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First published online August 9, 2007
Journal of Experimental Biology 210, 2781-2794 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.006981

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Temperature influences the coordinated expression of myogenic regulatory factors during embryonic myogenesis in Atlantic salmon (Salmo salar L.)

Daniel J. Macqueen¹, David Robb² and Ian A. Johnston^1,*

¹ Gatty Marine Laboratory, School of Biology, University of St Andrews, St Andrews, Fife, KY16 8LB, UK
² EWOS Innovation, EWOS Ltd, Westfield, Bathgate, EH48 3BP, UK

View larger version (49K): [in this window] [in a new window]   Fig. 1. Amino acid sequence alignment of all known salmonid myoD family members with a myoD orthologue in the cephalochordate-amphioxus (amphi-myoD1). Accession numbers are identical to those described in phylogeny methodology. Within the alignment, dots mark residues identical to smyoD1a and dashes indicate a gap. Below the alignment, asterisks show residues conserved globally and colons highlight conserved amino acid substitutions. The basic (red underlined) and helix-loop-helix (blue underlined) regions are highly conserved. Also shown is the cysteine–histidine rich region (green underlined) and helix-III domain, where residues identical and different to smyoD1a are respectively highlighted yellow and red. A highly conserved region present in vertebrate myoD genes, (but not other MRFs and less so with amphi-myoD) is shown in bold italics on smyoD1a.

View larger version (17K): [in this window] [in a new window]   Fig. 2. Intron–exon structures of all known Atlantic salmon myoD family member genes. Each gene is represented by three exons (black boxes) and two introns (lines). The known sizes of exons and introns are shown. Introns with a double line are of unknown size (but in each case are greater than 1 kb). However, all intron–exon boundaries are supported experimentally.

View larger version (6K): [in this window] [in a new window]   Fig. 3. Maximum likelihood tree produced in PHYML (see Materials and methods) displaying the phylogenetic relationships of all known salmonid myoD family proteins. The cephalochordate myoD homologue amphi-myoD1 is included as an outgroup. All confidence values are shown and were obtained using 500 bootstrap psuedoreplicates. The scale bar shows the number of substitutions per site.

View larger version (46K): [in this window] [in a new window]   Fig. 4. Schematic diagram illustrating the mRNA expression patterns recorded for six MRF genes, s-smlc1 and Pax7 in the most anterior somite of the Atlantic salmon developmental stages numbered 1–6 (shown at the top of the figure). (A) smyoD1a, (B) smyf5, (C) smyoD1b, (D) smyoD1c, (E) smyoG, (F) sMRF4, (G) s-smlc1, (H) Pax7. The left of each box shows an expression field as viewed from either a dorsal of lateral perspective (indicated) of the most anterior somite using DIC optics. The right of each box shows a corresponding cross section through the region of expression in that somite. ad, adaxial cells; ecl, external cell layer; n, notochord; nt, neural tube; spc, spinal cord; So-1, somite 1.

View larger version (64K): [in this window] [in a new window]   Fig. 5. mRNA expression patterns of myoD family member genes and a schematic representation of s-smlc1 expression during the 30–45 ss. Numbers i-v represent the following cRNA probes: (i) smyf5, (ii) smyoD1a, (iii) smyoG, (iv) sMRF4 and (v) s-smlc1. Letters A–D represent specific regions marked on schematic drawings of whole embryos from different stages (diagram on left side of figure). Images on the left of each box are dorsal perspective flat-mounts. Images on the right of each box are 18 µm cryosections from the region identified by a black arrow. Red arrows show the position of the last somite. *Magnified flat-mount of smyf5 expression to show the lack of expression in adaxial myoblasts. Abbreviations are as in Fig. 4 with the addition of s, somite; psm, presomitic mesoderm. Scale bars, 50 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 6. mRNA expression patterns of myoD family member genes, s-smlc1 and Pax7 at the 65 ss and during the eyed stage. The numbering and lettering system is equivalent to that used in Fig. 5, except that (vi) represents the Pax7 cRNA probe. Images on the left of each box are lateral perspective flat-mounts, except for Av–Avi and Bv–Bvi, which are mounted from the dorsal perspective. Images on the right of each box are 18 µm cryosections from the region identified by a black arrow. Abbreviations are as in Figs 4 and 5 and red arrows as in Fig. 5. Scale bars, 50 µm.

View larger version (5K): [in this window] [in a new window]   Fig. 7. Rate of somitogenesis in Atlantic salmon reared at three embryonic temperatures, 2°C, 5°C and 8°C. First order linear regressions were fitted to each group and the following equations were obtained: 2°C: somite number=–54.32+ 0.0696xh.p.f., R2=99.5% (N=23). 5°C: somite number=–54.7+0.123xh.p.f., R2=98.2% (N=21). 8°C: somite number=–51.8+0.192xh.p.f., R2=99.3%(N=28).

View larger version (60K): [in this window] [in a new window]   Fig. 8. Representative images showing the temperature associated heterochronies observed in smyf5 expression. Images A and B correspond to the boxed regions labelled A and B on the schematic embryos below and temperatures (2°C and 8°C) are identified above each panel. Flat-mount images are viewed from the dorsal perspective except where otherwise indicated (by an asterisk) in boxes in B. Somite number is shown as s(n) where s=somite, n=number and the most caudal somite is the last numerically. Abbreviations are as in Fig. 5. Scale bars, 50 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 9. Representative images showing the temperature associated heterochronies observed in sMRF4 expression. Lettering and numbering system is the same as in Fig. 8. Flat-mount images are viewed from the dorsal perspective. Scale bars, 50 µm.

View larger version (55K): [in this window] [in a new window]   Fig. 10. Representative images showing the temperature-associated heterochronies observed in s-smlc1 expression. Lettering and numbering system is the same as in Fig. 8. Flat-mount images are viewed from the dorsal perspective. The blue arrow shows the last somite considered to have s-smlc1 expression. Abbreviations are as in Fig. 5. Scale bars, 50 µm.