a) Immunofluorescence stainings for pERK and TBXT and their radial intensity profiles in 2D gastruloids at different times. Error bars in graphs are standard deviations over N=4 colonies. b,c) Effect FGF receptor inhibition (FGFRi, b) or MEK inhibition (MEKi, c) on pERK and TBXT in colonies fixed at 42h after BMP treatment. Inhibition for either 30 minutes at 41.5h (rounded to 42 in figure) or throughout differentiation. d) BMP only control and FGFRi inhibition in cells expressing doxycycline-inducible SOScat with and without addition of doxycycline. e-f) Cell numbers (e) and radial profiles of TBXT positive cells for conditions in (d). g) PS marker TBXT, pERK and pluripotency marker SOX2 in Nodal knockout cells with inhibitors of Wnt secretion (WntSeci) and BMP receptors (BMPRi) with or without MEKi or FGFRi. All scale bars 50um.

a) Schematic of the FGF/ERK signaling pathway. b) Distribution of pERK levels in TBXT+ cells versus ISL1+ and other cells based on data and thresholds in Supp. Fig. 2b. c) Expression of key markers in PHATE39 projection of scRNA-seq data for 42h colony. d) Cell type annotation. PS-LC: primitive streak-like cells, NasMe: nascent mesoderm, AE-LC: amniotic ectoderm-like cells: PGC-LC: primordial germ cell-like cells, endo: definitive endoderm. e) Expression of canonical FGF ligands (log transformed and smoothened). f) Comparison of FGF ligand expression in 2D human gastruloids to human, monkey, and mouse embryo, normalized within each type of sample. g-i) Expression of several genes involved in FGF signaling with strong differential expression between PS-like cells or nascent mesoderm and pluripotent cells, split by gene category.

a) phospho-FGFR1 stain with and without 1h FGFRi treatment at 41h, radial intensity profile on the right. b) FGFR1 with and without 1h FGFRi treatment at 41h, radial intensity profile on the right. c) qPCR data for relative expression of FGF receptor 1 isoforms IIIb, IIIc and total FGFR1 in pluripotent cells, gastruloids at 42h and directly differentiated PS-like cells. d) RNA-seq analysis of absolute FGFR1 isoform expression. All scale bars 50 um.

a) pERK and pFGFR1 response of pluripotent colonies in standard culture grown in FGF-free (E6) medium for 24 hrs and then treated with FGF2 for 30 minutes. b) Fluorescent dextran in micropatterned pluripotent colony with quantification. c) pERK response in micropatterned pluripotent colony after 42h in E6 with TGFβ and FGF2. d) Radial intensity profile of dextran and pERK for conditions in b,c), averaged over 2 colonies. e) Cross-section of 2D gastruloid shows FGF receptor 1 predominantly below the tight junction marked by ZO-1. White box marks are magnified in inset e’. f) pERK and pFGFR1 response in transwell experiment with cell treated with FGF either apically or basally. g) ERK signaling in scratched colonies with or without FGFRi. h) Quantification of pERK intensity as a function of distance from the scratch. Error bars represent standard deviation over colonies. Scale bars 50 micron.

Illustrations in panel f created with BioRender.com/v73i147.

a) Pattern at 42h with exogenous FGF2 in the culture medium for different amounts of time. b) Pseudotime analysis of FGFs and TBXT, TBX6 expression. c,f) FGF4 (c), and FGF17 (f) FISH co-stained for TBXT and pERK at 32h and 40h after BMP4 treatment. d,g) Radial intensity profiles for conditions in (c,f) respectively. e,h) Scatterplots of FGF4 (e), and FGF17 (h) mRNA versus pERK colored for TBXT. i) FGF4 FISH co-stained for TBXT and pERK in ctrl (scramble) shRNA versus FGF4 shRNA cells and corresponding radial intensity profiles.j) pERK and TBXT expression in WT versus FGF17+/- cells. k) SOX17 and TFAP2C expression in WT versus FGF17+/- cells.

Cell signaling reagents.

Primary antibodies used for immunofluorescence.

Secondary antibodies.

Marker genes for cell fate annotation

qPCR primers

Probes for Fluorescent In Situ Hybridization

a) DAPI images and ISL1 stains corresponding to Fig. 1a. b) Scatter plots of TBXT and ISL1 expression. c) Staining for three primitive streak markers with or with FGFRi or MEKi. d) DAPI stain corresponding to Fig. 1d. e) Radial intensity profile for TBXT corresponding to Fig.1d. f) Quantification of conditions in Fig.1g for five images each, N indicates total number of cells.

a) PHATE projection of scRNA-seq data colored for sample. b,c) Expression of canonical FGF ligands across different cell types in 2D gastruloids (b) and human CS7 embryo (c). Expression across clusters is normalized by row, while total expression is shown in a separate column to the directly to left. The leftmost column indicates differential expression between PS-like and pluripotent cells. The color scale of (differential) expression is cut off for better contrast. d) Threshold-based annotation (left) for human, monkey, and mouse embryos used in interspecies comparison (Fig. 2f), compared to original annotation (right). e) Interspecies comparison as in Fig. 2f but for nascent mesoderm and PS-LC + nascent mesoderm. f) Bulk RNA-seq data by Loh et al38 for expression of different FGFs in directed differentiation to mid primitive streak (MPS) or anterior primitive streak (APS) relative the human embryonic stem cells (hESCs). g) Expression of genes involved in FGF signaling.

a) DAPI image corresponding to Fig. 3a. b-c) pFGFR1 (b) or pERK (c) and TBXT stains with and without continuous MEKi treatment. d) DAPI image corresponding to Fig. 3b. e) Design of qPCR primers to detect FGFR1 isoforms, diagram adapted from Eswarakumar et al63. f) Primer sequence and properties. g) Amplicon sizes match prediction. Scale bars 50um.

a) DAPI image corresponding to Fig. 5a. b) Pseudotime dynamics from Fig.5b with each gene normalized individually. c) Correlations between FGFs and TBXT, TBX6 colored for clusters, with cluster colors matching Fig. 2d. d,e) DAPI images and overlays including DAPI corresponding to Fig.5c,f respectively. f) Representative stainings and quantification for FGF4 siRNA versus controls. g) DAPI stain, individual channels and overlay with DAPI for images corresponding to Fig. 5k. h) Scatterplot of fraction of TBXT-positive cells in repeated experiments with FGF17+/- cells colored for genotype (left) or experiment (right). i) Individual channels including DAPI and FOXA2 for images corresponding to Fig.5l. j) Radial profiles of cells positive for different markers in FGF17+/- versus WT.

a) Genotyping for FGF17+/- cells.