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. 2026 Jun 30;29(3):49.
doi: 10.1007/s10456-026-10062-8.

Endothelial Notch1 drives multicellular remodelling during hyaloid vessel regression

Shaymaa Khazaal #  1   2 , Abdoul-Razak Sango #  1   2 , Kawtar Zouine #  1   2 , Alexa Silva Sosa #  1   2 , Ariane Vanessa Megne  1   2 , Rony Chidiac  3 , Kiran Bora  4 , Gaelle Mawambo  1   2 , Jing Chen  4 , Malika Oubaha  5   6
Affiliations

Endothelial Notch1 drives multicellular remodelling during hyaloid vessel regression

Shaymaa Khazaal et al. Angiogenesis. .

Abstract

Hyaloid vessel regression is essential for vitreous transparency and normal vision, yet how this transient vascular network is dismantled remains unclear. Here we show that postnatal hyaloid regression in mice is not driven by a measurable increase in apoptosis, but instead by coordinated endothelial and mural cell delamination, extravascular redistribution and a transient plasticity program marked by Snail1 and Slug induction. Notch1 signalling peaks during the regression window, and vascular endothelial-specific Notch1 deletion causes persistent hyaloid vessels, with excessive proliferation and failure of endothelial and mural cell disengagement. Mechanistically, loss of Notch1 preserves endothelial identity, suppresses an endothelial-mesenchymal transition-like transcriptional program and reduces expression of the Wnt co-recepteors Lrp5 and Lrp6. Wnt pathway mutant phenocopies the delamination defect, supporting functional convergence between Notch1 and Wnt signalling during vessel involution. Together, these findings identify Notch1 as a key driver of developmental vascular pruning. They also redefine hyaloid regression as an apoptosis-non-exclusive remodelling process, with broader implications for physiological and pathological vascular remodelling, and may guide therapeutic strategies to modulate vascular regression in ocular disorders.

Keywords: Endothelial plasticity; Endothelial-to-mesenchymal transition; Hyaloid regression; Notch signaling; Vascular remodeling; Vision.

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Conflict of interest statement

