Michael Melnick

Submandibular gland morphogenesis: Stage‐specific expression of TGF‐α/EGF, IGF, TGF‐β, TNF, and IL‐6 signal transduction in normal embryonic mice and the phenotypic effects of TGF‐β2, TGF‐β3, and EGF‐r null mutations

Branching morphogenesis of the mouse submandibular gland (SMG) is dependent on cell‐cell conversations between and within epithelium and mesenchyme. Such conversations are typically mediated in other branching organs (lung, mammary glands, etc.) by hormones, growth factors, cytokines, and the like in such a way as to translate endocrine, autocrine, and paracrine signals into specific gene responses regulating cell division, apoptosis, and histodifferentiation. We report here the protein expression in embryonic SMGs of four signal transduction pathways: TGF‐α/EGF/EGF‐R; IGF‐II/IGF‐IR/IGF‐IIR; TGF‐βs and cognate receptors; TNF, IL‐6, and cognate receptors. Their in vivo spatiotemporal expression is correlated with specific stages of progressive SMG development and particular patterns of cell proliferation, apoptosis, and mucin expression. Functional necessity regarding several of these pathways was assessed in mice with relevant null mutations (TGF‐β2, TGF‐β3, EGF‐R). Among many observations, the following seem of particular importance: (1) TGF‐α and EGF‐R, but not EGF, are found in the Initial and Pseudoglandular Stages of SMG development; (2) ductal and presumptive acini lumena formation was associated with apoptosis and TNF/TNF‐R1 signalling; (3) TGF‐β2 and TGF‐β3 null mice have normal SMG phenotypes, suggesting the presence of other pathways of mitostasis; (4) EGF‐R null mice displayed an abnormal SMG phenotype consisting of decreased branching. These and other findings provide insight into the design of future functional studies. Anat Rec 256:252–268, 1999.

Embryonic Submandibular Gland Morphogenesis: Stage-Specific Protein Localization of FGFs, BMPs, Pax6 and Pax9 in Normal Mice and Abnormal SMG Phenotypes in FgfR2-IIIc+/Δ, BMP7–/– and Pax6–/–Mice

Embryonic submandibular salivary gland (SMG) initiation and branching morphogenesis are dependent on cell-cell communications between and within epithelium and mesenchyme. Such communications are typically mediated in other organs (teeth, lung, lacrimal glands) by growth factors in such a way as to translate autocrine, juxtacrine and paracrine signals into specific gene responses regulating cell division and histodifferentiation. Using Wnt1-Cre/R26R transgenic mice, we demonstrate that embryonic SMG mesenchyme is derived exclusively from cranial neural crest. This origin contrasts to that known for tooth mesenchyme, previously shown to be derived from both neural crest and nonneural crest cells. Thus, although both SMGs and teeth are mandibular derivatives, we can expect overlap and differences in the details of their early inductive interactions. In addition, since embryonic SMG branching morphogenesis is analogous to that seen in other branching organs, we also expect similarities of expression regarding those molecules known to be ubiquitous regulators of morphogenesis. In this study, we performed an analysis of the distribution of specific fibroblast growth factors (FGFs), FGF receptors, bone morphogenetic proteins (BMPs) and Pax transcription factors, previously shown to be important for tooth development and/or branching morphogenesis, from the time of initiation of embryonic SMG development until early branching morphogenesis. In addition, we report abnormal SMG phenotypes in FgfR2- IIIc+/Δ, BMP7–/–and Pax6–/– mice. Our results, in comparison with functional studies in other systems, suggest that FGF-2/FGFR-1, FGF-8/FGFR-2(IIIc) and FGF-10/FGFR-2(IIIb) signaling have different paracrine and juxtacrine functions during SMG initial bud formation and branching. Finally, our observations of abnormal SMGs in BMP7–/– and Pax6–/–indicate that both BMP7 and Pax6 play important roles during embryonic SMG branching morphogenesis.

