' 2005 Wiley-Liss, Inc.
genesis 41:116–121 (2005)
TECHNOLOGY REPORT
MyoD-cre Transgenic Mice: A Model for Conditional Mutagenesis and Lineage Tracing of Skeletal Muscle Jennifer C. J. Chen, Justin Mortimer, Jason Marley, and David J. Goldhamer* Center for Regenerative Biology, Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut Received 26 July 2004; Accepted 21 December 2004
Summary: The Cre-loxP recombination system has been used to great advantage in vivo for conditional gene targeting, lineage tracing, and other applications. To express cre in skeletal myoblasts and muscle fibers, we utilized the well-characterized transcriptional regulatory regions of the muscle determination gene, MyoD. Transgenic mouse lines were produced (F3/-2.5cre) in which the cre gene is driven by the MyoD promoter and core enhancer, which directs the early activation of MyoD. Specificity of cre expression and efficiency of recombination was determined by monitoring reporter gene expression after crossing to the Cre-dependent reporter lines, R26R and Z/AP. Efficient labeling of embryonic and fetal myoblasts and muscle fibers was observed, with timing that was similar (branchial arches and limb buds) or slightly delayed (myotomes) relative to the endogenous MyoD gene. In satellite cell cultures, a strict concordance between MyoD protein and reporter gene expression was observed, demonstrating the muscle specificity and efficiency of Cre-mediated recombination. Nascent muscle fibers were labeled following injury of adult muscle, indicating recombination in satellite cells or their daughters c 2005 Wiley-Liss, Inc. in vivo. genesis 41:116–121, 2005. Key words: MyoD; skeletal muscle; myogenesis; myoblast; muscle precursor; lineage tracing; conditional knockout; mouse; Cre recombination; regeneration; transgenic; satellite cell
Transgenic mouse lines that express the cre recombinase gene (cre; Sauer and Henderson, 1988) in skeletal muscle precursor cells and muscle fibers have been generated for the purpose of lineage tracing and conditional genetic modification of skeletal muscle. Most existing muscle-specific cre transgenic lines utilize regulatory elements that are specific for differentiated skeletal muscle fibers (Bothe et al., 2000), or for both cardiac and skeletal muscle fibers (e.g., Bruning et al., 1998; Chen et al., 1998; Miniou et al., 1999). In this report, regulatory regions that control the expression of the muscle determination gene, MyoD (reviewed by Chen and Goldhamer, 1999), were used to direct cre expression in skeletal muscle precursor cells as well as muscle fibers. MyoD is expressed in skeletal muscle progenitors beginning at mouse embryonic day 9.5 (E9.5), and high levels are maintained throughout
development (Sassoon et al., 1989; Faerman and Shani, 1993; Chen et al., 2001). In adults, MyoD is expressed at low levels in mature muscle fibers, but is upregulated in satellite cells after muscle injury (reviewed by Hawke and Garry, 2001). The MyoD core enhancer directs reporter gene expression in myogenic cells of the somites, limb buds, and branchial arches in a spatiotemporal pattern that is similar to that of the endogenous MyoD gene (Goldhamer et al., 1992, 1995; Faerman et al., 1995; Chen et al., 2002; Chen and Goldhamer, 2004). A 4-kb genomic fragment that contains the core enhancer (F3; Goldhamer et al., 1992) was used together with 2.5 kb of MyoD promoter-proximal sequences to direct cre expression. Four F3/-2.5cre transgenic lines were characterized in detail. The following observations reflect analysis of line M23, unless otherwise noted. b-Galactosidase (b-gal) staining of whole-mount embryos generated by crossing F3/-2.5cre and R26R Credependent reporter mice (Soriano, 1999) is shown in Figure 1. In limb buds, X-gal staining was observed in myoblasts of the dorsal and ventral premuscle masses coincident with MyoD activation between E10 and E10.5 (Fig. 1b,f). At E11.5, muscle anlagen in forelimb and hindlimb buds stained in a pattern indistinguishable from MyoD mRNA (Fig. 1c,g). Essentially every limb muscle fiber was X-gal-stained at E12.5 (Figs. 1d, 2d), E13.5 (Fig. 2f), and E17.5 (Fig. 2i–m). Facial muscles develop from muscle precursors in the occipital somites and unsegmented paraxial mesoderm that migrate into the branchial arches (Noden et al., 1999). We first detected b-gal staining in the branchial arches at E9.5, similar to the timing and pattern of endogenous MyoD expression (Fig. 1a,e; data not
