Gene regulatory networks, axis specification and morphogenesis of the sea urchin embryo

Overview of research themes

The main goal of our research is to answer fundamental questions such as how an embryo with multiple differentiated cell types can develop from a single cell and how is the linear information encoded in the DNA used to shape the embryo during gastrulation and morphogenesis. More specifically, we try to understand how the dorsal-ventral and left-right axes of polarity are specified starting from an egg with a much simpler organization. We also analyze the mechanisms regulating formation and morphogenesis of the skeleton of the larva, a process that involves epithelial-mesenchymal transition and oriented cell migration and dissect the gene regulatory networks that orchestrate these processes. As a broad approach to sea urchin development, we are doing a large scale in situ screen which has generated a number of useful markers for studying specification of cell fates and patterning of the embryo.

Figure 1. Early development and morphogenesis of the sea urchin embryo.


Dorsal-ventral patterning of the sea urchin embryo

In the sea urchin, as in vertebrates, the TGF-beta signals Nodal and BMP2/4 play crucial roles in the establishment of dorsal-ventral polarity of the embryo (Duboc and Lepage, 2006; Duboc et al., 2004). Expression of nodal is initiated around the 32-60-cell stage in most cells of the presumptive ectoderm and is rapidly restricted to the presumptive ventral ectoderm by mechanisms that are not well understood. Nodal function is absolutely required for establishment of dorsal-ventral polarity. Rescue experiments revealed that Nodal expressing cells have a long range organizing activity on the ectoderm and are capable of restoring D/V polarity over the whole embryo (Duboc et al., 2004; Duboc et al., 2005b) (Fig. 3). The long-range organizing activity of Nodal is assured by BMP2/4, which acts as a relay molecule synthesized in the ventral ectoderm, then translocated to the opposite side of the embryo (Lapraz et al., 2009). Finally, Chordin, which is co-expressed with bmp2/4 downstream of Nodal, is largely responsible for the spatial restriction of BMP2/4 signaling to the dorsal side (Lapraz et al., 2009). How the gradient of BMP2/4 is formed in this unusual situation and what are the factors that regulate this process is largely unknown.

Figure 2. Nodal expression and phenotypes resulting from perturbations of the Nodal and BMP2/4 pathways.

Our current research on D/V patterning is focused on several interrelated questions:

What are the maternal factors that regulate Nodal activity?
Several mechanisms and factors have been implicated in specification of the dorsal-ventral axis upstream of nodal expression including the p38 MAP kinase pathway, Redox gradients and mitochondria. However, the links between mitochondria, Redox gradients, p38 signaling, and the transcriptional machinery responsible for initiating nodal expression, remain unclear. We are characterizing the role of transcription factors of the ETS family that are good candidate as factors that may link p38 and nodal expression. We are also analyzing the function of extracellular proteins that we recently characterized and that may cooperate with Lefty to restrict the spatial expression of nodal.

Dissection of the D/V GRN activated by Nodal
To better understand how the ectoderm of the sea urchin embryo is patterned by Nodal and BMP2/4 signals we recently conducted a gene regulatory network analysis (Saudemont et al., 2010). Using a combination of gain and loss of function studies, we analyzed at high spatial resolution the expression and regulation by Nodal and BMP2/4 of 18 transcription factors and 8 signaling molecules that displayed a restricted expression along the D/V axis. Using an assay with recombinant proteins, we identified direct targets of Nodal and BMP2/4. Finally, by conducting a large-scale analysis of the epistatic relationships between these genes, we started ordering them into a hierarchy and identified key regulators acting

Figure 3. Provisional gene regulatory network governing dorsal ventral patterning of the sea urchin embryo (from Molina et al.
Current Opin Dev. Biol. 2013).

downstream of Nodal and BMP2/4. We are currently continuing our analysis of the D/V GRN by characterizing recently discovered novel components of the GRN and searching for missing components downstream of Nodal and BMP2/4.


Establishment of Left-Right asymmetry

Echinoderms offer a remarkable example of left-right asymmetry. Their larvae undergo a metamorphosis during which most larval tissues are replaced by adult tissues that are generated from an imaginal disk, called the adult rudiment, that forms exclusively on the left side of an otherwise bilaterally symmetric larva. We showed that Nodal is expressed on the right side of the larva and that one key function of Nodal signals on the right side is to repress formation of the adult rudiment. Furthermore, we showed that inhibition of the H+/K+-ATPase disrupts left-right asymmetry and randomizes both nodal expression and positioning of the rudiment. Recently, we identified a nodal expressing left-right organizing centre in the archenteron of the gastrula. We further showed that formation of this centre and subsequent positioning of the rudiment requires a combination of inhibitory signals from the Notch pathway and inductive signals from the FGF and BMP pathways. We are continuing our dissection of the early events responsible for establishment of left right asymmetry. Using nodal expression in the left-right organizer as an entry point, we try to dissect the mechanism of symmetry breaking and attempt to identify the transcription factors and signaling molecules that are required to restrict the expression of nodal to this right-sided organizer as well as those that act downstream of Nodal to repress rudiment formation on the right side.

