Extended Data Fig. 10: Axial extension and spatio-temporal Hox expression patterns in iPSC-derived gastruloids.
From: Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids
a. Dot plot representing the progression in the measured longitudinal extension of gastruloids produced either from ESCs or from iPSCs. In each case, 10 different gastruloids were measured at the different time points indicated. The median (round points) and the interquartile range (vertical bars) are reported. b, Light microscopy images showing representative examples of gastruloids at the different time points analysed in a. Zoom: 10×. Note that iPSC-derived gastruloids exhibit delay in their longitudinal extension rate and at 120 h AA they are markedly smaller than their ESC-derived counterparts. For this analysis, gastruloids were produced starting from the same number of cells (800 cells per well). c, Dot plots representing the Bra mRNA levels, showing comparable dynamics of this gene in both types of gastruloids. Circles represent individual data points and the short horizontal line represents the mean. The number of biological independent replicates (n) per condition is indicated. d, Confocal images showing the expression of Oct4, SOX2 and BRA (top) or of Oct4, SOX1 and CDX2 (bottom) in 120 h AA gastruloids derived from the iPSC line Oct4::Gfp (IpSL40N). iPSC-derived gastruloids were fixed and stained for SOX2 and BRA (top) and CDX2 and SOX1 (bottom). Oct4::GFP signal is shown in grey. Scale bar, 200 μm. In each case, data are representative of one experiment with three independent biological replicates. e, Dot plots representing the Hoxd mRNA levels in ESC- or iPSC-derived gastruloids collected at different time points AA. Each circle represents an independent biological replicate, the horizontal bars represent the mean value of the replicates. Both sets of gastruloids sequentially activated Hoxd gene expression. However, their temporal activation seemed to be delayed in iPSC gastruloids (especially that of the most 3′ Hoxd paralogues). f, WISH of 144 h AA gastruloids showing the expression of different Hoxd paralogues. Even though iPSC-derived gastruloids reproduced the antero-posterior Hoxd collinear expression, the Hoxd9 expression domain often extended more anteriorly in comparison to that in ESC-derived gastruloids (see Fig. 4c), occupying roughly the same domain as Hoxd4. Patches of Hoxd-negative cells were often observed within the Hoxd4/Hoxd9 expression domain (white). The fraction of gastruloids displaying the reported expression pattern is indicated in the upper right corner of each image. Experimental statistics are provided in Supplementary Information dataset 3. Scale bar, 100 µm.
