Bud specific N-sulfation of heparan sulfate regulates Shp2-dependent FGF signaling during lacrimal gland induction

Preferential outgrowth of the bud cells forms the basis of branching morphogenesis. Here, we show that lacrimal gland development requires specific modification of heparan sulfates by Ndst genes at the tip of the lacrimal gland bud. Systemic and conditional knockout experiments demonstrate the tissue specific requirement of Ndst1 and Ndst2 in the lacrimal gland epithelial, but not mesenchymal, cells, and the functional importance of Ndst1 in Fgf10-Fgfr2b, but not of Fgf1-Fgfr2b, complex formation. Consistent with this, Fgf10-induced ectopic lacrimal gland budding in explant cultures is dependent upon Ndst gene dose, and epithelial deletion of Fgfr2 abolishes lacrimal gland budding, its specific modification of heparan sulfate and its phosphorylation of Shp2 (Ptpn11 - Mouse Genome Informatics). Finally, we show that genetic ablation of Ndst1, Fgfr2 or Shp2 disrupts ERK signaling in lacrimal gland budding. Given the evolutionarily conserved roles of these genes, the localized activation of the Ndst-Fgfr-Shp2 genetic cascade is probably a general regulatory mechanism of FGF signaling in branching morphogenesis.     

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.     

Lacrimal gland development and Fgf10-Fgfr2b signaling are controlled by 2-O- and 6-O-sulfated heparan sulfate

Heparan sulfate, an extensively sulfated glycosaminoglycan abundant on cell surface proteoglycans, regulates intercellular signaling through its binding to various growth factors and receptors. In the lacrimal gland, branching morphogenesis depends on the interaction of heparan sulfate with Fgf10-Fgfr2b. To address if lacrimal gland development and FGF signaling depends on 2-O-sulfation of uronic acids and 6-O-sulfation of glucosamine residues, we genetically ablated heparan sulfate 2-O and 6-O sulfotransferases (Hs2st, Hs6st1, and Hs6st2) in developing lacrimal gland. Using a panel of phage display antibodies, we confirmed that these mutations disrupted 2-O and/or 6-O but not N-sulfation of heparan sulfate. The Hs6st mutants exhibited significant lacrimal gland hypoplasia and a strong genetic interaction with Fgf10, demonstrating the importance of heparan sulfate 6-O sulfation in lacrimal gland FGF signaling. Altering Hs2st caused a much less severe phenotype, but the Hs2st;Hs6st double mutants completely abolished lacrimal gland development, suggesting that both 2-O and 6-O sulfation of heparan sulfate contribute to FGF signaling. Combined Hs2st;Hs6st deficiency synergistically disrupted the formation of Fgf10-Fgfr2b-heparan sulfate complex on the cell surface and prevented lacrimal gland induction by Fgf10 in explant cultures. Importantly, the Hs2st;Hs6st double mutants abrogated FGF downstream ERK signaling. Therefore, Fgf10-Fgfr2b signaling during lacrimal gland development is sensitive to the content or arrangement of O-sulfate groups in heparan sulfate. To our knowledge, this is the first study to show that simultaneous deletion of Hs2st and Hs6st exhibits profound FGF signaling defects in mammalian development.     

Site-specific inductive and inhibitory activities of MMP-2 and MMP-3 orchestrate mammary gland branching morphogenesis

During puberty, mouse mammary epithelial ducts invade the stromal mammary fat pad in a wave of branching morphogenesis to form a complex ductal tree. Using pharmacologic and genetic approaches, we find that mammary gland branching morphogenesis requires transient matrix metalloproteinase (MMP) activity for invasion and branch point selection. MMP-2, but not MMP-9, facilitates terminal end bud invasion by inhibiting epithelial cell apoptosis at the start of puberty. Unexpectedly, MMP-2 also represses precocious lateral branching during mid-puberty. In contrast, MMP-3 induces secondary and tertiary lateral branching of ducts during mid-puberty and early pregnancy. Nevertheless, the mammary gland is able to develop lactational competence in MMP mutant mice. Thus, specific MMPs refine the mammary branching pattern by distinct mechanisms during mammary gland branching morphogenesis.     

Glycosaminoglycan-dependent restriction of FGF diffusion is necessary for lacrimal gland development

Glycosaminoglycans (GAGs) play a central role in embryonic development by regulating the movement and signaling of morphogens. We have previously demonstrated that GAGs are the co-receptors for Fgf10 signaling in the lacrimal gland epithelium, but their function in the Fgf10-producing periocular mesenchyme is still poorly understood. In this study, we have generated a mesenchymal ablation of UDP-glucose dehydrogenase (Ugdh), an essential biosynthetic enzyme for GAGs. Although Fgf10 RNA is expressed normally in the periocular mesenchyme, Ugdh mutation leads to excessive dispersion of Fgf10 protein, which fails to elicit an FGF signaling response or budding morphogenesis in the presumptive lacrimal gland epithelium. This is supported by genetic rescue experiments in which the Ugdh lacrimal gland defect is ameliorated by constitutive Ras activation in the epithelium but not in the mesenchyme. We further show that lacrimal gland development requires the mesenchymal expression of the heparan sulfate N-sulfation genes Ndst1 and Ndst2 but not the 6-O and 2-O-sulfation genes Hs6st1, Hs6st2 and Hs2st. Taken together, these results demonstrate that mesenchymal GAG controls lacrimal gland induction by restricting the diffusion of Fgf10.     

FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis

Branching morphogenesis of mouse submandibular glands is regulated by multiple growth factors. Here, we report that ex vivo branching of intact submandibular glands decreases when either FGFR2 expression is downregulated or soluble recombinant FGFR2b competes out the endogenous growth factors. However, a combination of neutralizing antibodies to FGF1, FGF7 and FGF10 is required to inhibit branching in the intact gland, suggesting that multiple FGF isoforms are required for branching. Exogenous FGFs added to submandibular epithelial rudiments cultured without mesenchyme induce distinct morphologies. FGF7 induces epithelial budding, whereas FGF10 induces duct elongation, and both are inhibited by FGFR or ERK1/2 signaling inhibitors. However, a PI3-kinase inhibitor also decreases FGF7-mediated epithelial budding, suggesting that multiple signaling pathways exist. We immunolocalized FGF receptors and analyzed changes in FGFR, FGF and MMP gene expression to identify the mechanisms of FGF-mediated morphogenesis. FGFR1b and FGFR2b are present throughout the epithelium, although FGFR1b is more highly expressed around the periphery of the buds and the duct tips. FGF7 signaling increases FGFR1b and FGF1 expression, and MMP2 activity, when compared with FGF10, resulting in increased cell proliferation and expansion of the epithelial bud, whereas FGF10 stimulates localized proliferation at the tip of the duct. FGF7- and FGF10-mediated morphogenesis is inhibited by an MMP inhibitor and a neutralizing antibody to FGF1, suggesting that both FGF1 and MMPs are essential downstream mediators of epithelial morphogenesis. Taken together, our data suggests that FGFR2b signaling involves a regulatory network of FGFR1b/FGF1/MMP2 expression that mediates budding and duct elongation during branching morphogenesis.     

Gene expression profiles of mouse submandibular gland development: FGFR1 regulates branching morphogenesis in vitro through BMP- and FGF-dependent mechanisms

Analyses of gene expression profiles at five different stages of mouse submandibular salivary gland development provide insight into gland organogenesis and identify genes that may be critical at different stages. Genes with similar expression profiles were clustered, and RT-PCR was used to confirm the developmental changes. We focused on fibroblast growth factor receptor 1 (FGFR1), as its expression is highest early in gland development. We extended our array results and analyzed the developmental expression patterns of other FGFR and FGF isoforms. The functional significance of FGFR1 was confirmed by submandibular gland organ culture. Antisense oligonucleotides decreased expression of FGFR1 and reduced branching morphogenesis of the glands. Inhibiting FGFR1 signaling with SU5402, a FGFR1 tyrosine kinase inhibitor, reduced branching morphogenesis. SU5402 treatment decreased cell proliferation but did not increase apoptosis. Fgfr, Fgf and Bmp gene expression was localized to either the mesenchyme or the epithelium by PCR, and then measured over time by real time PCR after SU5402 treatment. FGFR1 signaling regulates Fgfr1, Fgf1, Fgf3 and Bmp7 expression and indirectly regulates Fgf7, Fgf10 and Bmp4. Exogenous FGFs and BMPs added to glands in culture reveal distinct effects on gland morphology. Glands cultured with SU5402 were then rescued with exogenous BMP7, FGF7 or FGF10. Taken together, our results suggest specific FGFs and BMPs play reciprocal roles in regulating branching morphogenesis and FGFR1 signaling plays a central role by regulating both FGF and BMP expression.     

