DPP homologs: morphogenesis and organanogenesis

Apart from the gut, which expresses both Sonic hedgehhog and Indian hedgehhog, there is no overlap in the various Hh expression domains. Shh is predominantly expressed in epithelia at numerous sites of epithelial-mesenchymal interactions, including the tooth, hair, whisker, rugae, gut, bladder, urethra, vas deferens, and lung, Dhh in Schwann and Sertoli cell precursors, and Ihh in gut and cartilage. Thus, it is likely that Hh signaling plays a central role in a diverse array of morphogenetic processes. Hh expression was compared with that of a second family of signaling molecules, the Bone morphogenetic proteins (Bmps), vertebrate relatives of Decapentaplegic, a target of the Drosophila Hh signaling pathway. The frequent expression of Bmp-2, -4, and -6 in similar or adjacent cell populations suggests a conserved role for Hh/Bmp interactions in vertebrate development (Bitgood, 1995).

An antibody specific for the phosphorylated and activated form of Smad1 has been used to examine endogenous patterns of BMP signaling in chick embryos during early development. Complex spatial and temporal distributions of BMP signaling are found that elucidate how BMPs may function in multiple patterning events in the early chick embryo. In the pregastrula embryo, BMP signaling is initially ubiquitous and is extinguished in the epiblast at the onset of primitive streak formation. At the head process stage, BMP signaling is inactivated in prospective neural plate, while it is strongly activated at the neural plate border, a region which is populated by cells that will give rise to neural crest. During later development, a dynamic spatiotemporal activation of BMP signaling is found along the rostrocaudal axis, in the dorsal neural tube, in the notochord, and in the somites during their maturation process (Faure, 2002).

Models for where and when BMPs signal during development have in the past been based primarily on inferences from the pattern of expression of BMP ligands and their inhibitors. While this examination of phosphoSmad1 levels during chick development confirms that, in most instances, patterns of Smad1 activation are broadly consistent with the expression of ligands and inhibitors, two types of exceptions to this consistency emerge from this work. First, at several points in early chick embryogenesis, Smad1 activation is detected when the known BMP ligands are not expressed. This is observed in the notochord at stage 14 and in the somites as soon as stage 10. While a large number of vertebrate BMP ligands have been identified, their expression and role in embryogenesis have not been systematically examined. These observations indicate that the ligands studied to date are not sufficient to fully explain endogenous patterns of BMP signaling. Second, at several developmental stages in chick, the BMP antagonist Noggin is clearly localized to a region of Smad1 activation. This is observed in Hensen’s node at stage 5, in the dorsal neural tube at stage 10, and in the dorsomedial differentiated somite at stage 14. This is not consistent with the simple equation of Noggin expression with BMP antagonism. One explanation for the colocalization of BMP signal activation and Noggin expression is that Noggin is activated downstream of BMP signaling as part of a negative feedback loop. If so, however, this feedback loop is clearly restricted by additional mechanisms to only a small subset of the cell types in which BMP signaling is activated, since Noggin expression is not generally correlated with BMP signaling during development. The fact that Smad1 phosphorylation and Noggin expression can persist in the same set of cells also suggests that there are ligands present in these regions that are resistant to Noggin inhibition (Faure, 2002).

Vertebrate Bmp2 and Bmp4 diverged from a common ancestral gene and encode closely related proteins. Mice homozygous for null mutations in either gene show early embryonic lethality, thereby precluding analysis of shared functions. The current studies present phenotypic analysis of compound mutant mice heterozygous for a null allele of Bmp2 in combination with null or hypomorphic alleles of Bmp4. Whereas mice lacking a single copy of Bmp2 or Bmp4 are viable and have subtle developmental defects, compound mutants show embryonic and postnatal lethality due to defects in multiple organ systems including the allantois, placental vasculature, ventral body wall, skeleton, eye and heart. Within the heart, BMP2 and BMP4 function coordinately to direct normal lengthening of the outflow tract, proper positioning of the outflow vessels, and septation of the atria, ventricle and atrioventricular canal. These results identify numerous BMP4-dependent developmental processes that are also very sensitive to BMP2 dosage, thus revealing novel functions of Bmp2 (Goldman, 2009).

DPP homologs and morphogenesis: Maintenance of the boundaries of the neural plate

An investigation was carried out of the cell interactions and signaling molecules involved in setting up and maintaining the border between the neural plate and the adjacent non-neural ectoderm in the chick embryo at primitive streak stages. msx-1, a target of BMP signaling, is expressed in this border at a very early stage. It is induced by FGF and by signals from the organizer, Hensen's node. The node also induces a ring of BMP-4, some distance away. By the early neurula stage, the edge of the neural plate is the only major site of BMP-4 and msx-1 expression, and is also the only site that responds to BMP inhibition or overexpression. At this time, the neural plate appears to have a low level of BMP antagonist activity. Using in vivo grafts and in vitro assays, it has been shown that the position of the border is further maintained by interactions between non-neural and neural ectoderm. It is concluded that the border develops by integration of signals from the organizer, the developing neural plate, the paraxial mesoderm and the non-neural epiblast, involving FGFs, BMPs and their inhibitors. It is suggested that BMPs act in an autocrine way to maintain the border state (Streit, 1999).

The non-neural ectoderm is divided into neural plate border and epidermal cells. At early blastula stages, Wnt and BMP signals interact to induce epidermal fate, but when and how cells initially acquire neural plate border fate remains poorly defined. Evidence is provided in chick that the specification of neural plate border cells is initiated at the late blastula stage and requires both Wnt and BMP signals. The results indicate, however, that at this stage BMP signals can induce neural plate border cells only when Wnt activity is blocked, and that the two signals in combination generate epidermal cells. Evidence that Wnt signals do not play an instructive role in the generation of neural plate border cells, but promote their generation by inducing BMP gene expression, which avoids early simultaneous exposure to the two signals and generates neural plate border instead of epidermal cells. Thus, specification of neural plate border cells is mediated by a novel Wnt-regulated BMP-mediated temporal patterning mechanism (Patthey, 2009).

DPP homologs and morphogenesis: mesodermal induction, patterning and growth

The visceral yolk sac plays a critical role in normal embryogenesis, yet little is known about the specific molecules that regulate its development. Four winged-helix genes (HNF-3alpha, HNF-3beta, HNF-3gamma and HFH-4) are restricted to visceral endoderm. In the absence of HNF-3beta, visceral endoderm forms but the morphogenetic movements by which the embryo becomes enclosed within its yolk sac are disrupted and serum protein gene transcription is greatly reduced. Hedgehog and Bmp genes, which encode signaling molecules known to play multiple roles in embryonic development, are also differentially expressed in the closely apposed yolk sac mesoderm and endoderm layers. It is thought that Indian hedgehog signals from the visceral mesoderm to establish BMP2, BMP4 and BMP6 in the yolk sac mesoderm. All three BMPs may amplify their own transcription by an autoregulatory mechanism and participate in the differentiation of mesodermal cells. In an autocrine role, Indian hedgehog also signals to establish BMP6 in visceral mesoderm. Desert hedgehog may signal back from yolk sac mesoderm to induce BMP6 in visceral endoderm. These results suggest that similar mechanisms may be utilized to mediate inductive interactions in both extraembryonic and embryonic tissues (Farrington, 1997).

