Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb
The olive flounder (Paralichthys olivaceus) Pax3 homologues are highly conserved, encode multiple isoforms and show unique expression patterns Shuang Jiao a, Xungang Tan a,⁎, Qian Wang a,b, Meijie Li a,b, Shao Jun Du c a b c
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, Shandong, People's Republic of China University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, 701 E. Pratt St, Baltimore, MD 21202, USA
a r t i c l e
i n f o
Article history: Received 5 June 2014 Received in revised form 3 October 2014 Accepted 3 October 2014 Available online 15 October 2014 Keywords: Pax3a Pax3b Spliced isoforms Somite Muscle Neural crest
a b s t r a c t Pax genes encode a highly conserved family of transcription factors that play crucial roles in the formation of tissues and organs during development. Pax3 plays crucial roles in patterning of the dorsal central nervous system (CNS), neural crest and skeletal muscle. Here, we identified two spliced isoforms of Pax3a and three spliced isoforms of Pax3b and characterized their expression patterns. Both of flounder Pax3a-1 and Pax3b-1 contain the conserved paired domain (PD), an octapeptide motif (OP), and a paired type homeodomain (HD). But the PD domain in Pax3a-2 and Pax3b-3 is not intact and there is no HD in Pax3b-2 and Pax3b-3. Pax3a and Pax3b show distinct temporal expression patterns during embryogenesis. Whole-mount in situ hybridization demonstrates that Pax3a and Pax3b are expressed in overlapping patterns in the dorsal central nervous system, with some subtle regional differences between the two genes. In addition, Pax3a is scattered in the somites while Pax3b is specifically expressed in the newly forming somites. RT-PCR results have shown that there were different expression patterns between the different isoforms. These results indicate subfunction partitioning of the duplicated Pax3 genes. The duplicated Pax3 may provide additional flexibility in fine-tuning neurogenesis and somitogenesis. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Pax genes encode a highly conserved family of transcription factors that play crucial roles in the formation of tissues and organs during development. The most characteristic feature of these genes is the presence of a highly conserved 128 amino acid DNA-binding domain, known as the paired box domain (PD). However, the different Pax proteins contain a variable number of additional conserved sequences that include an octapeptide motif (OP) and a paired type homeodomain (HD) (Gruss and Walther, 1992; Chi and Epstein, 2002; Lang et al., 2007). According to the additional domain, Pax have been divided into four subfamilies, Pax1/9, Pax2/5/8, Pax3/7 and Pax4/6, and the last two subfamilies contain both PD and HD (Gruss and Walther, 1992). The PD is a bipartite DNA-binding domain composed of two subdomains (PAI and RED), with a similar folding pattern as the HD (Czerny et al., 1993; Xu et al., 1995; Jun and Desplan, 1996). During vertebrate evolution, Pax3 and Pax7 were duplicated from a unique ancestral Pax3/7 gene (Paixao-Cortes et al., 2013). Amphibians, birds and mammals have one Pax3 and one Pax7 gene, whereas there were two Pax3 and two Pax7 in zebrafish, which has a two round ⁎ Corresponding author. Tel.: +86 532 82898559. E-mail address: [email protected] (X. Tan).
http://dx.doi.org/10.1016/j.cbpb.2014.10.002 1096-4959/© 2014 Elsevier Inc. All rights reserved.
genome duplication during its evolution (Thompson et al., 2008). Alternative splicing variants of Pax3 and Pax7 have been identified in human, mouse, zebrafish and salmon, although their specific distribution and function remain poorly understood (Vogan et al., 1996; Ziman et al., 1997; Seo et al., 1998; Barber et al., 1999; Gotensparre et al., 2006). The existence of multiple isoforms is likely to increase the functional diversity of Pax proteins. Pax3 plays important roles in central nervous system, neural crest and paraxial mesoderm patterning and differentiation during embryogenesis (Mansouri and Gruss, 1998; Schubert et al., 2001) as well as in muscle homeostasis and repair in adults (Buckingham, 2006; Le Grand and Rudnicki, 2007). During mouse embryonic development, Pax3 is expressed in the dorsal neural tube, the neural crest and the presomitic mesoderm as somites are formed and progressively restricted to dermomyotome as somites mature. Pax3 mutant mice (Splotch) have severe defects in dorsal neural tube closure, neural crest cell migration, and somitogenesis. In adult skeletal muscles of mouse, there were few Pax3 positive satellite cells (Relaix et al., 2006). During zebrafish embryogenesis, the expression patterns of Pax3a/Pax3b are very similar to their murine homologues, although several subtle differences, particularly with respect to neural crest and mesodermal expression are also detected (Seo et al., 1998; Minchin and Hughes, 2008; Minchin et al., 2013). Pax3 is sufficient to induce MyoD and Myf5 expression in vitro
8
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15
through directly binding and transactivating their enhancers (Maroto et al., 1997; Bajard et al., 2006; Hu et al., 2008). Olive flounder (Paralichthys olivaceus) is an important fish in aquaculture of Asian region, but information regarding Pax3 in olive flounder has remained unknown. In the present study, the open reading frames (ORFs) encoding Pax3a and Pax3b were cloned from embryos of olive flounder and their expression patterns were investigated during embryonic development. In flounder, Pax3a and Pax3b are highly conserved, encode multiple spliced isoforms and show distinct and overlapping spatiotemporal expression pattern, which provides novel insights into the functional conservation and divergence of the duplicated Pax3 genes.
2.4. Whole mount in situ hybridization
2. Materials and methods
Using zebrafish homologous genes as queries, we BLAST with olive flounder genomic sequences, and predicted the possible ORFs of Pax3a and Pax3b. Two distinct Pax3 genes, named Pax3a and Pax3b, were identified in olive flounder by RT-PCR, respectively. The genomic structures of Pax3a and Pax3b without the untranslated regions (UTRs) are shown in Fig. 1. Two spliced isoforms of Pax3a and three spliced isoforms of Pax3b were obtained and named as Pax3a-1, Pax3a-2, Pax3b-1, Pax3b2, and Pax3b-3, in accordance with their length from long to short, respectively (Fig. 1). The open reading frame (ORF) of Pax3a-1 spans about 16.9 kb and contains nine exons and eight introns, while that of Pax3b-1 spans about 23.4 kb and contains nine exons and eight introns (Fig. 1). The existence of Pax3a isoforms was due to alternative splicing of splice donor site at the end of exon3 which causes a deletion of 72 nucleotides (Fig. 2A). Similarly, the existence of Pax3b isoforms was due to alternative splicing of splice donor site at the end of exon4 which causes a premature stop codon (Fig. 2B-b) without/with alternative splicing of splice donor site at the end of exon3 which causes a deletion of 72 nucleotides (Fig. 2B-a).
2.1. Fish and embryos culture Olive flounder were cultured at a local fish farm (Rongcheng, Weihai, China) under controlled conditions (photoperiod, 14: 10-h light-dark; temperature, 15 ± 1 °C; seawater; aeration). Fish were fed a commercial particle diet twice a day. The fertilized eggs were obtained by mixing sperm and eggs collected from matured males and females by artificially gently stripping, respectively. The embryos were cultured at 15 ± 1 °C in 1 m3 tank under the same condition as the fish culture.
2.2. Phylogenetic analysis Phylogenetic trees were constructed using the Neighbor Joining method (Saitou and Nei, 1987) with the MEGA6.0 software (Tamura et al., 2013). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method (Zuckerkandl and Pauling, 1965) and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated.
2.3. RT-PCR Total RNA was isolated from olive flounder embryos using TRIzol reagent (Invitrogen Life Technologies, Inc., Carlsbad, CA) following the manufacturer's instruction. After DNase treatment, 1 μg RNA was reverse transcribed with Oligo(dT) primers and M-MLV reverse transcriptase (Promega, USA). For isolation of Pax3a and Pax3b, PCR was performed to isolate the predicted ORFs using gene specific primers (Table S1) and the template was cDNA prepared from embryos at segmentation stage. 2 × Es Taq Master Mix (CWBIO, Ltd. China) was used. PCR cycles were as follows: 94 °C for 2 min, 38 cycles (Pax3a-1, Pax3a-2, Pax3b-1, Pax3b-2, and Pax3b-3) of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s. The target fragment was purified, cloned into pEASY-T3 (TransGen Biotech, China) vector and sequenced. For the analysis of the expressions in different stages, PCR was performed using 2 × Es Taq Master Mix (CWBIO, Ltd. China). PCR cycles were as follows: 94 °C for 2 min, 38 cycles (Pax3a-1, Pax3a-2, Pax3b-1, Pax3b-2, and Pax3b-3) or 30 cycles (β-actin) of 94 °C for 30 s, 55 °C or 58 °C for 30 s, and 72 °C for X s (1 kb/30 s). PCR products were analyzed by electrophoresis followed by ethidium bromide staining. The levels of β-actin mRNA were measured and used as an internal control. Sequences of primers used were listed in Table S2 and primer positions were indicated in Fig. 4.
