Gdf11 is a negative regulator of chondrogenesis and myogenesis in the developing chick limb.

GDF11, a new member of the TGF-beta gene superfamily, regulates anterior/posterior patterning in the axial skeleton during mouse embryogenesis. Gdf11 null mice display skeletal abnormalities that appear to represent anterior homeotic transformations of vertebrae consistent with high levels of Gdf11 expression in the primitive streak, presomitic mesoderm, and tail bud. However, despite strong Gdf11 expression in the limb throughout development, this structure does not appear to be affected in the knockout mice. In order to understand this dichotomy of Gdf11 expression versus Gdf11 function, we identified the chicken Gdf11 gene and studied its role during limb formation. In the early limb bud, Gdf11 transcripts are detected in the subectodermal mesoderm at the distal tip, in a region overlapping the progress zone. At these stages, Gdf11 is excluded from the central core mesenchyme where precartilaginous condensations will form. Later in development, Gdf11 continues to be expressed in the distal most mesenchyme and can also be detected more proximally, in between the forming skeletal elements. When beads incubated in GDF11 protein were implanted into the early wing bud, GDF11 caused severe truncations of the limb that affected both the cartilage elements and the muscle. Limb shortening appeared to be the result of an inhibition of chondrogenesis and myogenesis and using an in vitro micromass assay, we confirmed the negative effects of GDF11 on both myogenic and chondrogenic cell differentiation. Analysis of molecular markers of skeletal patterning revealed that GDF11 induced ectopic expression of Hoxd-11 and Hoxd-13, but not of Hoxa-11, Hoxa-13, or the Msx genes. These data suggest that GDF11 may be involved in controlling the late distal expression of the Hoxd genes during limb development and that misregulation of these Hox genes by excess GDF11 may cause some of the observed alterations in skeletal element shape. In addition, GDF11 induced the expression of its own antagonist follistatin, indicating that the activity of GFD11 may be limited by a negative feedback mechanism. The data from our studies in the chick suggest that Gdf11 plays a role in the formation and development of the avian limb skeleton.

Bone morphogenetic protein-3 is a negative regulator of bone density.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-beta) superfamily. Many BMPs are produced in bone and show osteogenic activity, suggesting that they may be determinants of bone mass. BMP3 was originally purified from bone as osteogenin, which induces osteogenic differentiation. Recombinant BMP3 (rhBMP3) has no biological activity, however, leaving its role in skeletal growth unclear. Here we show that BMP3 is an antagonist of osteogenic BMPs: BMP3 dorsalizes Xenopus laevis embryos, inhibits BMP2-mediated induction of Msx2 and blocks BMP2-mediated differentiation of osteoprogenitor cells into osteoblasts. These effects appear to be mediated through activin receptors. Finally, Bmp3(-/-) mice have twice as much trabecular bone as wild-type littermates, indicating that BMP3, the most abundant BMP in adult bone, is a negative determinant of bone density.

Regulatory regions driving developmental and tissue-specific expression of the essential pancreatic gene pdx1.

pdx1 (pancreatic and duodenal homeobox gene-1), which is expressed broadly in the embryonic pancreas and, later, in a more restricted manner in the mature beta cells in the islets of Langerhans, is essential both for organ formation and beta cell gene expression and function. We carried out a transgenic reporter gene analysis to identify region- and cell type-specific regulatory regions in pdx1. A 14.5-kb pdx1 genomic fragment corrected the glucose intolerance of pdx1(+/-) animals but, moreover, fully rescued the severe gut and pancreas defects in pdx1(-/-) embryos. Sequences sufficient to direct reporter expression to the entire endogenous pdx1 expression domain lie within 4.3 kb of 5' flanking DNA. In this region, we identified two distinct fragments that drive reporter gene expression to different sets of islet neuroendocrine cells. One shows pan-endocrine cell specificity, the other is selectively activated in insulin-producing beta cells. The endocrine-specific regulatory regions overlap a localized region of 5' flanking DNA that is remarkably conserved in sequence between vertebrate pdx1 genes, and which has been associated with beta cell-selective expression in cultured cell lines. This region contains potential binding sites for several transcription factors implicated in endodermal development and the pathogenesis of some forms of type-2 diabetes. These results are consistent with our previous proposal that conserved upstream pdx1 sequences exert control over pdx1 during embryonic organogenesis and islet endocrine cell differentiation. We propose that mutations affecting the expression and/or activity of transcription factors operating via these sequences may predispose towards diabetes, at least in part by direct effects on endocrine pdx1 expression.

