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BackgroundAnalyses of Fgf10 and Fgfr2b mutant mice, as well as human studies, suggest that FGF10/FGFR2b signaling may play an essential, nonredundant role during embryonic SMG development. To address this question, we have analyzed the SMG phenotype in Fgf10 and Fgfr2b heterozygous and null mutant mice. In addition, although previous studies suggest that the FGF10/FGFR2b and FGF8/FGFR2c signaling pathways are functionally interrelated, little is known about the functional relationship between these two pathways during SMG development. We have designed in vivo and in vitro experiments to address this question.ResultsWe analyzed Fgf10 and Fgfr2b heterozygous mutant and null mice and demonstrate dose-dependent SMG phenotypic differences. Hypoplastic SMGs are seen in Fgf10 and Fgfr2b heterozygotes whereas SMG aplasia is seen in Fgf10 and Fgfr2b null embryos. Complementary in vitro studies further indicate that FGF10/FGFR2b signaling regulates SMG epithelial branching and cell proliferation. To delineate the functional relationship between the FGF10/FGFR2b and FGF8/FGFR2c pathways, we compared the SMG phenotype in Fgfr2c+/Δ/Fgf10+/- double heterozygous mice to that seen in wildtype, Fgf10+/- (Fgfr2c+/+/Fgf10+/-) and Fgfr2c+/Δ (Fgfr2c+/Δ/Fgf10+/+) single heterozygous mutant littermates and demonstrate genotype-specific SMG phenotypes. In addition, exogenous FGF8 was able to rescue the abnormal SMG phenotype associated with abrogated FGFR2b signaling in vitro and restore branching to normal levels.ConclusionOur data indicates that FGF10/FGFR2b signaling is essential for the SMG epithelial branching and histodifferentiation, but not earliest initial bud formation. The functional presence of other endogenous signaling pathways could not prevent complete death of embryonic SMG cells in Fgf10 and Fgfr2b null mice. Though we were able to rescue the abnormal phenotype associated with reduced in vitro FGF10/FGFR2b signaling with exogenous FGF8 supplementation, our results indicate that the FGF10/FGFR2b and FGF8/FGFR2c are nonredundant signaling pathways essential for in vivo embryonic SMG development. What remains to be determined is the in vivo functional relationship between the FGF10/FGFR2b signal transduction pathway and other key signaling pathways, and how these pathways are integrated during embryonic SMG development to compose the functional epigenome.

The hedgehog (Hh) signaling pathway has been shown to be essential for craniofacial development. Although mandibular arch derivatives are largely absent in Shh null mice, little is known about the role of Hh signaling during Meckel’s cartilage development per se. Mandible development is dependent on the morphogenesis of Meckel’s cartilage, which then serves as a template for subsequent skeletal differentiation. In this study, we examine the biological function of Hh signaling during Meckel’s cartilage development in vivo and in vitro. E13.5 Shh null mice present a small mesenchymal condensation in the region of a presumptive Meckel’s cartilage in the hypoplastic mandibular arch. By E15.5, the Shh mutant exhibits a mere remnant of the mandibular arch, without evidence of Meckel’s cartilage differentiation. Further, wild-type embryonic (E11 or E12) mandibular explants cultured for up to 5 days in the presence of cyclopamine, a steroidal alkaloid that specifically disrupts the Hh signaling pathway, exhibit a stage-dependent inhibition of Meckel’s cartilage chondroblast differentiation to mature chondrocytes. This phenotype can be rescued by exogenous FGF8, a downstream effector of Hh signaling. Taken together, our results indicate that the Hh signaling pathway is critical to Meckel’s cartilage ontogenesis and the rate of chondrogenesis, but not to initial primordium formation. The reliance on Hh signaling is stage dependent.

Objectives-Submandibular vasodilatory responses are impaired in male streptozotocin-diabetic rats. However, the effects of diabetes on submandibular vascular reactivity in female rats have not been examined. The purpose of this study was to determine whether there are gender differences in the effects of diabetes on parasympathetic vasodilatation in the rat submandibular gland.Methods-Diabetes was induced using streptozotocin, and vascular responses (calculated as the % increase in submandibular vascular conductance) to parasympathetic stimulation (1-10 Hz) were measured using laser-Doppler flowmetry. To estimate the relative contributions of nitric oxide (NO), prostacyclin (PGI 2 ) and endothelium-derived hyperpolarizing factor (EDHF), vascular conductance was measured before and after inhibition of cyclooxygenase (COX) and NO synthase (NOS).Results-Frequency dependent increases in blood flow were observed in both male and female rats, but the contribution of EDHF was greater in females than in males. Further, PGI 2 appeared to play a role only in males. Vasodilatory responses were diminished in all diabetic animals, and when compared with their respective controls the degree of impairment was similar in males and females. However, in diabetic males inhibition of COX and NOS had little or no effect, whereas inhibition of NO, but not COX, resulted in a further significant decrease in vascular responses in diabetic females.Conclusions-Parasympathetic vasodilatation in the rat submandibular gland is diminished equally in diabetic males and females. However, in males diabetes predominantly impairs PGI 2 -and NO-dependent vasodilatation, whereas in females the contribution of EDHF-mediated pathways are affected and NO-dependent vasodilatation is preferentially maintained.