In the chick embryo, the primitive streak is the first axial structure to develop. The initiation of primitive streak formation in the posterior area pellucida is influenced by the adjacent posterior marginal zone (PMZ). Chick Vg1 (cVg1), a member of the TGFbeta family of signaling molecules, whose homolog in Xenopus is implicated in mesoderm induction, is expressed in the PMZ of prestreak embryos. Vg1 is a member of the 60A branch of the BMP family, more distantly related to Dpp than is BMP2 and BMP4. Vg1 is even more distantly related to Drosophila Screw and vertebrate Nodal and Nodal related. Early in development, Vg1 expression is detected before formation of the primitive streak. Expression becomes localized to the epiblast of the posterior marginal zone and is finally concentrated in cells of the primitive streak. Later, VG1 is expressed throughout the unsegmented paraxial mesoderm and is also expressed in the mesenchyme of the branchial arches and in the myocardium of the heart. VG1 has a patterned distribution in the nervous system. Ectopic expression of cVg1 protein in the marginal zone chick blastoderms directs the formation of a secondary primitive streak, which subsequently develops into an ectopic embryo. The cells that contribute to the ectopic primitive streak undergo a change of fate; they acquire two distinct properties of primitive streak cells, as defined by gene expression and cell movements. Naive epiblast explants exposed to cVg1 protein in vitro acquire axial mesodermal properties. Together, these results show that cVg1 can mediate ectopic axis formation in the chick by inducing new cell fates and they permit the analysis of distinct events that occur during primitive streak formation (Shah, 1997).

Homozygous BMP-4 knockout embryos die between 6.5 and 9.5 days p.c., with a variable phenotype. Most knockouts do not proceed beyond the egg cylinder stage, do not express the mesodermal marker T (Brachyury), and show little or no mesodermal differentiation. Some homozygous mutants develop to the head fold or beating heart/early somite stage or beyond. However, they are developmentally retarded and have truncated or disorganized posterior structures and a reduction in extraembryonic mesoderm, including blood islands. These results provide direct genetic evidence that BMP-4 is essential for several different processes in early mouse development, beginning with gastrulation and mesoderm formation. Moreover, in the presumed absence of zygotic ligand, it appears that homozygous mutants can be rescued partially by related proteins or by maternal BMP-4 (Winnier, 1995).

Establishment of the dorsoventral axis is central to animal embryonic organization. In Xenopus two different classes of signaling molecules function in the dorsoventral patterning of the mesoderm. Both the TGF-beta-related products of the BMP-2 and BMP-4 genes and the Wnt molecule encoded by Xenopus Wnt-8 specify ventral fate and appear to inhibit dorsal mesodermal development. The similar functions of these molecularly very different classes of signaling molecules prompted a study of their mutual regulation and a comparison of their roles in mesoderm patterning. Wnt-8 and BMP-4 are indistinguishable in their abilities to induce expression of ventral genes. Although BMP-2/-4 signaling regulates Wnt-8 expression, these genes do not function in a linear pathway because Wnt-8 overexpression cannot compensate for an inhibition of BMP-2/-4 function; rather, BMP-4 overexpression rescues ventral gene expression in embryos with inhibited Wnt-8 function. Wnt-8 and BMP-2/-4 differ in their abilities to regulate dorsal gene expression. While BMP-4 appears to generally inhibit the expression of dorsal genes, Xenopus Wnt-8 only inhibits the expression of the notochord marker Xnot. Whereas the inhibitory effect of BMP-2/-4 localizes dorsal mesodermal fate, these results suggest that Xenopus Wnt-8 functions in the further patterning of the dorsal mesoderm into the most dorsal sector (from where the notochord develops) and the dorsolateral sector (from where the somites differentiate) (Hoppler, 1998).

Shortly after their formation, somites of vertebrate embryos differentiate along the dorsoventral axis into sclerotome, myotome and dermomyotome. The dermomyotome is then patterned along its mediolateral axis into medial, central and lateral compartments, which contain progenitors of epaxial muscle, dermis and hypaxial muscle, respectively. Wnt-11 was used as a molecular marker for the medial compartment of dermomyotome (the 'medial lip') to demonstrate that BMP in the dorsal neural tube indirectly induces formation of the medial lip by up-regulating Wnt-1 and Wnt-3a (but not Wnt-4) expression in the neural tube. Noggin in the dorsal somite may inhibit the direct action of BMP on this tissue. Wnt-11 induction is antagonized by Sonic Hedgehog, secreted by the notochord and the floor plate. Together, these results show that the coordinated actions of the dorsal neural tube (via BMP and Wnts), the ventral neural tube/notochord (via Shh) and the somite itself (via noggin) mediates patterning of the dorsal compartment of the somite (Marcelle, 1997).

Lbx1 (Drosophila homolog: Ladybird) staining resides within the expression domain of the lateral marker Sim1, a basic helix-loop-helix transcription factor, indicating that Lbx1 belongs to the lateral program of the somite. Lbx1 marks a sub-population of lateral somite cells only. Within the lateral somite, expression of Lbx1 co-localizes with elevated signals for the paired and homeobox containing transcription factor Pax3, and with signals for the receptor tyrosine kinase c-Met, both thought to be indicative of prospective hypaxial myoblasts. while the mechanisms leading to the dorsally restricted expression pattern of Sim1, the lateral up-regulation of Pax3 and the lateral activation of c-Met may be related to those stimulating Lbx1, Lbx1 is the only known marker that is exclusive for the lateral dermomyotomal lip. To study the mechanisms that lead to the formation of this musculature, Lbx1 was used as a marker for the anatomical structures that produce the signals necessary for the specification of the hypaxial musculature. These signals have been characterized by ablating muscles or transplanting them to ectopic locations in the chick embryo. In addition, BMP4 soaked beads were inserted medial to the somite. The data suggest that lateralizing signals from intermediate and lateral mesoderm have to synergize with dorsalizing signals from the surface ectoderm to induce the formation of the hypaxial musculature. However, the lateralizing function of the lateral mesoderm can only in part be mimicked by BMP4. The following model is proposed for the induction of hypaxial musculature: in the paraxial mesoderm, lateral identities are induced and medial identities are repressed by intermediate and lateral mesoderm, involving BMP4 as a general lateralizing signal, and possibly additional factors involved in the specification of the hypaxial musculature. Within the lateral somite half, dorsal identities are induced by the surface ectoderm, possibly via WNT signalling. The dorsalizing signals may be antagonized by signals released by the underlying aorta or endoderm. However, where the lateralizing and dorsalizing signals synergize, formation of the hypaxial musculature is induced, as monitored by the upregulation of Pax3, and at occipital, cervical and limb levels, by the expression of the novel marker Lbx1 (Dietrich, 1998).

