Rhomboid protease Rhbdl2 regulates macrophage recruitment and wound regeneration in zebrafish
Saroj GourkantiiD, Gayathri RamakrishnaniD, Jacqueline CheungiD, Taylor J. SchoeniD, Yazmin MunoziD, Rosa M. ChaveziD, Jan DohnálekiD, Katie MartiniD, Matthew Lovett-BarroniD, Thomas C. WhisenantiD, Kvido Střı́šovskýiD, Sonya E. NealiD
Abstract
Tissue regeneration requires tight control of immune cell behavior, yet the mechanisms that restrain immune-driven regenerative responses remain poorly defined. Here, we identify the rhomboid intramembrane serine protease Rhbdl2 as a critical regulator of regeneration in zebrafish. We generated rhbdl2 mutants by CRISPR Cas9 and found that it does not affect normal development, but triggers enhanced regenerative growth following injury, accompanied by increased macrophage accumulation at the wound site, which is accompanied by increased early apoptosis and proliferation. Proteomic analyses reveal increased Rac2 protein levels in rhbdl2 mutants, indicating dysregulated immune signaling. Functionally, Rac2 morpholino oligonucleotides mediated knockdown in rhbdl2 mutant larvae suppresses the elevated macrophage recruitment and enhanced tissue regenerative phenotype. Together, these findings uncover Rhbdl2 as an immune checkpoint that constrains macrophage driven enhanced regeneration, with vast implications for inflammatory disease, fibrosis, and tumor immune interactions.
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In-depth summary
Claude Opus 5.5 · xhigh effort · 7 Nov 2026 · from the full text of version 2 (17 Sept 2026) · CC BY 4.0
Loss of the rhomboid protease Rhbdl2 in zebrafish larvae speeds tail fin regrowth after injury, raises macrophage accumulation and Rac2 protein, and Rac2 knockdown reverses both effects.
The mechanisms that restrain immune-driven wound repair are poorly defined. The paper says earlier Rhbdl2 work relied mostly on cell culture and no vertebrate knockout existed, so it builds a zebrafish RNA-null mutant to study Rhbdl2 in vivo.
Key results
ShownRhbdl2 mutants regrow more tail fin tissue after transection, but unwounded fin size and overall development are normal. The chemical inhibitor STS1650 reproduces the effect in wild-type larvae.
Wound repair area was significantly larger in rhbdl2-/- than in WT at 72 and 96 hpw (n = 59 vs 61 at 72 hpw; 52 vs 46 at 96 hpw). STS1650 raised regrowth dose-dependently: no significant effect at 10 µM, significant at 15 µM, comparable to mutants at 25 µM. Unwounded 6 dpf fin area did not differ (n = 52 vs 68). · An RNA-less rhbdl2−/− allele does not impact…; Loss of Rhbdl2 induces enhanced tail fin…
ShownMutant wounds show more cell proliferation and more apoptosis. Proliferation rises only after wounding. Blocking caspases did not remove the macrophage or regrowth phenotype.
EdU-positive cells in the dorsal fin and pH3-positive cells at 72 hpw were significantly higher in mutants after wounding, with no difference in unwounded fins. Active caspase-3 area was higher at 24 and 72 hpw. The pan-caspase inhibitor QVD-OPH (50 µM) did not suppress macrophage recruitment or outgrowth (n = 44 to 48 per group). · Rhbdl2-deficient larvae have increased…; Loss of Rhbdl2 increases macrophage accumulation…
SuggestedLabel-free proteomics of 2 dpf larvae found 48 differentially abundant proteins in mutants. Rac2 was higher while Rac1a, Rac1b, Rac3a and Rac3b were lower. Rac mRNA did not change, which the authors read as post-transcriptional regulation.
8,932 proteins were identified and 2,425 entered the statistics. Of the 48 differential proteins, 26 were lower and 22 higher in mutants (limma, P < 0.05). Three replicates per genotype, each pooling about 50 to 60 larvae. RT-qPCR showed no significant change in Rac gene mRNA. · Proteome profile of rhbdl2−/− zebrafish reveals…; Mass spectrometry analysis
ShownMacrophage accumulation at the wound is higher in mutants at 24 and 48 hpw and returns to WT levels by 72 hpw. Neutrophil recruitment is mostly unchanged. Mutant macrophages move somewhat faster, and total macrophage numbers do not differ.