* Correspondence to: David J. Goldhamer, Center for Regenerative Biology, Department of Molecular and Cell Biology, University of Connecticut, 1392 Storrs Road, Unit 4243, Storrs, CT 06269-4243. E-mail:
[email protected] Contract grant sponsors: NIH, the Muscular Dystrophy Association (to D.J.G.) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/gene.20104
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FIG. 1. Comparison of X-gal staining of F3/-2.5cre;R26R embryos (a–d) and embryos processed for MyoD in situ hybridization (e–h). a: At E9.75, F3/-2.5cre;R26R transgenic embryos exhibited b-gal activity in the mandibular arch (black arrow), consistent with the presence of MyoD mRNA at this stage (e). At E10.5 (b) and E11.5 (c), X-gal staining was detected throughout the mandibular arch (black arrows), extending beyond the endogenous MyoD pattern (f,g). By E12.5 (d), the ectopic staining was restricted to a band of cells extending from the developing ear to the mandible (black arrows). Forelimb buds showed X-gal staining at E10.5, the stage at which MyoD mRNA is first detected (b,f, red arrowheads). X-gal and MyoD staining in the premuscle masses of forelimb and hindlimb buds at E11.5 (c,g) and E12.5 (d,h) were indistinguishable. In somites, the appearance of b-gal activity was slightly delayed compared to MyoD mRNA (a,b,e; black arrowheads). X-gal staining was observed in the ventrolateral (hypaxial) myotome at E10.5 (b; black arrowhead), whereas MyoD was expressed in both ventrolateral and dorsomedial (epaxial) myotomal domains of interlimb somites (f; black arrowheads). By E11.5, X-gal staining in the myotomes closely resembled the endogenous MyoD pattern (c,g), although staining intensity was weak compared to other muscle populations. By E12.5, the staining intensity of the myotomally-derived trunk muscles approached that of other muscle populations (d). Ectopic X-gal staining of the liver was observed in this transgenic line (b; red arrow).
shown). At E10.5 and E11.5, however, the entire mesenchymal core of the mandibular arch stained strongly (Figs. 1b,c, 2a,b), contrasting with the restricted expression of MyoD in muscle precursors (Fig. 1f,g). At E12.5, a band of stained mesenchyme extended from the lower jaw to a position ventral to the developing ear (Fig. 1d). The generalized staining of the mandibular arch and the mesenchymal band were observed in most F3/-2.5cre lines, but has not been observed with any MyoD-lacZ transgenic constructs, including F3/-2.5lacZ (Goldhamer et al., 1992, 1995; Faerman et al., 1995; Chen et al., 2002). Transient activity of F3 or promoterproximal flanking sequences in branchial arch mesenchyme is a possible explanation for this result. b-Gal staining of F3/-2.5cre;R26R embryos in the somitic myotomes was first detected at E10.5 (Fig. 1a),
representing a delay of 0.5 to 1 day relative to the endogenous MyoD gene (Fig. 1e; Chen et al., 2001, 2002; Chen and Goldhamer, 2004). This delay likely reflects the activity of the regulatory elements rather than inefficient Cremediated recombination, as a similar delay was observed with a F3/-2.5lacZ transgene (Faerman et al., 1995). At E10.5, staining was clearly observed in the lateralmost hypaxial myotome of interlimb somites, whereas only weak staining was detected in the epaxial myotome of somites anterior to the forelimb buds (Fig. 1b, data not shown). At E11.5, embryos exhibited reporter expression throughout most of the myotome (Fig. 1c); the relatively weak myotomal staining at this stage probably is the consequence of delayed transgene expression. At E12.5, b-gal activity was detected in all trunk musculature, including the abdominal muscles, diaphragm, and
