Characterization of jam2bGt(2A-Gal4);UAS:GFP expression at 1-5 dpf.

(A-F) jam2bGt(2A-Gal4);UAS:GFP shows strong GFP expression within the lateral plate mesoderm (LPM) along the yolk extension at 26 hpf (white arrows, A,B), which becomes presumptive mesothelium at 3-5 dpf (white arrows, C-F). GFP expression is also apparent in a subset of skeletal muscle cells (white arrowheads, A,C,E), craniofacial muscle (yellow arrows, E,F) and in the cells surrounding the eye (yellow arrowheads, E,F). (G-I) Co-expression of jam2bGt(2A-Gal4);UAS:GFP and etv2 (red) in the lateral plate mesoderm of the trunk region at 30 hpf. Note that GFP and etv2 expression overlaps in the SVF cells (arrowheads), located in the most dorsal region of GFP expression domain. (J-R) Expression of jam2bGt(2A-Gal4);UAS:GFP in the LPM along the yolk extension and in muscle cells from 30 to 66 hpf. A few GFP-positive cells are present in the PCV and overlap with vascular endothelial marker kdrl:mCherry expression (white arrowheads and insets). (S-U) Analysis of jam2bGt(2A-Gal4);UAS:GFP expression in lyve1b:dsRed reporter background at 4 dpf, which labels PCV and the thoracic duct. Note GFP-positive cells in the TD (arrowheads). Bright GFP expression in the skeletal muscle fibers is also apparent. PCV, posterior cardinal vein; DA, dorsal aorta; TD, thoracic duct. Lateral views of the trunk region, anterior is to the left. Scale bars: 100 µm.

Jam2b-positive cells are derived from the lateral plate mesoderm and contribute to the vasculature.

(A-F) Time-lapse images of jam2bGt(2A-Gal4);UAS:GFP ; Tg(kdrl:mCherry) embryo starting at 24 hpf show two GFP-positive cells (arrowheads), which migrate dorsally, proliferate, integrate into the PCV and initiate kdrl:mCherry expression (see Movie 1). Time is in minutes after 24 hpf. PCV, posterior cardinal vein; DA, dorsal aorta. Scale bar: 50 µm. (G-I), HCR against etv2 in a jam2bGt(2A-Gal4);UAS:GFP embryo at the 18-somite stage showing jam2b expression (green) lateral and ventral to etv2 positive vascular progenitors (red). (J-L) HCR against prrx1a in a jam2bGt(2A-Gal4);UAS:GFP embryo at the 18-somite stage showing colocalization of prrx1a mRNA and GFP fluorescence. Dorsolateral view of the trunk region is shown, anterior is to the left.

Genetic lineage tracing analysis of jam2b+ cell contribution to vasculature.

(A) Schematic diagram of recombination in jam2bGt(2A-Gal4); UAS:Cre; actb2-loxP-BFP-loxP-dsRed embryos. Ubiquitous BFP expression switches to dsRed in Cre-positive cells. (B-J) Lineage tracing analysis in embryos at 3 dpf obtained by crossing jam2bGt(2A-Gal4)and UAS:Cre; actb2:loxP-BFP-loxP-dsRed; fli1:GFP parents. Red cells, indicating the genetic switch, are observed in the mesothelial layer surrounding the yolk (arrow, B,E), skeletal muscle (yellow arrowheads, B), and the epicardial (arrows, C,D) and myocardial (arrowheads, C,D) heart layers. Switched cells in the vasculature are observed in the venous ISVs (white arrowheads, B,E,F,G), the PCV (blue arrowheads, F,G) and SIA (arrow, I,J). A control embryo negative for jam2bGt(2A-Gal4) is shown in (H) to indicate autofluorescence. (F,G) show larger magnification views of (B,E). (K) Quantification of endothelial cell labeling in different vascular beds in jam2bGt(2A-Gal4); UAS:Cre; actb2-loxP-BFP-loxP-dsRed embryos at 3 dpf. (L) Quantification of endothelial cell labeling in different vascular beds in jam2bGt(2A-Gal4); UAS:CreERT2; actb2-loxP-BFP-loxP-dsRed embryos at 3 dpf. 4-OHT was added starting at 7 hpf or 22 hpf stages. In a third group of embryos, 4-OHT was added at 7 hpf and washed out at 22 hpf stages. Note that cell contribution to SIV could not be analyzed reliably due to very strong red fluorescence in the mesothelial layer surrounding the yolk.

Intestinal vasculature in the middle and posterior trunk region forms largely by new vasculogenesis.

