ΔNp63 regulates Sfrp1 expression to direct salivary gland branching morphogenesis.

Branching morphogenesis is a complex process shared by many organs including the lungs, kidney, prostate, as well as several exocrine organs including the salivary, mammary and lacrimal glands. This critical developmental program ensures the expansion of an organ's surface area thereby maximizing processes of cellular secretion or absorption. It is guided by reciprocal signaling from the epithelial and mesenchymal cells. While signaling pathways driving salivary gland branching morphogenesis have been relatively well-studied, our understanding of the underlying transcriptional regulatory mechanisms directing this program, is limited. Here, we performed in vivo and ex vivo studies of the embryonic mouse submandibular gland to determine the function of the transcription factor ΔNp63, in directing branching morphogenesis. Our studies show that loss of ΔNp63 results in alterations in the differentiation program of the ductal cells which is accompanied by a dramatic reduction in branching morphogenesis that is mediated by dysregulation of WNT signaling. We show that ΔNp63 modulates WNT signaling to promote branching morphogenesis by directly regulating Sfrp1 expression. Collectively, our findings have revealed a novel role for ΔNp63 in the regulation of this critical process and offers a better understanding of the transcriptional networks involved in branching morphogenesis.

Salivary Gland Tissue Recombination Can Modify Cell Fate.

Although mesenchyme is essential for inducing the epithelium of ectodermal organs, its precise role in organ-specific epithelial fate determination remains poorly understood. To elucidate the roles of tissue interactions in cellular differentiation, we performed single-cell RNA sequencing and imaging analyses on recombined tissues, where mesenchyme and epithelium were switched ex vivo between two types of embryonic mouse salivary glands: the parotid gland (a serous gland) and the submandibular gland (a predominantly mucous gland). We found partial induction of molecules that define gland-specific acinar and myoepithelial cells in recombined salivary epithelium. The parotid epithelium recombined with submandibular mesenchyme began to express mucous acinar genes not intrinsic to the parotid gland. While myoepithelial cells do not normally line parotid acini, newly induced myoepithelial cells densely populated recombined parotid acini. However, mucous acinar and myoepithelial markers continued to be expressed in submandibular epithelial cells recombined with parotid mesenchyme. Consequently, some epithelial cells appeared to be plastic, such that their fate could still be modified in response to mesenchymal signaling, whereas other epithelial cells appeared to be already committed to a specific fate. We also discovered evidence for bidirectional induction: transcriptional changes were observed not only in the epithelium but also in the mesenchyme after heterotypic tissue recombination. For example, parotid epithelium induced the expression of muscle-related genes in submandibular fibroblasts that began to mimic parotid fibroblast gene expression. These studies provide the first comprehensive unbiased molecular characterization of tissue recombination approaches exploring the regulation of cell fate.

Adamts18 Deficiency Causes Spontaneous SMG Fibrogenesis in Adult Mice.

Chronic sclerosing sialadenitis of the submandibular gland (also known as Küttner tumor) is characterized by concomitant swelling of the submandibular glands secondary to strong lymphocytic infiltration and fibrosis. The pathogenesis of this disease has been unclear, but it is associated with immune disorders. ADAMTS18 is a member of the ADAMTS superfamily of extracellular proteinases. In this study, we showed that Adamts18 is highly expressed in submandibular salivary gland (SMG) during embryonic development and decreases but is retained in adult SMG tissue in mice. Adamts18 deficiency led to reduced cleft formation and epithelial branching in embryonic SMG before embryonic day 15.5 in mice. No significant histologic changes in the later stages of branching or the morphology of SMG were detected in Adamts18-/- mice. However, Adamts18 deficiency causes spontaneous SMG fibrogenesis and fibrosis in adult mice. At 8 wk of age, Adamts18-/- mice began to manifest the first signs of pathologic changes of mild fibrosis and CD11b+ cell infiltration in SMG tissues. At ≥8 mo, all male and female Adamts18-/- mice developed unilateral or bilateral SMG scleroma that is similar to patients with chronic sclerosing sialadenitis of the submandibular gland. Adamts18-/- mice also showed secretory dysfunction and severe dental caries. Histologically, SMG scleroma is characterized by progressive periductal fibrosis, acinar atrophy, irregular duct ectasis, and dense infiltration of IgG-positive plasma cells. A significant infiltration of CD4+ T lymphocytes and CD11b+ monocytes and macrophages was also detected in the SMG scleroma of Adamts18-/- mice. The levels of TGF-ß1, IL-6, and IL-33 were significantly increased in Adamts18-/- SMGs, which induces chronic inflammation and myofibroblast activation, ultimately leading to fibrosis. This study indicates that Adamts18 regulates the early branching morphogenesis of embryonic SMG and plays a role in protecting from spontaneous SMG fibrogenesis via modulating local inflammation, autoimmune reaction, and myofibroblast activation in adult mice.