A novel BMP expressed in developing mouse limb, spinal cord, and tail bud is a potent mesoderm inducer in Xenopus embryos.

The bone morphogenetic proteins (BMPs) play critical roles in patterning the early embryo and in the development of many organs and tissues. We have identified a new member of this multifunctional gene family, BMP-11, which 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, we tested the protein in the Xenopus ectodermal explant (animal cap) assay. BMP-11 induced axial mesodermal tissue (muscle and notochord) in a dose-dependent fashion. At higher concentrations, BMP-11 also induced neural tissue. Interestingly, the activin antagonist, follistatin, but not noggin, an antagonist of BMPs 2 and 4, inhibited BMP-11 activity on animal caps. Our 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.

Hepatocyte nuclear factor 3beta is involved in pancreatic beta-cell-specific transcription of the pdx-1 gene.

The mammalian homeobox gene pdx-1 is expressed in pluripotent precursor cells in the dorsal and ventral pancreatic bud and duodenal endoderm, which will produce the pancreas and the rostral duodenum. In the adult, pdr-1 is expressed principally within insulin-secreting pancreatic islet beta cells and cells of the duodenal epithelium. Our objective in this study was to localize sequences within the mouse pdx-1 gene mediating selective expression within the islet. Studies of transgenic mice in which a genomic fragment of the mouse pdx-1 gene from kb -4.5 to +8.2 was used to drive a beta-galactosidase reporter showed that the control sequences sufficient for appropriate developmental and adult specific expression were contained within this region. Three nuclease-hypersensitive sites, located between bp -2560 and -1880 (site 1), bp -1330 and -800 (site 2), and bp -260 and +180 (site 3), were identified within the 5'-flanking region of the endogenous pdx-1 gene. Pancreatic beta-cell-specific expression was shown to be controlled by sequences within site 1 from an analysis of the expression pattern of various pdr-1-herpes simplex virus thymidine kinase promoter expression constructs in transfected beta-cell and non-beta-cell lines. Furthermore, we also established that this region was important in vivo by demonstrating that expression from a site 1-driven beta-galactosidase reporter construct was directed to islet beta-cells in transgenic mice. The activity of the site 1-driven constructs was reduced substantially in beta-cell lines by mutating a hepatocyte nuclear factor 3 (HNF3)-like site located between nucleotides -2007 and -1996. Gel shift analysis indicated that HNF3beta present in islet beta cells binds to this element. Immunohistochemical studies revealed that HNF3beta was present within the nuclei of almost all islet beta cells and subsets of pancreatic acinar cells. Together, these results suggest that HNF3beta, a key regulator of endodermal cell lineage development, plays an essential role in the cell-type-specific transcription of the pdx-1 gene in the pancreas.

The Evi1 proto-oncogene is required at midgestation for neural, heart, and paraxial mesenchyme development.

The ecotropic viral integration site-1 (Evi1) locus was initially identified as a common site of retroviral integration in myeloid tumors of the AKXD-23 recombinant inbred mouse strain. The full-length Evi1 transcript encodes a putative transcription factor, containing ten zinc finger motifs found within two domains of the protein. To determine the biological function of the Evi1 proto-oncogene, the full-length, but not an alternately spliced, transcript was disrupted using targeted mutagenesis in embryonic stem cells. Evi1 homozygous mutant embryos die at approximately 10.5 days post coitum. Mutants were distinguished at 10.5 days post coitum by widespread hypocellularity, hemorrhaging, and disruption in the development of paraxial mesenchyme. In addition, defects in the heart, somites, and cranial ganglia were detected and the peripheral nervous system failed to develop. These results correlated with whole-mount in situ hybridization analyses of embryos which showed expression of the Evi1 proto-oncogene in embryonic mesoderm and neural crest-derived cells associated with the peripheral nervous system. These data suggest that Evi1 has important roles in general cell proliferation, vascularization, and cell-specific developmental signaling, at midgestation.

