Genetic targeting of Purkinje fibres by Sema3a-CreERT2

The maintenance of the heart rhythm and the conduction of excitatory signals require changing excitatory signals via electrical activity and coordination by communication between working and conductive cardiomyocytes. Understanding how the ventricular conduction system is established provides novel insights into the pathophysiological progress of cardiac arrhythmias. However, the major hurdle in this field is the lack of a specific genetic tool that targets the Purkinje fibres of the ventricular conduction system and no other types of cardiomyocytes or coronary vessels. Here, we generated a Sema3a-CreERT2 knock-in mouse line to test its specificity for genetically labelled Purkinje fibres. We found that Sema3a was expressed in the subendocardial layer of the trabecular myocardium in the embryonic heart and was restricted to the Purkinje fibres in the adult heart. A fate mapping study based on the Sema3a-CreERT2 line revealed that the Sema3a+ cardiomyocytes were restricted to the fate of Purkinje fibres in the perinatal but not the embryonic stage. Collectively, our study provides a new genetic tool, i.e., Sema3a-CreERT2, for studying the molecular mechanisms that regulate the function of Purkinje fibres.

defects in Sema3a result in abnormal neuronal innervation 11,12 . Moreover, Sema3a also plays physiological roles in immunoregulation 13 and cancer development 14 . A recent study indicates that Sema3a can maintain a normal heart rhythm by mediating the patterning of the sympathetic innervation 15 . Interestingly, the expression of Sema3a is detected in the Purkinje fibres and a subset of trabecular cardiomyocytes, but to our knowledge, lineage tracing results regarding Sema3a in the cardiac conduction system have not yet been reported. In our study, we delineated the expression patterns of Sema3a in the developing and adult heart. The genetic lineage tracing analysis with Sema3a-CreERT2 revealed that Sema3a specifically labels Purkinje fibres in the adult heart, and the fate of these specialized cardiomyocytes is actually determined in the perinatal stage. These results expand our knowledge about the development of the VCS and provide a new genetic mouse tool for the study of Purkinje fibre function.

Results
Generation and characterization of the Sema3a-CreERT2 mouse line. We generated a Sema3a-CreERT2 knock-in mouse line by homologous recombination using a conventional ES cell-based targeting strategy. These Sema3a-CreERT2 mice expressed the tamoxifen-inducible CreERT2 recombinase (CreER T2 ) under the endogenous transcriptional control of the Sema3a gene (Fig. 1a). DNA from the tails of heterozygous mice (Sema3a-CreERT2) and wild-type littermates (Sema3a +/+ ) was extracted for polymerase chain reaction (PCR) experiments. The Sema3a-CreERT2 mice had two bands: one band corresponded to the mutant knock-in allele, and the other band corresponded to the wild-type allele (Fig. 1b). Un-cropped PCR gel pictures are presented in Supplementary Fig. 1. Homozygous mutant mice were not obtained in our study due to the high postnatal mortality of Sema3a null mutations 11 . To detect the function of these heterozygous mice, we tested the ejection fraction and the fractional shortening of 8-week-old mice and found no significant difference between the Sema3a-CreERT2 and the Sema3a +/+ littermate control mice ( Supplementary  Fig. 2a,b). ECG and echocardiography revealed that the Sema3a-CreERT2 mice exhibited normal heart function at the age of 8 weeks (Supplementary Fig. 2c). To test whether the knock-in allele was expressed as expected, we first used oestrogen receptor (ESR) antibody staining as a surrogate for Sema3a expression in embryonic lung and bone tissue. ESR/Sema3a was highly expressed in both organs ( Supplementary Fig. 3), which is consistent with a previous study 16 . Then, we co-stained for ESR and TNNI3 in E12.5, E14.5 and E19.5 embryonic heart sections. The immunostaining results revealed that ESR/Sema3a expression was largely restricted to the trabecular myocardium, but minimal staining was detected in the compact layer in the E12.5 and E14.5 embryonic hearts (Fig. 1c). At E19.5, ESR/Sema3a was expressed in a subset of subendocardial trabecular cardiomyocytes that were located along the side of the ventricular chamber (Fig. 1c). ESR/Sema3a was not detected in the cardiomyocytes or the coronary arteries of the compact myocardium (Fig. 1c), which is consistent with a previous study of Sema3a expression 15 . Taken together, these data demonstrated the successful generation of the Sema3a-CreERT2 mouse line.
