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First published online September 14, 2007
Journal of Experimental Biology 210, 3461-3472 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.009183

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FoxK1 splice variants show developmental stage-specific plasticity of expression with temperature in the tiger pufferfish

Jorge M. O. Fernandes^1,2, Matthew G. MacKenzie^1,3, James R. Kinghorn¹ and Ian A. Johnston^1,*

¹ School of Biology, University of St Andrews, St Andrews, Fife KY16 8LB, UK
² Department of Fisheries and Natural Sciences, Bodø University College, No-8049 Bodø, Norway
³ School of Life Sciences Research, University of Dundee, Dundee DD1 5EH, UK

View larger version (57K): [in this window] [in a new window]   Fig. 1. Multiple sequence alignment of FoxK1 protein sequences from T. rubripes (TFoxK1) and their orthologues from mouse (MFoxK1) and zebrafish (DFoxK1). The accession numbers are as follows: TFoxK1- (GenPept: AAS68037), TFoxK1- (GenPept: AAS68039), TFoxK1- (GenPept: AAS68038), MFoxK1- (GenPept: NP_951031), MFoxK1-ß (GenPept: NP_034942) and DFoxK1- (GenPept: NP_956196). Amino acid residues identical to TFoxK1- are represented by a dot, gaps are indicated by a dash, global identity is marked by an asterisk and conserved substitutions are represented by a colon. The forkhead and forkhead-associated domains are indicated by red and blue rectangles, respectively.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Structure of the FoxK1 transcripts from T. rubripes and zebrafish. (A) Gene structure of FoxK1 from T. rubripes (TFoxK1) and schematic representation of its three alternatively spliced transcripts (, and ). Exons (blue boxes) are indicated by Arabic numbers and introns labelled with Roman numerals; introns, yellow boxes; stop codons, asterisks. The positions of the primers used for qPCR are indicated by arrows (not to scale). Red bars represent the location of the riboprobes used for in situ hybridisation. Scale bar, 400 bp. (B) Exon–intron structure of zebrafish FoxK1 (DFoxK1; GenBank: NM_199902). Scale bar, 1.5 kb. Introns I and II of DFoxk1 are not depicted to scale; the size of these introns is 4.7 kb and 10.5 kb, respectively. The last exon comprises a 463 kb untranslated region (white rectangle).

View larger version (36K): [in this window] [in a new window]   Fig. 3. Partial synteny map of the genomic neighbourhood of FoxK1 in T. rubripes, zebrafish and mouse. Synteny between TFoxK1 located in scaffold 40 and the corresponding regions in chromosomes 5 and 17 of mouse (A) and chromosomes 1 and 3 of zebrafish (B). Genes are represented by pointed coloured bars. Broken lines drawn between chromosomes indicate homology between individual genes. Genes that are coloured light green have no known orthologues in the genomic regions shown.

View larger version (62K): [in this window] [in a new window]   Fig. 4. Spatial expression pattern of TFoxK1- in T. rubripes embryos during development at 18°C. TFoxK1- transcripts were detected by whole-mount in situ hybridisation with a DIG-labelled cRNA probe. The different developmental stages are (A) 57 h.p.f., bud stage of gastrulation, (B) 63 h.p.f., 5-somite stage, (C) 76 h.p.f., 10-somite stage, (D,E) 86 h.p.f., 16-somite stage. A sense (negative) control of a 10-somite stage embryo is shown in F. Anterior is top. Scale bar, 0.5 mm. h, tubular heart; hb, hindbrain; l, lens; mb, midbrain; ov, optic vesicle; psm, pre-somitic mesoderm; r, retina; rmf, rhombo-mesencephalic fissure; s, somites; tb, tail bud.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Influence of embryonic temperature on expression of TFoxK1 splice variants. Relative expression of TFoxK1-, TFoxK1- and TFoxK1- during development of T. rubripes embryos at 15°C (blue bars), 18°C (green bars) or 21°C (red bars). The different developmental stages are indicated on the bottom x axis. Unfortunately, the 28-somite-stage samples from embryos incubated at 15°C were not available, owing to technical difficulties. For each data point, TFoxK1 mRNA levels measured by qPCR were normalised (using RNA polymerase II as standard) and expressed as ratios in relation to the expression level of TFoxK1- during mid-gastrulation at 18°C. Values are means ± s.e.m. (N=3).

View larger version (23K): [in this window] [in a new window]   Fig. 6. Tissue distribution of the three alternatively spliced transcripts of FoxK1 in adult T. rubripes. Relative expression levels of TFoxK1- (blue bars), TFoxK1- (yellow bars) or TFoxK1- (red bars) in fast muscle (WM), slow muscle (RM), heart (H), liver (L), skin (SK), brain (B) and gonads (G) are represented as ratios against the expression level of TFoxK1- in fast muscle. RNA polymerase II was used as standard. Values are means ± s.e.m. (N=4). Statistically significant differences (P<0.05) between splice variants within a tissue are labelled with the same letter; significant differences in expression of an isoform across different tissues are indicated by the same number. (Inset) Expression of FoxK1- in fast myotomal muscle of adult fish. Transverse sections of fast muscle were immunostained for FoxK1-, a molecular marker of muscle progenitor cells (MPCs). FoxK1--positive nuclei (indicated by arrowheads) occupy a sublaminar position that is typical of MPCs.