A new autosomal Myh11-CreER^T2 smooth muscle cell lineage tracing and gene knockout mouse model

Abstract

Background:

The Myh11 promoter is extensively used as a smooth muscle cell (SMC) Cre driver and is regarded as the most restrictive and specific promoter available to study SMCs. Unfortunately, in the existing Myh11-CreER^T2 mouse, the transgene was inserted on the Y chromosome precluding the study of female mice. Given the importance of including sex as a biological variable and that numerous SMC-based diseases have a sex-dependent bias, the field has been tremendously limited by the lack of a model to study both sexes. Herein, we describe a new autosomal Myh11-CreER^T2 mouse (referred to as Myh11-CreER^T2-RAD), which allows for SMC-specific lineage tracing and gene knockout studies in vivo using both male and female mice

Methods:

A Myh11-CreER^T2-RAD transgenic C57BL/6 mouse line was generated using bacterial artificial chromosome (BAC) clone RP23-151J22 modified to contain a Cre-ER^T2 after the Myh11 start codon. Myh11-CreER^T2-RAD mice were crossed with two different fluorescent reporter mice and tested for SMC-specific labeling by flow cytometric and immunofluorescence analyses.

Results:

Myh11-CreER^T2-RAD transgene insertion was determined to be on mouse chromosome 2. Myh11-CreER^T2-RAD fluorescent reporter mice showed Cre-dependent, tamoxifen-inducible labeling of SMCs equivalent to the widely used Myh11-CreER^T2 mice. Labeling was equivalent in both male and female Cre⁺ mice and was limited to vascular and visceral SMCs and pericytes in various tissues as assessed by immunofluorescence.

Conclusions:

We generated and validated the function of an autosomal Myh11-CreER^T2-RAD mouse that can be used to assess sex as a biological variable with respect to the normal and pathophysiological functions of SMCs.

Graphical Abstract

Introduction

The ability to drive conditional, tissue-specific expression of Cre recombinase has greatly enhanced scientific discovery across many fields. The CreER^T2-loxP system allows for inducible cell type-specific lineage tracing and testing of cell type-specific gene functions when coupled with floxed (flanked by loxP) fluorescent reporter genes and/or floxed gene knockouts, respectively. For these systems to be reliable, there are several critical requirements that must be met. The first is Cre-driver specificity: i.e., the Cre driver must only be expressed in the cell type of interest as aberrant labeling or gene knockout in unintended cell types would confound data interpretation and possibly lead to incorrect conclusions. The second is Tamoxifen dependence: i.e., the Cre driver used must be tested for both tamoxifen-dependent and -independent recombination efficiency with respect to each floxed target (including both fluorescent reporters and gene knockouts)^1,2. An example of the importance of Cre-driver specificity in SMCs was highlighted by Sui et al. who reported that knockout of IKKβ (the activator of the master regulator of inflammation NFκB) in SMCs driven by an SM22Cre-Ikkβ-flox system rendered low density lipoprotein receptor null mice resistant to diet-induced obesity and metabolic disorders³. However, through a series of innovative subsequent experiments, they provided evidence that this was due to unanticipated SM22Cre mediated knockout of IKKβ in adipose stromal vascular cells and accumulation of adipocyte precursor cells³. Numerous examples of the importance of testing for tamoxifen independent recombination have been reported with respect to both fluorescent reporters as well as floxed genes of interest ^4–7.

A comprehensive review of the different promoters used as Cre drivers to study SMC function was recently published, including a detailed discussion of their distinct advantages and limitations⁸. MYH11 is widely considered to be the most SMC-specific marker in differentiated SMCs^9,10. The Myh11-CreER^T2 transgenic mouse generated by Stefan Offermanns lab has been extensively used for SMC-specific lineage tracing and gene knockouts in hundreds of scientific reports^11–20. The Myh11-CreER^T2 mouse specifically labels vascular and visceral SMCs as well as pericytes ^11,21,22. The major limitation of this mouse is that the Myh11-CreER^T2 transgene is located on the Y chromosome, which only allows for the study of male mice. Moreover, the Myh11-CreER^T2 transgene on the Y chromosome has been reported to translocate to the X chromosome resulting in a Myh11-CreER^T2 positive female mouse at a reported observed rate of ~3%^23,24. Indeed, Liao et al. established an X-linked Myh11-CreER^T2 line from such a recombination event, however X-linked inactivation in female mice leads to mosaicism in labeling of SMC requiring the females to be used as homozygotes while the males can only be hemizygotes²³.

