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doi: 10.1242/10.1242/jcs.00364

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Migration of adult myogenic precursor cells as revealed by GFP/nLacZ labelling of mouse transplantation chimeras

Developmental Biology and Molecular Pathology, W7, University of Bielefeld, D-33501 Bielefeld, Germany

View larger version (111K): [in a new window]   Fig. 1. Complementary muscle grafts with GFP-labelled host, 0 to GFP (A,C,D) and GFP labelled donor, GFP to 0 (B). (A,B) Macroscopic appearance under UV light of grafts of tibialis anterior (TA) muscle to the TA bed. (A) 49 days p.o., (B) 70 days p.o. Bar in B, 2 mm. (C,D) Complementary labelling in a 0/nLacZ to GFP/0 graft, 50 days p.o. Cross-section at graft(g)/host(h) border. (C) X-gal plus eosin staining, (D) GFP (green) and Hoechst (blue) nuclear fluorescence. The donor was homozygous for the nLacZ knock-in (and for DesKO). There are several GFP-positive muscle fibres that have nLacZ-positive nuclei (arrows), indicating co-fusion of donor myoblasts with host muscle fibres. Bar in C, 100 µm.

View larger version (103K): [in a new window]   Fig. 2. Unlabelled graft to a double-labelled host, 0/0 to GFP/nLacZ, 77 days p.o. (A-D), and nLacZ-labelled graft to a GFP host (complementary label), 0/nLacZ to GFP/0, 75 days p.o. (E,F). Migration of host cells into the graft. The same region of the host(h)/graft(g) border in adjacent sections is shown in fluorescence (A,C,E) and after Xgal/eosin staining (B,D,F). (A,B,E,F) Cross-sections of proximal parts, (C,D) longitudinal sections of distal parts of the graft site. (A,E) Within the graft there are numerous fibers with host label (GFP) that can be followed through serial sections, as indicated by green contours in B,F. In B,D, a few nLacZ-positive nuclei are found in GFP-positive fibres close to the host (arrows). In F, nearly all of the GFP-positive fibres show blue nuclei. C,D,F indicate immigration of host stem cells and co-fusion with graft muscle fibres rather than ingrowth of host muscle fibres. In C, a green margin along bent muscle fibres is due to a local GFP overspill during thawing of the section (Jockusch et al., 2003). Bar, 100 µm.

View larger version (26K): [in a new window]   Fig. 3. Quantitative evaluation of the colonization of muscle grafts by host myogenic cells: results of six independent 0 to GFP transplantation experiments. Numbers of GFP-positive fibres in the graft were counted in eight to 10 sections in segments of 250 µm across the section and plotted as a function of the distance from the host/graft (h/g) border; symbols indicate average numbers between proximal and distal counts, with ends of bars indicating proximal and distal averages. The experiment with 77 days regeneration time is identical to the one shown in Fig. 2A-D. No immigration was observed in short-term experiments (filled symbols).

View larger version (136K): [in a new window]   Fig. 4. Emigration of graft cells into the host: GFP to 0, 55 days p.o. The border between the host gastrocnemius (h, left) and the grafted anterior tibial muscle (g, right) is shown. (A) Fluorescence shows GFP. (B) The adjacent section is haematoxylin/eosin-stained, with contours to show GFP-positive fibres. Inset, enlargement of the framed region to show that there are no central nuclei in the GFP-positive fibres. (C) Region of host with GFP-positive fibres. (D) Same region as in C, with SDH staining to show fibre types and black contours to indicate GFP-positive host muscle fibres. Insets in C and D, with comparable regions in the contralateral leg to which no GFP-positive TA had been grafted, to show lack of autofluorescence of oxidative fibres. In C', blue fluorescence indicates Hoechst-stained nuclei, and in D', SDH enzyme histochemistry indicates a fibre-type pattern. Bar, 100 µm.

View larger version (21K): [in a new window]   Fig. 5. Quantitative evaluation of the migration of graft myogenic cells into adjacent host muscle: results of four GFP to 0 experiments. In cross-sections through adjacent host and graft muscles, GFP-positive fibres in the host were counted. Numbers are given as a function of the distance from the graft. Averages (symbols) and highest and lowest counts (ends of bars) are given separately for proximal and distal sections (cf. Fig. 3 for additional explanations). Empty circles correspond to the experiment shown in Fig. 4. No emigration was observed in short-term experiments (filled symbols).

View larger version (129K): [in a new window]   Fig. 6. Early stages of graft cell migration: double-labelled donor grafted into unlabelled host, GFP/nLacZ to 0/0, two different experiments (A-D and E,F), both 52 days p.o. The same region of the host (h)/graft(g) border is shown in the adjacent cross (A-D) and longitudinal (E,F) sections. (A,C,E) Fluorescence to show donor GFP; (B,D,F) Xgal plus eosin, to show donor-labelled nuclei. (A-D) Arrows indicate GFP/nLacZ-positive donor cells `on their way' into host muscle; arrow heads indicate a cell in which no blue nucleus was seen. (E,F) GFP-positive fibres in the host muscle with no nLacZ-positive nucleus visible. In the graft, low GFP level and strong ß-galactosidase activity indicate myotubes and immature muscle fibres. Bars, 100 µm.

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