Characterization and expression of a presomitic mesoderm-specific<Emphasis Type="Italic"> mespo</Emphasis> gene in zebrafish

A complete zebrafish mespo cDNA encoding a protein of 131 amino acids with a bHLH domain in the C-terminal has been isolated. The bHLH domain of zebrafish Mespo is highly similar to those in the mouse, chick and Xenopus, sharing 82.4%, 80.4% and 74.5% amino acid identity, respectively. At 50% epiboly, the zebrafish mespo is first detected in the marginal zone of the blastoderm but excluding the prospective shield. Subsequently, mespo expression is intensified in the involuting mesoderm at 60% epiboly, and then restricted to the presomitic mesoderm (PSM) at 95% epiboly. At the 1-somite stage, mespo expression becomes reduced in the most rostral PSM. During segmentation, mespo expression is gradually downregulated at the most rostral segmental plate where cells are being coalesced to form somites. In spadetail mutant embryos, most of mespo-expressing cells were missing.     

Wnt/beta-catenin signaling controls Mespo expression to regulate segmentation during Xenopus somitogenesis

The vertebral column is derived from somites, which are transient segments of the paraxial mesoderm that are present in developing vertebrates. The strict spatial and temporal regulation of somitogenesis is of crucial developmental importance. Signals such as Wnt and FGF play roles in somitogenesis, but details regarding how Wnt signaling functions in this process remain unclear. In this study, we report that Wnt/beta-catenin signaling regulates the expression of Mespo, a basic-helix-loop-helix (bHLH) gene critical for segmental patterning in Xenopus somitogenesis. Transgenic analysis of the Mespo promoter identifies Mespo as a direct downstream target of Wnt/beta-catenin signaling pathway. We also demonstrate that activity of Wnt/beta-catenin signaling in somitogenesis can be enhanced by the PI3-K/AKT pathway. Our results illustrate that Wnt/beta-catenin signaling in conjunction with PI3-K/AKT pathway plays a key role in controlling development of the paraxial mesoderm.     

Cyclic expression of esr9 gene in Xenopus presomitic mesoderm

During somitogenesis, the cycling expression of members of the Notch signalling cascade is involved in a segmentation clock that regulates the periodic budding of somites in chicken, mouse, and zebrafish. In frog, genes with cycling expression in the presomitic mesoderm have not been reported. Here, we describe the expression of Xenopus esr9 and esr10, two new members of the Hairy/Enhancer of split related family of bHLH proteins. We show that they are expressed in a highly dynamic fashion, with their mRNA levels oscillating periodically in the presomitic mesoderm during somitogenesis. This dynamic expression is independent of de novo protein synthesis. Thus, expression of esr9 and esr10 is an indicator of the segmentation clock in the amphibian embryo. This confirms the evolutionary conservation of a molecular pathway involved in vertebrate segmentation clock.     

Surface ectoderm is necessary for the morphogenesis of somites

The paraxial mesoderm of the neck and trunk of mouse embryos undergoes extensive morphogenesis in forming somites. Paraxial mesoderm is divided into segments, it elongates along its anterior posterior axis, and its cells organize into epithelia. Experiments were performed to determine if these processes are autonomous to the mesoderm that gives rise to the somites. Presomitic mesoderm at the tailbud stage was cultured in the presence and absence of its adjacent tissues. Somite segmentation occurred in the absence of neural tube, notochord, gut and surface ectoderm, and occurred in posterior fragments in the absence of anterior presomitic mesoderm. Mesodermal expression of Dll1 and Notch1, genes with roles in segmentation, was largely independent of other tissues, consistent with autonomous segmentation. However, surface ectoderm was found to be necessary for elongation of the mesoderm along the anterior-posterior axis and for somite epithelialization. To determine if there is specificity in the interaction between ectoderm and mesoderm, ectoderm from different sources was recombined with presomitic mesoderm. Surface ectoderm from only certain parts of the embryo supported somite epithelialization and elongation. Somite epithelialization induced by ectoderm was correlated with expression of the basic-helix-loop-helix gene Paraxis in the mesoderm. This is consistent with the genetically defined requirement for Paraxis in somite epithelialization. However, trunk ectoderm was able to induce somite epithelialization in the absence of strong Paraxis expression. We conclude that somitogenesis consists of autonomous segmentation patterned by Notch signaling and nonautonomous induction of elongation and epithelialization by surface ectoderm.     

Bmp inhibition is necessary for post-gastrulation patterning and morphogenesis of the zebrafish tailbud

Intricate interactions between the Wnt and Bmp signaling pathways pattern the gastrulating vertebrate embryo using a network of secreted protein ligands and inhibitors. While many of these proteins are expressed post-gastrula, their later roles have typically remained unclear, obscured by the effects of early perturbation. We find that Bmp signaling continues during somitogenesis in zebrafish embryos, with high activity in a small region of the mesodermal progenitor zone at the posterior end of the embryo. To test the hypothesis that Bmp inhibitors expressed just anterior to the tailbud are important to restrain Bmp signaling we produced a new zebrafish transgenic line, allowing temporal cell-autonomous activation of Bmp signaling and thereby bypassing the effects of the Bmp inhibitors. Ectopic activation of Bmp signaling during somitogenesis results in severe defects in the tailbud, including altered morphogenesis and gene expression. We show that these defects are due to non-autonomous effects on the tailbud, and present evidence that the tailbud defects are caused by alterations in Wnt signaling. We present a model in which the posteriorly expressed Bmp inhibitors function during somitogenesis to constrain Bmp signaling in the tailbud in order to allow normal expression of Wnt inhibitors in the presomitic mesoderm, which in turn constrain the levels of canonical and non-canonical Wnt signaling in the tailbud.     