One-color or two-color in situ hybridization using digoxigeninlabeled or fluorescein-labeled antisense riboprobes was performed as reported previously (Tan and Du, 2002). The specificity and strength of the riboprobes were validated by dot-blot analysis. Images were captured with a Leica DFC420C camera mounted on a Leica DMLB2 microscope (Leica, Wetzlar, Germany). 3. Results 3.1. Identification multiple spliced isoforms of Pax3a and Pax3b genes in olive flounder
3.2. Structural and phylogenetic analysis of Pax3a and Pax3b in olive flounder The ORFs of Pax3a-1 and Pax3a-2 are 1485 bp and 1413 bp, which encode a protein of 494 amino acid (aa) and 470 aa, respectively. The ORFs of Pax3b-1, Pax3b-2 and Pax3b-3 are 1482 bp, 603 bp and 531 bp, which encode a protein of 493 aa, 200 aa and 176 aa, respectively (Figs. 1, 3 and 4). Analysis of the deduced amino acid sequences suggested that Pax3a-1 and Pax3b-1 contain a paired box domain (PD) with specific DNA binding properties, an octapeptide motif (OP) and a homeodomain (HD) (Figs. 3 and 4). However, Pax3a-2 lacks 24 aa in the PD domain, Pax3b-2 lacks the whole HD domain, and Pax3b-3 lacks both of them. The proteins encoded by flounder Pax3a-1 and Pax3b-1 genes share 84.62% and 71.88% similarity with their corresponding zebrafish homologues, respectively (data not shown). We performed phylogenetic analysis using the deduced amino acid sequences of teleost and other Pax3 species. It was shown that flounder Pax3a and Pax3b were placed into the teleost Pax3a and Pax3b clades, respectively (Fig. 5), indicating that they are co-orthologs to the mammalian Pax3 gene. In addition, all teleost Pax3a and Pax3b are grouped into two different clades. This result provides further evidence that an additional gene duplication event occurred in Ray-finned Fishes. 3.3. The two Pax3 genes exhibit specific spatiotemporal expression patterns The temporal expression patterns of olive flounder Pax3a and Pax3b were determined by RT-PCR using gene-specific primers (Table S2 and Fig. 4) and in situ hybridization during early development. Pax3a and Pax3b were expressed from the early stage of embryogenesis (Fig. 6). The expression patterns of Pax3a-1 and Pax3a-2 were basically consistent, except for 36 h after neurula stage and hatching stages,
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15
9
Fig. 1. Genomic organization of olive flounder Pax3a and Pax3b genes without the UTRs. Exons are shown as rectangles and introns are shown as lines. The sizes of exons and introns are shown.
when Pax3a-1 transcript level was much weaker than that of Pax3a-2. Although Pax3b-2 and Pax3b-3 showed similar expression patterns, the expression patterns of Pax3b-1 showed some differences from Pax3b-2 and Pax3b-3. We did not detect Pax3b-1 expression at the hatching stage. Pax3a and Pax3b mRNA signals were also detected by whole mount in situ hybridization during early development. At neurula stage, transcripts for Pax3a were detected as bilateral stripes along the lateral margins of the neural plate during early stages of neurulation (Fig. 7A) and presumptive brain region. Its expression levels at bilateral stripes gradually decreased, contrarily its expression level at brain gradually
increased as embryos develop (Fig. 7B–H). From 12 h after neurula stage (Fig. 7D), the Pax3a expression area in the brain could be divided into diencephalon, midbrain, and rhombomere. Further analysis indicated that Pax3a signals were also scattered in the somites (Fig. S1, Fig. 10). Besides, weak expression levels of pax3a could be detected in the neural tube. At neurula stage, transcripts of Pax3b were detected in the presumptive brain and bilateral stripes along the lateral margins of the neural plate. Moreover, its expression in the presumptive brain showed a much stronger signal which resembled segment-like stripes which extend towards the midline from the longitudinal stripes (Fig. 8A).
Fig. 2. Alternative splice variants of olive flounder Pax3a and Pax3b. (A) The nucleotide sequences at the end of exon 3 and beginning of intron 4 of Pax3a. (B-a) The nucleotide sequences at the end of exon 3 and beginning of intron 4 of Pax3b. (B-b) The nucleotide sequences at the end of exon 4 of Pax3b. Splice donor sites and stop codon are marked in red color. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
10
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15
Fig. 3. Amino acid sequence alignment of olive flounder (f) Pax3a (GenBank accession no. KM675813 and KM675814 for 3a-1 and 3a-2) and Pax3b (GenBank accession no. KM675815, KM675816, and KM675817 for 3b-1, 3b-2, and 3b-3) with human (h) pax3 (NP852122.1). Sequence alignment was obtained by the ClustalW method. Paired box domain (PD), an octapeptide (OP) motif and homeodomain (HD) are indicated in box. Identical amino acid residues are indicated by dots and the differing amino acids are shown. Gaps required for optimal alignments are represented by hyphens.
Fig. 4. Schematic overview of olive flounder Pax3a and Pax3b putative isoforms. Abbreviations: N, amino terminal; C, carboxy terminal; PD, paired box domain; OP, octapeptide; HD, homeodomian. The forward and reverse arrows denote forward and reverse primers for analyzing each gene expression, respectively. R3, R4, and R5 were located in 3' UTR of their corresponding genes.
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15
11
Fig. 5. Phylogenetic tree of Pax3a and Pax3b. The tree was rooted with fruitfly Gsb and Gsb-n. Values on branches are the percentages of times that the two clades grouped as sisters. The following sequences were used: Fruitfly gooseberry (Gsb): AAF47315.2; Fruitfly gooseberry-neuro (Gsb-n): NP_523862.1; Amphioxus Pax3/7: XP_002610806.1; Xenopus Pax3: NP_001006776.2; Chicken Pax3: ENSGALP00000008414; Mouse Pax3: ENSMUSP00000084320; Human Pax3 (isoform c): ENSP00000343052; Stickleback Pax3: ENSGACP00000018514; Fugu Pax3a: ENSTRUP00000034277; Fugu Pax3b: ENSTRUP00000023137; Tetraodon Pax3a: ENSTNIP00000018338; Tetraodon Pax3b: ENSTNIP00000017329; Medaka Pax3a: ENSORLP00000019947; Medaka Pax3b: ENSORLP00000011326; Zebrafish Pax3a: NP_571352.1; Zebrafish Pax3b: ACN88554.1; olive flounder Pax3a-1: GenBank accession no. KM675813 and Pax3b-1: GenBank accession no.KM675815.
Concomitant with the formation of the neural keel the transverse band fused at the dorsal midline (Fig. 8B). At 4–8 h after neurula stage, additional signal was detected in the somites. As embryonic development proceeds, signals in somites extended posterior and signals in bilateral stripes gradually became weak (Fig. 8B–H). We deduced that Pax3a
was expressed in the newly forming somites. Besides, weak expression levels of pax3b could be detected in the neural tube, too. Although Pax3a and Pax3b had some subtle regional different expression patterns before hatching (Fig. 7 and 8), they showed similar expression patterns at hatching stage. Both of them were detected in the brain region, neural tube, and dermomyotome, however, no signals were detected in the formed somites at hatching stage (Fig. 9). 3.4. Pax3a and Pax3b genes exhibit different and specific expression patterns in the somites To further compare the expression patterns of Pax3a and Pax3b in the somites, two-color in situ hybridization was performed using Diglabeled Pax3a or Pax3b antisense riboprobe and fluorescein-labeled MyoD antisense riboprobe at 8 h after neurula stage. MyoD, expressed in developing somites, is essential for initiating the skeletal muscle program in the embryos (Weintraub, 1993). Both Pax3a and Pax3b mRNA signals were detected in presumptive brain region, lateral neural plate, and weakly in neural tube. Notably, Pax3b and MyoD mRNA signals showed overlapped expression in the newly forming somites (Fig. 10H, K, K′). On the contrary, Pax3a and MyoD mRNA signals were not overlapped and some Pax3a signals were scattered in the somites (Fig. 10E, E′, E″), suggesting that the Pax3b gene, not Pax3a gene, is specifically expressed in the newly forming somites. 4. Discussion
Fig. 6. Expression patterns of olive flounder Pax3a and Pax3b in early development by RTPCR. RNA samples were prepared at the indicated time points. 4–44 h represents hours after neurula stage. Twenty to thirty embryos were used for RNA isolation in each time point. N.C., negative control.