Canonical Wnt signaling regulates branching morphogenesis of submandibular gland by modulating levels of lama5.

Branching morphogenesis is a crucial developmental mechanism for the formation of the typical bush-like structure of the submandibular gland (SMG). However, the detailed mechanism underlying this process remains to be fully understood. Here, we have investigated whether cross-talk may exist between the Wnt/beta-catenin signaling pathway and lama5 during the branching process in SMG development. An embryonic mouse SMG organ culture model was established, and the validity of this model was confirmed. The roles and possible interactions of the Wnt/beta-catenin signaling pathway, FGF signaling, and lama5 in the branching process were investigated by morphogenesis assays and gene expression patterns. Here, we show that the E12 or E13 SMG organ culture model can be used as an ideal approach to study the process of branching morphogenesis. Our branching morphogenesis assay revealed that the epithelial branching process can be promoted when the canonical Wnt pathway is inhibited and significantly suppressed when the wnt pathway is over activated. Further experiments indicated that FGF signaling most likely acts upstream as a negative regulator of the canonical Wnt pathway during the branching process, whose effect could be partially reversed by Wnt3a. Finally, we show that Wnt/beta-catenin signaling regulates branching morphogenesis through Lama5. We conclude that the Wnt/beta-catenin signaling pathway acting downstream of FGF signaling can serve as a negative regulatory mechanism in the process of SMG branching morphogenesis through Lama5.

Comparing development and regeneration in the submandibular gland highlights distinct mechanisms.

A common question in organ regeneration is the extent to which regeneration recapitulates embryonic development. To investigate this concept, we compared the expression of two highly interlinked and essential genes for salivary gland development, Sox9 and Fgf10, during submandibular gland development, homeostasis and regeneration. Salivary gland duct ligation/deligation model was used as a regenerative model. Fgf10 and Sox9 expression changed during regeneration compared to homeostasis, suggesting that these key developmental genes play important roles during regeneration, however, significantly both displayed different patterns of expression in the regenerating gland compared to the developing gland. Regenerating glands, which during homeostasis had very few weakly expressing Sox9-positive cells in the striated/granular ducts, displayed elevated expression of Sox9 within these ducts. This pattern is in contrast to embryonic development, where Sox9 expression was absent in the proximally developing ducts. However, similar to the elevated expression at the distal tip of the epithelium in developing salivary glands, regenerating glands displayed elevated expression in a subpopulation of acinar cells, which during homeostasis expressed Sox9 at lower levels. A shift in expression of Fgf10 was observed from a widespread mesenchymal pattern during organogenesis to a more limited and predominantly epithelial pattern during homeostasis in the adult. This restricted expression in epithelial cells was maintained during regeneration, with no clear upregulation in the surrounding mesenchyme, as might be expected if regeneration recapitulated development. As both Fgf10 and Sox9 were upregulated in proximal ducts during regeneration, this suggests that the positive regulation of Sox9 by Fgf10, essential during development, is partially reawakened during regeneration using this model. Together these data suggest that developmentally important genes play a key role in salivary gland regeneration but do not precisely mimic the roles observed during development.

Specification of the patterning of a ductal tree during branching morphogenesis of the submandibular gland.

The development of ductal structures during branching morphogenesis relies on signals that specify ductal progenitors to set up a pattern for the ductal network. Here, we identify cellular asymmetries defined by the F-actin cytoskeleton and the cell adhesion protein ZO-1 as the earliest determinants of duct specification in the embryonic submandibular gland (SMG). Apical polarity protein aPKCζ is then recruited to the sites of asymmetry in a ZO-1-dependent manner and collaborates with ROCK signaling to set up apical-basal polarity of ductal progenitors and further define the path of duct specification. Moreover, the motor protein myosin IIB, a mediator of mechanical force transmission along actin filaments, becomes localized to vertices linking the apical domains of multiple ductal epithelial cells during the formation of ductal lumens and drives duct maturation. These studies identify cytoskeletal, junctional and polarity proteins as the early determinants of duct specification and the patterning of a ductal tree during branching morphogenesis of the SMG.