Declarations. Conflict of interest: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Cell relocation, apoptosis, and proliferation during postnatal hyaloid vessel pruning. A Schematic diagram of mouse hyaloid vessels (HV) regression and retinal vascular outgrowth at embryonic day 18.5 (E18.5) and postnatal days 0 (P0), 4 (P4), and 8 (P8). B Representative Isolectin B4 (IB4) and DAPI immunofluorescence staining of E18.5, P0, P4 and P8 HV flatmounts. C–E Quantification of HV density, vessel diameter and vascular branch points (n = 5–6). F Representative confocal micrographs of P0, P4 and P8 HV flatmounts labeled with cleaved caspase 3 (C-Casp3), IB4 and DAPI. G–I 3D reconstructions of HV at P0, P4, and P8 show the top and side views. Quantification of apoptotic cells (C-Casp3+) per 100 μm of HV length (J), and extravascular (extrav.) apoptotic cells among total extravascular cells K (n = 3). L Representative images of Ki67, IB4 and DAPI immunofluorescence staining of P0, P4 and P8 HV flatmounts show proliferating cells within (white arrowheads) and outside (yellow arrowheads) the vascular compartment. M–O 3D reconstructions show proliferating cells in vascular and extravascular regions of HV at P0, P4 and P8. Quantification of proliferating cells (Ki67+) per 100 μm of HV length (P), and extravascular proliferating cells among total extravascular cells (Q) (n = 3). Results are expressed as fold change (FC) relative to E18.5 (C–E) and as percentages (%) normalize to P0 (J, K, P, Q). Data are means ± SEM. Represented p values are *≤ 0.05, **≤ 0.01, ***≤ 0.001, ****≤0.0001 from ordinary one-way ANOVA test with Dunnett’s multiple comparison. Non-significant (ns). Scale bars, 500 μm (B, F, L) and 50 μm [for higher magnification images in (F–I, L–O)]
Fig. 2
Fig. 2
Proliferative endothelial and mural cells delamination accompanies hyaloid vessels regression. A Representative confocal micrographs of P8 HV flatmounts from Rosa-mT/mG; Cdh5-CreERT2 mice in the absence (-Tam) or following tamoxifen induction (+ Tam), showing tdTomato (tdT), GFP, and DAPI labeling. B 3D reconstructions of HV illustrating tdT⁺, GFP⁺, and tdT⁺/GFP⁺ and their extravascular localization. Arrowheads indicate tdT⁺ cells (red), GFP⁺ cells (green), and tdT⁺/GFP⁺ cells (yellow). C Quantification of extravascular tdT⁺, GFP⁺, and tdT⁺/GFP⁺ cells, expressed as a percentage of total extravascular cells in -Tam and + Tam conditions (n = 3). D Representative confocal images of P0 and P8 HV flatmounts showing EdU incorporation in ECs (ERG1/2/3⁺). Nuclei are labeled with DAPI. E 3D reconstructions showing proliferating ECs (white circle) and non-proliferating ECs (green circle) in vascular and extravascular regions of HV at P0 and P8. F Quantification of EdU⁺ ECs relative to total number of extravascular ERG1/2/3⁺ cells in HV (n = 3). G Representative confocal images of P0 and P8 HV flatmounts showing EdU incorporation in mural cells (MCs; Desmin⁺). Nuclei are labeled with DAPI. H 3D reconstruction of HV at P0 and P8 showing proliferating MCs (white circle) and non-proliferating MCs (green circle). I Quantification of extravascular proliferating MCs among total extravascular MCs (n = 3–4). Results are expressed as percentages (%) from total cells (C) or as % normalized to P0 stage (F, I). Data are shown as means ± SEM. Represented p-values are ***≤0.001 from two-tailed parametric unpaired t-test. Scale bars, 50 μm
Fig. 3
Fig. 3
Phagocytic uptake of ECs and MCs by hyalocytes and steady state apoptosis during hyaloid regression. A Flow cytometry (FACS) analysis of ECs and non-ECs apoptosis from HV at P0 and P8. Representative FACS plots show the gating strategy followed. Numerals shown in the plots indicate the percentages of the indicated cell population within each plot. B Quantification of total ECs (7-AAD−PE-CD31⁺) and non-ECs (7-AAD− PE-CD31⁻), C CD31 median fluorescence intensity (MFI) and D apoptotic (FITC-AnnexinV+) cells within each population at P0 and P8 (n = 3). E Representative confocal micrographs and F 3D reconstructions of ERG1/2/3, F4/80, and DAPI immunofluorescence in