DPP homologs and morphogenesis: chondrogenesis and bone growth

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of skeletal growth. Activating mutations in Fgfr3cause achondroplasia, the most common genetic form of dwarfism in humans. Little is known about the mechanism by which FGFR3 inhibits bone growth and how FGFR3 signaling interacts with other signaling pathways that regulate endochondral ossification. To understand these mechanisms, the expression of an activated FGFR3 was targeted to growth plate cartilage in mice using regulatory elements from the collagen II gene. As with humans carrying the achondroplasia mutation, the resulting transgenic mice are dwarfed, with axial, appendicular and craniofacial skeletal hypoplasia. FGFR3 inhibits endochondral bone growth by markedly inhibiting chondrocyte proliferation and by slowing chondrocyte differentiation. Significantly, FGFR3 downregulates the Indian hedgehog (Ihh) signaling pathway and Bmp4 expression in both growth plate chondrocytes and in the perichondrium. Conversely, Bmp4 expression is upregulated in the perichondrium of Fgfr3-/- mice. These data support a model in which Fgfr3 is an upstream negative regulator of the Hedgehog (Hh) signaling pathway. Additionally, Fgfr3 may coordinate the growth and differentiation of chondrocytes with the growth and differentiation of osteoprogenitor cells by simultaneously modulating Bmp4 and patched expression in both growth plate cartilage and in the perichondrium (Naski, 1998).

Bone morphogenetic protein-2 (BMP-2) inhibits terminal differentiation of C2C12 myoblasts and converts them into osteoblast lineage cells. The possible involvement of Smad proteins, vertebrate homologs of Drosophila Mothers against decapentaplegic, has been examined in the BMP effects on the differentiation of C2C12 myoblasts. C2C12 cells express Smad1, Smad2, Smad4, and Smad5 mRNAs, and expression levels are not altered by treatment with BMP-2 or TGF-beta1. When Smads are transiently transfected into C2C12 cells, both Smad1 and Smad5 induce alkaline phosphatase (ALP) activity and decrease the activity of myogenin promoter/chloramphenicol acetyltransferase (myogenin-CAT) without BMP-2. When C-terminal-truncated Smad1 and Smad5 are transfected into constitutively active BMP receptor type IB (BMPR-IB)-expressing C2C12 cells, BMP signals are blocked, resulting in an increase in myogenin-CAT activity. In contrast, Smad1 and Smad5 decrease myogenin-CAT activity but do not induce ALP activity in MyoD-transfected NIH3T3 fibroblasts. These results suggest that both Smad1 and Smad5 are involved in the intracellular BMP signals that inhibit myogenic differentiation and induce osteoblast differentiation in C2C12 cells, and that the conversion of the two differentiation pathways is regulated independently at a transcriptional level (Yamamoto, 1997).

Notochord grafted laterally to the neural tube enhances the differentiation of the vertebral cartilage at the expense of the derivatives of the dermomyotome. In contrast, the dorsomedial graft of a notochord inhibits cartilagedifferentiation of the dorsal part of the vertebra carrying the spinous process. Cartilage differentiation is preceded by the expression of Pax family (Pax1/Pax9, Drosophila homolog: Pox meso) transcription factors in the ventrolateral domain, and Msx family transcription factors in the dorsal domain. The proliferation and differentiation of Msx-expressing cells in the dorsal precartilaginous domain of the vertebra are stimulated by BMP4, which acts upstream of Msx genes. SHH protein arising from the notochord (and floor plate) is necessary for the survival and further development of Pax1/Pax9-expressing sclerotomal cells. Shh acts antagonistically to Bmp4. SHH-producing cells grafted dorsally to the neural tube at E2 inhibit expression of Bmp4 and Msx genes and also inhibit the differentiation of the spinous process (Watanabe, 1998).

