Polarity and morphogenesis of the eye epithelium requires the adhesion junction associated adaptor protein Traf4

<p>During development, neuroepithelial progenitors acquire apico-basal polarity and adhere to one another via apically located tight and adherens junction complexes. This polarized neuroepithelium must continue to integrate cells arising through cell divisions and intercalation, and allow for cell movements, at the same time as undergoing morphogenesis. Cell proliferation, migration and intercalation all occur in the morphing embryonic eye. To understand how eye development might depend on dynamic epithelial adhesion, we investigated the function of a known regulator of junctional plasticity, Tumour necrosis factor receptor-associated factor 4 (Traf4). <i>traf4a</i> mRNA is expressed in the developing eye vesicle over the period of optic cup morphogenesis, and Traf4a loss leads to disrupted evagination and elongation of the eye vesicles, and aberrant organization and apico-basal polarity of the eye epithelium. We propose a model whereby Traf4a regulates apical junction plasticity in nascent eye epithelium, allowing for its polarization and morphogenesis.</p> <p><b>Symbols and Abbreviations</b>: AB: apico-basal; aPKC: atypical protein kinase-C; CRISPR: clustered regularly-interspaced short palindromic repeats; GFP: green fluorescent protein; hpf: hours post-fertilization; MO: antisense morpholino oligonucleotide; pHH3: phospho histone H3; ss: somite stage; Traf4: Tumour necrosis factor receptor-associated factor 4; ZO-1: zona occludens-1</p

Angioblast migration distance and speed.

(DOCX)</p

sema3fb mutants display aberrant and persistent filopodia in the dorsal ISA.

A) Lateral images of the trunk vasculature with mosaic endothelial expression of the transgene fli1ep: Lifeact-EGFP highlighting actin (green) and endothelial cytoplasm using Tg(kdrl:mCherry; white) in ISAs at 30 hpf. DLAV gaps (blue asterisks) and truncated ISA sprouts (yellow arrowheads) are marked. Inset shows an enlarged view of single ISAs with Lifeact-EGFP expression that have reached the level of DLAV at 30hpf. Scale bar, 100 μm. B) Representative still images from time-lapse imaging from 28–30 hpf. Enlarged still images of stage-matched embryos with mosaic Lifeact-EGFP (green) in endothelial cells spanning the ISA and reaching the level of the DLAV by 28 hpf in both wild type and sema3fbca305 embryo. Endothelial cytoplasm is shown in red Tg(kdrl:mCherry). White arrowheads indicate filopodia present in connecting ISA sprouts within the boxed regions below the DLAV. C) Quantification of number Lifeact-EGFP positive filopodia on ISAs from 28–30 hpf from embryos of the indicated genotypes. N = 3: WT (14 EGFP positive ISAs/ 30 ISAs total, 6 embryos, mean of 4 filopodia/ISA) and homozygous sema3fbca305 (18 EGFP positive ISAs/35s ISAs total, 7 embryos, mean of 7 filopodia/ISA). Unpaired t-test with Welch’s correction,*p = 0.03 and ***p = 0.0002. Error bars = ±SD.</p

<i>sema3fb</i> mutants have increased VEGF receptor expression and activity.

A) RT-qPCR analysis of key endothelial markers in wild type and sema3fbca305 FACS isolated Tg(kdrl:mCherry) positive endothelial cells at 26hpf (inset). N = 2, 2-Way ANOVA Tukey’s multiple comparisons test, *p = 0.0184, **p = 0.0021, and ****pS3 Table for fold-change details). B) Fluorescent HCR in situ of 30 hpf whole-mount wild type and embryos sema3fbca305 embryos. Representative images show punctate overlapping expression of vegfr2 (white) and sflt1 (red) mRNA transcripts within the DA and ISAs (dashed white outline). C) Quantification of HCR in situ pixel density in ISAs and DA, wild type (WT, n = 3 embryos, 15 ISAs) and sema3fbca305 (n = 3 embryos, 15 ISAs), Unpaired Student’s t-test with Welch’s correction WT vs. sema3fbca305: vegfr2 *p = 0.047 and sflt1 *p = 0.036. D) Whole-mount Immunostaining for phosphoERK (pERK) in WT and sema3fbca305 embryos fixed at 30 hpf. Representative images show Tg(kdrl:mCherry) positive ISAs (purple) and pERK positive ECs (green). Inset: pERK positive ISAs are traced using kdrl:mCherry expression (dashed white line) and dashed oval outlines highlight individual ECs with pERK staining within each ISA. E) Number of pERK positive ISAs at 30hpf. F) Quantification of average pERK fluorescence intensity in embryos at 30 hpf. D-E) N = 3, WT (n = 21 embryos, mean of 5 pERK positive ISAs), and homzygous sema3fbca305 (n = 19 embryos, mean of 5 pERK positive ISAs). 2-Way ANOVA Tukey’s multiple comparisons test, *p = 0.012. G) Schematic of Vegfr2 inhibition time course, embryos are treated at 20 hpf with either 0.1%DMSO or Vegfr2 inhibitors and removed from treatment for live imaging at 30hpf. H) Representative confocal images of trunk vasculature (black) of 30 hpf embryos treated with DMSO control or 0.2 μM SU5416. DLAV gaps (blue asterisks) and truncated ISA (yellow arrowheads) are marked. Scale bar, 100 μm. I) Length of ISA sprouts in treated embryos at 30 hpf: WT + DMSO (n = 25 ISAs, mean of 104±9 μm), WT + 0.2 μM SU5416 (n = 25 ISAs, mean of 92±17 μm), sema3fbca305 + DMSO (n = 30 ISAs, mean of 85±17 μm), and sema3fbca305 + 0.2μm SU5416 (n = 30 ISAs, mean of 98±11 μm) **p = 0.0039 and ****psema3fbca305 + DMSO (n = 30 ISA-DLAV, mean 46±16%), and sema3fbca305 +0.2 μm SU5416 (n = 30 ISA-DLAV, mean 73±10%), **p = 0.0084, ***p = 0.0002, and ****psema3fbca305 + DMSO = 6 embryos, sema3fbca305 + 0.2 μm SU5416 = 6 embryos,. 2-Way ANOVA Tukey’s multiple comparisons test. Error Bars = ±SD.</p

