Spatial gene expression in the T-stage mouse metanephros

The E11.5 mouse metanephros is comprised of a T-stage ureteric epithelial tubule sub-divided into tip and trunk cells surrounded by metanephric mesenchyme (MM). Tip cells are induced to undergo branching morphogenesis by the MM. In contrast, signals within the mesenchyme surrounding the trunk prevent ectopic branching of this region. In order to identify novel genes involved in the molecular regulation of branching morphogenesis we compared the gene expression profiles of isolated tip, trunk and MM cells using Compugen mouse long oligo microarrays. We identified genes enriched in the tip epithelium, sim-1, Arg2, Tacstd1, Crlf-1 and BMP7; genes enriched in the trunk epithelium, Innp1, Itm2b, Mkrn1, SPARC, Emu2 and Gsta3 and genes spatially restricted to the mesenchyme surrounding the trunk, CSPG2 and CV-2, with overlapping and complimentary expression to BMP4, respectively. This study has identified genes spatially expressed in regions of the developing kidney involved in branching morphogenesis, nephrogenesis and the development of the collecting duct system, calyces, renal pelvis and ureter.     

Six1 regulates Grem1 expression in the metanephric mesenchyme to initiate branching morphogenesis

Urinary tract morphogenesis requires subdivision of the ureteric bud (UB) into the intra-renal collecting system and the extra-renal ureter, by responding to signals in its surrounding mesenchyme. BMP4 is a mesenchymal regulator promoting ureter development, while GREM1 is necessary to negatively regulate BMP4 activity to induce UB branching. However, the mechanisms that regulate the GREM1-BMP4 signaling are unknown. Previous studies have shown that Six1-deficient mice lack kidneys, but form ureters. Here, we show that the tip cells of Six1^−/− UB fail to form an ampulla for branching. Instead, the UB elongates within Tbx18- and Bmp4-expressing mesenchyme. We find that the expression of Grem1 in the metanephric mesenchyme (MM) is Six1-dependent. Treatment of Six1^−/− kidney rudiments with GREM1 protein restores ampulla formation and branching morphogenesis. Furthermore, we demonstrate that genetic reduction of BMP4 levels in Six1^−/− (Six1^−/−; Bmp4^+/−) embryos restores urinary tract morphogenesis and kidney formation. This study uncovers an essential function for Six1 in the MM as an upstream regulator of Grem1 in initiating branching morphogenesis.     

Time-lapse observation of branching morphogenesis of the lung bud epithelium in mesenchyme-free culture and its relationship with the localization of actin filaments

It has been shown that branching morphogenesis of the lung bud is mediated by epithelial-mesenchymal interaction via such molecules as FGF10, BMP4 and Shh. However, a recent study showed that the isolated lung epithelium still undergoes branching morphogenesis in vitro even in the absence of mesenchyme (Nogawa and Ito, 1995). In the present study, we observed in vitro the dynamic movement of the isolated lung epithelium of the fetal mouse using time-lapse recording, and investigatedthe roles of actinfilaments in branching of the lung bud. First, time-lapse observation of the initial phase of lung branching morphogenesis revealed that at the sites of cleft formation, the epithelial surface was retracted inward from its original position. From this observation we assumed that there should be some structures which exert a physical force on the epithelium, and the localization and arrangement of actin fibers in the cultured lung epithelium were examined at various stages of branching morphogenesis. At the prebudding (6 h) and onset-budding (24 h) stages, no specific localization of actin filaments was observed in the lung bud epithelium, but at the postbudding stage (48 h) they were localized densely in the cells at the tip of the branched lung epithelium. The cell density was not different between the tip and cleft regions of the lung bud epithelium. When cultured with FGF-soaked beads, an actin-rich region was induced at the tip of the lung bud which was growing toward an FGF-soaked bead. These results indicate that actin fibers do not play a significant part in cleft formation but can be secondarily induced by FGF in the surrounding matrix and play some roles at later shaping of the branch in lung 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.     

