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Molecular characterization of three muscle alpha actin genes in mud loach (Misgurnus mizolepis; Cypriniformes)

Molecular characterization of three muscle alpha actin genes in mud loach (Misgurnus mizolepis;... Background: Teleosts represent unique features in the regulation of muscle development and growth, with a great deal of myogenic plasticity. Muscle actins are major components to compose muscle fibers, and they play essential roles in cellular mobility and other related functions. In order to understand isoform-dependent roles of muscle actins in the mud loach (Misgurnus mizolepis), this study was aimed to characterize gene structure and expression pattern of the three muscle actin isoforms (α-skeletal, α-cardiac, and α-smooth muscle actins) isolated from this benthic fish species. Results: Mud loach α-actin isoforms (ACTA1, ACTC1, and ACTA2) were fairly conserved in their primary structures and shared a high-sequence identity one another. At genomic level also, all the three isoforms exhibited the same exon-intron organization pattern characterized by eight translated exons. However, in mRNA expression patterns, these three actin isoforms were found to display an apparent isoform-dependency in tissue distribution (i.e., ACTA1 in the skeletal muscles, ACTC1 in the heart, and ACTA2 in the intestines) and developmental regulation (i.e., increased expression of ACTA1 and ACTC1 with the progress of myogenesis, and the significant elevation of ACTA2 during organ development in early larvae). Conclusion: Data from this study suggest that three muscle actin isoforms have undergone certain functional differentiation with regard to their roles in the development and adult musculature, in spite of high structural similarity. Genetic information on muscle actins obtained in this study could provide a useful base to extend our understanding on evolutionary diversification of the myogenic regulation in teleosts. Keywords: Mud loach Misgurnus mizolepis, Alpha actins, Gene structure, mRNA expression Background and amniote vertebrates is the indeterminate growth For the adaptation to aquatic locomotion, teleosts ex- pattern of fish muscles. Most teleosts display a contin- hibit the distinct separation of muscle fiber types (slow- ued growth throughout most of their lifespan, with body twitch fibers to red muscle and fast-twitch fibers to size and muscle mass increasing, albeit at a slow rate white muscle) into discrete layers in their striated mus- until mortality or senescence occurs (Johnston et al. cles, which has not been seen in other amniote verte- 2011). Muscle growth in teleosts is signified by the com- brates (Watabe 1999; Kiessling et al. 2006). White bined contribution of a long-lasting hyperplasia (increase muscle (glycolytic, anaerobic metabolism; for fast-start, in fiber number) as well as the hypertrophy (increase in burst swimming) and red muscle (oxidative metabolism; fiber size), which is hardly observable in many other for slow, sustained movement) occupy distinct axial re- higher vertebrates (Kiessling et al. 2006; Johnston et al. gions (Kiessling et al. 2006; Chong et al. 2009). Another 2011). Furthermore, metabolic and contractile character- striking difference in a skeletal musculature between fish istics of teleost muscles also represent significant plasti- city and flexibility with regard to not only environmental conditions (e.g., temperature (Hall et al. 2003; Johnston * Correspondence: yoonknam@pknu.ac.kr et al. 2008; Stickland et al. 1988; Johnston 2006)) but Department of Marine Bio-Materials and Aquaculture, Pukyong National University, Busan 48513, Korea © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 2 of 11 also reproductive status (e.g., gonad maturation development stages and adult tissues (Perrin and Ervasti (Johnston et al. 2011; Mathana et al. 2012)). 2010; Bertola et al. 2008). However, in contrast to rich Taken together, teleosts have developed unique or spe- information on mammalian counterparts, the compara- cialized features in the architecture and physiology of tive expression profiles of the muscle actin isoforms in a muscles during their evolutionary history, and such adap- given single species have remained to be further ex- tive processes undoubtedly have resulted in modifications plored in teleosts. of the genetic pathways modulating muscle growth and Mud loach (Misgurnus mizolepis Günther; Cyprini- functions in teleosts, as compared to terrestrial vertebrates formes) is an aquaculture-relevant food fish in Korea (Kim (Morita 2000; Mudalige et al. 2007). For this reason, iden- et al. 1994). This species has various advantageous merits tification of genetic determinants for muscle-specific as a candidate model system for muscle research in teleosts. genes from teleosts may be an important basis to study As a model fish, it possesses general merits such as easiness the evolutionary diversification of the musculature in the of laboratory maintenance, transparent embryonic develop- vertebrate lineage. Also, from an aquaculture viewpoint, ment, and relatively short generation time (Nam et al. myogenic genes are potentially of