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1.  Pax3 and Zic1 trigger the early neural crest gene regulatory network by the direct activation of multiple key neural crest specifiers 
Developmental biology  2013;386(2):461-472.
Neural crest development is orchestrated by a complex and still poorly understood gene regulatory network. Premigratory neural crest is induced at the lateral border of the neural plate by the combined action of signaling molecules and transcription factors such as AP2, Gbx2, Pax3 and Zic1. Among them, Pax3 and Zic1 are both necessary and sufficient to trigger a complete neural crest developmental program. However, their gene targets in the neural crest regulatory network remain unknown. Here, through a transcriptome analysis of frog microdissected neural border, we identified an extended gene signature for the premigratory neural crest, and we defined novel potential members of the regulatory network. This signature includes 34 novel genes, as well as 44 known genes expressed at the neural border. Using another microarray analysis which combined Pax3 and Zic1 gain-of-function and protein translation blockade, we uncovered 25 Pax3 and Zic1 direct targets within this signature. We demonstrated that the neural border specifiers Pax3 and Zic1 are direct upstream regulators of neural crest specifiers Snail1/2, Foxd3, Twist1, and Tfap2b. In addition, they may modulate the transcriptional output of multiple signaling pathways involved in neural crest development (Wnt, Retinoic Acid) through the induction of key pathway regulators (Axin2 and Cyp26c1). We also found that Pax3 could maintain its own expression through a positive autoregulatory feedback loop. These hierarchical inductions, feedback loops, and pathway modulations provide novel tools to understand the neural crest induction network.
PMCID: PMC3962137  PMID: 24360906
Neural crest; Pax3; Zic1; gene regulatory network; transcriptome; microarray; embryo; Xenopus laevis
2.  Systems control of BMP morphogen flow in vertebrate embryos 
Embryonic morphogenetic programs coordinate cell behavior to ensure robust pattern formation. Having identified components of those programs by molecular genetics, developmental biology is now borrowing concepts and tools from systems biology to decode their regulatory logic. Dorsal-ventral (D-V) patterning of the frog gastrula by Bone Morphogenetic Proteins (BMPs) is one of the best studied examples of a self-regulating embryonic patterning system. Embryological analyses and mathematical modeling are revealing that the BMP activity gradient is maintained by a directed flow of BMP ligands towards the ventral side. Pattern robustness is ensured through feedback control of the levels of extracellular BMP pathway modulators that adjust the flow to the dimensions of the embryonic field.
PMCID: PMC3224208  PMID: 21937218
3.  Wnt Signaling Requires the Sequestration of Glycogen Synthase Kinase 3 inside Multivesicular Endosomes 
Cell  2010;143(7):1136-1148.
Canonical Wnt signaling requires inhibition of Glycogen Synthase Kinase 3 (GSK3) activity, but the molecular mechanism by which this is achieved remains unclear. Here we report that Wnt signaling triggers the sequestration of GSK3 from the cytosol into multivesicular bodies (MVBs), so that this enzyme becomes separated from its many cytosolic substrates. Endocytosed Wnt co-localized with GSK3 in acidic vesicles positive for endosomal markers. After Wnt addition, endogenous GSK3 activity decreased in the cytosol, and GSK3 became protected from protease treatment inside membrane-bounded organelles. Cryoimmuno electron microscopy showed that these corresponded to multivesicular bodies. Two proteins essential for MVB formation, HRS/Vps27 and Vps4, were required for Wnt signaling. The sequestration of GSK3 extended the half-life of many other proteins in addition to β-Catenin, including an artificial Wnt-regulated reporter protein containing GSK3 phosphorylation sites. We conclude that multivesicular endosomes are essential components of the Wnt signal transduction pathway.
PMCID: PMC3022472  PMID: 21183076
4.  Crossveinless-2 is required for the relocalization of Chordin protein within the vertebral field in mouse embryos 
Developmental biology  2010;347(1):204-215.