CXCR4 Regulates Temporal Differentiation via PRC1 Complex in Organogenesis of Epithelial Glands.

CXC-chemokine receptor type 4 (CXCR4), a 7-transmembrane receptor family member, displays multifaceted roles, participating in immune cell migration, angiogenesis, and even adipocyte metabolism. However, the activity of such a ubiquitously expressed receptor in epithelial gland organogenesis has not yet been fully explored. To investigate the relationship between CXCL12/CXCR4 signaling and embryonic glandular organogenesis, we used an ex vivo culture system with live imaging and RNA sequencing to elucidate the transcriptome and protein-level signatures of AMD3100, a potent abrogating reagent of the CXCR4-CXCL12 axis, imprinted on the developing organs. Immunostaining results showed that CXCR4 was highly expressed in embryonic submandibular gland, lung, and pancreas, especially at the periphery of end buds containing numerous embryonic stem/progenitor cells. Despite no significant increase in apoptosis, AMD3100-treated epithelial organs showed a retarded growth with significantly slower branching and expansion. Further analyses with submandibular glands revealed that such responses resulted from the AMD3100-induced precocious differentiation of embryonic epithelial cells, losing mitotic activity. RNA sequencing analysis revealed that inhibition of CXCR4 significantly down-regulated polycomb repressive complex (PRC) components, known as regulators of DNA methylation. Treatment with PRC inhibitor recapitulated the AMD3100-induced precocious differentiation. Our results indicate that the epigenetic modulation by the PRC-CXCR12/CXCR4 signaling axis is crucial for the spatiotemporal regulation of proliferation and differentiation of embryonic epithelial cells during embryonic glandular organogenesis.

Morphological differences between regenerating salivary glands after salivary gland duct ligation and embryonic salivary glands.

BACKGROUND: Most animals and organs have regenerative capabilities. Whether regeneration is a developmental process or a distinct phenomenon that is independent of development is debatable. METHOD: We examined the differences between developing and regenerating salivary glands using duct-ligation models. We performed morphological analyses comparing submandibular gland regeneration and development. To reveal the proliferation processes that occur during salivary gland regeneration and development, we counted the number of Ki67-positive cells over time. In addition, we examined the expression of the following markers: aquaporin 5, smooth muscle actin, cytokeratin 7, and tubulin beta 3. RESULT: The proliferation patterns seen during regeneration differed from those observed during development. Different salivary gland marker expression patterns were seen during development and regeneration. CONCLUSION: This study showed that regenerating salivary glands do not follow the same growth process as developing salivary glands.

Dual Sympathetic Input into Developing Salivary Glands.

Neuronal signaling is known to be required for salivary gland development, with parasympathetic nerves interacting with the surrounding tissues from early stages to maintain a progenitor cell population and control morphogenesis. In contrast, postganglionic sympathetic nerves arrive late in salivary gland development to perform a secretory function; however, no previous report has shown their role during development. Here, we show that a subset of neuronal cells within the parasympathetic submandibular ganglion (PSG) express the catecholaminergic marker tyrosine hydroxylase (TH) in developing murine and human submandibular glands. This sympathetic phenotype coincided with the expression of transcription factor Hand2 within the PSG from the bud stage (E12.5) of mouse embryonic salivary gland development. Hand2 was previously associated with the decision of neural crest cells to become sympathetic in other systems, suggesting a role in controlling neuronal fate in the salivary gland. The PSG therefore provides a population of TH-expressing neurons prior to the arrival of the postganglionic sympathetic axons from the superior cervical ganglion at E15.5. In culture, in the absence of nerves from the superior cervical ganglion, these PSG-derived TH neurons were clearly evident forming a network around the gland. Chemical ablation of dopamine receptors in explant culture with the neurotoxin 6-hydroxydopamine at early stages of gland development resulted in specific loss of the TH-positive neurons from the PSG, and subsequent branching was inhibited. Taken altogether, these results highlight for the first time the detailed developmental time course of TH-expressing neurons during murine salivary gland development and suggest a role for these neurons in branching morphogenesis.

Initial stages of development of the submandibular gland (human embryos at 5.5-8 weeks of development).