biotechnological interest 2000). Besides, mud loach undergoes characteristic changes from the perspective of growth regulation and quality con- in locomotory behavior during early larval development, trol of the flesh product. Understanding these key genes suggesting that this species is a good subject to visualize the could provide the base in order to develop future pro- interrelationships between the differentiation of different grams involving genetic manipulation and selection of de- muscle types and larval swimming behavior. Furthermore, sired growth trait(s) in commercially important fish with this species, dramatically induced muscle hyperplasia species (Johnston et al. 2008). has been demonstrated by a GH-transgenesis (Nam et al. Actins are one of major components of muscle tissues 2001; Nam et al. 2008), which can provide useful platform to compose the myofibrils of musculature. As an evolu- to examine differential regulation of genetic pathways asso- tionary conserved protein, actins play essential roles in ciated with muscle growth in hyperplasia/hypertrophy-ac- maintaining cytoskeletal structure, cellular mobility, cell celerated fishes. Taken together, our long-term goal is to list division and differentiation, intracellular movement, and this species as a research model for myogenesis of teleosts. contractile processes, which are associated with a wide This study, as an initial step toward our goal, was aimed to spectrum of physiological aspects in vertebrates (Perrin isolate genetic determinants for three representative muscle and Ervasti 2010). The actin multigene family has been actins (skeletal, cardiac, and smooth muscle types) from known to be evolved through duplication and divergence mud loach and to examine the isoform-dependent from a common ancestral gene, leading diverse isoforms expression patterns in adult tissues and developing embryos (and subisoforms) in the extant actin genes (Miwa et al. and larvae. 1991). Although actin isoforms share remarkably high sequence identity one another, each isoform exhibits dis- Methods tinct regulation pattern for its spatial and temporal ex- Molecular cloning of α-actin isoform cDNAs and genomic pression (Perrin and Ervasti 2010). Further, teleosts have genes been known to retain multiple paralog copies of actin From next-generation sequencing (NGS) database of mud isoform genes depending on taxonomic lineages as a re- loach whole fry transcriptome (unpublished data), expressed sult of a whole-genome duplication (WGD) event (Kim sequence tag clones showing significant homology to previ- and Nam 2009; Glasauer and Neuhauss 2014). It poten- ously known vertebrate muscle actins were collected. The tially implies that certain subfunctionalization and/or fragment sequences were assembled into contigs using neofunctionalization might have occurred in teleostean Sequencher software (Gene Codes, Ann Arbor, MI, USA) actin isoform groups (Venhatesh et al. 1996). Muscle ac- and subjected to NCBI GenBank BLASTx search to verify tins (alpha actins) in teleosts can be broadly classified their potential annotations as muscle alpha actin members. into three primary types, α-skeletal, α-cardiac, and α- From this pilot analysis, three distinct actin isoforms were smooth muscle actins. Alpha-skeletal (ACTA1) and α- identified to be potentially designated α-skeletal, α-cardiac, cardiac (ACTC1) actins are expressed in striated and α-smooth muscle actins, respectively. muscles, importantly involved in locomotory action of In order to get full-length complementary DNA (cDNA) skeletal muscles and cardiac contractility, respectively. for each isoform, vectorette polymerase chain reaction On the other hand, α-smooth muscle actin (ACTA2)is (PCR) was conducted to both 5′-and 3′-directions using expressed in vascular and visceral smooth muscles (Kim the excised phagemid stock of the mud loach fry whole and Nam 2009; Venhatesh et al. 1996). Mostly from body cDNA library (Agilent, Santa Clara CA, USA) as a mammalian studies, the expression of these three actin template for PCR amplification. Oligonucleotides used in isoforms has been known to be regulated through both this study were listed in Additional file 1: Table S1. PCR- isoform-specific and cooperative ways depending on isolated fragments of each isoform were cloned into Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 3 of 11 pGEM-T easy vector (Promega, Madison, WI, USA), se- [BW] = 30.1 ± 3.5 g) were selected. From each individual, quenced and assembled into contigs. Based on the contig the brain, eye, fin (caudal), gill, heart, intestine, kidney, liver, sequence for each actin isoform, full-length continuous muscle (skeletal muscle), spleen, skin, and gonad (ovary or fragment of each isoform was re-isolated from mud loach testis) were surgically removed. Upon removal, tissues were total RNA (mixed total RNA from muscles and internal or- immediately frozen on dry ice and stored at − 80 °C deep gans) by reverse transcription-PCR (RT-PCR). Amplified freezer until used for RNA extraction. RT-PCR products were purified and sequenced directly in In order to prepare embryo and larvae samples, artificial order to determine the representative cDNA sequence for induced spawning was performed according to the each α-actin isoform. methods described previously (Kim et al. 1994; Nam et al. Based on the cDNA sequence, genomic gene of each iso- 2001). Briefly, carp pituitary extract (Sigma-Aldrich, St. form was PCR-amplified from a mud loach genomic DNA Louis, MO, USA) was delivered to six female and six male template prepared with caudal fin. Amplified fragment(s) broodfish via intraperitoneal injection at the dose levels of for each isoform gene was directly sequenced at both direc- 10 μg/g BW and 2 μg/g BW, respectively. Pooled eggs tions by primer walking method. From the genomic gene (from three females based on egg quality) were divided sequence, the genome walking to 5′-upstream region was into three egg batches. Each egg batch was inseminated by carried out in order to identify putative non-translated sperm (saline-diluted) from each of three individual males exon. Genome walking was conducted using Universal in order to prepare three fertilized egg groups. Insemin- Genome Walker Kit (Clontech Laboratories Inc., Mountain ation was done using wet method. Fertilized eggs were View, CA, USA) according to the manufacturer’sinstruc- rinsed with clean tap water (1-μm-filtered) and placed on tions. Depending on isoform genes, two to five rounds of an incubator at 25 ± 1 °C with a constant aeration to ad- genome walking were carried out. Assembled 5′-upstream just the dissolved oxygen level to be 7 ± 1 ppm through- region for each isoform was re-isolated together with its out the experiment. Fertilization rates and hatching downstream coding region in a continuous fragment, and success of the each replicate group were higher than 98 finally, the representative sequence of each isoform gene and 85%, respectively. During the embryonic develop- was determined by direct sequencing. ment, approximately 300 embryos were randomly sam- pled at 0 (just fertilized), 2, 4, 6, 8, 12, 16, 20, 24, and 28 Bioinformatic sequence analysis (hatch-out) hours post fertilization (HPF). Information on Open reading frame (ORF) in each isoform cDNA was pre- the embryogenesis of mud loach can be referred to previ- dicted using the ORF finder tool in NCBI page (https:// ous report (Kim et al. 1987). After hatching, the hatched www.ncbi.nlm.nih.gov/orffinder/), and the ORF sequence larvae from each fertilization group were transferred to was deduced into amino acid sequence using the same tool. one of three larval incubation tanks (each 60-L rectangu- Homology of each isoform to previously known orthologs lar plastic container) equipped with thermostat-assisted was examined by BLAST searches against NCBI GenBank heater and aeration apparatus. Larvae were reared at the database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple same temperature as above. After yolk sac absorption sequence alignments of nucleotide and amino acid se- (around 60 h after hatch), larvae were fed with 100-μm quences were carried out using the ClustalW tool (http:// commercial diet powder for flounder larvae (Woosung www.genome.jp/tools-bin/clustalw). Physicochemical pa- Feed Cor., Korea) and live Artemia nauplii (INVE Aqua- rameters of protein (with deduced amino acid sequence) culture Inc., Salt Lake City, UT, USA). During the larval were computed using ExPASy ProtParam tool (http:// rearing period, about 100 larvae from each replicate group web.expasy.org/protparam/). Phylogenetic analysis of actin were randomly sampled every day until 7 days post- isoforms was conducted by MEGA 7 (ver. 7.0.26) software hatching (DPH). Sampled larvae were frozen on dry ice using neighbor-joining algorithm (http://www.megasoftwar- and kept in deep freezer until used for RNA extraction. e.net/). In order to compare genomic structure of teleost ac- tins, actin genes were searched against Gene database in RT-qPCR assay NCBI (https://www.ncbi.nlm.nih.gov/gene/), and genomic Total RNA was extracted using TriPure Reagent (Roche context of each output was filed for further comparison. Applied Science, Mannheim, Germany) and RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to manufac- Preparation of tissue and developmental samples turers’ recommendations. Quality and quantity of total Mud loach specimens used in this study were laboratory- RNA extracted from tissues were verified by MOPS- bred strains that have been maintained in Experimental formaldehyde agarose gel electrophoresis and NanoDrop Fish Culture Station, Pukyong National University, Busan, Microvolume spectrophotometer (Thermo Fisher Scientific, South Korea. In order to examine the expression pattern of Waltham, MA, USA). Two microgram of total RNA from actin isoforms in adult tissues, 12 normally grown healthy each sample was reverse transcribed into cDNA by using individuals (six females and six males; average body weight Omniscript® Reverse Transcription Kit (Qiagen, Germany) Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 4 of 11 according to the manufacturer’s protocol. During reverse Three mud loach α-actin isoforms represented highly transcription (RT) reaction, a mud loach 18S rRNA reverse similar calculated molecular masses (41,959–41,995 Da) primer was included in order to prepare the normalization and theoretical isoelectric points (pI = 5.22–5.23). Se- control for quantitative reverse transcription PCR (RT- quence identity among them at the amino acid level was qPCR) assay (Nam et al. 2011). The synthesized cDNAs at least more than 98%, in which there were only eight were fourfold diluted with sterile distilled water, and 2 μL non-conservative amino acid substitutions out of 377 of diluted cDNA was used for each quantitative PCR residues (Fig. 1). Thereby, our sequence data consistently (qPCR) amplification. PCR was run with LightCyler480® II confirm the known appraisal on the divergence of actin Real-Time PCR System (Roche Applied Science, Germany) isoform genes from a common ancestral form through and LightCycler® DNA Master SYBR Green I (Roche Ap- duplication events (Miwa et al. 1991; Venhatesh et al. plied Science, Germany). Primer pairs for each actin iso- 1996). In addition, from the multiple sequence align- form and 18S rRNA normalization control were confirmed ments of each isoform with its representative orthologs to amplify the specific band based on the melting curve also showed a considerably high degree of amino acid analysis after the thermal cycling. PCR efficiency of each sequence identity ranging from 97 to 100% among spe- primer pair was validated to be higher than at least 92% cies (data not shown). based on the standard curve for each gene. Quantitative A closer look into the sequence alignment among the PCR assay of each cDNA sample was carried out in tripli- three mud loach α-actin isoforms indicates that substitution cates. After each thermal cycle, amplification signals were patterns in several non-conserved residues are similar with analyzed with LightCyler480® II Software (ver. 1.5; Roche those observed in other teleost actin isoforms. For example, Applied Science, Germany). Relative mRNA expression the alanine/serine substitution found at position 157 (Ala/ levels of each actin isoform in each tissue and developmen- Ser-157) is also found (1) between pufferfish α-skeletal actin tal stages were estimated by ΔCt method based on the isoform 1 and isoform 2 (Venhatesh et al. 1996), (2) be- normalization against its own level of 18S rRNA control tween rattail fish (Coryphaenoides acrolepis and C. cinereus) (Schmittgen and Livak 2008). Significantly different expres- α-skeletal actin isoforms (Morita 2000), and also (3) be- sion levels among tissues and developmental stages were tween the two types of Leporinus macrocephalus skeletal α- addressed by one-way ANOVA followed by Duncan’smul- actin isoforms (Alves-Costa et al. 2015). In particular, the tiple ranged test at the level of P =0.05. substitution in L. macrocephalus α-actin isoforms was ob- served between actin isoforms isolated respectively from Results and discussion white muscle (Ser-157) and red (Ala-157) muscle (Alves- Characteristics of cDNA and deduced amino acid Costa et al. 2015). This amino acid substitution occurs in sequences the vicinity of actin ATP-binding site and has been pro- The three α-actin isoforms isolated from this species posed to influence the actin’s affinity to ATP (also to diva- were designated, ACTA1 [1268 bp excluding the poly(A lent cation) based on the assumption that Ser has a higher +) tail], ACTC1 (1273 bp), and ACTA2 (1607 bp) based hydrogen bonding capacity than Ala (Morita 2000; Alves- on the homology search and molecular phylogeny (see Costa et al. 2015). Affinity of actin to ATP has also been re- below). The three isoform cDNAs showed the same stop ported to affect the thermal stability of fish actins (more codon (TAA). Canonical putative polyadenylation signal stable in ATP-bound forms) extracted from myofibril pow- (AATAAA) was predicted in the three isoform, 23– der (Torigai and Konno 1997). Similarly, the substitutions 26 bp prior to the poly(A+) tail. However, ACTA2 among mud loach α-actin isoforms Thr/Ser-91, Val/Ile-167, showed an additional polyadenylation signal (same and Ala/Thr-280 are observed among subisoforms of puf- AATAAA sequence) in the vicinity of the poly(A+) tail ferfish α-skeletal actin (Venhatesh et al. 1996). (10 bp prior to the tail). The 5′- and 3′-untranslated re- gions (UTRs) of ACTA2 are relatively longer than that of Molecular phylogeny ACTA1, similarly reported in other cypriniform fish In order to designate the nomenclature of three α-actin (Kim and Nam 2009). All of the three isoforms revealed isoforms cloned in mud loach, a molecular phylogenetic a single ORF (each 1131 bp excluding stop codon) to en- analysis was carried out with the taxa sampled from rep- code the same number of amino acids (377 amino acids, resentative teleost and amniote species (Fig. 2). Molecu- including the first two Met-Cys residues known to be lar phylogenetic analysis resulted in a monophyletic processed after translation). Sequences of the three iso- status of three α-actin isoforms (skeletal, cardiac, and forms are available in GenBank under the accession smooth muscle forms), although the topology was not numbers KX347544 (ACTA1), KX347545 (ACTC1), supported by the high bootstrap value. Teleost α-skeletal and KX347546 (ACTA2). Summarized information on actins and α-smooth muscle actins formed the two the sequence characteristics of mud loach α-actin iso- monophyletic clades, where mud loach ACTA1 and forms is also provided in Additional file 2: Table S2. ACTA2 branched off, respectively, in each of the two Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 5 of 11 Fig. 1 Primary polypeptide structures (a) and genomic organization (b) of mud loach α-actin isoforms ACTA1 (skeletal muscle actin), ACTC1 (cardiac muscle actin), and ACTA2 (smooth muscle actin). In the multiple sequence alignment (a), non-identical amino acid residues are indicated by boldface, red letters, and the positions for seven introns are noted by arrowheads. In the schematic representation for genomic architecture (b), all the three actin isoforms possess a non-translated exon 1 (NTE1) and eight translated exons (E2–E9). Translation initiation codon (ATG) in the first translated exon is indicated by arrow in ACTA2. Lengths of translated regions in exons (vertical boxes) are identical for three isoforms, while size of introns (horizontal line) is variable among isoforms clusters (skeletal and cardiac). Mud loach ACTA1 iso- Inconsistency between phylogenetic affiliation and form showed the closest phylogenetic affiliation to previously known taxonomic appraisal of the species Cyprinus carpio ACTA1, while mud loach ACTA2 dis- has been reported in previous attempt on the recon- played the closest relationship with Hemibarbus mylo- struction of phylogenetic tree of teleost muscle actins, don and Danio rerio ACTA2 orthologs. However, in the in which complex pattern of phylogenetic relationships present analysis, teleost α-cardiac actins did not form a would be apparent due to significant gene duplications monophyletic clade. Rather, they were divided into two and diversifications of actin paralogs in teleost genomes subclades, in which the C. carpio and D. rerio ACTC1s (Kim and Nam 2009). Gene duplications following formed a small, their own subclade, although the node differential subfunctionalization(s) seemed to have did not receive the statistical support. Furthermore, the occurred in an isoform- and species (or lineage)- mud loach ACTC1 was not closely affiliated with ortho- dependent fashion, as particularly noticed in Salmoni- logs from the two cypriniform species (C. carpio and D. formes, Gadiformes, Cypriniformes, and Tetraodonti- rerio); instead, it is closely related with α-cardiac ortho- formes (Johnston et al. 2008; Kim and Nam 2009). logs from distantly related species such as Takifugu Furthermore, certain muscle actin isoforms observed in rubripes and Oreochromis niloticus. Because we did not these taxa exhibited no clear affiliation to currently sample all the presumed α-cardiac actins from the tele- established orthology in vertebrate actin multigene osts according to our primary objective of molecular family, making the phylogenetic explanation more com- phylogeny (i.e., type identification of three mud loach plicated (Kim and Nam 2009; Venhatesh et al. 1996). isoforms for their proper nomenclatures), extensive re- Whatever the evolutionary mechanism(s), teleostean constructions of the tree with much more complete se- muscle actins have experienced different evolutionary quence sampling might also be needed in future. Also, history in comparison to amniote counterparts, and di- the further mining of additional paralog actin isoforms versified paralogs could be the potential source of from the mud loach genome might be helpful to explain muscle plasticity to interact with their environmental the reason behind this unexpected finding. changes or variations. Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 6 of 11 Fig. 2 Phylogenetic neighbor-joining tree showing the genetic affiliations of mud loach actin isoforms with either teleost α-skeletal (ACTA1), α- cardiac (ACTC1), or α-smooth muscle (ACTA2) actins. Tree was drawn with amino acid sequences of selected muscle α-actin isoforms using MEGA7 software (ver. 7.0.26), including amniote γ-cytoplasmic actins as outgroups. Each node was tested by bootstrap method (1000 replicates), and only bootstrap values of 50% or above were shown here Gene structure and organization translated exon (E2–E9) was perfectly identical among the From the genomic cloning, the coding sequence of each three isoform genes (129, 129, 111, 85, 162, 192, 182, and isoform gene was clearly matched to its corresponding 144 bp, respectively for E2 to E9). On the other hand, cDNA counterpart, and the GT/AG exon-intron splicing lengths of non-coding, intronic lengths are variable among rule was well conserved at each boundary region. Genomic the three isoform genes (Fig. 1; Additional file 2: Table S2). architectures (exon-intron organization) of the three actin Actins are one of the most conserved protein groups, isoforms were found to be identical (eight translated exons), and extremely high sequence similarly of actin proteins again strongly suggesting that they have derived from a are commonly found among almost eukaryotic organ- common ancestral gene that had gone through duplications isms. However, in contrast to such a homogenous fea- and conversions. Based on the