In this study, we identify two co-orthologs of mammalian Pax3 in olive flounder, namely Pax3a and Pax3b. Pax3 and Pax7 are two different genes in mammals, birds and xenopus, whereas a single ancestral gene Pax3/7 is retained as cognates of vertebrate Pax3 and Pax7 in protostomes, ascidians and amphioxus (Paixao-Cortes et al., 2013). An additional fish specific genome duplication (FSGD) has taken place in the
12
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15
Fig. 7. Spatial distribution of Pax3a transcripts during early embryonic stages. Head to the left, dorsal view. A, embryos at about neurula stage. B–H, 4 h (B), 8 h (C), 12 h (D), 16 h (E), 20 h (F), 24 h (G) and 36 h (H) after neurula stage. I, Cross-section from the dash line of B. Black arrowhead indicates isthmus of decreasing Pax3a expression that separates midbrain and hindbrain boundary. White arrowhead indicates the presumptive first rhombomere primordia. Black arrow indicates the primordial cerebellum. Black asterisk indicates the lateral neural plate. White asterisk indicates cranial neural crest (NC). Bars: 50 μm (A–H) and 20 μm (I).
teleost lineage before the beginning of the teleost radiation (Venkatesh, 2003). In zebrafish, medaka (Oryzias latipes), tetraodon (Tetraodon nigroviridis), and fugu (Takifugu rubripes), the four duplicated genes, Pax3a/Pax3b and Pax7a/Pax7b have been already characterized (Minchin and Hughes, 2008; Minchin et al., 2013). Our findings of olive flounder Pax3a and Pax3b also support the additional FSGD theory. Interestingly, two alternatively spliced isoforms of Pax3a and three alternatively spliced isoforms of Pax3b were discovered in flounder. All of these alternative splice forms are due to use of alternative splice donor sites (Fig. 2). In Pax3a-2, 24 amino acids (GMFSWEIRDKLLKDGICDRNNVPS) are missing between helix 4 and helix 6 of C-terminal subdomain due to the use of an alternation 5′ donor site downstream of exon3. This 24 amino acid deletion results in the absence of the entire helix 5 and partial helix 6. This region is supposed to form a helix–turn–helix motif that plays a major role in the DNA-binding of this subdomain (Xu et al., 1995). In Pax3b, the two additional spliced isoforms Pax3b-2 and Pax3b-3 are produced by a premature stop codon between the octapeptide motif and homeodomain without or with a 24 amino acid deletion as that in Pax3a-2. Both paired box domain and homeodomain are required for the DNA binding properties of Pax3 (Galli et al., 2008). So, Pax3a-2, Pax3b-2 and Pax3b-3 might lack the DNA binding properties, and they might have a role in negative modulation of target genes recognized by Pax3a and/or Pax3b. Further studies on the function of these isoform would be more interesting. Pax3 transcripts are initially detected at the neural plate stage during gastrulation (Goulding et al., 1991; Seo et al., 1998; Maczkowiak et al., 2010). Our results (Fig. 6) are consistent with the data in the zebrafish, Xenopus and mouse homologues. Although different spliced isoforms of
Pax3a and Pax3b displayed specific expression patterns, the early onset of Pax3a and Pax3b expression was consistent with the data in the zebrafish, Xenopus and mouse homologues. However, our in situ hybridization data showed some differences from the corresponding zebrafish homologues. In zebrafish, both Pax3a and Pax3b mRNAs coincide in cranial neural crest (NC), dorsal central nervous system (CNS), and lateral neural plate. Besides, as embryos grow, Pax3a signals are also detected in the somites. Somatic Pax3b displays a dynamic expression pattern from all somites to dermomytome (Minchin and Hughes, 2008; Minchin et al., 2013). Furthermore, in zebrafish, somitic Pax3a was expressed in all somites at 15 and 18 somites, nevertheless, somitic pax3b mRNA was restricted to the anterior somites at 15 and 18 somites (Minchin et al., 2013). In flounder embryos, the Pax3a transcripts are expressed in somites, and it will be scattered in the somite as it matured. Moreover, we also noted several differences, particularly with respect to the somites between olive flounder Pax3b and zebrafish Pax3b. Unlike in zebrafish, olive flounder Pax3b signals specifically appear in the newly forming somites. In flounder, Pax3a seems to be scattered in somites, whereas pax3b shows very unique expression in newly forming somites. This provides a unique opportunity to assess the subfunctionalization of duplicated pax3 genes in ray-finned fish. Although olive flounder and zebrafish are both ray-finned fishes, the habits and size are obviously different. The flounder is an ocean-dwelling flatfish species that is found in coastal lagoons and estuaries of the Northern Atlantic and Pacific Oceans. It has very strong muscle explosive force, which could instantaneously burst into the water surface from the water bottom. We deduce that flounder have very strong musculature, especially fast twitch
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15
13
Fig. 8. Spatial distribution of Pax3b transcripts during early embryonic stages. Head to the left, dorsal view. A, embryos at about neurula stage. B–H, 4 h (B), 8 h (C), 12 h (D), 16 h (E), 20 h (F), 24 h (G) and 36 h (H) after neurula stage. I, Cross-section from the dash line of D. Black arrowhead indicates isthmus of decreasing Pax3b expression that separates midbrain and hindbrain boundary. White arrowhead indicates the presumptive first rhombomere primordia. Black arrow indicates the primordial cerebellum. White arrow indicates the newly forming somites. Black asterisk indicates the lateral neural plate. White asterisk indicates cranial neural crest (NC). Bars: 50 μm (A–H) and 5 μm (I).
muscles, which are important for rapid movements. Also, the flounder can grow much larger than zebrafish and more muscle cells will be needed during their growth, which might be derived from muscle satellite cells. Therefore, during flounder embryogenesis, Pax3a and Pax3b play different roles in the patterning of skeletal muscle. Perhaps the different expression patterns of Pax3a and Pax3b in somites between flounder and zebrafish play a key role in causing their different movements and growth.
In summary, we have characterized Pax3a and Pax3b in olive flounder and shown their specific expression pattern during embryogenesis. Our discovery of several alternative spliced isoforms of these two genes further enriches our understanding of Pax gene in teleost. The olive flounder is one of the most commercially important marine fish species for aquaculture in East Asia. Because of its good taste and nutritional value, the olive flounder farming industry is now expanding rapidly throughout the coastal areas of North China. Promoting olive flounder
Fig. 9. Pax3a and Pax3b mRNA show similar expression patterns at hatching stage. Head to the left; head, dorsal view; trunk, lateral view. A, Whole-mount in situ hybridization showing Pax3a expression in the central nervous system. B, C, D, Cross-section from the dash lines of A in sequential order from left to right. E, Whole-mount in situ hybridization showing Pax3b expression in the central nervous system. F, G, H, Cross-section from the dash lines of E in sequential order from left to right. Black arrowhead indicates isthmus of decreasing Pax3a or Pax3b expression that separates midbrain and hindbrain boundary. Black arrow indicates the primordial cerebellum. White arrowhead indicates the presumptive first rhombomere primordia. Black star indicates the neural tube. Black diamond indicates the dermomyotome. Abbreviation: d, diencephalon; r, rhombomere; v, the fourth ventricle. Bars: 50 μm (A, E) and 5 μm (B, C, D, F, G, H).
