The formation of IRV by angiogenic sprouting from the DA.

(A) Transverse sections of Tg(kdrl:GFP)^s843 embryos at 34, 48 and 72 hpf visualized for 3β-Hsd activity (black), GFP (green) and Fibronectin (red). All sections are oriented with the posterior end toward top of page. While the IRV sprouting from the DA (yellow arrows) invades the interrenal tissue, steroidogenic interrenal cells form a protruding extension which migrates toward and across the central midline. Fibronectin (white arrowheads) accumulates at the interface between the endothelium and the interrenal tissue, and around the tip of growing IRV endothelium. (B) fn1 transcripts (black; indicated by white arrowhead) were detected around and ventral to the DA on the transverse section at the level of ff1b-expressing interrenal tissue (red) in a 2 dpf embryo. Abbreviations: notochord (NC), interrenal tissue (IR), arterial endothelial cell (AEC), venous endothelial cell (VEC), dorsal aorta (DA), swim bladder (SB). Scale bar is 50 μM.

The vascular phenotype and interrenal-endothelial interaction in the fn1 morphant.

Sets of confocal images display the interrenal tissue as detected by 3β-Hsd activity staining (orange arrows), and the midtrunk vasculature by green fluorescence, of 45 hpf and 3 dpf Tg(kdrl:EGFP)^s843 embryos injected with either STD-MO or fn1MO. Panels (A-F, A′-F′, A′′-F′′) are dorsal views showing the midtrunk of the representative embryo for each phenotypic class, with anterior oriented to the top; while panels (A′′′-F′′′) lateral views of the same embryo with anterior to the right. The outlined peri-interrenal areas in E′′ and F′′ indicate enlarged DA segments. The outlined glomerular (G) and interrenal regions in (A′′′-F′′′) are magnified and annotated in the insets. The angiogenesis of ISV and kidney glomerulus, as well as the association between interrenal and endothelial cells, are not inhibited upon the absence of Fn. Abbreviations: dorsal aorta (DA), posterior cardinal vein (PCV), intersegmental vessel (ISV), glomerulus (G), the third somite (S3), the fourth somite (S4). Scale bar is 50 μM.

The effect of fn1 knockdown on the interrenal tissue and the peri-interrenal vasculature.

Transverse sections of Tg(kdrl:GFP)^s843 embryos injected with either STD-MO (A-A′′, D-D′′) or fn1MO (B-B′′, C-C′′, E-E′′, F-F′′) were assayed for 3β-Hsd activity (black), GFP (green) and Fn expression (red). All sections are oriented with the posterior end toward top of page. The IRV structure of a 3 dpf control embryo (D′-D′′) appeared to be discontinuous on a single confocal section, because the IRV at this stage extended slightly anteriorward and then posteriorward, after spanning ventrally through the interrenal tissue and before connecting to the AMA segment (Video S1). The formation of IRV is defective in the absence of peri-vascular Fn. Yellow arrows indicate the IRV sprouting from the DA. Orange arrow denotes the interrenal tissue. Abbreviations: dorsal aorta (DA), interrenal vessel (IRV), notochord (NC). Scale bar is 50 μM.

The interrenal tissue and peri-interrenal vasculature phenotypes in the fn1 mutant.

Transverse sections of fn^kt259;Tg(kdrl:EGFP)^s843 embryos and their wild-type siblings at 3 dpf were visualized for 3β-Hsd activity (black) and GFP (green). All sections are oriented with the posterior end toward top of page. The images for wild-type siblings (A–A′′) were acquired by focusing on the sprouting point of the IRV from the DA, and hence its surrounding steroidogenic cells were masked by the endothelial structure on panel A. The IRV angiogenesis and interrenal morphogenetic movements are severely disrupted in the fn mutant. Yellow arrow, interrenal tissue (IR). Abbreviations: notochord (NC), interrenal tissue (IR), dorsal aorta (DA), interrenal vessel (IRV).

The effect of fn1b knockdown on the interrenal tissue and the peri-interrenal vasculature.

