Abstract
All current smooth muscle cell (SMC) Cre mice similarly recombine floxed alleles in vascular and visceral SMCs. Here we present an Itga8-CreERT2 knock-in mouse and compare its activity with a Myh11-CreERT2 mouse. Both Cre drivers demonstrate equivalent recombination in vascular SMCs. However, Myh11-CreERT2 mice, but not Itga8-CreERT2 mice, display high activity in visceral SMC-containing tissues such as intestine, show early tamoxifen-independent activity and produce high levels of CreERT2 protein. Whereas Myh11-CreERT2-mediated knockout of serum response factor (Srf) causes a lethal intestinal phenotype precluding analysis of the vasculature, loss of Srf with Itga8-CreERT2 (SrfItga8) yields viable mice with no evidence of intestinal pathology. Male and female SrfItga8 mice exhibit vascular contractile incompetence, and angiotensin II causes elevated blood pressure in wild-type, but not SrfItga8, male mice. These findings establish the Itga8-CreERT2 mouse as an alternative to existing SMC Cre mice for unfettered phenotyping of vascular SMCs after selective gene loss.
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Data availability
RNA-seq data are available through the Gene Expression Omnibus (GSE138824 and GSE199244). Long-read data (informative reads only) are available through the National Center of Biotechnology Informationʼs Sequence Read Archive database, which can be downloaded from www.ncbi.nlm.nih.gov/sra, under BioProject number PRJNA825511. Original source data are included in the Supplementary Materials. Updates to Supplementary Table 1 are available upon reasonable request.
Change history
09 June 2023
A Correction to this paper has been published: https://doi.org/10.1038/s44161-023-00301-2
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Acknowledgements
We thank members of the University of Rochester Medical Center Genomics Core for carrying out the RNA-seq experiments and generating Fig. 2e, and we thank the Electron Microscopy and Histology Core at Augusta University for performing the immunogold studies and generating Fig. 1b. This work was supported by American Heart Association postdoctoral grants 17POST3360938 to Q.L. and CDA858380 to J.L.F. and by National Institutes of Health grants R00HL146948 to J.F.L.; HL155265 to E.B.C.; HL122578 to M.J.D.; HL122686 and HL139794 to X.L.; HL138987, HL136224 and HL147476 to J.M.M.; EY026614 to L.G.; and R00-HL143198 to S.D.Z.
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G.D.W. helped develop the figures and performed western blotting studies in Extended Data Figs. 2e, 4d and 8a,b and Figs. 2b and 3g,h; bulk RNA-seq studies in Fig. 2e and Supplementary Table 2; and Fig. 3a,b. G.D.W. also assisted with the blood pressure studies of Figs. 4 and 5 and with performing the myography studies of Fig. 6. J.L.F. performed and/or analyzed vascular function and blood pressure data of Figs. 4, 5 and 6a–c and wrote the Methods subsections related to these experiments. J.D. performed the studies in Fig. 3c,d and Extended Data Fig. 10 and conducted tamoxifen injections for studies of blood pressure and myography measurements. A.R.G. provided foundational work and helped generate Supplementary Fig. 1. P.G. assisted with western blotting studies of Extended Data Fig. 2a,b. A.C.Y. generated the data for Supplementary Fig. 2. O.J.S. performed all histology, immunostaining, confocal immunofluorescence and bright-field microscopy and generated all of the images, save lymphatic vessels. M.J.D. and S.D.Z. planned and performed experiments in Fig. 3e,f and Extended Data Figs. 6 and 7; they also contributed to the writing and editing of the Methods and Results sections. C.K.C., A.C.Y. and S.H.G. did all breeding and genotyping of mice. C.K.C. provided images to Extended Data Fig. 8c and Supplementary Fig. 4a. C.T.B. helped perform the telemetry experiments of Figs. 4 and 5. T.C.K. isolated aortas and helped run myography experiments in Fig. 6. W.B.B. prepared and analyzed the Sanger sequencing of the Cre in Myh11-CreERT2 of Supplementary Fig. 3 and the long-read sequence mapping of Myh11-CreERT2 in Fig. 2c,d. A.A. helped with the interpretation of Myh11-CreERT2 mapping and the design of Fig. 2d. A.K. contributed to western blotting studies of Fig. 2b. X.L. contributed to data in Fig. 2a and provided intellectual input on study design and interpretation. L.G. and X.X. designed and generated the Itga8-CreERT2 mouse and developed Extended Data Fig. 1a. E.B.C. helped with the acquisition and interpretation of the blood pressure and vascular reactivity data. Q.R.L. performed studies in Extended Data Figs. 2a–d, 3 and 9b–g, Supplementary Fig. 4b,d and Fig. 7; he also provided intellectual input throughout the study and finalized the figures. J.M.M. conceived and supervised the entire project, analyzed all data, developed Extended Data Fig. 1b and Supplementary Fig. 5, assembled original western blots in Extended Data Figs. 4a–c and 5 and Supplementary Fig. 6 and outlined, wrote and edited the manuscript.
