HPD differentiation requires EphrinB1-controlled actomyosin contractility. a Experimental scheme for 65 μM blebbistatin incubation during HPD remodeling from 52–60 hpf. b–d Blebbistatin-treated embryos show two types of EHB epithelial defects: c extensive luminal loops (45.5%, n = 11, N = 2) and d gaps in the emerging apical lumen (45.5%, n = 11, N = 2; and 9% wild-type like). The nascent EPD lumen is mostly resolved, consistent with the treatment window; scale bars = 15 μm. PanCadherin staining (magenta) of magnified views of CBD (red dashed box) shows rounder epithelial cells (red arrowheads), a multi-layered configuration with loops and cells exhibiting extended apical domains (green arrowheads) or gaps and cells with multiple apical domains (blue arrowhead) in treated embryos compared to controls; scale bars = 5 μm. e Quantification of CBD length shows significant increase upon blebbistatin treatment compared to controls. f, g Actin, pMLC and ZO-1 localize apically (white arrowheads) in the forming CBD; whether pMLC is at the apical membrane and/or the junctional belt cannot be distinguished. pMLC is at the duct mesoderm border (yellow arrowheads) and lower levels at the lateral membrane (orange arrowheads) 60 hpf; scale bars = 5 μm. h Quantification of apical pMLC expression in CBD cells of 60 hpf control (n = 3) and ephrinb1 embryos (n = 3). For quantification strategy see Supplementary Fig. 5. Statistical test: e, h = Student’s t-test. Error bars show SEM; *p < 0,05, ***p < 0,001. Source data are provided as a Source Data file
|
