Lydia Besnardeau

Nodal and BMP2/4 Signaling Organizes the Oral-Aboral Axis of the Sea Urchin Embryo

In the sea urchin embryo, the oral-aboral axis is specified after fertilization by mechanisms that are largely unknown. We report that early sea urchin embryos express Nodal and Antivin in the presumptive oral ectoderm and demonstrate that these genes control formation of the oral-aboral axis. Overexpression of nodal converted the whole ectoderm into oral ectoderm and induced ectopic expression of the orally expressed genes goosecoid, brachyury, BMP2/4, and antivin. Conversely, when the function of Nodal was blocked, by injection of an antisense Morpholino oligonucleotide or by injection of antivin mRNA, neither the oral nor the aboral ectoderm were specified. Injection of nodal mRNA into Nodal-deficient embryos induced an oral-aboral axis in a largely non-cell-autonomous manner. These observations suggest that the mechanisms responsible for patterning the oral-aboral axis of the sea urchin embryo may share similarities with mechanisms that pattern the dorsoventral axis of other deuterostomes.

Patterning of the dorsal-ventral axis in echinoderms: insights into the evolution of the BMP-chordin signaling network.

Deciphering the process of dorsal-ventral patterning in the sea urchin reveals an extreme case of BMP translocation and an unusual configuration of the BMP-Chordin axis in echinoderms.

FGF signals guide migration of mesenchymal cells, control skeletal morphogenesis and regulate gastrulation during sea urchin development

The sea urchin embryo is emerging as an attractive model to study morphogenetic processes such as directed migration of mesenchyme cells and cell sheet invagination, but surprisingly, few of the genes regulating these processes have yet been characterized. We present evidence that FGFA, the first FGF family member characterized in the sea urchin, regulates directed migration of mesenchyme cells, morphogenesis of the skeleton and gastrulation during early development. We found that at blastula stages, FGFA and a novel putative FGF receptor are expressed in a pattern that prefigures morphogenesis of the skeletogenic mesoderm and that suggests that FGFA is one of the elusive signals that guide migration of primary mesenchyme cells (PMCs). We first show that fgfA expression is correlated with abnormal migration and patterning of the PMCs following treatments that perturb specification of the ectoderm along the oral-aboral and animal-vegetal axes. Specification of the ectoderm initiated by Nodal is required to restrict fgfA to the lateral ectoderm, and in the absence of Nodal, fgfA is expressed ectopically throughout most of the ectoderm. Inhibition of either FGFA, FGFR1 or FGFR2 function severely affects morphogenesis of the skeleton. Furthermore, inhibition of FGFA and FGFR1 signaling dramatically delays invagination of the archenteron, prevents regionalization of the gut and abrogates formation of the stomodeum. We identified several genes acting downstream of fgfA in these processes, including the transcription factors pea3 and pax2/5/8 and the signaling molecule sprouty in the lateral ectoderm and SM30 and SM50 in the primary mesenchyme cells. This study identifies the FGF signaling pathway as an essential regulator of gastrulation and directed cell migration in the sea urchin embryo and as a key player in the gene regulatory network directing morphogenesis of the skeleton.

A Raf/MEK/ERK signaling pathway is required for development of the sea urchin embryo micromere lineage through phosphorylation of the transcription factor Ets

In the sea urchin embryo, the skeleton of the larva is built from a population of mesenchymal cells known as the primary mesenchyme cells (PMCs). These derive from the large micromeres that originate from the vegetal pole at fourth cleavage. At the blastula stage, the 32 cells of this lineage detach from the epithelium and ingress into the blastocoel by a process of epithelial-mesenchymal transition. We report that shortly before ingression, there is a transient and highly localized activation of the MAP-kinase ERK in the micromere lineage. We show that ingression of the PMCs requires the activity of ERK, MEK and Raf, and depends on the maternal Wnt/β-catenin pathway. Dissociation experiments and injection of mRNA encoding a dominant-negative form of Ras indicated that this activation is probably cell autonomous. We identified the transcription factors Ets1 and Alx1 as putative targets of the phosphorylation by ERK. Both proteins contain a single consensus site for phosphorylation by the MAP kinase ERK. In addition, the Ets1 protein sequence contains a putative ERK docking site. Overexpression of ets1 by injection of synthetic mRNA in the egg caused a dramatic increase in the number of cells becoming mesenchymal at the blastula stage. This effect could be largely inhibited by treating embryos with the MEK inhibitor U0126. Moreover, mutations in the consensus phosphorylation motif substituting threonine 107 by an aspartic or an alanine residue resulted respectively in a constitutively active form of Ets1 that could not be inhibited by U0126 or in an inactive form of Ets1. These results show that the MAP kinase pathway, working through phosphorylation of Ets1, is required for full specification of the PMCs and their subsequent transition from epithelial to mesenchymal state.