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A.A. is a co-founder of Tango Therapeutics, Azkarra Therapeutics, Ovibio Corporation and Kytarro; a member of the board of Cytomx and Cambridge Science Corporation; a member of the scientific advisory board of Genentech, GLAdiator, Circle, Bluestar, Earli, Ambagon, Phoenix Molecular Designs and Trial Library; a consultant for SPARC, ProLynx and GlaxoSmithKline; receives grant or research support from SPARC and AstraZeneca; and holds patents on the use of PARP inhibitors held jointly with AstraZeneca from which he has benefited financially (and may do so in the future). The patents are unrelated to any aspect of the current study. All other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Strategy and sequence validation of Itga8-CreERT2.
a, Partial Itga8 locus, overlapping long noncoding RNA, and general strategy for mouse ESC targeting. The 22 amino acid P2A self-cleaving cassette (GSGATNFSLLKQAGDVEENPGP) allows for concurrent detection of the CreERT2 fusion and the mCherry reporter; however, mCherry fluorescence was not easily detected and therefore not pursued in this study. Small horizontal arrows (p1-p4) denote primers for PCR genotyping of mice. b, Nucleotide sequence of targeted knock-in cassette following Flippase-mediated removal of the Neo gene. Colors correspond to the schematic in a. Bases highlighted in black boxes represent codon-optimized substitutions from original sequences. HR, homologous recombination.
Extended Data Fig. 2 ITGA8 protein expression in heterozygous Itga8-CreERT2 mice.
a, Western blots of ITGA8 protein in two independent experiments performed by two independent investigators with quantitative data at right. n = 6 independent male aortas for each condition. b, qRT-PCR analysis of Itga8 mRNA and c, the antisense Ak086420 long noncoding RNA in wild type versus Itga8-CreERT2 heterozygous aorta. n = 6 male aortas per genotype. d, qRT-PCR of cytosolic versus nuclear Actb and Ak086420 expression in wild type aorta (n = 4 male aortas). Error bars represent the mean ± standard deviation; p-values determined by two-tailed, unpaired Student’s t-test. e, Western blot of ITGA8 and CreERT2 in aorta of seven week-old male and female Itga8-CreERT2 heterozygous mice (n = 3 independent mice of each sex). Molecular weight markers at right here and below are in kilodaltons.
Extended Data Fig. 3 Recombination activity of different SMC Cre mice in myeloid cells.