Autonomous endodermal determination in Xenopus: regulation of expression of the pancreatic gene XlHbox 8.

In neural plate stage Xenopus embryos, XlHbox 8 expression marks anterior endodermal cells fated to develop into pancreas/duodenum, and expression continues in adult pancreas in exocrine duct, acinar, and islet cells. Here, XlHbox 8 is used as a marker in experiments addressing the mechanisms of early endodermal patterning, particularly with respect to the role of specific polypeptide growth factors. When mesoderm-free vegetal explants (VEs) from early blastula stage embryos are cultured in isolation, XlHbox 8 expression develops autonomously in the dorsal region, strongly suggesting that endodermal region-specific determination occurs before MBT. Data from microinjection experiments using RNA encoding the activin and FGF dominant negative receptors and growth factor treatments of isolated VEs suggest that activin positively regulates XlHbox 8 expression, whereas bFGF is a potent negative regulator. Moreover, bFGF induces mesodermal marker expression in VEs. This suggests that the early endodermal determination state is plastic and that elevated levels of bFGF may convert vegetal (endodermal) cells into mesoderm. We propose a model for XlHbox 8 regulation in which an early signal from the Nieuwkoop center (whose eventual fate is endoderm) predisposes dorsovegetal cells for autonomous XlHbox 8 expression, in an area of high local activin (or activin-like) ligand concentration, and low relative concentrations of bFGF.

Expression of murine STF-1, a putative insulin gene transcription factor, in beta cells of pancreas, duodenal epithelium and pancreatic exocrine and endocrine progenitors during ontogeny.

The XlHbox 8 homeodomain protein of Xenopus and STF-1, its mammalian homolog, are selectively expressed by beta cells of adult mouse pancreatic islets, where they are likely to regulate insulin expression. We sought to determine whether the expression of the homeobox protein/s during mouse embryonic development was specific to beta cells or, alternatively, whether XlHbox 8/STF-1 protein/s were initially expressed by multipotential precursors and only later became restricted to the insulin-containing cells. With two antibodies, we studied the localization of STF-1 during murine pancreatic development. In embryos, as in adults, STF-1 was expressed by most beta cells, by subsets of the other islet cell types and by mucosal epithelial cells of the duodenum. In addition, most epithelial cells of the pancreatic duct and exocrine cells of the pancreas transiently contained STF-1. We conclude that in mouse, STF-1 not only labels a domain of intestinal epithelial cells but also provides a spatial and temporal marker of endodermal commitment to a pancreatic and subsequently, to an endocrine beta cell fate. We propose a model of pancreatic cell development that suggests that exocrine and endocrine (alpha, beta, delta and PP) cells arise from a common precursor pool of STF-1+ cells and that progression towards a defined monospecific non-beta cell type is correlated with loss of STF-1 expression.

Murine Cdx-4 bears striking similarities to the Drosophila caudal gene in its homeodomain sequence and early expression pattern.

A third member of the murine caudal-like gene family, Cdx-4, has been isolated. In situ hybridization and immunohistochemistry have been used to study the localization of mRNA and protein during murine embryogenesis. Cdx-4 is expressed transiently from 7.0 d.p.c. (days post coitum) until 10 d.p.c., starting at the beginning of gastrulation (7.0-7.5 d.p.c.) in the allantois and posterior tip of the primitive streak. At the mid-streak stage, Cdx-4 expression moves rostrally, and protein and mRNA are detected in all cells over the posterior half of the primitive streak. As development proceeds, Cdx-4 gene products continue to be restricted to the posterior of the embryo, including the remnants of the primitive streak. Cdx-4 is expressed in neurectoderm, presomitic and lateral plate mesoderm, and hindgut endoderm, but the anterior boundary in the paraxial mesoderm is staggered with respect to the other germ layers. At all stages analyzed, Cdx-4 exhibits a graded expression pattern with a posterior maximum, a distribution highly reminiscent of the Drosophila caudal gene. These data add to the recently described distributions of other vertebrate caudal-like genes, and further support the idea that members of this homeobox gene subfamily have regulatory roles in the specification of anteroposterior axial polarity in early embryos.