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First published online August 8, 2003

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Temperature and the expression of seven muscle-specific protein genes during embryogenesis in the Atlantic cod Gadus morhua L.

Thomas E. Hall^1,*, Nicholas J. Cole² and Ian A. Johnston¹

¹ Gatty Marine Laboratory, School of Biology, University of St Andrews, Fife, KY16 8LB, UK
² Division of Cell and Developmental Biology, MSI/WTB Complex, University of Dundee, Dow Street, Dundee, DD1 5EH, UK

View larger version (25K): [in a new window]   Fig. 1. Expression of myofibrillar muscle-specific protein mRNAs at different temperatures according to somite stage. (A) -actin, (B) myosin heavy chain (MyHC), (C) troponin T (TnT), (D) troponin I (TnI), (E) muscle creatine kinase (CK-M) (F) troponin C (TnC). Blue, 4°C; green, 7°C; red, 10°C.

View larger version (19K): [in a new window]   Fig. 2. Comparison of the regression lines of muscle-specific protein expression with somite stage, together with the regression lines of myotube and myofibril synthesis (from Hall and Johnston, 2003) against somite stage.

View larger version (15K): [in a new window]   Fig. 3. Multiple sequence alignment of the deduced amino acid sequence of Atlantic cod Gadus morhua troponin I (TnI), with those of Atlantic salmon Salmo salar (sequence identity 62%) and Atlantic herring Clupea harengus (sequence identity 55%). The actin\Troponin C (TnC) binding site is boxed. Identical regions are shaded.

View larger version (18K): [in a new window]   Fig. 4. Multiple sequence alignment of the deduced amino acid sequence of Atlantic cod Gadus morhua troponin T (TnT), with those of Atlantic salmon Salmo salar (sequence identity 86%) and zebrafish Danio rerio (sequence identity 81%). A region of low homology near to the N terminus is boxed. Identical regions are shaded.

View larger version (30K): [in a new window]   Fig. 8. Whole-mount in situ hybridization of muscle-specific protein mRNAs in developing cod embryos at 4°C at sequential somite stages. Patterns at 7°C and 10°C were identical (Fig. 1).MyHC, myosin heavy chain; TnT, troponin T; TnI, troponin I; CK-M, muscle creatine kinase; TnC, troponin C. Scale bar, 200 µm.

View larger version (12K): [in a new window]   Fig. 5. Multiple sequence alignment of the deduced amino acid sequence of Atlantic cod Gadus morhua troponin C (TnC), with those of rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar (sequence identity 83% in both cases). Ca2+ binding sites II, III and IV are boxed. Identical regions are shaded.

View larger version (19K): [in a new window]   Fig. 6. Multiple sequence alignment of the deduced amino acid sequence of Atlantic cod Gadus morhua creatine kinase (CK-M), with those of Mozambique tilapia Oreochromis mossabicus (sequence identity 89%) and zebrafish Danio rerio (sequence identity 87%). The active site motif is boxed. Identical regions are shaded.

View larger version (25K): [in a new window]   Fig. 7. Multiple sequence alignment of the deduced amino acid sequence of Atlantic cod Gadus morhua MyoD, with those of zebrafish Danio rerio and rainbow trout Oncorhynchus mykiss (sequence identity 64% in both cases). Basic and helix–loop–helix domains are boxed. Identical regions are shaded.

View larger version (109K): [in a new window]   Fig. 9. MyoD expression in the developing somites of a 15-somite embryo. Expression can be seen the posteriormost seven somites, and in a single band in the presomitic mesoderm at the site of imminent somite formation. The anteriormost somites are by this time unstained for MyoD. Arrowhead, most recent somite furrow. nm, notochord/mesoderm boundary; Ad, unstained adaxial cells. Scale bar, 100 µm.

View larger version (42K): [in a new window]   Fig. 10. RT-PCR analysis of MyoD expression in different stage embryos developing at different temperatures. At all temperatures, MyoD transcripts are switched on as the somites began to form. Note that despite the lack of in situ staining for MyoD beyond approximately the 35-somite stage (Fig. 10), transcripts are still present. L15 is a 60S ribosomal subunit `housekeeping' gene, expressed at all temperatures and in all stages, used as an internal standard. The positions of marker nucleotides (bp) are shown at left.

View larger version (35K): [in a new window]   Fig. 11. MyoD expression in 5-somite (A–C) and 35-somite (D–F) embryos at 4, 7 and 10°C. Note that the 7 and 10°C embryos are shorter than the 4°C embryos, reflecting differences in the extent of epiboly between temperature groups (Hall and Johnston, 2003). However, the expression of MyoD in relation to somite development remains constant. Scale bar, 300 µm.

View larger version (19K): [in a new window]   Fig. 12. Phylogenetic relationships of the vertebrate MyoD sequences published to date. Note that the cod sequence clusters with the seabream MyoD2 sequence. Similarities are also evident in the in situ expression patterns. The tree was constructed in PHYLIP (Felsenstein 1995) by the neighbour-joining method. Node numbers refer to the percentage of bootstrap trials supporting a clade. Bootstrap confidence is based on 1000 pseudoreplications. The amino acid sequence for the single myogenic factor (AMD1) of the ascidian Halocynthia roretzi (accession no. D13507) was used as the outgroup. Other accession numbers are seabream Sparus aurata MyoD1, AF478568, MyoD2 AF478569; blue tilapia Oreochromis aureus, AF270790; Atlantic cod Gadus morhua, AF329903; pig Sus scrofa, U12574; human Homo sapiens, NM_002478; sheep Ovis aries, X62102; rat Rattus norvegicus, M84176; mouse Mus musculus, XM_124916; chicken Gallus gallus, L34006; Cotornix cotornix, L16686; Xenopus laevis xlmf1, M31116, xlmf25 M31118; rainbow trout Oncorhynchus mykiss MyoD1, X75798, MyoD2, Z46924; zebrafish Danio rerio, NM_131262; common carp Cyprinus carpio, AB012882.