Multiscale quantification of tissue behavior during amniote embryo axis elongation

Abstract

Embryonic axis extension is a complex multi-tissue morphogenetic process responsible for the formation of the posterior part of the amniote body. Cells located in the caudal part of the embryo divide and rearrange to participate in the elongation of the different embryonic tissues (e.g. neural tube, axial and paraxial mesoderm, lateral plate, ectoderm, endoderm). We previously identified the paraxial mesoderm as a crucial player of axis elongation, but how movements and growth are coordinated between the different posterior tissues to drive morphogenesis remain largely unknown. Here we use the quail embryo as a model system to quantify cell behavior and movements in the various tissues of the elongating embryo. We first quantify the tissue-specific contribution to axis elongation by using 3D volumetric techniques, then quantify tissuespecific parameters such as cell density and proliferation at different embryonic stages. To be able to study cell behavior at a multi-tissue scale we used high-resolution 4D imaging of transgenic quail embryos expressing constitutively expressed fluorescent proteins. We developed specific tracking and image analysis techniques to analyze cell motion and compute tissue deformations in 4D. This analysis reveals extensive sliding between tissues during axis extension. Further quantification of "tissue tectonics" showed patterns of rotations, contractions and expansions, which are coherent with the multi-tissue behavior observed previously. Our results confirm the central role of the PSM in axis extension; we propose that the PSM specific cell proliferation and migration programs control the coordination of elongation between tissues during axis extension. Material and methodsQuail embryo and embryo culture Wild-type quail embryos (Japonica coturnix) were obtained from commercial sources and from the USC aviary. The PGK1:H2B-chFP quail line generation was described previously (Huss et al. 2015) and is maintained in the USC aviary. Embryos were staged according to (Ainsworth et al. 2010;Hamburger andHamilton 1992) Hamburger andHamilton, 1951). Embryos were cultured ex ovo with filter paper on albumen agar plates according to the EC (Early chick) technique (Chapman et al. 2001). Staining and immunodetectionsEmbryo were collected at the desired stages and fixed overnight in 4% formaldehyde [36% formaldehyde (47608, Sigma) diluted to 4% in PBS]. Blocking and tissues permeabilization were carried out for 2 hours in PBS/0.5% Triton/1% donkey serum. Primary antibodies against Sox2 (1/5000, millipore ab5603) and Bra (1/500, R&D AF2085) were incubated overnight at 4°C. After washing off primary antibody in PBT (PBS/O.1% Triton), embryos were incubated with secondary antibodies (donkey anti goat Alexa 594 and goat anti rabbit Alexa 488, 1/1000, Molecular probes) and DAPI (1/1000, D1306, Molecular probes) overnight at 4°C. The embryos were cleared in U2 scale (Hama et al. 2011) for at least 48h at 4°C and then mounted between slide and coverslip and imaged by confocal/2P microscopy. Proliferation ...

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