BMP and Wnt specify hematopoietic fate by activation of the Cdx-Hox pathway

The formation of blood in the embryo is dependent on bone morphogenetic protein (BMP), but how BMP signaling intersects with other regulators of hematopoietic development is unclear. Using embryonic stem (ES) cells, we show that BMP4 first induces ventral-posterior (V-P) mesoderm and subsequently directs mesodermal cells toward blood fate by activating Wnt3a and upregulating Cdx and Hox genes. When BMP signaling is blocked during this latter phase, enforced expression of either Cdx1 or Cdx4 rescues hematopoietic development, thereby placing BMP4 signaling upstream of the Cdx-Hox pathway. Wnt signaling cooperates in BMP-induced hemogenesis, and the Wnt effector LEF1 mediates BMP4 activation of Cdx genes. Our data suggest that BMP signaling plays two distinct and sequential roles during blood formation, initially as an inducer of mesoderm, and later to specify blood via activation of Wnt signaling and the Cdx-Hox pathway.     

BMP signaling in the epiblast is required for proper recruitment of the prospective paraxial mesoderm and development of the somites

Bmpr1a encodes the BMP type IA receptor for bone morphogenetic proteins (BMPs), including 2 and 4. Here, we use mosaic inactivation of Bmpr1a in the epiblast of the mouse embryo (Bmpr-MORE embryos) to assess functions of this gene in mesoderm development. Unlike Bmpr1a-null embryos, which fail to gastrulate, Bmpr-MORE embryos initiate gastrulation, but the recruitment of prospective paraxial mesoderm cells to the primitive streak is delayed. This delay causes a more proximal distribution of cells with paraxial mesoderm character within the primitive streak, resulting in a lateral expansion of somitic mesoderm to form multiple columns. Inhibition of FGF signaling restores the normal timing of recruitment of prospective paraxial mesoderm and partially rescues the development of somites. This suggests that BMP and FGF signaling function antagonistically during paraxial mesoderm development.     

Defining early lineage specification of human embryonic stem cells by the orchestrated balance of canonical Wnt/beta-catenin, Activin/Nodal and BMP signaling

The canonical Wnt/beta-catenin signaling has remarkably diverse roles in embryonic development, stem cell self-renewal and cancer progression. Here, we show that stabilized expression of beta-catenin perturbed human embryonic stem (hES)-cell self-renewal, such that up to 80% of the hES cells developed into the primitive streak (PS)/mesoderm progenitors, reminiscent of early mammalian embryogenesis. The formation of the PS/mesoderm progenitors essentially depended on the cooperative action of beta-catenin together with Activin/Nodal and BMP signaling pathways. Intriguingly, blockade of BMP signaling completely abolished mesoderm generation, and induced a cell fate change towards the anterior PS progenitors. The PI3-kinase/Akt, but not MAPK, signaling pathway had a crucial role in the anterior PS specification, at least in part, by enhancing beta-catenin stability. In addition, Activin/Nodal and Wnt/beta-catenin signaling synergistically induced the generation and specification of the anterior PS/endoderm. Taken together, our findings clearly demonstrate that the orchestrated balance of Activin/Nodal and BMP signaling defines the cell fate of the nascent PS induced by canonical Wnt/beta-catenin signaling in hES cells.     

Defining early hematopoietic-fated primitive streak specification of human pluripotent stem cells by the orchestrated balance of Wnt,activin, and BMP signaling