Transverse sections of Tg(kdrl:EGFP)^s843 embryos were injected with either STD-MO or fn1b antisense morpholino, harvested at 3 dpf and visualized for 3β-Hsd activity (black), GFP (green) and Fn expression (red). All sections are oriented with the posterior end toward top of page. The IRV structure (yellow arrows) of a 3 dpf control embryo appeared discontinuous on a single confocal section because the IRV at this stage, after spanning through the interrenal tissue (IR), curved anteriorward and then posteriorward before connecting to the AMA segment. Fn doposition in the interrenal region, as well as the interrenal migration and angiogenesis, remain unperturbed in the fn1b morphant. The Fn expression and IRV morphology in the fn1b morphant shown here is a representative of 10 samples exhibiting a disruption of somite boundaries characteristic of the Fn1b-deficient embryo. Abbreviations: notochord (NC), interrenal tissue (IR), dorsal aorta (DA), interrenal vessel (IRV).

The expression and phenotype of itga5 at the level of interrenal tissue at 2 dpf.

(A) The double ISH assay revealed that itga5 was expressed in the interrenal area and at the non-somitic mesodermal cells around the DA, the swim bladder (SB) and the gut. (B) The effect of itga5 knockdown on the interrenal tissue (IR, white arrows) and the peri-interrenal vasculature. Transverse sections of Tg(kdrl:GFP)^s843 embryos injected with either STD-MO (a-a′′) or itga5 MOs (b-b′′, c-c′′) and assayed for 3β-Hsd activity (black), GFP (green) and Fn expression (red). All sections are oriented with the posterior end toward top of page. The accumulation of peri-vascular Fn and the formation of IRV (yellow arrows) are both disrupted upon the knockdown of itga5 expression. Abbreviations: notochord (NC), somite (S), dorsal aorta (DA), swim bladder (SB), interrenal tissue (IR), interrenal vessel (IRV), posterior cardinal vein (PCV).

The effect of fn1 and itga5 double knockdown on the interrenal tissue and the peri-interrenal vasculature.

Transverse sections of Tg(kdrl:EGFP)^s843 embryos injected with either STD-MO or fn1/itga5 antisense morpholinos, harvested at 2 dpf and visualized for 3β-Hsd activity (black) and GFP (green). All sections are oriented with the posterior end toward top of page. The fn1/itga5 double morphant phenocopied either fn1 or itga5 morphant in both migration and vessel formation of the interrenal tissue (IR, white arrow). Yellow arrows indicate the IRV sprouting from the DA. Abbreviations: notochord (NC), interrenal tissue (IR), interrenal vessel (IRV), dorsal aorta (DA).

The effect of tnnt2aMO and RGD peptide treatment on the formation of IRV.

(A) Blockage of blood flow by the tnnt2aMO injection into Tg(kdrl:GFP)^s843 embryos led to a collapsed and enlarged phenotype of the DA at 48 hpf, yet did not inhibit the formation of IRV (white arrows). (B) While Fn (red) was tightly aggregated around the IRV in the control embryo at 48 hpf, in the tnnt2a morphant it was loosely deposited around the IRV which extended toward the lateral rather than the ventral side. (C) RGD treatment via microangiography into Tg(kdrl:GFP)^s843 embryos at 1.5 dpf and harvested at 52 hpf showed that antagonizing the Fn function led to stunted IRV formation and retarded steroidogenic cell migration as compared with the BSA injected control embryo, which was confirmed by quantification of the length of IRV in (D) and of the distance between the midline and the tip of migrating interrenal tissue in (E). (D) The length of IRV was checked with confocal Z-stacks covering the full range of IRV growth, and measured from single focal planes displaying the maximal range of ventrally extending IRV. (E) The distance between the midline and the migrating tip of steroidogenic tissue is designated as positive if the tip of migrating cells has not reached the midline, and as negative if the tip has migrated across the midline. Abbreviations: interrenal tissue (IR), dorsal aorta (DA), interrenal vessel (IRV), notochord (NC), posterior cardinal vein (PCV). Scale bar is 50 μM.

