Regulation of prostate branching morphogenesis by activin A and follistatin

Ventral prostate development occurs by branching morphogenesis and is an androgen-dependent process modulated by growth factors. Many growth factors have been implicated in branching morphogenesis including activins (dimers of beta(A) and beta(B) subunits); activin A inhibited branching of lung and kidney in vitro. Our aim was to examine the role of activins on prostatic development in vitro and their localization in vivo. Organ culture of day 0 rat ventral prostates for 6 days with activin A (+/- testosterone) inhibited prostatic branching and growth without increasing apoptosis. The activin-binding protein follistatin increased branching in vitro in the absence (but not presence) of testosterone, suggesting endogenous activins may reduce prostatic branching morphogenesis. In vivo, inhibin alpha subunit was not expressed until puberty, therefore inhibins (dimers of alpha and beta subunits) are not involved in prostatic development. Activin beta(A) was immunolocalized to developing prostatic epithelium and mesenchymal aggregates at ductal tips. Activin beta(B) immunoreactivity was weak during development, but was upregulated in prostatic epithelium during puberty. Activin receptors were expressed throughout the prostatic epithelium. Follistatin mRNA and protein were expressed throughout the prostatic epithelium. The in vitro evidence that activin and follistatin have opposing effects on ductal branching suggests a role for activin as a negative regulator of prostatic ductal branching morphogenesis.     

The activin-follistatin system in the neonatal ovine uterus

Uterine gland development or adenogenesis in the neonatal ovine uterus involves budding and tubulogenesis followed by coiling and branching morphogenesis of the glandular epithelium (GE) from the luminal epithelium (LE) between birth (Postnatal Day [PND] 0) and PND 56. Activins, which are members of the transforming growth factor beta superfamily, and follistatin, an inhibitor of activins, regulate epithelial branching morphogenesis in other organs. The objective of the present study was to determine effects of postnatal age on expression of follistatin, inhibin alpha subunit, betaA subunit, betaB subunit, activin receptor (ActR) type IA, ActRIB, and ActRII in the developing ovine uterus. Ewes were ovariohysterectomized on PND 0, 7, 14, 21, 28, 35, 42, 49, or 56. The uterus was analyzed by in situ hybridization and immunohistochemistry. Neither inhibin alpha subunit mRNA or protein was detected in the neonatal uterus. Expression of betaA and betaB subunits was detected predominantly in the endometrial LE and GE and myometrium between PND 0 and PND 56. In all uterine cell types, ActRIA, ActRIB, and ActRII were expressed, with the highest levels observed in the endometrial LE and GE and myometrium. Between PND 0 and PND 14, follistatin was detected in all uterine cell types. However, between PND 21 and PND 56, follistatin was only detected in the stroma and myometrium and not in the developing GE. Collectively, the present results indicate that components of the activin-follistatin system are expressed in the developing neonatal ovine uterus and are potential regulators of endometrial gland morphogenesis.     

The role of GDNF in patterning the excretory system

Mesenchymal-epithelial interactions are an important source of information for pattern formation during organogenesis. In the developing excretory system, one of the secreted mesenchymal factors thought to play a critical role in patterning the growth and branching of the epithelial ureteric bud is GDNF. We have tested the requirement for GDNF as a paracrine chemoattractive factor by altering its site of expression during excretory system development. Normally, GDNF is secreted by the metanephric mesenchyme and acts via receptors on the Wolffian duct and ureteric bud epithelium. Misexpression of GDNF in the Wolffian duct and ureteric buds resulted in formation of multiple, ectopic buds, which branched independently of the metanephric mesenchyme. This confirmed the ability of GDNF to induce ureter outgrowth and epithelial branching in vivo. However, in mutant mice lacking endogenous GDNF, kidney development was rescued to a substantial degree by GDNF supplied only by the Wolffian duct and ureteric bud. These results indicate that mesenchymal GDNF is not required as a chemoattractive factor to pattern the growth of the ureteric bud within the developing kidney, and that any positional information provided by the mesenchymal expression of GDNF may provide for renal branching morphogenesis is redundant with other signals.     

Extracellular matrix and cytoskeletal dynamics during branching morphogenesis

Branching morphogenesis is a fundamental developmental process which results in amplification of epithelial surface area for exchanging molecules in organs including the lung, kidney, mammary gland and salivary gland. These complex tree-like structures are built by iterative rounds of simple routines of epithelial morphogenesis, including bud formation, extension, and bifurcation, that require constant remodeling of the extracellular matrix (ECM) and the cytoskeleton. In this review, we highlight the current understanding of the role of the ECM and cytoskeletal dynamics in branching morphogenesis across these different organs. The cellular and molecular mechanisms shared during this morphogenetic process provide insight into the development of other branching organs.     

