Inducible lineage tracing of Pax7-descendant cells reveals embryonic origin of adult satellite cells

Abstract

We have generated a mouse strain carrying a Cre-ER(T2) knock-in allele at the Pax7 locus, the Pax7(CE) allele (Lepper et al., 2009, Nature 460:627-631). Combining Pax7(CE) and the R26R(LacZ) Cre reporter allele, here we describe temporal-specific tamoxifen (tmx)-inducible lineage tracing of embryonic Pax7-expressing cells. In particular, we focus on the somitic lineage. Tmx-inducible Cre activity directed by the Pax7(CE) allele is similar to the endogenous Pax7 expression pattern. The somitic Pax7-expressing cells selectively marked at embryonic day 9.5 (E9.5) give rise to dorsal dermis and brown adipose tissue, in addition to dorsal aspects of trunk muscles and the diaphragm muscle. However, they do not contribute to ventral body wall and limb muscles. After E12.5, marked Pax7-expressing cells become lineage restricted to muscles. Descendants of these early marked Pax7-expressing cells begin to occupy sublaminal positions associated with the myofibers around E16.5, characteristic of embryonic satellite cells. Furthermore, they contribute to adult myofibers and regeneration competent satellite cells in the tibialis anterior muscle, providing evidence that some adult satellite cells are of embryonic origin.

Figure 1. Pax7^CE allele is a null allele

a, schema for generating the Pax7^CE allele via recombineering; modified from Lepper et al. (2009). Oligonucleotide primers, vectors, selection cassettes, 3′ probe, as well as locations of loxP sites and Cre-ERT2 cassette are detailed in Materials and Methods; E, exon; BamHI is used for Southern Blot analysis; pink arrows 1 and 2, location of genotyping PCR primers. b, Southern blot using the 3′ probe (yellow box in a). c and d, immunofluorescence of Pax7 on E10.5 Pax7^+/CE and Pax7^CE/CE embryos, respectively. From left to right for both panels: DAPI, Pax7, and merged image; transverse sections at the fore limb level; arrows, secondary myogenic domain; stars, dorsal spinal cord; scale bar = 100 μm.

Figure 2. Pax7^CE allele allows tightly controlled β-gal labeling by Tamoxifen administration

A–H, whole mount X-gal histochemical staining of Pax7^+/CE;R26R^+/LacZ embryos one day after tmx administration; stages as indicated. The upper right insets are Pax7^+/CE;R26R^+/LacZ control embryos without tmx treatment, showing no X-gal signal. In a, the lower right inset is a magnified image of boxed cervical somites; lines demarcate somite boundaries. b–e, stars, staining in the spinal cord; d–h, black arrows, staining in hind limb muscles. i, cross section of E15.5->16 hind limb; t, tibia, f, fibula; TA, tibialis anterior muscles. j, longitudinal section of E15.5->16 TA muscle; white arrows, single round β-gal⁺ cells present in muscle fibers.

Figure 3. Pax7^CE directs β-gal labeling in the dermomyotome and secondary myogenic domain

a–d, transverse sections of X-gal stained Pax7^+/CE;R26R^+/LacZ embryos of stages indicated; a, cervical level; b–d, forelimb level; stages as indicated; arrows, β-gal⁺ cells in the trunk and limb; stars, β-gal⁺ cells in the dorsal spinal cord; scale bar = 50 μm. e and f, Pax7/β-gal double immunofluorescence on transverse sections of E8.5->9.5 (cervical level) and E9.5->10.5 (fore limb level) samples, respectively; white arrows, co-labeled cells; white arrowheads, β-gal+Pax7⁻ cells at the dorsal somite; white dashed lines demarcate the ventro-medial Pax7⁺β-gal⁻ population; scale bar = 50μm.

Figure 4. Inducible long-term lineage tracing reveals progressive changes of the developmental potential of Pax7-expressing cells

a–d, lineage tracing as indicated. a, from left to right: First, transverse section at the branchial level; Top for dermis (d), bottom for brown adipose tissue (bat); upper fore limb; upper hind limb. Dashed line demarcates the ventral boundary of X-gal signal; m, muscles; e, epidermis. Panels b–d, organization of subfigures is the essentially the same as in panel a with following differences: 1) for Panel b fore limb figure, the mid level and toes (inset) are shown; 2) for c, mid levels of fore limb and hind limb are shown, as well as the toes (insets in each); 3) for d, toes are shown to demonstrate distal expansion of β-gal labeling.

Figure 5. β-gal+ cells contribute to myofibers

Double immunofluorescence for β-gal (in red, first column) and myosin heavy chain (MF-20 in green, second column) were performed on E9.5->16.5 (top) and E11.5->16.5 (bottom) samples. Merged images with DAPI (blue) stain are in the third column. The muscle groups shown are: a, the back (back) muscles; b, the intercostal (in cos) muscles; c, the panniculus carnovous (pan car) or subcutaneous muscle; d, the diaphragm (dia) muscle; e and g, the abdominal (ab) muscles; f and h, the TA muscles. Scale bar = 50 μm.

Figure 6. Marked β-gal⁺ cells occupy sub-laminal space at E16.5

Triple immunofluorescence of Pax7 (in green, first column), β-gal (in red, second column), and Laminin (in white, third column). Merged images are in the fourth column. a, b, and c are E9.5->16.5 samples of the back muscle, intercostal muscle and diaphragm muscle, respectively. d, TA muscle of E11.5->16.5. Solid arrows, sub-laminal β-gal⁺Pax7⁺ cells; lined arrows, β-gal⁺Pax7⁺ cells outside of the Laminin boundary; round arrows, βgal⁺Pax7⁻ staining within the Laminin boundary, likely recently fused myocytes. Scale bar = 20 μm.

Figure 7. Embryonically marked β-gal+ cells can become functional adult satellite cells

a, postnatal day 60 (P60) TA muscle from a non-tmx (−tmx) treated Pax7^+/CE;R26R^+/LacZ animal (control). b, P60 TA sample from a tmx treated (+tmx) embryo at E11.5. c, a magnified image from boxed area in b to show strong X-gal staining at the periphery of the myofiber (white arrow) and uneven X-gal signal within myofibers. d, X-gal staining of TA muscle regenerates from an E11.5->P70 animal; arrows, central nuclei of regenerated myofibers. Injury was performed at P60 by CTX and the sample was harvested 10 days (10d) later at P70. e, E11.5->P60 TA muscles subjected to immunofluorescent staining for β-gal, Pax7, and Laminin; merged image with DAPI stain to the right. f, same as in e, except staining for M-cadherin (M-cad). Arrowheads indicate β-gal+ satellite cells. g and h are fluorescent images of β-gal/Pax7/EdU and β-gal/M-cad/EdU, respectively, of 10-day TA muscle regenerates. EdU was administered daily on days 2–5 post injury. Scale bars = 20 μm.