François Lapraz

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.

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.

Nodal and BMP2/4 pattern the mesoderm and endoderm during development of the sea urchin embryo.

Nodal factors play fundamental roles in induction and patterning of the mesoderm and endoderm in vertebrates, but whether this reflects an ancient role or one that evolved recently in vertebrates is not known. Here, we report that in addition to its primary role in patterning the ectoderm, sea urchin Nodal is crucial for patterning of the endoderm and skeletogenic mesoderm through the regulation of the expression of key transcription factors and signalling molecules, including BMP2/4 and FGFA. In addition, we uncovered an essential role for Nodal and BMP2/4 in the formation and patterning of the non-skeletogenic mesoderm. By comparing the effects of misexpressing Nodal or an activated Nodal receptor in clones of cells, we provide evidence that Nodal acts over a long range in the endomesoderm and that its effects on the blastocoelar cell precursors are likely to be direct. The activity of Nodal and BMP2/4 are antagonistic, and although bmp2/4 is transcribed in the ventral ectoderm downstream of Nodal, the BMP2/4 ligand is translocated to the dorsal side, where it activates signalling in the dorsal primary mesenchyme cells, the dorsal endoderm and in pigment cell precursors. Therefore, correct patterning of the endomesoderm depends on a balance between ventralising Nodal signals and dorsalising BMP2/4 signals. These experiments confirm that Nodal is a key regulator of dorsal-ventral polarity in the sea urchin and support the idea that the ventral ectoderm, like the Spemann organiser in vertebrates, is an organising centre that is required for patterning all three germ layers of the 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.

A deuterostome origin of the Spemann organiser suggested by Nodal and ADMPs functions in Echinoderms

During development of chordates, establishment of the body plan relies on the activity of an organizing centre located on the dorsal side of the embryo that patterns the embryo and induces neural tissue. Intriguingly, the evolutionary origin of this crucial signalling centre remains unclear and whether analogous organizers regulate D/V patterning in other deuterostome or protostome phyla is not known. Here we provide evidence that the ventral ectoderm of the sea urchin embryo is a long-range organizing centre that shares several fundamental properties with the Spemann organizer: the ability to induce duplicated embryonic axes when ectopically induced, the ability to induce neural fate in neighbouring tissues and the ability to finely regulate the level of BMP signalling by using an autoregulatory expansion–repression mechanism. These findings suggest that the evolutionary origin of the Spemann organizer is more ancient than previously thought and that it may possibly be traced back to the common ancestor of deuterostomes.

The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo

Specification of the dorsal-ventral axis in the highly regulative sea urchin embryo critically relies on the zygotic expression of nodal, but whether maternal factors provide the initial spatial cue to orient this axis is not known. Although redox gradients have been proposed to entrain the dorsal-ventral axis by acting upstream of nodal, manipulating the activity of redox gradients only has modest consequences, suggesting that other factors are responsible for orienting nodal expression and defining the dorsal-ventral axis. Here we uncover the function of Panda, a maternally provided transforming growth factor beta (TGF-β) ligand that requires the activin receptor-like kinases (Alk) Alk3/6 and Alk1/2 receptors to break the radial symmetry of the embryo and orient the dorsal-ventral axis by restricting nodal expression. We found that the double inhibition of the bone morphogenetic protein (BMP) type I receptors Alk3/6 and Alk1/2 causes a phenotype dramatically more severe than the BMP2/4 loss-of-function phenotype, leading to extreme ventralization of the embryo through massive ectopic expression of nodal, suggesting that an unidentified signal acting through BMP type I receptors cooperates with BMP2/4 to restrict nodal expression. We identified this ligand as the product of maternal Panda mRNA. Double inactivation of panda and bmp2/4 led to extreme ventralization, mimicking the phenotype caused by inactivation of the two BMP receptors. Inhibition of maternal panda mRNA translation disrupted the early spatial restriction of nodal, leading to persistent massive ectopic expression of nodal on the dorsal side despite the presence of Lefty. Phylogenetic analysis indicates that Panda is not a prototypical BMP ligand but a member of a subfamily of TGF-β distantly related to Inhibins, Lefty, and TGF-β that includes Maverick from Drosophila and GDF15 from vertebrates. Indeed, overexpression of Panda does not appear to directly or strongly activate phosphoSmad1/5/8 signaling, suggesting that although this TGF-β may require Alk1/2 and/or Alk3/6 to antagonize nodal expression, it may do so by sequestering a factor essential for Nodal signaling, by activating a non-Smad pathway downstream of the type I receptors, or by activating extremely low levels of pSmad1/5/8. We provide evidence that, although panda mRNA is broadly distributed in the early embryo, local expression of panda mRNA efficiently orients the dorsal-ventral axis and that Panda activity is required locally in the early embryo to specify this axis. Taken together, these findings demonstrate that maternal panda mRNA is both necessary and sufficient to orient the dorsal-ventral axis. These results therefore provide evidence that in the highly regulative sea urchin embryo, the activity of spatially restricted maternal factors regulates patterning along the dorsal-ventral axis.

The mitochondrial genomes of the acoelomorph worms Paratomella rubra and Isodiametra pulchra

Acoels are small, ubiquitous, but understudied, marine worms with a very simple body plan. Their internal phylogeny is still in parts unresolved, and the position of their proposed phylum Xenacoelomorpha (Xenoturbella+Acoela) is still debated. Here we describe mitochondrial genome sequences from two acoel species: Paratomella rubra and Isodiametra pulchra. The 14,954 nucleotide-long P. rubra sequence is typical for metazoans in size and gene content. The larger I. pulchra mitochondrial genome contains both ribosomal genes, 21 tRNAs, but only 11 protein-coding genes. We find evidence suggesting a duplicated sequence in the I. pulchra mitochondrial genome. Mitochondrial sequences for both P. rubra and I. pulchra have a unique genome organisation in comparison to other published metazoan mitochondrial genomes. We found a large degree of protein-coding gene and tRNA overlap in P. rubra, with little non-coding sequence making the genome compact. Conversely, the I. pulchra mitochondrial genome has many long non-coding sequences between genes, likely driving the genome size expansion. Phylogenetic trees inferred from concatenated alignments of mitochondrial genes grouped the fast-evolving Acoela and Tunicata, almost certainly due to the systematic error of long branch attraction: a reconstruction artefact that is probably compounded by the fast substitution rate of mitochondrial genes in this taxon.

Left-Right asymmetry in Drosophila: from molecular to organism chirality

SOX2 is expressed throughout the endoderm component. We investigated the involvement of SOX2 in the foregut development using a conditional Sox2-KO mouse line, where Sox2 is inactivated specifically in the endoderm by tamoxifen administration. Endoderm specific Sox2 inactivation resulted in the fusion of esophagus and trachea, bronchi branching directly from the fused duct, and shortening of esophagus. Based on the Sox2-KO embryo phenotypes, we are investigating the following points. 1) Is the loss of Sox2 expression in foregut affect the boundary between foregut and hindgut? 2) How does the loss of SOX2 cause fusion of trachea and esophagus? 3) Are the mesenchymal components involved in the Sox2-KO phenotypes? The complementary or inversely related expression patterns of transcription factors CDX2, NKX2.1, and SOX9 with SOX2 during lung and intestine development have been reported (Que et al., 2007, Gao et al., 2009, Mahony et al., 2014). In addition, the axial identity of digestive tract is considered to be regulated by Hox genes expressed in the mesenchyme at the circumference of the endodermal duct. Investigation is underway to clarify how expression of these transcription factors are affected in the Sox2-KO embryo.