BMP7 inhibits branching morphogenesis in the prostate gland and interferes with Notch signaling

The mouse prostate gland develops by branching morphogenesis from the urogenital epithelium and mesenchyme. Androgens and developmental factors, including FGF10 and SHH, promote prostate growth (Berman, D.M., Desai, N., Wang, X., Karhadkar, S.S., Reynon, M., Abate-Shen, C., Beachy, P.A., Shen, M.M., 2004. Roles for Hedgehog signaling in androgen production and prostate ductal morphogenesis. Dev. Biol. 267, 387-398; Donjacour, A.A., Thomson, A.A., Cunha, G.R., 2003. FGF-10 plays an essential role in the growth of the fetal prostate. Dev. Biol. 261, 39-54), while BMP4 signaling from the mesenchyme has been shown to suppresses prostate branching (Lamm, M.L., Podlasek, C.A., Barnett, D.H., Lee, J., Clemens, J.Q., Hebner, C.M., Bushman, W., 2001. Mesenchymal factor bone morphogenetic protein 4 restricts ductal budding and branching morphogenesis in the developing prostate. Dev. Biol. 232, 301-314). Here, we show that Bone Morphogenetic Protein 7 (BMP7) restricts branching of the prostate epithelium. BMP7 is expressed in the periurethral urogenital mesenchyme prior to formation of the prostate buds and, subsequently, in the prostate epithelium. We show that BMP7(lacZ/lacZ) null prostates show a two-fold increase in prostate branching, while recombinant BMP7 inhibits prostate morphogenesis in organ culture in a concentration-dependent manner. We further explore the mechanisms by which the developmental signals may be interpreted in the urogenital epithelium to regulate branching morphogenesis. We show that Notch1 activity is associated with the formation of the prostate buds, and that Notch1 signaling is derepressed in BMP7 null urogenital epithelium. Based on our studies, we propose a model that BMP7 inhibits branching morphogenesis in the prostate and limits the number of domains with high Notch1/Hes1 activity.     

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.     

Mesenchymal factor bone morphogenetic protein 4 restricts ductal budding and branching morphogenesis in the developing prostate

The budding of the urogenital sinus epithelium into the surrounding mesenchyme signals the onset of prostate morphogenesis. The epithelial and mesenchymal factors that regulate ductal budding and the ensuing process of ductal growth and branching are not fully known. We provide evidence that bone morphogenetic protein 4 (BMP4) is a mesenchymal factor that regulates ductal morphogenesis. The Bmp4 gene was most highly expressed in the male urogenital sinus from embryonic day 14 through birth, a period marked by formation of main prostatic ducts and initiation of ductal branching. From an initial wide distribution throughout the prostatic anlage of the urogenital sinus, Bmp4 expression became progressively restricted to the mesenchyme immediately surrounding the nascent prostatic ducts and branches. Exogenous BMP4 inhibited epithelial cell proliferation and exhibited a dose-dependent inhibition of ductal budding in urogenital sinus tissues cultured in vitro. Adult Bmp4 haploinsufficient mice exhibited an increased number of duct tips in both the ventral prostate and coagulating gland. Taken together, our data indicate that BMP4 is a urogenital sinus mesenchymal factor that restricts prostate ductal budding and branching morphogenesis.     

FGF-10 induces SP-C and Bmp4 and regulates proximal-distal patterning in embryonic tracheal epithelium

The induction, growth, and differentiation of epithelial lung buds are regulated by the interaction of signals between the lung epithelium and its surrounding mesenchyme. Fibroblast growth factor-10 (FGF-10), which is expressed in the mesenchyme near the distal tips, and bone morphogenetic protein 4 (BMP4), which is expressed in the most distal regions of the epithelium, are important molecules in lung morphogenesis. In the present study, we used two in vitro systems to examine the induction, growth, and differentiation of lung epithelium. Transfilter cultures were used to determine the effect of diffusible factors from the distal lung mesenchyme (LgM) on epithelial branching, and FGF-10 bead cultures were used to ascertain the effect of a high local concentration of a single diffusible molecule on the epithelium. Embryonic tracheal epithelium (TrE) was induced to grow in both culture systems and to express the distal epithelial marker surfactant protein C at the tips nearest the diffusible protein source. TrE cultured on the opposite side of a filter to LgM branched in a pattern resembling intact lungs, whereas TrE cultured in apposition to an FGF-10 bead resembled a single elongating epithelial bud. Examination of the role of BMP4 on lung bud morphogenesis revealed that BMP4 signaling suppressed expression of the proximal epithelial genes Ccsp and Foxj1 in both types of culture and upregulated the expression of Sprouty 2 in TrE cultured with an FGF-10 bead. Antagonizing BMP signaling with Noggin, however, increased expression of both Ccsp and Foxj1.     