Given that numerous diseases that affect SMC rich tissues show sex-dependent differences or biases including atherosclerosis²⁵, Abdominal Aortic Aneurysm (AAA)²⁶, Inflammatory Bowel Disease (IBD)²⁷, Fibromuscular Dysplasia (FMD)²⁸ and Spontaneous Coronary Artery Dissection (SCAD)²⁸, the lack of a SMC-specific Cre driver that allows for equal study of both male and female mice has been a major limitation to the field. Herein, we describe the generation and validation of a new autosomal Myh11 Cre-driver mouse, Myh11-CreER^T2-RAD, that replicates floxed reporter gene recombination driven by the Offermanns Myh11-CreER^T2 (herein referred to as Myh11-CreER^T2-Off) but allows for the study of sex as a biological variable in smooth muscle lineage tracing and gene knockout studies.

Methods

The data generated and analyzed for this study are available from the corresponding author upon reasonable request.

Mice

All experiments adhered to animal protocols approved by the University of Virginia Animal Care and Use Committee (Animal Protocol 2400). Myh11-CreER^T2-RAD mice were made by Cyagen. Briefly, BAC clone RP23-151J22, which contains the mouse Myh11 promoter in the BAC cloning vector pBACe3.6, was modified to insert a Cre-ER^T2 cassette at the start codon of mouse Myh11 gene and remove loxP and loxP511 sites on BAC vector backbone. The modified BAC was then injected into the pronucleus of fertilized C57BL/6 eggs where it randomly inserted into the genome. Fertilized eggs were then implanted into surrogate mothers to obtain offspring carrying the transgene which served as founders for the new line. The Myh11-CreER^T2-RAD mouse has been deposited at The Jackson Laboratory and will be available as strain 037658. For the generation of SMC lineage tracing mice, Myh11-CreER^T2-RAD mice were crossed with either a floxed tdTomato reporter mouse [B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J, The Jackson Laboratory, strain 007914, also known as Ai14] or a floxed eYFP reporter mouse [B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J, The Jackson Laboratory Strain 006148] to generate Myh11-CreER^T2-RAD/ROSA26-STOP^flox-tdTomato or Myh11-CreER^T2-RAD/ROSA26-STOP^flox-eYFP (herein referred to as Myh11-CreER^T2-RAD/tdTom or Myh11-CreER^T2-RAD/eYFP respectively)^29,30. The Myh11-CreER^T2-Off/ROSA26-STOP^flox-eYFP and Myh11-CreER^T2-Off/ROSA26-STOP^flox-tdTom using the Offermanns Myh11-CreER^T2 (The Jackson Laboratory Strain 019079) have been previously described^21,31. All experimental mice using the Myh11-CreER^T2-RAD lines were Cre^+/− to avoid any unanticipated effects from transgene insertion. Male Cre⁺ and female Cre⁻ experimental mice from the Myh11-CreER^T2-Off lines were used due to the transgene location on the Y chromosome.

Expanded Methods can be found in the Supplemental Material.

Results

The Myh11-CreER^T2-RAD transgene integrated on Mouse Chromosome 2.

Both male and female Cre^+/− founders for the Myh11-CreER^T2-RAD were generated, proved fertile and bred well. The line was maintained as heterozygotes by crossing with C57BL/6J mice. Founders produced both male and female Cre^+/− and Cre^−/− mice. To determine the chromosomal insertion site of the Myh11-CreER^T2-RAD transgene, Targeted Locus Amplification (TLA) analysis and Next Generation Sequencing (NGS) was performed³². Five independent primer sets were employed due to the large size of the BAC used to generate these transgenic mice (Table S1). Data using primer set 1 showed that the Myh11-CreER^T2-RAD transgene integrated on chromosome 2 (Figure S1A). Similar results were obtained using primer set 2–5 (data not shown). Notably, TLA analysis also picked up the endogenous Myh11 promoter location on Chromosome 16 (Figure S1A). Further analysis of the breakpoint sequences showed that the transgene was inserted into Chr2:178,826,578-178,826,802 based on the GRCm39 Mus Musculus genome assembly (Figure S1B). The closest annotated genes to the insertion site are 7×10³ base pairs upstream (pseudo-gene Gm14292) and 56×10³ base pairs downstream (pseudo-gene Gm14293). Given the position, it is unlikely to result in gene disruption based on available data to date. Moreover, no sequence variants were detected in the CreER^T2 region, and no large structural variants were detected in the integrated BAC. These results are summarized in Figure S1C and confirm that the Myh11-CreER^T2-RAD transgene is integrated on an autosome which will allow for Cre expression in both male and female mice.