In spite of the fact that vertebrae are formed by a single cell type, cartilage, their development involves different molecular pathways according to the vertebral region considered. The ventrolateral part of the vertebra (i.e. vertebral body and neural arches) develops from the ventral sclerotomal cells that express the transcription factor Pax1 before the onset of chondrogenesis. Previous work has shown that chondrogenesis of the ventrolateral part of the vertebra takes place under the influence of the notochord: a supernumerary notochord grafted dorsomedially to the somite extends the Pax1-expressing somitic domain dorsally, and subsequently its differentiation into cartilage takes place to the point that the development of the dorsal somitic derivatives (i.e. the dermomyotome) can be totally suppressed. The most dorsal part of the vertebra that closes the vertebral arch differentiates from mesenchymal cells of somitic origin. This occurs between two ectodermal layers: the superficial ectoderm and the roof plate. Thus, the unilateral graft of quail somites into chick embryos results in the formation of chimeric vertebrae with a hemivertebral body and hemispinous process, and a neural arch made up of donor cells on the operated side and host cells on the intact side. The limit between the host's and donor's territories corresponds strikingly to the sagittal plane of the embryo. Therefore, somitic cells with a chondrogenic fate must migrate medially in order to surround the neural tube and form the vertebral body ventrally and the spinous process dorsally. The cells that migrate dorsally from E3 onward fail to express Pax1 but start to express Msx1 and Msx2 as they become positioned between the superficial ectoderm and the roof plate, which produces BMP4. Moreover, the lateral graft of a roof plate or of cells producing BMP4 induces ectopic expression of Msx genes in the host somitic mesenchyme. Such an induction, however, can occur only if the inducer (e.g. the roof plate) is placed in close proximity to the superficial ectoderm. This supports the contention that bone formation in the subcutaneous site, where the spinous process is formed, is under the control of BMP4, and that Msx genes are involved in the pathway leading to chondrogenesis. This view was confirmed by the fact that overexpression of BMP4 (or of the closely related compound BMP2) dorsal to the neural tube results in the expansion of the Msx1- and Msx2-positive mesenchymal territory and subsequently in the enlargement of the spinous process. Duality in vertebral chondrogenesis was further underlined by the opposite effect of BMPs on the development of the ventrolateral part of the vertebra. Chondrogenesis was strongly inhibited by the graft of BMP2/4-producing cells in a ventrolateral position, with respect to the neural tube (Watanabe, 1998 and references).

These observations raised the question of the nature of the factor of notochord/floor plate origin that is responsible for chondrogenesis in the ventrolateral domain of the vertebra. The most obvious candidate was the protein SHH. Lateral grafts of SHH-producing cells do indeed enhance Pax1 expression in sclerotomal cells and induce the over-development of cartilage laterally at the level of the neural arches. The positive influence of SHH protein on Pax1 expression by somitic cells has already been demonstrated by in vitro experiments and in vivo by the use of retroviral vectors controlling Shh gene expression. This paper demonstrates that enhancement of the number of Pax1-expressing cells by SHH is followed in vivo by the increase in size of the ventrolateral part of the vertebral cartilage. In contrast, dorso-medial grafts of notochord and of SHH-QT6 cells inhibit the expression of the Bmp4 gene in dorsal ectoderm, dorsal mesenchyme and roof plate. Since Msx gene expression has been shown to be controlled by BMP signaling in several induction systems, it is probable that, under the experimental conditions described here, the inhibition of Bmp4 expression is primarily responsible for that of Msx1 and Msx2 and for the failure of chondrogenesis in the dorsal part of the vertebra. This leads to the identification of two molecular pathways in bone development. They concern cartilage and bone formation in 'deep' and 'subectodermal' positions, respectively. Ectoderm has previously been shown to reduce or inhibit chondrogenesis in somitic explant cultures. Such an inhibition is proposed to be relieved by the local production of BMP4 by the dorsal ectoderm and neural tube, thus allowing the formation of superficial bony structures from mesodermal (or mesectodermal) mesenchyme to take place. The deep vertebral cartilage that develops at a distance from the ectoderm and surrounds the notochord and the ventrolateral part of the neural tube requires SHH signaling to differentiate from the sclerotome (Watanabe, 1998 and references).