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FIG. 2. Histological analysis of Xgal-stained F3/-2.5cre;R26R embryos. a: E10.5 embryo showing staining throughout the mesenchymal core of the mandibular arch. b: Higher magnification of the boxed region of (a). c: Ectopic liver staining in an E10.5 embryo, likely due to position effects. Sections of E12.5 (d) and E13.5 (e–g) embryos exhibited staining in skeletal muscle beds of limb buds, body wall muscles, and the diaphragm, whereas nonskeletal muscle cells were unstained. h,i,k: Sections of E17.5 F3/-2.5cre;R26R hindlimbs showing muscle-specific X-gal staining. j: Boxed region of (i) showing rare staining of two osteocytes in developing bone (arrows). l: Boxed region of (k) showing a few rare X-gal-stained cells in the fibrous layer of the perichondrium (arrowhead). m: Section through ossifying cartilage at E17.5, showing that all skeletal elements, as well as the bone marrow, were unstained. In a–g, embryos were stained as wholemounts and either cryostat (a–c) or paraffin (d–g) sectioned. In h–m, limbs were frozen, cryostat sectioned, and stained with X-gal. Cu, cutaneous muscle; Di, diaphragm; Es, esophagus; H, heart; In, intercostal muscle LA, left atrium; LFL, left forelimb; Li, liver; Lu, lung; RA, right atrium; Ri, rib primordium; Sk. M, skeletal muscle; V, blood vessel.
intercostal muscles (Figs. 1d,h, 2d,e,g) and staining intensity approached that of the limb musculature. Ectopic transgene expression was detected at several sites in the embryo and adult. Staining of the developing liver (Figs. 1b–d, 2c) is specific to line M23 and is likely due to transgene position effects. Sporadic staining in single cells or small groups of cells was also observed. On rare occasions, for example, single X-gal-stained chondrocytes and osteocytes were observed in cryostat sections of E17.5 limbs (Fig. 2i,j). Individual labeled cells or small clusters of labeled cells were infrequently observed in the fibrous layer of the perichondrium and periosteum (Fig. 2k,l). In addition, some cells directly subjacent to the mesothelium of the gastrointestinal tract (i.e., in the position of the muscularis externae of the mature intestine) exhibited X-gal staining (data not
shown). In the adult, small clusters of labeled cells in the adult heart, lung, and some blood vessels were detected on occasion (data not shown). The sporadic occurrence of these labeled clusters is most consistent with clonal expansion of individual cells in which the enhancer/promoter is transiently active. While the biological significance of this apparently stochastic activity is unknown, these findings are consistent with the detection of rare, MyoD-positive cells in a number of nonskeletal muscle tissues (Gerhart et al., 2001). Examination of leg musculature, including the tibialis anterior (TA) and gastrocnemius muscle, revealed labeling of all muscle fibers (Fig. 3a), consistent with the high efficiency of myoblast and muscle fiber labeling in the embryo. Upon injury, quiescent muscle stem cells (satellite cells) are stimulated to reenter the cell cycle
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FIG. 3. AP staining of cryostat sections of normal and injured TA muscles derived from F3/-2.5cre;Z/AP mice. a: Section through uninjured muscle showing AP staining of all muscle fibers. b: Section through regenerating muscle 11 days after cardiotoxin injection. All nascent fibers are APþ. c: Subsequent to AP staining, the muscle section shown in (b) was counterstained with hematoxylin to visualize centrally located nuclei (two shown at arrows), a hallmark of newly regenerated muscle fibers.
