Extended Data Fig. 9: Explanation for how elongation and differentiation-front speed were measured, and HCR stainings and kymographs of gastruloids embedded in 10% Matrigel.
From: Single-cell and spatial transcriptomics reveal somitogenesis in gastruloids
a, Kymographs (space–time plots) of bright field channel and LfngT2AVenus signal along the anterior–posterior axis of a DMSO-treated (control) and a PD03 (MEKinhibitor)-treated LfngT2AVenus gastruloid. Gastruloids were embedded in 100% Matrigel at 96 h; DMSO or PD03 (66.7 μM) was added at 96.5 h. Kymographs were used to measure the elongation speed of the gastruloid (angle of blue dashed line; VPSM) (Methods) and the speed of the differentiation front (angle of red dashed line; VDIFF). Similar results were obtained in n = 4 independent experiments. b, Illustration explaining the effect of FGF inhibition, which increases the speed of the differentiation front (red arrows, VDiff) without altering the elongation rate (blue arrows, VPSM) of gastruloids. Three time points (t1, t2 and t3) are depicted. White tissue, nondifferentiated tissue (presomitic mesoderm); grey tissue, differentiated tissue. c, In situ hybridization staining for Uncx4.1 on 120-h LfngT2AVenus gastruloids that were not embedded in Matrigel (0%) (standard, previously published protocol1,20) or that were embedded in 25% or 100% Matrigel at 96 h. Numbers below the panels indicate the number of gastruloids in which stripy Uncx4.1 expression patterns were observed. Similar results were obtained in n = 3 independent experiments. d, LfngT2AVenus gastruloids that were embedded in 10% Matrigel (Methods) at 96 h and stained for Uncx4.1 using HCR21 at 120 h. A magnified view of the left gastruloid is shown in Fig. 3a. Similar results were obtained in n = 5 independent experiments. e, Kymographs of LfngT2AVenus signal and bright field channel along the anterior–posterior axis of gastruloids that were embedded in 10% Matrigel at 96 h, and subsequently imaged for 20 h (Supplementary Video 6). Top kymograph belongs to the gastruloid that is shown in Fig. 3b. Similar results were obtained in n = 2 independent experiments. Scale bars, 200 μm.
