Spatial segregation of Nanog mRNA domains in pluripotent colonies and the blastocyst.

Fluorescence in situ hybridization (ISH) for Nanog using 3’UTR specific (red) and CDS specific (green) probes in mouse and human embryonic stem cells (ESCs) and blastocyst stage embryos. (A) Representative images showing spatial distribution of Nanog 3’UTR and CDS expression patterned ESC colonies. Nanog exhibits prominent most pronounced spatial segregation of 3’UTR and CDS biased expression compared with other pluripotency associated genes. (See Figure 1-figure supplement 1A for Sox2 and Oct4). (B) Higher magnification image of Nanog expression at colony border highlighting cells with high 3’UTR signal and little or no detectable CDS signal (arrows). (C) Nanog 3’UTR/CDS spatial patterning is maintained across colony diameters ranging from 80-1000 um (images from different micropatterned chips). (D) Quantification of the fraction of high Nanog 3’UTR cells as a function of colony size after plating of a single pool of hESCs (mean +/- SEM; n=3 biological replicates; Student’s t-test). (E) Nanog 3’UTR/CDS expression patterns differ between aggregated (>3 cells) and sparsely distributed ESCs (means of three biological replicates: Chi-square analysis (X2, p<0.01)). (F) Nanog 3’UTR/CDS expression in blastocyst stage mouse and human embryos. Additional markers are shown in Figure 1-figure supplement 1A. Scale bar: A, C; 100 um, B; left 100 um, right 20 um, C; 160 um, E; 130 um, F; mouse 20 um, human, 45 um.

Differential Nanog mRNA domain usage stratifies pluripotent cell states

(A) t-SNE projections of hESCs cultured under indicated growth conditions, colored by growth condition (left), normalized Nanog expression (middle), and relative Nanog mRNA domain usage (3’UTR vs CDS; right). Red indicates cells with higher relative 3’UTR usage and green indicates cells with higher relative CDS usage. (B) Gene Ontology (GO) terms associated with hESC clusters characterized by higher relative Nanog 3’UTR or CDS usage. (C) t-SNE projections of mESCs under indicated growth conditions, colored as in (A). (D) GO terms associated with mESC clusters characterized by higher relative Nanog 3’UTR or CDS usage. (E) Combined ISH and immunofluorescence showing spatial organization of Nanog mRNA domain usage together with indicated chromatin and junctional markers. IF signals align with Nanog 3’UTR/CDS expression in concentric regions. Additional IF analyses are shown in Figure 2-figure supplement 1D-H). Scale bar E; 100 μm.

Nanog 3’UTR deletion impairs ESC colony dispersal

(A) Schematic of selective CRISPR knockout of the Nanog CDS or Nanog 3’UTR in mESCs with validation by genomic PCR, western blot, and Nanog IF. The CDS was targeted with a single gRNA and the Nanog 3’UTR (excluding the polyadenylation site) was deleted using two flanking gRNAs. (B) Western blot analysis of Oct4 and Sox2 expression across control (Ctl), Nanog CDS knockout (NCKO), and Nanog 3’UTR knockout (NUKO) lines indicating maintenance of pluripotency associated protein expression. (C) Low-density clonal cultures stained for alkaline phosphatase demonstrating pluripotent identity across lines. (D) IF for phalloidin and α-tubulin in planar culture. NUKO colonies show increased circularity compared to Ctl and NCKO colonies. Data are shown as mean +/- SEM (n=3 biological replicates; n>5 colonies per condition; Student’s t-test). (E) On CYTOOTM micropatterned circles, NUKO cells showed reduced spreading across the circle compared to Ctl; NCKO spread across the circle but left gaps while Ctl cells formed a cohesive monolayer. Orange arrows indicate differential border patterning; purple arrows indicate gaps. (F) Live imaging for 48 h shows reduced spreading and increased post-division clustering in NUKO cells relative to Ctl and NCKO cells. Data are shown as mean +/- SEM (n=3 biological replicates; Student’s t-test). (G) IF of 8-day colonies stained for H3K27me3 (border associated), Sox2 (non-border), and phalloidin (F-actin). All genotypes establish concentric organization, indicating that Nanog 3’UTR deletion impairs spreading behavior without abolishing spatial organization. Scale bar (yellow, bottom right); A;150 um, C; 8.3 mm, D; 110 um, inset 25 um, E; left, 250 um, middle 125 um, right, 65 um, F; 15 um, G; left, 120 um, right, 40 um.

Nanog 3’UTR and Nanog CDS deletions uncouple transcriptional and spatial behaviors in pluripotent ESCs.