to generate a pool of myogenic precursors that ultimately differentiate and fuse into nascent myofibers (reviewed by Chen and Goldhamer, 2003). To examine F3/-2.5cre activity in regenerating muscle, we used both R26R and Z/AP reporter mice. Z/AP mice express lacZ prior to Cre-mediated recombination and alkaline phosphatase (AP) after recombination (Lobe et al., 1999). Following cardiotoxin injection of the TA muscle, recombination of both reporter loci was detected in nascent myofibers (Fig. 3b), indicating transgene activity in satellite cells and/or their daughter cells. However, due to apparent promoter inactivity in mononuclear cells at early stages of regeneration (data not shown), the timing of recombination could not be determined. To investigate satellite cell labeling, primary cultures were established from adult hindlimb muscles and cells were coimmunostained for b-gal and MyoD. In mixed cultures comprised predominantly of myogenic cells and fibroblasts, b-gal was only detected in cells that were also positive for MyoD, demonstrating the specificity of Cre-mediated recombination (data not shown). Further, essentially all MyoDþ cells were also b-galþ (Fig. 4a,b), indicating that recombination within the satellite cell population was highly efficient. MyoD-cre transgenic mice will be an important resource for manipulating gene expression in skeletal muscle, not only at differentiation stages, but also in undifferentiated myogenic precursor cells. Further, the ability to permanently label satellite cells provides an experimental approach to address their developmental potential and other biological questions relevant to normal and abnormal regenerative processes. MATERIALS AND METHODS DNA Constructs Regulatory sequences from the human MyoD locus were used to direct expression of the cre gene. These
sequences include a 4-kb genomic fragment that contains the MyoD core enhancer (F3; Goldhamer et al., 1992, 1995) and 2.5-kb of promoter-proximal 50 flanking sequences. F3/-2.5cre was produced by excising F3/-2.5 genomic sequences from F3/-2.5CAT (Goldhamer et al., 1992) using Not I and Xho I (Klenow-filled) and cloning into the Not I and Eco RI (Klenow-filled) sites of the plasmid CreKSþ (kindly provided by Michael Parmacek). The cre gene was modified to contain a nuclear localization signal, an SV40 T-antigen intron, and an HSV-TK polyadenylation sequence (Bunting et al., 1999). Production of Transgenic Mice, Genotyping, and Generation of Embryos for Analysis DNA was prepared for transgenic injection as previously described (Chen et al., 2001). F3/-2.5cre transgenic lines were produced by the University of Pennsylvania Transgenic and Chimeric Mouse Facility by pronuclear injection of BL6SJLF2/J one-cell embryos. A total of 20 transgenic lines were generated. Genotyping for the presence of the cre gene was done by PCR of tail DNA (30 cycles of 948C (1 min), 558C (1 min), 728C (1 min)). A band of 355 bp was generated using the following primers: Cre.forward: CAT CGT CGG TCC GGG CTG CC; Cre.reverse: CCC CCA GGC TAA GTG CCT TC. Lines were maintained by breeding to FVB mice. Specificity of cre expression was assessed by crossing cre lines to two reporter mouse lines. The majority of the analyses reported here were performed using the R26R line, in which lacZ is expressed from the Rosa26 promoter following Cre-mediated recombination (Soriano, 1999). R26R mice were of a mixed BALB/c and FVB background. The second reporter line (Z/AP; Lobe et al., 1999) uses a strong CMV enhancer and the b-actin promoter to express lacZ prior to Cre-mediated recombination and human placental alkaline phosphatase (AP) after recombination.
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FIG. 4. Indirect immunofluorescence localization of MyoD and b-gal in 5-day satellite cell cultures derived from F3/-2.5cre;R26R hindlimb muscles. a,b: All MyoDþ cells were also b-galþ. d,e: Parallel control cultures in which the primary antibodies were omitted. c,f: Cultures were counterstained with DAPI to reveal nuclei.