Macrophage counts were markedly higher in rhbdl2-/- at 24 and 48 hpw (24 hpw: n = 66 WT, 70 mutant). Neutrophils were comparable at 24 and 48 hpw, with a modest rise at 72 hpw. Early recruitment (1 to 6 hpw) showed only a non-significant trend. Whole-larva macrophage counts at 2 and 3 dpf were similar between genotypes. · Loss of Rhbdl2 increases macrophage accumulation…; Figure legends
ShownRac2 morpholino knockdown brings mutant macrophage recruitment and wound area back to WT-like levels. Clodronate macrophage depletion lowers repair in both genotypes, but mutants keep a slightly larger repair area.
With control morpholino, mutants had more wound macrophages than WT at 24 and 48 hpw. Rac2 morpholino restored levels indistinguishable from WT and also reduced mutant wound area to WT-like (n = 40 control, 52 Rac2 morpholino for mutants). Clodronate cut macrophage counts in both genotypes. · Rac2 contributes to enhanced macrophage…; Figure legends
Methods, data and reproducibility
The authors made a promoterless, RNA-null rhbdl2 zebrafish allele with CRISPR-Cas9 and compared mutant and WT cousin larvae after tail transection. They used confocal imaging, label-free mass spectrometry, a chemical inhibitor, Rac2 morpholino knockdown and clodronate macrophage depletion.
- rhbdl2-/- and WT cousin larvae in wound repair, proliferation, apoptosis and leukocyte imaging assays · zebrafish · n = per-assay n from about 40 to 70 larvae in three to four replicates
- Label-free LC-MS/MS proteome of 2 dpf WT vs rhbdl2-/- larvae · zebrafish · n = 3 replicates per genotype (about 50 to 60 larvae pooled each) · 10.17632/gwh3j3rrjr.1
- Cleavage assays of Spint1 (HEK293T) and endogenous EGFR (rhbdl2-deficient HaCaT cells) with human or zebrafish Rhbdl2 · human (cell lines) · n = N = 3 replicates
- Methods & tools
- CRISPR-Cas9, CRISPRscan, AlphaFold, Jalview (MUSCLE), timsTOF 2 HT mass spectrometer, Peaks Studio 12.5, Perseus, limma (R), g:Profiler, Imaris 11.0, FIJI, GraphPad Prism 10
- Data & accessions
- https://doi.org/10.17632/gwh3j3rrjr.1
- Reagents & environment
- R 4.5.2, GraphPad Prism 10, Imaris 11.0, NIS Elements Ar 6.10.01, Peaks Studio 12.5; no container or requirements file stated.
- Reproducibility gaps
- No analysis code is stated. Several results are marked 'data not shown' (rhbdl2 expression in epidermis, mRNA rescue toxicity).
Limitations (4)
- The whole-body knockout cannot separate epithelial-cell-autonomous from macrophage-autonomous Rhbdl2 function. The Rhbdl2 substrate acting on Rac2 is not identified. · stated by the authors
- Rac2 knockdown by morpholino was global rather than macrophage-specific. Macrophage-depleted mutants still showed slightly enhanced repair, pointing to macrophage-independent effects. · stated by the authors
- Macrophage polarization markers were measured by RT-qPCR on whole-tail RNA, so cell-type-restricted changes could be masked. Rescue by rhbdl2 mRNA injection failed because of dose-dependent toxicity. · stated by the authors
- The proteomic hits use a nominal P < 0.05 cutoff with three replicates per genotype. The Rac2 increase is shown by mass spectrometry only in the text, so the Rac2 mechanism rests on this and the morpholino rescue. · noted in this summary
Who should read:Read this if you study wound repair, macrophage recruitment or intramembrane proteases in vivo, or want a zebrafish Rhbdl2 loss-of-function model with a Rac2 link.
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