Neuropilin 1 mediates epicardial activation and revascularization in the regenerating zebrafish heart

ABSTRACT Unlike adult mammals, zebrafish can regenerate their heart. A key mechanism for regeneration is the activation of the epicardium, leading to the establishment of a supporting scaffold for new cardiomyocytes, angiogenesis and cytokine secretion. Neuropilins are co-receptors that mediate signaling of kinase receptors for cytokines with crucial roles in zebrafish heart regeneration. We investigated the role of neuropilins in response to cardiac injury and heart regeneration. All four neuropilin isoforms (nrp1a, nrp1b, nrp2a and nrp2b) were upregulated by the activated epicardium and an nrp1a-knockout mutant showed a significant delay in heart regeneration and displayed persistent collagen deposition. The regenerating hearts of nrp1a mutants were less vascularized, and epicardial-derived cell migration and re-expression of the developmental gene wt1b was impaired. Moreover, cryoinjury-induced activation and migration of epicardial cells in heart explants were reduced in nrp1a mutants. These results identify a key role for Nrp1 in zebrafish heart regeneration, mediated through epicardial activation, migration and revascularization.

Anti-sense riboprobes differential staining patterns were compared to previous reports (43) to confirm specific nrp isoform detection. All neuropilin isoforms are observed in the brain with additional differential expression patterns observed between different isoforms. Nrp1a is observed in the fin buds and otic vesicles, nrp1b is expressed in the dorsal aorta and intersegmental vessels, nrp2a is observed in the hind brain and fin buds, whereas nrp2b is largely restricted to the brain and hind brain, n ≥ 8.

Development • Supplementary information
Activated epicardial cells were identified with Wt1 antibody (red) and assessed for Nrp1 expression (green). White arrows indicate regions of colocalization. Scale bars = 20µm.

Development • Supplementary information
Results are presented as mean GFP+ signal area in mm 2 ± S.E.M (two-tailed t-test of n = 4, p=0.0713).

RT-qPCR
Five ventricles from corresponding time points and treatments were pooled for RNA extraction and homogenized in lysis buffer (Qiagen) in 1.4mm ceramic bead-containing tubes and mechanically disrupted in a Minilys homogenizer (Peqlab). Homogenates were passed through a QIAshredder spin column (Qiagen) then total RNA was extracted using the RNeasy Mini Kit (Qiagen). The quantity and purity of RNA were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) and RNA integrity was assessed with a Bioanalyzer (Agilent). 250-500 ng of total RNA was reverse transcribed using the QuantiTect® Reverse Transcription Kit (Qiagen).
All primers (see table S1) and standards were purchased from qStandard: Absolute RT-qPCR values were measured using Brilliant III Ultra-Fast QPCR Master Mix (Agilent Technologies Inc.) and the Rotor-Gene PCR thermocycler (Qiagen). Values were normalized using a normalization factor generated using GeNorm software from the following three reference genes: gapdh, rpl13a and eef1a1a to calculate the expression of the genes of interest. Images were captured on the Leica TCS SPE1 confocal microscope system and processed using the publicly available ImageJ software.

AFOG staining
Deparaffinized and rehydrated sections were fixed in Bouins fixative (Thermo Fisher Scientific) for 2 hours at 60 o C followed by overnight incubation at room temperature. The following day, slides were washed for 10 minutes in constant tap water stream. Nuclei were Development: doi:10.1242/dev.174482
Previously established probes, such as: cmlc2, raldh2, wt1b and tbx18 (gift from Nadia Mercader, Universität Bern, Switzerland) were synthesized in a similar manner. Primers were used in a PCR reaction to amplify probe sequence region and amplicons cloned to pGEM®-T plasmid vectors. Each plasmid was used in two RNA polymerase reactions (either SP6 or T7) to generate the sense and the anti-sense probes.
After deparaffinization in xylene, sections were rehydrated, fixed with 4% (wt/vol) paraformaldehyde at room temperature for 10 minutes and digested with proteinase K (10μg/ml in PBS) at 37 o C for 10 minutes. Sections were then post-fixed with 4% (wt/vol) paraformaldehyde for 5 minutes at room temperature, washed twice in PBS, and acetylated for 10 minutes with 0.25% acetic anhydride in 0.1 M triethanolamine (DEPC-treated), then

In vitro epicardial cell culture (heart explants)
Thrombin/fibrin reaction in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) was performed as described previously (Kim et al., 2012) to produce a fibrin gel matrix in either 24-well plates or Lab-Tek™II 4-chamber slides (Thermo Fisher Scientific).
The apex of cryoinjured and sham-operated zebrafish hearts 5 days post-surgery were isolated and rinsed several times with HDMEM (23mM HEPES, 15mM NaCl in DMEM) to remove residual blood. One apex per well/chamber was placed firmly on set fibrin gel matrices, ensuring epicardial surface contact with the gel. Excess HDMEM was removed from heart tissue and left to adhere for 1 hour in a tissue culture incubator (28°C, 5% CO2) before careful addition of DMEM supplemented with 0.5% fetal bovine serum (FBS), Normocin™ (invivoGen) and penicillin/streptomycin antibiotics (Sigma-Aldrich) into the wells or slide chambers. Medium was changed every 2 days and cells were cultured for 7 days before harvesting epicardial outgrowths for immunofluorescence imaging. For immunofluorescence analysis, heart tissue was discarded and cells were fixed in 4% (wt/vol) paraformaldehyde for 15 mins and permeabilized with 0.1% Triton X-100 for 10 mins before blocking in PBST (1% BSA, 10% donkey serum) at room temperature for 1 hour and incubated overnight at 4°C with primary antibody diluted in blocking solution. The antibodies used to stain epicardial cells from heart explants are the following: anti-Neuropilin 1,

Development • Supplementary information
The following day, samples were incubated for one hour at room temperature with fluorescent-tagged secondary antibodies (anti-rabbit Alexa 488 and Alexa Fluor 555 Phalloidin, both Thermo Fisher Scientific). Slides were mounted with ProLong DAPI mounting medium (Thermo Fisher Scientific).

Immunoblotting
Lysates were obtained from zebrafish hearts by homogenizing 3 ventricles in RIPA buffer (Sigma-Aldrich) supplemented with TCEP, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails (Sigma-Aldrich) in 1.4mm ceramic bead-containing tubes using the Minilys homogenizer (Peqlab). Lysates were supplemented with Lithium dodecyl sulfate anionic detergent (Thermo Fisher Scientific) and RIPA buffer to load equal amount of proteins. All samples were then denatured by heating for 3 minutes at 95°C before loading to gels. Proteins were separated by electrophoresis on 4-12% Bis-Tris polyacrylamide gels, C2206; all used at 1:100 dilution. Proteins were detected using the ECL Plus™ Western blotting detection system and Hyperfilm (both Amersham).