Bone morphogenetic proteins (BMPs), members of the transforming growth factor beta superfamily, have been identified by their ability to induce cartilage and bone from nonskeletal cells and have been shown to act as a ventral morphogen in Xenopus mesoderm. A murine homeobox-containing gene, distal-less 5 (mDlx5), has been isolated as a BMP-inducible gene in osteoblastic MC3T3-E1 cells. Stable transfectants of MC3T3-E1 that overexpress mDlx5 mRNA show increases in various osteogenic markers; a fourfold increase in alkaline phosphatase activity; a sixfold increase in osteocalcin production, and the appearance of mineralization in the extracellular matrix. Furthermore, mDlx5 is induced orthotopically in mouse embryos treated with BMP-4 and in the fractured bone of adult mice. Consistent with these observations, it has been found that injection of mDlx5 mRNA into dorsal blastomeres enhances the ventralization of Xenopus embryos. These findings suggest that mDlx5 is a target gene of the BMP signaling pathway and acts as an important regulator of both osteogenesis and dorsoventral patterning of embryonic axis (Miyama, 1999).

The bone morphogenetic proteins (BMPs) play critical roles in patterning the early embryo and in the development of many organs and tissues. A recently identified new member of this multifunctional gene family, BMP-11, is most closely related to GDF-8/myostatin. During mouse embryogenesis, BMP-11 is first detected at 9.5 dpc in the tail bud with expression becoming stronger as development proceeds. At 10.0 dpc, BMP-11 is expressed in the distal and posterior region of the limb bud and later localizes to the mesenchyme between the skeletal elements. BMP-11 is also expressed in the developing nervous system, in the dorsal root ganglia, and dorsal lateral region of the spinal cord. To assess the biological activity of BMP-11, the protein was tested in the Xenopus ectodermal explant (animal cap) assay. BMP-11 induces axial mesodermal tissue (muscle and notochord) in a dose-dependent fashion. At higher concentrations, BMP-11 also induces neural tissue. Interestingly, the activin antagonist, follistatin (but not noggin, an antagonist of BMPs 2 and 4) inhibits BMP-11 activity on animal caps. These data suggest that in Xenopus embryos, BMP-11 acts more like activin, inducing dorsal mesoderm and neural tissue, and less like other family members such as BMPs 2, 4, and 7, which are ventralizing and anti-neuralizing signals. Taken together, these data suggest that during vertebrate embryogenesis, BMP-11 plays a unique role in patterning both mesodermal and neural tissues (Gamer, 1999).

Differentiation of mouse embryonic stem (ES) cells via embryoid bodies has been established as a suitable model to study development in vitro. Differentiation of ES cells in vitro into chondrocytes can be modulated by members of the transforming growth factor-beta family (TGF-beta, BMP-2 and -4). ES cell differentiation into chondrocytes is characterized by the appearance of Alcian blue-stained areas and the expression of cartilage-associated genes and proteins. Different stages of cartilage differentiation can be distinguished according to the expression pattern of the transcription factor scleraxis, and the cartilage matrix protein collagen II. The number of Alcian-blue-stained areas decreases slightly after application of TGF-beta, whereas BMP-2 or -4 induces chondrogenic differentiation. The inducing effect of BMP-2 is dependent on the time of application, consistent with its role to recruit precursor cells to the chondrogenic fate. It is proposed that BMP-2 and -4 are early inducers of chondrogenic differentiation. These results are in line with reports showing that BMPs induce the formation of new cartilage and bone in vivo when implanted subcutaneously or in muscle tissue. Furthermore, BMP-2 and -4 induce differentiation of murine mesenchymal progenitor cells into osteogenic cells as well as into chondrocytes and adipocytes. The mesenchymal cell line C3H10T1/2 differentiates into the chondrogenic lineage after treatment with BMP-2, and BMP-2 and -4 have been found to be responsible for the initiation of both cartilage and bone cell lineages (Kramer, 2000).

Two populations of axial mesoderm cells can be recognized in the chick embryo: posterior notochord and anterior prechordal mesoderm. The cellular and molecular events that govern the specification of prechordal mesoderm have been examined. Notochord and prechordal mesoderm cells are intermingled and share expression of many markers as they initially extend out of Hensen’s node. In vitro culture studies, together with in vivo grafting experiments, reveal that early extending axial mesoderm cells are labile and that their character may be defined subsequently through signals that derive from anterior endodermal tissues. Anterior endoderm elicits aspects of prechordal mesoderm identity in extending axial mesoderm by repressing notochord characteristics, briefly maintaining gsc expression and inducing BMP7 expression. Other notochord markers assayed at early stages, including netrin-1, brachyury and cnot-1 appear to be expressed by both notochord and prechordal mesoderm, becoming exclusive to notochord only at stage 6-7. Together these experiments suggest that, in vivo, signaling by anterior endoderm may determine the extent of prechordal mesoderm. The transforming growth factor beta superfamily members BMP2, BMP4, BMP7 and activin, all of which are transiently expressed in anterior endoderm mimic distinct aspects of its patterning actions. Together these results suggest that anterior endoderm-derived TGFbetas may specify prechordal mesoderm character in chick axial mesoderm (Vesque, 2000).

These experiments suggest a model in which the differentiation of chick axial mesoderm occurs in a stepwise fashion, in which TGFbeta signaling operates sequentially. In the first step, notochord and prechordal mesoderm precursor cells form within an area of the embryo devoid of BMP signaling, in response to Vg1-like and activin-like signals. As they migrate anteriorly, axial mesoderm cells continue to express markers indicative of their exposure to activin-like signaling, including gsc, chordin and 3B9. It is suggested that, in the second step, the convergent extension of axial mesoderm results in the exposure of anterior and posterior regions to distinct signals. The continued absence of BMP signaling is a prerequisite for the maintenance of notochord character in posterior regions of the axial mesoderm, but the anterior migration of axial mesoderm results in its re-exposure to TGFbeta signals that are now confined to anterior endoderm. These findings are consistent with the idea that the exposure of anteriormost regions of axial mesoderm to BMPs and activin-like signals are required for its specification to a prechordal mesoderm identity (Vesque, 2000).