Extracellular matrix and cytoskeletal dynamics during branching morphogenesis

Branching morphogenesis is a fundamental developmental process which results in amplification of epithelial surface area for exchanging molecules in organs including the lung, kidney, mammary gland and salivary gland. These complex tree-like structures are built by iterative rounds of simple routines of epithelial morphogenesis, including bud formation, extension, and bifurcation, that require constant remodeling of the extracellular matrix (ECM) and the cytoskeleton. In this review, we highlight the current understanding of the role of the ECM and cytoskeletal dynamics in branching morphogenesis across these different organs. The cellular and molecular mechanisms shared during this morphogenetic process provide insight into the development of other branching organs.     

Patterning parameters associated with the branching of the ureteric bud regulated by epithelial-mesenchymal interactions

The mechanisms by which the branching of epithelial tissue occurs and is regulated to generate different organ structures are not well understood. In this work, image analyses of the organ rudiments demonstrate specific epithelial branching patterns for the early lung and kidney; the lung type typically generating several side branches, whereas kidney branching was mainly dichotomous. Parameters such as the number of epithelial tips, the angle of the first branch, the position index of the first branch (PIFB) in a module, and the percentage of epithelial module type (PMT) were analysed. The branching patterns in the cultured lung and kidney, and in homotypic tissue recombinants recapitulated their early in vivo branching patterns. The parameters were applied to heterotypic tissue recombinants between lung mesenchyme and ureteric bud, and tip number, PIFB and PMT values qualified the change in ureter morphogenesis and the reprogramming of the ureteric bud with lung mesenchyme. All the values for the heterotypic recombinant between ureteric bud and lung mesenchyme were significantly different from those for kidney samples but similar to those of the lung samples. Hence, lung mesenchyme can instruct the ureteric bud to undergo aspects of early lung-type epithelial morphogenesis. Different areas of the lung mesenchyme, except the tracheal region, were sufficient to promote ureteric bud growth and branching. In conclusion, our findings provide morphogenetic parameters for monitoring epithelial development in early embryonic lung and kidney and demonstrate the use of heterotypic tissue recombinants as a model for studying tissue-specific epithelial branching during organogenesis.     

Dystroglycan binding to laminin α1LG4 module influences epithelial morphogenesis of salivary gland and lung in vitro

Dystroglycan is a receptor for the basement membrane components laminin-1, -2, perlecan, and agrin. Genetic studies have revealed a role for dystroglycan in basement membrane formation of the early embryo. Dystroglycan binding to the E3 fragment of laminin-1 is involved in kidney epithelial cell development, as revealed by antibody perturbation experiments. E3 is the most distal part of the carboxyterminus of laminin alpha1 chain, and is composed of two laminin globular (LG) domains (LG4 and LG5). Dystroglycan-E3 interactions are mediated solely by discrete domains within LG4. Here we examined the role of this interaction for the development of mouse embryonic salivary gland and lung. Dystroglycan mRNA was expressed in epithelium of developing salivary gland and lung. Immunofluorescence demonstrated dystroglycan on the basal side of epithelial cells in these tissues. Antibodies against dystroglycan that block binding of alpha-dystroglycan to laminin-1 perturbed epithelial branching morphogenesis in salivary gland and lung organ cultures. Inhibition of branching morphogenesis was also seen in cultures treated with polyclonal anti-E3 antibodies. One monoclonal antibody (mAb 200) against LG4 blocked interactions between a-dystroglycan and recombinant laminin alpha1LG4-5, and also inhibited salivary gland and lung branching morphogenesis. Three other mAbs, also specific for the alpha1 carboxyterminus and known not to block branching morphogenesis, failed to block binding of alpha-dystroglycan to recombinant laminin alpha1LG4-5. These findings clarify why mAbs against the carboxyterminus of laminin alpha1 differ in their capacity to block epithelial morphogenesis and suggest that dystroglycan binding to alpha1LG4 is important for epithelial morphogenesis of several organs.     