The aim of this study was to determine the main stages of submandibular salivary gland development during the embryonic period in humans. In addition, we studied submandibular salivary gland development in rats on embryonic days 14-16 and expression in the submandibular salivary gland region with the monoclonal antibody HNK-1. Serial sections from 25 human embryos with a greatest length ranging from 10 to 31 mm (Carnegie stages 16-23; weeks 5.5-8 of development) and Wistar rats of embryonic days (E) 14-16 were analysed with light microscopy. Five stages of submandibular salivary gland development were identified. The prospective stage (1), between weeks 5.5 and early week 6, is characterized by a thickening of the epithelium of the medial paralingual groove in the floor of the mouth corresponding to the primordium of the submandibular salivary gland parenchyma. At this stage, the primordium of the parasympathetic ganglion lies below the lingual nerve. The primordium of the submandibular salivary gland parenchyma is observed in rats on E14 in the medial paralingual groove with mesenchymal cells, underlying the lingual nerve. These cells are HNK-1-positive, corresponding to the primordium of the parasympathetic ganglion. The bud stage (2), at the end of week 6 in humans and on E15 in rats, is characterized by the proliferation and invagination of the epithelial condensation, surrounded by an important condensation of the mesenchyme. The pseudoglandular stage (3) at week 6.5 is characterized by the beginning of the formation of lobes in the condensed mesenchyme. The canalicular stage (4), between week 7 and 7.5, is characterized by the appearance of a lumen in the proximal part of the submandibular duct. The innervation stage (5) occurs during week 8, with the innervation of the submandibular and interlobular ducts. Nervous branches arriving from the parasympathetic ganglion innervate the glandular parenchyma. Numerous blood vessels are observed nearby. Our results suggest that submandibular salivary gland development requires interactions among epithelium, mesenchyme, parasympathetic ganglion and blood vessels.

Grhl2 regulation of SPINT1 expression controls salivary gland development.

Development of the salivary gland is characterized by extensive branching morphogenesis and lumen formation, the latter of which is closely associated with differentiation into acinar and ductal cells. Although various molecules, including signaling and cell adhesion molecules, have been implicated in salivary gland development, transcription factors (TFs) regulating the expression of those molecules and morphological development of the gland are largely unknown. Here we show that knockdown of the epithelial TF, Grainyhead-like 2 (Grhl2), with siRNA in developing mouse submandibular salivary gland (SMG) cultured ex vivo resulted in retardation of epithelial development. This retardation was concomitant with suppression of gene expression for the cell adhesion molecules, such as E-cadherin and the extracellular protease inhibitor SPINT1, and with the disorganized deposition of the basal lamina protein laminin. ChIP-PCR demonstrated the binding of Grhl2 protein to the Spint1 gene in the SMG. Notably, addition of recombinant SPINT1 protein in cultured SMG overcame the suppressive effects of Grhl2 siRNA on epithelial development and laminin deposition. These findings show that Grhl2 regulation of SPINT1 expression controls salivary gland development.

RDH10-mediated retinol metabolism and RARα-mediated retinoic acid signaling are required for submandibular salivary gland initiation.

In mammals, the epithelial tissues of major salivary glands generate saliva and drain it into the oral cavity. For submandibular salivary glands (SMGs), the epithelial tissues arise during embryogenesis from naïve oral ectoderm adjacent to the base of the tongue, which begins to thicken, express SOX9 and invaginate into underlying mesenchyme. The developmental mechanisms initiating salivary gland development remain unexplored. In this study, we show that retinoic acid (RA) signaling activity at the site of gland initiation is colocalized with expression of retinol metabolic genes Rdh10 and Aldh1a2 in the underlying SMG mesenchyme. Utilizing a novel ex vivo assay for SMG initiation developed for this study, we show that RDH10 and RA are required for salivary gland initiation. Moreover, we show that the requirement for RA in gland initiation involves canonical signaling through retinoic acid receptors (RAR). Finally, we show that RA signaling essential for gland initiation is transduced specifically through RARα, with no contribution from other RAR isoforms. This is the first study to identify a molecular signal regulating mammalian salivary gland initiation.

A novel role for cilia-dependent sonic hedgehog signaling during submandibular gland development.