(A) Principal component analysis (PCA) of bulk RNA-seq showing separation of control (Ctl), NUKO, and NCKO lines, with independent clonal replicates clustering by genotype. (B) Heatmap of pluripotency and differentiation associated gene expression showing broad retention of pluripotency markers across all lines. (C,D) Gene Ontology (GO) analysis of genes differentially expressed in NUKO and NCKO cells relative to Ctl, highlighting distinct biological processes associated with genes upregulated (C) or downregulated (D) in each genotype. NUKO cells show enrichment for processes related to early extracellular matrix (ECM) remodeling and pattern specification, whereas NCKO cells are enriched for developmentally associated and morphogenetic gene categories. (E) Heatmaps of ECM structural and ECM remodeling genes showing reduced expression of early ECM structural genes in NUKO cells and increased expression of ECM-related remodeling genes associated with epithelial organization and matrix stabilization in NCKO cells. (F) Heatmap of chromatin modifying gene expression showing broad alterations in NCKO cells, including reduced expression of TET family dioxygenases, compared with Ctl and NUKO cells. (G) Heatmap of pattern specification associated genes showing increased expression in NUKO cells relative to Ctl and NCKO cells, while pluripotency marker expression remains broadly maintained. (H) Mixed label assay comparing Ctl (red) and NUKO (green) cells showing preferential localization of Ctl cells to colony border regions. (I) Mixed label assay comparing Ctl cells and Ctl cells overexpressing the Nanog 3’UTR (Ctl:NUOE), showing enrichment of Nanog 3’UTR overexpressing cells at colony borders. (J) Mixed label assay comparing NUKO cells and NUKO cells overexpressing the Nanog 3’UTR (NUKO:NUOE), showing preferential localization of Nanog 3’UTR overexpressing cells to border regions. Data represent mean +/- SEM (n=3 biological replicates; Student’s t-test. Heatmap analysis for curated genes according to references in methods. Scale bar (yellow, bottom right); H; left,70 um, right, 100 um, I; left, 45 um, right, 120 um, J; left, 45 um, right, 45 um.

ROCK inhibition modulates cytoskeletal organization in Nanog 3’UTR deleted cells.

(A) Embryoid body (EB) formation from Ctl, NCKO, NUKO and full-length knockout (FLKO) mESCs cultured for 2 days under non-adherent conditions. Ctl and NCKO cells form predominantly spherical EBs, whereas NUKO and FLKO cells form elongated, chainlike structures. Yellow arrowheads show “spheres”. Validation of FLKO lines in Figure 5-figure supplement 1A-E. (B) IF staining for cytoskeletal and adhesion-associated proteins, including Talin, integrin a5, and Vinculin, showing altered localization patterns in NUKO and FLKO compared to Ctl and NCKO cells. Summary schematics indicate representative protein distributions. Additional markers in Figure 5-figure supplement 1A-E; Supplementary Table 2. (C) Colony morphology and Talin localization in Ctl and NUKO cells treated with vehicle (DMSO) or the ROCK inhibitor Thiazovivin (TZV, 4μM). TZV treatment alters colony morphology and Talin distribution in NUKO cells. Insets show representative colonies. (D) IF staining for Yap in Ctl and NUKO cells treated with DMSO or TZV. YAP localization is predominantly cytoplasmic (white arrows) in NUKO and shifts following (yellow arrows) TZV (4 um) treatment. NCKO and FLKO cells shown in Figure 5-figure supplement 1H. (E) Schematic summary illustrating colony level morphological and cytoskeletal changes associated with Nanog 3’UTR deletion and their modulation by ROCK inhibition. Scale bar (yellow, bottom right); A; left, 980 um, right, 300 um, B; 60 um, C; 90 um, inset 30 um, D; 55 um, inset 25 um.

Nanog CDS and Nanog 3’UTR deletions differentially bias differentiation trajectories

(A) LIF withdrawal assays in Ctl, NCKO, NUKO and FLKO mESCs cultured without LIF for 8 days (left and middle columns) or following extended passaging under LIF depleted conditions (right column). Representative images are shown. Colony size was quantified. Data represent +/- SEM (n=3 biological replicates; > 5 colonies/replicate per condition; Student’s t-test). (B) Embryoid body differentiation followed by explant plating (10 days) showing expression of mesoderm/endoderm markers (FoxA2, Gata4) and ectoderm markers (Sox2, Pax6), indicating retained competence to generate derivatives of all three germ layers across genotypes. (C) Heatmap of bulk RNA-seq from plated 10-day differentiation cultures showing expression of representative endodermal (Endo), mesodermal (Meso) and ectodermal (Ecto), markers in Ctl, NCKO and NUKO cells. (D) qPCR analysis of sister cultures showing significant changes in representative endodermal (Gata4, Sox17) and mesodermal (Eomes, T) markers in NCKO cells relative to Ctl and NCKO cells. Additional markers tested did not show significant differences (Supplemental Table 3). Scale bar (yellow, bottom right); A; 160 um, B; 100 um.

Spatial patterning of additional pluripotent gene 3’UTR and CDS expression in hESCs cultured on micropatterned substrates and in blastocyst embryos.

(A) Fluorescence in situ hybridization (ISH) for Oct4 and Sox2 using 3’UTR specific (red) and CDS specific (green) probes in mouse and human embryonic stem cells (ESCs). (B) Schematic of early embryonic development from the 4-cell stage to the blastocyst. (C) ISH for Nanog and Oct4 3’UTR (red) and CDS (green) and Nanog and Oct4 protein (blue) in blastocyst stage mouse embryos. (D) Nanog 3’UTR/CDS expression together with Oct4 protein (showing ICM) in human blastocyst, the first two panels are reproduced from Figure 1 for reference. Scale bar (yellow, bottom right); A; 100 um, C; 110 um, D; 200 um.