Sema3a-CreERT2 labels Purkinje fibres in the adult mouse heart. According to the ESR staining analysis of the Sema3a-CreERT2 mice, Sema3a was primarily expressed in the trabecular cardiomyocytes and not in the compact layer. Since Purkinje fibres are restricted in the innermost layer of the ventricular myocardial wall 5 , we next determined whether Sema3a-CreERT2 could specifically target these specialized cardiomyocytes in the adult mouse heart. We crossed Sema3a-CreERT2 mice with R26-tdTomato 17 mice to generate Sema3a-CreERT2; R26-tdTomato double-positive mice (Fig. 2a). CreERT2 is retained in the cytoplasm, and, only in the presence of tamoxifen 18 , CreERT2 translocates into the nucleus for Cre-loxP recombination (Fig. 2b), which leads to a permanent tdTomato expression in the Sema3a + cells and their descendants 19 . We treated 8-12-week-old mice with tamoxifen and collected the hearts 48 hours after the tamoxifen treatment. We found that very sparse RFP + cells were detected among the atrial cardiomyocytes (Fig. 2c,d). To determine whether Sema3a was expressed in the SA node and the AV node, we co-stained for HCN4 and RFP and found that Sema3a was not expressed in the SA node and was sparsely expressed in the AV node (Fig. 2e,f). With the purpose of detecting whether Sema3a was expressed in the stellate ganglia or cardiac nerves, we co-stained for RFP and tyrosine hydroxylase (TH, which is a marker of the stellate ganglia) and co-stained for RFP and Tuj1 (a marker of cardiac nerves) and found that Sema3a was not expressed in the stellate ganglia or in the cardiac nerves ( Supplementary Fig. 4a,b). Because Cx40 has been reported to mark the VCS, we generated Sema3a CreERT2; R26-tdTomato; Cx40 GFP 5 triple-positive mice to test for the expression of Sema3a in the Purkinje fibres. By immunostaining heart sections for RFP and GFP, we found that RFP was co-localized with GFP + cells in the subendocardial layer of the ventricular wall, whereas RFP was not detected in the GFP + coronary arteries in the compact myocardium (Fig. 2g). We next dissected the ventricles for whole-mount fluorescence views of the inside of the Sema3a-CreERT2; R26-tdTomato; Cx40-GFP mouse hearts ( Fig. 2h and Supplementary Fig. 5). Cx40 exhibited gross expression in the atrium, whereas Sema3a was hardly detected. Cx40 was also expressed in the bundle of His (HB), in the left and right bundle branches, and in the Purkinje fibres, which is consistent with a previous report 5 . Compared with Cx40 (GFP signal), Sema3a (RFP signal) was largely restricted to the Purkinje fibres and was not detected in the bundle of His or the bundle branches (Fig. 2h,i). Z-stack confocal microscopy confirmed the expression of Sema3a (RFP signal) in the Cx40 + Purkinje fibres that reside in the subendocardial location of the ventricular myocardium (Fig. 2j). The same pattern of Sema3a expression was observed in the right ventricle ( Supplementary Fig. 5). These data demonstrated that Sema3a-CreERT2 specifically targeted the ventricular Purkinje fibres in the adult mouse heart.
