Species-specific contribution of volumetric growth and tissue convergence to posterior body elongation in vertebrates.

Posterior body elongation is a widespread mechanism propelling the generation of the metazoan body plan. The posterior growth model predicts that a posterior growth zone generates sufficient tissue volume to elongate the posterior body. However, there are energy supply-related differences between vertebrates in the degree to which growth occurs concomitantly with embryogenesis. By applying a multi-scalar morphometric analysis in zebrafish embryos, we show that posterior body elongation is generated by an influx of cells from lateral regions, by convergence-extension of cells as they exit the tailbud, and finally by a late volumetric growth in the spinal cord and notochord. Importantly, the unsegmented region does not generate additional tissue volume. Fibroblast growth factor inhibition blocks tissue convergence rather than volumetric growth, showing that a conserved molecular mechanism can control convergent morphogenesis through different cell behaviours. Finally, via a comparative morphometric analysis in lamprey, dogfish, zebrafish and mouse, we propose that elongation via posterior volumetric growth is linked to increased energy supply and is associated with an overall increase in volumetric growth and elongation.Jean-François Nicolas, Estelle Hirsinger: Core funding from the Institut Pasteur and Agence Nationale de la Recherche (ANR-10-BLAN-121801 DEVPROCESS). Estelle Hirsinger and Sylvie Mazan are from the Centre National de la Recherche Scientifique (CNRS). Benjamin Steventon was funded by the Agence Nationale de la Recherche (ANR- 10-BLAN-121801 DEVPROCESS), then a Roux fellowship (Institut Pasteur) then an AFM-Téléthon fellowship (number 16829).This is the author accepted manuscript. The final version is available from The Company of Biologists via http://dx.doi.org/10.1242/dev.12637

Régionalisation du somite et ségrégation des différents lignages somitiques

Le somite est une structure mésodermique embryonnaire, caractéristique des vertébrés. Chez les amniotes, elle donne naissance au derme du dos, aux muscles striés squelettiques du tronc et des membres ainsi qu’à la colonne vertébrale et aux côtes. La ségrégation de ces différents lignages est liée à la mise en place, au sein du somite, de deux axes de polarité, l’axe dorso-ventral et l’axe médio-latéral. Alors que la mise en place du premier paraît dépendre essentiellement de signaux émanant des structures environnantes, celle du second repose à la fois sur des mécanismes intrinsèques et sur des signaux extérieurs au somite. En ce qui concerne les mécanismes extrinsèques régulant la mise en place de chaque axe, la régionalisation somitique est le résultat d’activités antagonistes ou combinatoires de facteurs diffusibles, tels que Sonic Hedgehog, Wnt et Bmp-4, agissant en gradients

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

International audienceZebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development

Somite formation and patterning

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Delta–Notch Signaling in Odontogenesis: Correlation with Cytodifferentiation and Evidence for Feedback Regulation

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Delta 1-activated Notch inhibits muscle differentiation without affecting Myf5 and Pax3 expression in chick limb myogenesis

International audienceThe myogenic basic helix-loop-helix (bHLH) transcription factors, Myf5, MyoD, myogenin and MRF4, are unique in their ability to direct a program of specific gene transcription leading to skeletal muscle phenotype. The observation that Myf5 and MyoD can force myogenic conversion in non-muscle cells in vitro does not imply that they are equivalent. In this paper, we show that Myf5 transcripts are detected before those of MyoD during chick limb development. The Myf5 expression domain resembles that of Pax3 and is larger than that of MyoD. Moreover, Myf5 and Pax3 expression is correlated with myoblast proliferation, while MyoD is detected in post-mitotic myoblasts. These data indicate that Myf5 and MyoD are involved in different steps during chick limb bud myogenesis, Myf5 acting upstream of MyoD. The progression of myoblasts through the differentiation steps must be carefully controlled to ensure myogenesis at the right place and time during wing development. Because Notch signalling is known to prevent differentiation in different systems and species, we sought to determine whether these molecules regulate the steps occurring during chick limb myogenesis. Notch1 transcripts are associated with immature myoblasts, while cells expressing the ligands Delta1 and Serrate2 are more advanced in myogenesis. Misexpression of Delta1 using a replication-competent retrovirus activates the Notch pathway. After activation of this pathway, myoblasts still express Myf5 and Pax3 but have downregulated MyoD, resulting in inhibition of terminal muscle differentiation. We conclude that activation of Notch signalling during chick limb myogenesis prevents Myf5-expressing myoblasts from progressing to the MyoD-expressing stag

Noggin acts downstream of Wnt and Sonic Hedgehog to antagonize BMP4 in avian somite patterning

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Maintenance of neuroepithelial progenitor cells by Delta–Notch signalling in the embryonic chick retina

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Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

International audienceWe report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution