eLife Assessment

This important study addresses the question of how organ-specific blood vessels form during different stages of development, and how specific genes may regulate these processes. New genetic tools were developed to label distinct endothelial cell populations and track them over time in different mutant backgrounds. The results are solid; however, additional data quantification, lineage tracing, and cell autonomy experiments would further strengthen the conclusions.

Reviewer #1 (Public review):

The manuscript by Griciunaite et al. explores jam2b functions in the formation of late vascular precursors in what is termed the secondary heart field. The authors nicely show that expression of jam2b defines these cells in the lateral plate mesoderm and the intestinal vasculature using a target integration of Gal4 into the jam2b locus. This analysis is followed by using a UAS:cre approach to follow the lineage of jam2b expressing cells, demonstrating their contributions to the vasculature during a second round of specification of vascular precursors. This is confirmed with single-cell analysis of jam2b-gal4 expressing cells. The authors then explore the genetic requirements of jam2a and b in zebrafish and also show that hand2 functions in the secondary heart field upstream of jam2b.

Overall, the experimental evidence and results support the major conclusions. The study elucidates a novel role for jam2 in the specification of vascular precursors at later stages of development.

This understanding has important implications for treating vascular disease and regenerative therapies. The manuscript is very clearly written, and the major conclusions are likely to have a lasting impact on the field.

Reviewer #2 (Public review):

Summary:

Griciunaite et al. report on the function of jam2b and hand2 in the formation of the intestinal vasculature derived from late-forming endothelial cells (ECs) within the secondary vascular field (SVF). They generate transgenic lines that allow for the tracking of jam2b-expressing cells, both with fluorescent proteins and through Cre-mediated recombination in reporter lines. They also show that double maternal zygotic mutants in jam2a and jam2b, as well as hand2 mutants, display defects in the formation of the intestinal vasculature.

Strengths:

The results are interesting, as they address the important question of how blood vessels form during later developmental time points and potentially identify specific genes regulating this process.

Weaknesses:

(1) The authors generate a new tool, a Gal4 knock-in of the jam2b locus, to track EGFP-expressing cells over time and follow the developmental trajectory of jam2b-expressing cells. Figure 1 characterizes the line. However, it lacks quantification, e.g., how many etv2-expressing cells also show EGFP expression or the contribution of EGFP-expressing cells to different types of blood vessels. This type of quantification would be useful, as it would also allow for comparison of their findings to their previous data examining the contribution of SVF cells to different types of blood vessels. All the authors state that at 30 hpf, EGFP-expressing cells can be seen in the vasculature (apparently the PCV).

It is not clear why the authors do not use a nuclear marker for both ECs (as they did in their previous publication) and for jam2b-expressing cells. UAS:nEGFP and UAS:NLS-mcherry (e.g. pt424tg) transgenic lines are available. This would circumvent the problem the authors encounter with the strong fluorescence visible in the yolk extension. It would also facilitate quantifying the contribution of jam2b cells to different types of blood vessels.

(2) The time-lapse movie in Figure 2 is not very informative, as it just provides a single example of a dividing cell contributing to the PCV. Also, quantifications are needed. As SVF cells appear to expand significantly after their initial specification, it would be informative to know how many cell divisions and which types of blood vessels jam2b-expressing cells contribute to. Can the authors observe cells that give rise to different types of blood vessels? Jam2b expression in LPM cells apparently precedes expression of etv2. Is etv2 needed for maintenance, or do Jam2b-expressing cells contribute to different types of tissues in etv2 mutant embryos? Comparing time-lapse analysis in wildtype and etv2 mutant embryos would address this question.

(3) In Figure 3, the authors generate UAS:Cre and UAS:Cre-ERT2 transgenic lines to lineage trace the jam2b-expressing cells. It is again not clear why the authors do not use a responder line containing nuclear-localized fluorescent proteins to circumvent the strong expression of fluorescent proteins in the yolk extension. It is also unclear why the two transgenic lines give very different results regarding the number of cells being labelled. The ERT2 fusions label around 3 cells in the SIA, while the Cre line labels only about 1.5 cells per embryo, with very little contribution of labelled cells to other blood vessels. One would expect the Cre line requiring tamoxifen induction to label fewer cells when compared to the constitutive Cre line. What is the reason for this discrepancy? Are the lines single integration? Is there silencing? This needs to be better characterized, also regarding the reproducibility of the experiments. If the Cre lines were to be multiple copy integrations, outcrossing the line might lead to lower expression levels in future generations.

It is also not clear how the authors conclude from these findings that "SVF cells show major contribution to the SIA and SIV" when only 1.5 or 3 cells of the SIA are labelled, with even fewer cells labelled in other blood vessels. They speculate that this might be due to low recombination efficiency, a question they then set out to answer using photoconversion of etv2:KAEDE expressing cells, an experiment that they also performed in their 2014 and 2022 publications. To check for low recombination efficiency, the authors could examine the expression of Cre mRNA in their transgenic embryos. Do many more jam2b expressing cells express Cre mRNA than they observe in their switch lines? They could also compare their experiments using Cre recombinase with those using EGFP expression in jam2b cells. EGFP is relatively stable, and the time frames the authors analyze are short. As no quantification of EGFP-expressing cells is provided in Figure 1, this comparison is currently not possible. Do these two different approaches answer different questions here?

