BMPs and heart development

The ability to regenerate a heart after ablation of cardiogenic mesoderm has been demonstrated in early stage fish and amphibian embryos but this type of regulation of the heart field has not been seen in avians or mammals. The regulative potential of the cardiogenic mesoderm was examined in avian embryos and related to the spatial expression of genes implicated in early cardiogenesis. With the identification of early cardiac regulators such as bmp-2 and nkx-2.5, it is now possible to reconcile classical embryological studies with molecular mechanisms of cardiac lineage determination in vivo. The most anterior lateral embryonic cells have been identified as the region that becomes the heart; removal of all or any subset of these cells results in the loss of corresponding cardiac structures. In addition, removal of the lateral heart forming mesoderm while leaving the lateral endoderm intact also results in loss of cardiac structures. Thus the medial anterior mesoderm cannot be recruited into the heart lineage in vivo even in the presence of potential cardiac inducing endoderm. In situ analysis demonstrates that genes involved in early events of cardiogenesis such as bone morphogenetic protein 2 (bmp-2) and nkx-2.5 are expressed coincidentally with the mapped far lateral heart forming region. The activin type IIa receptor (actR-IIa) is a potential mediator of BMP signaling since it is expressed throughout the anterior mesoderm with the highest level of expression occurring in the lateral prospective heart cells. The posterior boundary of actR-IIa is consistent with the posterior boundary of nkx-2.5 expression, supporting a model whereby ActR-IIa is involved in restricting the heart forming region to an anterior subset of lateral cells exposed to BMP-2. Analysis of the cardiogenic potential of the lateral plate mesoderm posterior to nkx-2.5 and actR-IIa expression demonstrates that these cells are not cardiogenic in vitro and that removal of these cells from the embryo does not result in loss of heart tissue in vivo. Thus, the region of the avian embryo that will become the heart is defined medially, laterally, and posteriorly by nkx-2.5 gene expression. Removal of all or part of the nkx-2.5 expressing region results in the loss of corresponding heart structures, demonstrating the inability of the chick embryo to regenerate cardiac tissue in vivo at stages after nkx-2.5 expression is initiated (Ehrman, 1999).

Bone morphogenetic protein (BMP) signaling plays a central role in the induction of cardiac myogenesis in the chick embryo. At the time when chick precardiac cells become committed to the cardiac muscle lineage, they are in contact with tissues expressing BMP-2, BMP-4, and BMP-7. Application of BMP-2-soaked beads in vivo elicits ectopic expression of the cardiac transcription factors CNkx-2.5 (homolog of Drosophila Tinman) and GATA-4. Furthermore, administration of soluble BMP-2 or BMP-4 to explant cultures induces full cardiac differentiation in stage 5 to 7 anterior medial mesoderm, a tissue that is normally not cardiogenic. The competence to undergo cardiogenesis in response to BMPs is restricted to mesoderm located in the anterior regions of gastrula- to neurula-stage embryos. The secreted protein Noggin, which binds to BMPs and antagonizes BMP activity, completely inhibits differentiation of the precardiac mesoderm, indicating that BMP activity is required for myocardial differentiation in this tissue. Together, these data imply that a cardiogenic field exists in the anterior mesoderm and that localized expression of BMPs selects which cells within this field enter the cardiac myocyte lineage (Schultheiss, 1997).

Drosophila induction of the homeobox gene tinman, and subsequent heart formation, are both dependent on dpp signaling from overlying ectoderm. In order to define vertebrate heart-inducing signals dpp-homologs have been sought that are expressed in stage 4 chicken embryos. The majority of transcripts are found to be BMP-2 among several other members of the BMP family. From embryonic stage 4 onwards cardiogenic mesoderm appears to be in close contact to BMP-2 expressing cells that initially are present in lateral mesoderm and subsequently after headfold formation in the pharyngeal endoderm. In order to assess the role of BMP-2 for heart formation, gastrulating chick embryos in culture were implanted with BMP-2 producing cells. BMP-2 implantation results in ectopic cardiac mesoderm specification. BMP-2 is able to induce Nkx2-5 expression ectopically within the anterior head domain, while GATA-4 is induced more caudally. However, cardiogenic induction by BMP-2 remains incomplete, since neither Nkx2-8 nor the cardiac-restricted structural gene VMHC-1 become ectopically induced. BMP-2 expressing cells implanted adjacent to paraxial mesoderm result in impaired somite formation and block the expression of marker genes, such as paraxis, Pax-3, and the forkhead gene cFKH-1. These results suggest that BMP-2 is part of the complex of cardiogenic signals and is involved in the patterning of early mesoderm similar to the role of dpp in Drosophila (Andrée, 1998).