There is a striking parallel between the expression patterns of the Bmp4, Msx1 and Msx2 genes in the lateral ridges of the neural plate before neural tube closure and later on, in the dorsal neural tube and superficial midline ectoderm. The spinous process of the vertebra is formed from Msx1- and 2-expressing mesenchyme and that the dorsal neural tube can induce the differentiation of subcutaneous cartilage from the somitic mesenchyme. Mouse BMP4- or human BMP2-producing cells grafted dorsally to the neural tube at E2 or E3 increase considerably the amount of Msx-expressing mesenchymal cells which are normally recruited from the somite to form the spinous process of the vertebra. Later on, the dorsal part of the vertebra is enlarged, resulting in vertebral fusion and, in some cases (e.g. grafts made at E3), in the formation of a 'giant' spinous process-like structure dorsally. In strong contrast, BMP-producing cells grafted laterally to the neural tube at E2 exerted a negative effect on the expression of Pax1 and Pax3 genes in the somitic mesenchyme, which then turned on Msx genes. Moreover, sclerotomal cell growth and differentiation into cartilage were then inhibited. Dorsalization of the neural tube, manifested by expression of Msx and Pax3 genes in the basal plate contacting the BMP-producing cells, was also observed. In conclusion, this study demonstrates that differentiation of the ventrolateral and dorsal parts of the vertebral cartilage is controlled by different molecular mechanisms. The former develops under the influence of signals arising from the floor plate-notochord complex. These signals inhibit the development of dorsal subcutaneous cartilage forming the spinous process, which requires the influence of BMP4 to differentiate (Monsoro-Burg, 1996).

Since the bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily that induce the differentiation of mesenchymal precursor cells into the osteogenic cells, the relevant signaling molecules responsible for mediating BMP-2 effects on mesenchymal precursor cells have been identified. BMP-2 induces osteoblastic differentiation of the pluripotent mesenchymal cell line C2C12 by increasing alkaline phosphatase activity and osteocalcin production. Because recent studies have demonstrated that cytoplasmic Smad proteins are involved in TGF-beta superfamily signaling, the relevant Smad family members involved in osteoblastic differentiation have been identifed. Human Smad5 is highly homologous to Smad1. BMP-2 causes serine phosphorylation of Smad5 as well as Smad1. In contrast, TGF-beta fails to cause serine phosphorylation of Smad1 and Smad5. Smad5 is directly activated by BMP type Ia or Ib receptors through physical association with these receptors. Following phosphorylation, Smad5 binds to DPC4, another Smad family member, and the complex is translocated to the nucleus. Overexpression of point-mutated Smad5 (G419S) or a C-terminal deletion mutant DPC4 (DPC4 delta C) blocks the induction of alkaline phosphatase activity, osteocalcin production, and Smad5-DPC4 signaling cascades upon BMP-2 treatment in C2C12 cells. These data suggest that activation of Smad5 and subsequent Smad5-DPC4 complex formation are key steps in the BMP signaling pathway, which mediates BMP-2-induced osteoblastic differentiation of the C2C12 mesenchymal cells (Nishimura, 1998).

Noggin is a bone morphogenetic protein (BMP) antagonist expressed in Spemann's organizer. Murine Noggin is expressed in condensing cartilage and immature chondrocytes, as are many BMPs. In mice lacking Noggin, cartilage condensations initiate normally but develop hyperplasia, and initiation of joint development fails as measured by the expression of growth and differentiation factor-5. The maturation of cartilage and Hoxd expression are unaffected. Excess BMP activity in the absence of Noggin antagonism may enhance the recruitment of cells into cartilage, resulting in oversized growth plates; chondrocytes are also refractory to joint-inducing positional cues (Brunet, 1998).