Bone morphogenetic proteins (BMPs), as well as the BMP-binding molecules Chordin (Chd), Crossveinless-2 (CV2) and Twisted Gastrulation (Tsg), are essential for axial skeletal development in the mouse embryo. We previously reported a strong genetic interaction between CV2 and Tsg and proposed a role for this interaction in the shaping of the BMP morphogenetic field during vertebral development. In the present study we investigated the roles of CV2 and Chd in the formation of the vertebral morphogenetic field. We performed immunostainings for CV2 and Chd protein on wild-type, CV2−/− or Chd−/− mouse embryo sections at the stage of onset of the vertebral phenotypes. By comparing mRNA and protein localizations we found that CV2 does not diffuse away from its place of synthesis, the vertebral body. The most interesting finding of this study was that Chd synthesized in the intervertebral disc accumulates in the vertebral body. This relocalization does not take place in CV2−/− mutants. Instead, Chd was found to accumulate at its site of synthesis in CV2−/− embryos. These results indicate a CV2-dependent flow of Chd protein from the intervertebral disc to the vertebral body. Smad1/5/8 phosphorylation was decreased in CV2−/−vertebral bodies. This impaired BMP signaling may result from the decreased levels of Chd/BMP complexes diffusing from the intervertebral region. The data indicate a role for CV2 and Chd in the establishment of the vertebral morphogenetic field through the long-range relocalization of Chd/BMP complexes. The results may have general implications for the formation of embryonic organ-forming morphogenetic fields.
PMCID: PMC2975673  PMID: 20807528
BMP signaling; CV2; Chd; Chdl-1; Chdl-2; long-range signaling; morphogenetic field; vertebral development; Tolloid; Twisted gastrulation
5.  Systems Biology of the Self-regulating Morphogenetic Gradient of the Xenopus Gastrula 
The morphogenetic field concept was proposed by experimental embryologists to account for the self-regulative behavior of embryos. Such fields have remained an abstract concept until the recent identification of their molecular components using a combination of genetics, biochemistry, and theoretical modeling. One of the best studied models of a morphogenetic field is the Dorsal-Ventral (D-V) patterning of the early frog embryo. This patterning system is regulated by the bone morphogenetic protein (BMP) signaling pathway and an intricate network of secreted protein antagonists. This biochemical pathway of interacting proteins functions in the extracellular space to generate a D-V gradient of BMP signaling, which is maintained during extensive morphogenetic movements of cell layers during gastrulation. The D-V field is divided into a dorsal and a ventral center, in regions of low and high BMP signaling respectively, under opposite transcriptional control by BMPs. The robustness of the patterning is assured at two different levels. First, in the extracellular space by secreted BMP antagonists that generate a directional flow of BMP ligands to the ventral side. The flow is driven by the regulated proteolysis of the Chordin inhibitor and by the presence of a molecular sink on the ventral side that concentrates BMP signals. The tolloid metalloproteinases and the Chordin-binding protein Crossveinless-2 (CV2) are key components of this ventral sink. Second, by transcriptional feedback at the cellular level: The dorsal and ventral signaling centers adjust their size and level of BMP signaling by transcriptional feedback. This allows cells on one side of a gastrula containing about 10,000 cells to communicate with cells in the opposite pole of the embryo.
A network of BMP ligands and antagonists regulates embryonic patterning in frogs. Proteolysis of inhibitors, a molecular sink, and transcriptional feedback loops ensure its robustness.
PMCID: PMC2742089  PMID: 20066084
6.  Molecular Characterization of the Gastrula in the Turtle Emys orbicularis: An Evolutionary Perspective on Gastrulation 
PLoS ONE  2008;3(7):e2676.
Due to the presence of a blastopore as in amphibians, the turtle has been suggested to exemplify a transition form from an amphibian- to an avian-type gastrulation pattern. In order to test this hypothesis and gain insight into the emergence of the unique characteristics of amniotes during gastrulation, we have performed the first molecular characterization of the gastrula in a reptile, the turtle Emys orbicularis. The study of Brachyury, Lim1, Otx2 and Otx5 expression patterns points to a highly conserved dynamic of expression with amniote model organisms and makes it possible to identify the site of mesoderm internalization, which is a long-standing issue in reptiles. Analysis of Brachyury expression also highlights the presence of two distinct phases, less easily recognizable in model organisms and respectively characterized by an early ring-shaped and a later bilateral symmetrical territory. Systematic comparisons with tetrapod model organisms lead to new insights into the relationships of the blastopore/blastoporal plate system shared by all reptiles, with the blastopore of amphibians and the primitive streak of birds and mammals. The biphasic Brachyury expression pattern is also consistent with recent models of emergence of bilateral symmetry, which raises the question of its evolutionary significance.