Distinct regions of the primitive streak (PS) have diverse potential to differentiate into several tissues, including the hematopoietic lineage originated from the posterior region of PS. Although various signaling pathways have been identified to promote the development of PS and its mesoderm derivatives, there is a large gap in our understanding of signaling pathways that regulate the hematopoietic fate of PS. Here, we defined the roles of Wnt, activin, and bone morphogenetic protein (BMP) signaling pathways in generating hematopoietic-fated PS from human pluripotent stem cells (hPSCs). We found that the synergistic balance of these signaling pathways was crucial for controlling the PS fate determination towards hematopoietic lineage via mesodermal progenitors. Although the induction of PS depends largely on the Wnt and activin signaling, the PS generated without BMP4 lacks the hematopoietic potential, indicating that the BMP signaling is necessary for the PS to acquire hematopoietic property. Appropriate levels of Wnt signaling is crucial for the development of PS and its specification to the hematopoietic lineage. Although the development of PS is less sensitive to activin or BMP signaling, the fate of PS to mesoderm progenitors and subsequent hematopoietic lineage is determined by appropriate levels of activin or BMP signaling. Collectively, our study demonstrates that Wnt, activin, and BMP signaling pathways play cooperative and distinct roles in regulating the fate determination of PS for hematopoietic development. Our understanding of the regulatory networks of hematopoietic-fated PS would provide important insights into early hematopoietic patterning and possible guidance for generating functional hematopoietic cells from hPSCs in vitro.     

BMP2 is a positive regulator of Nodal signaling during left-right axis formation in the chicken embryo

A model of left-right axis formation in the chick involves inhibition of bone morphogenetic proteins by the antagonist Car as a mechanism of upregulating Nodal in the left lateral plate mesoderm. By contrast, expression of CFC, a competence factor, which is absolutely required for Nodal signaling in the lateral plate mesoderm is dependent on a functional BMP signaling pathway. We have therefore investigated the relationship between BMP and Nodal in further detail. We implanted BMP2 and Noggin-expressing cells into the left lateral plate and paraxial mesoderm and observed a strong upregulation of Nodal and its target genes Pitx2 and Nkx3.2. In addition Cfc, the Nodal type II receptor ActrIIa and Snr were found to depend on BMP signaling for their expression. Comparison of the expression domains of Nodal, Bmp2, Car and Cfc revealed co-expression of Nodal, Cfc and Bmp2, while Car and Nodal only partially overlapped. Ectopic application of BMP2, Nodal, and Car as well as combinations of this signaling molecules to the right lateral plate mesoderm revealed that BMP2 and Car need to synergize in order to specify left identity. We propose a novel model of left-right axis formation, which involves BMP as a positive regulator of Nodal signaling in the chick embryo.     

Zebrafish Bmp4 regulates left-right asymmetry at two distinct developmental time points

Left-right (LR) asymmetry is regulated by early asymmetric signals within the embryo. Even though the role of the bone morphogenetic protein (BMP) pathway in this process has been reported extensively in various model organisms, opposing models for the mechanism by which BMP signaling operates still prevail. Here we show that in zebrafish embryos there are two distinct phases during LR patterning in which BMP signaling is required. Using transgenic lines that ectopically express either noggin3 or bmp2b, we show a requirement for BMP signaling during early segmentation to repress southpaw expression in the right lateral plate mesoderm and regulate both visceral and heart laterality. A second phase was identified during late segmentation, when BMP signaling is required in the left lateral plate mesoderm to regulate left-sided gene expression and heart laterality. Using morpholino knock down experiments, we identified Bmp4 as the ligand responsible for both phases of BMP signaling. In addition, we detected bmp4 expression in Kupffer's vesicle and show that restricted knock down of bmp4 in this structure results in LR patterning defects. The identification of these two distinct and opposing activities of BMP signaling provides new insight into how BMP signaling can regulate LR patterning.     

Bone morphogenetic protein acts as a ventral mesoderm modifier in early Xenopus embryos

Mesoderm of early vertebrate embryos gradually acquires dorsal–ventral polarity during embryogenesis. This specification of mesoderm is thought to be regulated by several polypeptide growth factors. Bone morphogenetic protein (BMP), a member of the TGF-β family, is one of the regulators suggested to be involved in the formation of ventral mesoderm. In this paper, the nature of the endogenous BMP signal in dorsal–ventral specification was assessed in early Xenopus embryos using a dominant negative mutant of the Xenopus BMP receptor. In ectodermal explant assays, disruption of endogenous BMP signaling by the mutant receptor changed the competence of the explant cells to mesoderm-inducing factors, activin and basic fibroblast growth factor (bFGF), and led to formation of neural tissue without mesoderm induction. This result suggests that endogenous BMP acts as a ventral mesoderm modifier rather than a ventral mesoderm inducer, and that interactions between endogenous BMP and mesoderm-inducing factors may be important in dorsal–ventral patterning of embryonic mesoderm. In addition, the induction of neural tissue by inhibition of the BMP signaling pathway also suggests involvement of BMP in neural induction.     