HV at P0 and P8 reveal two EC populations: ERG1/2/3⁺ cells positive for the hyalocyte (HC) marker F4/80 (white circles; ERG1/2/3⁺F4/80⁺) and ERG1/2/3+cells negative for F4/80 (green circles; ERG1/2/3⁺F4/80⁻). G Quantification of extravascular ERG1/2/3+ cells among total extravascular cells, and H extravascular ERG1/2/3+F4/80+ cells, expressed relative to the total number of extravascular ERG1/2/3+ cells (n = 3). I Representative confocal micrographs and J 3D reconstructions of P0 and P8 HV flatmounts labeled for CD13, IBA1, and DAPI reveal two MCs (CD13⁺) populations: MCs co-expressing the HC marker IBA1 (white circles; CD13⁺IBA1⁺) and MCs lacking IBA1 expression (green circles; CD13⁺IBA1⁻). K Quantification of extravascular CD13⁺ cells among total extravascular cells, and L extravascular CD13⁺IBA1⁺ cells, expressed relative to the total number of extravascular CD13⁺ cells (n = 3). Results are expressed as percentages (%) (B, D) and normalized to P0 stage (G, H, K, L) or as arbitrary units (a.u.) (C). Data are shown as means ± SEM. Represented p-values are **≤0.01 from two-tailed parametric unpaired t-test. Non-significant (ns). Scale bars, 50 μm
Fig. 4
Fig. 4
Notch1 drives endothelial identity loss and plasticity-associated gene expression during hyaloid regression. A Quantitative reverse transcription polymerase chain reaction (qRT-PCR) shows downregulation of endothelial cell (EC) identity markers Vegfr2, Pecam1, Vwf and Tie-2 during hyaloid regression. B qRT-PCR analysis shows induction of endothelial mesenchymal transition (EndoMT)-associated transcription factors: Snail1, Slug and Acta2 in hyaloid from P0 to P8. Twist expression remains unchanged. C Western blot analysis of hyaloid cell lysates at P0, P4 and P8 showing expression of Notch1, its active form (Notch1 intracellular domain, N1ICD), the ligands DLL4 and JAG-1 and the downstream target gene HES1 (n = 2–3). Densitometric quantification of each protein is shown relative to the loading control, β-ACTIN. D qRT-PCR analysis shows the levels of Notch1, E Dll4, F Jag1 and G Hes1 transcripts in HV at P0, P4, and P8. H Schematic diagram of tamoxifen-induced Notch1 deletion in ECs in Notch1fl/fl; Cdh5- CreERT2 C57BL/6 transgenic mice. I Validation of Notch1 deletion by qRT-PCR in Notch1cKO versus Notch1cWT P8 HV. J Western blot analysis of P8 hyaloid cell lysates from Notch1cWT and Notch1cKO shows the protein expression of Notch1 and VEGFR2 (n = 2–3). Densitometric quantification of each protein is shown relative to the loading control, β-ACTIN. K qRT-PCR shows upregulation of EC identity markers Vegfr2, Pecam1, Vwf and Tie2 in Notch1cKO versus Notch1cWT P8 HV. L qRT-PCR analysis shows reduction of EndoMT-associated transcription factors Snail1, Slug, Acta2 and Twist in Notch1cKO P8 HV versus Notch1cWT. β-actin was used as a reference gene. Results are presented as fold change (FC) normalize to P0 stage (A, B, D–G) or P8 Notch1cWT (I, K, L). Data are shown as means ± SEM. Represented p values are *≤ 0.05, **≤ 0.01, ***≤ 0.001, ****≤0.0001 from ordinary one-way ANOVA test with Dunnett’s multiple comparison (A, B, D, E, G), Kruskal-Wallis test with Dunn’s multiple comparisons (F) or two-tailed parametric unpaired t-test (I, K, L). n = 3–5 for Vegfr2 and Tie2; n = 3 for Pecam1 and Twist; n = 3–4 for Vwf, Snail1 and Slug; n = 4 for Acta2 (A, B); n = 3–4 for Notch1 D and Hes1 (G); n = 3–6 for Dll4 (E); n = 4–6 Jag1 (F); n = 3 for Notch1 (I); n = 3 for Vegfr2 and Pecam1; n = 4–6 for Vwf; n = 4–5 for Tie2; n = 3–4 for Snail1; n = 3 for Slug and Twist1; n = 4–6 for Acta2 (K, L). Non-significant (ns)
Fig. 5
Fig. 5