Ancestral Regulatory Circuits Governing Ectoderm Patterning Downstream of Nodal and BMP2/4 Revealed by Gene Regulatory Network Analysis in an Echinoderm

Echinoderms, which are phylogenetically related to vertebrates and produce large numbers of transparent embryos that can be experimentally manipulated, offer many advantages for the analysis of the gene regulatory networks (GRN) regulating germ layer formation. During development of the sea urchin embryo, the ectoderm is the source of signals that pattern all three germ layers along the dorsal-ventral axis. How this signaling center controls patterning and morphogenesis of the embryo is not understood. Here, we report a large-scale analysis of the GRN deployed in response to the activity of this signaling center in the embryos of the Mediterranean sea urchin Paracentrotus lividus, in which studies with high spatial resolution are possible. By using a combination of in situ hybridization screening, overexpression of mRNA, recombinant ligand treatments, and morpholino-based loss-of-function studies, we identified a cohort of transcription factors and signaling molecules expressed in the ventral ectoderm, dorsal ectoderm, and interposed neurogenic (“ciliary band”) region in response to the known key signaling molecules Nodal and BMP2/4 and defined the epistatic relationships between the most important genes. The resultant GRN showed a number of striking features. First, Nodal was found to be essential for the expression of all ventral and dorsal marker genes, and BMP2/4 for all dorsal genes. Second, goosecoid was identified as a central player in a regulatory sub-circuit controlling mouth formation, while tbx2/3 emerged as a critical factor for differentiation of the dorsal ectoderm. Finally, and unexpectedly, a neurogenic ectoderm regulatory circuit characterized by expression of “ciliary band” genes was triggered in the absence of TGF beta signaling. We propose a novel model for ectoderm regionalization, in which neural ectoderm is the default fate in the absence of TGF beta signaling, and suggest that the stomodeal and neural subcircuits that we uncovered may represent ancient regulatory pathways controlling embryonic patterning.

Cis-regulatory analysis of nodal and maternal control of dorsal-ventral axis formation by Univin, a TGF-beta related to Vg1.

The TGF-β family member Nodal is essential for specification of the dorsal-ventral axis of the sea urchin embryo, but the molecular factors regulating its expression are not known. Analysis of the nodal promoter is an excellent entry point to identify these factors and to dissect the regulatory logic driving dorsal-ventral axis specification. Using phylogenetic footprinting, we delineated two regulatory regions located in the 5′ region of the nodal promoter and in the intron that are required for correct spatial expression and for autoregulation. The 5′ regulatory region contains essential binding sites for homeodomain, bZIP, Oct, Tcf/Lef, Sox and Smad transcription factors, and a binding site for an unidentified spatial repressor possibly related to Myb. Soon after its initiation, nodal expression critically requires autoregulation by Nodal and signaling by the maternal TGF-β Univin. We show that Univin is related to Vg1, that both Nodal and Univin signal through Alk4/5/7, and that zygotic expression of univin, like that of nodal, is dependent on SoxB1 function and Tcf/β-catenin signaling. This work shows that Tcf, SoxB1 and Univin play essential roles in the regulation of nodal expression in the sea urchin and suggests that some of the regulatory interactions controlling nodal expression predate the chordates. The data are consistent with a model of nodal regulation in which a maternal TGF-β acts in synergy with maternal transcription factors and with spatial repressors to establish the dorsal-ventral axis of the sea urchin embryo.

