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

<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

<i>smad1</i> overexpression in endothelial cells results in increased vSMC coverage.

A) Vector construct for overexpression of smad1 under the endothelial cell promoter kdrla. B) RT-qPCR fold change in smad1 and id-1 expression levels in endothelial specific smad1 overexpressing embryos (smad1ECOE) embryos at 4 dpf (n = 3). RT-qPCR data show the mean ± SEM, Student's two-tailed t-test *p Tg(BRE:EGFP); Tg(acta2:mCherry) embryos. Control embryos (C-C”) and smad1ECOE embryos (D-D”) showing endothelial BRE:EGFP and vSMC acta2:mCherry expression in the ventral aorta (VA) and pharyngeal arch arteries (PAA). E) Quantification of green fluorescent marker (BRE:EGFP) along the VA, highlighted within the yellow region in C” and D”, as corrected total cell fluorescence (CTCF). F) Quantification of acta2 positive cell number on VA, within area outlined in C and D. Number of acta2 positive cells is significantly increased in smad1ECOE embryos. G) Quantification of length of VA, within area outlined in C and D. H) Quantification of the percent vSMC coverage of ventral aorta. For each quantification, N = 3, smad1ECOE embryos n = 8, Control n = 8, Student's two-tailed t-test, *-***p< 0.01–0.0001 as compared to control. Error bars = SEM, Scale bar represents 50μm.</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

<i>miR26a</i> is expressed in blood vessels; endothelial cells have active BMP signalling.

A) Model of how miR26a controls BMP signaling via direct targeting of smad1. B) Lateral view of whole mount in situ expression of miR26a at 48 hpf shows ubiquitous expression pattern, with strong expression in the ventral head of the embryo. B’) Cross section of the head at 48 hpf. C) At 4 dpf miR26a is expressed in the pharyngeal arches, bulbous arteriosus and ventral aorta. C’) Cross section of the head showing miR26a expression in blood vessels (purple; punctate stain) compared with endothelial stain (brown; kdrl:GFP transgenic). Inset is an enlargement of image in C’. D) Ventral view of the pharyngeal region of a 4 dpf double transgenic Tg(BRE:EGFP);Tg(kdrl:mCherry) embryo shows BRE:EGFP (green) expression within endothelial cells in aortic arches (red, white arrowheads in E’-E”‘) and ventral aorta (red, white arrowheads F’-F”‘). G-H) Ventral and lateral views of a 4 dpf double transgenic Tg(BRE:EGFP); Tg(acta2:mCherry) zebrafish shows that acta2 positive cells are in direct contact with BMP-responsive endothelial cells but do not express BRE:EGFP. Scale bar represents 50μm.</p

Angioblast migration distance and speed.

(DOCX)</p

Mechanistic model by which <i>miR26a</i> modulates BMP signaling to promote vSMC differentiation via interactions with endothelial cells.

miR26a modulates vascular stability by directly targeting smad1. At developmental stages when smooth muscle appears, the endothelium has active BMP signaling. Loss of miR26a results in increased BMP signaling in endothelial cells where Smad1 becomes phosphorylated. Increased pSmad1 in endothelial cells leads to increased differentiation (acta2 expression) and increased vSMC cell number, while blocking BMP signaling leads to a decrease of both. (Dashed arrows indicated the indirect effect on vSMC marker expression and cell number).</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

Increased levels of <i>smad1</i> result in defects in the vascular system and body axis.

A-C) Representative 48 hpf miR26a knockdown embryos with hemorrhage, as indicated by arrows. D) Quantification of average rates of hemorrhage. (Error bars = SD Unpaired t test, miR26a MO *psmad1 overexpression. G-I) miR26a and smad1 double knockdown experiments. G) Representative 48 hpf smad1 MO embryos with mild (V1) and severe (V2) ventralization phenotypes. H) Representative 48 hpf double miR26a and smad1 knockdown embryos with rescued hemorrhage and normal body axis showing only mild (V1-WT) ventralization phenotypes. I) Quantification of observed phenotypes double knockdown experiments (N = 4, total n wildtype = 193, Scr. Control MO = 157, smad1 MO = 175, smad1 mRNA = 95, miR26a MO = 190, and miR26a MO + smad1 MO = 190. One Way ANOVA of hemorrhage phenotype; Wildtype/Scr. Control MO vs. miR26a MO pSMAD1 mRNA pmiR26a MO vs. miR26a MO+ smad1 MO psmad1 MO pmiR26a MO+ smad1 MO p< 0.0001. Error Bars = SEM. Scale bar represents 500μm.</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