Fig 2: Circuit-mediated BMP4 secretion enables control over cell composition and pattern formation in micropatterned hPSC colonies A, BDiscrete circuit-mediated control of BMP4 by endogenous miRNAs. (A) Circuit schematic. Minimal circuit controlling BMP4 in response to the endogenous miRNAs miR-302a/b target sites (Off) or mock target sites TFF3/TFF6 (On). (B) Micropatterned RUES2 have been transfected with On and Off circuits. Bar chart shows BMP4 concentration as determined by ELISA and average iRFP expression as measured by Flow Cytometry (left). Corresponding bar chart showing the average fraction of cells expressing a given germ-layer marker (right).C–FCircuit-mediated tuning of BMP4 = using miSFITs library. (C) Circuit schematic. Minimal circuit in mock On state controlling untargeted iRFP and miSFITs-targeted BMP4. Five miSFITs variants have been transfected on micropatterned RUES2 cells. (D). Bar chart on the left shows BMP4 concentration for each circuit variant determined from culture media by ELISA. Bar chart on the right shows the same secreted BMP4 concentration along cell internal iRFP expression signal, measured by Flow Cytometry in comparison to untransfected controls in the absence (-BMP4) and presence (+BMP4) of BMP4 (right). (E) Bar chart showing the relative fraction of cells expressing a given germ-layer marker as analyzed from confocal microscopy images (right). Dose response chart of the marker fraction in response to measured BMP4 concentration (left). See also representative Flow Cytometry scatter plots in Fig EV5C. (F) Radial marker profiles of each germ-layer marker analyzed from confocal microscopy images. Charts on the left show normalized intensity of marker expression at a given colony position, where x = 0 is the center of the colony and x = 1 is the edge of the colony.Representative microscopy image of a single colony (top) showing composite of Sox2/mCitrine (yellow), Sox17/mCherry (red) and TBXT/AF647 (blue). Data information: Each bar show mean ± s.d. from at least three biological replicates for BMP4 and iRFP data and from at least 20 colonies for germ-layer data. Scale bar represents 200 µm. See also Fig EV5. Source data are available online for this figure.

Fig 3: Single cell RNA‐Seq reveal subpopulation difference between the wild type and ZIC2 mutants during mesoderm formation. A, tSNE representation of the transcription profiles of the day 3 cells from wild type and ZIC2 mutants hESCs. B, tSNE plots with the expression distribution of the classical developmental stage markers, including pluripotency markers: POU5F1, NANOG, SOX2; primitive markers: NODAL, MIXL1, T(TBXT); mesoderm stage markers: EOMES, MESP1, TBX6. C, Subpopulation clusters based on the transcription profile. There are in total nine clusters, but clusters 1‐3 are very similar, so seven distinct populations can be identified. D, Heat map showing the marker genes of each cluster. Horizontal axis shows the composition of the cells from different groups. The vertical axis on the left shows the color code for each cluster, and the vertical axis on the right shows the names of marker genes. E, tSNE plots with the expression of example marker genes for different clusters

Fig 4: Effect of TBXT reduction on human pembryonic stem cell (hES)-derived neuromesodermal progenitors (NMPs).(A) Differentiation/tetracycline (Tet) treatment scheme. (B) Immunofluorescence analysis of the expression of TBXT in shRNA hEScell-derived NMPs in the presence and absence of Tet. Scale bar = 100 μm. (C) Mean fluorescence intensity of TBXT protein in Tet-treated and control NMP cultures. (D) Normalised expression values of TBXT transcripts in control, Tet-treated NMPs and undifferentiated hESC samples following RNA sequencing (RNA-seq) analysis. (E) Gene ontology (GO) term analysis of differentially expressed genes in hESC-derived NMPs following TBXT knockdown. (F) Representative significantly downregulated and upregulated transcripts following TBXT depletion. (G) Quantitative PCR (qPCR) expression analysis of indicated HOX genes in control vs Tet-treated NMPs. Error bars represent SD (n=3). *p<0.05, **p<0.005, ***p<0.0005 (paired t-test). n.s. not significant.

Fig 5: TBXT depletion impairs posterior axial identity acquisition by neural crestNC.(A) Differentiation/tetracycline (Tet) treatment scheme. (B) qPCR expression analysis of indicated HOX genes in control vs Tet-treated NMP-derived trunk NC cells. Error bars represent SD (n=4–6). *p<0.05, **p<0.005, ***p<0.0005 (paired t-test). (C) Immunofluorescence analysis of the expression of HOXC9 in control vs Tet-treated NMP-derived trunk NC cells. Quantification of HOXC9+ cells in the presence and absence of Tet is also shown. Error bars represent SD (n=3). *p<0.05 (paired t-test). Scale bar = 100 μm. (D) qPCR expression analysis of indicated markers in control vs Tet-treated NMP-derived trunk NC cells. Error bars represent SD (n=6). *p<0.05, **p<0.01, n.s. not significant (paired t-test). (E) Immunofluorescence analysis of the expression of SOX10 in control vs Tet-treated NMP-derived trunk NC cells. Quantification of SOX10+ cells in the presence and absence of Tet is also shown. Error bars represent SD (n=3). Scale bar = 100 μm. n.s. not significant (paired t-test).