14
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15
Fig. 10. Two-color in situ hybridization showing Pax3a or Pax3b (blue) and MyoD (red) expression. Embryos at 8 h after neurula stage. A, B, G, H, Head to the left, dorsal view. C, D, E, E′, E″, F, F′, I, J, K, K′, L, transverse cryosections have dorsal towards the top. A, B, Two-color in situ hybridization showing Pax3a expression in the lateral neural plate not in the newly forming somites. Sarcomere area is magnified in B. C–F, Cross-section from the dash lines of A in sequential order from left to right (E, E′ and E″ are cross-sections from sarcomere area. F and F ′ are cross-sections from posterior area of sarcomere area,). G, H, Two-color in situ hybridization showing Pax3b expression in the newly forming somites. Sarcomere area is magnified in H. I–L, Cross-section from the dash lines of G in sequential order from left to right (both K and K′ are cross-sections from sarcomere area). White arrow indicates the newly forming somites. Black asterisk indicates the lateral neural plate. Bars: 50 μm (A, G), 10 μm (B, H) and 5 μm (C–F, I–L). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
growth rate, especially the muscle growth rates is of great economic significance. Our study has presented the opportunity to further elucidate the functional role of Pax3a and Pax3b in the formation of neural system and myogenesis in olive flounder. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbpb.2014.10.002.
Acknowledgments We thank Prof. Songlin Chen (Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences) for providing genomic sequences of olive flounder Pax3a and Pax3b. This study was supported by the Natural Science Foundation of China (31128017), the National High Technology Research and Development Program of China (863 Program, 2012AA092203), and the Natural Science Foundation of China (30970423).
References Bajard, L., Relaix, F., Lagha, M., Rocancourt, D., Daubas, P., Buckingham, M.E., 2006. A novel genetic hierarchy functions during hypaxial myogenesis: Pax3 directly activates Myf5 in muscle progenitor cells in the limb. Genes Dev. 20, 2450–2464. Barber, T.D., Barber, M.C., Cloutier, T.E., Friedman, T.B., 1999. PAX3 gene structure, alternative splicing and evolution. Gene 237, 311–319. Buckingham, M., 2006. Myogenic progenitor cells and skeletal myogenesis in vertebrates. Curr. Opin. Genet. Dev. 16, 525–532. Chi, N., Epstein, J.A., 2002. Getting your Pax straight: Pax proteins in development and disease. Trends Genet. 18, 41–47. Czerny, T., Schaffner, G., Busslinger, M., 1993. DNA sequence recognition by Pax proteins: bipartite structure of the paired domain and its binding site. Genes Dev. 7, 2048–2061. Felsenstein, J., 1985. Confidence-limits on phylogenies — an approach using the bootstrap. Evolution 39, 783–791.
Galli, L.M., Knight, S.R., Barnes, T.L., Doak, A.K., Kadzik, R.S., Burrus, L.W., 2008. Identification and characterization of subpopulations of Pax3 and Pax7 expressing cells in developing chick somites and limb buds. Dev. Dyn. 237, 1862–1874. Gotensparre, S.M., Andersson, E., Wargelius, A., Hansen, T., Johnston, I.A., 2006. Insight into the complex genetic network of tetraploid Atlantic salmon (Salmo salar L.): description of multiple novel Pax-7 splice variants. Gene 373, 8–15. Goulding, M.D., Chalepakis, G., Deutsch, U., Erselius, J.R., Gruss, P., 1991. Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. EMBO J. 10, 1135–1147. Gruss, P., Walther, C., 1992. Pax in development. Cell 69, 719–722. Hu, P., Geles, K.G., Paik, J.H., DePinho, R.A., Tjian, R., 2008. Codependent activators direct myoblast-specific MyoD transcription. Dev. Cell 15, 534–546. Jun, S., Desplan, C., 1996. Cooperative interactions between paired domain and homeodomain. Development 122, 2639–2650. Lang, D., Powell, S.K., Plummer, R.S., Young, K.P., Ruggeri, B.A., 2007. PAX genes: roles in development, pathophysiology, and cancer. Biochem. Pharmacol. 73, 1–14. Le Grand, F., Rudnicki, M.A., 2007. Skeletal muscle satellite cells and adult myogenesis. Curr. Opin. Cell Biol. 19, 628–633. Maczkowiak, F., Mateos, S., Wang, E., Roche, D., Harland, R., Monsoro-Burq, A.H., 2010. The Pax3 and Pax7 paralogs cooperate in neural and neural crest patterning using distinct molecular mechanisms, in Xenopus laevis embryos. Dev. Biol. 340, 381–396. Mansouri, A., Gruss, P., 1998. Pax3 and Pax7 are expressed in commissural neurons and restrict ventral neuronal identity in the spinal cord. Mech. Dev. 78, 171–178. Maroto, M., Reshef, R., Munsterberg, A.E., Koester, S., Goulding, M., Lassar, A.B., 1997. Ectopic Pax-3 activates MyoD and Myf-5 expression in embryonic mesoderm and neural tissue. Cell 89, 139–148. Minchin, J.E., Hughes, S.M., 2008. Sequential actions of Pax3 and Pax7 drive xanthophore development in zebrafish neural crest. Dev. Biol. 317, 508–522. Minchin, J.E., Williams, V.C., Hinits, Y., Low, S., Tandon, P., Fan, C.M., Rawls, J.F., Hughes, S.M., 2013. Oesophageal and sternohyal muscle fibres are novel Pax3-dependent migratory somite derivatives essential for ingestion. Development 140, 2972–2984. Paixao-Cortes, V.R., Salzano, F.M., Bortolini, M.C., 2013. Evolutionary history of chordate PAX genes: dynamics of change in a complex gene family. PLoS ONE 8, e73560. Relaix, F., Montarras, D., Zaffran, S., Gayraud-Morel, B., Rocancourt, D., Tajbakhsh, S., Mansouri, A., Cumano, A., Buckingham, M., 2006. Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells. J. Cell Biol. 172, 91–102. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Schubert, F.R., Tremblay, P., Mansouri, A., Faisst, A.M., Kammandel, B., Lumsden, A., Gruss, P., Dietrich, S., 2001. Early mesodermal phenotypes in splotch suggest a role for Pax3 in the formation of epithelial somites. Dev. Dyn. 222, 506–521.
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15 Seo, H.C., Saetre, B.O., Havik, B., Ellingsen, S., Fjose, A., 1998. The zebrafish Pax3 and Pax7 homologues are highly conserved, encode multiple isoforms and show dynamic segment-like expression in the developing brain. Mech. Dev. 70, 49–63. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Tan, X., Du, S.J., 2002. Differential expression of two MyoD genes in fast and slow muscles of gilthead seabream (Sparus aurata). Dev. Genes Evol. 212, 207–217. Thompson, J.A., Zembrzycki, A., Mansouri, A., Ziman, M., 2008. Pax7 is requisite for maintenance of a subpopulation of superior collicular neurons and shows a diverging expression pattern to Pax3 during superior collicular development. BMC Dev. Biol. 8, 62. Venkatesh, B., 2003. Evolution and diversity of fish genomes. Curr. Opin. Genet. Dev. 13, 588–592.
15
Vogan, K.J., Underhill, D.A., Gros, P., 1996. An alternative splicing event in the Pax-3 paired domain identifies the linker region as a key determinant of paired domain DNAbinding activity. Mol. Cell. Biol. 16, 6677–6686. Weintraub, H., 1993. The MyoD family and myogenesis: redundancy, networks, and thresholds. Cell 75, 1241–1244. Xu, W., Rould, M.A., Jun, S., Desplan, C., Pabo, C.O., 1995. Crystal structure of a paired domain-DNA complex at 2.5 A resolution reveals structural basis for Pax developmental mutations. Cell 80, 639–650. Ziman, M.R., Fletcher, S., Kay, P.H., 1997. Alternate Pax7 transcripts are expressed specifically in skeletal muscle, brain and other organs of adult mice. Int. J. Biochem. Cell Biol. 29, 1029–1036. Zuckerkandl, E., Pauling, L., 1965. Evolutionary divergence and convergence in proteins. In: Bryson, V., Vogel, H.J. (Eds.), Evolving Genes and Proteins. Academic Press, New York, pp. 97–166.