Differential requirement for BMP signaling in atrial and ventricular lineages establishes cardiac chamber proportionality

The function of an organ relies upon the proper relative proportions of its individual operational components. For example, effective embryonic circulation requires the appropriate relative sizes of each of the distinct pumps created by the atrial and ventricular cardiac chambers. Although the differences between atrial and ventricular cardiomyocytes are well established, little is known about the mechanisms regulating production of proportional numbers of each cell type. We find that mutation of the zebrafish type I BMP receptor gene alk8 causes reduction of atrial size without affecting the ventricle. Loss of atrial tissue is evident in the lateral mesoderm prior to heart tube formation and results from the inhibition of BMP signaling during cardiac progenitor specification stages. Comparison of the effects of decreased and increased BMP signaling further demonstrates that atrial cardiomyocyte production correlates with levels of BMP signaling while ventricular cardiomyocyte production is less susceptible to manipulation of BMP signaling. Additionally, mosaic analysis provides evidence for a cell-autonomous requirement for BMP signaling during cardiomyocyte formation and chamber fate assignment. Together, our studies uncover a new role for BMP signaling in the regulation of chamber size, supporting a model in which differential reception of cardiac inductive signals establishes chamber proportion.     

Tailbud-derived Bmp4 drives proliferation and inhibits maturation of zebrafish chordamesoderm

In zebrafish, BMP signaling establishes cell identity along the dorsoventral (DV) axis during gastrulation. Owing to the early requirements of BMP activity in DV patterning, it has been difficult to assign later roles in cell fate specification to specific BMP ligands. In this study, we have taken advantage of two follistatin-like genes (fstl1 and fstl2), as well as a transgenic zebrafish line carrying an inducible truncated form of the BMP-type 1 receptor to study the role of Bmp4 outside of the context of DV specification. Characterization of fstl1/2 suggests that they exert a redundant role as BMP antagonists during late gastrulation, regulating BMP activity in axial mesoderm. Maintenance of appropriate levels of BMP signaling is crucial for the proper development of chordamesoderm, a subset of axial mesoderm that gives rise to the notochord, but not prechordal mesoderm, which gives rise to the prechordal plate. Bmp4 activity in particular is required during a crucial window beginning at late gastrulation and lasting through early somitogenesis to promote chordamesoderm proliferation. In the absence of Bmp4, the notochord precursor pool is depleted, and the notochord differentiates prematurely. Our results illustrate a role for Bmp4 in the proliferation and timely differentiation of axial tissue after DV axis specification.     

Inhibition of FGF signaling converts dorsal mesoderm to ventral mesoderm in early Xenopus embryos

In early vertebrate development, mesoderm induction is a crucial event regulated by several factors including the activin, BMP and FGF signaling pathways. While the requirement of FGF in Nodal/activin-induced mesoderm formation has been reported, the fate of the tissue modulated by these signals is not fully understood. Here, we examined the fate of tissues when exogenous activin was added and FGF signaling was inhibited in animal cap explants of Xenopus embryos. Activin-induced dorsal mesoderm was converted to ventral mesoderm by inhibition of FGF signaling. We also found that inhibiting FGF signaling in the dorsal marginal zone, in vegetal-animal cap conjugates or in the presence of the activin signaling component Smad2, converted dorsal mesoderm to ventral mesoderm. The expression and promoter activities of a BMP responsive molecule, PV.1 and a Spemann organizer, noggin, were investigated while FGF signaling was inhibited. PV.1 expression increased, while noggin decreased. In addition, inhibiting BMP-4 signaling abolished ventral mesoderm formation induced by exogenous activin and FGF inhibition. Taken together, these results suggest that the formation of dorso-ventral mesoderm in early Xenopus embryos is regulated by a combination of FGF, activin and BMP signaling.     

