FIGURE SUMMARY
Title

Species tailoured contribution of volumetric growth and tissue convergence to posterior body elongation in vertebrates

Authors
Steventon, B., Duarte, F., Lagadec, R., Mazan, S., Nicolas, J.F., Hirsinger, E.
Source
Full text @ Development

Convergence of lateral cells contributes to the elongating posterior body. (A-C) Tracks (A) and displacement vectors (B,C) of spinal cord (yellow), tailbud (blue) and PSM (red) precursors from automated tracking of photolabelled nuclei from the 90% epiboly to the 12-somite stage. (D-F) Tracks of cells from the 90% epiboly to the 12-somite stage. (G) Proliferation rate, expressed as the number of additional cells generated divided by the time interval (5h), within clones fated towards the epidermis (Epi; mean 0.13 cells/h, n=53), tailbud (mean 0.36 cells/h, n=53), spinal cord (mean 0.04 cells/h, n=85) and PSM (mean 0.2 cells/h, n=74). (H) Index of dispersion (mean increase in inter-nuclear distance between neighbouring cells) for cells fated towards the epidermis (Epi; mean 5.56µm, n=53), tailbud (mean 18µm, n=53), spinal cord (mean 50µm, n=85) and PSM (mean 11µm, n=74). Small dashes show values from five independent movies. Large dashes mark the mean.

Posterior body elongation in zebrafish occurs firstly in the absence of volumetric growth followed by a later volumetric growth phase. (A) Surface reconstructions (grey region) of the posterior body through posterior body elongation. Embryos previously injected with nuclear mCherry and membrane eGFP mRNAs in lateral view with A and P marking anterior and posterior poles. (B,C) Plots of volume (B; n=21) and length (C; n=21) against time (number of somites). Grey points/trendlines: three- to 24-somite stages. Black points/trendlines: 24- to 32-somite stages. (D,E) Width (D) and height (E) against distance from posterior tip of the tail to where each measurement was taken. Each curve corresponds to a developmental stage (number of somites). Colour code: purple, 12 somites (s) (n=5); dark blue, 15s (n=8); light blue, 18s (n=7); green, 20s (n=12); yellow, 24s (n=17). Data points show individual measurements; n=total number of measurements from three embryos per stage. Black arrows show direction of trend over time.

Volumetric growth only occurs within already segmented regions of the zebrafish posterior body. (A,D,G) Stills from time-lapse movies of embryos injected at the one-cell stage with KikGR mRNA and photolabelled within the unsegmented region at the 15-somite (A) or 21-somite (D) stage or within the segmented region at the 15-somite stage (G). Embryos in lateral view with posterior to the top. (B,E,H) Photoactivated region length (percentage of initial length) plotted against time (number of somites). (B) n=25, (E) n=51, (I)  n=29. (C,F,I). Photoactivated region volume (percentage of initial volume) plotted against time (number of somites), (C) n=25, (F) n=25, (I) n=31. White arrows in A and G indicate the displacement of labels within the somitic mesoderm (lower arrows) relative to the spinal cord (upper arrows). Data points show individual measurements; n=total number of measurements from three independent experiments.

Proliferation rapidly reaches a low level in the unsegmented region. (A) Stills from a time-lapse of zFUCCI green embryos injected with nuclear mCherry mRNA. (B) Plot of the percentage of nuclei in S/M/G2 (green in A) with respect to total nuclei (red in A) against time (number of somites), showing the posterior body as a whole (black line) and the tailbud (blue line). This trend was observed in three independent time-lapse movies. (C) Percentage of nuclei in S/M/G2 (green) with respect to total nuclei (red) in tailbud (blue), PSM (red), notochord (purple), spinal cord (yellow), blood precursors (green) and somites (orange) at 20-, 25- and 30-somite stages. Small dashes show counts from four embryos fixed at each stage, large dashes indicate mean value.