Endothelial-specific Notch1 deletion in vivo disrupts hyaloid vascular regression and retards retinal vascular superficial layer formation A Representative confocal micrographs of P8 HV from Notch1cKO and Notch1cWT mice immunostained with IB4 and DAPI. B Quantification of hyaloid vascular density, C vessels diameter, and D hyaloid vascular branch points of confocal microscopy data in (A) (n = 6). E Representative IB4 staining and F quantification of retinal vascular area, and G vascular branch points in P8 flatmounted retinal vasculature in Notch1cKO versus Notch1cWT (n = 5). H, I 3D reconstructions of P14 and P21 retinal vasculature labeled with IB4 show three vascular layers [first superficial layer (yellow), second deep layer (blue) and third intermediate layer (green)] in Notch1cKO versus Notch1cWT (n = 3). Results are presented as fold change (FC) relative to Notch1cWT control HV. Data are shown as means ± SEM. Represented p-values are **≤ 0.01, ****≤0.0001 from two-tailed parametric unpaired t-test (B, C, F, G) or Mann-Whitney test (D). Scale bar, 500 μm (A, E) and 200 μm [for higher magnification images in (A)] and 100 μm [for higher magnification images in (E)], and 50 μm (H, I),
Fig. 6
Fig. 6
Loss of endothelial Notch1 alters hyaloid endothelial and mural cells positioning and proliferation. A Representative confocal micrographs of P8 HV flatmounts from Notch1cWT and Notch1cKO mice showing EdU incorporation in ERG1/2/3⁺ cells. Nuclei are labeled with DAPI. B 3D reconstructions show ERG1/2/3+EdU⁺ cells (white circle) and ERG1/2/3+EdU− cells (green circle) in P8 HV from Notch1cWT and Notch1cKO mice (n = 3). C Quantification of extravascular ERG1/2/3+ among total extravascular cells and D ERG1/2/3+EdU+cells, expressed relative to total ERG1/2/3+ in P8 HV in Notch1cKO versus Notch1cWT (n = 3). E Representative confocal micrographs and F 3D reconstructions of Desmin, Ki67 and DAPI immunofluorescence in P8 from Notch1cWT and Notch1cKO mice, showing distinction between proliferating MCs (Desmin+Ki67+) (white circle) and non-proliferating MCs (Desmin+Ki67−) (green circle). G Quantification of extravascular Desmin+ among total extravascular cells and H Desmin+Ki67+, expressed relative to total Desmin+ of confocal microscopy data in (E) (n = 3). I Representative confocal micrographs and J 3D reconstructions of F4/80, Ki67 and DAPI immunofluorescence in P8 from Notch1cWT and Notch1cKO mice, showing distinction between proliferating HCs (F4/80+Ki67+) (white circle) and non-proliferating HCs (F4/80+Ki67−) (green circle). K Quantification of extravascular F4/80+Ki67+ of confocal microscopy data in (I) (n = 3). Results are expressed as percentages normalized to Notch1cWT control HV. Data are shown as means ± SEM. Represented p-values are *≤0.05, **≤0.01 and ***≤ 0.001 from two-tailed parametric unpaired T-test. Scale bars, 50 μm
Fig. 7
Fig. 7
Notch1-Wnt transcriptional coupling orchestrates endothelial and mural plasticity during hyaloid vessel regression. A qRT-PCR analysis shows transcripts expression of Lrp5 and B Lrp6 in P8 Notch1cKO versus Notch1cWT HV. C, D Gene Set Enrichment Analysis (GSEA) plots showing normalized enrichment scores (NES), False Discovery Rate (FDR) and P-value for Notch 1-associated Reactome pathway analysis of EC cluster scRNA-seq data. Genes were ranked based on differential expression between postnatal day (P3) Fz5−/− and Fz5+/+ control. In Fz5−/−, pathways associated with pre-Notch expression and processing, and Notch1 signaling are downregulated relative to Fz5+/+ controls. E Representative confocal micrographs showing ERG1/2/3, IB4 and DAPI immunofluorescence of P8 HV from Ndp−/− and Ndp+/+mice. F Quantification of HV density, G total and H extravascular ERG1/2/3+ cells of confocal microscopy data in (E) (n = 3–4). I Representative confocal micrographs of P8 HV flatmounts from Ndp−/− and Ndp+/+ mice immunostained with CD13, IB4 and DAPI. J Quantification of total and K extravascular CD13+ cells of confocal microscopy data in (I) (n = 3). White arrowheads indicate ERG1/2/3+ or CD13+ cells within the vasculature, and yellow arrowheads indicate extravascular ERG1/2/3+ or CD13+ cells. Results are expressed as fold change (FC) relative to Notch1cWT (A, B, F) and as percentages (%) normalized to Ndp+/+ (G, H, J, K) control HV. Data are means ± SEM. Represented p-values are *≤ 0.05, **≤ 0.01 and ***≤ 0.001 from two-tailed parametric unpaired t-test. Non-significant (ns). Scale bars, 500 μm and 50 μm [for higher-magnification images in (E, I)]
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