Mitotic cell rounding and epithelial thinning regulate lumen

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1 Mitotic cell rounding and epithelial thinning regulate lumen growth and shape Esteban Hoijman, Davide Rubbini, Julien Colombelli and Berta Alsina SUPPLEMENTARY FIGURES Supplementary Figure 1 (a-c) Localization of Lifeact-RFP (a) and hzo1-gfp (b) at 14 h.p.f. (c) Merge image. M:medial, A:Anterior (d and e). Distribution of Pard3-GFP (green) at 15 h.p.f (d) and 20.5 h.p.f (e). Pard3GFP mrna was co-injected in embryos with memb-mcherry mrna(red). M:medial, A:Anterior (f) z-projection of F-actin mesh in Tg(actb1:lifeact-GFP) (green) injected with membmcherry mrna (red) in an embryo at 23 h.p.f. M:medial, A:Anterior Magnification in (a-f) is the same. Scale bar in (b), 20 µm.

2 (g and h) Sagital (g) and transverse (h) views made by reslices of a coronal z-stack of the lumen at 23 h.p.f. Embryo was co-injected with membrane-gfp (gray) and H2BmCherry (red) mrnas. D: Dorsal, A: Anterior, M:Medial. Magnification in (g) and (h) is the same. Scale bar in (g), 20 µm.

3 Supplementary Figure 2 (a) Examples of individual cytosolic labeled cells at early and late time points obtained by mosaic labeling of otic vesicles. Pard3, H2B and membrane fluorescent fusion

4 proteins are ubiquitously expressed in the vesicle. The signal of the Kaede protein corresponds to the photoconverted pool. (b) Dynamics of cell shape changes (ratio of the apical and the lateral membrane length). Data are mean ± sem (n=12 cells, 5 embryos). (c) Apical/lateral membrane length ratio at the indicated times in control and ouabain treated embryos. Data are mean ± sem, n=12 cells from 5 embryos. ANOVA, *** p<0.001 and * p<0.05. (d) Coronal view of the hindbrain during opening of the ventricles at 17 (left) and 20 (right) h.p.f taken from a time-lapse movie. We observed that as lumen grows, the neuroepithelium thins. The violet bar indicates the location from where the apicobasal length of the neuroepithelium was calculated overtime and plotted in (e). Moreover, at 20 h.p.f there is a clear spatial association between the thinning and the ML lumen length, being the lumen bigger where the epithelium is thinner (f).. Scale bar, 40 µm.

5 Supplementary Figure 3 Distribution of Pard3-RFP (a, b and c ), hzo1-gfp (a, b and c ) or merged images of both proteins (a, b, c, d, e, f, g, h and i) in vesicles from control embryos at 17 (a-a, d and e) or 23 h.p.f (b-b, f and g), and from ouabain-treated embryos at 23 h.p.f (c-c, h and i). The z-slice of each lumen was selected to highlight the hzo1-gfp localization. (d, e, f, g, h and i) merged images at higher magnification of coronal sections located at the ventral or dorsal end of the lumen (apical region of the cells). The images were taken from vesicles from 17 (d and e) or 23 h.p.f (f and g) control embryos, and from ouabain-treated embryos at 23 h.p.f (h and i). In higher magnification images, two examples are shown for each experimental group. Scale bars: 10 µm.

6 Supplementary Figure 4 Examples of quantification of pulling events taken from different movies. Changes in the apico-basal epithelial length, the cell body height and width over time are shown.

7 Supplementary Figure 5 (a) Schematic representation of a region of zebrafish ect2 pre-mrna. Exons (E) are depicted in squares and introns (I) as lines. Bars indicate the location of the splice site targeted by the MOs (E3-I3MO and I6-E7MO). Arrows indicate the location of primers used to amplify the cdna in the PCR. The ATG is located in exon 2. The exon 4 (96 b) is endogenously alternative according to sequences ENSDART and ENSDART The exon 3 (80 b) is skipped by the E3-I3MO injection leading to a frame shift and a premature stop codon in exon 5. The exon 7 (111 b) is skipped by the I6-E7MO injection and leads to a deletion on a BCRT domain.

8 (b) RT-PCR from 19h.p.f embryos injected with controlmo or a combination of E3- I3MO and I6-E7MO. Primers F2-R5 were used to check the effect on splicing of the ect2 gene of the E3-I3MO, and primers F6-R8 to check for the effect of I6-E7MO. Numbers in the gel correspond to drawings below depicting the exon composition of every PCR product. The size of the MW marker bands is (from top to bottom): 1000 bp, 850 bp, 650 bp, 500 bp, 400 bp, 300 bp, 200 bp and 100 bp.

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10 Supplementary Figure 6 (a) Otic vesicles from hi3820atg embryos (ect2 mutant line) at 19 h.p.f. The cellular phenotype is the same than in ect2mos injected embryos, with similar number of defective cytokinesis events leading to binucleated cells (white arrows). At this stage lumen formation is disrupted, compared to wild type embryos with a lumen already formed and expanding (see Figure 1). These luminal defects are not present in the ect2mos injected embryos, in which cytokinesis defects appear later in development than in the mutant. Green: Lyn- GFP, red: H2B- mcherry. Scale bar, 20 mm (magnification is the same in all panels).. (b) Quantification of AP lumen length at 22 h.p.f from embryos of the experimental groups indicated. Values are fold change to DMSO group. Data are mean ± sem (n=8 embryos). ANOVA, * p<0.05. (c) Change in the number of cells at the coronal plane of higher luminal area after cell cycle interfering experiments in mounted embryos during expansion. Data are mean ± sem (n=3), ANOVA, ** p<0.01. Supplementary Note 1. Molecular candidates for fluid movement during lumen expansion. According to the expression data from Thisse, B., Pflumio, S., Fürthauer, M., Loppin, B., Heyer, V., Degrave, A., Woehl, R., Lux, A., Steffan, T., Charbonnier, X.Q. and Thisse, C. Expression of the zebrafish genome during embryogenesis, ZFIN, 2001, and the function described for the genes in the Genecards encyclopedia ( some transporters, ion channels and ion binding proteins are being expressed in the otic vesicle during the stages spanning the lumen expansion (18-24 hpf), making them candidates to mediate the fluid movement required for lumen growth. The list includes: 1) slc9a3r1 (or nherf1) a sodium/hydrogen exchanger regulatory cofactor involved in the regulation of the CFTR conductance, 2) slc26a10, a putative chloride/bicarbonate exchanger, 3) cahz, the carbonic anhydrase, which also plays an important role in sodium uptake; 4) trpm7, a calcium channel; 5) cadps2, a calcium-dependent activator of secretion protein and 6) calm2a, a calmodulin that can control the function of several aquaporins and ion channels. By contrast, aquaporin genes do not seem to be expressed at these early stages of development in the inner ear. We also performed a screen of drugs blocking several channels and transporters during

11 lumen expansion but none of them induced a phenotype in lumen expansion. We treated embryos from 15 to 23 h.p.f with inhibitors that block (higher concentration tested): potasium channels (TEA 100mM), chloride channels (DIDS 50µM), calcium signalling (thapsigargin 15 µm) and the CFTR channel (CFTR µm).