When female cre transgenic mice were bred to male R26R reporters, recombination at the R26R locus was observed in offspring irrespective of cre cotransmission, as previously noted with other cre transgenic lines (e.g., Sakai and Miyazaki, 1997; Logan et al., 2002; Vincent and Robertson, 2003). This observation is consistent with cre transgene expression in the female germline (Sakai and Miyazaki, 1997). Therefore, experimental animals were generated by crossing males hemizygous for the cre transgene with females hemizygous for the reporter allele. For embryonic stages, noon on the day of the vaginal plug was considered E0.5. Two or more litters were analyzed for each developmental timepoint.
as above for 1 h, rinsed five times for 20 min each in PBS (pH 7.4), equilibrated sequentially in 1:3, 1:2, 1:1 of 30% sucrose and PBS, and incubated in 30% sucrose overnight at 48C. Muscles were then equilibrated in 1:1 of 30% sucrose and OCT and mounted in OCT using liquid nitrogen-cooled isopentane. Twenty to 40mm sections were collected, air-dried, postfixed with 2% paraformaldehyde / 0.25% glutaraldehyde, and processed for staining with X-gal (R26R and Z/AP reporters) or BM Purple (Roche; Z/AP reporters). After capturing images, sections were counterstained with hematoxylin and rephotographed to identify muscle fiber nuclei.
Histochemistry
Satellite Cell Cultures and Immunohistochemistry
For paraffin histology, R26R embryos and adult tissues were fixed in 2% paraformaldehyde / 0.25% glutaraldehyde on ice and processed for X-gal staining as previously described (Chen et al., 2001). Z/AP embryos and tissues were prepared and stained according to Lobe et al. (1999). Paraffin histology was performed by conventional methods, and sections were counterstained with nuclear fast red (Vector, Burlingame, CA) or hematoxylin/eosin. For cryostat sectioning of whole-mount embryos and fetal limbs, tissues were fixed as above, cryoprotected in 30% sucrose overnight at 48C, sequentially equilibrated in 1:1 of 30% sucrose and OCT (Tissue-Tek) and 100% OCT, embedded in OCT, and frozen in liquid nitrogen-cooled isopentane. Adult muscles were fixed
Satellite cell cultures were established from adult hindlimb muscles of F3/-2.5;R26R mice as described (Rando and Blau, 1994) and maintained in growth medium (Ham’s F10, 20% FBS, 2.5 ng/ml FGF, 2% chick embryo extract) on collagen-coated plates. After 5 days in culture, cells were fixed for 5 min in 4% paraformaldehyde, rinsed three times for 5 min each in PBS, permeabilized with 0.1% Triton X-100 for 4 min, and blocked with 5% normal goat serum in PBS for 30 min. Cells were incubated for 1 h at room temperature with a rabbit antiserum to mouse MyoD (1:20, Santa Cruz Biologicals, Santa Cruz, CA, sc-760) and a mouse monoclonal antibody to b-gal (1:50, Promega, Madison, WI, Z3781) diluted in blocking solution. Cells were washed three times for 5 min each in PBS and incubated with Alexa Fluor secondary antibodies
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(goat a-mouse 488 and goat a-rabbit 546 (1:200, Molecular Probes, Eugene, OR)) for 30 min. Cells were washed in PBS as above, treated with 0.1% DAPI for 1 min, and mounted in 90% glycerol in PBS. Cardiotoxin-Induced Muscle Injury Adult mice were anesthetized with isoflurane and shaved. The TA muscle was injected with 100 mL of 10 mM cardiotoxin (Calbiochem, La Jolla, CA) in PBS (pH 7.4). Injected and uninjected contralateral muscles were harvested 11 days after injection and processed for cryostat sectioning as described above. Photomicroscopy Images were photographed, captured and assembled as previously described (Chen et al., 2001). ACKNOWLEDGMENTS We thank Raga Ramachandran and Rachel Benedetto for technical assistance and members of the Goldhamer lab for critical review of the manuscript. LITERATURE CITED Bothe GW, Haspel JA, Smith CL, Wiener HH, Burden SJ. 2000. Selective expression of Cre recombinase in skeletal muscle fibers. genesis 26:165–166. Bruning JC, Michael MD, Winnay JN, Hayashi T, Horsch D, Accili D, Goodyear LJ, Kahn CR. 1998. A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance. Mol Cell 2:559–569. Bunting M, Bernstein KE, Greer JM, Capecchi MR, Thomas KR. 1999. Targeting genes for self-excision in the germ line. Genes Dev 13:1524–1528. Chen JC, Goldhamer DJ. 1999. Transcriptional mechanisms regulating MyoD expression in the mouse. Cell Tissue Res 296:213–219. Chen JC, Goldhamer DJ. 2003. Skeletal muscle stem cells. Reprod Biol Endocrinol 1:101. Chen JC, Goldhamer DJ. 2004. The core enhancer is essential for proper timing of MyoD activation in limb buds and branchial arches. Dev Biol 265:502–512. Chen J, Kubalak SW, Chien KR. 1998. Ventricular muscle-restricted targeting of the RXRalpha gene reveals a non-cell-autonomous requirement in cardiac chamber morphogenesis. Development 125:1943–1949. Chen JCJ, Love CM, Goldhamer DJ. 2001. Two upstream enhancers collaborate to regulate the spatial patterning and timing of MyoD transcription during mouse development. Dev Dyn 221:274–288.
