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doi: 10.1242/10.1242/dev.00394

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Knock-in of integrin ß1D affects primary but not secondary myogenesis in mice

Ana Sofia Cachaço1,2,*, Susana M. Chuva de Sousa Lopes3,*, Ingrid Kuikman4, Fernanda Bajanca1,2, Kuniya Abe5, Christian Baudoin4,{dagger}, Arnoud Sonnenberg4, Christine L. Mummery3 and Sólveig Thorsteinsdóttir1,2,{ddagger}

1 Department of Animal Biology and Centre for Environmental Biology, Faculty of Sciences, University of Lisbon, 1749-016 Lisbon, Portugal
2 Gulbenkian Institute of Science, Oeiras, Portugal
3 Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Utrecht, The Netherlands
4 Department of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands
5 Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto, Japan
{dagger} Present address: Faculty of Medicine, INSERM U385, Nice, France



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  		Fig. 1. Different migratory cell types behaved normally in ß1D1D ki/ki embryos. (A-D) Transverse sections showing (A,D) snail expression in migratory neural crest cells (arrows) and (B,E) pax3 expression in migratory limb muscle precursor cells (arrows) of E9.5 heterozygous (A,B) and ß1D1D ki/ki (D,E embryos). (C,F) PGC distribution was analysed in transverse serial sections of E10.5 wild-type (C) and ß1D1D ki/ki (F) embryos. An enlargement of the right gonadal ridge is shown. (G,H) At E10.5, PGCs were detected by AP staining in the hindgut (hg), mesenterium (m), gonadal ridges (gr) and ectopic regions (o) of ß1D1D ki/ki, heterozygous and wild-type embryos in the quantities indicated. (I) RT-PCR detection of ß1A and ß1D in isolated PGCs from different stages. Oct4 is a PGC marker at these stages. HPRT is a loading control. ba, first branchial arch; fb, forebrain; gr, gonadal ridges; h, newborn heart; l, limb bud; nt, neural tube; s, E11.5 gonadal somatic tissue. Scale bars: 200 µm (A,B,D,E), 400 µm (C,F).

 


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  		Fig. 2. At E14.5, abnormal ß1D1D ki/ki embryos showed defective vascularisation and increased apoptosis of endothelial cells in the placental labyrinth. (A,D,G) Heterozygous, (J) wild-type (B,E,H,K) normal ß1D1D ki/ki and (C,F,I,L) abnormal ß1D1D ki/ki embryos. (A-C) Laminin immunostaining, in transverse sections of E14.5 placentae, showing the basal lamina of foetal blood vessels. (D-F) Higher magnification of the labyrinth in A-C showing reduced branching and obstructed blood vessels in abnormal ß1D1D ki/ki embryos (arrow). (G-I) Haematoxylin and Eosin staining. (J-L) Transverse sections showing MECA32- (red) and TUNEL- (green) positive cells (arrows in L). cp, chorionic plate; lb, labyrinth. Scale bar: 200 µm.

 


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  		Fig. 3. Late gestation ß1D1D ki/ki embryos and newborn pups are thinner/shorter than their littermates and exhibit a reduction in muscle mass. (A,B) Gross morphology of E18.5 embryos (A) and newborns (B). Note the curved posture of the E18.5 ß1D1D ki/ki embryo. (C-L) Transverse Haematoxylin and Eosin-stained sections of E18.5 wild-type (C,E,G,I,K) and ß1D1D ki/ki (D,F,H,J,L) embryos showing lung (C,D), heart (E,F), thoraxic body wall (G,H), shoulder (I,J) and diaphragm (K,L) muscles. Dg, diaphragm; Dl, deltoideus; e, esophagus; I, intercostal; lg, lung; P, pectoral; r, rib; s, scapula. Scale bar: 200 µm.