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb
The olive flounder (Paralichthys olivaceus) Pax3 homologues are highly conserved, encode multiple isoforms and show unique expression patterns Shuang Jiao a, Xungang Tan a,⁎, Qian Wang a,b, Meijie Li a,b, Shao Jun Du c a b c
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, Shandong, People's Republic of China University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, 701 E. Pratt St, Baltimore, MD 21202, USA
a r t i c l e
i n f o
Article history: Received 5 June 2014 Received in revised form 3 October 2014 Accepted 3 October 2014 Available online 15 October 2014 Keywords: Pax3a Pax3b Spliced isoforms Somite Muscle Neural crest
a b s t r a c t Pax genes encode a highly conserved family of transcription factors that play crucial roles in the formation of tissues and organs during development. Pax3 plays crucial roles in patterning of the dorsal central nervous system (CNS), neural crest and skeletal muscle. Here, we identified two spliced isoforms of Pax3a and three spliced isoforms of Pax3b and characterized their expression patterns. Both of flounder Pax3a-1 and Pax3b-1 contain the conserved paired domain (PD), an octapeptide motif (OP), and a paired type homeodomain (HD). But the PD domain in Pax3a-2 and Pax3b-3 is not intact and there is no HD in Pax3b-2 and Pax3b-3. Pax3a and Pax3b show distinct temporal expression patterns during embryogenesis. Whole-mount in situ hybridization demonstrates that Pax3a and Pax3b are expressed in overlapping patterns in the dorsal central nervous system, with some subtle regional differences between the two genes. In addition, Pax3a is scattered in the somites while Pax3b is specifically expressed in the newly forming somites. RT-PCR results have shown that there were different expression patterns between the different isoforms. These results indicate subfunction partitioning of the duplicated Pax3 genes. The duplicated Pax3 may provide additional flexibility in fine-tuning neurogenesis and somitogenesis. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Pax genes encode a highly conserved family of transcription factors that play crucial roles in the formation of tissues and organs during development. The most characteristic feature of these genes is the presence of a highly conserved 128 amino acid DNA-binding domain, known as the paired box domain (PD). However, the different Pax proteins contain a variable number of additional conserved sequences that include an octapeptide motif (OP) and a paired type homeodomain (HD) (Gruss and Walther, 1992; Chi and Epstein, 2002; Lang et al., 2007). According to the additional domain, Pax have been divided into four subfamilies, Pax1/9, Pax2/5/8, Pax3/7 and Pax4/6, and the last two subfamilies contain both PD and HD (Gruss and Walther, 1992). The PD is a bipartite DNA-binding domain composed of two subdomains (PAI and RED), with a similar folding pattern as the HD (Czerny et al., 1993; Xu et al., 1995; Jun and Desplan, 1996). During vertebrate evolution, Pax3 and Pax7 were duplicated from a unique ancestral Pax3/7 gene (Paixao-Cortes et al., 2013). Amphibians, birds and mammals have one Pax3 and one Pax7 gene, whereas there were two Pax3 and two Pax7 in zebrafish, which has a two round ⁎ Corresponding author. Tel.: +86 532 82898559. E-mail address: [email protected] (X. Tan).
http://dx.doi.org/10.1016/j.cbpb.2014.10.002 1096-4959/© 2014 Elsevier Inc. All rights reserved.
genome duplication during its evolution (Thompson et al., 2008). Alternative splicing variants of Pax3 and Pax7 have been identified in human, mouse, zebrafish and salmon, although their specific distribution and function remain poorly understood (Vogan et al., 1996; Ziman et al., 1997; Seo et al., 1998; Barber et al., 1999; Gotensparre et al., 2006). The existence of multiple isoforms is likely to increase the functional diversity of Pax proteins. Pax3 plays important roles in central nervous system, neural crest and paraxial mesoderm patterning and differentiation during embryogenesis (Mansouri and Gruss, 1998; Schubert et al., 2001) as well as in muscle homeostasis and repair in adults (Buckingham, 2006; Le Grand and Rudnicki, 2007). During mouse embryonic development, Pax3 is expressed in the dorsal neural tube, the neural crest and the presomitic mesoderm as somites are formed and progressively restricted to dermomyotome as somites mature. Pax3 mutant mice (Splotch) have severe defects in dorsal neural tube closure, neural crest cell migration, and somitogenesis. In adult skeletal muscles of mouse, there were few Pax3 positive satellite cells (Relaix et al., 2006). During zebrafish embryogenesis, the expression patterns of Pax3a/Pax3b are very similar to their murine homologues, although several subtle differences, particularly with respect to neural crest and mesodermal expression are also detected (Seo et al., 1998; Minchin and Hughes, 2008; Minchin et al., 2013). Pax3 is sufficient to induce MyoD and Myf5 expression in vitro
8
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15
through directly binding and transactivating their enhancers (Maroto et al., 1997; Bajard et al., 2006; Hu et al., 2008). Olive flounder (Paralichthys olivaceus) is an important fish in aquaculture of Asian region, but information regarding Pax3 in olive flounder has remained unknown. In the present study, the open reading frames (ORFs) encoding Pax3a and Pax3b were cloned from embryos of olive flounder and their expression patterns were investigated during embryonic development. In flounder, Pax3a and Pax3b are highly conserved, encode multiple spliced isoforms and show distinct and overlapping spatiotemporal expression pattern, which provides novel insights into the functional conservation and divergence of the duplicated Pax3 genes.
2.4. Whole mount in situ hybridization
2. Materials and methods
Using zebrafish homologous genes as queries, we BLAST with olive flounder genomic sequences, and predicted the possible ORFs of Pax3a and Pax3b. Two distinct Pax3 genes, named Pax3a and Pax3b, were identified in olive flounder by RT-PCR, respectively. The genomic structures of Pax3a and Pax3b without the untranslated regions (UTRs) are shown in Fig. 1. Two spliced isoforms of Pax3a and three spliced isoforms of Pax3b were obtained and named as Pax3a-1, Pax3a-2, Pax3b-1, Pax3b2, and Pax3b-3, in accordance with their length from long to short, respectively (Fig. 1). The open reading frame (ORF) of Pax3a-1 spans about 16.9 kb and contains nine exons and eight introns, while that of Pax3b-1 spans about 23.4 kb and contains nine exons and eight introns (Fig. 1). The existence of Pax3a isoforms was due to alternative splicing of splice donor site at the end of exon3 which causes a deletion of 72 nucleotides (Fig. 2A). Similarly, the existence of Pax3b isoforms was due to alternative splicing of splice donor site at the end of exon4 which causes a premature stop codon (Fig. 2B-b) without/with alternative splicing of splice donor site at the end of exon3 which causes a deletion of 72 nucleotides (Fig. 2B-a).
2.1. Fish and embryos culture Olive flounder were cultured at a local fish farm (Rongcheng, Weihai, China) under controlled conditions (photoperiod, 14: 10-h light-dark; temperature, 15 ± 1 °C; seawater; aeration). Fish were fed a commercial particle diet twice a day. The fertilized eggs were obtained by mixing sperm and eggs collected from matured males and females by artificially gently stripping, respectively. The embryos were cultured at 15 ± 1 °C in 1 m3 tank under the same condition as the fish culture.
2.2. Phylogenetic analysis Phylogenetic trees were constructed using the Neighbor Joining method (Saitou and Nei, 1987) with the MEGA6.0 software (Tamura et al., 2013). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method (Zuckerkandl and Pauling, 1965) and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated.
2.3. RT-PCR Total RNA was isolated from olive flounder embryos using TRIzol reagent (Invitrogen Life Technologies, Inc., Carlsbad, CA) following the manufacturer's instruction. After DNase treatment, 1 μg RNA was reverse transcribed with Oligo(dT) primers and M-MLV reverse transcriptase (Promega, USA). For isolation of Pax3a and Pax3b, PCR was performed to isolate the predicted ORFs using gene specific primers (Table S1) and the template was cDNA prepared from embryos at segmentation stage. 2 × Es Taq Master Mix (CWBIO, Ltd. China) was used. PCR cycles were as follows: 94 °C for 2 min, 38 cycles (Pax3a-1, Pax3a-2, Pax3b-1, Pax3b-2, and Pax3b-3) of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s. The target fragment was purified, cloned into pEASY-T3 (TransGen Biotech, China) vector and sequenced. For the analysis of the expressions in different stages, PCR was performed using 2 × Es Taq Master Mix (CWBIO, Ltd. China). PCR cycles were as follows: 94 °C for 2 min, 38 cycles (Pax3a-1, Pax3a-2, Pax3b-1, Pax3b-2, and Pax3b-3) or 30 cycles (β-actin) of 94 °C for 30 s, 55 °C or 58 °C for 30 s, and 72 °C for X s (1 kb/30 s). PCR products were analyzed by electrophoresis followed by ethidium bromide staining. The levels of β-actin mRNA were measured and used as an internal control. Sequences of primers used were listed in Table S2 and primer positions were indicated in Fig. 4.