Loss of <i>sema3fb</i> disrupts ISA migration.

A) Lateral confocal time-lapse images from 25–29 hpf double transgenic Tg(kdrl:mCherry;fli1a:nEGFP) endothelial cells (magenta) and nuclei (white). The location of the horizontal myoseptum (green dashed line) and DLAV (blue dashed line) are noted to highlight ISA growth over time. Scale bar, 50 μm. B) Average ISA Sprout Length at 30-minute intervals from 25–29 hpf: WT vs sema3fbca305 at 25.0 hpf, p = 0.474; at 25.5 hpf p = 0.262; at 26.0 hpf p = 0.081; at 26.5 hpf *p = 0.023; at 27.0 hpf *p = 0.020; at 27.5 hpf **p = 0.030; at 28.0 hpf **p = 0.008; at 28.5 hpf **p = 0.007; at 29.0 hpf *p = 0.024. C-E) Quantification of ISA migration speeds (μm/min). C) At 26–27 hpf WT = 0.15 μm/min and sema3fbca305 = 0.12 μm/min, p = 0.157. D) At 27–28 hpf WT = 0.19 μm/min and sema3fbca305 = 0.13 μm/min, *p = 0.020. E) At 28–29 hpf: WT = 0.16 μm/min and sema3fbca305 = 0.19 μm/min, p = 0.461. B-E) N = 2; WT = 7 embryos (n = 33 ISAs) and sema3fbca305 = 7 embryos (n = 35 ISAs), Unpaired t-test with Welch’s correction. F-H) Lead angioblast distance from DA at 1hr intervals. F) At 27 hpf mean distance from DA: WT = 55.12±14.06 μm and sema3fbca305 = 47.18±5.75 μm, *p = 0.030. G) At 28 hpf mean distance from DA: WT = 71.57±15.47 μm and sema3fbca305 = 55.47±9.65 μm, ***p = 0.0008. H) At 29 hpf mean distance from DA: WT = 85.19±18.03 μm and sema3fbca305 = 68.36±16.64 μm, **p = 0.008. F-H) N = 1: WT = 4 embryos (20 ISAs) and sema3fbca305 = 3 embryos (15 ISAs), Unpaired t-test with Welch’s correction. I) Lateral confocal images of 30hpf double transgenic Tg(kdrl:mCherry;fli1a:nEGFP) endothelial cells (ECs, magenta) and nuclei (white). EC nuclei clumps (blue arrows/arrowheads) are noted. Scale bar, 100 μm. Inset: Schematics show method for measuring distance between EC nuclei and highlight EC nuclei clumps in ISAs. J) Number of EC nuclei (angioblasts) per ISAs at 30 hpf. WT (mean of 3 nuclei/ISA), heterozygous (het) sema3fbca305/+ (3 nuclei/ISA), and homozygous (hom) sema3fbca305 (3 nuclei/ISA). K) Quantification of inter-endothelial nuclei spacing per ISA at 30 hpf. WT (mean 28±13 μm), het sema3fbca305/+ (23±13μm), and hom sema3fbca305 (22±14 μm), ***p = 0.0002 and ****p2), het sema3fbca305/+ (mean 60±24 μm2), and hom sema3fbca305 (mean 56±23 μm2),**p = 0.0069 and ****psema3fbca305/+ = 19 embryos (n = 190 ISAs), and homsema3fbca305 = 11 embryos (n = 110 ISAs),. 2-Way ANOVA Tukey’s multiple comparisons test Error bars = ±SD.</p

<i>sema3fb</i> mutants display aberrant and persistent filopodia.