Spatial pattering of Nanog 3’UTR and CDS expression in hESCs cultured on micropatterned substrates.

Nanog 3’UTR (red) and CDS (green) fluorescence in situ hybridization (ISH) in hESCs cultured on CYTOOTM micropatterned circles. (A) Border cells show enriched nuclear Nanog 3’UTR and low or undetectable Nanog CDS, DAPI (blue). (B,C) Similar spatial patterning is observed in irregular and rectangular geometries with Nanog 3’UTR enriched at colony borders and higher Nanog CDS expression in interior regions. A band of combined 3’UTR/CDS expression of ∼100 um in width is consistently observed. Many DAPI positive cells show little or no detectable Nanog 3’UTR or CDS expression. A difference in confluence across circles due to cell shifting during plating reveals that Nanog 3’UTR expression is visible in border cells before Nanog CDS is detected in interior cells (white arrows). (D) Low magnification view of CYTOOTM chips showing reproducible patterning across multiple circle sizes. In larger (500 μm) circles, pattern formation is delayed relative to smaller circles, suggesting confluency dependent pattern. (E) Pattern is reproducible across multiple circles, with delayed expression in slower growing outer circles presumably due to cell confluence. In these circles Nanog 3’UTR border cells are visible before interior high CDS cells. Scale bar (yellow; bottom right): (A) 30 um, (B,C,C’)150 um, (D,E) 250 um.

scRNA-seq datasets and additional ISH and IF on micropatterned cultures.

(A) Transcriptome wide analysis in mESCs showing no correlation between overall gene expression level (Normalized Counts; NormC) and 3’UTR/CDS ratio. Each dot (top) represents one gene, (bottom), each circle represents one cell. (B,C) Gene Ontology (GO) analysis of scRNA-seq comparing clusters enriched for Nanog 3’UTR vs cells enriched for Nanog CDS expression in human (B) and mouse (C) datasets. (D-M) Additional markers in relation to Nanog 3’UTR/CDS expression on CYTOOTM micropatterned circles. (D’-H’) Co-expression of histone and chromatin-associated proteins with Nanog 3’UTR/CDS ISH. (I-K) HP1α is not detected in Nanog mRNA expressing cells. (L,M) H3K9ac is not detected (red in P; green in Q) in either Oct4 3’UTR or CDS expressing cells. Scale bar (yellow; bottom right): D-H, 100 um; D’–H’ 30 um; I-K, 110 um; L,M, 60 um.

Genomic validation and Nanog protein stability in Nanog 3′UTR knockout mESC lines.

(A) IGV traces showing deletion of the Nanog 3’UTR in independent Nanog 3’UTR knockout (NUKO) clones. (B) IGV traces showing point mutations or insertions in independent Nanog CDS knockout (NCKO) clones. (C) Western blot time-course analysis of Nanog protein levels in control (Ctl), and NUKO lines after cycloheximide (CHX) treatment for up to 8 hours, showing no detectable differences in Nanog protein stability in cells lacking the Nanog 3’UTR.

Blastocyst integration of Nanog CDS and 3’UTR knockout lines.

(A) Cells (tdTomato-labeled) from independent NCKO and NUKO mESC lines integrate into the inner cell mass (ICM) of mouse host blastocysts. (B-D) Individual data points from mixed cell border localization assays corresponding to mean values summarized in Figure 4H-J. Scale bar (yellow; bottom right): A; 100 um.

Validation of full length Nanog knockout (FLKO) lines and cytoskeletal protein responses to ROCK inhibition.

(A) IGV trace showing deletion of the Nanog 3’UTR and point mutations in the Nanog CDS in FLKO clones FL58 and FL84. (B) Left, qPCR showing absence of Nanog 3’UTR (1.5 kb) transcripts, right; western blot confirming loss of Nanog protein in FLKO lines. (C) GO categories for FLKO cells compared to Ctl cells. (D) Comparison of ECM remodeling gene expression shown in Figure 4E with expression in FLKO cells. Approximately half of genes downregulated in NUKO cells are similarly reduced in FLKO cells, while others show mixed or increased expression. (E) In contrast only ∼20% of genes upregulated in NCKO cells are upregulated in FLKO cells, with many showing reduced expression. Overall FLKO cells display a mixed transcriptional profile relative to NUKO and NCKO cells. (F) Additional actin regulator protein expression in Ctl and NUKO cells. (G,H) Effects of TZV (4 mM) on Talin and YAP (white arrows in G show intact Talin, yellow show puncta), (white arrows in H show perinuclear, cytoplasmic YAP localization, yellow arrows show nuclear localization). (I) Western blot showing increased Talin protein levels in NUKO cells relative to Ctl cells and near complete restoration following TZV treatment. Scale bar (yellow; bottom right): F-H; 100 um.