Fate-mapping analysis of Purkinje fibre development with Sema3a-CreERT2. Since Sema3a was expressed in a broader domain during the embryonic stages than in the adults, we asked when Sema3a + cardiomyocytes were specialized to the fate of Purkinje fibres in the developing hearts. We injected tamoxifen at E12.5, E14.5 or E18.5 into Sema3a-CreERT2; R26-tdTomato; Cx40-GFP triple-positive mice and collected the hearts at P7 or P21 for analysis. The lineage tracing data revealed that the Sema3a + cells at E12.5 gave rise to both the Cx40 + Purkinje fibres (minority) and Cx40 − working cardiomyocytes (majority) at P7 and P21 (Fig. 3a,b). The Sema3a + cells at E14.5 were more restricted to the subendocardial layer of the ventricular myocardium and gave rise to the Cx40 + Purkinje fibres and Cx40 − working cardiomyocytes at P7 and P21 (Fig. 3c,d). The Sema3a + cells at E18.5 gave rise to almost all of the Cx40 + Purkinje fibres in the subendocardial surface of the ventricular myocardium at P7 and P21 and gave rise to few if any Cx40 − working cardiomyocytes (Fig. 3e,f). These data demonstrated that the fate of Sema3a + cardiomyocytes was specialized to Purkinje fibres in the perinatal stage (e.g., E18.5) when their cell fate was restricted to the ventricular conduction system (Fig. 3g).
Quantification of the Sema3a + cardiomyocytes. Finally, we quantified the percentage of cardiomyocytes that were labelled with Sema3a-CreERT2; R26-tdTomato in the adult mouse hearts. Tamoxifen was injected into the adult mice, and the hearts were collected for analysis 48 hours after the tamoxifen treatment. Immunostaining of the Sema3a-CreERT2; R26-tdTomato hearts for RFP and TNNI3 revealed RFP + cardiomyocytes in the subendocardial surface of the ventricular myocardium (Fig. 4a). Quantification of the percentage of RFP + cardiomyocytes in the heart sections revealed that 1.02 ± 0.13% of the cardiomyocytes were labelled with Sema3a-CreERT2, and there were almost no cardiomyocytes that were labelled without the tamoxifen treatment (Fig. 4c). We also isolated cardiomyocytes and placed them in dishes for quantification (Fig. 4b). We found that 1.10 ± 0.18% of the cardiomyocytes were RFP + in the tamoxifen-treated mouse hearts, and very few RFP + cardiomyocytes were present in the mice without the tamoxifen treatment (Fig. 4d). These findings were consistent with the quantification obtained following immunostaining of the heart sections (Fig. 4c).

Discussion
In this study, we generated a new genetic tool, i.e., Sema3a-CreERT2, which specifically targets Purkinje fibres in the adult mouse heart. In contrast, Hcn4-CreERT2 targets the sinoatrial node, atrioventricular node, the bundle of His, the bundle branches and the Purkinje fibres, and Cntn2-Cre is detected in the SA node, AV node, and His-Purkinje system 4 . In contrast to the Cx40-CreERT2 tool, Sema3a-CreERT2 does not label coronary arterial endothelial cells or atrial cardiomyocytes. The fate mapping study also revealed that the Sema3a + cells were trabecular cardiomyocytes in the early embryonic stage, and during the perinatal period, the Sema3a + cardiomyocytes specialized into the Purkinje fibre fate. Since our CreERT2 is inducible, and its efficiency is not 100%, the Sema3a-CreERT2 labelled most, but not all, of the Cx40-GFP cells. In the heart, we detected RFP + GFP + labelled cells and RFP − GFP + unlabelled cells in the P7 and P21 hearts. No significant dilution of RFP labelling efficiency was observed from P7 to P21. It might be possible, although unlikely, that a few Cx40-GFP + cells could be recruited from myocytes at a later stage. Furthermore, tamoxifen treatment lasts 1-2 days for the labelling of cells; we, therefore, cannot formally exclude the possibility that a small number of CreERT2 + cells were labelled at a later stage due to incomplete clearance of the tamoxifen from the body. We believe this possibility is unlikely because the majority of tamoxifen is cleared after 1-2 days, and the labelling of the CreERT2 + cells afterwards should be minimal. Additionally, the expression domain of CreERT2 (Sema3a) in the myocardium decreases rather than increases during embryonic development (e.g., compare E12.5, E14.5 and E19.5 in Fig. 1c). A broader Cre-loxP recombination domain is, therefore, unlikely even if there is residual tamoxifen remaining after 1-2 days. In conclusion, Sema3a-CreERT2 is the first genetic mouse tool for the specific targeting of Purkinje fibres and provides a means for the in vivo functional study of Purkinje fibres during ventricular conduction system formation and cardiac arrhythmias.