PMCID: PMC2442194  PMID: 18628985
7.  Systems biology of embryonic morphogens 
Molecular bioSystems  2007;3(7):454-457.
A network of secreted proteins that interact with each other in the extracellular space regulates embryonic morphogenesis. Mathematical modeling offers an excellent opportunity to understand how morphogens signal and self-regenerate pattern.
PMCID: PMC2412835  PMID: 17579769
8.  The metazoan history of the COE transcription factors. Selection of a variant HLH motif by mandatory inclusion of a duplicated exon in vertebrates 
The increasing number of available genomic sequences makes it now possible to study the evolutionary history of specific genes or gene families. Transcription factors (TFs) involved in regulation of gene-specific expression are key players in the evolution of metazoan development. The low complexity COE (Collier/Olfactory-1/Early B-Cell Factor) family of transcription factors constitutes a well-suited paradigm for studying evolution of TF structure and function, including the specific question of protein modularity. Here, we compare the structure of coe genes within the metazoan kingdom and report on the mechanism behind a vertebrate-specific exon duplication.
COE proteins display a modular organisation, with three highly conserved domains : a COE-specific DNA-binding domain (DBD), an Immunoglobulin/Plexin/transcription (IPT) domain and an atypical Helix-Loop-Helix (HLH) motif. Comparison of the splice structure of coe genes between cnidariae and bilateriae shows that the ancestral COE DBD was built from 7 separate exons, with no evidence for exon shuffling with other metazoan gene families. It also confirms the presence of an ancestral H1LH2 motif present in all COE proteins which partly overlaps the repeated H2d-H2a motif first identified in rodent EBF. Electrophoretic Mobility Shift Assays show that formation of COE dimers is mediated by this ancestral motif. The H2d-H2a α-helical repetition appears to be a vertebrate characteristic that originated from a tandem exon duplication having taken place prior to the splitting between gnathostomes and cyclostomes. We put-forward a two-step model for the inclusion of this exon in the vertebrate transcripts.
Three main features in the history of the coe gene family can be inferred from these analyses: (i) each conserved domain of the ancestral coe gene was built from multiple exons and the same scattered structure has been maintained throughout metazoan evolution. (ii) There exists a single coe gene copy per metazoan genome except in vertebrates. The H2a-H2d duplication that is specific to vertebrate proteins provides an example of a novel vertebrate characteristic, which may have been fixed early in the gnathostome lineage. (iii) This duplication provides an interesting example of counter-selection of alternative splicing.
PMCID: PMC2394523  PMID: 18454855
9.  Evolution of Axis Specification Mechanisms in Jawed Vertebrates: Insights from a Chondrichthyan 
PLoS ONE  2007;2(4):e374.
The genetic mechanisms that control the establishment of early polarities and their link with embryonic axis specification and patterning seem to substantially diverge across vertebrates. In amphibians and teleosts, the establishment of an early dorso-ventral polarity determines both the site of axis formation and its rostro-caudal orientation. In contrast, amniotes retain a considerable plasticity for their site of axis formation until blastula stages and rely on signals secreted by extraembryonic tissues, which have no clear equivalents in the former, for the establishment of their rostro-caudal pattern. The rationale for these differences remains unknown. Through detailed expression analyses of key development genes in a chondrichthyan, the dogfish Scyliorhinus canicula, we have reconstructed the ancestral pattern of axis specification in jawed vertebrates. We show that the dogfish displays compelling similarities with amniotes at blastula and early gastrula stages, including the presence of clear homologs of the hypoblast and extraembryonic ectoderm. In the ancestral state, these territories are specified at opposite poles of an early axis of bilateral symmetry, homologous to the dorso-ventral axis of amphibians or teleosts, and aligned with the later forming embryonic axis, from head to tail. Comparisons with amniotes suggest that a dorsal expansion of extraembryonic ectoderm, resulting in an apparently radial symmetry at late blastula stages, has taken place in their lineage. The synthesis of these results with those of functional analyses in model organisms supports an evolutionary link between the dorso-ventral polarity of amphibians and teleosts and the embryonic-extraembryonic organisation of amniotes. It leads to a general model of axis specification in gnathostomes, which provides a comparative framework for a reassessment of conservations both among vertebrates and with more distant metazoans.
PMCID: PMC1847705  PMID: 17440610

Results 1-9 (9)