Figure 8.

Figure 8. Interfering with the function of ArhGEF11/PDZ-RhoGEF suggests an activity at the interface between endothelial and hemogenic cells that relies on restricting the mobility of junctional pools at tri-cellular junctions, with a prominent role of the +exon 38 variant during endothelial cell intercalation.

(A) 2D-cartography of an aortic segment of a Tg(kdrl:eGFP-Jam3b; kdrl:nls-mkate2) embryo injected at the one cell stage with the ArhGEF11 exon 38 splicing morpholino. All the arrows pointing at reinforced junctional contacts between cells were bleached and imaged for FRAP analysis (the bleached areas correspond to the spots of highest intensities in the regions of interest, as visualized at the fluorescent confocal microscope, see Materials and methods). Black and Magenta arrows point at he-he-ec and he-ec-ec tri-cellular junctions, respectively (he: hemogenic cell; ec: endothelial cell). Green arrows point at he-ec bi-cellular junctions. ec: endothelial cell, he: hemogenic cell. Scale bar = 20 µm. (B, C) FRAP analysis of bleached eGFP-Jam3b localized in regions of interest in controls (grey) and ArhGEF11 exon 38 splicing morpholino injected embryos (blue) or homozygous ArhGEF11^{CRISPR-Cterdel+/+} mutants (green). The number of biological replicates (n) is stated on the plots. Statistical tests: two-sided unpaired two samples Wilcoxon test. Bb, Cc, evolution, after photobleaching (at t=0 s), of the mean fluorescence intensity per condition at HE-EC, HE-EC-EC and HE-HE-EC bi- and tri-junctions over time (10 min). Bb’, Cc’, median values for maximum amplitude of recovery (maximum of simple exponential fitted curves, see Materials and methods). Bb’’, Cc’’, early evolution, after photobleaching (at t=0 s), of the mean fluorescence intensity per condition over time (for the first 30 s). The fitted lines correspond to the linear regression of the mean fluorescence intensities. Bb’’’, Cc’’’, median fluorescence recovery slopes (linear regression). (D) Model (2D deployment of the aortic wall) representing the endothelial/hemogenic dynamic interplay and the proposed function of ArhGEF11 and of its + exon 38 peptide encoding variant at junctional and membrane interfaces. This interplay involves 2 essential dynamic events requiring junctional remodeling: 1 (left cartoon, magenta arrows), the mobility of he-ec-ec tri-junctional contacts accompanying the movement of endothelial cells (ex: for ec1 and ec2) along lateral sides of hemogenic cells which is required to decrease the number of adjoining cells (see Lancino et al., 2018). This takes place contemporarily to - or in alternance with -, the contraction of HE and EHT cells as they are progressing throughout the emergence and reducing contacting membrane surfaces along the longitudinal axis (the reduction in contacting membrane surfaces also involves membrane retrieval, hypothetically via endocytosis). Our data obtained with the CRISPR deletion mutants (a slight tendency to increase, on average, the turnover and mobile pool of these he-ec-ec junctions) suggest that ArhGEF11, in the wild type condition, should be slowing down the recycling of junctional components at tri-junctions, which hypothetically should contribute to increase adhesion strength. This may be required also to stabilize the junction-cytoskeleton interface involved in controlling the contraction/shrinkage of HE cells along the longitudinal axis, a hypothesis that is compatible with the mutant phenotype observed in this study, that is the increase in frequency of more elongated HE cells and the decrease in HE cell progression throughout EHT (see Figure 7); 2 (right cartoon, cyan arrow and bottom cartoons a-c), the intercalation of an endothelial cell to isolate two adjacent hemogenic cells or two daughter cells after mitosis (not depicted). This is mandatory for EHT progression and completion which requires adjoining endothelial cells to protrude membrane extensions that will anastomose to seal the aortic floor (see Lancino et al., 2018). The accumulation of adjoining cells of rather small length and apparently impaired in EHT progression that we describe in Figure 7 upon MO interference (that may also indicate impairment in abscission completion) suggests that the ArhGEF11 + exon 38 peptide encoding variant is more specifically involved in controlling the remodeling of the he/he interface that leads to endothelial cell intercalation (bottom cartoons, the remodeling of he-he-ec junctions is leading to he-ec-ec junctions and takes place between b and c). The increase in the recycling parameters that we measure in this interfering condition (mobile pool and early speed of recovery) indicates that the activity of ArhGEF11, and in particular of its + exon 38 peptide encoding variant, negatively controls junctional recycling. Hypothetically, the junctional adhesion strengthening triggered by reducing the recycling of junctional components at the he/he interface may be required during the early phase and progression of intercalation to support increase in membrane tension and environmental constraints (intercalation contributes to reducing the length/surface of the he/he membrane interface preceding junctional remodeling (cartoons b and c); this reducing membrane interface is in addition submitted to the shear stress imposed by blood flow).