Myh11-CreER^T2-RAD efficiently induces floxed reporter gene recombination and activation in SMCs in the brachiocephalic artery (BCA) and aorta.

To validate and compare the activity of Myh11-CreER^T2-RAD with Myh11-CreER^T2-Off, mice were crossed to either tdTomato or eYFP reporter mice as shown in the schematic in Figure 1A and compared to Myh11-CreER^T2-Off mice crossed to the same reporter mice^21,31. Following tamoxifen administration, reporter gene recombination and subsequent fluorescent protein expression in the BCA was assessed by confocal microscopy or in aortic cells by flow cytometry as shown in the schematic in Figure 1B. Tamoxifen diet was used in lieu of intraperitoneal injected tamoxifen based on our previous finding that intraperitoneal injection of tamoxifen in peanut oil caused autofluorescence of adipose tissue macrophages causing false identification as eYFP⁺ cells¹². Oral tamoxifen administration induced expression of tdTomato or eYFP in medial SMCs of the BCA in all Cre⁺ mice as determined by coincident ACTA2 expression (Figure 1C and Figure S2A, lower panels). No tdTomato or eYFP was detected in Cre⁻ mice (Figure 1C and Figure S2A, upper panels). Cell counting quantification demonstrated that 97% ±1.06% SEM (standard error of the mean) and 95% ±1.37% SEM of ACTA2⁺ cells inside the external elastic lamina (EEL) were tdTomato⁺ in Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD BCAs respectively (Figure 1D). Similar results were found for mice coupled to the eYFP reporter (95% ±1.10% SEM and 94% ±1.46% SEM of ACTA2⁺ cells were eYFP⁺ in Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD BCAs respectively) (Figure S2B). This experiment was powered to detect differences in reporter expression of 10% or greater. We show here that the difference between reporter expression in Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD mice is less than 10%. There was no significant difference with respect to the percentage of tdTomato⁺/ACTA2⁺ or eYFP⁺/ACTA2⁺ cells between the two Myh11 Cre drivers (Figure 1D and Figure S2B). Importantly, there was no detectable labeling of endothelial cells lining the lumen or adventitial cells located outside the EEL (Figure 1E and Figure S2C). Flow cytometric analysis of cells isolated from whole intact aortae (containing SMCs, ECs and adventitial cells) showed that 61% ±2.60% SEM and 53% ±3.29% SEM of cells isolated were tdTomato⁺ (or 60% ±1.80% SEM and 58% ±1.75% SEM were eYFP⁺) in the Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD respectively and <1.5% tdTomato⁺ or eYFP⁺ cells were detected in Cre⁻ mice from either line (Figure 1F and Figure S2D). There were no significant differences in the %tdTomato⁺ aortic cells (Figure 1F) or %eYFP⁺ aortic cells (Figure S2D) between the two Myh11 Cre drivers. When analyzed by sex, there were no significant differences in %tdTomato⁺/ACTA2⁺ cells in male and female Myh11-CreER^T2-RAD/tdTom mice (96% ±1.63% SEM and 94% ±2.04% SEM respectively) (Figure S3A–B). There were also no significant differences in the %tdTomato⁺ aortic cells in male and female Myh11-CreER^T2-RAD/tdTom mice (53% ±4.68% SEM and 53% ±5.05% SEM respectively) (Figure S3C). Similar results were found in Myh11-CreER^T2-RAD/eYFP mice with no significant differences in %eYFP⁺/ACTA2⁺ cells in male and female mice (93% ±1.78% SEM and 95% ±2.66% SEM respectively) (Figure S4A–B) nor significant differences in %eYFP⁺ aortic cells (60% ±1.64% SEM and 54% ±2.79% SEM respectively) (Figure S4C). Taken together, these results demonstrate that the Myh11-CreER^T2-RAD transgene drives reporter recombination in SMCs in the BCA and aorta of both male and female mice in a manner comparable to male Myh11-CreER^T2-Off mice.

