Microinjection of synthetic Xhox-1A homeobox mRNA disrupts somite formation in developing Xenopus embryos

The structural similarity between Drosophila and vertebrate homeobox genes begs the question of whether the vertebrate gene products affect cell fate and pattern formation. To study the function of the Xenopus homeobox protein, Xhox-1A, we microinjected fertilized Xenopus eggs with an excess of synthetic Xhox-RNA and assayed for effects on development. The predominant phenotype is a disturbance in somite formation. When embryos are injected with Xhox-1A mRNA, but not with control mRNAs, morphogenesis of somites occurs chaotically and individual segments are lost. Histological staining, in situ hybridization, and immunohistochemistry indicate that the disorganized somitic tissue has differentiated into muscle cells. Overall, these results suggest that correct regulation of the Xhox-1A gene may be important for the normal development of the segmented somite pattern in early embryos. Moreover, the inferred role of Xhox-1A in somite formation indicates that there may be molecular parallels between mechanisms of segmentation in flies and vertebrates.     

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.     

A Xenopus mRNA related to Drosophila twist is expressed in response to induction in the mesoderm and the neural crest

We have cloned a Xenopus cDNA related to the twist gene, which is required for mesodermal differentiation in Drosophila. Northern blots of dissected embryos and in situ hybridization show that the corresponding mRNA, called Xtwi, first appears in early gastrulae, and is present only in mesodermal cells. Within the mesoderm, Xtwi is expressed in the notochord and lateral plate, but not in the myotome; therefore there is a complementary pattern of Xtwi and muscle-specific gene expression in the mesoderm. Xtwi expression therefore marks the subdivision of the mesoderm. Xtwi is also activated a few hours later in the early development of the neural crest. This gene is thus expressed in response to two sequential early inductions in frog development.     

Dmbx1, a novel evolutionarily conserved paired-like homeobox gene expressed in the brain of mouse embryos.

To identify novel homeobox genes expressed during mouse embryogenesis, we searched the databases and found a novel mouse paired-like homeobox gene, Dmbx1(diencephalon/mesencephalon-expressed brain homeobox gene 1), that is also conserved in zebrafish and human. Linkage analysis mapped mouse Dmbx1 to the mid-portion of chromosome 4 that is the homologous gene cluster region of human chromosome 1, where human DMBX1 is located. Both mouse and human Dmbx1/DMBX1 have four coding exons and their gene structures are conserved. Whole-mount in situ hybridization revealed that Dmbx1 expression is detected in 7.5-9.5 dpc mouse embryos. At 7.5 and 8.5 dpc, Dmbx1 is expressed in a sub-region of the anterior head folds. At 9.5 dpc, expression is observed in the caudal diencephalon as well as in the mesencephalon and is restricted to the neuroepithelium. Expression in adult tissues was detected in brain, stomach, and testis. Dmbx1 provides a unique marker of the developing anterior nervous system and should provide a useful molecular resource to elucidate the mechanisms that pattern the vertebrate brain.     

The lipocalin Xlcpl1 expressed in the neural plate of Xenopus laevis embryos is a secreted retinaldehyde binding protein.

The cellular and structural properties and binding capabilities of a lipocalin expressed in the early neural plate of Xenopus laevis embryos and the adult choroid plexus have been investigated. It was found that this lipocalin, termed Xlcpl1, binds retinal at a nanomolar concentration, retinoic acid in the micromolar range, but does not show binding to retinol. Furthermore, this protein also binds D/L thyroxine. The Xlcpl1 cDNA was expressed in cell culture using the vaccinia virus expression system. In AtT20 cells, Xlcpl1 was secreted via the constitutive secretory pathway. We therefore assume that cpl1 binds retinaldehyde during the transport through the compartments of the secretory pathway that are considered to be the storage compartments of retinoids. Therefore, cpl1-expressing cells will secrete the precursors of active retinoids such as retinoic acid isomers. These retinoids may enter the cytosol by diffusion or receptor-controlled mechanisms, as has been shown for exogenously applied retinoids. Based on these data, it is suggested that cpl1 is an integral member of the retinoid signaling pathway and, therefore, it plays a key role in pattern formation in early embryonic development.     

Pattern and morphogenesis of presumptive superficial mesoderm in two closely related species, Xenopus laevis and Xenopus tropicalis

The mesoderm, comprising the tissues that come to lie entirely in the deep layer, originates in both the superficial epithelial and the deep mesenchymal layers of the early amphibian embryo. Here, we characterize the mechanisms by which the superficial component of the presumptive mesoderm ingresses into the underlying deep mesenchymal layer in Xenopus tropicalis and extend our previous findings for Xenopus laevis. Fate mapping the superficial epithelium of pregastrula stage embryos demonstrates ingression of surface cells into both paraxial and axial mesoderm (including hypochord), in similar patterns and amounts in both species. Superficial presumptive notochord lies medially, flanked by presumptive hypochord and both overlie the deep region of the presumptive notochord. These tissues are flanked laterally by superficial presumptive somitic mesoderm, the anterior tip of which also appears to overlay the presumptive deep notochord. Time-lapse recordings show that presumptive somitic and notochordal cells move out of the roof of the gastrocoel and into the deep region during neurulation, whereas hypochordal cells ingress after neurulation. Scanning electron microscopy at the stage and position where ingression occurs suggests that superficial presumptive somitic cells in X. laevis ingress into the deep region as bottle cells whereas those in X. tropicalis ingress by "relamination" (e.g., [Dev. Biol. 174 (1996) 92]). In both species, the superficially derived presumptive somitic cells come to lie in the medial region of the presumptive somites during neurulation. By the early tailbud stages, these cells lie at the horizontal myoseptum of the somites. The morphogenic pathway of these cells strongly resembles that of the primary slow muscle pioneer cells of the zebrafish. We present a revised fate map of Xenopus, and we discuss the conservation of superficial mesoderm within amphibians and across the chordates and its implications for the role of this tissue in patterning the mesoderm.     

Vsx1, a rapidly evolving paired-like homeobox gene expressed in cone bipolar cells.

The paired-like homeodomain (HD) protein Chx10 is distinguished by the presence of the CVC domain, a conserved 56 amino acid sequence C-terminal to the HD. In mammals, Chx10 is essential both for the proliferation of retinal progenitor cells and for the formation or survival of retinal bipolar interneurons. We describe the cloning and characterization of a mouse Chx10 homologue, Vsx1; phylogenetic analysis suggests that Vsx1 and its putative vertebrate orthologues have evolved rapidly. Vsx1 expression in the adult is predominantly retinal. Whereas Chx10 is expressed both in retinal progenitors in the developing eye and apparently in all bipolar cells of the mature retina, Vsx1 expression is first detected in the eye at postnatal day 5, where it is restricted to cone bipolar cells.     

Differential antero-posterior expression of two proteins encoded by a homeobox gene in Xenopus and mouse embryos.

The X.laevis XlHbox 1 gene uses two functional promoters to produce a short and a long protein, both containing the same homeodomain. In this report we use specific antibodies to localize both proteins in frog embryos. The antibodies also recognize the homologous proteins in mouse embryos. In both mammalian and amphibian embryos, expression of the long protein starts more posteriorly than that of the short protein. This difference in spatial expression applies to the nervous system, the segmented mesoderm and the internal organs. This suggests that each promoter from this gene has precisely restricted regions of expression along the anterior-posterior axis of the embryo. Because the long and short proteins share a common DNA-binding specificity but differ by an 82 amino acid domain, their differential distribution may have distinct developmental consequences.