During endochondral ossification, two secreted signals, Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP), have been shown to form a negative feedback loop regulating the onset of hypertrophic differentiation of chondrocytes. Bone morphogenetic proteins (BMPs), another family of secreted factors regulating bone formation, have been implicated as potential interactors of the Ihh/PTHrP feedback loop. BMP and Ihh/PTHrP signaling were found to interact to coordinate chondrocyte proliferation and differentiation. To analyze the relationship between the two signaling pathways, an organ culture system was used for limb explants of mouse and chick embryos. Chondrocyte differentiation was manipulated by supplementing these cultures either with BMP2, PTHrP and Sonic hedgehog as activators or with Noggin and cyclopamine as inhibitors of the BMP and Ihh/PTHrP signaling systems. Overexpression of Ihh in the cartilage elements of transgenic mice results in an upregulation of PTHrP expression and a delayed onset of hypertrophic differentiation. Noggin treatment of limbs from these mice does not antagonize the effects of Ihh overexpression. Conversely, the promotion of chondrocyte maturation induced by cyclopamine, which blocks Ihh signaling, can not be rescued with BMP2. Thus BMP signaling does not act as a secondary signal of Ihh to induce PTHrP expression or to delay the onset of hypertrophic differentiation. Similar results were obtained using cultures of chick limbs. The role of BMP signaling in regulating proliferation and hypertrophic differentiation of chondrocytes was further investigated and three functions of BMP signaling in this process were identified: (1) maintaining a normal proliferation rate requires BMP and Ihh signaling acting in parallel; (2) a role for BMP signaling in modulating the expression of Ihh has been identified. Third, the application of Noggin to mouse limb explants results in advanced differentiation of terminally hypertrophic cells, implicating BMP signaling in delaying the process of hypertrophic differentiation itself. This role of BMP signaling is independent of the Ihh/PTHrP pathway (Minina, 2001).

These results suggest the following model for the regulation of chondrogenesis by the Ihh/PTHrP and BMP signaling pathways. Ihh produced by prehypertrophic chondrocytes promotes proliferation of the adjacent chondrocytes and, in addition, induces the expression of several Bmp genes in the perichondrium and in the proliferating chondrocytes. Ihh, furthermore, induces the expression of PTHrP in the periarticular region. PTHrP, in turn, negatively regulates the onset of hypertrophic differentiation. The range of PTHrP activity determines the distance from the joint region at which chondrocytes initiate the hypertrophic differentiation program and thereby the size of the domain of chondrocytes that are competent to proliferate. Ihh and BMP signaling together regulate the level of chondrocyte proliferation thereby pushing some cells out of the PTHrP signaling range. These cells are then released from the block of hypertrophic differentiation and activate the expression of Ihh, which, as discussed above, might be directly or indirectly regulated by BMP signaling. Since Ihh signaling regulates the expression of both Bmp genes and PTHrP, it tightly controls its own activation (Minina, 2001).

Because a temporal arrest in the G(1) phase of the cell cycle is thought to be a prerequisite for cell differentiation, cell cycle factors were investigated that critically influence the differentiation of mouse osteoblastic MC3T3-E1 cells induced by bone morphogenetic protein 2 (BMP-2), a potent inducer of osteoblast differentiation. Of the G(1) cell cycle factors examined, the expression of cyclin-dependent kinase 6 (Cdk6) was found to be strongly down-regulated by BMP-2/Smads signaling, mainly via transcriptional repression. The enforced expression of Cdk6 blocked BMP-2-induced osteoblast differentiation to various degrees, depending on the level of its overexpression. However, neither BMP-2 treatment nor Cdk6 overexpression significantly affected cell proliferation, suggesting that the inhibitory effect of Cdk6 on cell differentiation was exerted by a mechanism that is largely independent of its cell cycle regulation. These results indicate that Cdk6 is a critical regulator of BMP-2-induced osteoblast differentiation and that its Smads-mediated down-regulation is essential for efficient osteoblast differentiation (Ogasawara, 2004).