The spinal cord and notochord are the principal contributors to volumetric growth and the TB-to-PSM transiting cells to thinning and lengthening. (A) Different tissues are colour coded according to the key shown. (B,C) Stills from time-lapse movies of embryos injected at the one-cell stage with KikGR mRNA and photolabelled in the spinal cord and in the PSM (B) or in the tailbud (TB) (C) at the 14-somite stage. (D,E) Plots of volume fold increase (D; spinal cord, n=51, notochord; n=31; PSM to somites, n=58; TB to PSM, n=23) and length:width ratio (E; spinal cord, n=43; notochord, n=30; PSM to somites, n=17; TB to PSM, n=12) of each photolabelled region against time (number of somites). The red curve in D corresponds to the volume increase of the KikGR-labelled cytoplasm of notochord cells. Data points show individual measurements; n=total number of measurements from embryos from three experiments.

FGF signalling is required for thinning and lengthening, but not volumetric growth of the zebrafish posterior body. (A-D) Embryos injected at the one-cell stage with nuclear mCherry mRNA and incubated in either 500µM DMSO (A), 200µM SU5402 (B), 500µM SU5402 (C) or 20µM PD173074 (D) from the ten-somite stage until the 32-somite stage and imaged by confocal microscopy at the 32-somite stage. (E-G) Mean value of posterior body volume (E), length (F) and width (G) for each treatment group at the 32-somite stage. Error bars indicate s.d. (H,I) Stills from time-lapse movies of embryos in which small regions within the tailbud are photolabelled at the 12-somite stage in the presence of 500µM DMSO (H) or 20µM PD173074 (I). (J,K) Quantification of the volume (J) and length:width ratio (K) of the photolabelled region over time (number of somites). Green lines correspond to the control situation (500µM DMSO), red lines indicate treatment with PD173074.

FGF signalling is required for cell movements but not cell proliferation. (A-D) Embryos previously injected with nls-KikGR mRNA were incubated in either 500µM DMSO (A,B) or 200µM SU5402 (C,D). Colours indicate cells in the PSM (yellow), tailbud (red) and spinal cord (blue). (E,F) Photolabels in tailbud (TB-PSM; red), PSM (PSM-Som; yellow) or spinal cord (SC; blue) were measured to display mean length:width ratio (E) and cell number (F). Error bars indicate s.d. (G,H). zFUCCI green embryos injected with nuclear mCherry mRNA and incubated in either 500µM DMSO (G) or 200µM SU5402 (H) from the ten- to the 32-somite stage. (I) Mean percentage of nuclei in S/M/G2 are plotted for each treatment group at the 18- and 31-somite stages. Error bars indicate s.d. Three to five embryos were analysed for each condition shown. (J,K) Fluorescent in situ hybridisations for the immediate FGF response gene sprouty4 (green), together with counter-staining for nuclei with DAPI (yellow) in the presence of 200µM SU5402 (K) or with DMSO only (J). Images are maximum projections of 20× 0.5683µm z-planes through the embryonic midline to determine FGF inhibition deep within the tissue of interest.

Comparative 3D morphometric analysis of posterior body elongation across vertebrates. (A-D) Maximal projections of tiled z-stacks of lamprey (A; n=7), dogfish (B; n=11), zebrafish (C; n=15) and mouse (D; n=10) embryos. All embryos were labelled with DAPI and phalloidin and are shown in lateral view with posterior to the right. Grey surface shows the segmented region, yellow the two most rostral segments of the posterior body and the red surface shows the unsegmented region. Plots of the unsegmented region volume over time (number of somites) are shown to the right for each species. (E) Simplified phylogeny for each species studied together with total somite number. (F-H) Fold change from tailbud stage until the completion of somitogenesis in length (F) and in volume (G) of the whole posterior body and fold change in volume of the two-most anterior segments (H) for each species. Purple bar, lamprey; blue, dogfish; green, zebrafish; red, mouse.

Proportional volumes of the spinal cord, paraxial mesoderm and notochord with respect to the whole posterior body. (A,B) Confocal stacks of 15-somite stage embryos injected at the one-cell stage with nuclear mCherry and membrane-bound eGFP mRNAs were used to build surface reconstructions of the spinal cord (blue), paraxial mesoderm (green) and notochord (purple). (C) Proportional volume of each tissue with respect to whole posterior body volume. Measurements are from three embryos.

Acknowledgments
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