The first evident break in left-right symmetry of the primitive zebrafish heart tube is the shift in pattern of BMP4 expression from radially symmetric to left-predominant. The midline heart tube then 'jogs' to the left and subsequently loops to the right. 279 mutations, affecting more than 200 genes, were examined; 21 mutations were found that perturb this process. Some cause BMP4 to remain radially symmetric. Others randomize the asymmetric BMP4 pattern. Retention of BMP4 symmetry is associated with failure to jog: right-predominance of the BMP4 pattern is associated with reversal of the direction of jogging and looping. Raising BMP4 diffusely throughout the heart, via Sonic hedgehog injection, or the blocking of its action by injection of a dominant negative BMP4 receptor, prevent directional jogging or looping. The genes crucial to directing cardiac asymmetry include a subset of those needed for patterning the dorsoventral axis and for notochord and ventral spinal cord development. Thus, the pattern of cardiac BMP4 appears to be in the pathway by which the heart interprets lateralizing signals from the midline and that BMP4 may drive asymmetry of the heart in zebrafish (Chen, 1997).

Previous studies have identified two signaling interactions regulating cardiac myogenesis in avians, a hypoblast-derived signal acting on epiblast and mediated by activin or a related molecule and an endoderm-derived signal acting on mesoderm and involving BMP-2. In this study, experiments were designed to investigate the temporal relationship between these signaling events and the potential role of other TGFbeta superfamily members in regulating early steps of heart muscle development. While activin or TGFbeta can potently induce cardiac myogenesis in pregastrula epiblast, they show no capacity to convert noncardiogenic mesoderm toward a myocardial phenotype. Conversely, BMP-2 or BMP-4, in combination with FGF-4, can readily induce cardiac myocyte formation in posterior mesoderm, but shows no capacity to induce cardiac myogenesis in epiblast cells. Activin/TGFbeta and BMP-2/BMP-4 therefore have distinct and reciprocal cardiac-inducing capacities that mimic the tissues in which they are expressed, the pregastrula hypoblast and anterior lateral endoderm, respectively. Experiments with noggin and follistatin provide additional evidence indicating that BMP signaling lies downstream of an activin/TGFbeta signal in the cardiac myogenesis pathway. In contrast to the cardiogenic-inducing capacities of BMP-2/BMP-4 in mesoderm, however, BMP-2 or BMP-4 inhibits cardiac myogenesis prior to stage 3, demonstrating multiple roles for BMPs in mesoderm induction. These and other published studies suggest a signaling cascade in which a hypoblast-derived activin/TGFbeta signal is required prior to and during early stages of gastrulation, regulated both spatially and temporally by an interplay between BMPs and their antagonists. Later cardiogenic signals arising from endoderm, and perhaps transiently from ectoderm, and mediated in part by BMPs, act on emerging mesoderm within cardiogenic regions to activate or enhance expression of cardiogenic genes such as GATA and cNkx family members, leading to cardiac myocyte differentiation (Ladd, 1998).

Formation of the long bones requires a cartilage template. Cartilage formation (chondrogenesis) proceeds through determination of cells and their aggregation into prechondrogenic condensations, differentiation into chondrocytes, and later maturation. Several studies indicate that members of the bone morphogenetic protein (BMP) family promote cartilage formation, but the exact step(s) in which BMPs are involved during this process remains undefined. To resolve this issue, a retroviral vector was used to misexpress the BMP antagonist Noggin in the embryonic chick limb. The resulting phenotype was characterized in depth, analyzing histological and early chondrogenic markers, as well as the patterns of cell death and proliferation. Misexpression of Noggin prior to the onset of chondrogenesis leads to the total absence of skeletal elements. Noggin inhibits cartilage formation at two distinct steps: (1) mesenchymal cells do not aggregate into prechondrogenic condensations, and additional results suggest that these cells persist in an undifferentiated state; (2) differentiation of chondroprogenitors into chondrocytes can also be blocked, concurrent with expanded expression of a presumptive joint region marker. In addition, alterations in muscle and tendon morphogenesis have been observed. These studies therefore provide in vivo evidence that BMPs are necessary for different steps of chondrogenesis: chondroprogenitor determination and/or condensation and subsequent differentiation into chondrocytes (Pizette, 2000).

Advantage has been taken of a transient transgenic strategy in Xenopus embryos to demonstrate that BMP signaling is required in vivo for heart formation in vertebrates. Ectopic expression of dominant negative Type I (tALK3) or Type II (tBMPRII) BMP receptors in developing Xenopus embryos results in reduction or absence of heart formation. Additionally, blocking BMP signaling in this manner downregulates expression of XNkx2-5, a homeobox gene required for cardiac specification, prior to differentiation. Notably, however, initial expression of XNkx2-5 is not affected. Mutant phenotypes can be rescued by co-injection of mutant with wild-type receptors or co-injection of mutant receptors with XSmad1, a downstream mediator of BMP signaling. Whole-mount in situ analyses indicate that ALK3 and XSmad1 are coexpressed in cardiogenic regions. Together, these results demonstrate that BMP signaling is required for maintenance of XNkx2-5 expression and heart formation and suggest that ALK3, BMPRII, and XSmad1 may mediate this signaling (Shi, 2000).