Descriptive Epidemiology of Nonsyndromic Cleft Lip with or without Cleft Palate in Shanghai, China, from 1980 to 1989

OBJECTIVE The purpose of this study was to characterize nonsyndromic cleft lip with or without cleft palate (CL+/- CP) in an Asian population. DESIGN Birth prevalence was assessed in a large birth series in Shanghai, China. A 1:3 sex-age-hospital matched case-control design was used to assess the effects of parental ages and pregnancy history on risk of CL+/- CP. PARTICIPANTS Records of live births from 1980 to 1989 in 22 hospitals in Shanghai, China, were reviewed, comprising 541,504 consecutive births, which is by far the largest such Chinese sample ever investigated. The case-control study included 528 (308 male, 220 female) nonsyndromic CL+/- CP cases and 1,563 (912 male, 651 female) controls. RESULTS From 1980 to 1989, the overall birth prevalence was 1.2 per 1,000 live births with statistically significant seasonal variation (more CL+/- CP births in January to July). The overall male:female ratio was 1.40:1. For males, statistically significant associations were identified with maternal age for the most severe clefts (bilateral overall, and also bilateral CL+CP subgroup). For females, statistically significant association was shown for pregnancy age with birth order (overall and in most subgroups). CONCLUSIONS The birth prevalence of CL+/- CP in this Asian population was similar to published Caucasian rates. The observed seasonal variation would be consistent with possible environmental factors. Significant associations with maternal age, pregnancy age, and birth order warrant additional study of pregnancy history in Asian CL+/- CP.

FGF10/FGFR2b signaling plays essential roles during in vivo embryonic submandibular salivary gland morphogenesis

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.

Sonic Hedgehog Signaling Plays an Essential Role During Embryonic Salivary Gland Epithelial Branching Morphogenesis

Gene targeting studies indicate that sonic hedgehog (Shh) signaling plays an essential role during craniofacial development. Because numerous mandibular derivatives (e.g., teeth, tongue, Meckels 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 (∼E14). 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. Developmental Dynamics 229:722–732, 2004.

Salivary gland branching morphogenesis: a quantitative systems analysis of the Eda/Edar/NFκB paradigm

BackgroundEctodysplasin-A appears to be a critical component of branching morphogenesis. Mutations in mouse Eda or human EDA are associated with absent or hypoplastic sweat glands, sebaceous glands, lacrimal glands, salivary glands (SMGs), mammary glands and/or nipples, and mucous glands of the bronchial, esophageal and colonic mucosa. In this study, we utilized EdaTa(Tabby) mutant mice to investigate how a marked reduction in functional Eda propagates with time through a defined genetic subcircuit and to test the proposition that canonical NFκB signaling is sufficient to account for the differential expression of developmentally regulated genes in the context of Eda polymorphism.ResultsThe quantitative systems analyses do not support the stated hypothesis. For most NFκB-regulated genes, the observed time course of gene expression is nearly unchanged in Tabby (EdaTa) as compared to wildtype mice, as is NFκB itself. Importantly, a subset of genes is dramatically differentially expressed in Tabby (Edar, Fgf8, Shh, Egf, Tgfa, Egfr), strongly suggesting the existence of an alternative Eda-mediated transcriptional pathway pivotal for SMG ontogeny. Experimental and in silico investigations have identified C/EBPα as a promising candidate.ConclusionIn Tabby SMGs, upregulation of the Egf/Tgfα/Egfr pathway appears to mitigate the potentially severe abnormal phenotype predicted by the downregulation of Fgf8 and Shh. Others have suggested that the buffering of the phenotypic outcome that is coincident with variant Eda signaling could be a common mechanism that permits viable and diverse phenotypes, normal and abnormal. Our results support this proposition. Further, if branching epithelia use variations of a canonical developmental program, our results are likely applicable to understanding the phenotypes of other branching organs affected by Eda (EDA) mutation.

The glucocorticoid-glucocorticoid receptor signal transduction pathway, transforming growth factor-beta, and embryonic mouse lung development in vivo.

Lung morphogenesis has been shown to be regulated by glucocorticoids(CORT). Because CORT has been primarily thought to affect fetal lung development, previous studies have focused on the role of CORT receptor(GR)-mediated regulation of fetal lung development. Although endogenous CORT increases during embryonic and fetal stages and exogenous CORT treatmentin vivo and in vitro clearly accelerates embryonic lung development, little is known about the morphoregulatory role of theembryonic CORT-GR signal transduction pathway during lung development. In this study, we characterize the embryonic mouse CORT-GR pathway and demonstrate: stage-specific in situ patterns of GR immunolocalization; similarity in GR relative mobility with progressive (E13→ E17) development; that embryonic GR can be activated to bind a GR response element (GRE); significantly increasing levels of functional GR with increasing lung maturation; and the presence of heat shock protein (hsp) 70 and hsp90 from early (E13) to late (E17) developmental stages. These results support the purported importance of the embryonic CORT-GR signal transduction pathway in progressive lung differentiation. To demonstrate that the embryonic CORT-GR directed pathway plays a role in lung development, early embryonic(E12) lungs were exposed to CORT in utero and surfactant-associated protein A (SP-A) expression was analyzed; CORT treatment up-regulates SP-A mRNA expression and spatiotemporal protein distribution. Finally, to determine whether CORT-GR-directed pulmonary morphogenesis in vivo involves the modulation of growth factors, we studied the effect of CORT on TGF-β gene expression. Northern analysis of TGF-β1, TGF-β2, and TGF-β3 transcript levels in vivo indicates that CORT regulates the rate of lung morpho- and histodifferentiation by down-regulating TGF-β3 gene expression.