Bone morphogenetic proteins (BMPs) can either promote growth of embryonic muscle by expanding the Pax-3-expressing muscle precursor population or restrict its development by inducing apoptosis. Follistatin, a proposed BMP antagonist, is expressed in embryonic muscle. Deficiency in Follistatin results in muscle defects and postnatal asphyxia. During chick limb development Follistatin enhances BMP-7 action to induce muscle growth but prevents the ability of BMP-7 to induce apoptosis and muscle loss. Follistatin, unlike another BMP-binding protein, Noggin, promotes Pax-3 expression and transiently delays muscle differentiation and thus exerts proliferative signaling during muscle development. Data is provided which show that Follistatin binds BMP-7 and BMP-2 at low affinities and that the binding is reversible. These data suggest that Follistatin acts to present BMPs to myogenic cells at a concentration that permits stimulation of embryonic muscle growth; Follistatin binds BMPs, and in the bound form, the activity of the BMP is neutralized. However, the binding seems to be reversible and released BMP regains biological activity. It is proposed that Follistatin stores and presents BMPs in a subapoptotic concentration which promotes continuous muscle growth (Amthor, 2002).

During early vertebrate development, members of the transforming growth factor beta (TGFß) family play important roles in a variety of processes, including germ layer specification, patterning, cell differentiation, migration, and organogenesis. The activities of TGFßs need to be tightly controlled to ensure their function at the right time and place. Despite identification of multiple regulators of Bone Morphogenetic Protein (BMP) subfamily ligands, modulators of the activin/nodal class of TGFß ligands are limited, and include follistatin, Cerberus, and Lefty. Recently, a membrane protein, tomoregulin-1 (TMEFF1, originally named X7365), was isolated and found to contain two follistatin modules in addition to an Epidermal growth factor (EGF) domain, suggesting that TMEFF1 may participate in regulation of TGFß function. Unlike follistatin and follistatin-related gene (FLRG), TMEFF1 inhibits nodal but not activin in Xenopus. Interestingly, both the follistatin modules and the EGF motif contribute to nodal inhibition. A soluble protein containing the follistatin and the EGF domains, however, is not sufficient for nodal inhibition; the location of TMEFF1 at the membrane is essential for its function. These results suggest that TMEFF1 inhibits nodal through a novel mechanism. TMEFF1 also blocks mesodermal, but not epidermal induction by BMP2. Unlike nodal inhibition, regulation of BMP activities by TMEFF1 requires the latter’s cytoplasmic tail, while deletion of either the follistatin modules or the EGF motif does not interfere with the BMP inhibitory function of TMEFF1. These results imply that TMEFF1 may employ different mechanisms in the regulation of nodal and BMP signals. In Xenopus, TMEFF1 is expressed from midgastrula stages onward and is enriched in neural tissue derivatives. This expression pattern suggests that TMEFF1 may modulate nodal and BMP activities during neural patterning. In summary, these data demonstrate that tomoregulin-1 is a novel regulator of nodal and BMP signaling during early vertebrate embryogenesis (Chang, 2003).

The bone morphogenetic protein (BMP) and Notch signaling pathways are crucial for cellular differentiation. In many cases, the two pathways act similarly; for example, to inhibit myogenic differentiation. It is not known whether this inhibition is caused by distinct mechanisms or by an interplay between Notch and BMP signaling. Functional Notch signaling is shown to be required for BMP4-mediated block of differentiation of muscle stem cells, i.e., satellite cells and the myogenic cell line C2C12. Addition of BMP4 during induction of differentiation dramatically reduces the number of differentiated satellite and C2C12 cells. Differentiation is substantially restored in BMP4-treated cultures by blocking Notch signaling using either the gamma-secretase inhibitor L-685,458 or by introduction of a dominant-negative version of the Notch signal mediator CSL. BMP4 addition to C2C12 cells increases transcription of two immediate Notch responsive genes, Hes1 and Hey1 (Drosophila homolog: Hairy/E(spl)-related with YRPW motif), an effect that is abrogated by L-685,458. A 3 kb Hey1-promoter reporter construct is synergistically activated by the Notch 1 intracellular domain (Notch 1 ICD) and BMP4. The BMP4 mediator SMAD1 mimics BMP activation of the Hey1 promoter. A synthetic Notch-responsive promoter containing no SMAD1 binding sites responds to SMAD1, indicating that DNA-binding activity of SMAD1 is not required for activation. Accordingly, Notch 1 ICD and SMAD1 interacts in binding experiments in vitro. Thus, the data presented here provide evidence for a direct interaction between the Notch and BMP signaling pathways, and indicate that Notch has a crucial role in the execution of certain aspects of BMP-mediated differentiation control (Dahlqvist, 2003).

Recent studies have postulated that distinct regulatory cascades control myogenic differentiation in the head and the trunk. However, although the tissues and signaling molecules that induce skeletal myogenesis in the trunk have been identified, the source of the signals that trigger skeletal muscle formation in the head remains obscure. Although myogenesis in the trunk paraxial mesoderm is induced by Wnt signals from the dorsal neural tube, myogenesis in the cranial paraxial mesoderm is blocked by these same signals. In addition, BMP family members that are expressed in both the dorsal neural tube and surface ectoderm are also potent inhibitors of myogenesis in the cranial paraxial mesoderm. Evidence is provided suggesting that skeletal myogenesis in the head is induced by the BMP inhibitors, Noggin and Gremlin, and the Wnt inhibitor, Frzb. These molecules are secreted by both cranial neural crest cells and by other tissues surrounding the cranial muscle anlagen. These findings demonstrate that head muscle formation is locally repressed by Wnt and BMP signals and induced by antagonists of these signaling pathways secreted by adjacent tissues (Tzahor, 2003).

The paired-like homeobox gene Mixl1 is expressed in the primitive streak of the gastrulating embryo, and marks cells destined to form mesoderm and endoderm. The role of Mixl1 in development of haematopoietic mesoderm was investigated by analysing the differentiation of ES cells in which GFP was targeted to one (Mixl1^GFP/w) or both (Mixl1^GFP/GFP) alleles of the Mixl1 locus. In either case, GFP was transiently expressed, with over 80% of cells in day 4 embryoid bodies (EBs) being GFP⁺. Up to 45% of Mixl1^GFP/w day 4 EB cells co-expressed GFP and the haemangioblast marker FLK1, and this doubly-positive population was enriched for blast colony forming cells (BL-CFCs). Mixl1-null ES cells, however, displayed a haematopoietic defect characterised by reduced and delayed Flk1 expression and a decrease in the frequency of haematopoietic CFCs. These data indicated that Mixl1 is required for efficient differentiation of cells from the primitive streak stage to blood. Differentiation of ES cells under serum-free conditions demonstrate that induction of Mixl1- and Flk1-expressing haematopoietic mesoderm requires medium supplemented with BMP4 or activin A. In conclusion, this study has revealed an important role for Mixl1 in haematopoietic development and demonstrates the utility of the Mixl1^GFP/w ES cells for evaluating growth factors influencing mesendodermal differentiation (Ng, 2005).