alignment of cDNA and ture at protein level, genomic exon-intron organization genomic sequences, each of three mud loach α-actin iso- of actin genes (i.e., number of exons) has been known to forms was proven to possess a non-translated exon 1 be variable among species and/or taxonomic lineages (NTE1). Lengths of NTE1s found in ACTA1 (KX347541), (Bertola et al. 2008; Kusakabe et al. 1999). For example, ACTC1 (KX347542), and ACTA2 (KX347543)were24, most mammalian α-skeletal and α-cardiac actin genes 22, and 33 bp, respectively. Although the presence of NTE1 reveal six translated exons with the conserved intron po- has not been extensively characterized in fish α-actin genes, sitions, while α-vascular and γ-enteric smooth muscle our bioinformatic survey has shown that similar NTE1s actin genes exhibit eight translated exons in mammals. exist in many other teleost α-actin genes (data not shown) Ascidian muscle actins show four or five exons depend- as reported in human actin genes (Miwa et al. 1991). Be- ing on larval and adult muscle types. Many insect spe- sides the NTE1, three α-actin isoform genes from mud cies lost all introns in their α-skeletal actin genes. loach represented equally eight translated exons (exon E2 Caenorhabditis elegans (nematode) have three or four to exon E9) and the length of coding sequence in each exons in its actin genes (Bertola et al. 2008; Kusakabe et Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 7 of 11 al. 1999). Further, teleost species, which have experi- mud loach tissues (Fig. 3). Mud loach ACTA1 transcripts enced WGD in their evolutionary history, are known to were detectable in all the tissues examined. As expected, retain more diverse paralogs in actin multigene family. skeletal muscle exhibited the most robust expression of With this context, the gene architecture (exon-intron ACTA1 among tissues (P < 0.05). Besides the muscular organization) of teleost actins is thought to be relatively expression, the intestine, skin, gill, and eye revealed the more diversified, compared to ones established in other moderate expression of ACTA1, whereas other vertebrates including mammals. However, barring only a remaining tissues displayed weak expression levels few examples, the comparative information on genomic (P < 0.05). The lowest expression of ACTA1 was found organization patterns of teleost actin genes has not been in the ovary (P < 0.05). On the other hand, ACTC1 tran- sufficiently described. scripts were not clearly detectable in several tissues in- As mentioned above, mud loach displays a uniform cluding the brain, fin, kidney, liver, and gonads under pattern of genomic organization (eight translated exons the present RT-qPCR conditions. The heart expressed interrupted by seven introns) for all the three actin para- the highest amount of ACTC1 transcripts. Although logs. However, our bioinformatic survey to other teleost skeletal muscle showed the second highest expression actin loci strongly indicates that such a uniform pattern level of ACTC1, the expressed amount in skeletal muscle observed in mud loach may not be conserved in all was significantly lower than that in the heart (P < 0.05). other teleostean genomes. Rather, the exon-intron Gill and spleen exhibited quite a low expression of organization is found to be highly species- and isoform- ACTC1 in this species, while the eye, intestine, and skin dependent in the teleost lineage (Additional file 3: Table showed a modest expression of ACTC1 (P < 0.05). S3). For ACTA1 (skeletal actin), the gene organization Meanwhile, the expression pattern of ACTA2 was also with six translated exons is the most frequently found ubiquitously observable in all the tissues. The highest form, which is corresponding to the organization pattern expression was found in the intestine, followed by the of all known mammalian α-skeletal actin genes. ACTA1s spleen (P < 0.05). Interestingly, testis revealed an active with six translated exons are exemplified in channel cat- transcription of ACTA2, of which expression level corre- fish Ictalurus punctatus (GenBank Gene ID = sponded to the third highest among all the tissues exam- 108263396), pufferfish Takifugu rubripes (Gene ID = ined. The heart also expressed a moderate level of 101069447), large yellow croaker Larimichthys crocea ACTA2 transcripts while the other remaining tissues (Gene ID = 104920157), zebrafish Danio rerio (Gene ID were found to show a weak or minute expression of = 550445), and tilapia Oreochromis niloticus (Gene ID = ACTA2 (P < 0.05). 