Insulin-like growth factor II receptor, transforming growth factor-β, and Cdk4 expression and the developmental epigenetics of mouse palate morphogenesis and dysmorphogenesis

The B10/B10.A congenic mouse pair serves as a model for identifying specific genes related to morphogenesis and dysmorphogenesis of the embryonic palate and other organs. The present report describes our initial investigation of the Fraser‐Juriloff paradigm, which proposes that susceptibility to malformation results from genetically determined differences in normal developmental patterns. Specifically, we evaluated the relationship between Igf2r gene expression, transforming growth factor‐β (TGF‐β) activation, and cdk4 gene expression. By using in situ hybridization, RNase protection assays, indirect immunofluorescence, Western blots, and bioassays, we show 1) the presence of insulin‐like growth factor II (IGF‐II), IGF‐II receptor (IGF‐IIR), IGF‐IR, TGF‐β, plasminogen, plasminogen activators [urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA)], and Cdk4 in developing palates; 2) on embryonic day 14 (E14), which is a critical day for palatal growth, B10.A embryos have 82% greater IGF‐IIR mRNA than B10; 3) on E14, B10.A embryonic palates have a 57% greater level of active TGF‐β2 than B10, although the total TGF‐β2 is nearly identical; and 4) on E14, B10 embryonic palates have a 52% greater level of Cdk4 mRNA than B10.A palates, a measure of cell cycle progression. Because cellular activation of latent TGF‐β appears to require binding to the mannose‐6‐phosphate (M6P) binding site of the IGF‐IIR and is plasmin and plasminogen activator dependent, the positive correlation of IGF‐IIR levels and active TGF‐β2 levels seems to be key. Thus, the strain variation of TGF‐β2/IGF‐IIR‐mediated growth inhibition in late G1 phase would appear to account for the slower growth and development of B10.A palates relative to B10. Elevated corticosteroid (CORT) exposure in E14 B10.A embryos significantly increases TGF‐β levels, 87% of which is TGF‐β2, as well as the levels of active TGF‐β, 64% of which is TGF‐β2. Without exogenous CORT, B10.A embryos do not have clefts; hence, we present an outline of pathogenesis: slower growing B10.A embryos have an up‐regulation of IGF‐IIR, which serves to sequester IGF‐II from the growth‐promoting IGF‐IR and to bind more CORT‐up‐regulated, latent TGF‐β2 for subsequent plasmin‐dependent activation; higher levels of TGF‐β2 signaling down‐regulate Cdk4 and result in greater palatal growth inhibition at a critical stage of palatogenesis and, thus, cleft palate. We present an epigenetic model of information processing related to cell proliferation. The model is a dynamical network that uses continuous logic to learn its rules from changing conditions. Dev. Dyn. 1998;211:11–25.

Bone remodeling in W/Wv mast cell deficient mice

Strong experimental evidence exists for a relationship between mast cells and bone disease, but the role of mast cells in the regulation of bone remodeling is unknown. In order to address this question, mast cell deficient mice (W/Wv) were paired with their mast cell sufficient (+/+) littermates and evaluated for differences in response to an induced cycle of bone remodeling. This was achieved using a tooth egression protocol, in which a synchronous cycle of bone remodeling was induced in the mandibular buccal alveolar periosteum by extraction of the opposing dentition. Quantitative histomorphometric changes during the activation, resorption, reversal, and formation phases of bone remodeling were documented using standard techniques. Most cell deficient mutants exhibited the following defects in response to an induced cycle of bone remodeling: (a) the onset of the remodeling cycle was delayed by a prolonged activation phase, (b) the duration and extent of the active formation phase was decreased, and (c) the amount of new bone matrix synthesized was diminished while mineralization rates were found to be normal. These results suggest that mast cells and their mediators provide a paracrine mechanism which influences the recruitment of osteoclast and osteoblast progenitors and their participation in bone remodeling. Nonetheless, since bone remodeling occurs in mast cell deficient mice, albeit less efficiently, this mechanism is most likely one of several redundant mechanisms that provide for adequate skeletal homeostasis.

Meckel's cartilage differentiation is dependent on hedgehog signaling

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.