EGF-receptor regulates salivary gland branching morphogenesis by supporting proliferation and maturation of epithelial cells and survival of mesenchymal cells

Epidermal growth factor receptor (EGF-R) regulates epithelial morphogenesis during development and is important for the proper branching of the lung, mammary gland, and pancreas. We analyzed the salivary gland phenotype of EGF-R-deficient mice and showed impaired growth, branching, and maturation of the epithelium. Furthermore, treatment of wild-type E13 salivary glands with gefitinib, a small molecular inhibitor of EGF-R, led to apoptosis of the mesenchyme. Interestingly, MMP2 and plasminogen activators were upregulated upon inhibition of EGF-R signaling. To summarize, we show that EGF-R is a physiological regulator of salivary gland development and its main function is to support the proliferation and maturation of the epithelium and the survival of the mesenchyme.     

Identification of pleiotrophin as a mesenchymal factor involved in ureteric bud branching morphogenesis

Branching morphogenesis is central to epithelial organogenesis. In the developing kidney, the epithelial ureteric bud invades the metanephric mesenchyme, which directs the ureteric bud to undergo repeated branching. A soluble factor(s) in the conditioned medium of a metanephric mesenchyme cell line is essential for multiple branching morphogenesis of the isolated ureteric bud. The identity of this factor had proved elusive, but it appeared distinct from factors such as HGF and EGF receptor ligands that have been previously implicated in branching morphogenesis of mature epithelial cell lines. Using sequential column chromatography, we have now purified to apparent homogeneity an 18 kDa protein, pleiotrophin, from the conditioned medium of a metanephric mesenchyme cell line that induces isolated ureteric bud branching morphogenesis in the presence of glial cell-derived neurotrophic factor. Pleiotrophin alone was also found to induce the formation of branching tubules in an immortalized ureteric bud cell line cultured three-dimensionally in an extracellular matrix gel. Consistent with an important role in ureteric bud morphogenesis during kidney development, pleiotrophin was found to localize to the basement membrane of the developing ureteric bud in the embryonic kidney. We suggest that pleiotrophin could act as a key mesenchymally derived factor regulating branching morphogenesis of the ureteric bud and perhaps other embryonic epithelial structures.     

Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43^−/− salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43^−/− samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43^−/− phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.     

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity^1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood ⁴. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo⁵. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-111^6,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands⁸. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.     

Melatonin Inhibits Embryonic Salivary Gland Branching Morphogenesis by Regulating Both Epithelial Cell Adhesion and Morphology

Many organs, including salivary glands, lung, and kidney, are formed by epithelial branching during embryonic development. Branching morphogenesis occurs via either local outgrowths or the formation of clefts that subdivide epithelia into buds. This process is promoted by various factors, but the mechanism of branching morphogenesis is not fully understood. Here we have defined melatonin as a potential negative regulator or “brake” of branching morphogenesis, shown that the levels of it and its receptors decline when branching morphogenesis begins, and identified the process that it regulates. Melatonin has various physiological functions, including circadian rhythm regulation, free-radical scavenging, and gonadal development. Furthermore, melatonin is present in saliva and may have an important physiological role in the oral cavity. In this study, we found that the melatonin receptor is highly expressed on the acinar epithelium of the embryonic submandibular gland. We also found that exogenous melatonin reduces salivary gland size and inhibits branching morphogenesis. We suggest that this inhibition does not depend on changes in either proliferation or apoptosis, but rather relates to changes in epithelial cell adhesion and morphology. In summary, we have demonstrated a novel function of melatonin in organ formation during embryonic development.     

Morphogenesis of epithelial tubes: Insights into tube formation, elongation, and elaboration

Epithelial tubes are a fundamental tissue across the metazoan phyla and provide an essential functional component of many of the major organs. Recent work in flies and mammals has begun to elucidate the cellular mechanisms driving the formation, elongation, and branching morphogenesis of epithelial tubes during development. Both forward and reverse genetic techniques have begun to identify critical molecular regulators for these processes and have revealed the conserved role of key pathways in regulating the growth and elaboration of tubular networks. In this review, we discuss the developmental programs driving the formation of branched epithelial networks, with specific emphasis on the trachea and salivary gland of Drosophila melanogaster and the mammalian lung, mammary gland, kidney, and salivary gland. We both highlight similarities in the development of these organs and attempt to identify tissue and organism specific strategies. Finally, we briefly consider how our understanding of the regulation of proliferation, apicobasal polarity, and epithelial motility during branching morphogenesis can be applied to understand the pathologic dysregulation of these same processes during metastatic cancer progression.     

