Christian Gache

Localized VEGF signaling from ectoderm to mesenchyme cells controls morphogenesis of the sea urchin embryo skeleton.

During development, cell migration plays an important role in morphogenetic processes. The construction of the skeleton of the sea urchin embryo by a small number of cells, the primary mesenchyme cells (PMCs), offers a remarkable model to study cell migration and its involvement in morphogenesis. During gastrulation, PMCs migrate and become positioned along the ectodermal wall following a stereotypical pattern that determines skeleton morphology. Previous studies have shown that interactions between ectoderm and PMCs regulate several aspects of skeletal morphogenesis, but little is known at the molecular level. Here we show that VEGF signaling between ectoderm and PMCs is crucial in this process. The VEGF receptor (VEGFR) is expressed exclusively in PMCs, whereas VEGF expression is restricted to two small areas of the ectoderm, in front of the positions where the ventrolateral PMC clusters that initiate skeletogenesis will form. Overexpression of VEGF leads to skeletal abnormalities, whereas inhibition of VEGF/VEGFR signaling results in incorrect positioning of the PMCs, downregulation of PMC-specific genes and loss of skeleton. We present evidence that localized VEGF acts as both a guidance cue and a differentiation signal, providing a crucial link between the positioning and differentiation of the migrating PMCs and leading to morphogenesis of the embryonic skeleton.

Spatial expression of the hatching enzyme gene in the sea urchin embryo

The sea urchin embryo at the blastula stage hatches from its protective fertilization envelope which is degraded by a secreted protease, the hatching enzyme. We have previously purified the hatching enzyme from Paracentrotus lividus (Lepage and Gache (1989). J. Biol. Chem. 264, 4787-4793), cloned its cDNA, and analyzed the temporal expression of its gene (Lepage and Gache (1990). EMBO J. 9, 3003-3012). We study here the temporal and spatial expression of the hatching enzyme gene in whole embryos by immunolabeling with an affinity-purified polyclonal antibody and by in situ hybridization using nonradioactive RNA probes. The timing of expression is consistent with our data on the activation of the gene, the mRNA accumulation in the blastula, and the role of the enzyme. Immunolabeling was observed only in blastula stage embryos; neither before the 128-cell stage nor after hatching. The distribution of the enzyme varies with time from a diffuse labeling around the nucleus to a punctate localization between the nucleus and the apical face of the blastomeres, and finally at the time of hatching, to a submembranous apical location. Not all the cells of an embryo are labeled. The presence of the hatching enzyme is restricted to a sharply delimited continuous territory spanning about two-thirds of the blastula. The orientation of this territory has been determined with respect to the animal-vegetal axis of the embryo using as a landmark the subequatorial pigmented band of the P. lividus species. The synthesis of the hatching enzyme only takes place in the animal-most two-thirds of the blastula. By in situ hybridization, the mRNA coding for the hatching enzyme is only detected in early blastulas, in a limited area having the same size and shape as the territory in which the protein is found. Thus the hatching enzyme gene is likely to be spatially controlled at the transcriptional level: its expression is restricted to a region of the blastula that corresponds roughly to the presumptive ectoderm territory. To date, the hatching enzyme gene products constitute the earliest molecular markers of the sea urchin embryo spatial organization along the primordial egg axis.

Using reporter genes to study cis-regulatory elements

This chapter summarizes four powerful assays for analyzing gene expression in cis-regulatory studies. The enzymatic assays (CAT, luciferase, lacZ) are currently limited by their application to embryo homogenates or fixed samples, but offer more robust analysis of gene activity than GFP. Assays based on CAT enzymatic activity or on CAT mRNA detection by WMISH are laborious but are well established for accurately quantifying gene expression and to determine spatial patterns at defined timepoints during development. LacZ assays are the current standard for spatially visualizing gene products in whole-mount fixed embryos. They are very sensitive but they provide limited temporal or quantitative information due to the perdurance of beta-galactosidase and the subtleties of the staining technique. Recently developed luciferase assays promise to be even more sensitive and accurate than the CAT and lacZ assays, and applicable to living cells and embryos. But, they have not yet been well established in invertebrate deuterostome research. GFP allows visualization of gene expression within living embryos. But because this is not an enzymatic assay, sensitivity can be a problem, particularly for weak promoters. Furthermore, imaging live embryos and quantifying gene expression in space and time (due to scattering of light by tissue, the perdurance of GFP, and other experimental details) is currently fraught with challenges. Ongoing improvements in imaging technology and the advent of multiple fluorescent proteins, as well as fluorescent and luminescent assays for vital imaging, will dramatically facilitate studies of gene expression in the coming decade.

Frizzled5/8 is required in secondary mesenchyme cells to initiate archenteron invagination during sea urchin development.

Wnt signaling pathways play key roles in numerous developmental processes both in vertebrates and invertebrates. Their signals are transduced by Frizzled proteins, the cognate receptors of the Wnt ligands. This study focuses on the role of a member of the Frizzled family, Fz5/8, during sea urchin embryogenesis. During development, Fz5/8 displays restricted expression, beginning at the 60-cell stage in the animal domain and then from mesenchyme blastula stage, in both the animal domain and a subset of secondary mesenchyme cells (SMCs). Loss-of-function analyses in whole embryos and chimeras reveal that Fz5/8 is not involved in the specification of the main embryonic territories. Rather, it appears to be required in SMCs for primary invagination of the archenteron, maintenance of endodermal marker expression and apical localization of Notch receptors in endodermal cells. Furthermore, among the three known Wnt pathways, Fz5/8 appears to signal via the planar cell polarity pathway. Taken together, the results suggest that Fz5/8 plays a crucial role specifically in SMCs to control primary invagination during sea urchin gastrulation.

