Jin et al., 2007 - A transgene-assisted genetic screen identifies essential regulators of vascular development in vertebrate embryos. Developmental Biology   307(1):29-42 Full text @ Dev. Biol.

Fig. 1 Mutations affecting specification of endothelial precursors. (A–D) Epifluorescent micrographs of 36 hpf wild-type (A), clos5 mutant (B), mins202 mutant (C), and grcs635 mutant (D) embryos visualized by Tg(flk1:EGFP)s843 expression. Morphologically, grcs635 and mins202 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 clos5 mutant embryos (B). White arrows point to region with Tg(flk1:EGFP)s843 positive endothelial cells are conspicuously missing.

Fig. 2 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 mins202 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 mins202 mutant embryos (arrows). (I–J) Micrographs of 96 hpf mins202 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 mins202 mutant embryos, visualized for Tg(flk1:EGFP)s843 expression (green), β-Catenin (red), and TOPRO (blue). Although the vasculature of mins202 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 mins202 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 mins202 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 mins202 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.

Fig. 3 santa negatively regulates vascular specific gene expression and endothelial cell morphology. (A–F) Bright-field micrographs of 60 hpf wild-type (A) and sans234 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 sans234 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 sans234 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 sans234 mutant embryos (white arrows). (M–P) Epifluorescent micrographs of 60 hpf wild-type (M and O) and sans234 mutant (N and P) Tg(flk1:EGFP)s843 larvae showing the subintestinal vessel (M and N) and posterior trunk (O and P). Vessels in sans234 mutant larvae (white brackets) appear dilated compared to those in wild-type siblings. Abbreviations: BA: bulbus arteriosus, V: ventricle, A: atrium.

Fig. 4 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), sols828 mutant (D), mtss233 mutant (E), logs231 mutant (F), and psns634 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 sols828 and mtss233 mutant embryos, and in the size and shape of the vascular lumens in logs231 and psns634 mutant embryos.

Fig. 5 Mutations affecting vascular patterning. (A–T) Bright-field and epifluorescent micrographs of Tg(flk1:EGFP)s843unfs808 mutant embryos (D, E, 30 hpf; F, 48 hpf), slvs887 mutant (J–L, 78 hpf) and qads840 mutant (Q–R, 78 hpf) larvae and their wild-type (wt) siblings. (A–F) unfs808 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 slvs887 mutant larvae. (M–S) Loop formation in the hindbrain capillary network in qads840 mutant larvae (P (wt) and T (qads840) are dorsal views focusing on the hindbrain capillary network). Instead of connecting lateral to medial as in wild-type (O, P), in qads840 mutant larvae capillaries connect back to their vessel of origin (arrowheads in panels S, T). Note that the tail vasculature in qads840 mutants (R) appears indistinguishable from wild-type (N). qads840 mutant embryos and their wild-type siblings were PTU treated to allow better visualization of the head vasculature.

Fig. 6 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. ints413 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. ints413 mutant embryos exhibit a pronounced dilation of the PCV (inset in panel D).

Fig. 7 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)s843adrs277 mutant larvae (D–F) and wild-type siblings (A–C), wdis631 mutant larvae (J–L) and wild-type siblings (G–I), and bars847 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 adrs277 mutant larvae (F arrowheads), regression of the dorsal aorta in wdis631 mutant larvae (asterisk in panels L; K, L), and apparently disappearing endothelial cells in bars847 mutant larvae (Q, R).

Fig. S1 The number of endothelial cells is reduced in mins202 and grcs635. (A–D) Transverse sections of 30 hpf Tg(flk1:EGFP)s843 wild-type (A) and mins202 mutant (B) embryos, and 36 hpf wild-type (C) and grcs635 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, mins202 mutant embryos have less than one endothelial cell per section (n = 10), and grcs635 mutant embryos have less than 3 endothelial cells per section (n = 14).

Fig. S2 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 logs231 mutant embryos (B and F) and wild-type siblings (A and E), and in 36 hpf mtss233 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.

EXPRESSION / LABELING:
Genes:
Fish:
Anatomical Terms:
Stage: Prim-25
PHENOTYPE:
Fish:
Observed In:
Stage: Day 4

Fig. S3 Circulation defects in adrs277 mutant embryos. (A–B) Microangiography of wild-type (A) and adrs277 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 adrs277 mutant larvae. (C–D) Circulatory patterns in wild-type (C) and adrs277 mutant (D) larvae at 100 hpf (lateral views, anterior to the right), captured via video microscopy. Although circulation in adrs277 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 adrs277 mutant larvae is limited to the loop shown in D.

PHENOTYPE:
Fish:
Observed In:
Stage: Day 4

Table 1 ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

Unillustrated author statements

Acknowledgments:
ZFIN wishes to thank the journal Developmental Biology for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Developmental Biology, 307(1), Jin, S.W., Herzog, W., Santoro, M.M., Mitchell, T.S., Frantsve, J., Jungblut, B., Beis, D., Scott, I.C., D'Amico, L.A., Ober, E.A., Verkade, H., Field, H.A., Chi, N.C., Wehman, A.M., Baier, H., and Stainier, D.Y., A transgene-assisted genetic screen identifies essential regulators of vascular development in vertebrate embryos, 29-42, Copyright (2007) with permission from Elsevier. Full text @ Dev. Biol.