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First published online February 4, 2009
doi: 10.1242/10.1242/jcs.038356

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Induced early expression of mrf4 but not myog rescues myogenesis in the myod/myf5 double-morphant zebrafish embryo

Esther Schnapp^1,*, Anna Silvia Pistocchi^2,*, Evangelia Karampetsou², Efrem Foglia², Carla Lora Lamia², Franco Cotelli^2, and Giulio Cossu^1,2,

¹ Stem Cell Research Institute, DiBiT, San Raffaele Scientific Institute, 58 via Olgettina, 20132 Milan, Italy
² Department of Biology, University of Milan, 26 via Celoria, 20133 Milan, Italy

View larger version (76K): [in this window] [in a new window]   Fig. 1. Myf5 and Myod knockdown results in immobile embryos and absence of skeletal myosin. (A) 24 h.p.f. embryos injected with standard control morpholino look and move similarly to uninjected embryos. (B) Embryos co-injected with myf5 and myod morpholinos (MO) look phenotypically normal but do not move. (C,D,E) Whole-mount myosin staining of control embryos (C), and myf5/myod morphants at 24 h.p.f. The embryo in D has no skeletal myosin and that in E very little skeletal myosin. The arrow in D indicates the heart, which expresses cardiac myosin independently of myf5 and myod. (C',D',E') Cross sections of control embryos (C') and myf5/myod morphants (D',E') at 20-somite stage, stained for myosin. Myosin is in red and nuclei are blue. Scale bars: 100 µm in C and 50 µm in C'.

View larger version (78K): [in this window] [in a new window]   Fig. 2. mrf4 and myog are expressed later than myod, after the onset of somitogenesis. (A-L) Whole-mount in situ hybridisation of mrf4 (A-D), myog (E-H) and myod (I-L). (A,B,E,F,I,J) 1- to 2-somite stage. (C,D,G,H,K,L) 6- to 8-somite stage. (A,C,E,G,I,K) dorsal view anterior to the left. (B,D,F,H,J,L) lateral view anterior to the left. No signal for mrf4 and myog can be detected at the 1- to 2-somite stage (A,B,E,F) when myod is already strongly expressed in adaxial cells and weakly expressed in the first somites (I,J). After the 5-somite stage, mrf4 and myog start to be expressed in the adaxial cells (C,D,G,H), whereas myod is expressed both in adaxial cells and somites (K,L). Scale bar: 100 µm.

View larger version (80K): [in this window] [in a new window]   Fig. 3. mrf4 and myog expression is unperturbed in single morphants but absent in double morphants. (A-H) Whole-mount in situ hybridisation for mrf4 (A,C,E,G) and myog (B,D,F,H) in 20-somite embryos, anterior to the left. In situ signal in uninjected embryos (A,B) is undistinguishable from that in myf5 (C,D) and myod (E,F) single morphants. No signal is detected in the majority of myf5/myod double morphants (G,H). Scale bar is 100 µm.

View larger version (73K): [in this window] [in a new window]   Fig. 4. Ectopic mrf4 can rescue the muscle phenotype in myf5/myod double morphants. (A) In situ hybridisation for myog in mrf4-rescued embryos at 20-somite stage. (B-C') Myosin staining in mrf4-rescued (B,B') and myog-coinjected (C,C') double morphants at 24 h.p.f. B' and C' show part of the tail at higher magnification. Myosin is in red and nuclei are in blue. The arrow in C indicates the heart. Scale bars: 100 µm.

View larger version (149K): [in this window] [in a new window]   Fig. 5. Semi-thin sections and electron microscopy reveal disturbed muscle in double morphants and rescued muscle in mrf4-injected double morphants. (A-C) Transverse sections of control (A), double morphant (B), and rescued double morphant (C) embryos at 24 h.p.f., stained with gentian violet. Arrows in B and C indicate apoptotic and/or disturbed muscle. (D-F) Electron microscopy of control (D), double morphant (E), and rescued double morphant (F) embryos at 24 h.p.f. Transverse sections monitor highly organised sarcomeric structures in control and rescued embryos, whereas the sarcomeres are disoriented and unorganised in double morphants. Scale bars: 50 µm in C and 500 nm in D-F.

View larger version (121K): [in this window] [in a new window]   Fig. 6. Myogenesis in the mrf4 rescue appears to proceed normally. The onset of expression of myog (A,B), pax3 (C,D), pax7 (E,F), slow Myosin (G,H) and fast Myosin (I,J) was examined in mrf4-rescued embryos (B,D,F,H,J) and uninjected embryos (A,C,E,G,I) at indicated developmental stages by in situ hybridisation (A-F, anterior to the left) or antibody staining (G-J, flat-mount views of tail region, anterior up). Except for myog, which is upregulated in the rescued embryos (compare B to A), all other genes seem to be expressed normally. The arrows in C and D indicate pax3 expression in muscle precursors, the strong anterior staining is in the central nervous system. Slow and fast Myosin in G-J is stained in red, nuclei are in blue.

View larger version (116K): [in this window] [in a new window]   Fig. 7. mrf4 rescues skeletal muscle via the activation of myod. (A-E) Flat mounts of whole-mount Myod antibody staining in myf5 morphant (A), myod morphant (B), uninjected (C), double morphant (D), and mrf4-rescued (E) embryos at the 12-somite stage. Myod is not expressed in myod single and double morphants (B and D), but it is expressed normally in mrf4-rescued embryos (E). Myod is in green, nuclei are in blue; anterior is to the top. Scale bar: 50 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 8. mrf4 is a strong activator of myod but not of myf5. Real-time PCR results show fold changes in mrf4, myod and myf5 mRNA expression in double morphants, rescued and mrf4 mRNA injected embryos normalised to a housekeeping gene and to their expression in uninjected embryos, which is set to 1. Error bars indicate s.d. RNA of all embryos was extracted at the 15-somite stage.

View larger version (47K): [in this window] [in a new window]   Fig. 9. The minority of double morphants recovers skeletal muscle 1 week after morpholino injection. (A-H) Whole-mount myosin staining of 3 d.p.f. (A-D), 6 d.p.f. (E,F) and 8 d.p.f. (G,H) in control (A,E), double morphant (B,C,F,G) and mrf4-rescued larvae (D,H). Control and rescued larvae show normal myosin expression (A,E,D,H), whereas most double morphants are still largely devoid of myosin at 3 d.p.f. and 6 d.p.f. (B,F) and remain immobile. Some double morphants recover and start to express myosin in an unorganised pattern 3 d.p.f. (C) and later regain mobility and improve myotome organisation (G), although skeletal muscle is still less organised compared with control (E) or rescued larvae (H). Scale bar: 100 µm.

View larger version (79K): [in this window] [in a new window]   Fig. 10. GFP expression in -actin-GFP transgenic embryos. (A-C) Embryo injected with the standard control morpholino and (D-I) two myf5/myod double-morphant embryos at 24 h.p.f. (A,D,G), 3 d.p.f. (B,E,H) and 5 d.p.f. (C,F,I). The majority of the double morphants never express GFP (D-F), however the double-morphant embryos that do express GFP at 24 h.p.f. (G, arrow points to weak GFP expression) increase GFP expression over time (see H,I). Each panel is composed of the normal light and the fluorescence image of the same embryo. A and G show a part of the tail at higher magnification.

View larger version (3K): [in this window] [in a new window]   Fig. 11. Model of muscle gene interactions during zebrafish development.

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