The mature heart valves and septa are derived from the cardiac cushions that initially form as local outgrowths of mesenchymal cells within the outflow tract and atrioventricular regions. Endocardial cells respond to signals from the overlying myocardium and undergo an epithelial-to-mesenchymal transformation to invade the intervening extracellular matrix. The molecules that can induce and maintain these cell populations are not known, but many candidates, including several TGFßs and BMPs, have been proposed based on their expression patterns and activities in other systems. In the present study, the expression of Bmp6 and Bmp7 in overlapping and adjacent sites is described, including the cardiac cushions during mouse embryonic development. Previous analyses have demonstrated that neither of these BMPs is required during cardiogenesis, but analysis of Bmp6;Bmp7 double mutants has uncovered a marked delay in the formation of the outflow tract endocardial cushions. A proportion of Bmp6;Bmp7 mutants also displays defects in valve morphogenesis and chamber septation, and the embryos die between 10.5 and 15.5 dpc due to cardiac insufficiency. These data provide the first genetic evidence that BMPs are involved in the formation of the cardiac cushions (Kim, 2001).

The heart is the first organ to form and function during vertebrate embryogenesis. The role played by BMP during the initial myofibrillogenesis was examined in chick cultured precardiac mesoendoderm (mesoderm + endoderm; ME) using a secreted protein, noggin, which specifically antagonizes bone morphogenetic protein (BMP)-2 and -4. Conditioned medium from COS7 cells transfected with Xenopus noggin cDNA inhibits the expression of sarcomeric proteins (such as sarcomeric alpha-actinin, Z-line titin, and sarcomeric myosin), and so myofibrillogenesis is perturbed in cultured stage 4 precardiac ME; however, noggin does not inhibit the expression of smooth muscle alpha-actin (the first isoform of alpha-actin expressed during cardiogenesis). In cultured stage 5 precardiac ME, noggin does not inhibit either the formation of I-Z-I components or the expression of sarcomeric myosin, but it does inhibit the formation of A-bands. Smooth muscle alpha-actin is expressed without the addition of BMP4, although BMP4 is required to induce the expressions of sarcomeric alpha-actinin, titin, and sarcomeric myosin in cultured stage 6 posterolateral mesoderm (noncardio-genic mesoderm). Interestingly, in cultured stage 6 posterolateral mesoderm, BMP2 induces the expressions of sarcomeric alpha-actinin and titin, but not of sarcomeric myosin. These results suggest that (1) BMP4 function lies upstream of the initial formation of I-Z-I components and A-bands separately in a stage-dependent manner, and (2) at least two signaling pathways are involved in the initial cardiac myofibrillogenesis: one is an unknown pathway responsible for the expression of smooth muscle alpha-actin; the other is BMP signaling, which is involved in the expression of sarcomeric alpha-actinin, titin, and sarcomeric myosin (Nakajima, 2002).

During cardiogenesis, perturbation of a key transition at mid-gestation from cardiac patterning to cardiac growth and chamber maturation often leads to diverse types of congenital heart disease, such as ventricular septal defect (VSD), myocardium noncompaction, and ventricular hypertrabeculation. This transition, which occurs at embryonic day (E) 9.0-9.5 in murine embryos and E24-28 in human embryos, is crucial for the developing heart to maintain normal cardiac growth and function in response to an increasing hemodynamic load. Although, ventricular trabeculation and compaction are key morphogenetic events associated with this transition, the molecular and cellular mechanisms are currently unclear. Initially, cardiac restricted cytokine bone morphogenetic protein 10 (BMP10) was identified as being upregulated in hypertrabeculated hearts from mutant embryos deficient in FK506 binding protein 12 (FKBP12). To determine the biological function of BMP10 during cardiac development, BMP10-deficient mice were generated. An essential role of BMP10 in regulating cardiac growth and chamber maturation is described. BMP10 null mice display ectopic and elevated expression of p57kip2 and a dramatic reduction in proliferative activity in cardiomyocytes at E9.0-E9.5. BMP10 is also required for maintaining normal expression levels of several key cardiogenic factors (e.g. NKX2.5 and MEF2C) in the developing myocardium at mid-gestation. Furthermore, BMP10-conditioned medium is able to rescue BMP10-deficient hearts in culture. The data suggest an important pathway that involves a genetic interaction between BMP10, cell cycle regulatory proteins and several major cardiac transcription factors in orchestrating this transition in cardiogenesis at mid-gestation. This may provide an underlying mechanism for understanding the pathogenesis of both structural and functional congenital heart defects (Chen, 2004).