XHRT-1, a hairy and Enhancer of split related gene with expression in floor plate and hypochord during early Xenopus embryogenesis

We have isolated a Xenopus homologue of the mammalian hairy and Enhancer of split related gene HRT1. XHRT1 expression in late gastrula and early neurula embryos is restricted to two stripes of cells in the medial neural plate and in dorsal endodermal cells. At later stages, XHRT1 is expressed in the floor plate, in hypochord cells and in the somitogenic and anterior presomitic mesoderm. By tailbud stage, XHRT1 is also highly expressed in the dorsal hindbrain, telencephalon and eye vesicles, olfactory placodes, pronephros, branchial arches and tail fin. We also show that XHRT1 expression in medial neural cells is induced by Notch signaling and that there are differences in the way XHRT1 and other H/E(spl) genes are regulated.     

Developmental expression of Shisa-2 in Xenopus laevis

Shisa is an antagonist of Wnt and FGF signaling, that functions cell autonomously in the endoplasmic reticulum (ER) to inhibit the post-translational maturation of Wnt and FGF receptors. In this paper we report the isolation of a second Xenopus shisa gene (Xshisa-2). Xenopus Shisa-2 shows 30.7% identity to Xshisa. RT-PCR analysis indicated that Xshisa-2 mRNA is present throughout early development and shows an increased expression during neurula and tailbud stages. At neurula stages Xenopus shisa-2 is initially expressed in the presomitic paraxial mesoderm and later in the developing somites. The expression profiles and pattern of Xshisa and Xshisa-2 differ significantly. During gastrulation only Xshisa mRNA is present in the Spemann-Mangold organizer and later on becomes restricted to the neuroectoderm and the prechordal plate.     

Priming, initiation and synchronization of the segmentation clock by deltaD and deltaC

Zebrafish somitogenesis is governed by a segmentation clock that generates oscillations in expression of several Notch pathway genes, including her1, her7 and deltaC. Using a combination of pharmacological inhibition and Mendelian genetics, we show that DeltaD and DeltaC, two Notch ligands, represent functionally distinct signals within the segmentation clock. Using high-resolution fluorescent in situ hybridization, the oscillations were divided into phases based on eight distinct subcellular patterns of mRNA localization for 140,000 cells. her1, her7 and deltaC expression was examined in wild-type, deltaD(-/-) and deltaC(-/-) embryos. We identified areas within the tailbud where the clock is set up in the progenitor cells (priming), where the clock starts running (initiation), and where the clocks of neighbouring cells are entrained (synchronization). We find that the clocks of motile cells are primed by deltaD in a progenitor zone in the posterior tailbud and that deltaD is required for cells to initiate oscillations on exiting this zone. Oscillations of adjacent cells are synchronized and amplified by deltaC in the posterior presomitic mesoderm as cell movement subsides and cells maintain stable neighbour relationships.     

The role of presenilin 1 during somite segmentation

The Notch signalling pathway plays essential roles during the specification of the rostral and caudal somite halves and subsequent segmentation of the paraxial mesoderm. We have re-investigated the role of presenilin 1 (Ps1; encoded by Psen1) during segmentation using newly generated alleles of the Psen1 mutation. In Psen1-deficient mice, proteolytic activation of Notch1 was significantly affected and the expression of several genes involved in the Notch signalling pathway was altered, including Delta-like3, Hes5, lunatic fringe (Lfng) and Mesp2. Thus, Ps1-dependent activation of the Notch pathway is essential for caudal half somite development. We observed defects in Notch signalling in both the caudal and rostral region of the presomitic mesoderm. In the caudal presomitic mesoderm, Ps1 was involved in maintaining the amplitude of cyclic activation of the Notch pathway, as represented by significant reduction of Lfng expression in Psen1-deficient mice. In the rostral presomitic mesoderm, rapid downregulation of the Mesp2 expression in the presumptive caudal half somite depends on Ps1 and is a prerequisite for caudal somite half specification. Chimaera analysis between Psen1-deficient and wild-type cells revealed that condensation of the wild-type cells in the caudal half somite was concordant with the formation of segment boundaries, while mutant and wild-type cells intermingled in the presomitic mesoderm. This implies that periodic activation of the Notch pathway in the presomitic mesoderm is still latent to segregate the presumptive rostral and caudal somite. A transient episode of Mesp2 expression might be needed for Notch activation by Ps1 to confer rostral or caudal properties. In summary, we propose that Ps1 is involved in the functional manifestation of the segmentation clock in the presomitic mesoderm.