli2 function revealing a different requirement for Gli proteins in this rhombomere. Taken together, these observations indicate that Gli2 and Gli3 perform rhombomere-specific function during DV patterning of the hindbrain.     

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.     

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.     

Role of the hindbrain in dorsoventral but not anteroposterior axial specification of the inner ear

An early and crucial event in vertebrate inner ear development is the acquisition of axial identities that in turn dictate the positions of all subsequent inner ear components. Here, we focus on the role of the hindbrain in establishment of inner ear axes and show that axial specification occurs well after otic placode formation in chicken. Anteroposterior (AP) rotation of the hindbrain prior to specification of this axis does not affect the normal AP orientation and morphogenesis of the inner ear. By contrast, reversing the dorsoventral (DV) axis of the hindbrain results in changing the DV axial identity of the inner ear. Expression patterns of several ventrally expressed otic genes such as NeuroD, Lunatic fringe (Lfng) and Six1 are shifted dorsally, whereas the expression pattern of a normally dorsal-specific gene, Gbx2, is abolished. Removing the source of Sonic Hedgehog (SHH) by ablating the floor plate and/or notochord, or inhibiting SHH function using an antibody that blocks SHH bioactivity results in loss of ventral inner ear structures. Our results indicate that SHH, together with other signals from the hindbrain, are important for patterning the ventral axis of the inner ear. Taken together, our studies suggest that tissue(s) other than the hindbrain confer AP axial information whereas signals from the hindbrain are necessary and sufficient for the DV axial patterning of the inner ear.     

Identification of sonic hedgehog as a candidate gene responsible for the polydactylous mouse mutant Sasquatch

The mouse mutants of the hemimelia-luxate group (lx, lu, lst, Dh, Xt, and the more recently identified Hx, Xpl and Rim4; [1] [2] [3] [4] [5]) have in common preaxial polydactyly and longbone abnormalities. Associated with the duplication of digits are changes in the regulation of development of the anterior limb bud resulting in ectopic expression of signalling components such as Sonic hedgehog (Shh) and fibroblast growth factor-4 (Fgf4), but little is known about the molecular causes of this misregulation. We generated, by a transgene insertion event, a new member of this group of mutants, Sasquatch (Ssq), which disrupted aspects of both anteroposterior (AP) and dorsoventral (DV) patterning. The mutant displayed preaxial polydactyly in the hindlimbs of heterozygous embryos, and in both hindlimbs and forelimbs of homozygotes. The Shh, Fgf4, Fgf8, Hoxd12 and Hoxd13 genes were all ectopically expressed in the anterior region of affected limb buds. The insertion site was found to lie close to the Shh locus. Furthermore, expression from the transgene reporter has come under the control of a regulatory element that directs a pattern mirroring the endogenous expression pattern of Shh in limbs. In abnormal limbs, both Shh and the reporter were ectopically induced in the anterior region, whereas in normal limbs the reporter and Shh were restricted to the zone of polarising activity (ZPA). These data strongly suggest that Ssq is caused by direct interference with the cis regulation of the Shh gene.     

Sonic hedgehog signaling regulates Gli3 processing, mesenchymal proliferation, and differentiation during mouse lung organogenesis

Lack of Sonic hedgehog (Shh) signaling, mediated by the Gli proteins, leads to severe pulmonary hypoplasia. However, the precise role of Gli genes in lung development is not well established. We show Shh signaling prevents Gli3 proteolysis to generate its repressor forms (Gli3R) in the developing murine lung. In Shh(-/-) or cyclopamine-treated wild-type (WT) lung, we found that Gli3R level is elevated, and this upregulation appears to contribute to defects in proliferation and differentiation observed in the Shh(-/-) mesenchyme, where Gli3 is normally expressed. In agreement, we found Shh(-/-);Gli3(-/-) lungs exhibit enhanced growth potential. Vasculogenesis is also enhanced; in contrast, bronchial myogenesis remains absent in Shh(-/-);Gli3(-/-) compared with Shh(-/-) lungs. Genes upregulated in Shh(-/-);Gli3(-/-) relative to Shh(-/-) lung include Wnt2 and, surprisingly, Foxf1 whose expression has been reported to be Shh-dependent. Cyclins D1, D2, and D3 antibody labelings also reveal distinct expression patterns in the normal and mutant lungs. We found significant repression of Tbx2 and Tbx3, both linked to inhibition of cellular senescence, in Shh(-/-) and partial derepression in Shh(-/-); Gli3(-/-) lungs, while Tbx4 and Tbx5 expressions are less affected in the mutants. Our findings shed light on the role of Shh signaling on Gli3 processing in lung growth and differentiation by regulating several critical genes.     

The BMP signaling gradient patterns dorsoventral tissues in a temporally progressive manner along the anteroposterior axis

Patterning of the vertebrate anteroposterior (AP) axis proceeds temporally from anterior to posterior. How dorsoventral (DV) axial patterning relates to AP temporal patterning is unknown. We examined the temporal activity of BMP signaling in patterning ventrolateral cell fates along the AP axis, using transgenes that rapidly turn "off" or "on" BMP signaling. We show that BMP signaling patterns rostral DV cell fates at the onset of gastrulation, whereas progressively more caudal DV cell fates are patterned at progressively later intervals during gastrulation. Increased BMP signal duration is not required to pattern more caudal DV cell fates; rather, distinct temporal intervals of signaling are required. This progressive action is regulated downstream of, or in parallel to, BMP signal transduction at the level of Smad1/5 phosphorylation. We propose that a temporal cue regulates a cell's competence to respond to BMP signaling, allowing the acquisition of a cell's DV and AP identity simultaneously.