During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. Feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state are upregulated, leading initially to progenitor overspecification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT are increased, while Smad1 deletion in Nkx2-5 mutants rescue SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies are also rescued by Smad1 deletion. These findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation, and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD (Prall, 2007).

BMPs and kidney development

Members of the bone morphogenetic protein (BMP) family exhibit overlapping and dynamic expression patterns throughout embryogenesis. However, little is known about the upstream regulators of these important signaling molecules. There is some evidence that BMP signaling may be autoregulative as demonstrated for BMP4 during tooth development. Analysis of BMP7 expression during kidney development, in conjunction with studies analyzing the effect of recombinant BMP7 on isolated kidney mesenchyme, suggest that a similar mechanism may operate for BMP7. A beta-gal-expressing reporter allele has been generated at the BMP7 locus to closely monitor expression of BMP7 during embryonic kidney development. In contrast to other studies, the current analysis of BMP7/lacZ homozygous mutant embryos shows that BMP7 expression is not subject to autoregulation in any tissue. In addition, this reporter allele was used to analyze the expression of BMP7 in response to several known survival factors (EGF, bFGF) and inducers of metanephric mesenchyme, including the ureteric bud, spinal cord and LiCl. These studies show that treatment of isolated mesenchyme with EGF or bFGF allows survival of the mesenchyme but neither factor is sufficient to maintain BMP7 expression in this population of cells. Rather, BMP7 expression in the mesenchyme is contingent on an inductive signal. Thus, the reporter allele provides a convenient marker for the induced mesenchyme. Interestingly LiCl has been shown to activate the Wnt signaling pathway, suggesting that BMP7 expression in the mesenchyme is regulated by a Wnt signal. Treatment of whole kidneys with sodium chlorate to disrupt proteoglycan synthesis results in the loss of BMP7 expression in the mesenchyme, whereas expression in the epithelial components of the kidney are unaffected. Heterologous recombinations of ureteric bud with either limb or lung mesenchyme demonstrate that expression of BMP7 is maintained in this epithelial structure. Taken together, these data indicate that BMP7 expression in the epithelial components of the kidney is not dependent on cell-cell or cell-ECM interactions with the metanephric mesenchyme. By contrast, BMP7 expression in the metanephric mesenchyme is dependent on proteoglycans and possibly Wnt signaling. Treatment of kidney explants with sodium chlorate inhibits proteoglycan synthesis and arrests branching of the ureteric bud. As Wnt-11 is expressed in the tips of the ureter and expression of both Wnt-11 and BMP7 in the mesenchyme are lost in chlorate-treated kidneys, these data are consistent with Wnt 11 functioning as an inductive signal emanating from the ureteric bud to initiate BMP7 expression in the mesenchyme. These data also suggest that expression of BMP7 in the condensed mesenchyme requires proteoglycans. In contrast, expression in the pre-tubular aggregates or in the epithelial components is not dependent on proteoglycans (Godin, 1998).

The kidney of the Gpc3-/- mouse, a novel model of human renal dysplasia, is characterized by selective degeneration of medullary collecting ducts preceded by enhanced cell proliferation and overgrowth during branching morphogenesis. Cellular and molecular mechanisms underlying this renal dysplasia have been identified. Glypican-3 (GPC3) deficiency is associated with abnormal and contrasting rates of proliferation and apoptosis in cortical (CCD) and medullary collecting duct (MCD) cells. In CCD, cell proliferation is increased threefold. In MCD, apoptosis was increased 16-fold. Expression of Gpc3 mRNA in ureteric bud and collecting duct cells suggests that GPC3 can exert direct effects in these cells. Indeed, GPC3 deficiency abrogates the inhibitory activity of BMP2 on branch formation in embryonic kidney explants, converts BMP7-dependent inhibition to stimulation, and enhances the stimulatory effects of KGF. Similar comparative differences are found in collecting duct cell lines derived from GPC3-deficient and wild type mice and induced to form tubular progenitors in vitro, suggesting that GPC3 directly controls collecting duct cell responses. It is proposed that GPC3 modulates the actions of stimulatory and inhibitory growth factors during branching morphogenesis (Grisaru, 2001).

The molecular basis for observations in ureteric bud and collecting duct cells remains to be determined. The demonstration that bFGF forms a molecular complex with cell surface heparan sulfate and the FGF cell surface receptor suggests that GPC3 may physically interact with receptors that bind BMP2, BMP7, and KGF. The opposite response of collecting duct cells to GPC3 deficiency, that is, inhibition of BMP2 activity and enhancement of KGF activity, suggests that the consequences of these interactions may differ. A second possibility is that GPC3 may function via independent signaling pathways that physically interact at the postreceptor level with BMP and KGF signaling intermediates. Alternatively, the GPC3 and growth factor-signaling pathways may interact indirectly by regulating competing or complementary gene products. Increasing evidence regarding the nature of inhibitory and stimulatory BMP-dependent signaling pathways in collecting duct cells provides a basis to determine the nature of GPC3 interactions with BMP2 and BMP7 (Grisaru, 2001).

