Mesendoderm induction and reversal of left-right pattern by mouse Gdf1, a Vg1-related gene.

TGFbeta signals play important roles in establishing the body axes and germ layers in the vertebrate embryo. Vg1 is a TGFbeta-related gene that, due to its maternal expression and vegetal localization in Xenopus, has received close examination as a potential regulator of development in Xenopus, zebrafish, and chick. However, a mammalian Vg1 ortholog has not been identified. To isolate mammalian Vg1 we screened a mouse expression library with a Vg1-specific monoclonal antibody and identified a single cross-reactive clone encoding mouse Gdf1. Gdf1 is expressed uniformly throughout the embryonic region at 5.5-6.5 days postcoitum and later in the node, midbrain, spinal cord, paraxial mesoderm, lateral plate mesoderm, and limb bud. When expressed in Xenopus embryos, native GDF1 is not processed, similar to Vg1. In contrast, a chimeric protein containing the prodomain of Xenopus BMP2 fused to the GDF1 mature domain is efficiently processed and signals via Smad2 to induce mesendoderm and axial duplication. Finally, right-sided expression of chimeric GDF1, but not native GDF1, reverses laterality and results in right-sided Xnr1 expression and reversal of intestinal and heart looping. Therefore, GDF1 can regulate left-right patterning, consistent with the Gdf1 loss-of-function analysis in the mouse (C. T. Rankin, T. Bunton, A. M. Lawler, and S. J. Lee, 2000, Nature Genet. 24, 262-265) and a proposed role for Vg1 in Xenopus. Our results establish that Gdf1 is posttranslationally regulated, that mature GDF1 activates a Smad2-dependent signaling pathway, and that mature GDF1 is sufficient to reverse the left-right axis. Moreover, these findings demonstrate that GDF1 and Vg1 are equivalent in biochemical and functional assays, suggesting that Gdf1 provides a Vg1-like function in the mammalian embryo.

Expression of bone morphogenetic protein-4 (BMP-4), bone morphogenetic protein-7 (BMP-7), fibroblast growth factor-8 (FGF-8) and sonic hedgehog (SHH) during branchial arch development in the chick.

Expression of Fgf-8, Bmp-4, Bmp-7, and shh in the branchial arches of the chick embryo is examined by in situ hybridization. Fgf-8 expression is initially broad and diffuse, becoming more tightly restricted, particularly in the epithelium of the posterior ectodermal margin (PEM) of the 2nd branchial arch. Bmp-7 transcripts, first seen at stage 12 in discrete regions corresponding to the developing branchial clefts, are later detected in both clefts and arches, including the PEM of the 2nd arch while Bmp-4 transcripts are detected at stage 18 in the distal tips of the arches. Shh expression remains localized, overlapping with both Bmp-7 and Fgf-8 in the PEM of the 2nd arch at stages 16 and 18. Based on these data, a model is proposed for the role of these signalling molecules in branchial arch development.

TGF-beta related genes in development.

Embryonic induction is the process by which signals from one cell population change the developmental fate of another. Polypeptides related to growth factors are one group of molecules mediating many inductive events. Recent data on the embryonic expression and function of signaling proteins related to transforming growth factor beta, in both vertebrate and invertebrate systems, have shown that these molecules play important roles in both pattern formation and tissue specification during embryogenesis.

Expression and growth inhibitory effect of decapentaplegic Vg-related protein 6: evidence for a regulatory role in keratinocyte differentiation.

