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. Author manuscript; available in PMC: 2023 Mar 25.
Published in final edited form as: Nature. 2022 Jun 16;609(7925):136–143. doi: 10.1038/s41586-022-04953-1

Extended Data Figure 6. Endogenous WNT required for posterior patterning in marmoset 2D gastruloids models.

Extended Data Figure 6

a-c, pSMAD1/5 (phosphorylated SMAD 1/5) activity in marmoset 2D-gastruloids detected by immunostaining at day 2 (a) or day 4 (b) compared to human 2D-gastruloids under conventional conditions at day 2 (c). Micropatterned colonies were treated with self-renewal conditions (10 ng/mL FGF + 20 ng/mL Activin A), conventional gastruloid conditions (10 ng/mL FGF + 20 ng/mL Activin A + 50 ng/mL BMP4) or WNT modulatory conditions (10 ng/mL FGF + 20 ng/mL Activin A + 3 μM IWP-2 or 10 ng/mL FGF + 20 ng/mL Activin A + 3 μM CHIR99021). Representative maximum projection of immunofluorescence images (left). Quantification plots (right) mean +/- SEM across a minimum of 10 gastruloids across 2 wells. F/A = FGF/Activin A. pSMAD1/5 gradient indicates that in the marmoset system, similar to the human, FGF/ActivinA/BMP4 induce a graded response to BMP signalling at day 2, with the highest signalling in the outermost ring of the colony.

d, Molecular characterisation of 2D-gastruloids. Representative immunofluorescence images of gastruloids differentiated in conventional gastruloid conditions (10 ng/mL FGF + 20 ng/mL Activin A + 50 ng/mL BMP4) for 2 days. Quantification plots (bottom) display mean +/- SEM across a minimum of 10 gastruloids across 2 experiments. Anterior domain (A, SOX2+, TBXT-, TFAP2C-), posterior domain (P, TBXT high, CDX2 heterogeneous, SOX17 sparse, LEF1 sparse), and amnion domain (Am, TFAP2C high, SOX2-) demarcated. ISL1 and TFAP2A are observed heterogeneously predominantly in the amnion region. PDGFRA expression is low, indicating lack of mature mesoderm.

e, Schematic of lineage identities present in 2D-gastruloids and marker patterns that define each region.

f, WNT-associated anterior/posterior patterning phenotypes of 2D-gastruloids. Representative immunofluorescence images of gastruloids differentiated in conditions listed at left for 2 days. Quantification plots (bottom) mean +/- SEM across a minimum of 10 gastruloids across 2 wells.

g, WNT-associated amniogenesis phenotypes of 2D-gastruloids. Representative immunofluorescence images of gastruloids differentiated in conditions listed at left for 2 days. CDX2 expression is lost upon 3 μM IWP2 treatment, but TFAP2C and TFAP2A remain expressed. 3 μM CHIR treatment (WNT agonist) leads to low CDX2 expression but does not support upregulation of TFAP2C or TFAP2A. Quantification plots (bottom) mean +/- SEM across a minimum of 10 gastruloids across 2 wells.

h, No evident change in expression profiles associated with Hedgehog signalling manipulation in 2D-gastruloids. Representative immunofluorescence images of gastruloids differentiated in conditions listed at left for 2 days. Exogenous Indian Hedgehog (IHH, 200 ng/mL) did not lead to loss of pluripotency under FGF/Activin self-renewal conditions, or change expression patterns under conventional gastruloids conditions. Inhibition of hedgehog signalling with Cyclopamine (5 μM) also did not lead to evident changes in expression patterns. Quantification plots (bottom) mean +/- SEM across a minimum of 10 gastruloids across 2 wells.