 


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  		Fig. 4. Different muscle types of E18.5 embryos stained with the anti-slow MHC antibody show a reduction in primary and secondary myotubes in ß1D1D ki/ki embryos (B,E,H,K) compared to wild-type littermates (A,D,G,J). Transverse sections of body wall (A,B), cleidomastoideus (D,E) and lower hindlimb (G,H) muscles. J and K are higher magnifications of EDL muscles in G and H. (C,F,I,L) Graphical representation of the differences in myotube number between wild-type and ß1D1D ki/ki E18.5 muscles. (C) Total, secondary and primary myotube numbers/mm2 in intercostal/serratus dorsalis muscles. (F) Total, secondary and primary myotube numbers in red area of cleidomastoideus muscle. (I) Total number of primary myotubes in lower hindlimb represents the sum of myotubes in TA, EDL, and P group. (L) Total, secondary and primary myotubes in EDL muscle. Bars represent means ± s.d.; **P<=0.01. EDL, extensor digitorum longus; f, fibula; FDF, flexor digitorum fibularis; I, intercostal; LS, latissimus dorsi/serratus dorsalis; P, peroneus group (including: PB, peroneus brevis; PD, peroneus digiti; PL, peroneus longus); r, rib; ra, red area of cleidomastoideus; SOL, soleus; t, tibia; TA, tibialis anterior; wa, white area of cleidomastoideus. Scale bars: 200 µm.

 


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  		Fig. 5. Hindlimb muscles of ß1D1D ki/ki E14.5 embryos (D,E,F,H) have reduced numbers of primary myotubes compared to wild-type embryos (A,B,C,G). Transverse sections of lower hindlimb: (A,D) upper (B,E) middle and (C,F) lower region, stained with anti-slow MHC, showing that muscle size is always reduced in ß1D1D ki/ki embryos. (G,H) Higher magnification of EDL in (B,E) showing a clear difference in morphology of the primary myotubes. (I) Graphical representation of the differences in primary myotube numbers in TA, EDL, P muscles and their totals in E14.5 wild-type and ß1D1D ki/ki embryos. (J) Primary myotube diameter in EDL, showing a difference in size between wild-type and ß1D1D ki/ki. (K-N) Transverse sections of E14.5 hindlimbs (middle region), stained for phospho-histone-H3 (K,L) and subjected to TUNEL assay (M,N) show no differences in proliferation or apoptosis in muscle regions between wild-type (K,M) and ß1D1D ki/ki (L,N) embryos. (O-R) Adjacent transverse sections of E17.5 lower hindlimbs stained for slow MHC (O,P) and exposed to the TUNEL assay (Q,R) show a reduction in primary myotubes and increased apoptosis (arrows) in ß1D1D ki/ki (P,R) compared with wild type (O,Q). (S-T) Graphical representation of primary, secondary and total myotube numbers in E17.5 EDL (S) and TUNEL-positive nuclei in E17.5 TA, EDL and P per section, in wild type and ß1D1D ki/ki (T). For abbreviations see Fig. 4. Legend. Bars represent means ± s.d. *P<=0.05, **P<=0.01. Scale bars: 200 µm (A-F,K-R), 50 µm (G,H).

 


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  		Fig. 6. Myogenic differentiation of C2C12/ß1D and C2C12/ß1A cells. (A,B) ß1D and ß1A expression during differentiation of C2C12/ß1D and C2C12/ß1A cells. (C-E) Expression of p21 (C), pRB (D) and connexin43 (E) analysed by western blotting. During differentiation, p21 is upregulated and pRB is dephosphorylated in all cell groups analysed. Connexin43 was downregulated in C2C12 and C2C12/ß1D, but not in C2C12/ß1A. (F-H,J-L) Immunostaining for MHC (green) and nuclear To-Pro3 staining (red). C2C12/ß1D and C2C12/ß1A had fewer MHC+ cells than C2C12 at day 3 (F-H) and day 5 (J-L) of differentiation and C2C12/ß1A myotube morphology was abnormal (H,L). (I) Percentage of MHC+ cells with >1 nucleus. After 3 days of differentiation, the number of C2C12/ß1D and C2C12/ß1A myotubes was less than the C2C12 control. After 5 days, the number of C2C12/ß1A myotubes is similar to the C2C12 control, in contrast to the number of C2C12/ß1D myotubes, which was still lower. (M) Fusion index (percent), being the ratio of number of nuclei in myotubes (cells with >3 nuclei) to the total number of nuclei in MHC+ cells. After 3 and 5 days of differentiation, both C2C12/ß1D and C2C12/ß1A myotubes have fewer nuclei than C2C12 myotubes. Bars represent means ± s.d. *P<=0.05; **P<=0.01.

 




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