Sirenomelia or mermaid-like phenotype is one of the principal human congenital malformations that can be traced back to the stage of gastrulation. Sirenomelia is characterized by the fusion of the two hindlimbs into a single one. In the mouse, sirens have been observed in crosses between specific strains and as the consequence of mutations that increase retinoic acid levels. The loss of Bmp7 in combination with a half dose or complete loss of twisted gastrulation (Tsg) causes sirenomelia in the mouse. Tsg is a Bmp- and chordin-binding protein that has multiple effects on Bmp metabolism in the extracellular space; Bmp7 binds to Tsg. In Xenopus, co-injection of Tsg and Bmp7 morpholino oligonucleotides (MO) has a synergistic effect, greatly inhibiting formation of ventral mesoderm and ventral fin tissue. In the mouse, molecular marker studies indicate that the sirenomelia phenotype is associated with a defect in the formation of ventroposterior mesoderm. These experiments demonstrate that dorsoventral patterning of the mouse posterior mesoderm is regulated by Bmp signaling, as is the case in other vertebrates. Sirens result from a fusion of the hindlimb buds caused by a defect in the formation of ventral mesoderm (Zakin, 2005).

Sirens were discovered in the mouse (Gluecksohn-Schoenheimer, 1945) among the progeny of parents carrying various combinations of the Short-tail (T locus), anury (t⁰), Fused and ur mutations. The siren pups obtained had no tail, various degrees of reduction and fusion of elements of the hindlimbs, abnormalities of the spine, and fusion of ribs. Even though Tsg^-/-;Bmp7^-/- and Tsg^+/–;Bmp7^-/- sirenomelic pups do form tails (albeit shorter), the limb bud phenotypes observed are very similar to those seen in the Gluecksohn-Schoenheimer study. Could the old and new mutations be linked in any way? It is noted that the T locus (including brachyury), Fused (corresponding to Axin) and Tsg are all located on chromosome 17. The us mutation (urogenital syndrome), which is phenotypically identical to the now extinct ur (urogenital) mutant, and Bmp7 are both located on chromosome 2. Although the respective locations of these genes on these chromosomes are distant from each other, mutations at the T locus correspond to important chromosomal rearrangements, often leading to duplications and deficiencies of chromosome segments upon cell division (Gluecksohn-Schoenheimer, 1945). Thus, it is conceivable, although perhaps unlikely, that the occurrence of sirens in the initial description was associated with disruptions of the Tsg and/or Bmp7 genes. Unfortunately, some of the original mutations have been lost, so this is not a testable proposition (Zakin, 2005 and references therein).

In subsequent work, Hoornbeek found sirenomelic neonates in crosses between SM/J and BUA strains studied for the incidence of the 'careener' phenotype (Hoornbeek, 1970). These sirens have the same phenotype as in this study (fused hindlimbs, a tail, an abnormal umbilical artery). The genes affected in these crosses are not known, but the carriers of the 'siren' mutation (Hoornbeek, 1970) had tightly twisted tails; this is relevant because Tsg^-/- or Bmp7^-/- mutants also have kinked tails (Zakin, 2005 and references therein).

In conclusion, in the absence of Bmp7, two copies of Tsg are required for the proper differentiation of ventral and posterior structures. In the mouse, when Tsg and Bmp7 are mutated, the siren phenotype results from the fusion of the limb buds in the ventroposterior midline owing to a paucity of posterior ventral mesoderm. In Xenopus, knockdown of Tsg and Bmp7 results in an analogous phenotype: loss of posteroventral cell fates associated with decreased Bmp activity. These results demonstrate a common mechanism, mediated by Bmp signaling, in mouse and frog in the patterning of the dorsoventral axis (Zakin, 2005).

Bone morphogenetic protein (BMP) signaling pathways are essential regulators of chondrogenesis. However, the roles of these pathways in vivo are not well understood. Limb-culture studies have provided a number of essential insights, including the demonstration that BMP pathways are required for chondrocyte proliferation and differentiation. However, limb-culture studies have yielded contradictory results; some studies indicate that BMPs exert stimulatory effects on differentiation, whereas others support inhibitory effects. Therefore, this study characterized the skeletal phenotypes of mice lacking Bmpr1a in chondrocytes (Bmpr1a^CKO) and Bmpr1a^CKO;Bmpr1b^+/- (Bmpr1a^CKO;1b^+/-) in order to test the roles of BMP pathways in the growth plate in vivo. These mice reveal requirements for BMP signaling in multiple aspects of chondrogenesis. They also demonstrate that the balance between signaling outputs from BMP and fibroblast growth factor (FGF) pathways plays a crucial role in the growth plate. These studies indicate that BMP signaling is required to promote Ihh expression, and to inhibit activation of STAT and ERK1/2 MAPK, key effectors of FGF signaling. BMP pathways inhibit FGF signaling, at least in part, by inhibiting the expression of FGFR1. These results provide a genetic in vivo demonstration that the progression of chondrocytes through the growth plate is controlled by antagonistic BMP and FGF signaling pathways (Yoon, 2006).

During embryonic development in amniotes, the extraembryonic mesoderm, where the earliest hematopoiesis and vasculogenesis take place, also generates smooth muscle cells (SMCs). It is not well understood how the differentiation of SMCs is linked to that of blood (BCs) and endothelial (ECs) cells. This study shows that, in the chick embryo, the SMC lineage is marked by the expression of a bHLH transcription factor, dHand. Notch activity in nascent ventral mesoderm cells promotes SMC progenitor formation and mediates the separation of SMC and BC/EC common progenitors marked by another bHLH factor, Scl. This is achieved by crosstalk with the BMP and Wnt pathways, which are involved in mesoderm ventralization and SMC lineage induction, respectively. These findings reveal a novel role of the Notch pathway in early ventral mesoderm differentiation, and suggest a stepwise separation among its three main lineages, first between SMC progenitors and BC/EC common progenitors, and then between BCs and ECs (Shin, 2009).