One-color or two-color in situ hybridization using digoxigeninlabeled or fluorescein-labeled antisense riboprobes was performed as reported previously (Tan and Du, 2002). The specificity and strength of the riboprobes were validated by dot-blot analysis. Images were captured with a Leica DFC420C camera mounted on a Leica DMLB2 microscope (Leica, Wetzlar, Germany). 3. Results 3.1. Identification multiple spliced isoforms of Pax3a and Pax3b genes in olive flounder
3.2. Structural and phylogenetic analysis of Pax3a and Pax3b in olive flounder The ORFs of Pax3a-1 and Pax3a-2 are 1485 bp and 1413 bp, which encode a protein of 494 amino acid (aa) and 470 aa, respectively. The ORFs of Pax3b-1, Pax3b-2 and Pax3b-3 are 1482 bp, 603 bp and 531 bp, which encode a protein of 493 aa, 200 aa and 176 aa, respectively (Figs. 1, 3 and 4). Analysis of the deduced amino acid sequences suggested that Pax3a-1 and Pax3b-1 contain a paired box domain (PD) with specific DNA binding properties, an octapeptide motif (OP) and a homeodomain (HD) (Figs. 3 and 4). However, Pax3a-2 lacks 24 aa in the PD domain, Pax3b-2 lacks the whole HD domain, and Pax3b-3 lacks both of them. The proteins encoded by flounder Pax3a-1 and Pax3b-1 genes share 84.62% and 71.88% similarity with their corresponding zebrafish homologues, respectively (data not shown). We performed phylogenetic analysis using the deduced amino acid sequences of teleost and other Pax3 species. It was shown that flounder Pax3a and Pax3b were placed into the teleost Pax3a and Pax3b clades, respectively (Fig. 5), indicating that they are co-orthologs to the mammalian Pax3 gene. In addition, all teleost Pax3a and Pax3b are grouped into two different clades. This result provides further evidence that an additional gene duplication event occurred in Ray-finned Fishes. 3.3. The two Pax3 genes exhibit specific spatiotemporal expression patterns The temporal expression patterns of olive flounder Pax3a and Pax3b were determined by RT-PCR using gene-specific primers (Table S2 and Fig. 4) and in situ hybridization during early development. Pax3a and Pax3b were expressed from the early stage of embryogenesis (Fig. 6). The expression patterns of Pax3a-1 and Pax3a-2 were basically consistent, except for 36 h after neurula stage and hatching stages,
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15
9
Fig. 1. Genomic organization of olive flounder Pax3a and Pax3b genes without the UTRs. Exons are shown as rectangles and introns are shown as lines. The sizes of exons and introns are shown.
when Pax3a-1 transcript level was much weaker than that of Pax3a-2. Although Pax3b-2 and Pax3b-3 showed similar expression patterns, the expression patterns of Pax3b-1 showed some differences from Pax3b-2 and Pax3b-3. We did not detect Pax3b-1 expression at the hatching stage. Pax3a and Pax3b mRNA signals were also detected by whole mount in situ hybridization during early development. At neurula stage, transcripts for Pax3a were detected as bilateral stripes along the lateral margins of the neural plate during early stages of neurulation (Fig. 7A) and presumptive brain region. Its expression levels at bilateral stripes gradually decreased, contrarily its expression level at brain gradually
increased as embryos develop (Fig. 7B–H). From 12 h after neurula stage (Fig. 7D), the Pax3a expression area in the brain could be divided into diencephalon, midbrain, and rhombomere. Further analysis indicated that Pax3a signals were also scattered in the somites (Fig. S1, Fig. 10). Besides, weak expression levels of pax3a could be detected in the neural tube. At neurula stage, transcripts of Pax3b were detected in the presumptive brain and bilateral stripes along the lateral margins of the neural plate. Moreover, its expression in the presumptive brain showed a much stronger signal which resembled segment-like stripes which extend towards the midline from the longitudinal stripes (Fig. 8A).
Fig. 2. Alternative splice variants of olive flounder Pax3a and Pax3b. (A) The nucleotide sequences at the end of exon 3 and beginning of intron 4 of Pax3a. (B-a) The nucleotide sequences at the end of exon 3 and beginning of intron 4 of Pax3b. (B-b) The nucleotide sequences at the end of exon 4 of Pax3b. Splice donor sites and stop codon are marked in red color. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
10
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15
Fig. 3. Amino acid sequence alignment of olive flounder (f) Pax3a (GenBank accession no. KM675813 and KM675814 for 3a-1 and 3a-2) and Pax3b (GenBank accession no. KM675815, KM675816, and KM675817 for 3b-1, 3b-2, and 3b-3) with human (h) pax3 (NP852122.1). Sequence alignment was obtained by the ClustalW method. Paired box domain (PD), an octapeptide (OP) motif and homeodomain (HD) are indicated in box. Identical amino acid residues are indicated by dots and the differing amino acids are shown. Gaps required for optimal alignments are represented by hyphens.
Fig. 4. Schematic overview of olive flounder Pax3a and Pax3b putative isoforms. Abbreviations: N, amino terminal; C, carboxy terminal; PD, paired box domain; OP, octapeptide; HD, homeodomian. The forward and reverse arrows denote forward and reverse primers for analyzing each gene expression, respectively. R3, R4, and R5 were located in 3' UTR of their corresponding genes.
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15
11
Fig. 5. Phylogenetic tree of Pax3a and Pax3b. The tree was rooted with fruitfly Gsb and Gsb-n. Values on branches are the percentages of times that the two clades grouped as sisters. The following sequences were used: Fruitfly gooseberry (Gsb): AAF47315.2; Fruitfly gooseberry-neuro (Gsb-n): NP_523862.1; Amphioxus Pax3/7: XP_002610806.1; Xenopus Pax3: NP_001006776.2; Chicken Pax3: ENSGALP00000008414; Mouse Pax3: ENSMUSP00000084320; Human Pax3 (isoform c): ENSP00000343052; Stickleback Pax3: ENSGACP00000018514; Fugu Pax3a: ENSTRUP00000034277; Fugu Pax3b: ENSTRUP00000023137; Tetraodon Pax3a: ENSTNIP00000018338; Tetraodon Pax3b: ENSTNIP00000017329; Medaka Pax3a: ENSORLP00000019947; Medaka Pax3b: ENSORLP00000011326; Zebrafish Pax3a: NP_571352.1; Zebrafish Pax3b: ACN88554.1; olive flounder Pax3a-1: GenBank accession no. KM675813 and Pax3b-1: GenBank accession no.KM675815.
Concomitant with the formation of the neural keel the transverse band fused at the dorsal midline (Fig. 8B). At 4–8 h after neurula stage, additional signal was detected in the somites. As embryonic development proceeds, signals in somites extended posterior and signals in bilateral stripes gradually became weak (Fig. 8B–H). We deduced that Pax3a
was expressed in the newly forming somites. Besides, weak expression levels of pax3b could be detected in the neural tube, too. Although Pax3a and Pax3b had some subtle regional different expression patterns before hatching (Fig. 7 and 8), they showed similar expression patterns at hatching stage. Both of them were detected in the brain region, neural tube, and dermomyotome, however, no signals were detected in the formed somites at hatching stage (Fig. 9). 3.4. Pax3a and Pax3b genes exhibit different and specific expression patterns in the somites To further compare the expression patterns of Pax3a and Pax3b in the somites, two-color in situ hybridization was performed using Diglabeled Pax3a or Pax3b antisense riboprobe and fluorescein-labeled MyoD antisense riboprobe at 8 h after neurula stage. MyoD, expressed in developing somites, is essential for initiating the skeletal muscle program in the embryos (Weintraub, 1993). Both Pax3a and Pax3b mRNA signals were detected in presumptive brain region, lateral neural plate, and weakly in neural tube. Notably, Pax3b and MyoD mRNA signals showed overlapped expression in the newly forming somites (Fig. 10H, K, K′). On the contrary, Pax3a and MyoD mRNA signals were not overlapped and some Pax3a signals were scattered in the somites (Fig. 10E, E′, E″), suggesting that the Pax3b gene, not Pax3a gene, is specifically expressed in the newly forming somites. 4. Discussion
Fig. 6. Expression patterns of olive flounder Pax3a and Pax3b in early development by RTPCR. RNA samples were prepared at the indicated time points. 4–44 h represents hours after neurula stage. Twenty to thirty embryos were used for RNA isolation in each time point. N.C., negative control.