A) Representative still images of single-cell expression of fli1ep: Lifeact-EGFP (green) in ISA endothelial cells from 28-30hpf wildtype and sema3fbca305 embryo time-lapse imaging. A dashed white line represents the horizontal myoseptum and selected areas for filopodia counts are highlighted in white boxes. B) Quantification of number Lifeact-EGFP positive filopodia on ISA at 28hpf from embryos of the indicated genotypes. Unpaired t-test, p = 0.3566. C) Quantification of number Lifeact-EGFP positive filopodia on ISA at 29hpf from embryos of the indicated genotypes. Unpaired t-test, p = 0.0029. D) Quantification of number Lifeact-EGFP positive filopodia on ISA at 30hpf from embryos of the indicated genotypes. N = 3 for each quantification: WT (14 ISAs, 6 embryos, mean of 3 filopodia/ISA) and homozygous sema3fbca305 (18 ISAs, 7 embryos, mean of 8 filopodia/ISA). Unpaired t-test, p = 0.0002. (TIF)</p

Endothelial expressed sema3fb promotes endothelial cell sprouting.

A) Lateral view of sema3fb expression at 30hpf by ISH. Inset shows expression in the dorsal aorta (DA) and intersegmental arteries (ISAs). B) Schematic representation of the zebrafish vasculature at 30 hpf. Inset: The ISAs sprout from the DA and connect to form the Dorsal Longitudinal Anastomotic Vessel (DLAV) by 30 hpf. C-E) Lateral confocal images of the trunk vasculature (black) of 30 hpf (C) wild type sibling (sib), (D) heterozygous (het) sema3fbca305/+ and (E) homozygous (hom) sema3fbca305 mutants. Gaps in the DLAV (blue asterisks) and truncated ISA sprouts (yellow arrowhead) are noted. Abbreviations: DA (Dorsal Aorta), and PCV (Posterior Cardinal Vein). Anterior, left; Dorsal, up. Scale bar, 100 μm. F) ISA Sprout length at 30 hpf in wild type (WT) sibs (mean length of 106±10 μm), het sema3fbca305/+ (92±19μm), and hom sema3fbca305 (91±18μm), ****pca305/+ (50% connected) and hom sema3fbca305 (42% connected), ****pca305/+ (8.5±2.7 μm), and hom sema3fbca305 (9.2±2.9 μm)., **pca305/+ = 22 embryos (n = 163 ISAs), and hom sema3fbca305 = 9 embryos (n = 75 ISAs), 2-Way ANOVA Tukey’s multiple comparisons test. Error bars = ±SD.</p

Loss of <i>sema3fb</i> angiogenic deficits are independent of blood flow.

A) Confocal lateral images of laminin-stained embryos at 30hpf. Tg(kdrl:mCherry) endothelium (red) and laminin (green). Embryos derived from a heterozygous sema3fbca305/+ incross. B) Quantification of the length of ISA sprouts at 30hpf, N = 1: wild type (WT) (40 ISAs, 4 embryos, mean of 102±1 μm2), sema3fbca305/+ (120 ISAs, 12 embryos, mean of 90±8 μm2), and sema3fbca305 (150 ISAs, 15 embryos, mean of 90±14 μm2). C) Confocal lateral images of the trunk endothelium (black) in blood flow-stopped tnnt2ATG-MO injected wild type siblings (WT) and sema3fbca305 mutants. DLAV gaps (blue asterisks) and truncated ISAs sprouts (yellow arrowheads) are marked. Scale bar, 100μm. D) Length of ISA sprouts at 30 hpf, N = 3: WT (80 ISAs, 8 embryos, mean length of 106±3 μm), WT + tnnt2aATG-MO (100 ISAs, 10 embryos, mean 98±8.4 μm), sema3fbca305 (80 ISAs, 8 embryos, mean 86±17 μm), and sema3fbca305 + tnnt2aATG-MO (100 ISAs, 10 embryos, mean 85±22 μm). E) Percentage of ISAs connected at DLAV at 30 hpf, N = 3: WT (mean 82±9% connected), WT + tnnt2aATG-MO (mean 86±7% connected), sema3fbca305 (80 ISAs, 8 embryos, mean 54±15% connected), and sema3fbca305 + tnnt2aATG-MO (mean 54±16% connected). F) Quantification of width of DA in 30 hpf embryos, N = 3: WT (8 embryos, 5 measurements per embryo/n = 40 total, mean width of 18±3 μm), WT + tnnt2aATG-MO (10 embryos, 5 measurements per embryo/n = 50 total, mean 9±1 μm), sema3fbca305 (8 embryos, 5 measurements per embryo/n = 40 total, mean 11±3 μm), and sema3fbca305 + tnnt2aATG-MO (10 embryos, 5 measurements per embryo/n = 50 total, mean 10±1 μm). G) Quantification of width of PCV in 30 hpf embryos, N = 3: WT (n = 40, mean width of 22±3 μm), WT + tnnt2aATG-MO (n = 50, mean 19±4 μm), sema3fbca305 (n = 40, mean 20±3 μm), and sema3fbca305 + tnnt2aATG-MO(n = 50, mean 19±4 μm). 2-Way ANOVA Tukey’s multiple comparisons test, **** means p = (TIF)</p

Quantitative PCR mean fold change data.

(DOCX)</p

Raw data underlying all graphs in the manuscript.

(XLSX)</p