The precise function of the Notch pathway in the process of muscle and BC/EC lineage separation remains to be elucidated. The data suggest that, during chick ventral mesoderm differentiation, the Notch pathway acts together with the BMP and Wnt pathways, and that it plays a 'permissive', rather than an 'instructive', role in mediating the separation of SMCs and BC/ECs. The Notch pathway does not control the induction of but rather the balance between these two populations. Evidence is provided that the induction of these lineages is controlled by the activities of both the BMP pathway, as a general ventral mesoderm inducer, and the canonical Wnt pathway, as a strong SMC lineage inducer. Ectopic activation of the BMP pathway can induce both SMC and BC/EC lineages, with the balance of SMCs and BC/ECs being regulated by Notch activity. It is not clear whether the induction of SMCs by the BMP pathway is a direct or indirect process, or whether it requires an active Wnt pathway. In this analysis, a stronger and wider ectopic dHand induction was observed by CA-β-Catenin than by CA-ALK6 around the anterior primitive streak where BMP antagonists are highly expressed, suggesting that the induction of SMCs by the Wnt pathway does not require active BMP signaling. A recent in vitro study suggested that Notch activity promotes the degradation of Scl by facilitating its ubiquitination, and that this process requires the transcriptional regulation of Notch pathway activity through Suppressor of Hairless. Although there is no direct evidence in support of a similar phenomenon in the current system, it could in principle act as a possible mechanism for the Notch activity-mediated segregation of SMCs and BC/ECs. Furthermore, Nrarp (an ankyrin-repeat protein that is transcriptionally regulated by the Notch signaling pathway), in addition to serving as a Notch-activity readout and a feedback regulator of the Notch pathway, has also been shown to positively regulate the canonical Wnt pathway by blocking the ubiquitination and increasing the stability of Lef1 in zebrafish. This might also serve as a possible mechanism for the Notch and Wnt pathway-mediated SMC specification observed in this system (Shin, 2009).

Muscle progenitors, labeled by the transcription factor Pax7, are responsible for muscle growth during development. The signals that regulate the muscle progenitor number during myogenesis are unknown. This study shows, through in vivo analysis, that Bmp signaling is involved in regulating fetal skeletal muscle growth. Ectopic activation of Bmp signaling in chick limbs increases the number of fetal muscle progenitors and fibers, while blocking Bmp signaling reduces their numbers, ultimately leading to small muscles. The Bmp effect that was observed during fetal myogenesis (muscle growth or secondary myogenesis) is diametrically opposed to that previously observed during embryonic myogenesis (primary myogenesis or formation of the first multinucleated muscle fibers from embryonic progenitors). Bmp signaling regulates the number of satellite cells during development. Finally, Bmp signaling was shown to be active in a subpopulation of fetal progenitors and satellite cells at the extremities of muscles. Overall, these results show that Bmp signaling plays differential roles in embryonic and fetal myogenesis (Wang, 2010).

The embryonic head mesoderm gives rise to cranial muscle and contributes to the skull and heart. Prior to differentiation, the tissue is regionalised by the means of molecular markers. This pattern is shown to be established in three discrete phases, all depending on extrinsic cues. Assaying for direct and first-wave indirect responses, it was found that the process, analyzed in the chicken, is controlled by dynamic combinatorial as well as antagonistic action of retinoic acid (RA), Bmp and Fgf signalling. In phase 1, the initial anteroposterior (a-p) subdivision of the head mesoderm is laid down in response to falling RA levels and activation of Fgf signalling. In phase 2, Bmp and Fgf signalling reinforce the a-p boundary and refine anterior marker gene expression. In phase 3, spreading Fgf signalling drives the a-p expansion of bHLH transcription factor MyoR (musculin) and Tbx1 expression along the pharynx, with RA limiting the expansion of MyoR. This establishes the mature head mesoderm pattern with markers distinguishing between the prospective extra-ocular and jaw skeletal muscles, the branchiomeric muscles and the cells for the outflow tract of the heart (Bothe, 2011).

Expression of Fgf and Bmp responsive molecules indicated that the anterior head mesoderm receives Fgf and Bmp signals for the first time during phase 2 when Alx4 and MyoR are upregulated. Suppression of Bmp signalling prevented, and elevated Bmp signalling advanced, Alx4 activation. Thus, Bmp is necessary and sufficient to control Alx4. MyoR, however, was repressed by suppression of either Bmp or Fgf signalling. Elevation of Bmp or Fgf signalling promoted MyoR, albeit only at the stage at which the gene is normally expressed; premature MyoR expression could only be achieved by combinatorial application of Bmp and Fgf. Thus, combined Fgf and Bmp activity is required to activate MyoR (Bothe, 2011).

Expression analysis showed that the onset of MyoR is rather sudden. The bead implantation experiments indicated that in the anterior head mesoderm, Fgf enhanced the expression of Bmp responsive genes and Bmp upregulated genes indicative of active Fgf signalling. This suggests that Bmp and Fgf reinforce each other, possibly creating the appropriate setting to activate MyoR. Studies on mouse mutants placed Pitx2 upstream of MyoR. Thus, it is conceivable that, in addition to Bmp and Fgf, the earlier activation of Pitx2 is a further prerequisite for the activation of MyoR (Bothe, 2011).

In the posterior head mesoderm, Bmp strongly suppressed Tbx1. Fgf signalling, however, was unaffected, suggesting that Bmp controls the anterior border of Tbx1 expression, possibly directly targeting Tbx1. Tbx1, by contrast, has recently been suggested to suppress Bmp signalling by preventing Smad1-Smad4 interaction. This suggests that Tbx1 indirectly controls the extension of Bmp dependent markers (Bothe, 2011).

When Bmp and Fgf signalling commences in the anterior head mesoderm, Fgf signalling levels increase significantly in the posterior domain, owing to the positive Fgf-Tbx1 feedback loop. After applying Fgf to the anterior head mesoderm, i.e. elevating the Fgf level beyond that which is normally found there, it was noticed that Pitx2 and Alx4 expression declined. Thus, although Fgf is necessary for the activation of MyoR, high Fgf levels prevent the molecular set-up of the anterior head mesoderm. This infers that, whereas Bmp controls the anterior border of the posterior head mesoderm marker, Fgf controls the posterior border of the two anterior markers Pitx2 and Alx4 (Bothe, 2011).

In phase 3, extension of MyoR and Tbx1 expression is concomitant with the spread of high-level Fgf signalling along the floor of the pharynx. Fgf application was found to accelerate the MyoR-Tbx1 spread, and suppression of Fgf signalling prevented it. This suggests that Fgf signalling is key to establishing the final head mesoderm pattern. Notably, MyoR remained sensitive to RA. In the embryo, however, the site of RA production continuously recedes posteriorly during phases 2 and 3, suggesting that the posterior extension of MyoR expression occurs at a rate set by RA (Bothe, 2011).

