Evidence that FGF receptor signaling is necessary for endoderm-regulated development of precardiac mesoderm.

Endoderm cells in the heart forming region (HFR endoderm) of stage 6 chicken embryos are required to support the proliferation and terminal differentiation of precardiac mesoderm cells in vitro. The endoderm's effect can be substituted by growth factors, including members of the fibroblast growth factor (FGF) family. However, direct implication of FGFs in this process requires evidence that inhibition of FGF signaling interferes with proliferation and/or terminal differentiation. This report examines the consequences of treating endoderm/precardiac mesoderm co-explants with agents that inactivate FGF receptors. Using sodium chlorate, which prevents FGF ligand-receptor interaction, it was observed that the percentage of S-phase precardiac mesoderm cells was markedly reduced, suggesting that cell proliferation was inhibited. To more specifically affect FGF signaling, the explants were treated with an antibody that recognizes an extracellular domain of FGF receptor-1 (FGFR-1). This treatment similarly inhibited cell proliferation. Although both agents modestly delayed cardiac myocyte differentiation as indicated by the contractile function, expression of alpha-sarcomeric actin was not affected. These findings provide additional evidence that an intact FGF signaling pathway is required during heart development.     

Ventral tail bud mesenchyme is a signaling center for tail paraxial mesoderm induction.

A large body of evidence from several systems indicates that formation of the vertebrate tail is morphogenetically continuous with gastrulation, including neural inducing activity in descendants of the gastrula organizer. However, the signaling centers and molecular events regulating tail mesoderm induction and its organized elongation remain poorly defined. In mammals, the ventral ectoderm ridge (VER) is essential to maintain ongoing formation of paraxial mesoderm and somitogenesis in cultures of intact tail. Avian tail buds contain a similar VER structure. Here, we report that the chick ventral tail bud operates as a signaling center for paraxial mesoderm induction. By using "organizer" style grafting assays to early host embryos, we found that ventral tail bud was able to induce elongated paraxial mesodermal extensions and that the ventral tail bud mesenchyme underlying the VER is both necessary and sufficient for the induction in this assay system. Our observations combined with those of others suggest that interplay between several different signaling centers in the amniote tail bud regulates the coordinate induction and elongation of axial and paraxial structures in the developing tail.     

Ecsit is required for Bmp signaling and mesoderm formation during mouse embryogenesis

Bone morphogenetic proteins (Bmps) are members of the transforming growth factor beta (TGFbeta) superfamily that play critical roles during mouse embryogenesis. Signaling by Bmp receptors is mediated mainly by Smad proteins. In this study, we show that a targeted null mutation of Ecsit, encoding a signaling intermediate of the Toll pathway, leads to reduced cell proliferation, altered epiblast patterning, impairment of mesoderm formation, and embryonic lethality at embryonic day 7.5 (E7.5), phenotypes that mimic the Bmp receptor type1a (Bmpr1a) null mutant. In addition, specific Bmp target gene expression is abolished in the absence of Ecsit. Biochemical analysis demonstrates that Ecsit associates constitutively with Smad4 and associates with Smad1 in a Bmp-inducible manner. Together with Smad1 and Smad4, Ecsit binds to the promoter of specific Bmp target genes. Finally, knock-down of Ecsit with Ecsit-specific short hairpin RNA inhibits both Bmp and Toll signaling. Therefore, these results show that Ecsit functions as an essential component in two important signal transduction pathways and establishes a novel role for Ecsit as a cofactor for Smad proteins in the Bmp signaling pathway.     

XSUMO-1 is required for normal mesoderm induction and axis elongation during early Xenopus development.

The small ubiquitin-related modifier (SUMO) is a member of the ubiquitin-like protein family, and SUMO conjugation (SUMOylation) resembles ubiquitination. Despite many SUMOylation target proteins being reported, the role of this system in vertebrate development remains unclear. We inhibited the function of Xenopus SUMO-1 (XSUMO-1) using a morpholino antisense oligo against XSUMO-1 (XSUMO-1-MO) to clarify the role of SUMOylation. XSUMO-1-MO inhibited normal axis formation in embryos and elongation of activin-treated animal caps. The expression of several mesoderm markers was reduced by XSUMO-1-MO. We measured activin-like activity by using a reporter construct containing a multimer of activin-responsive elements from the Goosecoid promoter, [DE(6x)Luc]. This assay showed that XSUMO-1-MO directly inhibited activin/nodal signaling. Furthermore, XSUMO-1-MO inhibited ectopic axis formation induced by XSmad2, and XSmad2/4 mRNA could not rescue the axis elongation defect induced by XSUMO-1-MO. These results suggested that XSUMO-1 is required for normal axis elongation, at least partly mediating activin/nodal signaling.     