Decapentaplegic Vg-related protein 6 (DVR-6 or bone morphogenetic protein BMP-6) is a member of the DVR subgroup of the transforming growth factor beta superfamily, a large group of multifunctional signaling polypeptides that are expressed as secreted disulfide-bonded dimers proteolytically cleaved from larger precursors. The predominant expression of DVR-6 in the differentiating postmitotic layers of stratified squamous epithelia strongly suggests a role for DVR-6 in regulation of epithelial differentiation. In primary mouse keratinocytes induced to differentiate by suspension culture in methylcellulose, new expression of DVR-6 mRNA and protein was detected within 8 h among a majority of the suspended cells, which preceded the induction of expression of the suprabasal keratins K1 and K10. To test the hypothesis that DVR-6 is a keratinocyte growth regulatory factor, a retroviral expression vector expressing human DVR-6 was used to infect attached cultures of undifferentiated basal cells. Expression of DVR-6 in primary mouse keratinocytes before differentiation resulted in the secretion of prepro and processed (pro region) forms in the conditioned medium and a dramatic inhibition of cell growth. These findings suggest that inhibition of cell growth by DVR-6 may be a primary step in keratinocyte differentiation.

Biosynthesis and in vivo localization of the decapentaplegic-Vg-related protein, DVR-6 (bone morphogenetic protein-6).

DVR-6 (BMP-6 or Vgr-1) is a member of the TGF-beta superfamily of polypeptide signaling molecules. In situ hybridization studies have previously shown that DVR-6 RNA is expressed in a variety of cell types in the mouse embryo, but no information has been available on protein localization and biosynthesis. We have produced a polyclonal antibody to the proregion of DVR-6 and used it to localize the protein in whole mount and sectioned embryonic, newborn, and adult mouse tissues. DVR-6 protein is expressed in the mouse nervous system beginning at 9.5 days postcoitum (d.p.c.) and continues through adulthood. A variety of epithelial tissues also produce DVR-6 protein, including the suprabasal layer of the skin, bronchiolar epithelium, and the cornea. Additionally, a stably transfected cell line, BMGE+H/D6c4, is used to study the biosynthesis of DVR-6 protein and evidence is presented for translational regulation of DVR-6 expression.

Expression of Hox 2.1 protein in restricted populations of neural crest cells and pharyngeal ectoderm.

A polyclonal antibody, alpha Hox 2.1a, was used to localize Hox 2.1 protein in presumptive neural crest cells and nodose ganglion of 8.5-10.0 day p.c. mouse embryos. The following results were obtained: (1) The nodose placode, in its epithelial state, first expresses Hox 2.1 protein at 9.0 d.p.c. By 9.5 d.p.c. presumptive migrating neuroblasts between the nodose placode and ganglion primordium also express Hox 2.1 protein. (2) At 9.5 d.p.c., presumptive crest cells lateral to the cephalic cardinal vein and within pharyngeal arches 4 and 6 are immunoreactive for alpha Hox 2.1a. In the arch 6 region, positive cells extend medially to a mesenchymal cell population on the lateral aspect of the foregut wall. (3) At 10.0 d.p.c., Hox 2.1 protein expression in putative crest cells is restricted to the arch 6 cell population. A similar staining pattern is seen using alpha Hox 2.1a with chick embryos. Comparison with the chicken embryo suggests that the Hox 2.1 positive cells in the pharyngeal arch and those on the lateral aspect of the foregut in the mouse embryo correspond to the caudalmost subpopulation of the circumpharyngeal crest (Kuratani and Kirby: Am. J. Anat. 191:215-227, 1991; Anat. Rec. 234:263-280, 1992). These results are consistent with a role for Hox 2.1 in pattern formation in the caudalmost region of the vertebrate head.

Expression and modification of Hox 2.1 protein in mouse embryos.

A polyclonal antibody, alpha Hox 2.1a, has been generated and used to immunolocalize Hox 2.1 protein in mouse embryos. Protein is present in nuclei of all tissues previously shown to express Hox 2.1 RNA. In addition, protein is seen in somites and proximal regions of the limb buds, tissues in which Hox 2.1 RNA expression was not clearly detected previously by in situ hybridization. At the 7 somite stage, protein is detectable in the neural tube up to the level of somite 1, but later retracts to a more posterior position. Immunoblot, in vitro translation, and immunoprecipitation experiments were carried out to characterize the Hox 2.1 protein. The results show that the Hox 2.1 gene produces at least two related phosphorylated proteins present in different proportions in different tissues.