(4) Concerning the etv2:KAEDE photoconversion experiments: The percentages the authors report for SVF cells' contribution to the SIV and SIA differ from their previous study (Dev Cell, 2022). In that publication, SVF cells contributed 28% to the SIA and 48% to the SIV. In the present study, the numbers are close to 80% for both vessels. The difference is that the previous study analyzed 2dpf old embryos and the new one 4dpf old embryos. Do SVF-derived cells proliferate more than PCV-derived cells, or is there another explanation for this change in percentage contribution?

(5) Single-cell sequencing data: Why do the authors not show jam2b expression in their single-cell sequencing data? They sorted for (presumably) jam2b-expressing cells and hypothesize that jam2b expression in ECs at this time point is important for the generation of intestinal vasculature. Do ECs in cluster 15 express jam2b? Why are no other top marker genes (tal1, etv2, egfl7, npas4l) included in the dot blot in Figure 5b?

(6) Concerns about cell autonomy of mutant phenotypes: The authors need to perform in situ hybridization to characterize jam2a expression. Can it be seen in SVF cells? The double mutants show a clear phenotype in intestinal vessel development; however, it is unclear whether this is due to a cell-autonomous function of jam2a/b within SVF cells. The authors need to address this issue, as jam2b and potentially also jam2a are expressed within the tissue surrounding the forming SVF. For instance, do transplanted mutant cells contribute to the intestinal vasculature to the same extent as wild-type cells do?

(7) Finally, the authors analyze the phenotypes of hand2 mutants and their impact on the expression of jam2b and etv2. They observe a reduction in jam2b and etv2 expression in SVF cells. However, they do not show the vascular phenotypes of hand2 mutants. Is the formation of the SIA and SIV disturbed? Is hand2 cell autonomously needed in ECs? The authors suggest that hand2 controls SVF development through the regulation of jam2b. However, they also show that jam2b mutants do not have a phenotype on their own. Clearly, hand2, if it were to be required in ECs, regulates other genes important for SVF development. These might then regulate jam2b expression. The clear linear relationship, as the title suggests, is not convincingly shown by the data.

Reviewer #3 (Public review):

Summary:

This study by Griciunaite et al. investigates the function of the adhesion molecule Jam2 in initiating the formation of organ (intestinal)-specific vasculature in zebrafish. Their previous studies identified a group of late-forming vascular progenitors from the lateral plate mesoderm along the yolk extension termed the secondary vascular field (SVF), which can contribute to intestinal vasculature. Transcriptomic analysis of the zebrafish trunk region identified SVF-enriched marker genes, which include jam2b. They then performed expression analysis of jam2b using whole-mount in situ hybridization and Gal4 knock-in transgenic line analysis. These analyses show that jam2b is expressed in the SVF cells that correspond to etv2 and kdrl expression past 24 hours. Lineage tracing combining jam2b:Gal4 and UAS:Cre or UAS:CreERT2 show the contribution of jam2b in SVF and intestinal vasculature formation. jam2b mutations did not cause observable defects in the vasculature, but combined jam2a; jam2b mutations led to impaired ISV, PCV, SIA, SIV and thoracic duct lymphatic vasculature formation. Finally, the authors show that mutations in the transcription factor hand2 led to reduced jam2b expression and impaired SVF formation.

Strengths:

The authors accomplished many feats in generating new reporter lines and mutations that are valuable to the community. The study provided an interesting perspective on organ-specific vascular development and origin heterogeneity. The genetic aspects of the study are clean, and the mutational phenotypes are convincing.

Several suggestions and major comments that can improve the manuscript include:

(1) Overall molecular mechanisms of Jam2 function are not fully uncovered in the study. How do the adhesion molecules Jam2a and Jam2b regulate SVF cell formation? Are they responsible for migration, adhesion or fate determination of these structures? The authors should provide a more in-depth study of the jam2a, jam2b mutations and assess the processes affected in these mutants. Combining these mutants with etv2:Kaede can also provide a stronger causative link between their functions and defects in SVF formation.

(2) Have the authors tested the specificity of the jam2b knock-in reporter line? This is an important experiment, as many of the conclusions derive from lineage tracing and fluorescence reporting from this knock-in line. One suggestion is to cross the jam2b:GFP or jam2b:Gal4, UAS:GFP line to the generated jam2b mutants, and examine the expression pattern of these lines. Considering that the ISH experiment showed lack of jam2b expression, the reporter line should not be expressed in the jam2b mutants.

(3) The rationale behind the regeneration study is not clear, and the mechanisms underlying the phenotype are not well described. How do the authors explain the phenotype with the impaired regeneration, and what is the significance of this finding as it relates to SVF formation and function?

(4) The authors need to include representative images of jam2b>CreERT2 with 4-OH activation at different timepoints in Figure 3.

(5) The etv2:Kaede photoconversion experiment to show that the majority of intestinal vasculature derives after 24 hours needs to be supplemented with additional data on photoconverted post-24-hour-old endothelial cells, with the expectation that the majority of intestinal endothelial cells at 4 days will then be labeled with red Kaede. In addition, there have been data that show the red Kaede protein is not stable past several days in vivo, and 3 days might be sufficient for the removal or degradation of this photoconverted protein. Thus, the statement that intestinal vasculature forms largely by new vasculogenesis might be too strong based on existing data.

(6) To strengthen the claim that hand2 acts upstream of jam2b, the authors can perform combinatorial genetic epistatic analysis and examine whether jam2b mutations worsen hand2 homozygous or heterozygous effects on the SVF. Similarly, overexpressing jam2b might rescue the loss of SVF/etv2 expression in hand2 mutants.