Activin A induces expression of rat Sel-1l mRNA, a negative regulator of notch signaling, in rat salivary gland-derived epithelial cells

We previously established a rat submandibular gland (SMG)-derived epithelial cell line (RSMG-1) to study the mechanism of morphogenesis in salivary gland development and regeneration. We found that activin A regulated the branching morphogenesis of RSMG-1 cells, suggesting that it is involved in SMG morphogenesis. We used a subtraction cloning procedure with activin-A-treated and untreated RSMG-1 cells to identify activin-A-induced genes. One of the genes detected encoded a rat homologue of Sel-1l (rSel-1l). rSel-1l is a mammalian homologue of C. elegans sel-1, which is a negative regulator of Notch signaling. In this study, we confirmed that activin A induces rSel-1l mRNA expression in RSMG-1 cells, and that rSel-1l is expressed in SMG acinar cells. These results suggest that activin A regulates the differentiation of RSMG-1 cells to acinar cells.     

Induced repatterning of type XVIII collagen expression in ureter bud from kidney to lung type: association with sonic hedgehog and ectopic surfactant protein C

Epithelial-mesenchymal tissue interactions regulate the formation of signaling centers that play a role in the coordination of organogenesis, but it is not clear how their activity leads to differences in organogenesis. We report that type XVIII collagen, which contains both a frizzled and an endostatin domain, is expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomes localized to the epithelial tips in the lung during the early stages of epithelial branching, while its expression in the kidney is confined to the epithelial stalk region and is lost from the nearly formed ureter tips, thus displaying the reverse pattern to that in the lung. In recombinants, between ureter bud and lung mesenchyme, type XVIII collagen expression pattern in the ureter bud shifts from the kidney to the lung type, accompanied by a shift in sonic hedgehog expression in the epithelium. The lung mesenchyme is also sufficient to induce ectopic lung surfactant protein C expression in the ureter bud. Moreover, the shift in type XVIII collagen expression is associated with changes in ureter development, thus resembling aspects of early lung type epigenesis in the recombinants. Respecification of collagen is necessary for the repatterning process, as type XVIII collagen antibody blocking had no effect on ureter development in the intact kidney, whereas it reduced the number of epithelial tips in the lung and completely blocked ureter development with lung mesenchyme. Type XVIII collagen antibody blocking also led to a notable reduction in the expression of Wnt2, which is expressed in the lung mesenchyme but not in that of the kidney, suggesting a regulatory interaction between this collagen and Wnt2. Respecification also occurred in a chimeric organ containing the ureter bud and both kidney and lung mesenchymes, indicating that the epithelial tips can integrate the morphogenetic signals independently. A glial cell line-derived neurotrophic factor signal induces loss of type XVIII collagen from the ureter tips and renders the ureter bud competent for repatterning by lung mesenchyme-derived signals. Our data suggest that differential organ morphogenesis is regulated by an intra-organ patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen.     

Epithelial-mesenchymal interactions regulate the stage-specific expression of a cell surface proteoglycan, syndecan, in the developing kidney

Morphogenesis of the kidney is regulated by reciprocal tissue interactions between the epithelial ureter bud and the metanephric mesenchyme. The differentiation of the kidney involves profound changes in the extracellular matrix, and therefore matrix receptors may have an important role in this process. We studied the expression of syndecan, a cell surface proteoglycan acting as a receptor for interstitial matrix materials, by using a monoclonal antibody against the core protein of the molecule. Syndecan was not detected in the uninduced metanephric mesenchyme. During the formation of the ureter bud from the Wolffian duct, syndecan appeared in the mesenchymal cells around the invaginating bud. Simultaneously with the first branching of the ureter bud, the whole nephric mesenchyme became syndecan positive, but a 3- to 10-cell-thick layer around the branching ureter bud, representing the presumptive tubular cells, was most intensely stained. During the assembly of the mesenchyme cells into pretubular aggregates, syndecan was detected in these aggregates and, to a lesser degree, in the morphologically undifferentiated mesenchyme. Thereafter syndecan was found only in the differentiating epithelium, from which it was gradually lost during maturation of the nephron. It was last detected in the periphery of the kidney, where tubulogenesis still continued. In transfilter cultures we showed that syndecan appeared in the nephric mesenchyme during the period when the mesenchyme becomes programmed to transform into epithelial structures. By using interspecies recombinations and a species-specific antibody we excluded the possibility that syndecan in the mesenchyme would originate from the inductor. We conclude that syndecan expression is regulated by epithelial-mesenchymal interactions. The findings that syndecan appeared as an early response to induction and that its distribution showed both spatial and temporal correlation with kidney morphogenesis suggest an important role for this molecule in development.     