Figure 4. Nodal expression on the right side controls positioning of the rudiment.


Control of Epithelial-Mesenchymal Transition (EMT)

Despite its crucial role in development and cancer, the mechanisms that allow cells to become motile during EMT and the gene regulatory networks activated during this process are incompletely described. The sea urchin embryo offers an attractive model to study the mechanisms and genes that regulate EMT. At the onset of gastrulation, about 32 cells of the vegetal pole detach from the epithelium and delaminate into the blastocoel to become the primary mesenchymal cells that will later build the skeleton of the larva.

The process of delamination of the PMCs shares striking similarities to EMT triggered by growth factors and receptor tyrosine kinase or to that observed during tumoral transformation. For example, it is regulated at the transcriptional level by factors such as ETS and Snail. Moreover, the MAP kinase ERK is activated specifically in the PMC precursors and the whole MAP kinase pathway including ERK, MEK and RAF, is required for delamination of the PMCs downstream of beta catenin. Finally we showed that the activity of ERK is critically for the activity of the transcription factors Ets1 and possibly of Alx1. We are currently trying to identify the signals that activate the ERK pathway and that trigger EMT. We are also characterizing additional factors that may participate in the process of EMT of the PMC precursors.

Figure 5. Inhibition of MEK/ERK signaling blocks epithelial mesenchymal transition of the skeletogenic precursors in the sea urchin. Gene regulatory network regulating EMT downstream of beta catenin and role of MAP kinase signaling.


Last publications

p38 MAPK as an essential regulator of dorsal-ventral axis specification and skeletogenesis during sea urchin development: a re-evaluation. - 2017 - Development (Cambridge, England) - 144 P2270-2281 - Molina MD, Quirin M, Haillot E, Jimenez F, Chessel A, and Lepage,T

A deuterostome origin of the Spemann organiser suggested by Nodal and ADMPs functions in Echinoderms. - 2015 - Nature communications - 6 P8434 - Lapraz F, Haillot E, and Lepage,T

The Maternal Maverick/GDF15-like TGF-β Ligand Panda Directs Dorsal-Ventral Axis Formation by Restricting Nodal Expression in the Sea Urchin Embryo. - 2015 - PLoS biology - 13 Pe1002247 - Haillot E, Molina MD, Lapraz F, and Lepage,T

Nodal: master and commander of the dorsal-ventral and left-right axes in the sea urchin embryo. - 2013 - Curr Opin Genet Dev - 23 P445-53 - Molina MD, de Croz N, Haillot E, and Lepage,T

Reciprocal signaling between the ectoderm and a mesendodermal left-right organizer directs left-right determination in the sea urchin embryo. - 2012 - PLoS Genet - 8 Pe1003121 - Bessodes N, Haillot E, Duboc V, Rttinger E, Lahaye F, and Lepage,T

Maternal Oct1/2 is required for Nodal and Vg1/Univin expression during dorsal-ventral axis specification in the sea urchin embryo. - 2011 - Dev Biol - 357 P440-9 - Range R, and Lepage,T

Wnt6 activates endoderm in the sea urchin gene regulatory network. - 2011 - Development (Cambridge, England) - 138 P3297-306 - Croce J, Range R, Wu SY, Miranda E, Lhomond G, Peng JC, Lepage T, and McClay,DR

Ancestral regulatory circuits governing ectoderm patterning downstream of Nodal and BMP2/4 revealed by gene regulatory network analysis in an echinoderm. - 2010 - PLoS Genet - 6 Pe1001259 - Saudemont A, Haillot E, Mekpoh F, Bessodes N, Quirin M, Lapraz F, Duboc V, Rttinger E, Range R, Oisel A, Besnardeau L, Wincker P, and Lepage,T

Nodal and BMP2/4 pattern the mesoderm and endoderm during development of the sea urchin embryo. - 2010 - Development - 137 P223-35 - Duboc V, Lapraz F, Saudemont A, Bessodes N, Mekpoh F, Haillot E, Quirin M, and Lepage,T

Patterning of the dorsal-ventral axis in echinoderms: insights into the evolution of the BMP-chordin signaling network. - 2009 - PLoS Biol - 7 Pe1000248 - Lapraz F, Besnardeau L, and Lepage,T

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Lepage Thierry
Group Leader

2009 Prize Tregouboff in Marine Biology from French Academy of Sciences

2007 Prize "Coup d'Elan pour la recherche" Bettencourt-Foundation 2007

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Members Team
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    September 2018 - T. Lepage Team
   1 Post-Doc Position - 2 ans

    September 2018 - T. Lepage Team
   1 Post-Doc Position - 2 ans

    September 2018 - T. Lepage Team
   1 PhD Position - 3 ans



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