a, Representative flow cytometry of GFP labeled cells from wild type C57BL/6J mice (i); mT/mG reporter mice (ii); and mT/mG in Sm22-Cre (iii); tamoxifen-treated male Myh11-CreERT2 (iv); tamoxifen-treated male Itga8-CreERT2 (v); tamoxifen-treated female Itga8-CreERT2 (vi) mice. Quantitative data for GFP + circulating cells in upper left quadrant Q1 (vii) and upper right quadrant Q2 (viii) are shown for each Cre driver line (n = 3 male mice per Cre line, save the Itga8CreERT2 line which represent male, n = 2, and female, n = 2, samples pooled for the graphs shown). The horizontal axis was set to the same threshold for all panels according to the apparent two populations of cells in the positive control (Sm22-Cre-mT/mG). The vertical axis divides two apparent populations of different sized cells in the Sm22-Cre-mT/mG strain. Note, as we did not gate for any surface markers in this study, we cannot label any quadrant as to cell type. Source of cells was from circulation cleared of red blood cells. Error bars represent the mean ± standard deviation. One-way ANOVA and posthoc testing revealed indicated p-values. See Supplementary Fig. 5 for more details. b, Bone marrow aspirates of 10-week-old male Itga8-CreERT2 (i, ii) and Myh11-CreERT2 (iii, iv) mice (n = 2 mice/genotype) carrying mT/mG reporter and treated with tamoxifen. Arrows indicate multi-nucleated megakaryocytes. Scale bars are 20 μm.
Extended Data Fig. 4 Comparative Cre activity in adult male and female blood vessels.
a, The GFP signal demonstrates recombination of the mT/mG reporter in medial SMCs of both the aorta and vena cava, whereas the red stained endothelium indicates the absence of recombination. Scale bar is 33 μm for aorta and 100 μm for vena cava. The aorta and vena cava of Itga8-CreERT2 was from a female mouse. b, Activity of each CreERT2 driver in indicated segments of male mouse aorta. Scale bars are 20 μm for all panels. Lu, lumen of vessel wall. c, Tamoxifen-treated 54-week-old female (i-iii) and male (iv-vi) mouse thoracic aorta (i, iv), brain (ii, v), and heart (iii, vi). Each panel is representative of at least two independent male or female mice. Scale bars are all 20 μm. d, Western blot of ITGA8 protein in 10-week-old versus 54-week-old female mouse aorta (n = 3 independent mice per time point).
Extended Data Fig. 5 Comparative Cre activity in adult mouse tissues.
a, GFP signal restricted to VSMCs in blood vessels (arrows) of each indicated tissue type. b, GFP signal in VSMCs and non-VSMCs of indicated tissue types. White arrowheads, blood vessels of kidney and thymus; white arrows, glomeruli of kidney; yellow arrows, sinusoids of liver. Itga8-CreERT2-mediated GFP signal was also present in myoepithelial cells of mammary gland and thecal cells around mature follicle (white asterisk) of ovary. All images were processed the same except for the Testis panel under Itga8-CreERT2, which was uniformly enhanced to bring out more of the signal that otherwise would be too dark to visualize. Scale bar in a is 100 μm for all images; scale bars in b are 50 μm for kidney and thymus and 20 μm for liver, mammary gland, and ovary. Data are representative of at least two independent male or female mice analyzed over the course of five years in two independent labs.
Extended Data Fig. 6 Leaky CreERT2 activity in aged mice.
Sections of aorta from 24-week-old or 54-week-old male Itga8-CreERT2 and 54-week-old Myh11-CreERT2 mice. The brightness in the 54-week Itga8-CreERT2 image was uniformly enhanced to better appreciate the GFP signal. Scale bars are 20 μm for all panels. Images are representative of two independent mice.
Extended Data Fig. 7 Quantitative activity of Myh11-CreERT2 versus Itga8-CreERT2 in popliteal blood vessels.
Representative 2D maximum projections from confocal imaging of live GFP and tdTomato fluorescence popliteal artery (a, b) and vein (c, d) from Myh11-CreERT2 versus Itga8-CreERT2 with quantitation of each in panels e and f, respectively. n = 4 male mice per genotype. Scale bars represent 50 μm (a, b) and 100 μm (c, d). Error bars in panels e-f represented by mean ± standard deviation; p-values determined by two-tailed, unpaired Student’s t-test.