Substitution for mesenchyme by basement-membrane-like substratum and epidermal growth factor in inducing branching morphogenesis of mouse salivary epithelium

Mouse salivary epithelium cannot undergo branching morphogenesis in the absence of the surrounding mesenchyme. To clarify the nature of the mesenchymal influence on the epithelium, we have investigated the culture conditions in which the epithelium could normally branch in the absence of mesenchymal cells. Combination of basement-membrane-like substratum (Matrigel) and epidermal growth factor (EGF) could substitute for the mesenchyme, the epithelium showing typical branching morphogenesis. Transforming growth factor alpha had the same effect as EGF. Matrigel plus basic fibroblast growth factor or transforming growth factor beta 1 and collagen gel plus EGF were not sufficient to support the branching of the epithelium. These results clearly reveal that the role of mesenchyme in salivary morphogenesis is both to provide the epithelium with an appropriate substratum and to accelerate growth of the epithelium.     

A network of Wnt, hedgehog and BMP signaling pathways regulates tooth replacement in snakes

Most dentate vertebrates, from fish to humans, replace their teeth and yet the molecular basis of tooth replacement is poorly understood. Canonical Wnt signaling regulates tooth number in mice and humans, but it is unclear what role it plays in tooth replacement as it naturally occurs. To clarify this, we characterized Wnt signaling activity in the dental tissues of the ball python Python regius. This species replaces teeth throughout life (polyphyodonty) and in the same manner as in humans, i.e., sequential budding of teeth from the tip of the dental lamina. From initiation stage onwards, canonical Wnt read-out genes (Lef1 and Axin2) are persistently expressed by cells in the dental lamina tip and surrounding mesenchyme. This implies that molecular signaling at work during dental initiation carries over to tooth replacement. We show that canonical Wnt signaling promotes cell proliferation in python dental tissues and that by confining Wnt activity in the dental lamina the structure extends instead of thickens. Presumably, lamina extension creates space between successive tooth buds, ensuring that tooth replacement occurs in an ordered manner. We suggest that hedgehog signaling confines Wnt activity in the dental epithelium by direct planar repression and, during tooth replacement stages, by negatively regulating BMP levels in the dental mesenchyme. Finally, we propose that Wnt-active cells at the extending tip of the python dental lamina represent the immediate descendents of putative stem cells housed in the lingual face of the lamina, similar to what we have recently described for another polyphyodont squamate species.     

Branching morphogenesis of mouse salivary epithelium in basement membrane-like substratum separated from mesenchyme by the membrane filter

Branching morphogenesis of mouse salivary gland has been studied with organ-culture system. We developed a novel transfilter culture system for analyzing branching morphogenesis of the salivary epithelium. The submandibular salivary epithelium from early 13-day mouse fetus, clotted with Matrigel and separated from the mesenchyme by membrane filter, showed extensive growth and branching morphogenesis, morphological differentiation of lobules and stalks, and a typical cleft shape. The epithelium showed little growth and no branching without Matrigel clot or without the mesenchyme. This branching morphogenesis was induced even when the pore size of the filter was reduced to 0.05 microns. Use of type I collagen gel instead of Matrigel mostly induced incomplete morphogenesis with various histological abnormalities. These results suggest that the salivary epithelium can undergo branching morphogenesis in the absence of the mechanical action of mesenchymal cells although it needs an appropriate extracellular matrix and some mesenchymal factors transmitted through the filter.     

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.     

