Esco2 restrains pathological endothelial-mesenchymal transition through hidden isoform regulation in a Roberts syndrome model
Roy Jung, Sherin Saheera, Chinmay M. Trivedi
Roy Jung, Sherin Saheera, Chinmay M. Trivedi
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Research Article Cell biology Development Vascular biology

Esco2 restrains pathological endothelial-mesenchymal transition through hidden isoform regulation in a Roberts syndrome model

Abstract

Roberts syndrome is caused by biallelic ESCO2 pathogenic variants, but the mechanisms linking ESCO2 loss to embryonic endothelial failure remain poorly defined. Endothelial cells must maintain identity and resist endothelial-mesenchymal transition (EndMT), yet conventional transcriptomics measures gene abundance rather than isoform composition. Here, using endothelial-specific Esco2 deletion in a mouse model relevant to Roberts syndrome, we show that Esco2 loss causes yolk sac vascular remodeling failure, pathological EndMT, diversion from hemogenic endothelial fate, hemogenic endothelial depletion, impaired hematopoietic output, and embryonic lethality. Short-read single-cell RNA-seq revealed broad endothelial reprogramming, whereas single-cell long-read isoform sequencing uncovered a hidden layer of isoform dysregulation that conventional gene-level analysis failed to resolve. Esco2-deficient endothelium exhibited transcriptome-wide isoform switching, disruption of a multi-tier splicing-factor network, and predicted broad loss or rewiring of protein-coding output. Chromatin occupancy profiling localized Esco2 to exonic regions near alternatively spliced loci, supporting a cotranscriptional route to spliceopathy. Whole-genome sequencing showed that local DNA variation was insufficient to explain these changes, supporting a predominantly posttranscriptional mechanism. These findings identify isoform control as a disease-relevant layer of endothelial identity maintenance in Roberts syndrome.

Authors

Roy Jung, Sherin Saheera, Chinmay M. Trivedi

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Figure 5

Hidden isoform switching predicts widespread protein loss in Esco2-deficient endothelial cells.

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Hidden isoform switching predicts widespread protein loss in Esco2-defic...
(A) Scatter plot of 1,827 genes comparing gene-level fold change with maximum single-isoform log2 fold change, defined as the largest absolute log2 fold change of any isoform. Most altered genes (77.7%) were detected only at the isoform level. (B) Distribution of hidden isoform changes across 4 endothelial clusters. (C) Top 20 isoform-switched genes across endothelial clusters. (D) Functional classification of isoform-switch events. (E) Predicted protein fate among 1,827 altered genes: lost, 60.6% (1,107 genes); gained, 26.4% (482 genes); rewired, 10.6% (193 genes); noncoding, 2.5% (45 genes). (F) Dab2 switchPlot and AlphaFold3 pLDDT analysis showing predicted phosphotyrosine-binding domain replacement (dIF = −0.699, q = 4.0 × 10–²4). (G) Cald1 switchPlot and AlphaFold3 pLDDT comparison. The control-enriched l-CaD isoform (534 aa) was lost, whereas h-CaD (768 aa) was gained in Esco2-deficient endothelial cells (dIF = −0.214 and +0.076; q = 5.0 × 10–³9 and 2.2 × 10–¹²). The switch introduces a 234 aa central repeat domain. switchPlots used endothelial cell–only pseudobulk data. (H) COL4A1 immunofluorescence in control and Esco2F/F;Lyve1-Cre tissues. Top, E9.5 yolk sacs stained for Lyve1 (green), COL4A1 (red), GFP (gray), and Hoechst (blue); bottom, E9.5 embryo proper stained for GFP (green), COL4A1 (red), and Hoechst (blue). COL4A1 protein expression increased in GFP+ Lyve1-lineage cells in mutant yolk sacs and embryos. Images are representative of 3 control and 4 mutant biological replicates; quantification is shown in Supplemental Figure 2. Scale bars: 10 μm. For F and G, isoform fraction (IF) is individual transcript expression divided by summed expression of all detected transcripts from the same gene; dIF = IFmutant − IFcontrol. Full transcript identifiers are provided in Supplemental Table 3.