Fig 2: Apico-basal polarity of single maternal-zygotic Myh9 or Myh10 mutant embryos.Representative images of WT, mzMyh9, and mzMyh10 embryos stained for apico-basal polarity markers. From left to right: PRKCz, F-actin, ATP1A1, CDH1, and AQP3 shown in grey. Nuclei stained with DAPI are shown in yellow. Intensity profiles of a representative 15 µm line drawn from the embryo surface towards the interior are shown next to each marker and genotype (arrows). Intensities are normalized to each minimal and maximal signals. Scale bar, 20 µm.

Fig 3: Reprogramming-Specific Gene Expression Patterns Are Important for Conversion to iPSCs(A) t-SNE plots based on Figure 2A highlighting the expression of MEF-associated mesenchymal genes that are downregulated as cells transition from MEFs to pluripotency. Top schematic indicates the pattern of expression.(B) Percentage of Cdh1+ cells that also co-express the indicated MEF genes on the x axis. The percentage of MEF gene-expressing cells that express Cdh1 is presented in brackets on the x axis. Note that because of the limit of detection of single-cell transcriptional analysis, co-expression may be underestimated.(C) (i) t-SNE plots based on Figure 2A illustrating co-expression of Cdh1 with Twist1. Note that because of the limit of detection of single-cell transcriptional analysis, co-expression may be underestimated. (ii) Immunofluorescent staining for CDH1 and TWIST1. Percentage of CDH1+/TWIST1+ colonies on A2S day 4 shown below image. Scale bar, 10 µm.(D) t-SNE plots based on Figure 2A highlighting the expression of DNA replication and cell-cycle-associated genes. Top schematic indicates the pattern of expression.(E) Left: percentage of cells that are Ki67+ at each indicated reprogramming time point in FBS or A2S systems. Right: immunofluorescent staining of Ki67 during FBS and A2S reprogramming (day 9 and day 4, respectively). Scale bar, 50 µm.(F) t-SNE plot based on Figure 2A for the anti-proliferation gene Cdkn1c. Top schematic indicates the pattern of expression.(G) Percentage of Cdh1+ cells that co-express cell cycle or anti-proliferative genes. Note that because of the limit of detection of single-cell transcriptional analysis co-expression may be underestimated.(H) Number of NANOG+ colonies on day 4 of A2S reprogramming after small interfering RNA (siRNA)-mediated knock down of Ehf. Error bars represent SD of two replicates.

Fig 4: Apico-basal polarity of maternal Myh9;Myh10 mutant embryos composed of multiple cells or a single cell.Representative images of WT, multiple- or single-celled Myh9;Myh10 mutant embryos stained for apico-basal polarity markers. From left to right: PRKCz, F-actin, ATP1A1, CDH1, and AQP3 shown in grey. Nuclei stained with DAPI shown in yellow. Intensity profiles of a representative 15 µm line drawn from the embryo surface towards the interior are shown next to each marker and genotype (arrows). Intensities are normalized to each minimal and maximal signals. Scale bar, 20 µm.

Fig 5: Measurements to describe branched organoid morphology. (A) To determine organoid area, the borders of branched organoids were marked (yellow dotted line) using maximum intensity projections and the size of the yellow marked area was determined. Organoid length was measured in the direction of branch elongation (white arrow). (B) Organoid depth was measured using the depth coding function in LasX 3D visualization software (Leica Microsystems) or derived from the color scheme of the depth scale bar. (C) Representative 3D rendering of a branched organoid lacking a central core after elongation and branching. The primary branch was defined as the center of the organoid (yellow line), connected to secondary branches (red lines) and tertiary branches (green lines). Bifurcation point is indicated with asterisk; side branches/buds are indicated with white arrows. Branch length was calculated as the distance between two branch points or the distance between branch tip and previous branch point. (D) Representative 3D rendering of an organoid partially retaining a central core (yellow) after elongation. Primary branches are indicated with red lines, and secondary branches are indicated with green lines. Asterisks refer to bifurcation points; white arrows indicate side branches. (E,F) Zoom images of a side branch (E) and a bifurcation point (F) of the organoid depicted in (C). Cdh1-CFP is shown in cyan; smooth muscle actin labeling is shown in magenta. Scale bars represent 100 µm.