Mutations affecting specification of endothelial precursors. (A–D) Epifluorescent micrographs of 36 hpf wild-type (A), clo^s5 mutant (B), min^s202 mutant (C), and grc^s635 mutant (D) embryos visualized by Tg(flk1:EGFP)^s843 expression. Morphologically, grc^s635 and min^s202 mutant embryos show no obvious defects, but the number of endothelial cells appears significantly reduced (C and D), a reduction comparable to that seen in clo^s5 mutant embryos (B). White arrows point to region with Tg(flk1:EGFP)^s843 positive endothelial cells are conspicuously missing.

mirinay regulates endothelial and hematopoietic lineages. (A–H) Bright-field (A, C, E, and G) and epifluorescent (B, D, F, and H) micrographs of 20 hpf wild-type (A, B, E, and F) and min^s202 mutant (C, D, G, and H) embryos, shown in dorsal (A to D) and lateral (E to H) views. Note the reduction of endothelial cells in the posterior region of min^s202 mutant embryos (arrows). (I–J) Micrographs of 96 hpf min^s202 mutant larvae stained for endogenous alkaline phosphatase activity (I, bright-field) and visualized for Tg(flk1:EGFP)^s843 expression (J). Black arrows point to arrested intersegmental vessels (SEs) (I), white arrow points to discontinuous axial vessel (J). (K, L) Transverse sections from anterior (K) and posterior (L) trunk of 36 hpf min^s202 mutant embryos, visualized for Tg(flk1:EGFP)^s843 expression (green), β-Catenin (red), and TOPRO (blue). Although the vasculature of min^s202 mutant embryos eventually recovers, a drastically reduced number of endothelial cells is observed at this stage (white arrows point to the region of axial vessels). (M–R) Defective endothelial cell specification in 24 hpf min^s202 mutant embryos (N and P) compared to wild-type embryos (M and O), as assessed by in situ hybridization with the arterial endothelial marker ephrinB2a (M and N), and the venous endothelial marker flt4 (O and P); and defective erythropoiesis in 18 hpf min^s202 mutant embryos (R) compared to wild-type (Q), as assessed by examining gata1 expression in dorsal views. Black arrows in panels N and P point to the reduction in endothelial marker expression in min^s202 mutant embryos, and black arrow in panel R points to the region of the lateral plate mesoderm where erythrocytes form in wild-type embryos. Abbreviations: NT: neural tube, NC: notochord.

santa negatively regulates vascular specific gene expression and endothelial cell morphology. (A–F) Bright-field micrographs of 60 hpf wild-type (A) and san^s234 mutant (B) larvae. Black arrow in panel B points to enlarged heart and pericardial cavity. Endothelial cells visualized by in situ hybridization with tie2 in 60 hpf wild-type (C), and san^s234 mutant (D) larvae, shown in ventral view of the heart region. Black bracket in panel D demarcates the expansion of tie2 expression in this region. Micrographs of cmlc2 (E) and tie2 (F) expression in 60 hpf san mutant larvae. The enlarged heart in san^s234 mutant larvae shows an apparent increase of tie2 positive cells (black arrow in panel E and black bracket in panel F). (G–L) Epifluorescent micrographs of 36 hpf wild-type (G, I, and K) and san mutant (H, J, and L) Tg(tie2:GFP)^s849 embryos showing the aortic arches (G and H), trunk vessels (I and J), and heart (K and L). Note the elevated Tg(tie2:GFP)^s849 expression in san^s234 mutant embryos (white arrows). (M–P) Epifluorescent micrographs of 60 hpf wild-type (M and O) and san^s234 mutant (N and P) Tg(flk1:EGFP)^s843 larvae showing the subintestinal vessel (M and N) and posterior trunk (O and P). Vessels in san^s234 mutant larvae (white brackets) appear dilated compared to those in wild-type siblings. Abbreviations: BA: bulbus arteriosus, V: ventricle, A: atrium.

Mutations affecting vascular tube formation. (A) Epifluorescent micrograph of 36 hpf Tg(flk1:EGFP)^s843 embryo in lateral view (A). White line marks the location of the transverse sections shown in panels B to F. (B–G) Transverse sections of wild-type (B), sih/cmc2 MO injected (C), sol^s828 mutant (D), mts^s233 mutant (E), log^s231 mutant (F), and psn^s634 mutant (G) Tg(flk1:EGFP)^s843 embryos. Lack of circulation does not affect vascular lumen formation (C). Note changes in the number of vascular lumens in sol^s828 and mts^s233 mutant embryos, and in the size and shape of the vascular lumens in log^s231 and psn^s634 mutant embryos.