Materials and Methods
Animals. All mouse lines used in this study were handled according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) at the Institute for Nutritional Sciences, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Science, Chinese Academy of Science (approved protocol number 2015-AN-8). We generated the Sema3a-CreERT2 knock-in mouse line by knocking a cDNA encoding Cre recombinase fused with a mutant form of the oestrogen receptor hormone-binding domain into the Sema3a locus 20 . Briefly, a 129-bp mouse BAC clone containing the complete mouse Sema3a gene was obtained from the Sanger Institute (UK). The targeting vector was generated using the following cassettes: CreERT2 cDNA, SV40 polyA sequence, and Frt site-flanked PGK-EM7-Neo resistant gene. The neo cassettes were detected by PCR (Fig. 1b). All mice were maintained on a C129/C57BL6/J mixed background. Sema3a-CreERT2 mice were crossed with R26-tdTomato (Ai9) 17 and Cx40-GFP 5 mice to generate Sema3a-CreERT2; R26-tdTomato and Sema3a-CreERT2; R26-tdTomato; Cx40-GFP mouse lines for genetic lineage tracing of the Sema3a cells. Tamoxifen (Sigma, T5648) was dissolved in corn oil and introduced by gavage at the indicated times (0.1-0.15 mg/g mouse body weight). To determine the exact timing of the embryonic stages, we checked for a vaginal plug and recorded the result as embryonic day 0.5, then we injected tamoxifen at embryonic days 12.5, 14.5 and 18.5. The day of the mouse birthday was postnatal day 0, and the hearts were collected at postnatal days 7 and 21. To assess the leakiness of the inducible Cre in the absence of tamoxifen, we introduced corn oil by gavage to the Sema3a-CreERT2; R26-tdTomato mice in parallel. We designed PCR primers that spanned the genomic DNA and inserted cassette as follows: F1: CCTTTCATTCT GCTTCCTCGG, R1: TGCGAACCTCATCACTCGTTG (to test for the correct target allele of 533 bp); and F1: CCTTTC ATTCTGCTTCCTCGG, R2: GCTTTGATGGTTAG CGTTGGG (to test for the wild-type allele of 658 bp); and F2: CGACCACCAAGCGAAAC ATC, R3: AAAGACAGGCAATCCCAGTGAAC (to test for the Neo allele of 790 bp). All mice were bred on a C129/C57BL6/J mixed background. We thank Shanghai Biomodel Organism Co., Ltd. for the mouse generation.