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.     

Endogenous and ectopic gland induction by FGF-10

FGF-10, a member of the fibroblast growth factor family, is expressed in mesodermally derived cell populations during embryogenesis. During normal ocular development, FGF-10 is expressed in the perioptic mesenchyme adjacent to the Harderian and lacrimal gland primordia. In this report, we provide evidence that FGF-10 is both necessary and sufficient to initiate glandular morphogenesis. Lens-specific expression of FGF-10 was sufficient to induce ectopic ocular glands within the cornea. In addition, lacrimal and Harderian glands were not seen in FGF-10 null fetuses. Based on these results we propose that FGF-10 is an inductive signal that initiates ocular gland morphogenesis.     

Canonical Wnt signaling negatively regulates branching morphogenesis of the lung and lacrimal gland

Key gene families such as FGFs and BMPs are important mediators of branching morphogenesis. To understand whether Wnt genes, and in particular, the canonical Wnt signaling pathway also function in the branching process, we have used a combination of experimental and genetic gain and loss of function approaches to perturb the levels of canonical Wnt signaling in two arborized structures, the lung and the lacrimal gland. Here, we show that the addition of Wnt3a conditioned medium or LiCl strongly represses growth and proliferation of the lung and lacrimal gland, a result that was confirmed in vivo using a dominant stable mutation of beta-catenin conditionally expressed in the lacrimal gland epithelium. In agreement with these data, knockdown of Wnt signaling with beta-catenin morpholinos results in a greater number of branches and increased cell proliferation. In addition, we show that canonical Wnt signaling is able to modulate the levels of Fgf10 and suppress BMP-induced proliferation in the lacrimal gland. Thus, canonical Wnt signaling negatively regulates branching morphogenesis providing a balance to FGFs and BMPs which positively regulate this process. This multilayered control of growth and proliferation ensures that branched structures attain the morphology required to function efficiently.     

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.     

Specific heparan sulfate structures modulate FGF10-mediated submandibular gland epithelial morphogenesis and differentiation

FGF10, a heparan sulfate (HS)-binding growth factor, is required for branching morphogenesis of mouse submandibular glands (SMGs). HS increases the affinity of FGF10 for FGFR2b, which forms an FGF10.FGFR2b.HS ternary signaling complex, and results in diverse biological outcomes, including proliferation and epithelial morphogenesis. Defining the HS structures involved in specific FGF10-mediated events is critical to understand how HS modulates growth factor signaling in specific developmental contexts. We used HS-deficient BaF3/FGFR2b cells, which require exogenous HS to proliferate, to investigate the HS requirements for FGF10-mediated proliferation and primary SMG epithelia to investigate the structural requirements of HS for FGF10-mediated epithelial morphogenesis. In BaF3/FGFR2b cells, heparin with at least 10 saccharides and 6-O-, 2-O-, and N-sulfates were required for maximal proliferation. During FGF10-mediated SMG epithelial morphogenesis, HS increased proliferation and end bud expansion. Defined heparin decasaccharide libraries showed that 2-O-sulfation with either an N-or 6-O-sulfate induced end bud expansion, whereas decasaccharides with 6-O-sulfation alone induced duct elongation. End bud expansion resulted from increased FGFR1b signaling, with increased FGFR1b, Fgf1, and Spry1 as well as increased Aqp5 expression, a marker of end bud differentiation. Duct elongation was associated with expression of Cp2L1, a marker of developing ducts. Collectively, these findings show that the size and sulfate patterns of HS modulate specific FGF10-mediated events, such as proliferation, duct elongation, end bud expansion, and differentiation, and provide mechanistic insight as to how the developmental localization of specific HS structures in tissues influences FGF10-mediated morphogenesis and differentiation.     