Extended Data Fig. 8 Intestinal phenotype in Myh11-CreERT2 versus Itga8-CreERT2 mediated knockout of serum response factor (Srf).
a, Western blot showing lack of effect of tamoxifen on SRF in wild type aorta (n = 3 independent male mice per treatment). b, Quantitation of panel a represented by mean ± standard deviation; p-value determined by two-tailed, unpaired Student’s t-test. c, Anatomy of abdominal cavity in the indicated CreERT2 driver mice used for Srf inactivation. The two SrfMyh11 images were from mice 14 days following tamoxifen administration, whereas the SrfItga8 image was from a mouse 8 weeks after tamoxifen administration. d, Oil (panels i-iii) and Tamoxifen (panels iv-vi) treated SrfMyh11 mice with dissected gross intestine (i, iv), H&E stained intestine (ii, v), and immunostaining for SRF (green) and ACTA2 (red) in intestine (iii, vi). Scale bar is 100 μm for ii, iii, v, and vi or 1 mm for i and iv. Studies of panel d are representative of at least two independent male or female mice analyzed over the course of five years in two independent labs.
Extended Data Fig. 9 Itga8-CreERT2 mediated inactivation of Srf in adult mouse tissues.
a, Immunofluorescence confocal microscopy of sections of carotid artery (i, iv), bladder (ii, v), and intestine (iii, vi) from tamoxifen-treated male mice carrying homofloxed Srf alleles in the absence (i-iii) or presence (iv-vi) of Itga8-CreERT2 (abbreviated Cre). Sections were stained with antibodies to ACTA2 (red), SRF (green), and DAPI. Arrows and arrowheads point to blood vessels and visceral SMCs, respectively. Scale bar is 20 μm for all panels. Data from each panel are representative of at least two independent mice. b, SRF positive VSMCs were counted in sections of carotid arteries from Tam-administered homozygous floxed Srf mice without Cre (HoF + Tam, n = 6 male mice per condition) or homozygous floxed Srf mice with Cre (HoF + Cre+Tam, n = 5 male mice per condition). c, Same quantitative measures as in panel b only from intestine (n = 3 mice per condition). Western blots of SRF in aorta (d) and bladder (e) of indicated genotypes, all treated with the same schedule of tamoxifen. Corresponding quantitative data are shown for mouse aorta (f) and bladder (g) (n = 3 mice per genotype). HeF, heterozygous floxed Srf; HoF, homozygous floxed Srf. Error bars are mean ± standard deviation. Student unpaired, one-tailed t-test was applied in b and c, and one-way ANOVA with Tukey’s t-test for f and g to reveal indicated p-values in each graph.
Extended Data Fig. 10 SRF and contractile protein expression in mesenteric artery of SrfItga8 conditional knockout mice.
a, Confocal immunofluorescence microscopy of SRF and DAPI alone (i, iii) and merged with ACTA2 (ii, iv) in mesenteric arteries of Oil control (i, ii) versus tamoxifen-treated (iii, iv) homozygous SrfItga8 mice; scale bars are 20 μm. Results are representative of two independent mice for each condition. Western blots of SRF target, MYH11, in Oil versus Tam-induced SrfItga8 homozygous male (b) and female (c) mesenteric arteries. n = 3 independent animals for each condition. Error bars represented by mean ± standard deviation; p-values determined by two-tailed, unpaired Student’s t-test.
Supplementary information
Supplementary Information
Supplementary Figures 1-6, Supplementary Figure Legends
Supplementary Table
Supplementary Tables 1–6. Supplementary Table 1. Floxed alleles and associated SMC Cre drivers. Supplementary Table 2. Differentially expressed genes in aorta of wild-type versus Cre-containing mice. Supplementary Table 3. Mouse models. Supplementary Table 4. Oligonucleotide primers and single guide RNAs for mapping Myh11-Cre transgene. Supplementary Table 5. Antibodies. Supplementary Table 6. Cell lines.
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Warthi, G., Faulkner, J.L., Doja, J. et al. Generation and comparative analysis of an Itga8-CreERT2 mouse with preferential activity in vascular smooth muscle cells. Nat Cardiovasc Res 1, 1084–1100 (2022). https://doi.org/10.1038/s44161-022-00162-1
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DOI: https://doi.org/10.1038/s44161-022-00162-1
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