Figure 1: Myh11-CreER^T2-RAD efficiently induces floxed tdTomato reporter gene recombination and subsequent lineage tracing of SMC in the BCA and Aorta analogous to Myh11-CreER^T2.

A.) Schematic showing the experimental design of tamoxifen-induced recombination of floxed fluorescent reporter genes in Myh11-CreER^T2-RAD mice. B.) Schematic showing the experimental design to determine fluorescent reporter gene expression in the BCA and intact aorta driven by Myh11-CreER^T2-RAD using immunofluorescence imaging and flow cytometry respectively. C.) Representative images showing tamoxifen-induced tdTomato expression in SMCs of the BCA in Cre⁺ Myh11-CreER^T2-Off (left) and Myh11-CreER^T2-RAD (right) mice. D.) Quantification of % tdTomato⁺/ACTA2⁺ cells in the BCAs of Cre⁺ Myh11-CreER^T2-Off (n=5) and Myh11-CreER^T2-RAD (n=7) from immunofluorescence images. E.) Zoomed in Myh11-CreER^T2-RAD BCA image showing tdTomato expression (red) in the medial SMCs between the EEL and IEL (dashed lines) but not in the EC layer (arrow) or adventitia (arrowhead), L=lumen. F.) Quantification of flow cytometry results of aortic cells from Myh11-CreER^T2-Off (n=5 Cre⁻ females, n=5 Cre⁺ males) and Myh11-CreER^T2-RAD (n=8 Cre⁻, n=8 Cre⁺, both sexes) mice fed tamoxifen. Scale Bar=100μm. Schematics created with BioRender.com

Recombination of the reporter gene and fluorescent protein expression in Myh11-CreER^T2-RAD mice is SMC-specific.

To determine if the expression of Cre driven by the Myh11-CreER^T2-RAD transgene was SMC-specific, we examined various tissues for tamoxifen induced tdTomato expression by immunofluorescent confocal microscopy (Figure S5). tdTomato expression was seen in the coronary vessels within the heart, but not in the surrounding cardiac tissues (Figure S5A). tdTomato expression was also detected in the pulmonary arteries of the lung as well as in lung pericytes (which has been previously reported for Myh11-CreER^T2-Off)³³ (Figure S5B) as well as in the small vessels and pericytes of the retina and the spinotrapezius muscle (Figure S5C and S5D respectively). tdTomato expression was detected in mesenteric artery and vein (Figure S5E). Vessels in the thymus were also labeled by tdTomato in addition to sub-population of cells that are ACTA2⁻ (which has been previously reported for Myh11-CreER^T2-Off)²⁴ (Figure S5F). Lastly, Myh11-CreER^T2-RAD drove expression of tdTomato in visceral SMC within the small intestines, colon, bladder, and uterus (in female mice) as has been previously reported for Myh11-CreER^T2-Off (except for uterus which has not been examined in Myh11-CreER^T2-Off due to the inability to lineage trace female mice) (Figure S5G–J respectively)^11,24. Taken together, this data shows that the Myh11-CreER^T2-RAD transgene drives Cre-mediated recombination in vascular and visceral SMCs and pericytes in a manner similar to Myh11-CreER^T2-Off.

Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD lineage tracing mice exhibit detectable tamoxifen-independent but SMC-specific recombination of reporter genes which appears to be locus-dependent.

