Figure 1

Figure 1

(A) in situ hybridization chain reaction (HCR) was used to stain for msgn1 (yellow) and tbx6 (red), markers of the posterior and anterior PSM, respectively, from the 16 somite-stage to the end of somitogenesis. Nuclei were stained with DAPI (grey). (B) 2D contours (yellow outlines, left image) were manually drawn around the PSM at regular z-slices, up to the midline, to generate 3D surface reconstructions of the PSM (cyan, right image) and the nascent (i.e. most recently-formed) somite (Fig S1D) at each stage. (C) DAPI signal was isolated from each surface to show only nuclei in that tissue. (D) Spots were generated, marking the centre of each isolated nucleus (shown in slice view (left image) and 3D view (other images)), and providing cell number information. (E-J) The length, cell number, and volume of the PSM (E-G) and of each somite at its time of formation (H-J) was measured for a range of stages. Each point represents the PSM/nascent somite of a single embryo (n = 15 embryos for length data, n = 11 embryos for cell number data and n = 26 embryos for volume data). Possible trendline equations for each measurement were calculated in R, and AIC was used to determine the best statistical model (linear vs exponential). Solid trendlines (blue, PSM) indicate genuine change of one tissue over time, whereas dotted trendlines (red, nascent somites) indicate a trend based on separate tissues. The trendline equations are as follows (where x is somite stage): PSM length (E) = -14.1x + 155 (R² = 0.97); PSM cell number (F) = -139x + 4,620 (R² = 0.95); PSM volume (G) = 16,700,000e^-0.159x (R² = 0.98); Nascent somite length (H) = -1.58x + 75 (R² = 0.72); Nascent somite cell number (I) = -15.1x + 525 (R² = 0.83); Nascent somite volume (J) = 1,630,000e^-0.151x (R² = 0.94). (K-M) The above trendline equations were used to calculate how the length (K), cell number (L), and volume (M) of the paraxial mesoderm changed over time, by summing (for each stage) the current nascent somite and all previous nascent somite values to the current PSM value.