Nemo-like kinase (NLK) acts downstream of Notch/Delta signalling to downregulate TCF during mesoderm induction in the sea urchin embryo

Studies in Caenorhabditis elegans and vertebrates have established that the MAP kinase-related protein NLK counteracts Wnt signalling by downregulating the transcription factor TCF. Here, we present evidence that during early development of the sea urchin embryo, NLK is expressed in the mesodermal precursors in response to Notch signalling and directs their fate by downregulating TCF. The expression pattern of nlk is strikingly similar to that of Delta and the two genes regulate the expression of each other. nlk overexpression, like ectopic activation of Notch signalling, provoked massive formation of mesoderm and associated epithelial mesenchymal transition. NLK function was found to be redundant with that of the MAP kinase ERK during mesoderm formation and to require the activity of the activating kinase TAK1. In addition, the sea urchin NLK, like its vertebrate counterpart, antagonizes the activity of the transcription factor TCF. Finally, activating the expression of a TCF-VP16 construct at blastula stages strongly inhibits endoderm and mesoderm formation, indicating that while TCF activity is required early for launching the endomesoderm gene regulatory network, it has to be downregulated at blastula stage in the mesodermal lineage. Taken together, our results indicate that the evolutionarily conserved TAK/NLK regulatory pathway has been recruited downstream of the Notch/Delta pathway in the sea urchin to switch off TCF-β-catenin signalling in the mesodermal territory, allowing precursors of this germ layer to segregate from the endomesoderm.

Expression pattern of Brachyury in the embryo of the sea urchin Paracentrotus lividus

Abstract.Brachyury is a key transcription factor whose homologs have been identified in many animal species. Different Brachyury expression patterns have been observed amongst echinoderms. We have isolated PlBra, the Brachyury ortholog from the sea urchin Paracentrotus lividus and analyzed its expression during development. PlBra is first expressed at the end of cleavage in a ring of cells at the border between the presumptive endoderm and mesoderm territories. At later stages, PlBra is expressed around the blastopore and in the stomodaeum area as in most basal deuterostomes.

Coquillette, a sea urchin T-box gene of the Tbx2 subfamily, is expressed asymmetrically along the oral–aboral axis of the embryo and is involved in skeletogenesis

Transcription factors of the T-domain family regulate many developmental processes. We have isolated from the sea urchin a new member of the Tbx2 subfamily: coquillette. Coquillette has a late zygotic expression whose localization is dynamic: at the blastula stage it is restricted to the aboral side of most of the presumptive ectoderm and endoderm territories and from gastrulation on, to the aboral-most primary mesenchyme cells. Perturbation of coquillette function delays gastrulation and strongly disorganizes the skeleton of the larva. Coquillette is sensitive to alteration of the oral-aboral (OA) axis and we identify goosecoid, which controls oral and aboral fates in the ectoderm, as a probable upstream regulator. Coquillette appears to be an integral part of the patterning system along the OA axis.

Frizzled1/2/7 signaling directs β-catenin nuclearisation and initiates endoderm specification in macromeres during sea urchin embryogenesis

In sea urchins, the nuclear accumulation of β-catenin in micromeres and macromeres at 4th and 5th cleavage activates the developmental gene regulatory circuits that specify all of the vegetal tissues (i.e. skeletogenic mesoderm, endoderm and non-skeletogenic mesoderm). Here, through the analysis of maternal Frizzled receptors as potential contributors to these processes, we found that, in Paracentrotus lividus, the receptor Frizzled1/2/7 is required by 5th cleavage for β-catenin nuclearisation selectively in macromere daughter cells. Perturbation analyses established further that Frizzled1/2/7 signaling is required subsequently for the specification of the endomesoderm and then the endoderm but not for that of the non-skeletogenic mesoderm, even though this cell type also originates from the endomesoderm lineage. Complementary analyses on Wnt6 showed that this maternal ligand is similarly required at 5th cleavage for the nuclear accumulation of β-catenin exclusively in the macromeres and for endoderm but not for non-skeletogenic mesoderm specification. In addition, Wnt6 misexpression reverses Frizzled1/2/7 downregulation-induced phenotypes. Thus, the results indicate that Wnt6 and Frizzled1/2/7 are likely to behave as the ligand-receptor pair responsible for initiating β-catenin nuclearisation in macromeres at 5th cleavage and that event is necessary for endoderm specification. They show also that β-catenin nuclearisation in micromeres and macromeres takes place through a different mechanism, and that non-skeletogenic mesoderm specification occurs independently of the nuclear accumulation of β-catenin in macromeres at the 5th cleavage. Evolutionarily, this analysis outlines further the conserved involvement of the Frizzled1/2/7 subfamily, but not of specific Wnts, in the activation of canonical Wnt signaling during early animal development.

Expression of Exogenous mRNAs to Study Gene Function in the Sea Urchin Embryo

Expression of exogenous mRNAs has become part of the standard approach to studying gene function during development of the sea urchin. The method is simple and reliable, protocols for the preparation of synthetic mRNAs are well described, and the technique to transfer them into eggs is efficient. The protein encoded by these mRNAs can be designed to address a variety of biological questions and their DNA matrices are easily constructed by standard molecular biology techniques. The method aims to simulate gain or loss of gene function, and the phenotypes obtained are characterized using an increasing number of molecular markers. With the completion of the S. purpuratus genome project, the complete set of genes from the sea urchin will become available. Expression of mRNA will be an invaluable tool to study the function of newly identified genes and their protein products and to determine their positions within the networks of gene and protein interactions that control development.