The anteriorly spreading Fgf signals will eventually reach the Pitx2-Alx4 domain. Both genes were negatively regulated by high Fgf levels in phases 1 and 2; yet, in phase 3 the genes remain expressed. Likewise, Tbx1 spreads anteriorly although this territory is controlled by Bmp. Notably, Fgf levels vary along the anteroposterior extent of the pharynx; at HH13, for example, Fgf signalling appears lower in the anterior compared with the posterior pharyngeal arches. Thus, it is possible that in the anterior head mesoderm, Fgf levels might remain low enough to allow Pitx2 and Alx4 expression, but rise sufficiently to override the Bmp effect on Tbx1. Conversely, the Fgf levels in the posterior head mesoderm might by so high that MyoR expression can spread, whereas Pitx2 and Alx4 remain repressed. It cannot be excluded that additional signals restrict Pitx2 and Alx4 expression. Yet, the spread of MyoR outside of the Pitx2 territory indicates that in phase 3 MyoR expression has become independent from its former upstream regulator (Bothe, 2011).

RA, Bmp and Fgf signalling play multiple roles during development. RA, in many settings, promotes cell differentiation; in the head, RA first suppresses cardiac markers to set the posterior limit of the heart field, but then specifies the sinoatrial region of the heart. Moreover, RA has the capacity to provide cells with a more posterior positional identity. Bmp is a crucial regulator of cardiac development and has been suggested to recruit head mesodermal cells into the cardiac lineage. Fgf promotes the secondary heart field and keeps cells proliferative and undifferentiated. Therefore whether the observed changes in head mesodermal marker expression occurred because of cell recruitment into cardiac lineage, premature differentiation or posteriorisation was tested. RA or Fgf treatment was found not to change cell fate or differentiation status. Bmp induced cardiac marker gene expression only when applied during phase 0. When applied in phase 1, i.e. just before Bmp signalling is normally activated in the head mesoderm, Bmp did not induce cardiac markers unless the dosage was increased. This suggests that, possibly, cardiac induction can occur from exposure to higher Bmp levels and/or longer exposure times. Taken together, this study suggests that RA, Bmp and Fgf specifically control head mesoderm patterning with the cells remaining undifferentiated and competent to enter any of the possible mesodermal lineages (Bothe, 2011).

DPP homologs and morphogenesis: mesodermal patterning - somites

In the vertebrate embryo, the lateral compartment of the somite gives rise to muscles of the limb and body wall and is patterned in response to lateral-plate-derived BMP4. Activation of the myogenic program distinctive to the medial somite, i.e. relatively immediate development of the epaxial muscle lineage, requires neutralization of this lateral signal. The properties of molecules likely to play a role in opposing lateral somite specification by BMP4 were examined. It is proposed that the BMP4 antagonist Noggin plays an important role in promoting medial somite patterning in vivo. Noggin expression in the somite is under the control of a neural-tube-derived factor, whose effect can be mimicked experimentally by Wnt1. Wnt1 is appropriately expressed in the neural tube. It is shown that Sonic Hedgehog, expressed in both the notochord and neural tube is able to activate ectopic expression of Noggin resulting in the blocking of BMP4 specification of the lateral somite. These results are consistent with a model in which Noggin activation in the medial somite lies downstream of the SHH and Wnt pathways signaling from the notochord and neural tube (Hirsinger, 1997).

Signals from the neural tube, notochord, and surface ectoderm promote somitic myogenesis. Somitic myogenesis is under negative regulation as well; BMP signaling serves to inhibit the activation of MyoD and Myf5 in Pax3-expressing cells. BMP-4 is highly expressed in both the dorsal-neural tube and lateral plate mesoderm; when ectopically expressed, between the axial (nerve cord) and paraxial (lateral plate mesoderm) tissues, BMP-4 can block somitic expression of MyoD. BMP antagonist Noggin is expressed within the dorsomedial lip of the dermomyotome, where Pax3-expressing cells first initiate the expression of MyoD and Myf5 to give rise to myotomal cells in the medial somite. Consistent with the expression of Noggin in dorsomedial dermomyotomal cells that lie adjacent to the dorsal neural tube, coculture of somites with fibroblasts programmed to secrete Wnt1 (which is expressed in dorsal neural tube) can induce somitic Noggin expression. Ectopic expression of Noggin lateral to the somite dramatically expands MyoD expression into the lateral regions of the somite, represses Pax3 expression in this tissue, and induces formation of a lateral myotome. Together, these findings indicate that the timing and location of myogenesis within the somite are controlled by relative levels of BMP activity and localized expression of a BMP antagonist (Reshef, 1998).

In vertebrates, the dorsoventral patterning of somitic mesoderm is controlled by factors expressed in adjacent tissues. The ventral neural tube and the notochord function to promote the formation of the sclerotome, a ventral somite derivative, while the dorsal neural tube and the surface ectoderm have been shown to direct somite cells to a dorsal dermomyotomal fate. A number of signaling molecules are expressed in these inducing tissues during times of active cell fate specification, including members of the Hedgehog, Wnt, and BMP families. However, with the exception of the ventral determinant Sonic hedgehog (expressed in the notochord and floor plate of the nerve cord), the functions of these signaling molecules with respect to dorsoventral somite patterning have not been determined. The role of Wnt-1 (expressed in the dorsal neural tube), a candidate dorsalizing factor, has been investigated in the regulation of sclerotome and dermomyotome formation. When ectopically expressed in the presomitic mesoderm of chick embryos in ovo, Wnt-1 differentially affects the expression of dorsal and ventral markers. Specifically, ectopic Wnt-1 is able to completely repress ventral (sclerotomal) markers and to enhance and expand the expression of dorsal (dermomyotomal) markers. However, Wnt-1 appears to be unable to convert all somitic mesoderm to a dermomyotomal fate. Delivery of an activated form of beta-catenin to somitic mesoderm mimics the effects of Wnt-1, demonstrating that Wnt-1 likely acts directly on somitic mesoderm, and not through adjacent tissues via an indirect signal relay mechanism. In response to Shh expression in dorsal somitic tissues, a marked diminution of BMP-4 expression is observed. This finding is consistent with the notion that Shh influences myotome formation through the elimination of BMP-4, which is a known repressor of MyoD transcription. Since MyoD expression is not significantly affected in response to Wnt signaling, it is concluded that Wnt-mediated up-regulation of BMP-4 message is not sufficient to down-regulate MyoD expression. Taken together, these results support a model for somite patterning where sclerotome formation is controlled by the antagonistic activities of Shh and Wnt signaling pathways. Shh is clearly required to suppress dorsal cell fates and promote ventral cell fates (Capdevila, 1998a).