The IRV is sprouted from the DA and connected to the AMA. (A) Sets of confocal images display the interrenal tissues (IR, white arrows) as detected by 3β-Hsd activity staining, and the neighboring endothelium as labeled by green fluorescence, of Tg(kdrl:EGFP)^s843 embryos at 45, 69, and 84 hpf respectively. Panels (a, a′) are dorsolateral while panels (b, b′, c, c′) are lateral views, and all panels are oriented with anterior to the right. The fluorescent image of the vascular pattern for the 45 hpf embryo (a′) was acquired through a projection of a consecutive z-stack encompassing the peri-interrenal area, while single confocal images were shown for the peri-interrenal vascular patterns at 69 and 84 hpf (b′, c′). The IRV was formed caudal to and distinct from the pronephric glomerulus (G) and the AMA. Red and blue arrows denote arterial and venous structures, respectively. (B) The transverse view of the vascular structure neighboring the IRV. The fluorescent image represents a projection of a consecutive z-stack encompassing the IRV, and the more posterior swim bladder artery (SBA) segments. The IRV sprouted from the ventral DA and connected to the AMA segment near which two branches of SBA were branched out. The rotation view of this projection is shown in Video S1. (C) Microangiography by injecting rhodamine-dextran into the blood stream of a Tg(ff1bExon2:GFP) embryo at 3 dpf. The blood circulation through the developing interrenal tissue is established by 3 dpf. Abbreviations: interrenal tissue (IR), dorsal aorta (DA), intersegmental vessel (ISV), interrenal vessel (IRV), glomerulus (G), posterior cardinal vein (PCV), common cardinal vein (CCV), anterior mesenteric artery (AMA), SBA (swim bladder artery).

The phenotype of ISV and SIV in the fn1 morphant. Confocal images display lateral views of the ISV (5^th and 6^th pairs denoted by red arrows) and SIV (bracketed by red lines) of 3 dpf Tg(kdrl:EGFP)^s843 embryos injected with either STD-MO (upper panel) or fn1MO (lower panel), and the anterior is oriented to the right. The first four pairs of ISV (indicated by red arrowheads) and the SIV display aberrant angiogenic patterns in the absence of Fn. Abbreviations: intersegmental vessel (ISV), subintestinal vessel (SIV).

The expression of fn1b could not be detected in the vascular region during the growth of IRV. Unlike fn1 transcripts (black; indicated by white arrowhead in the upper panel) which could be detected around and ventral to the DA on the transverse section at the level of ff1b-expressing interrenal tissue (red) in a 2 dpf embryo, no fn1b mRNA is present at the same area (lower panel). Abbreviations: notochord (NC), dorsal aorta (DA), swim bladder (SB).

The distribution of pFAK in the interrenal and peri-interrenal regions of the fn1 and itga5 morphants as well as the wild-type control embryo at 2.5 dpf. Transverse sections of fn1 morphants (B, B′, C, C′) and itga5 morphants (D, D′, E, E′), as well as the wild type control embryo (A, A′), which were assayed for 3βHsd activity (black) and pFAK expression (red). All sections are oriented with the posterior end toward top of page. While pFAK could be readily detected in both interrenal and peri-interrenal regions of the wild-type embryo, its presence was disrupted in either fn1 or itga5 morphants. The 3βHsd-expressing interrenal tissues are indicated by organge arrows. Abbreviations: notochord (NC), swim bladder (SB).

The distribution of rhodamine dextran as coinjected with RGD peptides into the circulation. The rhodamine dextran (red) could be detected around the IRV in the Tg(kdrl:EGFP)^s843 embryo as harvested at 52 hpf (indicated by yellow arrowheads), after being co-injected with RGD peptides by microangiography at 1.5 dpf. Abbreviations: notochord (NC), interrenal tissue (IR), interrenal vessel (IRV).