Reduction of BMP4 activity by gremlin 1 enables ureteric bud outgrowth and GDNF/WNT11 feedback signalling during kidney branching morphogenesis

Antagonists act to restrict and negatively modulate the activity of secreted signals during progression of embryogenesis. In mouse embryos lacking the extra-cellular BMP antagonist gremlin 1 (Grem1), metanephric development is disrupted at the stage of initiating ureteric bud outgrowth. Treatment of mutant kidney rudiments in culture with recombinant gremlin 1 protein induces additional epithelial buds and restores outgrowth and branching. All epithelial buds express Wnt11, and Gdnf is significantly upregulated in the surrounding mesenchyme, indicating that epithelial-mesenchymal (e-m) feedback signalling is restored. In the wild type, Bmp4 is expressed by the mesenchyme enveloping the Wolffian duct and ureteric bud and Grem1 is upregulated in the mesenchyme around the nascent ureteric bud prior to initiation of its outgrowth. In agreement, BMP activity is reduced locally as revealed by lower levels of nuclear pSMAD protein in the mesenchyme. By contrast, in Grem1-deficient kidney rudiments, pSMAD proteins are detected in many cell nuclei in the metanephric mesenchyme, indicative of excessive BMP signal transduction. Indeed, genetic lowering of BMP4 levels in Grem1-deficient mouse embryos completely restores ureteric bud outgrowth and branching morphogenesis. The reduction of BMP4 levels in Grem1 mutant embryos enables normal progression of renal development and restores adult kidney morphology and functions. This study establishes that initiation of metanephric kidney development requires the reduction of BMP4 activity by the antagonist gremlin 1 in the mesenchyme, which in turn enables ureteric bud outgrowth and establishment of autoregulatory GDNF/WNT11 feedback signalling.     

VEGF-A signaling through Flk-1 is a critical facilitator of early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis

Vascular endothelial growth factor-A (VEGF-A) signaling directs both vasculogenesis and angiogenesis. However, the role of VEGF-A ligand signaling in the regulation of epithelial-mesenchymal interactions during early mouse lung morphogenesis remains incompletely characterized. Fetal liver kinase-1 (Flk-1) is a VEGF cognate receptor (VEGF-R2) expressed in the embryonic lung mesenchyme. VEGF-A, expressed in the epithelium, is a high affinity ligand for Flk-1. We have used both gain and loss of function approaches to investigate the role of this VEGF-A signaling pathway during lung morphogenesis. Herein, we demonstrate that exogenous VEGF 164, one of the 3 isoforms generated by alternative splicing of the Vegf-A gene, stimulates mouse embryonic lung branching morphogenesis in culture and increases the index of proliferation in both epithelium and mesenchyme. In addition, it induces differential gene and protein expression among several key lung morphogenetic genes, including up-regulation of BMP-4 and Sp-c expression as well as an increase in Flk-1-positive mesenchymal cells. Conversely, embryonic lung culture with an antisense oligodeoxynucleotide (ODN) to the Flk-1 receptor led to reduced epithelial branching, decreased epithelial and mesenchymal proliferation index as well as downregulating BMP-4 expression. These results demonstrate that the VEGF pathway is involved in driving epithelial to endothelial crosstalk in embryonic mouse lung morphogenesis.     

Persistence of responsiveness of adult mouse mammary gland to induction by embryonic mesenchyme

Persistence of the capacity for embryogenic morphogenesis in adult mammary epithelium was demonstrated by allowing it to interact with grafted embryonic mesenchyme in vivo. When 14-day embryonic mammary or salivary mesenchyme was transplanted in the mammary gland of syngeneic young adult virgin mice, organogenetic development of the mammary epithelial cells occurred responding to closely attached mesenchyme. An early change, within 2–4 days, that was observed equally in both types of the mesenchymes was proliferation of mammary epithelial cells in multiple layers resembling rudimental architecture. Subsequently, ductal branching occurred from the rudimental architecture by mesenchyme-dependent branching pattern, of mammary gland type with mammary mesenchyme and of salivary gland-like type with salivary mesenchyme. This developmental response did not require hormones secreted from ovaries since it was observed similarly in ovariectomized mice. The mammary epithelium at the lactating stage did not show such a potential to the transplanted salivary mesenchyme.     

Isolation and characterization of a novel plasma membrane protein, osteoblast induction factor (obif), associated with osteoblast differentiation

Background

The exocrine pancreas is composed of a branched network of ducts connected to acini. They are lined by a monolayered epithelium that derives from the endoderm and is surrounded by mesoderm-derived mesenchyme. The morphogenic mechanisms by which the ductal network is established as well as the signaling pathways involved in this process are poorly understood.

