Par complex is essential for NPT.

(A) Schematic illustration of the generation of ESCs without Crumbs, Par, or Scrib complex. (B-F) Crumbs, Par, or Scrib complex KO did not affect ESCs pluripotency, morphology, or proliferation. The expression levels of endogenous pluripotency markers Nanog, Oct4, Sox2, and Klf4 showed no significant differences (B-C). ESCs maintained a characteristic dome-shaped colony morphology over more than 30 passages (D). The initial cell number was 35,000 on day 0, and cell amounts were determined on day 3 (E-F). (G-I) Par KO yielded cells exhibiting a dome-shaped colony morphology during NPT, contrasting with the flat monolayer morphology of WT cells. The defining criteria for both morphological types are specified in (H). (J-K) Par KO resulted in aberrant subcellular localization of CDH1 and ZO1 in primed cells, as captured by LSM800 confocal microscopy. Experiments were repeated for at least three times unless otherwise mentioned. Error bars represent S.D. Two-way and one-way ANOVA analysis were performed in (C) and (E-F), respectively.

Suppression of AKT or FAK signaling rescues the defect induced by Par KO.

(A) Schematic illustration of RNA-seq analysis. Cells on day 0, 1, 3, 4, and 6 during NPT were collected for bulk RNA-seq during NPT. (B) Analysis of intercellular correlations revealed that the transcriptomic differences between Par KO and WT cells became progressively more significant as NPT proceeded. (C) Pseudotime analysis of the NPT process revealed that the differentiation progress of Par KO was slower than that of WT starting from day 4, with the difference reaching its maximum on day 6. (D) GO analysis of enriched pathways of DEGs on day 6. (E) Expression of key genes of the enriched pathways in (D) was analyzed, also see Figure S2B. (F) Cells were treated with agonists or inhibitors targeting the enriched pathways during NPT, followed by morphological analysis of dome-shaped colonies. Experiments were repeated for at least three times, with the exception of high-throughput sequencing, or if unless otherwise mentioned. Error bars represent S.D.

Conditioned medium from WT cells rescues the defect induced by Par KO.

(A-C) tdTomato-labeled Par KO ESCs were co-cultured with WT ESCs during NPT (cell number was 20,000 on day 0). Cell amounts and the proportion of tdTomato+ were determined on Day 6. (D-E) Based on the results from (B and C), the proportion of WT and Par KO cells in the co-culture systems was calculated, along with the proliferation efficiency of WT and Par KO cells. (F) Analysis of interactions between Par KO and WT cells using RNA-seq data. (G) Schematic illustration of component separation of WT CM. The total WT CM was separated into 7 groups. (H-I) Different components of WT CM were used to treat the Par KO cells respectively, and the cell morphology were analyzed. (J-K) Protein profile was conducted on WT and Par KO CM to analyze the differentially expressed proteins and their signaling pathways. Experiments were repeated for at least three times unless otherwise mentioned. Error bars represent S.D. One-way ANOVA analysis was performed in (B-C).

The AKT–FURIN–LEFTY–ECM–integrin–FAK signaling axis mediates Par complex-dependent morphological remodeling.

(A) Analyzed the transcriptome patterns of Par KO primed cells treated with MK2206 and WT CM on Day 6 during NPT. (B) Overlap analysis was performed on the upregulated genes from three comparisons: WT versus Par KO, MK2206- or WT CM-treated Par KO versus untreated Par KO. (C) KEGG analysis was then conducted on the 198 commonly upregulated genes in (B). (D) Par KO ESCs were treated with WT CM, MK2206, or PF562271 respectively during NPT, the p-FAK level in the cells was detected on Day 6. (E) Target proteins were overexpressed individually in Par KO ESCs, followed by subjecting the overexpressing ESCs to NPT and analyzing their cell morphology. (F) Analyzed the levels of LEFTY and THBS1 in WT and Par KO CM using proteomic data. (G) Par KO ESCs were treated with MK2206 or WT CM respectively during NPT, and the Furin mRNA expression was detected on Day 6. (H) Par KO ESCs were treated with WT CM, MK2206, or PF562271 respectively, and the protein level of the LEFTY and FURIN was detected. The position indicated by the arrow represents the LEFTY cut by FURIN. (I-J) Cell morphology of WT and Par KO cells treated with Furin inhibitor (BOS318) during NPT. Experiments were repeated for at least three times unless otherwise mentioned. Error bars represent S.D. Two-way and one-way ANOVA analysis were performed in (F) and (G), respectively.

Par KO impairs lineage differentiation via the AKT–FAK signaling axis.

(A) WT ESCs, Par KO ESCs, WT primed cells, and Par KO primed cells were subjected to EB differentiation via suspension culture. (B-E) Transcriptome profiling was performed on day 6 of EB differentiation separately for ESC-EBs (WT vs Par KO) and primed cell-EBs (WT vs Par KO). (F) Teratomas were generated by subcutaneous injection of WT and Par KO ESCs into immunodeficient mice, followed by histological assessment via H&E staining. H&E-stained sections were imaged by using the TG Tissue FAXS Plus ST. (G) Par KO ESCs were treated with MK2206 or PF562271 during EB differentiation, and the expression of three germ layers markers were detected. (H-I) Par KO ESCs were treated with MK2206 or PF562271 during the NSC differentiation. The proportion of NESTIN+ cells were detected by using the CytoFLEX. (J) Subcutaneous injection of WT, Par KO, MK2206- or WT CM-treated Par KO primed cells into immunodeficient mice to form teratoma, followed by H&E staining for histological assessment. Experiments were repeated for at least three times unless otherwise mentioned. Error bars represent S.D. Two-way ANOVA analysis was performed in (G).