FGFR2, FGF8, FGF10 and BMP7 as candidate genes for hypospadias

Hypospadias is a common malformation, which results from failure of urethral tube closure, and whose molecular mechanisms are still largely unknown. The normal genital development is orchestrated by the urethral plate epithelium (UPE), at the genital tubercle (GT), which has polarizing activity, controlling a network of epithelial-mesenchymal interactions, which, when disturbed, may lead to hypospadias. Homeobox proteins (HOXs), fibroblast growth factors (FGFs) and bone morphogenic proteins (BMPs) are essential in this process. Hypospadias in the Hoxa13 -/- mice occurs as a result of the combined loss of Fgf8 and Bmp7 expression in the UPE. In both Fgf10 and Fgfr2 deficient mutant hypospadic male mice, cell proliferation is arrested prematurely and the maturation of the urethral epithelium is disrupted. Fgf8, Fgf10, and their receptor Fgfr2 are downstream targets of androgens (AR) during external genital development, an important fact given the pivotal role of AR in male sex differentiation. Therefore, we examined FGFR2, FGF10, FGF8, and BMP7 as candidate genes for hypospadias. DNA from 60 boys with familial, isolated, hypospadias was screened for mutations in FGFR2, FGF10, FGF8, and BMP7 genes, using DHPLC and DNA sequence analysis. The sequence variations c.590C>G and c.582-62G>A in FGF8, and, c.550+27C>T, c.727+180T>G, c.830T>C (p.Me186Thr), and c.2454C>T in FGFR2 were found uniquely in patients with hypospadias, as compared with 96 controls. No genetic variant in the other genes was detected. These results indicate that mutations are rare in FGF8 and FGFR2 in hypospadias, but gene variants may influence the risk.     

FGF signaling segregates biliary cell-lineage from chick hepatoblasts cooperatively with BMP4 and ECM components in vitro.

Intrahepatic bile ducts (IHBDs) are indispensable for transporting bile secreted from hepatocytes to the hepatic duct. The biliary epithelial cells (BECs) of the IHBD arise from bipotent hepatoblasts around the portal vein, suggesting the portal mesenchyme is essential for their development. However, except for Notch or Activin/TGF-beta signaling molecules, it is not known which molecules regulate IHBD development. Here, we found that FGF receptors and BMP4 are specifically expressed in the developing IHBD and the hepatic mesenchyme, respectively. Using a mesenchyme-free culture of liver bud, we showed that bFGF and FGF7 induce the hepatoblasts to differentiate into BECs, and that BMP4 enhances bFGF-induced BEC differentiation. The extracellular matrix (ECM) components in the hepatic mesenchyme induced BEC differentiation. Forced expression of a constitutively active form of the FGF receptor partially induced BEC differentiation markers in vivo. These data strongly suggest that bFGF and FGF7 promote BEC differentiation cooperatively with BMP4 and ECMs in vivo.     

BMP and FGF regulatory pathways in semilunar valve precursor cells.

In the developing atrioventricular (AV) valve, limb bud, and somites, cartilage cell lineage differentiation is regulated by bone morphogenetic protein (BMP), while fibroblast growth factor (FGF) controls tendon cell fate. We observed aggrecan and sox9, characteristic of cartilage cell types, and scleraxis and tenascin, characteristic of tendon cell types, in developing avian semilunar valves. Addition of BMP4 to outflow tract (OFT) precursor cells of young (E4.5) but not older (E6) chick embryos activated Smad1/5/8 and induced sox9 and aggrecan expression, while FGF4 treatment increased phosphorylated MAPK (dpERK) signaling and promoted expression of scleraxis and tenascin. These results identify BMP and FGF pathways that promote expression of cartilage- or tendon-like characteristics in semilunar valve precursor cells. In contrast to AV valve precursor cells, which diversify into leaflets (cartilage-like) or chordae tendineae (tendon-like), semilunar valve cells exhibit both cartilage- and tendon-like characteristics in the developing and mature valve cusp.     

breathless, a Drosophila FGF receptor homolog, is essential for migration of tracheal and specific midline glial cells.

A Drosophila homolog of the fibroblast growth factor (FGF) receptor was isolated and structurally characterized. After EMS mutagenesis or imprecise excisions of marked P elements inserted upstream to the gene, a phenotypic series of mutations in the locus was isolated. The mutants exhibit defects in the two embryonic tissues in which the receptor is expressed: the tracheal system and the midline. The tracheal cells fail to migrate in severe mutants and remain within the tracheal pits. Hypomorphic alleles exhibit partial migration of all tracheal branches; thus, the locus was termed breathless (btl). In the midline of the mutant embryos, the posterior pair of midline glial cells begins to migrate anteriorly, but fails to reach the posterior commissure. Abnormalities in cell migration appear to be a common denominator for the btl defects in these two disparate tissues. In hypomorphic mutants the cells exhibit partial migration but follow the normal tracts, suggesting that the presence of this receptor is essential for the ability of the migrating cells to recognize external guiding cues.     