Members of both the bone morphogenetic protein (Bmp) and EGF-CFC families have been implicated in vertebrate myocardial development. Zebrafish swirl (swr) encodes Bmp2b, a member of the Bmp family required for patterning the dorsoventral axis. Zebrafish one-eyed pinhead (oep) encodes a maternally and zygotically expressed member of the EGF-CFC family essential for Nodal signaling. Both swr/bmp2b and oep mutants exhibit severe defects in myocardial development. swr/bmp2b mutants exhibit reduced or absent expression of nkx2.5, an early marker of the myocardial precursors. Embryos lacking zygotic oep (Zoep mutants) display cardia bifida and also display reduced or absent nkx2.5 expression. The zinc finger transcription factor Gata5 is an essential regulator of nkx2.5 expression. The relationships between bmp2b, oep, gata5, and nkx2.5 have been investigated. Both swr/bmp2b and Zoep mutants exhibit defects in gata5 expression in the myocardial precursors. Forced expression of gata5 in swr/bmp2b and Zoep mutants restores robust nkx2.5 expression. Moreover, overexpression of gata5 in Zoep mutants restores expression of cmlc1, a myocardial sarcomeric gene. These results indicate that both Bmp2b and Oep regulate gata5 expression in the myocardial precursors, and that Gata5 does not require Bmp2b or Oep to promote early myocardial differentiation. It is concluded that Bmp2b and Oep function at least partly through Gata5 to regulate nkx2.5 expression and promote myocardial differentiation. Thus Gata5 regulates nkx2.5 and cmlc1. Other work has suggested that Gata5 also regulates the expression of cmlc2, vmhc, gata4, gata6, and hand2. Although fgf8 is also expressed in the marginal zone of the gastrulating embryo, gata5 expression is normal in ace/fgf8 mutants and conversely, fgf8 is expressed normally in fau/gata5 mutants. Together, these data indicate that Gata5 and Fgf8 regulate myocardial differentiation independently of one another (Reiter, 2001).

The iterative formation of nephrons during embryonic development relies on continual replenishment of progenitor cells throughout nephrogenesis. Defining molecular mechanisms that maintain and regulate this progenitor pool is essential to understanding nephrogenesis in developmental and regenerative contexts. Maintenance of nephron progenitors is absolutely dependent on BMP7 signaling, and Bmp7-null mice exhibit rapid loss of progenitors. However, the signal transduction machinery operating downstream of BMP7 as well as the precise target cell remain undefined. Using a novel primary progenitor isolation system, signal transduction and biological outcomes elicited by BMP7 were investigated. It was found that BMP7 directly and rapidly activates JNK signaling in nephron progenitors resulting in phosphorylation of Jun and ATF2 transcription factors. This signaling results in the accumulation of cyclin D3 and subsequent proliferation of PAX2(+) progenitors, inversely correlating with the loss of nephron progenitors seen in the Bmp7-null kidney. Activation of Jun and ATF2 is severely diminished in Bmp7-null kidneys, providing an important in vivo correlate. BMP7 thus promotes proliferation directly in nephron progenitors by activating the JNK signaling circuitry (Blank, 2009).

Using a BMP-reporter mouse, it has been shown that the nephrogenic zone (NZ) is essentially unresponsive to SMAD-mediated transcription in vivo. Owing to the strong effect of Bmp7 inactivation on nephron progenitor maintenance, this surprising finding prompted an investigation of whether BMP7 acts through a SMAD-independent signaling mechanism in the NZ. The most extensively described alternative pathway downstream of TGFβ and BMP is activation of MAPK signaling through TGFβ-activated kinase 1 (TAK1). Activation of p38 and JNK pathways have both been reported downstream of TAK1, and immortalized Tak1 mutant embryonic fibroblasts exhibited impaired phosphorylation of JNK. Mice deficient in Tak1 die at mid-gestation, precluding analysis of TAK1-mediated signaling in metanephric kidney development. However, TAK1 is expressed in the NZ of the developing kidney, suggesting that activation of MAPK signaling downstream of BMPs might occur in this region of the kidney. Previous work has indicate that BMP7 can activate p38 signaling in collecting duct cells in vitro. Similarly, conditional deletion of Bmp7 using a podocyte-specific Cre, resulted in defective p38 signaling, but no effect on phosphorylation of SMAD1/SMAD5/SMAD8 was observed. Using primary cell purification system, this study found no evidence that BMP7 activates p38 in the NZ. Instead, several JNK isoforms were rapidly phosphorylated in response to BMP7, resulting in downstream phosphorylation of both Jun and ATF2. Importantly, it was shown that TAK1 is required for efficient activation of JNK in response to BMP7. SIX2 immunostaining shows that JNK activation takes place directly in the nephron progenitor compartment, indicating that these cells indeed do respond directly to BMP7. Interestingly, phosphorylated forms of the JNK-activated transcription factors Jun and ATF2 are both localized to cap mesenchyme in the developing E17.5 kidney. Furthermore, E12.5 Bmp7-null kidneys displayed sharply reduced JNK-Jun-ATF2 signaling in metanephric mesenchyme compared with the wild type, demonstrating that Bmp7 is required for activation of this signaling axis in nephron progenitors in vivo. Interestingly, phosphorylation of Jun or ATF2 does not appear to be significantly reduced in collecting duct cells of Bmp7-deficient embryos, suggesting that other growth factors function to activate the pathway in this cellular compartment (Blank, 2009).