Immunostaining. Immunostaining was performed according to previous protocols 21 . Briefly, we collected mouse hearts or other organs and washed them in cold PBS several times. Then, the heart tissue was fixed in 4% PFA (Sigma), and the fixation time depended on the size of the organ. In general, the embryonic hearts were fixed for approximately 30 minutes, and the adult hearts were fixed for approximately 1 hour. After fixation, the hearts were dehydrated in 30% sucrose at 4 °C until the tissue was fully penetrated. After soaking the hearts at an optimal cutting temperature (OCT, Sakura) for approximately 1 hour at 4 °C, the tissues were embedded in blocks and frozen in a −80 °C fridge. For the section orientation, the hearts were sectioned in the coronal plane (four chamber orientation). We focused on the trabecular muscle in the embryonic heart and the SA node, AV node, the bundle of His, the bundle branches and the Purkinje fibres in the adult hearts. Cyrosections of 8-10 µm thickness were collected on slides. The tissues were blocked in blocking solution (5% normal donkey serum in PBS with 0.1% Triton X-100) for 30 min at room temperature and subsequently incubated in the first antibodies overnight at 4 °C. After fully washing out the primary antibodies, the tissues were then developed with Alexa fluorescent antibodies (Invitrogen). For weak signals, we used HRP or biotin-conjugated antibodies (Jackson Immunoresearch) with a tyramide signal amplification kit (PerkinElmer). Before mounting the slides, the tissues were counterstained with 4′6-diamidino-2-phenylindole (DAPI, Vector Laboratories). The primary antibodies and dilution ratios were as follows: RFP (Rockland, 600-401-379,1:500); ESR (Abcam, ab27595, prediluted); GFP (Nacalai Tesque, 04404-84, 1:100); GFP (Abcam, ab6662, 1:200); TNNI3 (Abcam, ab56357, 1:100); Cx40 (Alpha Diagnostic, Cx40-A, 1:100); HCN4 (Abcam, ab32675, 1:100); tyrosine hydroxylase (TH, Millipore, AB152, 1:100); and Tuj1 (Covance, MMS-435P, 1:100). The images were acquired with a Zeiss confocal microscope (LSM510), an Olympus confocal microscope (FV1000), and a Zeiss stereo-microscope (AXIO Zoom. V16). To produce the Z-stack confocal images, we scanned 3-4 consecutive XY images on the z-axis with an Olympus confocal microscope (FV1000). Then, the images were analysed with ImageJ (NIH) software. In the stack data, an orthogonal view was used to reveal the merged signals on the XZ and YZ axes. The merged signals and split channels are presented to delineate the signals at a one-cell resolution. Each IHC data is representative of 3-5 individual samples.
Whole-mount heart imaging. The whole-mount images of the adult heart were collected with a Zeiss stereo-microscope (AXIO Zoom. V16). The hearts were collected from 8-12-week-old mice, and the blood was removed via stimulation with necropsy tools. Then, the heart tissues were washed in cold PBS several times until the solution became limpid. We fixed the hearts in 4% PFA (Sigma) for approximately 1 hour at 4 °C and then washed them with cold PBS 3 times for 5 minutes each time. We removed the atrium with surgical scissors and then split the left and right ventricle walls with a scalpel. To expose the Purkinje fibres, we fixed the hearts in agarose dishes with needles. Then, the images were collected with a Zeiss stereo-microscope. Dissociated adult cardiomyocytes. Cardiomyocytes were isolated from the adult hearts as described previously 22 . Briefly, we intraperitoneally injected Sema3a-CreERT2; R26-tdTomato mice with 200 µl of heparin (6.25 U/µl). Twenty minutes later, the mice were intraperitoneally anaesthetized with 10% chloral hydrate. The dissected heart was perfused with modified Tyrode's solution (MTS) for 5 min at a rate of 1 ml/min. The heart was next perfused for approximately 20-30 minutes at 37 °C with an enzyme buffer containing 250 U/ml collagenase type 2 (Worthington, LS004176) and 0.3 U/ml Protease XIV (Sigma, P5147) to dissociate the heart tissues. Micro-dissecting forceps were used to mince the ventricles into small pieces for dissociation and filtering with a 100-µm strainer. The isolated cells were centrifuged at 20× g for 3 minutes at 4 °C to allow for the collection of most of the cardiomyocytes.
Cardiomyocyte quantification. Cardiomyocytes were isolated from Sema3a-CreERT2; R26-tdTomato mice hearts within 48 hours of the injection of tamoxifen. All cardiomyocytes were collected in cell culture dishes, and the RFP + cardiomyocytes were quantified under a Zeiss stereo-microscope (AXIO Zoom. V16). The total number of RFP + cells from 5 hearts was divided by the total number of cardiomyocytes examined to obtain the percentage of RFP + cardiomyocytes. Statistical analysis. All data in this study were determined from 3-5 independent experiments and are presented as the mean values ± the s.e.m. All mice in this study were randomly assigned to the different experimental groups.