It has been previously reported that many CreER^T2 mouse lines display detectable tamoxifen-independent nuclear translocation of Cre and recombination of some (but not all) floxed gene loci⁶. To determine whether Myh11-CreER^T2-Off or Myh11-CreER^T2-RAD mice display tamoxifen-independent Cre-mediated recombination, BCAs and aortae from Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD mice that had not received tamoxifen were examined for expression of the tdTomato or eYFP reporters. Results from IF staining of BCA sections showed sporadic expression of tdTomato in SMCs in Cre⁺ Myh11-CreER^T2-Off/tdTom and Myh11-CreER^T2-RAD/tdTom mice in the absence of tamoxifen (Figure 2A, bottom panels). Interestingly, there was no detectable tamoxifen-independent eYFP expression in BCA SMCs from either Cre⁺ Myh11-CreER^T2-Off/eYFP or Myh11-CreER^T2-RAD/eYFP mice (Figure 2B, bottom panels) and no reporter expression was detected in Cre⁻ mice (Figure 2A and 2B, top panels). Moreover, we analyzed the percent of labeled aortic cells by flow cytometry in both Cre⁺ and Cre⁻ Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD mice crossed to either tdTomato or eYFP reporter mice. Cre⁺ mice from the Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD mice showed 21% ±0.81% SEM and 8% ±0.60% SEM tamoxifen-independent recombination of the tdTomato reporter respectively (Figure 2C) but ≤0.5% tamoxifen-independent recombination of the eYFP reporter (Figure 2D). Moreover, Cre⁻ mice from both lines displayed ≤0.1% tamoxifen-independent recombination of either reporter in aortic cells (Figure 2C and 2D). The tdTomato reporter is known to have a low recombination threshold and is therefore particularly susceptible to basal CreER^T2 recombination compared to other fluorescent reporters including eYFP, which has been shown to have a much higher recombination threshold^6,29. To determine whether disparity in the tamoxifen-independent Cre-mediated expression of tdTomato or eYFP in Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD mice was a result of differences in reporter gene recombination efficiency, we performed PCR analysis of genomic DNA from mice fed tamoxifen diet (TMX+) versus standard rodent diet (TMX−). The primers used produce a 291bp fragment for the recombined tdTomato locus and a 775bp fragment for the recombined eYFP locus as shown in the schematic in Figure 2E and 2F respectively and have been previously used to determine the recombination efficiency for these reporter genes⁶. No recombination was seen in the absence of Cre and tamoxifen for either reporter gene in either mouse line even after 41 cycles (Figure 2G and 2H). Abundant recombination product was obtained after 35 cycles for both tdTomato and eYFP in Cre⁺ Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD mice fed tamoxifen (Figure 2G and 2H) even with as little as five days of tamoxifen feeding (Figure S6). Tamoxifen-independent recombination of the tdTomato locus was detected after 35 and 38 cycles respectively in Cre⁺ Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD mice fed standard diet whereas no tamoxifen-independent recombination of the eYFP locus was seen in Cre⁺ mice of either line even up to 41 cycles. This demonstrates that tamoxifen-independent recombination of different floxed alleles occurs in a Cre driven manner and depends on the recombination threshold of the floxed gene of interest in both Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD mice.

Figure 2: Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD can induce tamoxifen independent recombination of floxed fluorescent reporter genes which is locus-dependent.

Mice were treated as shown in Figure 1B except mice were fed standard rodent diet (TMX−) at 6–8 weeks in lieu of tamoxifen diet (TMX+). A.) Representative immunofluorescent images of BCAs from Myh11-CreER^T2-Off/tdTom (left) and Myh11-CreER^T2-RAD/tdTom (right) showing tamoxifen-independent tdTomato expression in Cre⁺ (but not Cre⁻) mice. B.) Representative immunofluorescent images of BCAs from Myh11-CreER^T2-Off/eYFP (left) and Myh11-CreER^T2-RAD/eYFP (right) showing no detectable tamoxifen-independent eYFP expression in either Cre⁺ or Cre⁻ mice. C.) Quantification of flow cytometry results of cells dissociated from intact aortas from standard rodent diet fed (TMX−) Myh11-CreER^T2-Off/tdTom (n=5 Cre⁻ females, n=5 Cre⁺ males) and Myh11-CreER^T2-RAD/tdTom mice (n=8 Cre⁻, n=8 Cre⁺ both sexes). D.) Quantification of flow cytometry results of cells dissociated from intact aortas from standard rodent diet fed (−TMX) Myh11-CreER^T2-Off/eYFP (n=3 Cre⁻ females, n=5 Cre⁺ males) and Myh11-CreER^T2-RAD/eYFP mice (n=7 Cre⁻, n=6 Cre⁺ both sexes). E.) and F.) Schematics depicting the relative PCR recombination products generated after LoxP excision (black arrowheads) for tdTomato and eYFP reporter genes respectively. G.) PCR recombination analysis of the tdTomato reporter in Myh11-CreER^T2-Off/tdTom (top) and Myh11-CreER^T2-RAD/tdTom mice (bottom). H.) PCR recombination analysis of the eYFP reporter in Myh11-CreER^T2-Off/eYFP (top) and Myh11-CreER^T2-RAD/eYFP mice (bottom). Schematics created with BioRender.com.