BMPs and lung development

The mouse lung develops in a stereotypic pattern, with distinct dorsal-ventral, anterior-posterior and proximal-distal polarities. Lung development begins at embryonic day (E) 9.5 when a pair of primary buds evaginates from the ventral foregut endoderm into the surrounding splanchnic mesenchyme (mesoderm). The buds subsequently elongate and, in a series of reiterated branchings, give rise to the pulmonary tree. This process of branching morphogenesis begins at E10.5 and continues until birth. Branching morphogenesis is accompanied by the differentiation of epithelial cell types along the proximal-distal axis. Molecular differences between proximal and distal tissues are already evident at E10.5, when several genes are specifically expressed in the distal endoderm and mesoderm. Later, characteristic morphological differences distinguish the mature proximal and distal endoderm. The proximal endoderm of the bronchioles is columnar and ciliated. In contrast, by E17.5 the distal endoderm is low cuboidal or squamous. However, terminal differentiation of these distal cells into type I (squamous) or type II (cuboidal) pneumocytes does not occur until after birth, since markers for these cell types are not restricted until this time (Weaver, 1999).

Little is known about the signaling molecules that pattern the lung along the P-D axis. One candidate is bone morphogenetic protein 4 (Bmp4), which is expressed in a dynamic pattern in the epithelial cells in the tips of growing lung buds. To follow Bmp4 transcription during these events, a recently derived Bmp4-lacZ reporter mouse line was used that allows detection of Bmp4^lacZ expression with single cell resolution. Two distinct sites of Bmp4^lacZ expression were observed in the developing lung. In a 19- somite embryo, before morphological evidence of lung budding, Bmp4^lacZ expression is seen in two distinct domains in the ventral mesenchyme surrounding the gut tube. Even at this early stage, the right side domain is larger than the left. By 23 somites, the distinct domains have fused anteriorly. At 27 somites, when the primordial lung buds are first emerging from the forgut, X-gal positive cells are found throughout the ventral mesenchyme of the developing lung. This ventral mesenchymal expression is maintained at 30 somites, is particularly striking at E11.5, but is lost by E13.5. A second site of Bmp4^lacZ expression is observed in the endodermal compartment of the lung, beginning around E10. Removal of the mesenchymal tissue layer illustrates that Bmp4^lacZ expression is restricted to the distal endoderm at E11.5. This cap-like pattern of X-gal positive cells at the tips of extending buds is maintained as branching morphogenesis proceeds. In the E17.5 lung, Bmp4^lacZ is highly expressed in the distal lung endoderm, but not in the adjacent mesenchyme (Weaver, 1999).

Studies in which Bmp4 has been overexpressed in the lung endoderm suggest that this factor plays an important role in lung morphogenesis. To further investigate this question, two complementary approaches were utilized to inhibit Bmp signaling in vivo. The Bmp antagonist Xnoggin and, independently, a dominant negative Bmp receptor (dnAlk6), were overexpressed using the surfactant protein C (Sp-C) promoter/enhancer. Inhibiting Bmp signaling results in a severe reduction in distal epithelial cell types and a concurrent increase in proximal cell types, as indicated by morphology and expression of marker genes, including the proximally expressed hepatocyte nuclear factor/forkhead homolog 4 (Hfh4) and Clara cell marker CC10, and the distal marker Sp-C. In addition, electron microscopy demonstrates the presence of ciliated cells (a proximal cell type) in the most peripheral regions of the transgenic lungs. A model is proposed in which Bmp4 is a component of an apical signaling center controlling P-D patterning. Endodermal cells at the periphery of the lung, which are exposed to high levels of Bmp4, maintain or adopt a distal character, while cells receiving little or no Bmp4 signal initiate a proximal differentiation program. As the bud extends toward the mesenchymal Fgf10 signal, cells leave the region of high Bmp4 and Fgf10 signal and initiate proximal differentiation. In Sp-C-Xnoggin transgenic lungs, Fgf10 signaling remains high, but Bmp signaling is inhibited. This leads to a foreshortening of the apical signaling center, and an extension of proximal epithelium (Weaver, 1999).

