Sonic hedgehog signaling plays an essential role during embryonic salivary gland epithelial branching morphogenesis

Abstract

Gene targeting studies indicate that sonic hedgehog (Shh) signaling plays an essential role during craniofacial development. Because numerous mandibular derivatives (e.g., teeth, tongue, Meckel's cartilage) are absent in Shh null mice and the embryonic submandibular salivary gland (SMG) develops from the mandibular arch, we postulated that Shh signaling is important for embryonic SMG development. To address this question, we first determined the spatiotemporal distribution of Shh; two transmembrane proteins, patched 1 (Ptc) and Smoothened (Smo), which act as a negative or a positive regulator of the Shh signal, respectively; and the Gli 3 transcription factor, which is downstream of the Shh signal. The epithelial localization of Shh, Ptc, Smo, and Gli 3 suggests that Shh signaling may act within the epithelium in a juxtacrine manner. The SMG phenotype in our embryonic day (E) 18.5 Shh null mice can be characterized as "paedomorphic," that is, it fails to progress to ontogenic stages beyond the Early Pseudoglandular (ϳE14). In a complementary set of experiments, we used organ culture to evaluate the effect of enhanced or abrogated Shh signaling on embryonic SMG development in vitro. Paired E13 (Late Initial Bud stage) or E14 (Pseudoglandular stage) SMGs were cultured in the presence or absence of exogenous Shh peptide supplementation; Shh-supplemented explants exhibit a significant stage-dependent increase in branching morphogenesis compared with control explants. Furthermore, by using cyclopamine, a steroidal alkaloid that specifically disrupts the Shh pathway, to abrogate endogenous Shh signaling in vitro, we found a significant decrease in branching in cyclopamine-treated explants compared with controls, as well as a significant decrease in epithelial cell proliferation. Our results indicate that Shh signaling plays an essential role during embryonic SMG branching morphogenesis. Exogenous FGF8 peptide supplementation in vitro rescues the abnormal SMG phenotype seen in cyclopamine-treated explants, demonstrating that overexpression of a parallel, but related, downstream signaling pathway can compensate for diminished Shh signaling and restore embryonic SMG branching morphogenesis.

Recombinant EDA or Sonic Hedgehog rescue the branching defect in Ectodysplasin A pathway mutant salivary glands in vitro

“…Shh signaling is essential for SMG development; the SMGs of Shh À/À embryos do not develop beyond the onset of branching morphogenesis, and cyclopamine treatment of WT SMG explants results in less branching (Jaskoll et al, 2004a). Here, we show that exogenous Shh is sufficient to rescue the branching phenotype in Edar dlJ/dlJ and can do so in the same time scale as EDA A1.…”

Hedgehog-mediated mesenchymal–epithelial interactions modulate hepatic response to bile duct ligation

“…Antibody neutralization studies indicated that the myofibroblastic mesenchymal cells produced Hh ligands that enhanced the viability and proliferation of bile ductular epithelial cells, and vice versa (ie, that cholangiocyte-derived Hh ligands promoted growth of the myofibroblastic cells). To our knowledge, this is the first evidence that the Hh pathway, which is well accepted to regulate morphogenesis of various tissues during fetal life, [15][16][17][18][19][20][21][22] plays a role in adult liver repair. This discovery opens a novel area for future research that might help to clarify the pathogenesis of chronic cholangiopathies and lead to novel therapies to improve recovery from bile duct injury.…”

“…Two of the major cell types that accumulate during cholestatic liver damage, myofibroblastic cells and immature cholangiocytes (ie, bile ductular cells), 1 The possibility that Hh signaling might orchestrate injury responses in adult livers was somewhat unanticipated given present understanding of the Hh pathway. This signaling system is highly conserved across species, and regulates multiple, seemingly disparate, aspects of embryogenesis, including development of the nervous system, 22 heart, 19 thyroid, 20 lung, 16,53 proximal gastrointestinal tract, 17,18,21 and skeleton. 15 Although one study involving embryo explants suggested that Hh activity may also be required for ultimate hepatic specification of primitive cells in the ventral endoderm, 11 the lack of an obvious hepatic phenotype in mice with targeted disruption of various Hh pathway components has cast doubt about the importance of Hh signaling for fetal liver development.…”

Comparative Mechanisms of Branching Morphogenesis in Diverse Systems

“…Moreover, inhibition of Shh signaling by cyclopamine, a chemical inhibitor of this pathway, reduces branching in salivary gland organ cultures. These results suggest that HH signaling is required for salivary gland branching [78]. Surprisingly, Smoothened, a positive transducer of HH signaling, Gli3, an HHresponsive transcription activator, and Ptc-1 are expressed in the epithelium.…”

“…Surprisingly, Smoothened, a positive transducer of HH signaling, Gli3, an HHresponsive transcription activator, and Ptc-1 are expressed in the epithelium. In this context, therefore, Shh may function in an autocrine manner within the salivary epithelium compartment [78].…”