Molecular mechanisms by which the mesoderm is subdivided along the mediolateral axis have been studied in early chick embryos. When the presomitic mesoderm (medial mesoderm) is transplanted into the lateral plate, the graft is transformed into lateral plate tissue, indicating that the primitive somite is not fully committed and that the lateral plate provides a cue for mesodermal lateralization. Since the lateral plate expresses a high level of BMP-4 mRNA, a member of the TGF-ß family, it was hypothesized that BMP-4 is the molecule responsible for the lateralization of the somite. To test this, COS cells producing BMP-4 were transplanted into the presomitic region. Those cells locally prevent the presomitic cells from differentiating into somites, converting them instead into lateral plate mesoderm, which is revealed by expression of cytokeratin mRNA, a marker for the lateral plate. The effect is dependent on the level of effective BMP-4: with a high level of BMP-4, the somite is transformed completely to lateral plate; with a low level, the somite forms but is occupied by the lateral somitic component expressing cSim 1, a marker for the lateral somite. These results suggest that different thresholds of effective BMP-4 determine distinct subtypes of the mesoderm as a lateralizer during early development (Tonegawa, 1997).

Previous work has indicated that signals from the floor plate and notochord promote chondrogenesis of the somitic mesoderm. These tissues, acting through the secreted signaling molecule Sonic hedgehog (Shh), appear to be critical for the formation of the sclerotome. Later steps in the differentiation of sclerotome into cartilage may be independent of the influence of these axial tissues. Although the signals involved in these later steps have not yet been pinpointed, there is substantial evidence that the analogous stages of limb bud chondrogenesis require bone morphogenetic protein (BMP) signaling. Presomitic mesoderm (psm) cultured in the presence of Shh will differentiate into cartilage: the later stages of this differentiation process specifically depend on BMP signaling. Shh not only acts in collaboration with BMPs to induce cartilage, but it changes the competence of target cells to respond to BMPs. In the absence of Shh, BMP administration induces lateral plate gene expression in cultured psm. After exposure to Shh, BMP signaling no longer induces expression of lateral plate markers but now induces robust chondrogenesis in cultured psm. Shh signals are required only transiently for somitic chondrogenesis in vitro, and act to provide a window of competence during which time BMP signals can induce chondrogenic differentiation. These findings suggest that chondrogenesis of somitic tissues can be divided into two separate phases: Shh-mediated generation of precursor cells, which are competent to initiate chondrogenesis in response to BMP signaling, and later exposure to BMPs, which act to trigger chondrogenic differentiation (Murtaugh, 1999).

Tail bud formation in Xenopus depends on interaction between a dorsal domain (dorsal roof) expressing lunatic fringe and Notch, and a ventral domain (posterior wall) expressing the Notch ligand Delta. Ectopic expression of an activated form of Notch, Notch ICD, by means of an animal cap graft into the posterior neural plate, results in the formation of an ectopic tail-like structure containing a neural tube and fin. However, somites are never formed in these tails. BMP signaling is activated in the posterior wall of the tail bud and is involved in the formation of tail somites from this region. Grafts into the posterior neural plate, in which BMP signaling is activated, will form tail-like outgrowths. Unlike the Notch ICD tails, the BMP tails contain well-organized somites as well as neural tube and fin, with the graft contributing to both somites and neural tube. Through a variety of epistasis-type experiments, it has been shown that the most likely model involves a requirement for BMP signaling upstream of Notch activation, resulting in formation of the secondary neural tube, as well as a Notch-independent pathway leading to the formation of tail somites from the posterior wall (Beck, 2001).

Interactions between BMP4 and its inhibitor, noggin, regulate patterning of somites and neural crest. During mesoderm development, noggin mRNA is expressed in the intermediate mesoderm. Upon segmentation, it is detected in the lateral portion of epithelial somites becoming progressively medialized as they mature. In dissociated segments, noggin becomes transiently confined to the dorsomedial lip of the dermomyotome. The factor(s) that control this lateral-to-medial shift in transcription of somitic noggin has been investigated. Inhibition of BMP activity in the caudal lateral plate/intermediate mesoderm prevents noggin transcription in the lateral somite. Further rostrally (that is, later in development), inhibition of tube-derived BMP, but not of Wnt activity, prevents initial noggin expression in the dorsomedial lip of the dermomyotome. Moreover, BMP4 is sufficient to trigger initial expression of noggin even in the absence of ectoderm and/or neural tube, suggesting a direct action on the dorsomedial somite. Thus, the patterns of noggin transcription in somites are directly regulated by BMP4 activities emanating first from the mesoderm and later from the neural tube. Expression patterns of BMP4 and of type IA BMP receptors are spatiotemporally compatible with this lateral-to-medial shift. These results highlight the existence in the neural tube-mesoderm complex of a regulatory loop by which BMP positively regulates transcription of noggin, which in turn represses further ligand activity (Sela-Donenfeld, 2002).

DPP homologs and branching morphogenesis

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, bone morphogenetic protein 7 (Bmp7) is shown to be expressed with a complex pattern in the developing gland and has an important role in regulating branching. In loss-of-function analyses, Bmp7-null mice have been found to have distinctive reductions in lacrimal gland branch number, and 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, isolated mesenchyme and epithelium were tested. 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, a model is proposed suggesting that a discrete region of Bmp7-expressing head mesenchyme may be crucial in determining the location of the exorbital lobe of the gland (Dean, 2004).

DPP homologs and adipogenesis

Adipose tissue is central to the regulation of energy balance. Two functionally different types of fat are present in mammals: white adipose tissue, the primary site of triglyceride storage, and brown adipose tissue, which is specialized in energy expenditure and can counteract obesity. Factors that specify the developmental fate and function of white and brown adipose tissue remain poorly understood. This study demonstrates that whereas some members of the family of bone morphogenetic proteins (BMPs) support white adipocyte differentiation, BMP7 singularly promotes differentiation of brown preadipocytes even in the absence of the normally required hormonal induction cocktail. BMP7 activates a full program of brown adipogenesis including induction of early regulators of brown fat fate PRDM16 (PR-domain-containing 16) and PGC-1alpha (peroxisome proliferator-activated receptor-gamma (PPARgamma) coactivator-1alpha), increased expression of the brown-fat-defining marker uncoupling protein 1 (UCP1) and adipogenic transcription factors PPARgamma and CCAAT/enhancer-binding proteins (C/EBPs), and induction of mitochondrial biogenesis via p38 mitogen-activated protein (MAP) kinase-(also known as Mapk14) and PGC-1-dependent pathways. Moreover, BMP7 triggers commitment of mesenchymal progenitor cells to a brown adipocyte lineage, and implantation of these cells into nude mice results in development of adipose tissue containing mostly brown adipocytes. Bmp7 knockout embryos show a marked paucity of brown fat and an almost complete absence of UCP1. Adenoviral-mediated expression of BMP7 in mice results in a significant increase in brown, but not white, fat mass and leads to an increase in energy expenditure and a reduction in weight gain. These data reveal an important role of BMP7 in promoting brown adipocyte differentiation and thermogenesis in vivo and in vitro, and provide a potential new therapeutic approach for the treatment of obesity (Tseng, 2008).

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