Morphogenesis of the mouse lung involves reciprocal interactions between the epithelial endoderm and the surrounding mesenchyme, leading to an invariant early pattern of branching that forms the basis of the respiratory tree. There is evidence that Fgf10 and Bmp4, expressed in the distal mesenchyme and endoderm, respectively, play important roles in branching morphogenesis. To examine these roles in more detail, an in vitro culture system has been exploited in which isolated endoderm is incubated in Matrigel substratum with Fgf-loaded beads. In addition, a Bmp4 lacZ line of mice was used in which lacZ faithfully reports Bmp4 expression. Analysis of lung endoderm in vivo shows a dynamic pattern of Bmp4 lacZ expression during bud outgrowth, extension and branching. In vitro, Fgf10 induces both proliferation and chemotaxis of isolated endoderm, whether it is derived from the distal or proximal lung. Moreover, after 48 hours, Bmp4 lacZ expression is upregulated in the endoderm closest to the bead. Addition of 30-50 ng/ml of exogenous purified Bmp4 to the culture medium inhibits Fgf-induced budding or chemotaxis, and inhibits overall proliferation. By contrast, the Bmp-binding protein Noggin enhances Fgf-induced morphogenesis. Based on these and other results, a model is proposed for the combinatorial roles of Fgf10 and Bmp4 in branching morphogenesis of the lung (Weaver, 2000).

A model is presented for the dynamic interaction of growth factors in lung bud morphogenesis. Throughout early development Shh is expressed in the endoderm and patched1 in the adjacent mesoderm. (1) Shortly after initiation of bud growth, Fgf10 is transcribed at high levels in distal mesenchyme but only very low levels of Bmp4 expression are seen in the endoderm. Studies in transgenic embryos support the hypothesis that one function of Shh is to promote proliferation of the mesenchyme through Ptc and possibly Gli-dependent pathway(s). Shh may also downregulate Fgf10 expression. Present results suggest that Fgf10 promotes both the proliferation of the endoderm and its outward movement. (2) As bud outgrowth continues, endodermal Bmp4 expression increases. Meanwhile, Fgf10 expression gradually decreases at the tip but is upregulated laterally, in this case asymmetrically, by unknown mechanisms. (3) As the Fgf10 expression domain moves laterally, it overlies proximal endoderm. These results suggest that a lateral bud can only be induced where the level of Bmp4 falls below a threshold. (4) Before undergoing dichotomous branching, the distal endoderm expresses such high levels of Bmp4 that forward movement stops. (5) It is hypothesized that the mechanism that regulates Fgf10 at the tip now drives expression laterally and symmetrically, leading to the outgrowth of two new buds. The cycle of outgrowth, promotion of mesenchymal proliferation and endoderm movement begins again (Weaver, 2000).

Lineage formation in the lung mesenchyme is poorly understood. Using a transgenic mouse line expressing LacZ under the control of Fgf10 regulatory sequences, the pool of Fgf10-positive cells in the distal lung mesenchyme has been shown to contain progenitors of the parabronchial smooth muscle cells. Fgf10 gene expression is slightly repressed in this transgenic line. This allowed creation of a hypomorphic Fgf10 phenotype by expressing the LacZ transgene in a heterozygous Fgf10 background. Hypomorphic Fgf10 mutant lungs display a decrease in ß-galactosidase-positive cells around the bronchial epithelium associated with an accumulation of ß-galactosidase-expressing cells in the distal mesenchyme. This correlates with a marked reduction of alpha smooth muscle actin (SMA) expression, thereby demonstrating that FGF10 is mostly required for the entry of mesenchymal cells into the parabronchial smooth muscle cell lineage. The failure of exogenous FGF10 to phosphorylate its known downstream targets ERK and AKT in lung mesenchymal cultures strongly suggests that FGF10 acts indirectly on the progenitor population via an epithelial intermediate. This study provides support for a role of epithelial BMP4 in mediating the formation of parabronchial smooth muscle cells (Mailleux, 2005).

The results indicate a decrease in Bmp4 expression in Fgf10^LacZ/– embryos. This reduction in Bmp4 expression seems to primarily occur in the epithelium. These results are consistent with previous reports showing that FGF10 upregulates epithelial Bmp4 transcription. Overexpression of Bmp4 in the distal lung epithelium using the surfactant protein C promoter leads to ectopic expression of alpha-SMA in the distal mesenchyme. While addition of recombinant SHH induces alpha-SMA expression on isolated lung mesenchymal explants, overexpression of Shh in the distal lung epithelium in vivo does not modify alpha-SMA expression. This may be explained by the lack of upregulation of Bmp4 in the epithelium or the mesenchyme upon overexpression of Shh in vivo, by contrast to the induction of Bmp4 expression by SHH in vitro (Mailleux, 2005).