Lefty acts as an essential modulator of Nodal activity during sea urchin oral-aboral axis formation

Nodal is a key player in the process regulating oral-aboral axis formation in the sea urchin embryo. Expressed early within an oral organizing centre, it is required to specify both the oral and aboral ectoderm territories by driving an oral-aboral gene regulatory network. A model for oral-aboral axis specification has been proposed relying on the self activation of Nodal and the diffusion of the long-range antagonist Lefty resulting in a sharp restriction of Nodal activity within the oral field. Here, we describe the expression pattern of lefty and analyse its function in the process of secondary axis formation. lefty expression starts at the 128-cell stage immediately after that of nodal, is rapidly restricted to the presumptive oral ectoderm then shifted toward the right side after gastrulation. Consistently with previous work, neither the oral nor the aboral ectoderm are specified in embryos in which Lefty is overexpressed. Conversely, when Leftys function is blocked, most of the ectoderm is converted into oral ectoderm through ectopic expression of nodal. Reintroducing lefty mRNA in a restricted territory of Lefty depleted embryos caused a dose-dependent effect on nodal expression. Remarkably, injection of lefty mRNA into one blastomere at the 8-cell stage in Lefty depleted embryos blocked nodal expression in the whole ectoderm consistent with the highly diffusible character of Lefty in other models. Taken together, these results demonstrate that Lefty is essential for oral-aboral axis formation and suggest that Lefty acts as a long-range inhibitor of Nodal signalling in the sea urchin embryo.

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.

Beta-catenin patterns the cell cycle during maternal-to-zygotic transition in urochordate embryos.

During the transition from maternal to zygotic control of development, cell cycle length varies in different lineages, and this is important for their fates and functions. The maternal to zygotic transition (MZT) in metazoan embryos involves a profound remodeling of the cell cycle: S phase length increases then G2 is introduced. Although β-catenin is the master regulator of endomesoderm patterning at MZT in all metazoans, the influence of maternal β-catenin on the cell cycle at MZT remains poorly understood. By studying urochordate embryogenesis we found that cell cycle remodeling during MZT begins with the formation of 3 mitotic domains at the 16-cell stage arising from differential S phase lengthening, when endomesoderm is specified. Then, at the 64-cell stage, a G2 phase is introduced in the endoderm lineage during its specification. Strikingly, these two phases of cell cycle remodeling are patterned by β-catenin-dependent transcription. Functional analysis revealed that, at the 16-cell stage, β-catenin speeds up S phase in the endomesoderm. In contrast, two cell cycles later at gastrulation, nuclear β-catenin induces endoderm fate and delays cell division. Such interphase lengthening in invaginating cells is known to be a requisite for gastrulation movements. Therefore, in basal chordates β-catenin has a dual role to specify germ layers and remodel the cell cycle.

Ascidians: An Emerging Marine Model for Drug Discovery and Screening

Ascidians (tunicates; sea squirts) are marine animals which provide a source of diverse, bioactive natural products, and a model for toxicity screenings. Compounds isolated from ascidians comprise an approved anti-tumor drug and many others are potent drug leads. Furthermore, the use of invertebrate embryos for toxicological screening tests or analysis offers the possibility to image a large number of samples for high throughput screens. Ascidians are members of a sister clade to the vertebrates and make a vertebrate-like tadpole larva composed of less than 3000 cells in 18 hours. The neural complex of the ascidian larva is made of only 350 cells (of which 100 are neurons) and functional genomic studies have now uncovered numerous GRNs underpinning neural specification and differentiation. Numerous studies showed that brain formation in ascidians is sensitive to toxic insults especially from endocrine disruptors making them a suitable model to study neurodevelopmental defects. Modern techniques available for ascidians, including transgenic embryos where 3D time lapse imaging of GFPexpressing reporter constructs can be analyzed, now permit numerous end-points to be evaluated in order to test the specific mode of action of many compounds. This review summarizes the key evidence suggesting that ascidian embryos are a favorable embryological model to study neurodevelopmental toxicity of different compounds with molecular and cellular end-points. We predict that ascidians may become a significant source of marine blue biotechnologies in the 21st century.