Bmp7 regulates branching morphogenesis of the lacrimal gland by promoting mesenchymal proliferation and condensation

The lacrimal gland provides an excellent model with which to study the epithelial-mesenchymal interactions that are crucial to the process of branching morphogenesis. In the current study, we show that bone morphogenetic protein 7 (Bmp7) is expressed with a complex pattern in the developing gland and has an important role in regulating branching. In loss-of-function analyses, we find that Bmp7-null mice have distinctive reductions in lacrimal gland branch number, and that inhibition of Bmp activity in gland explant cultures has a very similar consequence. Consistent with this, exposure of whole-gland explants to recombinant Bmp7 results in increased branch number. In determining which cells of the gland respond directly to Bmp7, we have tested isolated mesenchyme and epithelium. We find that, as expected, Bmp4 can suppress bud extension in isolated epithelium stimulated by Fgf10, but interestingly, Bmp7 has no discernible effect. Bmp7 does, however, stimulate a distinct response in mesenchymal cells. This manifests as a promotion of cell division and formation of aggregates, and upregulation of cadherin adhesion molecules, the junctional protein connexin 43 and of alpha-smooth muscle actin. These data suggest that in this branching system, mesenchyme is the primary target of Bmp7 and that formation of mesenchymal condensations characteristic of signaling centers may be enhanced by Bmp7. Based on the activity of Bmp7 in promoting branching, we also propose a model suggesting that a discrete region of Bmp7-expressing head mesenchyme may be crucial in determining the location of the exorbital lobe of the gland.     

Role of FGF10/FGFR2b signaling during mammary gland development in the mouse embryo.

The mouse develops five pairs of mammary glands that arise during mid-gestation from five pairs of placodes of ectodermal origin. We have investigated the molecular mechanisms of mammary placode development using Lef1 as a marker for the epithelial component of the placode, and mice deficient for Fgf10 or Fgfr2b, both of which fail to develop normal mammary glands. Mammary placode induction involves two different signaling pathways, a FGF10/FGFR2b-dependent pathway for placodes 1, 2, 3 and 5 and a FGF10/FGFR2b-independent pathway for placode 4. Our results also suggest that FGF signaling is involved in the maintenance of mammary bud 4, and that Fgf10 deficient epithelium can undergo branching morphogenesis into the mammary fat pad precursor.     

Erk MAP kinase regulates branching morphogenesis in the developing mouse kidney.

Branching morphogenesis of epithelium is a common and important feature of organogenesis; it is, for example, responsible for development of renal collecting ducts, lung airways, milk ducts of mammary glands and seminal ducts of the prostate. In each case, epithelial development is controlled by a variety of mesenchyme-derived molecules, both soluble (e.g. growth factors) and insoluble (e.g. extracellular matrix). Little is known about how these varied influences are integrated to produce a coherent morphogenetic response, but integration is likely to be achieved at least partly by cytoplasmic signal transduction networks. Work in other systems (Drosophila tracheae, MDCK models) suggests that the mitogen-activated protein (MAP) kinase pathway might be important to epithelial branching. We have investigated the role of the MAP kinase pathway in one of the best characterised mammalian examples of branching morphogenesis, the ureteric bud of the metanephric kidney. We find that Erk MAP kinase is normally active in ureteric bud, and that inhibiting Erk activation with the MAP kinase kinase inhibitor, PD98059, reversibly inhibits branching in a dose-dependent manner, while allowing tubule elongation to continue. When Erk activation is inhibited, ureteric bud tips show less cell proliferation than controls and they also produce fewer laminin-rich processes penetrating the mesenchyme and fail to show the strong concentration of apical actin filaments typical of controls; apoptosis and expression of Ret and Ros, are, however, normal. The activity of the Erk MAP kinase pathway is dependent on at least two known regulators of ureteric bud branching; the GDNF-Ret signalling system and sulphated glycosaminoglycans. MAP kinase is therefore essential for normal branching morphogenesis of the ureteric bud, and lies downstream of significant extracellular regulators of ureteric bud development.     

The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells.

The mammary gland develops its adult form by a process referred to as branching morphogenesis. Many factors have been reported to affect this process. We have used cultured primary mammary epithelial organoids and mammary epithelial cell lines in three-dimensional collagen gels to elucidate which growth factors, matrix metalloproteinases (MMPs) and mammary morphogens interact in branching morphogenesis. Branching stimulated by stromal fibroblasts, epidermal growth factor, fibroblast growth factor 7, fibroblast growth factor 2 and hepatocyte growth factor was strongly reduced by inhibitors of MMPs, indicating the requirement of MMPs for three-dimensional growth involved in morphogenesis. Recombinant stromelysin 1/MMP3 alone was sufficient to drive branching in the absence of growth factors in the organoids. Plasmin also stimulated branching; however, plasmin-dependent branching was abolished by both inhibitors of plasmin and MMPs, suggesting that plasmin activates MMPs. To differentiate between signals for proliferation and morphogenesis, we used a cloned mammary epithelial cell line that lacks epimorphin, an essential mammary morphogen. Both epimorphin and MMPs were required for morphogenesis, but neither was required for epithelial cell proliferation. These results provide direct evidence for a crucial role of MMPs in branching in mammary epithelium and suggest that, in addition to epimorphin, MMP activity is a minimum requirement for branching morphogenesis in the mammary gland.     