BACKGROUND: Submandibular glands (SMGs) are specialized epithelial structures which generate saliva necessary for mastication and digestion. Loss of SMGs can lead to inflammation, oral lesions, fungal infections, problems with chewing/swallowing, and tooth decay. Understanding the development of the SMG is important for developing therapeutic options for patients with impaired SMG function. Recent studies have suggested Sonic hedgehog (Shh) signaling in the epithelium plays an integral role in SMG development; however, the mechanism by which Shh influences gland development remains nebulous. RESULTS: Using the Kif3af/f ;Wnt1-Cre ciliopathic mouse model to prevent Shh signal transduction by means of the loss of primary cilia in neural crest cells, we report that mesenchymal Shh activity is necessary for gland development. Furthermore, using a variety of murine transgenic lines with aberrant mesenchymal Shh signal transduction, we determine that loss of Shh activity, by means of loss of the Gli activator, rather than gain of Gli repressor, is sufficient to cause the SMG aplasia. Finally, we determine that loss of the SMG correlates with reduced Neuregulin1 (Nrg1) expression and lack of innervation of the SMG epithelium. CONCLUSIONS: Together, these data suggest a novel mechanistic role for mesenchymal Shh signaling during SMG development. Developmental Dynamics 247:818-831, 2018. © 2018 Wiley Periodicals, Inc.

TGF-ß1 attenuated branching morphogenesis of embryonic murine submandibular gland through Smad3 activation.

Transforming growth factor-ß1 (TGF-ß1) plays several crucial regulatory roles in multiple physiological and pathological processes. The aim of this work was to investigate the role of TGF-ß1 in branching morphogenesis of salivary gland. We harvested and cultured submandibular salivary glands (SMGs) from murine embryos, which were then treated with exogenous TGF-ß1, or its neutralized antibody, Smad3 inhibitor, or Smad3 small interfering RNA (siRNA). Our results suggested that TGF-ß1 attenuated branching morphogenesis of embryonic murine SMG via Smad3 activation, thus playing a negative regulatory role in salivary gland development.

The Hippo pathway effector YAP is an essential regulator of ductal progenitor patterning in the mouse submandibular gland.

Salivary glands, such as submandibular glands (SMGs), are composed of branched epithelial ductal networks that terminate in acini that together produce, transport and secrete saliva. Here, we show that the transcriptional regulator Yap, a key effector of the Hippo pathway, is required for the proper patterning and morphogenesis of SMG epithelium. Epithelial deletion of Yap in developing SMGs results in the loss of ductal structures, arising from reduced expression of the EGF family member Epiregulin, which we show is required for the expansion of Krt5/Krt14-positive ductal progenitors. We further show that epithelial deletion of the Lats1 and Lats2 genes, which encode kinases that restrict nuclear Yap localization, results in morphogenesis defects accompanied by an expansion of Krt5/Krt14-positive cells. Collectively, our data indicate that Yap-induced Epiregulin signaling promotes the identity of SMG ductal progenitors and that removal of nuclear Yap by Lats1/2-mediated signaling is critical for proper ductal maturation.

Fgf10 and Sox9 are essential for the establishment of distal progenitor cells during mouse salivary gland development.

Salivary glands are formed by branching morphogenesis with epithelial progenitors forming a network of ducts and acini (secretory cells). During this process, epithelial progenitors specialise into distal (tips of the gland) and proximal (the stalk region) identities that produce the acini and higher order ducts, respectively. Little is known about the factors that regulate progenitor expansion and specialisation in the different parts of the gland. Here, we show that Sox9 is involved in establishing the identity of the distal compartment before the initiation of branching morphogenesis. Sox9 is expressed throughout the gland at the initiation stage before becoming restricted to the distal epithelium from the bud stage and throughout branching morphogenesis. Deletion of Sox9 in the epithelium results in loss of the distal epithelial progenitors, a reduction in proliferation and a subsequent failure in branching. We demonstrate that Sox9 is positively regulated by mesenchymal Fgf10, a process that requires active Erk signalling. These results provide new insights into the factors required for the expansion of salivary gland epithelial progenitors, which can be useful for organ regeneration therapy.

Btbd7 is essential for region-specific epithelial cell dynamics and branching morphogenesis in vivo.