In this study, we identify two co-orthologs of mammalian Pax3 in olive flounder, namely Pax3a and Pax3b. Pax3 and Pax7 are two different genes in mammals, birds and xenopus, whereas a single ancestral gene Pax3/7 is retained as cognates of vertebrate Pax3 and Pax7 in protostomes, ascidians and amphioxus (Paixao-Cortes et al., 2013). An additional fish specific genome duplication (FSGD) has taken place in the
12
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15
Fig. 7. Spatial distribution of Pax3a transcripts during early embryonic stages. Head to the left, dorsal view. A, embryos at about neurula stage. B–H, 4 h (B), 8 h (C), 12 h (D), 16 h (E), 20 h (F), 24 h (G) and 36 h (H) after neurula stage. I, Cross-section from the dash line of B. Black arrowhead indicates isthmus of decreasing Pax3a expression that separates midbrain and hindbrain boundary. White arrowhead indicates the presumptive first rhombomere primordia. Black arrow indicates the primordial cerebellum. Black asterisk indicates the lateral neural plate. White asterisk indicates cranial neural crest (NC). Bars: 50 μm (A–H) and 20 μm (I).
teleost lineage before the beginning of the teleost radiation (Venkatesh, 2003). In zebrafish, medaka (Oryzias latipes), tetraodon (Tetraodon nigroviridis), and fugu (Takifugu rubripes), the four duplicated genes, Pax3a/Pax3b and Pax7a/Pax7b have been already characterized (Minchin and Hughes, 2008; Minchin et al., 2013). Our findings of olive flounder Pax3a and Pax3b also support the additional FSGD theory. Interestingly, two alternatively spliced isoforms of Pax3a and three alternatively spliced isoforms of Pax3b were discovered in flounder. All of these alternative splice forms are due to use of alternative splice donor sites (Fig. 2). In Pax3a-2, 24 amino acids (GMFSWEIRDKLLKDGICDRNNVPS) are missing between helix 4 and helix 6 of C-terminal subdomain due to the use of an alternation 5′ donor site downstream of exon3. This 24 amino acid deletion results in the absence of the entire helix 5 and partial helix 6. This region is supposed to form a helix–turn–helix motif that plays a major role in the DNA-binding of this subdomain (Xu et al., 1995). In Pax3b, the two additional spliced isoforms Pax3b-2 and Pax3b-3 are produced by a premature stop codon between the octapeptide motif and homeodomain without or with a 24 amino acid deletion as that in Pax3a-2. Both paired box domain and homeodomain are required for the DNA binding properties of Pax3 (Galli et al., 2008). So, Pax3a-2, Pax3b-2 and Pax3b-3 might lack the DNA binding properties, and they might have a role in negative modulation of target genes recognized by Pax3a and/or Pax3b. Further studies on the function of these isoform would be more interesting. Pax3 transcripts are initially detected at the neural plate stage during gastrulation (Goulding et al., 1991; Seo et al., 1998; Maczkowiak et al., 2010). Our results (Fig. 6) are consistent with the data in the zebrafish, Xenopus and mouse homologues. Although different spliced isoforms of
Pax3a and Pax3b displayed specific expression patterns, the early onset of Pax3a and Pax3b expression was consistent with the data in the zebrafish, Xenopus and mouse homologues. However, our in situ hybridization data showed some differences from the corresponding zebrafish homologues. In zebrafish, both Pax3a and Pax3b mRNAs coincide in cranial neural crest (NC), dorsal central nervous system (CNS), and lateral neural plate. Besides, as embryos grow, Pax3a signals are also detected in the somites. Somatic Pax3b displays a dynamic expression pattern from all somites to dermomytome (Minchin and Hughes, 2008; Minchin et al., 2013). Furthermore, in zebrafish, somitic Pax3a was expressed in all somites at 15 and 18 somites, nevertheless, somitic pax3b mRNA was restricted to the anterior somites at 15 and 18 somites (Minchin et al., 2013). In flounder embryos, the Pax3a transcripts are expressed in somites, and it will be scattered in the somite as it matured. Moreover, we also noted several differences, particularly with respect to the somites between olive flounder Pax3b and zebrafish Pax3b. Unlike in zebrafish, olive flounder Pax3b signals specifically appear in the newly forming somites. In flounder, Pax3a seems to be scattered in somites, whereas pax3b shows very unique expression in newly forming somites. This provides a unique opportunity to assess the subfunctionalization of duplicated pax3 genes in ray-finned fish. Although olive flounder and zebrafish are both ray-finned fishes, the habits and size are obviously different. The flounder is an ocean-dwelling flatfish species that is found in coastal lagoons and estuaries of the Northern Atlantic and Pacific Oceans. It has very strong muscle explosive force, which could instantaneously burst into the water surface from the water bottom. We deduce that flounder have very strong musculature, especially fast twitch
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15
13
Fig. 8. Spatial distribution of Pax3b transcripts during early embryonic stages. Head to the left, dorsal view. A, embryos at about neurula stage. B–H, 4 h (B), 8 h (C), 12 h (D), 16 h (E), 20 h (F), 24 h (G) and 36 h (H) after neurula stage. I, Cross-section from the dash line of D. Black arrowhead indicates isthmus of decreasing Pax3b expression that separates midbrain and hindbrain boundary. White arrowhead indicates the presumptive first rhombomere primordia. Black arrow indicates the primordial cerebellum. White arrow indicates the newly forming somites. Black asterisk indicates the lateral neural plate. White asterisk indicates cranial neural crest (NC). Bars: 50 μm (A–H) and 5 μm (I).
muscles, which are important for rapid movements. Also, the flounder can grow much larger than zebrafish and more muscle cells will be needed during their growth, which might be derived from muscle satellite cells. Therefore, during flounder embryogenesis, Pax3a and Pax3b play different roles in the patterning of skeletal muscle. Perhaps the different expression patterns of Pax3a and Pax3b in somites between flounder and zebrafish play a key role in causing their different movements and growth.
In summary, we have characterized Pax3a and Pax3b in olive flounder and shown their specific expression pattern during embryogenesis. Our discovery of several alternative spliced isoforms of these two genes further enriches our understanding of Pax gene in teleost. The olive flounder is one of the most commercially important marine fish species for aquaculture in East Asia. Because of its good taste and nutritional value, the olive flounder farming industry is now expanding rapidly throughout the coastal areas of North China. Promoting olive flounder
Fig. 9. Pax3a and Pax3b mRNA show similar expression patterns at hatching stage. Head to the left; head, dorsal view; trunk, lateral view. A, Whole-mount in situ hybridization showing Pax3a expression in the central nervous system. B, C, D, Cross-section from the dash lines of A in sequential order from left to right. E, Whole-mount in situ hybridization showing Pax3b expression in the central nervous system. F, G, H, Cross-section from the dash lines of E in sequential order from left to right. Black arrowhead indicates isthmus of decreasing Pax3a or Pax3b expression that separates midbrain and hindbrain boundary. Black arrow indicates the primordial cerebellum. White arrowhead indicates the presumptive first rhombomere primordia. Black star indicates the neural tube. Black diamond indicates the dermomyotome. Abbreviation: d, diencephalon; r, rhombomere; v, the fourth ventricle. Bars: 50 μm (A, E) and 5 μm (B, C, D, F, G, H).