FGF signaling is essential for ophthalmic trigeminal placode cell delamination and differentiation

The ophthalmic trigeminal (opV) placode gives rise exclusively to sensory neurons of the peripheral nervous system, providing an advantageous model for understanding neurogenesis. The signaling pathways governing opV placode development have only recently begun to be elucidated. Here, we investigate the fibroblast growth factor receptor‐4 (FGFR4), an opV expressed gene, to examine if and how FGF signaling regulates opV placode development. After inhibiting FGFR4, Pax3+ opV placode cells failed to delaminate from the ectoderm and did not contribute to the opV ganglion. Blocking FGF signaling also led to a loss of the early and late neuronal differentiation markers Ngn2, Islet‐1, NeuN, and Neurofilament. In addition, without FGF signaling, cells that stalled in the ectoderm lost their opV placode‐specific identity by down‐regulating Pax3. We conclude that FGF signaling, through FGFR4, is necessary for delamination and differentiation of opV placode cells. Developmental Dynamics 238:1073–1082, 2009. © 2009 Wiley‐Liss, Inc.     

The role of FGF and VEGF in angioblast induction and migration during vascular development.

The embryonic vasculature forms by the processes of vasculogenesis and angiogenesis. Angioblasts (endothelial cell precursors) appear to be induced by fibroblast growth factor 2 (FGF-2). The angioblasts contributing to the dorsal aortae arise by an epithelial to mesenchymal transformation of cells originating from the splanchnic mesoderm. QH-l and vascular endothelial growth factor receptor 2 (VEGFR-2) both appear to label these cells as they adopt a mesenchymal morphology. Since VEGFR-2 is the earliest known VEGF receptor this suggests that VEGF is not involved in angioblast induction. VEGF does appear to be critical, however, for growth and morphogenesis of angioblasts into the initial vascular pattern. Controlled delivery of FGF-2 from beads and aggregates of cells transfected with quail VEGF have been used in our laboratory to study the role of these growth factors in angioblast induction and migration. We have induced cells from the epithelial quail somite to differentiate into angioblasts with FGF-2 both in the embryo and in culture. This is a useful model system to study the origins of endothelial cells that are normally more diffusely induced during gastrulation by an obscure process probably involving signals from the embryonic endoderm. The origins of arterial versus venous endothelial cells is also poorly understood but recent findings on the distribution of ephrins and Eph receptors suggest that molecular differences exist prior to the onset of circulation. Finally, studies on the role of growth factors in such diverse phenomena as stem cell biology, angiogenesis, and molecular medicine in addition to vascular development suggest multiple roles for FGF-2 and VEGF in vascular development.     

BMP signaling in dermal papilla cells is required for their hair follicle-inductive properties

Hair follicle (HF) formation is initiated when epithelial stem cells receive cues from specialized mesenchymal dermal papilla (DP) cells. In culture, DP cells lose their HF-inducing properties, but during hair growth in vivo, they reside within the HF bulb and instruct surrounding epithelial progenitors to orchestrate the complex hair differentiation program. To gain insights into the molecular program that maintains DP cell fate, we previously purified DP cells and four neighboring populations and defined their cell-type-specific molecular signatures. Here, we exploit this information to show that the bulb microenvironment is rich in bone morphogenetic proteins (BMPs) that act on DP cells to maintain key signature features in vitro and hair-inducing activity in vivo. By employing a novel in vitro/in vivo hybrid knockout assay, we ablate BMP receptor 1a in purified DP cells. When DPs cannot receive BMP signals, they lose signature characteristics in vitro and fail to generate HFs when engrafted with epithelial stem cells in vivo. These results reveal that BMP signaling, in addition to its key role in epithelial stem cell maintenance and progenitor cell differentiation, is essential for DP cell function, and suggest that it is a critical feature of the complex epithelial-mesenchymal cross-talk necessary to make hair.     

BMP signaling pathways in cartilage and bone formation.

This review focuses on the molecular aspects of osteogenesis and chondrogenesis as they relate to cellular and developmental levels. We discuss BMP (bone morphogenetic protein)-mediated signaling pathways for the patterning of skeletal elements and mechanisms for the induction of cartilage and bone formation. We emphasize BMP-mediated initiation of chondrogenesis and osteogenesis and consider BMP-dependent signaling cascades in mesenchymal progenitor cells during the development of the axial and appendicular skeleton and during growth plate and joint formation. In particular, we discuss BMP-signaling mediators of the Smad and TAK family and the modulation of these signals by cross-talk and integration with other signaling pathways.     

Requirement for antigen in lipopolysaccharide-dependent induction of B cells.

The magnitude of the IgM response to a variety of antigens, induced on the in vivo administration of lipopolysaccharide (LPS) to mice, is approximately proportional to the magnitude of the background response observed in untreated animals. This striking correlation suggests that the administration of LPS alone cannot in general induce a specific response in the absence of a background response. Such is the case for the antigen rat erythrocytes against which no LPS-dependent or background response is found. The response to a marginally immunogenic dose of rat erythrocytes, however, can be considerably enhanced by the administration of LPS. These observations are expected on the hypothesis that both background and LPS-induced responses are due to ongoing antigen-dependent stimulation in normal mice. This hypothesis is further supported by evidence suggesting that the LPS-dependent anti-sheep erythrocyte response is due, at least in part, to a particular antigen present on degraded mouse erythrocytes.