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Chen JCJ, Ramachandran R, Goldhamer DJ. 2002. Essential and redundant functions of the MyoD distal regulatory region revealed by targeted mutagenesis. Dev Biol 245:213–223. Faerman A, Shani M. 1993. The expression of the regulatory myosin light chain 2 gene during mouse embryogenesis. Development 118:919–929. Faerman A, Goldhamer DJ, Puzis R, Emerson CP Jr, Shani M. 1995. The distal human myoD enhancer sequences direct unique muscle-specific patterns of lacZ expression during mouse development. Dev Biol 171:27–38. Gerhart J, Bast B, Neely C, Iem S, Amegbe P, Niewenhuis R, Miklasz S, Cheng PF, George-Weinstein M. 2001. MyoD-positive myoblasts are present in mature fetal organs lacking skeletal muscle. J Cell Biol 155:381–392. Goldhamer DJ, Faerman A, Shani M, Emerson CP Jr. 1992. Regulatory elements that control the lineage-specific expression of myoD. Science 256:538–542. Goldhamer DJ, Brunk BP, Faerman A, King A, Shani M, Emerson CP Jr. 1995. Embryonic activation of the myoD gene is regulated by a highly conserved distal control element. Development 121: 637–649. Hawke TJ, Garry DJ. 2001. Myogenic satellite cells: physiology to molecular biology. J Appl Physiol 91:534–551. Lobe CG, Koop KE, Kreppner W, Lomeli H, Gertsenstein M, Nagy A. 1999. Z/AP: a double reporter for Cre-mediated recombination. Dev Biol 208:281–292. Logan M, Martin JF, Nagy A, Lobe C, Olson EN, Tabin CJ. 2002. Expression of Cre Recombinase in the developing mouse limb bud driven by a Prxl enhancer. genesis 33:77–80. Miniou P, Tiziano D, Frugier T, Roblot N, Le Meur M, Melki J. 1999. Gene targeting restricted to mouse striated muscle lineage. Nucleic Acids Res 27:e27. Noden DM, Marcucio R, Borycki AG, Emerson CP Jr. 1999. Differentiation of avian craniofacial muscles. I. Patterns of early regulatory gene expression and myosin heavy chain synthesis. Dev Dyn 216: 96–112. Rando TA, Blau HM. 1994. Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. J Cell Biol 125:1275–1287. Sakai K, Miyazaki J. 1997. A transgenic mouse line that retains Cre recombinase activity in mature oocytes irrespective of the cre transgene transmission. Biochem Biophys Res Commun 237: 318–324. Sassoon DA, Lyons G, Wright WE, Lin VK, Lassar AB, Weintraub H, Buckingham M. 1989. Expression of two myogenic regulatory factors myogenin and MyoD1 during mouse embryogenesis. Nature 341:303–307. Sauer B, Henderson N. 1988. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A 85:5166–5170. Soriano P. 1999. Generalized lacZ expression with the ROSA26 Cre reporter strain [Letter]. Nat Genet 21:70–71. Vincent SD, Robertson EJ. 2003. Highly efficient transgene-independent recombination directed by a maternally derived SOX2CRE transgene. genesis 37:54–56.