14
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15
Fig. 10. Two-color in situ hybridization showing Pax3a or Pax3b (blue) and MyoD (red) expression. Embryos at 8 h after neurula stage. A, B, G, H, Head to the left, dorsal view. C, D, E, E′, E″, F, F′, I, J, K, K′, L, transverse cryosections have dorsal towards the top. A, B, Two-color in situ hybridization showing Pax3a expression in the lateral neural plate not in the newly forming somites. Sarcomere area is magnified in B. C–F, Cross-section from the dash lines of A in sequential order from left to right (E, E′ and E″ are cross-sections from sarcomere area. F and F ′ are cross-sections from posterior area of sarcomere area,). G, H, Two-color in situ hybridization showing Pax3b expression in the newly forming somites. Sarcomere area is magnified in H. I–L, Cross-section from the dash lines of G in sequential order from left to right (both K and K′ are cross-sections from sarcomere area). White arrow indicates the newly forming somites. Black asterisk indicates the lateral neural plate. Bars: 50 μm (A, G), 10 μm (B, H) and 5 μm (C–F, I–L). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
growth rate, especially the muscle growth rates is of great economic significance. Our study has presented the opportunity to further elucidate the functional role of Pax3a and Pax3b in the formation of neural system and myogenesis in olive flounder. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cbpb.2014.10.002.
Acknowledgments We thank Prof. Songlin Chen (Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences) for providing genomic sequences of olive flounder Pax3a and Pax3b. This study was supported by the Natural Science Foundation of China (31128017), the National High Technology Research and Development Program of China (863 Program, 2012AA092203), and the Natural Science Foundation of China (30970423).
References Bajard, L., Relaix, F., Lagha, M., Rocancourt, D., Daubas, P., Buckingham, M.E., 2006. A novel genetic hierarchy functions during hypaxial myogenesis: Pax3 directly activates Myf5 in muscle progenitor cells in the limb. Genes Dev. 20, 2450–2464. Barber, T.D., Barber, M.C., Cloutier, T.E., Friedman, T.B., 1999. PAX3 gene structure, alternative splicing and evolution. Gene 237, 311–319. Buckingham, M., 2006. Myogenic progenitor cells and skeletal myogenesis in vertebrates. Curr. Opin. Genet. Dev. 16, 525–532. Chi, N., Epstein, J.A., 2002. Getting your Pax straight: Pax proteins in development and disease. Trends Genet. 18, 41–47. Czerny, T., Schaffner, G., Busslinger, M., 1993. DNA sequence recognition by Pax proteins: bipartite structure of the paired domain and its binding site. Genes Dev. 7, 2048–2061. Felsenstein, J., 1985. Confidence-limits on phylogenies — an approach using the bootstrap. Evolution 39, 783–791.
Galli, L.M., Knight, S.R., Barnes, T.L., Doak, A.K., Kadzik, R.S., Burrus, L.W., 2008. Identification and characterization of subpopulations of Pax3 and Pax7 expressing cells in developing chick somites and limb buds. Dev. Dyn. 237, 1862–1874. Gotensparre, S.M., Andersson, E., Wargelius, A., Hansen, T., Johnston, I.A., 2006. Insight into the complex genetic network of tetraploid Atlantic salmon (Salmo salar L.): description of multiple novel Pax-7 splice variants. Gene 373, 8–15. Goulding, M.D., Chalepakis, G., Deutsch, U., Erselius, J.R., Gruss, P., 1991. Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. EMBO J. 10, 1135–1147. Gruss, P., Walther, C., 1992. Pax in development. Cell 69, 719–722. Hu, P., Geles, K.G., Paik, J.H., DePinho, R.A., Tjian, R., 2008. Codependent activators direct myoblast-specific MyoD transcription. Dev. Cell 15, 534–546. Jun, S., Desplan, C., 1996. Cooperative interactions between paired domain and homeodomain. Development 122, 2639–2650. Lang, D., Powell, S.K., Plummer, R.S., Young, K.P., Ruggeri, B.A., 2007. PAX genes: roles in development, pathophysiology, and cancer. Biochem. Pharmacol. 73, 1–14. Le Grand, F., Rudnicki, M.A., 2007. Skeletal muscle satellite cells and adult myogenesis. Curr. Opin. Cell Biol. 19, 628–633. Maczkowiak, F., Mateos, S., Wang, E., Roche, D., Harland, R., Monsoro-Burq, A.H., 2010. The Pax3 and Pax7 paralogs cooperate in neural and neural crest patterning using distinct molecular mechanisms, in Xenopus laevis embryos. Dev. Biol. 340, 381–396. Mansouri, A., Gruss, P., 1998. Pax3 and Pax7 are expressed in commissural neurons and restrict ventral neuronal identity in the spinal cord. Mech. Dev. 78, 171–178. Maroto, M., Reshef, R., Munsterberg, A.E., Koester, S., Goulding, M., Lassar, A.B., 1997. Ectopic Pax-3 activates MyoD and Myf-5 expression in embryonic mesoderm and neural tissue. Cell 89, 139–148. Minchin, J.E., Hughes, S.M., 2008. Sequential actions of Pax3 and Pax7 drive xanthophore development in zebrafish neural crest. Dev. Biol. 317, 508–522. Minchin, J.E., Williams, V.C., Hinits, Y., Low, S., Tandon, P., Fan, C.M., Rawls, J.F., Hughes, S.M., 2013. Oesophageal and sternohyal muscle fibres are novel Pax3-dependent migratory somite derivatives essential for ingestion. Development 140, 2972–2984. Paixao-Cortes, V.R., Salzano, F.M., Bortolini, M.C., 2013. Evolutionary history of chordate PAX genes: dynamics of change in a complex gene family. PLoS ONE 8, e73560. Relaix, F., Montarras, D., Zaffran, S., Gayraud-Morel, B., Rocancourt, D., Tajbakhsh, S., Mansouri, A., Cumano, A., Buckingham, M., 2006. Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells. J. Cell Biol. 172, 91–102. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Schubert, F.R., Tremblay, P., Mansouri, A., Faisst, A.M., Kammandel, B., Lumsden, A., Gruss, P., Dietrich, S., 2001. Early mesodermal phenotypes in splotch suggest a role for Pax3 in the formation of epithelial somites. Dev. Dyn. 222, 506–521.
S. Jiao et al. / Comparative Biochemistry and Physiology, Part B 180 (2015) 7–15 Seo, H.C., Saetre, B.O., Havik, B., Ellingsen, S., Fjose, A., 1998. The zebrafish Pax3 and Pax7 homologues are highly conserved, encode multiple isoforms and show dynamic segment-like expression in the developing brain. Mech. Dev. 70, 49–63. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729. Tan, X., Du, S.J., 2002. Differential expression of two MyoD genes in fast and slow muscles of gilthead seabream (Sparus aurata). Dev. Genes Evol. 212, 207–217. Thompson, J.A., Zembrzycki, A., Mansouri, A., Ziman, M., 2008. Pax7 is requisite for maintenance of a subpopulation of superior collicular neurons and shows a diverging expression pattern to Pax3 during superior collicular development. BMC Dev. Biol. 8, 62. Venkatesh, B., 2003. Evolution and diversity of fish genomes. Curr. Opin. Genet. Dev. 13, 588–592.
15
Vogan, K.J., Underhill, D.A., Gros, P., 1996. An alternative splicing event in the Pax-3 paired domain identifies the linker region as a key determinant of paired domain DNAbinding activity. Mol. Cell. Biol. 16, 6677–6686. Weintraub, H., 1993. The MyoD family and myogenesis: redundancy, networks, and thresholds. Cell 75, 1241–1244. Xu, W., Rould, M.A., Jun, S., Desplan, C., Pabo, C.O., 1995. Crystal structure of a paired domain-DNA complex at 2.5 A resolution reveals structural basis for Pax developmental mutations. Cell 80, 639–650. Ziman, M.R., Fletcher, S., Kay, P.H., 1997. Alternate Pax7 transcripts are expressed specifically in skeletal muscle, brain and other organs of adult mice. Int. J. Biochem. Cell Biol. 29, 1029–1036. Zuckerkandl, E., Pauling, L., 1965. Evolutionary divergence and convergence in proteins. In: Bryson, V., Vogel, H.J. (Eds.), Evolving Genes and Proteins. Academic Press, New York, pp. 97–166.