Results

By morphological analyzis of wild-type and mutant mouse embryos and using cultured embryonic explants we investigated how epithelial morphogenesis takes place and is regulated by chemokine signaling. Pancreas ontogenesis displayed a sequence of two opposite epithelial transitions. During the first transition, the monolayered and polarized endodermal cells give rise to tissue buds composed of a mass of non polarized epithelial cells. During the second transition the buds reorganize into branched and polarized epithelial monolayers that further differentiate into tubulo-acinar glands. We found that the second epithelial transition is controlled by the chemokine Stromal cell-Derived Factor (SDF)-1. The latter is expressed by the mesenchyme, whereas its receptor CXCR4 is expressed by the epithelium. Reorganization of cultured pancreatic buds into monolayered epithelia was blocked in the presence of AMD3100, a SDF-1 antagonist. Analyzis of sdf1 and cxcr4 knockout embryos at the stage of the second epithelial transition revealed transient defective morphogenesis of the ventral and dorsal pancreas. Reorganization of a globular mass of epithelial cells in polarized monolayers is also observed during submandibular glands development. We found that SDF-1 and CXCR4 are expressed in this organ and that AMD3100 treatment of submandibular gland explants blocks its branching morphogenesis.

Conclusion

In conclusion, our data show that the primitive pancreatic ductal network, which is lined by a monolayered and polarized epithelium, forms by remodeling of a globular mass of non polarized epithelial cells. Our data also suggest that SDF-1 controls the branching morphogenesis of several exocrine tissues.     

Epithelial morphogenesis in mouse embryonic submandibular gland: Its relationships to the tissue organization of epithelium and mesenchyme

Epithelial tissues in various organ rudiments undergo extensive shape changes during their development. The processes of epithelial shape change are controlled by tissue interactions with the surrounding mesenchyme which is kept in direct contact with the epithelium. One of the organs which has been extensively studied is the mouse embryonic submandibular gland, whose epithelium shows the characteristic branching morphogenesis beginning with the formation of narrow and deep clefts as well as changes in tissue organization. Various molecules in the mesenchyme, including growth factors and extracellular matrix components, affect changes of epithelial shape and tissue organization. Also, mesenchymal tissue exhibits dynamic properties such as directional movements in groups and rearrangement of collagen fibers coupled with force-generation by mesenchymal cells. The epithelium, during early branching morphogenesis, makes a cell mass where cell-cell adhesion systems are less developed. Such properties of both the mesenchyme and epithelium are significant for considering how clefts, which first appear as unstable tiny indentations on epithelial surfaces, are formed and stabilized.     

BMP4 and PTHrP interact to stimulate ductal outgrowth during embryonic mammary development and to inhibit hair follicle induction

The mammary glands develop initially as buds arising from the ventral embryonic epidermis. Recent work has shed light on signaling pathways leading to the patterning and formation of the mammary placodes and buds in mouse embryos. Relatively little is known of the signaling pathways that initiate branching morphogenesis and the formation of the ducts from the embryonic buds. Previous studies have shown that parathyroid hormone-related protein (PTHrP; also known as parathyroid hormone-like peptide, Pthlh) is produced by mammary epithelial cells and acts on surrounding mesenchymal cells to promote their differentiation into a mammary-specific dense mesenchyme. As a result of PTHrP signaling, the mammary mesenchyme supports mammary epithelial cell fate, initiates ductal development and patterns the overlying nipple sheath. In this report, we demonstrate that PTHrP acts, in part, by sensitizing mesenchymal cells to BMP signaling. PTHrP upregulates BMP receptor 1A expression in the mammary mesenchyme, enabling it to respond to BMP4, which is expressed within mesenchymal cells underlying the ventral epidermis during mammary bud formation. We demonstrate that BMP signaling is important for outgrowth of normal mammary buds and that BMP4 can rescue outgrowth of PTHrP(-/-) mammary buds. In addition, the combination of PTHrP and BMP signaling is responsible for upregulating Msx2 gene expression within the mammary mesenchyme, and disruption of the Msx2 gene rescues the induction of hair follicles on the ventral surface of mice overexpressing PTHrP in keratinocytes (K14-PTHrP). Our data suggest that PTHrP signaling sensitizes the mammary mesenchyme to the actions of BMP4, triggering outgrowth of the mammary buds and inducing MSX2 expression, which, in turn, leads to lateral inhibition of hair follicle formation within the developing nipple sheath.