(A,B). etv2:Kaede embryos were photoconverted from green to red at 24 hpf and analyzed at 4 dpf. Merged and red only channels are shown. (C-E) Higher magnification images of the boxed area in (A,B). Merged, red and green channels are shown. Note that SIA and SIV are comprised mostly from green only cells (white arrowheads), indicating that they have originated after the photoconversion. (F) Quantification of green only (new) and green+red (pre-existing) cells in the SIA and SIV at 4 dpf. The analysis focused on the middle and posterior trunk region over the yolk extension area.

scRNA-seq analysis of cells from jam2bGt(2A-Gal4);UAS:mCherry-NTR; etv2Gt(2A-Venus) embryos at 36 hpf.

(A) UMAP plot showing 37 cell clusters identified by Seurat analysis. LPM, lateral plate mesoderm; SVF, secondary vascular field; RBC, red blood cells. (B) Dot plot of selected marker gene expression level in each cell cluster. (C) 2D UMAP plot showing the distribution of Venus and mCherry positive cells. Note that Venus-positive cells mostly correspond to different vascular endothelial lineages, while mCherry-positive cells label different subsets of LPM. (D) Feature plots showing the expression of SVF cell markers, enriched in the cluster 15. (E) Violin plots show SVF cell marker expression in different clusters. (F) Pseudotime trajectory of vascular endothelial and SVF clusters made using Monocle 3. Note that SVF cells (cluster 15) can transition into both venous and arterial cell types.

SVF cell number is reduced in maternal-zygotic (MZ) jam2a; jam2b mutant embryos.

(A-C) In situ hybridization analysis (ISH) for etv2 expression in SVF cells (arrows, A,B) in MZ jam2a-/-; jam2b-/- embryos and control jam2a+/-; jam2b+/- embryos in kdrl:GFP background, obtained by crossing double homozygous jam2a-/-; jam2b-/-; kdrl:GFP adults to wild-type kdrl:GFP. (D-I) ISH analysis for tal1 and npas4l expression in SVF cells (arrows) in MZ jam2a-/-; jam2b-/- embryos and control embryos in fli1:GFP background. Controls were obtained by mating jam2a+/-; jam2b-/- embryos to wild-type fli1:GFP, resulting in a mixture of jam2a+/+; jam2b+/- and jam2a+/-; jam2b+/- genotypes. ***p<0.001, ****p<0.0001, two-tailed t-test, error bars show ±s.d. Data are combined from four (C) or two (F,I) independent replicate experiments. Het labels in (C,F,I) refer to double heterozygous in (C) and a mixture of jam2a+/+; jam2b+/- and jam2a+/-; jam2b+/- genotypes in (F,I), while Mut label refers to jam2a-/-; jam2b-/- MZ mutant embryos.

Analysis of vascular defects in MZ jam2a; jam2b mutant embryos.

(A-C) Intersegmental vessel extension analysis in MZ jam2a-/-;jam2b-/- and jam2a+/-;jam2b+/- embryos in kdrl:GFP background at 3 dpf. Note that some ISVs are truncated in MZ jam2a; jam2b mutant embryos (arrow, B, an inset shows higher magnification view of the boxed trunk region). (D-F) Endothelial cell number in the PCV is reduced in MZ jam2a; jam2b mutant embryos compared to jam2a+/-;jam2b+/- controls at 3 dpf. Endothelial cells were counted in the floor of the PCV (arrowheads) in the selected area of the trunk region based on fli1:GFP expression. (G-I) SIA and SIV are fragmented and show increased incidence of gaps in MZ jam2a;jam2b mutant embryos compared to heterozygous controls at 3 dpf. Analysis was performed in the background of etv2Gt(2A-Gal4); UAS:mCherry-NTR transgene, which shows expression in all vasculature. Note the underdeveloped SIA (arrowheads) and gaps in SIV (arrow) in jam2a;jam2b mutants. Higher magnification views are shown in the insets. Diagrams in (I) show percentage of embryos with gaps in SIA (left) and SIV (right). (J-L) Thoracic duct analysis in prox1:RFP; fli1:GFP transgenic embryos at 6 dpf. Note the gaps in the thoracic duct (arrows) observed in MZ jam2a;jam2b mutant embryos. (L) Shows the percentage of embryos with normal or fragmented thoracic duct. (M-O) Vascular recovery analysis after endothelial cell ablation. jam2a;jam2b mutant and control heterozygous embryos in etv2 Gt(2A-Gal4); UAS:NTR-mCherry background were treated with MTZ starting at 6 hpf to ablate ECs and subsequently washed out at 45 hpf. A partial vascular recovery is observed in the control embryos, while it is greatly reduced in MZ jam2a;jam2b mutants. (O) shows length measurements of the recovered vasculature. (C,F,O) graphs show two tailed t-test, while (I,L) show Fisher’s exact test. *p<0.05, **p<0.01, ****p<0.0001. DA, dorsal aorta; PCV, posterior cardinal vein; SIA, supraintestinal artery; SIV, subintestinal vein.

hand2 is required for jam2b expression in the lateral plate mesoderm and SVF cells.