Actin depolymerizing factors cofilin1 and destrin are required for ureteric bud branching morphogenesis

The actin depolymerizing factors (ADFs) play important roles in several cellular processes that require cytoskeletal rearrangements, such as cell migration, but little is known about the in vivo functions of ADFs in developmental events like branching morphogenesis. While the molecular control of ureteric bud (UB) branching during kidney development has been extensively studied, the detailed cellular events underlying this process remain poorly understood. To gain insight into the role of actin cytoskeletal dynamics during renal branching morphogenesis, we studied the functional requirements for the closely related ADFs cofilin1 (Cfl1) and destrin (Dstn) during mouse development. Either deletion of Cfl1 in UB epithelium or an inactivating mutation in Dstn has no effect on renal morphogenesis, but simultaneous lack of both genes arrests branching morphogenesis at an early stage, revealing considerable functional overlap between cofilin1 and destrin. Lack of Cfl1 and Dstn in the UB causes accumulation of filamentous actin, disruption of normal epithelial organization, and defects in cell migration. Animals with less severe combinations of mutant Cfl1 and Dstn alleles, which retain one wild-type Cfl1 or Dstn allele, display abnormalities including ureter duplication, renal hypoplasia, and abnormal kidney shape. The results indicate that ADF activity, provided by either cofilin1 or destrin, is essential in UB epithelial cells for normal growth and branching.     

Effects of hepatocyte growth factor (HGF) and activin a on the morphogenesis of rat submandibular gland-derived epithelial cells in serum-free collagen gel culture

Summary To study the mechanisms of morphogenesis in salivary gland regeneration, we have established the RSMG-1 cell line derived from submandibular gland (SMG) of 10-wk-old Wistar female rats in serum-free culture. Our finding that RSMG-1 cells originated from duct cells was based on morphology and immunohistochemical results. In three-dimensional serum-free collagen gel culture, HGF induced branching morphogenesis of RSMG-1 cells. Histological examination revealed that HGF-induced branching structure exhibited well-formed lumina. This morphology closely resembles that found in vivo. The cells also expressed activin A. Exogenously added activin A at a high concentration reduced HGF-induced branching morphogenesis. These findings suggest that the morphogenesis of the salivary gland is modulated by HGF and activin A. Our results show that the RSMG-1 cell line may be useful in studies of salivary gland regeneration.     

Xylose-linked proteoglycan synthesis does not have a primary role in the control of secretory cell differentiation in salivary glands

Xylose-linked proteoglycans, particularly chondroitin sulfate proteoglycan, have been shown to play a significant role in the regulation of salivary gland morphogenesis. The purpose of this study was to determine if xylose-linked proteoglycans are involved in the regulation of differentiation of salivary gland secretory cells. Embryonic rat submandibular salivary gland rudiments were cultured for 120 hr in the presence or absence of 0.75 to 1.0 mM p-nitrophenyl-beta-D-xylopyranoside (beta-D-xyloside), an inhibitor of xylose-linked proteoglycan assembly. beta-D-Xyloside has been shown to block submandibular gland morphogenesis (Thompson and Spooner, 1982). In the present study glandular morphogenesis was blocked in 93.3% of the rudiments cultured in the presence of beta-D-xyloside. However, secretory cell differentiation was observed in 71.4% of those rudiments in which morphogenesis had been inhibited. Biochemical evaluation confirmed that xylose-linked proteoglycan assembly had been inhibited by xyloside. These results indicate that while xylose-linked proteoglycans play a significant role in the control of salivary gland morphogenesis these molecules are not primary regulators for secretory cell differentiation within developing salivary glands.     

Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis

The mouse submandibular gland (SMG) epithelium undergoes extensive morphogenetic branching during embryonic development as the first step in the establishment of its glandular structure. However, the specific signaling pathways required for SMG branching morphogenesis are not well understood. Using E13 mouse SMG organ cultures, we showed that inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), wortmannin and LY294002, substantially inhibited branching morphogenesis in SMG. Branching morphogenesis of epithelial rudiments denuded of mesenchyme was inhibited similarly, indicating that PI 3-kinase inhibitors act directly on the epithelium. Immunostaining and Western analysis demonstrated that the p85 isoform of PI 3-kinase is expressed in epithelium at levels higher than in the mesenchyme. A target of PI 3-kinase, Akt/protein kinase B (PKB), showed decreased phosphorylation at Ser(473) by Western analysis in the presence of PI 3-kinase inhibitors. The major lipid product of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), was added exogenously to SMG via a membrane-transporting carrier in the presence of PI 3-kinase inhibitors and was found to stimulate cleft formation, the first step of branching morphogenesis. Together, these data indicate that PI 3-kinase plays a role in the regulation of epithelial branching morphogenesis in mouse SMG acting through a PIP(3) pathway.