DPP homologs: Blood cell induction

Cultures of Xenopus blastula animal caps were used to explore the hematopoietic effects of three candidate inducers of mesoderm: basic fibroblast growth factor (bFGF), bone morphogenetic proteins (BMPs) and activin A. In response to either bFGF or activin A, explants expand into egg-shaped structures: beneath an outer layer of epidermis, a ventral mesodermal lining surrounds a fluid-filled cavity containing 'blood-like cells'. Immunocytochemistry identifies some of these cells as early leukocytes, but erythrocytes are rare. BMP-2 or BMP-4 induces primitive erythrocytes as well as leukocytes, and a high concentration is required for these cells to differentiate in only a small proportion of explants. BMP-2 but not BMP-4 induces ventral mesoderm concomitantly. High concentrations of activin A dorsalizes explants, which contain infrequent leukocytes, and an optimal combination of activin A and bFGF causes differentiation of muscle with few blood cells. By contrast, BMP-2 or BMP-4 plus activin A synergistically increases the numbers of both leukocytes and erythrocytes. Explants treated with BMPs plus activin contain a well organized cell mass in which yolk-rich cells mix with blood cells and pigmented cells do not. BMP-2 plus bFGF also induces numerous leukocytes and fewer erythrocytes, but BMP-4 antagonizes the leukopoietic effect of bFGF. The data suggest that the signaling pathways these three factors use to induce leukopoiesis overlap and that erythropoiesis may be activated when inducers are present in combination (Miyanaga, 1999).

Blood and blood vessels develop in close association in vertebrate embryos and loss-of-function mutations suggest common genetic regulation. By the criteria of co-expression of blood and endothelial genes, and lineage tracing of progeny, two distinct populations of progenitors for blood and endothelial cells have been located in developing Xenopus embryos. The first population is located immediately posterior to the cement gland during neurula stages and gives rise to embryonic blood and vitelline veins in the anterior ventral blood island (aVBI), and to the endocardium of the heart. The second population resides in the dorsal lateral plate mesoderm, and contains precursors of adult blood stem cells and the major vessels. Both populations differentiate into endothelial cells in situ but migrate to new locations to differentiate into blood, suggesting that their micro-environments are unsuitable for hematopoietic differentiation. Both require BMP for their formation, even the Spemann organizer-derived aVBI, but individual genes are affected differentially. Thus, in the embryonic population, expression of the blood genes SCL and GATA2 depends on BMP signaling, while expression of the endothelial gene Xfli1 does not. By contrast, Xfli1 expression in the adult DLP population does require BMP. These results indicate that both adult and the anterior components of embryonic blood in Xenopus embryos derive from populations of progenitors that also give rise to endothelial cells. However, the two populations give rise to distinct regions of the vasculature and are programmed differentially by BMP (Walmsley, 2002).

In adult vertebrates, fibroblast growth factor (FGF) synergizes with many hematopoietic cytokines to stimulate the proliferation of hematopoietic progenitors. In vertebrate development, the FGF signaling pathway is important in the formation of some derivatives of ventroposterior mesoderm. However, the function of FGF in the specification of the embryonic erythropoietic lineage has remained unclear. The role of FGF in the specification of the erythropoietic lineage in the Xenopus embryo is addressed in this paper. Ventral injection of embryonic FGF (eFGF) mRNA at as little as 10 pg at the four-cell stage suppresses ventral blood island (VBI) formation, whereas expression of the dominant negative form of the FGF receptor in the lateral mesoderm, where physiologically no blood tissue is formed, results in a dramatic expansion of the VBI. Similar results were observed in isolated ventral marginal zones and animal caps. Bone morphogenetic protein-4 (BMP-4) is known to induce erythropoiesis in the Xenopus embryo. Therefore, an examination was carried out of how the BMP-4 and FGF signaling pathways might interact in the decision of ventral mesoderm to form blood. eFGF inhibits BMP-4-induced erythropoiesis by differentially regulating expression of the BMP-4 downstream effectors GATA-2 and PV.1. GATA-2, which stimulates erythropoiesis, is suppressed by FGF. PV.1, which inhibits blood development, is enhanced by FGF. Additionally, PV.1 and GATA-2 negatively regulate transcription of one another. Thus, BMP-4 induces two transcription factors that have opposing effects on blood development. The FGF and BMP-4 signaling pathways interact to regulate the specification of the erythropoietic lineage (Xu, 1999).