100534413). On the other hand, Japanese flounder Para- Overall, tissue distribution patterns of the three actin lichthys olivaceus and common carp Cyprinus carpio isoform mRNAs observed in the present study are well represent different numbers (six to eight) of translated in agreement with their annotated nomenclatures ad- exons depending upon paralog copies of ACTA1. Mean- dressed by phylogenetic analysis, as the highest or pre- while, many fish species examined in the present study dominant expression levels of skeletal (ACTA1)-, cardiac reveal various paralogs (subisoforms) with differential (ACTC1)-, and smooth muscle (ACTA2)-actin genes in exon numbers (six to eight) within the ACTC1 isoform the myotomal muscles, heart, and intestine, respectively. (cardiac type). Exceptional species is zebrafish, which Such an isoform-dependent expression pattern indicates highlights only the eight-exon-structured ACTC1 cur- a certain subfunctionalization of these three actin iso- rently. Unlike striated muscle actins (ACTA1 and forms with regard to their tissue-specific roles in the ACTC1), all the ACTA2 (smooth muscle type) isoforms musculature of adult mud loach (Perrin and Ervasti are comprised of eight translated exons, suggesting tele- 2010; Bertola et al. 2008). Within the isoform ACTA1, osts have retained the conserved gene organization pat- one notable finding is its active transcription in the in- tern for ACTA2 with mammalian ACTA2s (vascular, testine, which is not generally congruent with the ex- aorta actins). Taken together, the number of translated pected pattern of the skeletal muscle actins, since the exons of actin genes in teleosts is species- and isoform- intestine is a known smooth muscle tissue. Previously, a dependent, in which the organization pattern could also histological analysis has reported that loach would pos- be further diversified among paralog copies within a sess the proximal intestine consisting of intermingled given isoform. Collectively, the present analysis is striated and smooth muscle cells (Hara et al. 1989). One strongly suggestive of the complex evolutionary history plausible, but untested, assumption on this finding (i.e., of actin multigene family in the teleost lineage. the considerably active expression of ACTA1 in the in- testine) is that this species uses its intestine as an Tissue distribution and expression levels accessory air-breathing organ (Luo et al. 2016), and Based on the RT-qPCR assay, mRNA expression pat- thereby mud loach may require a unique contractility in terns of three actin isoforms were examined in adult order to eject air bubbles from its intestine. Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 8 of 11 a a’ b b’ c c’ Fig. 3 RT-qPCR analysis of mud loach ACTA1 (a), ACTC1 (b), and ACTA2 (c) transcripts in the brain (Br), eye (Ey), fin (Fi), gill (Gi), heart (He), intestine (In), kidney (Ki), liver (Li), skeletal muscle (Mu), spleen (Sp), skin (Sk), ovary (Ov), and testis (Te). Expression levels of each isoform were normalized against 18S rRNA control. Histograms excluding the muscle (for ACTA1), heart (for ACTC1), and intestine (for ACTA2) are also provided in a′, b′, and c′, respectively, for better resolution of expressed levels in other tissues. Within each isoform, means (± s.d.) with different letters indicate statistical difference based on one-way ANOVA followed by Duncan’s multiple ranged tests (P < 0.05). In b′, tissues showing undetectable mRNA expression levels of ACTC1 are indicated by nd As a main isoform playing essential roles in cardiac suggests certain putative roles of ACTA2 in the develop- contractility, the predominant expression of ACTC1 in ment of testis, which is also similar with previous identi- the mud loach heart is not surprising. Similar or redun- fication of a testis-specific anomalous actin isoform in dant expression pattern of ACTC1 with ACTA1 (i.e., ac- the pufferfish (Venhatesh et al. 1996). tive expression in skeletal muscle as well as the modest expression in the eye, intestine, and skin) has also been Developmental and ontogenic expression patterns examined previously (Bertola et al. 2008). On the other Three actin isoforms are differentially modulated during hand, the vigorous expression of ACTA2 in the intestine the period of embryonic development and larval early on- is in agreement with its functional involvements mainly togeny in mud loach (Fig. 4). During embryonic develop- in smooth muscle-contained tissues. In mammals, two ment, the onset expression of ACTA1 mRNAs was faintly differentiated isoforms of smooth muscle actins, ACTA2 detected by RT-qPCR at late gastrula stage (8 HPF; char- (vascular/aorta type) and ACTG2 (gastrointestinal type), acterized by 70% epiboly cover). The expression was grad- have been reported (Miwa et al. 1991). However, our ually increased with the progress of somitogenesis until 24 peer-review has been suggestive of no clearly character- HPF (corresponding to pre-hatching stage exhibiting more ized ACTG2 in teleosts. Rather, teleosts potentially rep- than 30 somites), suggesting the essential roles of ACTA1 resented novel and/or anomalous subisoforms/isoforms in embryonic myogenesis in mud loach embryos (Hall et with little orthology to mammalian actins (Kim and al. 2003; Xu et al. 2000; Moutou et al. 2001). Afterward, Nam 2009; Venhatesh et al. 1996). A noticeable finding the transcript level of ACTA1 in mud loach embryos was in the expression pattern of mud loach ACTA2 is the sharply increased until hatch-out and further rigorously prominently high expression in the testis, which is not until 2 DPH. Then, the expression of ACTA1 was rapidly seen with other two isoforms. The present finding dropped to the level observed at just hatching (28 HPF) Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 9 of 11 earlier initiation of myogenesis in fish has