Discussion

The Myh11-CreER^T2-Off mouse has been a powerhouse tool for the field of SMC biology having been used to knock out over 100 genes of interest²⁴. In combination with lineage tracing, our lab and others have used the Myh11-CreER^T2-Off mouse to reveal novel roles for SMC in diseases such as atherosclerosis and cancer that were previously unknown or underappreciated due to the loss of canonical marker gene expression^15,33,34. However, despite the advances made in SMC biology using the Myh11-CreER^T2-Off mouse, the inability to study mice of both sexes due to its insertion on the Y chromosome has been a major limitation. Given the known sex-dependent differences associated with cardiovascular and other SMC-based diseases and the requirement for inclusion of sex as a biological variable in preclinical research sponsored by the NIH and other funding agencies^35–37, we sought to create and validate a new SMC-specific Cre expressing mouse that works similarly to Myh11-CreER^T2-Off but allows for the inclusion of all mice regardless of sex.

In a side-by-side comparison, the Myh11-CreER^T2-RAD drives robust, SMC-specific expression of floxed fluorescent reporter genes similar to the original Myh11-CreER^T2-Off with the added benefit of being able to study mice of both sexes. The Myh11-CreER^T2-RAD transgene integrated into an autosome where no known annotated genes have been documented to date (Chr2:178,826,578-178,826,802). In comparison, the Myh11-CreER^T2-Off transgene is located on the Y chromosome and may have originally integrated on the X chromosome and translocated to the Y through homologous recombination based on the presence of two X-linked genes surrounding the transgene²⁴. An additional alternative Myh11-driven SMC lineage tracing mouse was also recently reported by Ruan et al, which was made by inserting a LoxP-nlacZ-stop-LoxP-H2B-GFP cassette into the endogenous Myh11 locus³⁸. This mouse is unique in that it labels all Myh11 expressing SMCs with LacZ. Upon crossing to a Cre-driver of interest, this mouse can be used to fate-map subpopulations of SMCs during development or disease states. However, it does not allow for the SMC-specific gene knockout studies that are capable in the Myh11-CreER^T2-Off or Myh11-CreER^T2-RAD mice (unless one of these mice is used as the Cre driver).

Like Myh11-CreER^T2-Off, Myh11-CreER^T2-RAD drives SMC-specific reporter expression in all vascular and visceral SMCs examined, thereby broadening its utility for fate mapping studies. By extension, gene deletions using the Myh11-CreER^T2-RAD mice will have the gene of interest knocked out in both SMC subpopulations. This could result in confounding variables if the effects of the gene knockout in the unintended SMC subtype impacts the disease state or phenotype of the SMC subtype of interest. Moreover, a lethal phenotype in one SMC subtype would preclude the study of that gene of interest in the other SMC subtype as occurred when SM22-CreER^T2 or Myh11-CreER^T2-Off driven knockout of the transcription factor SRF resulted in lethal visceral myopathies that precluded analysis of the loss of SRF in vascular SMCs^24,39,40. The Miano lab has recently generated a unique and valuable new inducible Cre driver using the alpha 8 integrin (Itga8) promoter that has preferential activity in vascular SMCs compared to visceral SMCs. This mouse will be useful in circumventing confounding visceral myopathies and allowing studies in vascular SMC that would otherwise be impossible²⁴. However, there are also important limitations of the Itga8-CreER^T2 mouse. First, Itga8 expression has been detected in non-SMC including neuronal axons, hepatic stellate cells, alveolar interstitial cells, and in glomerular cells of the kidney^41–44. The latter is particularly concerning for atherosclerosis studies given the clear association between chronic renal disease and exacerbation of atherosclerosis severity^45,46. In contrast, Myh11 expression appears to show near complete SMC specificity with the notable exception of expression in a subpopulation of cells in the thymus as detected in Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD mice²⁴. We speculate that these may be vascular mural cells (SMC and pericytes) or potentially myofibroblast-like cells⁴⁷. Second, there appears to be variable recombination of a reporter gene in visceral SMCs²⁴. This variable expression in visceral SMCs could obscure phenotypic changes and create more rather than less ambiguity. Conversely, highly consistent knockout in all SMC such as with Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD mice allows for straightforward interpretation of experimental results. Third, there is insufficient documentation of SMC specificity of Itga8 gene expression throughout the lifespan of the mouse. While complete temporal expression has not specifically been addressed using the Myh11-CreER^T2-Off or Myh11-CreER^T2-RAD mice, the SMC specificity of the Myh11 promoter-enhancer and MYH11 protein has been reproduced in hundreds of papers since their discovery over 30 years ago by our lab^48,49 and that of Ryozo Nagai^50,51. This includes our studies of Myh11-Cre-LacZ mice which provide an integral of all cells that express Myh11 at any time from fertilization of the egg until death of the mouse and thus extraordinary sensitivity in detecting possible expression of Myh11 in non-SMC^10,52. Notably, these results showed complete SMC-pericyte specificity. However, similar studies have not yet been done with an Itga8-Cre-LacZ mice. Lastly, the Itga8-CreER^T2 mouse was generated by targeted knock-in of the CreER^T2 cassette into the first exon of the endogenous Itga8 locus. While this approach has the benefit of ensuring the fidelity of Cre expression from the endogenous Itga8 promoter, it results in knockout of the Itga8 allele. When bred to homozygosity, this results in a lethal phenotype due to kidney agenesis²⁴. While they present evidence suggesting that the remaining Itga8 allele in heterozygous mice can compensate for the loss of one allele in young healthy mice, this may not be the case in the setting of stress including vascular injury, hypoxia-induced angiogenesis, or in disease states including atherosclerosis²⁴. Taken together, the Itga8-CreER^T2 mouse from the Miano lab is a valuable new model in the for studies in vascular SMCs, particularly in gene knockout studies where visceral lethality would preclude analysis of vascular SMC effects. However, it comes with its own set of limitations that require further analysis. We recommend that gene knockout studies using Myh11-CreER^T2-RAD mice should examine visceral SMC tissues to look for any confounding effects that may alter the phenotype seen in vascular SMC and vice versa.