(A-F) HCR analysis for hand2 (red) and jam2b (magenta) expression in kdrl:GFP embryos at 24 hpf. Anterior (A-C) and posterior (D-F) regions of the trunk are shown. Note the overlapping expression in the LPM along the yolk and yolk extension (arrows), which does not overlap with kdrl:GFP expression. (G-J) Whole-mount in situ hybridization analysis for jam2b expression in hand2 mutant and wt control embryos at 26 hpf. Lateral (G,H) and dorsal (I,J) views. Note that jam2b expression in hand2 mutants is absent from the posterior domain of the LPM along the yolk extension (black arrowheads), while the anterior portion of the LPM (white arrowheads) is only mildly affected. (K,L) Whole-mount in situ hybridization for etv2 expression in hand2 mutant and control wild-type embryos at 30 hpf. Note that etv2 expression in SVF cells (arrows) is absent in hand2 mutants. In addition, its expression in the PCV (arrowheads) is greatly reduced or absent. ISV, intersegmental vessel; DA, dorsal aorta; PCV, posterior cardinal vein. Scale bars: 100 μm.

A diagram illustrating the emergence of SVF cells from jam2b-positive lateral plate mesoderm.

SVF cells contribute to the intestinal vasculature, including the supraintestinal artery (SIA), subintestinal vein (SIV), as well as the posterior cardinal vein (PCV).

In situ hybridization analysis of jam2b expression between the 20-somite and 48 hpf stages.

Note strong jam2b expression in the lateral plate mesoderm along the yolk and yolk extension (arrows). (A,B,C,E) lateral view, (D) ventral view.

Generation of jam2bGt(2A-Gal4);UAS:GFP line using CRISPR-Cas9 mediated non-homologous recombination.

(A) A diagram of sgRNA-2A-Gal4 construct and its insertion into the jam2b genomic locus. The targeting construct contained jam2b sgRNA site, followed by in-frame fusion to the viral peptide P2A, Gal4 DNA-binding domain and the SV40 polyA sequence. jam2b sgRNA targets the second exon of jam2b genomic sequence. (B) Comparison of GFP fluorescence in jam2bGt(2A-Gal4);UAS:GFP embryo and jam2b mRNA expression analyzed by in situ hybridization at 24 hpf. Note the bilateral jam2b expression and GFP fluorescence observed in the lateral mesoderm along the yolk and yolk extension. Scale bars: 100 µm.

Quantification of jam2bGt(2A-Gal4);UAS:GFP cell contribution to different vascular beds.

All analysis at 3 dpf, except for TD, which is at 4 dpf. n=7 embryos except for TD, for which n=17. PCV, posterior cardinal vein, DA, dorsal aorta, ISV, intersegmental vessel, PL, parachordal lymphangioblast, SIA, supraintestinal artery, TD, thoracic duct.

Generation of jam2b and jam2a mutants using CRISPR-Cas9 genome editing.

(A) A schematic diagram illustrating generation of jam2b mutants using two sgRNAs designed to delete the promoter region. (B) A schematic diagram illustrating generation of jam2a mutants using two sgRNAs designed to delete the entire jam2a coding sequence. (C,D) In situ hybridization (ISH) analysis for jam2b expression in wild-type jam2b+/+ and jam2b-/- mutant embryos at 30 hpf. Note that jam2b expression along the lateral plate mesoderm is absent in jam2b mutants (arrows). Some residual staining is due to background and some pigment cells along the horizontal myoseptum. (E,F) ISH analysis for jam2a expression in wild-type jam2a+/+ and jam2a-/- mutants at 26 hpf. The strongest jam2a expression is observed in the posterior somites, which is absent in jam2a mutants (arrows).

jam2b mutant embryos do not show any apparent defects in SVF cell formation or vascular recovery after endothelial cell ablation.

(A-C) Quantification of SVF cells (white arrowheads) by in situ hybridization analysis for etv2 expression at 30 hpf. Experimental and control embryos were obtained from the incross of jam2b-/- or wild-type (jam2b+/+) sibling parents in kdrl:GFP background, respectively. (D,E) Quantification of vascular recovery after endothelial cell ablation. jam2b+/- and jam2b-/- adults in etv2Gt(2A-Gal4); UAS:mCherry-NTR; Tg(kdrl:GFP) background were crossed with each other. Embryos were treated with 10mM MTZ from 10 hpf to 45 hpf, which was then washed and embryos were allowed to undergo vascular recovery until 72 hpf, when they were imaged and genotyped. Control embryos were treated with 0.1% DMSO. The length of new vascular cords (white lines) at the position of the PCV and SIV was measured and compared in jam2b+/- and jam2b-/- mutant embryos. Note that the bright kdrl:GFP expression along the yolk extension corresponds to the pronephros and was not affected in these experiments. Lateral view is shown, anterior is to the left. Mean±sd is shown; ns, not significant. Scale bars: 100 µm.