Dynamic expression of zebrafish sema3e. (A, B). At 18 somites, sema3e is expressed in one primary motoneuron per somite (mn) and the dorsal aorta (da). (C–F) At 24 and 27 hpf, continued expression is observed in a single primary motoneuron and the dorsal aorta, with expression now observed in endodermal tissue ventral to the pronephric ducts (en). (G, H) By 48 hpf, only neural expression of sema3e is observed. Signal is seen in the otic vesicle (ov) and optic chiasm (oc); persisting through 72 hpf (I, J) where expression is also observed at the midbrain–hindbrain boundary (mhb) and in developing jaw structures (j). (A, C, E, G–J) are wholemount images while (B, D, F) are transverse sections. (A, C, E, G, I) are lateral views, (H) is dorsal view, (J) is ventral view. Scale bar in A is 325 μm, B is 10 μm, C, E, G–J is 400 μm, and D, F is 30 μm.

Intersegmental sprouting is delayed in sema3e^e3i3 morphants and is precocious in obd mutants. (A) Graphical representation of the mean number and standard deviation of angioblast sprouts observed in sema3e^e3i3 morphants and obd^fov1b mutants as compared to control embryos. Confocal (B, D, F) and corresponding white light images (C, E, G) of uninjected (B, C), sema3e^e3i3 morphants (D, E), and obd^fov1b mutants (F, G) at 24 hpf on a Tg(kdr:GFP)^la116 genetic background. Scale bar in B, D, F is 100 μm and C, E, G is 500 μm.

Angioblast sprouting is delayed in plxnB2^i10e11 and plxnB2^i10e11/sema3e^ATG morphants. (A–B) PlxnB2 expression analysis by in situ hybridization shows semi-ubiquitous expression with ventral enrichment in the region surrounding the axial vessels in wholemount (A) and cross section views (B). The dashed line in A indicates the level of the section in B. (C–D) White light images of control (C) and plxnB2^i10e11 morphant (D) at 24 hpf. (E–H) Confocal images of uninjected (E), plxnB2^i10e11 morphant (F), sema3e^ATG morphant (G), and plxnB2^i10e11/sema3e^ATG morphant (H) at 24 hpf on a Tg(kdr:GFP)^la116 genetic background. (I) Graphical representation of the mean number and standard deviation of angioblast sprouts observed in plxnB2^i10e11 morphants, sema3e^ATG morphants, and plxnB2^i10e11/sema3e^ATG morphants as compared to control embryos. Scale bar in A, C–H is 100 μm and 30 μm in B.

Loss of plxnD1 and sema3e/plxnB2 results in an intermediate angioblast sprouting phenotype (A) Graphical representation of the mean number and standard deviation of angioblast sprouts observed in plxnB2^i10e11/obd morphants as compared to control obd mutant embryos. (B) Graphical representation of the proportion of embryos exhibiting a given number of ISVs sprouts in uninjected obd wt sibs, sema3e^ATG MO/obd wt sib morphants, uninjected obd mutant embryos, and sema3e^ATG MO/obd morphants. (C–F) Confocal images of uninjected obd mutant (C), uninjected wt sibs (D), plxnB2^i10e11/obd morphants (E), and sema3e^ATG/obd morphants (F) at 24 hpf on a Tg(kdr:GFP)^la116 genetic background. Scale bars in (C–F) are 100 μm.

Sema3e acts both autonomously and non-autonomously in angioblasts while PlxnB2 acts autonomously. (A) Summary of the total proportion of cell types observed after transplantation given as a percentage of total cells counted. (B–E) Representative images of a Tg(kdr:GFP)la116 wild type donor embryo (B), recipient embryo from a wt > wt transplantation (C), recipient embryo from a wt > 3e MO transplantation (D), recipient embryo from a 3e MO > wt transplantation (E), recipient from a wt > plxnB2 MO transplantation (F), and recipient from a plxnB2 MO > wt transplantation (G). Arrows indicate cells classified as dorsal aorta (da), ISV cells (ISV). * denotes a probability of difference from wild type by Chi-square test of < 0.002. Scale bar is 100 μm.

RT-PCR of sema3e^e3i3 and plxnB2^i10e11 morphants.
(A) RT-PCR of sema3e^e3i3 morphants indicating complete mis-splicing of sema3e with 1.9 ng of morpholino. (B) RT-PCR of plxnB2i10e11 morphants indicating complete mis-splicing of plxnB2 with 16 ng of morpholino. EF1α was used as a loading control. (C-D) Schematic representation of the domain structures of both Sema3e (C) and PlxnB2 (D) and resulting protein likely present in sema3e^e3i3 and plxnB2^i10e11 morphants. Both Sema3e and PlxnB2 contain semaphorin and PSI (plexin, semaphorin, integrin) domains. In addition, PlxnB2 contains three IPT (integrin-like, plexin, transcription factors) domains, a transmembrane (TM) domain, and a cytoplasmic signalling domain.

Assessment of non-specific cell death in sema3ee3i3 and plxnB2i10e11 morphant by acridine orange staining.
(A-C) Acridine orange staining of Tg(kdr:GFP)^la116 uninjected (A), sema3e^ATG morphant (B), and plxnB2^i10e11 morphant (C) embryos at 24 hpf. Green dots represent cells undergoing apoptosis.

Rhodamine dextran images of representative transplanted embryos from Figure 5.
(A, C, E, G, I) Tg(kdr:GFP)^la116 donor angioblasts in non-transgenic recipient embryos from Fig. 5. (B, D, F, H, J) Corresponding rhodamine dextran images for embryos in A, C, E, G, I showing most transplanted cells residing in tissues of mesodermal origin. Scale bar is 100 μm.