Mutations affecting vascular patterning. (A–T) Bright-field and epifluorescent micrographs of Tg(flk1:EGFP)^s843unf^s808 mutant embryos (D, E, 30 hpf; F, 48 hpf), slv^s887 mutant (J–L, 78 hpf) and qad^s840 mutant (Q–R, 78 hpf) larvae and their wild-type (wt) siblings. (A–F) unf^s808 mutants have shortened intersegmental vessels (SEs, arrow in panel F) and fail to form the dorsal longitudinal anastomotic vessel (DLAV, arrowhead in panels C and F) and appear to have reduced sprouting angiogenesis in the head (E). (G–L) Mis-patterning of the intersegmental vessels (SEs, arrow in panel L) and failure to remodel the posterior cardinal vein (PCV, arrowhead in L) in slv^s887 mutant larvae. (M–S) Loop formation in the hindbrain capillary network in qad^s840 mutant larvae (P (wt) and T (qad^s840) are dorsal views focusing on the hindbrain capillary network). Instead of connecting lateral to medial as in wild-type (O, P), in qad^s840 mutant larvae capillaries connect back to their vessel of origin (arrowheads in panels S, T). Note that the tail vasculature in qad^s840 mutants (R) appears indistinguishable from wild-type (N). qad^s840 mutant embryos and their wild-type siblings were PTU treated to allow better visualization of the head vasculature.

intersection regulates vascular patterning. (A–D) Lateral bright-field and epifluorescent micrographs of 32 hpf embryos. Vascular patterning defects can be observed by visualizing Tg(flk1:EGFP)^s843 expression, but not via bright-field microscopy. int^s413 mutants fail to connect the lateral dorsal aortae (LDA) to form the dorsal aorta (DA), and the anterior and posterior cardinal veins (ACV and PCV) to the common cardinal vein (CCV). (E–H) Dorsal views of ve-cadherin and Tg(flk1:EGFP)^s843 expression. int^s413 mutant embryos exhibit a pronounced dilation of the PCV (inset in panel D).

Mutations affecting vascular maintenance and integrity. (A–R) Bright-field (A, D, G, J, M, and P) and epifluorescent (B, C, E, F, H, I, K, L, N, O, Q, and R) micrographs of Tg(flk1:EGFP)^s843adr^s277 mutant larvae (D–F) and wild-type siblings (A–C), wdi^s631 mutant larvae (J–L) and wild-type siblings (G–I), and bar^s847 mutant larvae (P–R) and wild-type siblings (M–O) at 72 hpf. Affected areas within epifluorescent micrographs are magnified and shown in panels C, F, I, L, O, and R. Note the massive vascular dilation throughout the head vasculature in adr^s277 mutant larvae (F arrowheads), regression of the dorsal aorta in wdi^s631 mutant larvae (asterisk in panels L; K, L), and apparently disappearing endothelial cells in bar^s847 mutant larvae (Q, R).

The number of endothelial cells is reduced in min^s202 and grc^s635. (A–D) Transverse sections of 30 hpf Tg(flk1:EGFP)^s843 wild-type (A) and min^s202 mutant (B) embryos, and 36 hpf wild-type (C) and grc^s635 mutant (D) embryos, visualized for Tg(flk1:EGFP)^s843 expression (green), β-Catenin (red), and TOPRO (blue). White arrows point to the apparent lack of endothelial cells in mutant embryos (as assessed by Tg(flk1:EGFP)^s843 expression). In wild-type embryos, 6 to 7 endothelial cells are present per any given optical section. In comparison, min^s202 mutant embryos have less than one endothelial cell per section (n = 10), and grc^s635 mutant embryos have less than 3 endothelial cells per section (n = 14).

Endothelial fate specification appears unaffected in mutants with defective lumen formation. (A–H) In situ hybridization with the arterial specific marker ephrinB2a (A to D) and the venous specific marker flt4 (E to H) in 36 hpf log^s231 mutant embryos (B and F) and wild-type siblings (A and E), and in 36 hpf mts^s233 mutant embryos (D and H) and wild-type siblings (C and G). Black arrows point to the vascular expression of ephrinB2a and flt4. Although these mutants display defective vascular lumen formation, the specification of endothelial cells appears relatively unaffected.

Circulation defects in adr^s277 mutant embryos. (A–B) Microangiography of wild-type (A) and adr^s277 mutant larvae (B) visualizing blood flow at 100 hpf. Note that the blood flow is largely confined to the area adjacent to the heart in adr^s277 mutant larvae. (C–D) Circulatory patterns in wild-type (C) and adr^s277 mutant (D) larvae at 100 hpf (lateral views, anterior to the right), captured via video microscopy. Although circulation in adr^s277 mutant larvae becomes gradually diminished, residual blood flow through the anterior vascular network can still be observed at this time point. Later, the circulation in adr^s277 mutant larvae is limited to the loop shown in D.

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