All Cre-drivers are subject to the limitations of the Cre-loxP system including tamoxifen-independent (but Cre-dependent) recombination of floxed genes. Promoters that drive high expression of Cre are likely to cause more tamoxifen-independent recombination compared to weaker Cre drivers. Both Myh11-CreER^T2-Off and Myh11-CreER^T2-RAD transgenes drive high levels of Cre expression in SMC and indeed show detectable recombination of the floxed tdTomato reporter gene in the absence of tamoxifen. However, this recombination remains SMC-specific and only occurs in presence of the Myh11-CreER^T2-Off or Myh11-CreER^T2-RAD transgene indicating the “leak” is likely due to tamoxifen-independent nuclear translocation of Cre. Our studies highlight that each floxed allele will vary with respect to its recombination threshold in the absence of tamoxifen as was seen when comparing Myh11-CreER^T2-Off or Myh11-CreER^T2-RAD mediated recombination of the tdTomato reporter (low recombination threshold) versus the eYFP reporter (high recombination threshold)⁶. It is therefore imperative that the rate of tamoxifen-independent recombination of each floxed locus be quantified regardless of the Cre-driver used along with consideration of the best experimental controls given these limitations.

In summary, we have generated and validated a new autosomal SMC-specific Cre-driver that performs similarly to the Offermanns Myh11-CreER^T2 but has the added benefit of being able to study both male and female mice. The Myh11-CreER^T2-RAD line is a valuable new tool that will help uncover sex-dependent differences in SMC function in both healthy and disease states.

Highlights.

• Myh11-CreER^T2-RAD is a new autosomal, SMC-specific Cre-driver mouse that allows for the inclusion of both male and female mice in lineage tracing and gene knockout studies.

• When crossed to floxed fluorescent reporter mice, Myh11-CreER^T2-RAD mice display near complete labeling (95%) of SMC in the BCA in both male and female mice with no detectable labeling of endothelial or adventitial cells.

• Myh11-CreER^T2-RAD expresses Cre in vascular and visceral SMCs in both large and small vessels similar to the widely used Offermanns Myh11-CreER^T2.

• Both Offermanns Myh11-CreER^T2 and Myh11-CreER^T2-RAD can induce tamoxifen-independent recombination of some floxed genes. This appears to depend on the recombination threshold of the floxed gene of interest and will need to be rigorously assessed with each floxed gene examined.

Non-standard Abbreviations and Acronyms

SMCs

smooth muscle cells

EC

endothelial cells

BAC

bacterial artificial chromosome

TMX

tamoxifen

PFA

paraformaldehyde

FMO

fluorescence minus one

BCA

brachiocephalic artery

TLA

targeted locus amplification

NGS

next generation sequencing

EEL

external elastic lamina

IEL

internal elastic lamina

tdTom

tdTomato

eYFP

enhanced yellow fluorescent protein

floxed

flanked by loxP

SEM

standard error of the mean