Consistent with a major role for Bmp4 in SMC differentiation, recombinant BMP4 induces alpha-SMA expression in lung mesenchyme explants in vitro after 48 hours of culture. These results strongly suggest that BMP4 induces SMC formation by acting directly on the mesenchyme. It is therefore proposed that FGF10 expressed by the distal mesenchyme may contribute to parabronchial SMC formation via the upregulation of BMP4 synthesis by the epithelium. The failure to induce alpha-SMA expression in all cells can be explained by the presence of other cell types in the mesenchymal explants, e.g. the endothelial cells. In addition, these explants also contain a layer of mesothelium, producing FGF9, which has been shown to prevent the differentiation of the smooth muscle cells (Mailleux, 2005).

BMPs and liver and gut development

Patterning of the gut into morphologically distinct regions results from the appropriate factors being expressed in strict spatial and temporal patterns to assign cells their fates in development. Often, the boundaries of gene expression early in development correspond to delineations between different regions of the adult gut. For example, Bmp4 is expressed throughout the hindgut and midgut, but is not expressed in the early gizzard. Ectopic BMP4 in the gizzard caused a thinning of the muscularis. To understand this phenotype the expression of the receptors transducing BMP signaling during gut development was examined. The BMP receptors are differentially expressed in distinct regions of the chicken embryonic gut. By using constitutively activated versions of the BMP type I receptors, it has been found that when ectopically expressed in the gizzard, the BMP receptors act in a manner similar to BMP4. The mesodermal thinning seen upon ectopic BMP signaling is due to an increase in apoptosis and a decrease in proliferation within the gizzard mesoderm. The mesodermal thinning is characterized by a disorganization and lack of differentiation of smooth muscle in the gizzard mesoderm. Further, ectopic BMP receptors cause an upregulation of Nkx2.5, the pyloric sphincter marker, similar to that seen with ectopic BMP4. This upregulation of Nkx2.5 is a cell-autonomous event within the mesoderm of the gizzard. Nkx2.5 is necessary and sufficient for establishing aspects of pyloric sphincter differentiation (Smith, 2000).

These data provide insight into the molecular controls of patterning events at the small intestinal-gizzard border. In the early stages of gut development, Shh expressed throughout the endoderm is secreted to signal to the overlying mesoderm, which expresses the SHH-receptor Ptc. This Shh signal causes an increase in proliferation to occur in the mesoderm throughout the gut tube, and it also causes the activation of BMP4 expression throughout the mesoderm of the intestine, but no BMP4 is activated in the gizzard. BMP4 then causes the mesoderm of the small intestine to decrease proliferation and increase apoptosis to antagonize the proliferation effects of SHH signaling, which results in the thin small intestinal mesoderm. The gizzard develops a thick mesodermal layer due both to the SHH signal and a lack of BMP signals. BMP4 expressed in the small intestinal mesoderm also diffuses across to the gizzard mesoderm to bind to BMPR1B to cause upregulation of Nkx2.5 within the posterior gizzard mesoderm and to specify the phenotype of the pyloric sphincter. Nkx2.5, a marker for the pyloric sphincter mesoderm, plays a direct role in this process, patterning the endoderm of the pyloric sphincter via some unknown signal(s). Meanwhile, BMP4 expressed in the small intestinal mesoderm, and later in the gizzard submucosa, delays smooth muscle differentiation in these tissues. Hence, BMP signaling has many important developmental roles in gut patterning. The universality of this anatomic boundary in vertebrates, expression of the molecules delineating this region, and conservation of the signaling pathways involved underscores the importance of gut development as a model system for understanding development (Smith, 2000).

Mesodermal signaling is critical for patterning the embryonic endoderm into different tissue domains. Classical tissue transplant experiments in the chick and recent studies in the mouse have indicated that interactions with the cardiogenic mesoderm are necessary and sufficient to induce the liver in the ventral foregut endoderm. Using molecular markers and functional assays, it has been shown that septum transversum mesenchyme cells, a distinct mesoderm cell type, are closely apposed to the ventral endoderm and contribute to hepatic induction. Specifically, using a mouse Bmp4 null mutation and an inhibitor of BMPs, it has been found that BMP signaling from the septum transversum mesenchyme is necessary to induce liver genes in the endoderm and to exclude a pancreatic fate. BMPs apparently function, in part, by affecting the levels of the GATA4 transcription factor, and work in parallel to FGF signaling from the cardiac mesoderm. BMP signaling also appears critical for morphogenetic growth of the hepatic endoderm into a liver bud. Thus, the endodermal domain for the liver is specified by simultaneous signaling from distinct mesodermal sources (Rossi, 2001).

BMPs and mammary development

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, it is demonstrated 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. BMP signaling is important for outgrowth of normal mammary buds and 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). These 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 (Hens, 2007).

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