Zebrafish Tbx16 regulates intermediate mesoderm cell fate by attenuating Fgf activity

Progenitors of the zebrafish pronephros, red blood and trunk endothelium all originate from the ventral mesoderm and often share lineage with one another, suggesting that their initial patterning is linked. Previous studies have shown that spadetail (spt) mutant embryos, defective in tbx16 gene function, fail to produce red blood cells, but retain the normal number of endothelial and pronephric cells. We report here that spt mutants are deficient in all the types of early blood, have fewer endothelial cells as well as far more pronephric cells compared to wildtype. In vivo cell tracing experiments reveal that blood and endothelium originate in spt mutants almost exclusive from the dorsal mesoderm whereas, pronephros and tail originate from both dorsal and ventral mesoderm. Together these findings suggest possible defects in posterior patterning. In accord with this, gene expression analysis shows that mesodermal derivatives within the trunk and tail of spt mutants have acquired more posterior identity. Secreted signaling molecules belonging to the Fgf, Wnt and Bmp families have been implicated as patterning factors of the posterior mesoderm. Further investigation demonstrates that Fgf and Wnt signaling are elevated throughout the nonaxial region of the spt gastrula. By manipulating Fgf signaling we show that Fgfs both promote pronephric fate and repress blood and endothelial fate. We conclude that Tbx16 plays an important role in regulating the balance of intermediate mesoderm fates by attenuating Fgf activity.     

Rotation and asymmetric development of the zebrafish heart requires directed migration of cardiac progenitor cells

We have used high-resolution 4D imaging of cardiac progenitor cells (CPCs) in zebrafish to investigate the earliest left-right asymmetric movements during cardiac morphogenesis. Differential migratory behavior within the heart field was observed, resulting in a rotation of the heart tube. The leftward displacement and rotation of the tube requires hyaluronan synthase 2 expression within the CPCs. Furthermore, by reducing or ectopically activating BMP signaling or by implantation of BMP beads we could demonstrate that BMP signaling, which is asymmetrically activated in the lateral plate mesoderm and regulated by early left-right signals, is required to direct CPC migration and cardiac rotation. Together, these results support a model in which CPCs migrate toward a BMP source during development of the linear heart tube, providing a mechanism by which the left-right axis drives asymmetric development of the vertebrate heart.     

BMP signaling through ACVRI is required for left-right patterning in the early mouse embryo

Vertebrate organisms are characterized by dorsal-ventral and left-right asymmetry. The process that establishes left-right asymmetry during vertebrate development involves bone morphogenetic protein (BMP)-dependent signaling, but the molecular details of this signaling pathway remain poorly defined. This study tests the role of the BMP type I receptor ACVRI in establishing left-right asymmetry in chimeric mouse embryos. Mouse embryonic stem (ES) cells with a homozygous deletion at Acvr1 were used to generate chimeric embryos. Chimeric embryos were rescued from the gastrulation defect of Acvr1 null embryos but exhibited abnormal heart looping and embryonic turning. High mutant contribution chimeras expressed left-side markers such as nodal bilaterally in the lateral plate mesoderm (LPM), indicating that loss of ACVRI signaling leads to left isomerism. Expression of lefty1 was absent in the midline of chimeric embryos, but shh, a midline marker, was expressed normally, suggesting that, despite formation of midline, its barrier function was abolished. High-contribution chimeras also lacked asymmetric expression of nodal in the node. These data suggest that ACVRI signaling negatively regulates left-side determinants such as nodal and positively regulates lefty1. These functions maintain the midline, restrict expression of left-side markers, and are required for left-right pattern formation during embryogenesis in the mouse.