Par KO impairs neural tube organoids formation and maturation via the AKT–FAK signaling axis.

(A) Schematic illustration of neural tube organoid induction. (B) The morphological changes of WT and Par KO cells during neural tube organoids induction. (C-D) The neural tube organoids were subjected to subsequent maturation culture, the morphological differences were recorded (C) and maturation rate were analyzed (D). (E) Mature neural tube organoids were assessed by immunofluorescence for markers of neural crest cells and neurons, such as SOX10, TUJ1, and NeuN. (F-G) Par KO ESCs were treated with co-culture, WT CM, MK2206, or PF562271 during neural tube organoid induction, the lumen formation and spontaneous elongation efficiency under different condition was assessed. (H) After treatment with co-culture, WT CM, MK2206, and PF562271, the expression of the lumen markers (ZO1 and CD133) as well as the NSC markers (PAX6 and NESTIN) was detected in Par KO neural tube organoids. (I-J) Par KO ESCs were treated with co-culture, WT CM, MK2206, or PF562271 during neural tube organoid maturation culture, the survival and maturation efficiency under different condition was assessed. Experiments were repeated for at least three times unless otherwise mentioned. Error bars represent S.D. One-way and two-way ANOVA analysis were performed in (D) and (G, J), respectively.

Par complex is essential for NPT, related to Figure 1.

(A) The sgRNA design strategy for knockout of Crumbs, Par, or Scrib complex. The plasmid was constructed using the CRISPR-cas9 technology. (B) Knockout efficiency for Crumbs, Par, and Scrib complexes was validated by WB. (C) Crumbs, Par, or Scrib complex KO did not affect ESCs pluripotency, the proportion of Oct4-GFP+ cells was not significantly different from that of the WT. (D) Par complex KO caused the cells to exhibit a dome-shaped colony morphology, and this defect can be stably maintained at least three passages of culture. (E) WT primed cells presented flat monolayer clusters, while Par KO primed cells exhibited dome colonies, as visualized by Phalloidin staining and imaged by LSM800 confocal microscopy with Z-stack acquisition. Experiments were repeated for at least three times unless otherwise mentioned. Error bars represent S.D.

AKT and FAK are key signal pathways that regulate cell morphology during NPT, related to Figure 2.

(A) The DEGs between Par KO and WT cells at different time points during NPT. The number of DEGs was the highest on Day 6. (B) Expression of key genes of the enriched pathways in (Figure 2D) was analyzed. (C) Cells were treated with agonists or inhibitors targeting the enriched pathways during NPT, followed by morphological analysis of dome colonies. (D) Par KO cells were treated with MK2206, PF562271, or PD0325901 during NPT and assessed Oct4-GFP+ expression. (E) The expression of ESC markers (such as Esrrb and Klf4) in cells treated with the MK2206, PF562271, and PD0325901 were analyzed during NPT. Experiments were repeated for at least three times unless otherwise mentioned. Error bars represent S.D. Two-way ANOVA analysis was performed in (E).

WT conditioned medium rescues the defect induced by Par KO, related to Figure 3.

(A-B) NPT in co-culture system was carried out by mixing Par KO ESCs and WT ESCs at ratio of 1:1 or 2:1. Cell morphology was analyzed at the end of NPT. (C-D) Par KO ESCs were treated with WT CM, KO CM, or WT CL during NPT, and the cell morphology was analyzed. (E) The rescue effect of WT CM on Par KO ESCs showed a time-dependent pattern. WT CM must be present during the period from Day 2 to Day 4 of NPT to exert the rescue effect. While any collection of WT CM taken on Days 1 to Day 5 during NPT has a rescue effect. Experiments were repeated for at least three times unless otherwise mentioned. Error bars represent S.D.

Par complex mediates cell morphology through the AKT-FAK signal axis, related to Figure 4.

(A) Par KO cells were treated with MK2206, or WT CM during NPT, the p-AKT level were investigated on day 6. (B) Transcriptomic analysis of FAK-related DEGs between WT and Par KO cells during NPT using RNA-seq data. (C) Based on the FAK signaling pathway, functional candidate proteins were screened and subsequently overexpressed in Par KO ESCs to validate their biological roles. (D) Overexpression of LEFTY resulted in flat monolayer clusters of Par KO cells resembling that of the WT cells during NPT. (E) Overexpression of LEFTY in Par KO cells reduced the p-FAK level during NPT. Experiments were repeated for at least three times unless otherwise mentioned. Error bars represent S.D.

Par KO impairs lineage differentiation via the AKT–FAK signaling axis, related to Figure 5.

(A) During EB differentiation from ESCs, expression of three germ layer markers was detected. (B-C) ESCs were induced to differentiate into NSCs. The cell morphology was analyzed, and the expression of NESTIN, a marker for NSCs, was detected. (D) Par KO ESCs were treated with MK2206 or PF562271 during the EB differentiation. The EB differentiation was performed using a suspension culture method. (E) Par KO ESCs were treated with MK2206 or PF562271 during the NSC differentiation. Experiments were repeated for at least three times unless otherwise mentioned. Error bars represent S.D. Two-way and one-way ANOVA analysis were performed in (A) and (C), respectively.

Uncropped gels, related to Figure 4, Figures S1 and S4.