Rhomboid protease Rhbdl2 regulates macrophage recruitment and wound regeneration in zebrafish
Gourkanti, S., Ramakrishnan, G., Cheung, J., Schoen, T. J., Munoz, Y., Chavez, R. M., Dohnalek, J., Martin, K., et al.
immunology
Quick verdict
This zebrafish study claims Rhbdl2 brakes macrophage-driven wound repair; KO+inhibitor data are solid, but the Rac2 mechanism rests on proteomics+MO.
Summary
This preprint establishes zebrafish rhbdl2, a conserved rhomboid intramembrane serine protease, as a negative regulator of tail fin wound repair. Using CRISPR-Cas9, the authors generated a promoterless RNA-null allele (sd73) that eliminates rhbdl2 mRNA without altering development, adult viability, or expression of other rhomboids. Homozygous mutants showed larger regenerated fin area at 72 and 96 hours post-wounding; the ketoamide inhibitor STS1650 phenocopied this dose-dependently, arguing against CRISPR off-targets. Mutants exhibited elevated wound-induced proliferation (EdU, pH3) and apoptosis (active caspase-3), yet pan-caspase inhibition (QVD-OPH) did not abolish macrophage accumulation or enhanced repair. Label-free proteomics of 2 dpf larvae identified 48 differentially abundant proteins enriched for leukocyte migration and Rac signaling; Rac2 protein rose while its mRNA did not. Live imaging and fixed-tissue counts showed increased macrophage migration speed and transient accumulation at wounds at 24-48 hpw, with neutrophils largely unchanged. Rac2 morpholino knockdown normalized macrophage recruitment and wound repair in mutants, and clodronate macrophage depletion reduced repair in both genotypes, though mutants retained slightly elevated outgrowth. Main limitations: the pivotal Rac2 test uses a single morpholino without rescue; Rhbdl2-to-Rac2 causality is correlative; whole-larva proteomics at an earlier, unwounded stage leaves cell-type and spatial resolution unresolved; and rhbdl2 mRNA rescue failed due to toxicity, so no gain-of-function exists.
Review complete
Assessment at a glance
5
risk flags identified
● high 0
● medium 3
● low 2
5 strengths · 5 weaknesses · 5 author questions
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Key claims under review
- Zebrafish rhbdl2 encodes a canonical rhomboid protease functionally conserved with human RHBDL2, cleaving Spint1 and the EGFR ectodomain and inhibited by STS1650.
- A promoterless RNA-null rhbdl2 allele leaves development and adult viability intact but enhances tail fin regeneration after injury, phenocopied by dose-dependent pharmacological inhibition.
- rhbdl2 mutants show elevated wound-induced proliferation (EdU, pH3) and apoptosis (active caspase-3), and the enhanced repair is not secondary to caspase-mediated apoptosis (QVD-OPH).
- Rac2 protein is post-transcriptionally elevated in mutants, and Rac2 morpholino knockdown normalizes both macrophage recruitment and the enhanced wound repair phenotype.
- Macrophage depletion with clodronate only partially reduces the mutant repair advantage, implying additional macrophage-independent Rhbdl2 functions.
Strengths
Allele design preempts common CRISPR artifacts
The sd73 allele deletes 2.1 kb covering the promoter, exon 1 and the start codon; RT-qPCR confirms absence of rhbdl2 mRNA at 2 and 6 dpf (Fig. 1C), and rhbdl1/3/4 levels are unchanged (Fig. 1D), excluding translational compensation and family redundancy.
Genetic and pharmacological convergence
STS1650 was first validated against zebrafish Rhbdl2 in a cell-based EGFR-shedding assay (Fig. S1F), then dose-dependently (15-25 uM) phenocopied the mutant repair phenotype in WT larvae (Fig. 2D), controlling both CRISPR off-targets and inhibitor specificity.
Systematic alternative-explanation controls
Transparent null results
Adequate scale and reproducibility scaffolding
Weaknesses
Pivotal Rac2 test lacks orthogonal validation
Rhbdl2-to-Rac2 link is correlative and mismatched in time and space
Proteomics statistics are uncorrected and unvalidated
No gain-of-function and unresolved cell autonomy
Abstract and title overreach relative to the body
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Scope & Claims
This bioRxiv preprint (Gourkanti et al., UCSD; corresponding author S. Neal) asks whether the rhomboid intramembrane serine protease Rhbdl2 - previously characterized only in cultured cells - regulates vertebrate wound repair in vivo. The central claims are: (1) zebrafish rhbdl2 encodes a canonical, functionally conserved rhomboid protease (64% identity to human; sheds Spint1 and EGFR ectodomain; Fig. S1); (2) a promoterless RNA-null CRISPR allele (sd73) leaves development and adult viability intact but enhances caudal fin regeneration after transection; (3) the phenotype runs through elevated macrophage accumulation and post-transcriptionally elevated Rac2 protein; (4) Rac2 knockdown normalizes both macrophage recruitment and repair. Note the deposited title/abstract say 'regulates' and 'immune checkpoint' with 'vast implications', while the body text says 'influences' and 'modulator'; this review follows the body.
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Frequently asked questions
Does losing the RHBDL2 gene help or hinder wound healing?
In this zebrafish study, losing Rhbdl2 does not harm normal development but enhances tail fin regeneration after injury: mutant larvae regrow a larger wounded fin area, and a chemical inhibitor of Rhbdl2 produces the same effect in normal larvae. This suggests Rhbdl2 normally acts as a brake on reparative growth, largely by restraining macrophage accumulation at wounds.
What is the connection between Rhbdl2 and Rac2?
Proteomic profiling of rhbdl2-mutant larvae showed elevated Rac2 protein - a small GTPase that drives immune cell motility - without changes in its mRNA. Blocking Rac2 with a morpholino restored macrophage recruitment and wound repair in the mutants to normal levels. This supports, but does not prove, that Rac2 mediates the phenotype; the link is correlative and the knockdown used a single morpholino without a rescue experiment.
Is rhbdl2 essential for zebrafish development?
No. A promoterless CRISPR knockout allele (sd73) that eliminates rhbdl2 mRNA left embryonic morphology, body length at 2 days post-fertilization, adult appearance at 6 months, and the expression of related rhomboid-family genes essentially unaffected.
Where can I get the data from this paper?
The proteomics datasets and the two leukocyte time-lapse videos are deposited in the Mendeley Data repository under DOI 10.17632/gwh3j3rrjr. No analysis code is stated to be available, and the zebrafish line (sd73) is presumably available from the corresponding author at UC San Diego.
About this review
This is an AI-generated preliminary review by Evolve.Pub, produced from the openly available text of the paper. It is not peer review and does not represent the authors' views. The canonical record of the paper is at the publisher link above.
- Figures and videos could not be inspected; all judgments rest on captions and in-text numbers.
- FDR status of the proteomics analysis is inferred from the stated nominal P<0.05 threshold; a correction may exist in the source data not shown here.
- This is a bioRxiv preprint and has presumably not completed peer review; the deposited title/abstract differ slightly from the body text.
- Statistical detail (exact F values, adjusted P values, effect sizes) is not present in the extracted text.
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