Branching morphogenesis of developing organs requires coordinated but poorly understood changes in epithelial cell-cell adhesion and cell motility. We report that Btbd7 is a crucial regulator of branching morphogenesis in vivo. Btbd7 levels are elevated in peripheral cells of branching epithelial end buds, where it enhances cell motility and cell-cell adhesion dynamics. Genetic ablation of Btbd7 in mice disrupts branching morphogenesis of salivary gland, lung and kidney. Btbd7 knockout results in more tightly packed outer bud cells, which display stronger E-cadherin localization, reduced cell motility and decreased dynamics of transient cell separations associated with cleft formation; inner bud cells remain unaffected. Mechanistic analyses using in vitro MDCK cells to mimic outer bud cell behavior establish that Btbd7 promotes loss of E-cadherin from cell-cell adhesions with enhanced migration and transient cell separation. Btbd7 can enhance E-cadherin ubiquitination, internalization, and degradation in MDCK and peripheral bud cells for regulating cell dynamics. These studies show how a specific regulatory molecule, Btbd7, can function at a local region of developing organs to regulate dynamics of cell adhesion and motility during epithelial branching morphogenesis.

Salivary glands and human congenital cytomegalovirus infection: What happens in early fetal life?

Salivary glands are a site of human cytomegalovirus (CMV) replication, latency, and persistence. Prolonged secretion of virus in saliva for months following a primary infection contribute to horizontal transmission. In order to better understand the early effects of CMV on salivary glands and the mechanisms of viral persistent replication, submandibular glands of six CMV congenitally infected fetuses at 21 weeks gestation were studied. Three fetuses at the same gestational age from CMV-seronegative women were compared as negative controls. Tissue viral load and the type of inflammatory infiltrate were evaluated. Moreover, development and branching of salivary glands, the number of myoepithelial cells, cellular proliferation, and expression of secretory proteins of the saliva (Gross Cystic Disease Fluid Protein-15 and lysozyme) were studied. A low viral load and rare CMV-positive cells associated with T CD8 cytotoxic lymphocytes were observed. Branching was impaired with a decrease in terminal acinar structures, the number of myoepithelial cells, and cellular proliferation were reduced. In addition, a compromised secretion of defense proteins involved in the oral humoral immunity was observed. These findings suggest that CMV may affect salivary glands, impairing structure development and secretion of defense proteins, probably responsible for the prolonged viral shedding in saliva. J. Med. Virol. 89:318-323, 2017. © 2016 Wiley Periodicals, Inc.

The WNT/MYB pathway suppresses KIT expression to control the timing of salivary proacinar differentiation and duct formation.

Growth factor signaling is involved in the development of various organs, but how signaling regulates organ morphogenesis and differentiation in a coordinated manner remains to be clarified. Here, we show how WNT signaling controls epithelial morphogenetic changes and differentiation using the salivary gland as a model. Experiments using genetically manipulated mice and organ cultures revealed that WNT signaling at an early stage (E12-E15) of submandibular salivary gland (SMG) development inhibits end bud morphogenesis and differentiation into proacini by suppressing Kit expression through the upregulation of the transcription factor MYB, and concomitantly increasing the expression of distal progenitor markers. In addition, WNT signaling at the early stage of SMG development promoted end bud cell proliferation, leading to duct formation. WNT signaling reduction at a late stage (E16-E18) of SMG development promoted end bud maturation and suppressed duct formation. Thus, WNT signaling controls the timing of SMG organogenesis by keeping end bud cells in an undifferentiated bipotent state.

Shh/Ptch and EGF/ErbB cooperatively regulate branching morphogenesis of fetal mouse submandibular glands.

The hedgehog family includes Sonic hedgehog (Shh), Desert hedgehog, and Indian hedgehog, which are well known as a morphogens that play many important roles during development of numerous organs such as the tongue, pancreas, kidney, cartilage, teeth and salivary glands (SMG). In Shh null mice, abnormal development of the salivary gland is seen after embryonic day 14 (E14). Shh also induced lobule formation and lumen formation in acini-like structures in cultured E14 SMG. In this study, we investigated the relationship between Shh and epidermal growth factor (EGF)/ErbB signaling in developing fetal mouse SMG. Administration of Shh to cultured E13 SMG stimulated branching morphogenesis (BrM) and induced synthesis of mRNAs for EGF ligands and receptors of the ErbB family. Shh also stimulated activation of ErbB signaling system such as ERK1/2. AG1478, a specific inhibitor of ErbB receptors, completely suppressed BrM and activation of EGF/ErbB/ERK1/2 cascade in E13 SMGs cultured with Shh. The expressions of mRNA for Egf in mesenchyme and mRNA for Erbb1, Erbb2 and Erbb3 in epithelium of E13 SMG were specifically induced by administration of Shh. These results show that Shh stimulates BrM of fetal mouse SMG, at least in part, through activation of the EGF/ErbB/ERK1/2 signaling system.