Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Abstract

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions).Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Tgfbr1 controls developmental plasticity between the hindlimb and external genitalia by remodeling their regulatory landscape

“…Optical projection tomography (OPT) and SPIM (Selective Illumination Plane Microscopy) were used to image E16.5 fetuses as previously described 81 with minor modifications. Briefly, fetuses were recovered by cesarean section in ice cold PBS, washed several times in PBS and fixed in 4% paraformaldehyde (PFA) in PBS at 4 °C for several days.…”

Tgfbr1 controls developmental plasticity between the hindlimb and external genitalia by remodeling their regulatory landscape

“…Optical projection tomography (OPT) and SPIM (Selective Illumination Plane Microscopy) were used to image E16.5 fetuses as previously described 77 with minor modifications. Briefly, fetuses were recovered by cesarean section in ice cold PBS, washed several times in PBS and fixed in 4% paraformaldehyde (PFA) in PBS at 4°C for several days.…”

Epha1 is a cell-surface marker for the neuromesodermal competent population

“…Whole-mount immunostaining was carried out according to Dias et al (2021). RapiClear 1.52 (SunJin Lab) was used for embryo clearing.…”

“…Secondary antibodies (all used at 1:1000 dilution) were as follows: donkey anti-goat 488 (Molecular Probes, A11055) and donkey anti-rabbit 568 (Molecular Probes, A10042). Images were acquired with a Prairie two-photon system, and the image dataset pre-processing was performed as described previously (Dias et al, 2021). No deconvolution or z-depth signal attenuation was performed in order to reduce possible preprocessing interferences that could misrepresent the real Epha1 expression levels in the tailbud.…”