Erythroid cell-specific gene regulation during terminal differentiation is controlled by transcriptional regulators, such as EKLF and GATA1, that themselves exhibit tissue-restricted expression patterns. Their early expression, already in evidence within multipotential hematopoietic cell lines, has made it difficult to determine what extracellular effectors and transduction mechanisms might be directing the onset of their own transcription during embryogenesis. To circumvent this problem, the novel approach has been taken of investigating whether the ability of embryonic stem (ES) cells to mimic early developmental patterns of cellular expression during embryoid body (EB) differentiation can address this issue. Conditions were established whereby EBs can form efficiently in the absence of serum. Surprisingly, in addition to mesoderm, these cells expressed hemangioblast and hematopoietic markers. However, they did not express the committed erythroid markers EKLF and GATA1, nor the terminally differentiated ß-like globin markers. Using this system, it has been determined that EB differentiation in BMP4 is necessary and sufficient to recover EKLF and GATA1 expression and differentiation can be further stimulated by the inclusion of VEGF, SCF, erythropoietin and thyroid hormone. EBs are competent to respond to BMP4 only until day 4 of differentiation, which coincides with the normal onset of EKLF expression. The direct involvement of the BMP/Smad pathway in this induction process was further verified by showing that erythroid expression of a dominant negative BMP1B receptor or of the inhibitory Smad6 protein prevents induction of EKLF or GATA1 even in the presence of serum. Although Smad1, Smad5 and Smad8 are all expressed in the EBs, BMP4 induction of EKLF and GATA1 transcription is not immediate. These data implicate the BMP/Smad induction system as being a crucial pathway to direct the onset of EKLF and GATA1 expression during hematopoietic differentiation and demonstrate that EB differentiation can be manipulated to study induction of specific genes that are expressed early within a lineage (Adelman, 2002).

Bone morphogenetic proteins (BMPs) play a role in hematopoiesis that is independent of their function in specifying ventral mesodermal fate. Based on the signaling specificity of the constitutively active and dominant mutant BMP receptors that were used in this study, BMP2, 4 and/or 7 are most likely required for hematopoiesis. Their genes are ubiquitously expressed during early gastrula stages. Thus, the spatial and temporal patterns of expression of these signaling molecules are appropriate for their proposed roles in hematopoiesis. When BMP activity is upregulated or inhibited in Xenopus embryos, hematopoietic precursors are specified properly but few mature erythrocytes are generated. Distinct cellular defects underlie this loss of erythrocytes: inhibition of BMP activity induces erythroid precursors to undergo apoptotic cell death, whereas constitutive activation of BMPs causes an increase in commitment of hematopoietic progenitors to myeloid differentiation and a concomitant decrease in erythrocytes that is not due to enhanced apoptosis. These blood defects are observed even when BMP activity is misregulated solely in non-hematopoietic (ectodermal) cells, demonstrating that BMPs generate extrinsic signals that regulate hematopoiesis independent of mesodermal patterning. Further analysis reveals that endogenous calmodulin-dependent protein kinase IV (CaM KIV) is required to negatively modulate hematopoietic functions of BMPs downstream of receptor activation. These data are consistent with a model in which CaM KIV inhibits BMP signals by activating a substrate, possibly cAMP-response element-binding protein (CREB), that recruits limiting amounts of CREB binding protein (CBP) away from transcriptional complexes functioning downstream of BMPs (Walters, 2002).