Vitamin D(3) receptor ablation alters mammary gland morphogenesis

Postnatal mammary gland morphogenesis is achieved through coordination of signaling networks in both the epithelial and stromal cells of the developing gland. While the major proliferative hormones driving pubertal mammary gland development are estrogen and progesterone, studies in transgenic and knockout mice have successfully identified other steroid and peptide hormones that impact on mammary gland development. The vitamin D(3) receptor (VDR), whose ligand 1,25-dihydroxyvitamin D(3) is the biologically active form of vitamin D(3), has been implicated in control of differentiation, cell cycle and apoptosis of mammary cells in culture, but little is known about the physiological relevance of the vitamin D(3) endocrine system in the developing gland. In these studies, we report the expression of the VDR in epithelial cells of the terminal end bud and subtending ducts, in stromal cells and in a subset of lymphocytes within the lymph node. In the terminal end bud, a distinct gradient of VDR expression is observed, with weak VDR staining in proliferative populations and strong VDR staining in differentiated populations. The role of the VDR in ductal morphogenesis was examined in Vdr knockout mice fed high dietary Ca(2+) which normalizes fertility, serum estrogen and neonatal growth. Our results indicate that mammary glands from virgin Vdr knockout mice are heavier and exhibit enhanced growth, as evidenced by higher numbers of terminal end buds, greater ductal outgrowth and enhanced secondary branch points, compared with glands from age- and weight-matched wild-type mice. In addition, glands from Vdr knockout mice exhibit enhanced growth in response to exogenous estrogen and progesterone, both in vivo and in organ culture, compared with glands from wild-type mice. Our data provide the first in vivo evidence that 1,25-dihydroxyvitamin D(3) and the VDR impact on ductal elongation and branching morphogenesis during pubertal development of the mammary gland. Collectively, these results suggest that the vitamin D(3) signaling pathway participates in negative growth regulation of the mammary gland.     

Platelet-derived growth factor receptor regulates salivary gland morphogenesis via fibroblast growth factor expression

A coordinated reciprocal interaction between epithelium and mesenchyme is involved in salivary gland morphogenesis. The submandibular glands (SMGs) of Wnt1-Cre/R26R mice have been shown positive for mesenchyme, whereas the epithelium is beta-galactosidase-negative, indicating that most mesenchymal cells are derived from cranial neural crest cells. Platelet-derived growth factor (PDGF) receptor alpha is one of the markers of neural crest-derived cells. In this study, we analyzed the roles of PDGFs and their receptors in the morphogenesis of mouse SMGs. PDGF-A was shown to be expressed in SMG epithelium, whereas PDGF-B, PDGFRalpha, and PDGFRbeta were expressed in mesenchyme. Exogenous PDGF-AA and -BB in SMG organ cultures demonstrated increased levels of branching and epithelial proliferation, although their receptors were found to be expressed in mesenchyme. In contrast, short interfering RNA for Pdgfa and -b as well as neutralizing antibodies for PDGF-AB and -BB showed decreased branching. PDGF-AA induced the expression of the fibroblast growth factor genes Fgf3 and -7, and PDGF-BB induced the expression of Fgf1, -3, -7, and -10, whereas short interfering RNA for Pdgfa and Pdgfb inhibited the expression of Fgf3, -7, and -10, indicating that PDGFs regulate Fgf gene expression in SMG mesenchyme. The PDGF receptor inhibitor AG-17 inhibited PDGF-induced branching, whereas exogenous FGF7 and -10 fully recovered. Together, these results indicate that fibroblast growth factors function downstream of PDGF signaling, which regulates Fgf expression in neural crest-derived mesenchymal cells and SMG branching morphogenesis. Thus, PDGF signaling is a possible mechanism involved in the interaction between epithelial and neural crest-derived mesenchyme.     

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.