Transient Shh signals from the notochord and floor plate confer a competence in somitic tissue for subsequent BMP signals to induce chondrogenesis. It has therefore been proposed that Shh induces a factor(s) that renders somitic cells competent to chondrify in response to subsequent BMP signals. Forced expression of Nkx3.2 (Drosophila homolog: Bagpipe), a transcriptional repressor induced by Shh, is able to confer chondrogenic competence in somites. Administration of Shh or forced Nkx3.2 expression induces the expression of the transcription factor Sox9 in the somitic tissue. Forced expression of Sox9 can, in turn, induce robust chondrogenesis in somitic mesoderm, provided that BMP signals are present. In the presence of BMP signals, Sox9 and Nkx3.2 induce each other's expression. Thus, Nkx3.2 may promote axial chondrogenesis by derepressing the expression of Sox9 in somitic mesoderm. Furthermore, forced expression of either Sox9 or Nkx3.2 not only activates expression of cartilage-specific genes in somitic mesoderm, but also promotes the proliferation and survival of the induced chondrocytes in the presence of BMP signals. However, unlike Nkx3.2, Sox9 is able to induce de novo cartilage formation in non-cartilage-forming tissues. These findings suggest that Shh and BMP signals work in sequence to establish a positive regulatory loop between Sox9 and Nkx3.2, and that Sox9 can subsequently initiate the chondrocyte differentiation program in a variety of cellular environments (Zeng, 2002).

Hematopoietic stem cells (HSCs) are first detected in the floor of the embryonic dorsal aorta (DA), this study investigated the signals that induce the HSC program there. While continued Hedgehog (Hh) signaling from the overlying midline structures maintains the arterial program characteristic of the DA roof, a ventral Bmp4 signal induces the blood stem cell program in the DA floor. This patterning of the DA by Hh and Bmp is the mirror image of that in the neural tube, with Hh favoring dorsal rather than ventral cell types, and Bmp favoring ventral rather than dorsal. With the majority of current data supporting a model whereby HSCs derive from arterial endothelium, these data identify the signal driving this conversion. These findings are important for the study of the production of HSCs from embryonic stem cells and establish a paradigm for the development of adult stem cells (Wilkinson, 2009).

DPP homologs in skeletogenesis and lymphopoesis

Dorsoventral patterning depends on the local concentrations of the morphogens. Twisted gastrulation (TSG) regulates the extracellular availability of a mesoderm inducer, bone morphogenetic protein 4 (BMP-4). However, TSG function in vivo is still unclear. A TSG cDNA was isolated as a secreted molecule from the mouse aorta-gonad-mesonephros region. TSG-deficient mice are born healthy, but more than half of the neonatal pups show severe growth retardation shortly after birth and display dwarfism with delayed endochondral ossification and lymphopenia, followed by death within a month. TSG-deficient thymus is atrophic, and phosphorylation of SMAD1 is augmented in the thymocytes, suggesting enhanced BMP-4 signaling in the thymus. Since BMP-4 promotes skeletogenesis and inhibits thymus development, these findings suggest that TSG acts as both a BMP-4 agonist in skeletogenesis and a BMP-4 antagonist in T-cell development. Although lymphopenia in TSG-deficient mice would partly be ascribed to systemic effects of runtiness and wasting, these findings may also provide a clue for understanding the pathogenesis of human dwarfism with combined immunodeficiency (Nosaka, 2003).

The evolutionarily conserved, secreted protein Twisted gastrulation (Tsg) modulates morphogenetic effects of Decapentaplegic and its orthologs, the Bone morphogenetic proteins 2 and 4 (BMP2/4), in early Drosophila and vertebrate embryos. A role has been uncovered for Tsg at a much later stage of mammalian development, during T cell differentiation in the thymus. BMP4 is expressed by thymic stroma and inhibits the proliferation of CD4(-)CD8(-) double-negative (DN) thymocytes and their differentiation to the CD4(+)CD8(+) double-positive (DP) stage in vitro. Tsg is expressed by thymocytes and up-regulated after T cell receptor signaling at two developmental checkpoints, the transition from the DN to the DP and from the DP to the CD4(+) or CD8(+) single-positive stage. Tsg can synergize with the BMP inhibitor chordin to block the BMP4-mediated inhibition of thymocyte proliferation and differentiation. These data suggest that the developmentally regulated expression of Tsg may allow thymocytes to temporarily withdraw from inhibitory BMP signals (Graf, 2003).

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