been proposed to reflect the requirement to generate swimming propul- sion at earlier life stages imposed by external fertilization (Johnston et al. 2011). Sharp increase of the expression from 24 DPH might be in relation with embryos’ prepar- ation to develop skeletal muscles for hatching event. After hatch, the mud loach yolk sac larvae rarely swim with the settlement at the bottom. However, in this period also, they seem to continue active transcriptions of skeletal muscle genes including ACTA1 in order to make them fully ready for the start-up of active swimming immedi- ately after yolk sac absorption (i.e., the first time full-scale swimming in their lives) (Fujimoto et al. 2006; Gap et al. 2014). Once larvae are fulfilled with transcripts, ACTA1 transcription is downregulated in a certain degree and then stabilized in subsequent phases. Transition to ex- ogenous feeding after yolk sac absorption usually occur at the period from 2 DPH to 3 DPH at 25 °C in mud loach, which is in accordance with our explanation on the larval ACTA1 gene expression pattern. On the other hand, the ACTC1 expression began to be apparent at 12 HPF (4-somite stage), continuously stimu- lated with the progressive development of embryonic myotomes until 24 HPF and quickly upregulated to reach a sharp peak at 28 HPF (just hatching). Redundant expres- sion of ACTA1 and ACTC1 in the development of striated muscle during embryogenesis is a known phenomenon in vertebrates (Bertola et al. 2008). Although ACTC1 eventu- ally becomes the main actin isoform in the heart in adult- hood, it has also been reported to be largely associated with early muscle development (Bertola et al. 2008). A steep increase of ACTC1 expression from the 24 DPH in mud loach embryo is congruent with the initial heart beat- ing and circulation. Afterward, the transcription of Fig. 4 Developmental regulations of mud loach ACTA1, ACTC1,and ACTC1 was rapidly downregulated until 5 DPH, although ACTA2 transcripts, as addressed by RT-qPCR assay. Expression levels of there was a lag period of downregulation from 1 DPH to 2 each isoform were normalized against the expression of 18S rRNA DPH. This is also similar with previous findings, in which control in each sample. Abbreviations for developmental stages are early the expression of α-cardiac actin would be downregulated blastula (EB), early gastrula (EG), late gastrula (LG), four-somite stage and α-skeletal actin becomes the dominant isoform in (4-SM), twelve- smote stage (12-SM), twenty-somite stage (20-SM), pre-hatching stage (PH), just hatching (JH) and yolk sac absorption (YSA). later developmental stages, eventually accounting for al- Based on ANOVA, statistically different means are indicated by different most proportion of the total striated muscle actins in letters (a–j for ACTA1 and ACTC1,and a–d for ACTA2 shown under the grown-up vertebrate animals (Sassoon et al. 1988). X-axis labels). To avoid complicated viewing, T-bars for standard Unlike the above two actin isoforms, mRNA expression deviations are omitted from the final illustration of ACTA2 was not clearly projected during embryonic de- velopment stages, and the initiation of its expression was and kept to be relatively constant in subsequent ages. observed at hatching (at 28 HPF) in a very low amount, Myogenesis has been known to initiate at an earlier stage which differs the previous demonstration that α-smooth of development in teleost embryos than in amniotes; the muscle actin would be also expressed during early cardiac myogenic precursor cells for slow and fast muscle in fish and skeletal muscle development in various myofibroblast- embryos have been reported to be spatially segregated be- like cells in mammalian embryogenesis (Bertola et al. 2008). fore somite formation (i.e., as early as gastrulation) Currently, it is not clear whether other paralog subiso- (Hirsinger et al. 2004). Hence, the onset expression of form(s) of mud loach ACTA2 may participate in the devel- ACTA1 transcripts at late gastrula stage in this study is opment of early embryonic muscles or not. However, our broadly in agreement with the previous observation. The finding on the absence of ACTA2 transcription in Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 10 of 11 embryogenesis is, at least in part, accordant with the previ- Additional files ous study on zebrafish ACTA2 to show that mural cells Additional file 1: Table S1. Oligonucleotide primers used for cloning (main expression site of zebrafish ACTA2)would develop and expression assay in this study. (PDF 69 kb) relatively late as compared to ones in mammals (Whitesell Additional file 2: Table S2. Summarized information on the mud loach et al. 2014). The authors have hypothesized that the slow α-actin isoform cDNAs and genomic gene. (PDF 75 kb) development of vascular mural cells might be a reflection Additional file 3: Table S3. Genomic exon-intron organization of muscle of the small body size of the zebrafish, and thus, there actin genes in teleost species examined in this study. (PDF 102 kb) would be little need to develop contractile smooth muscle at an early embryo stage (Whitesell et al. 2014). In this Acknowledgements Not applicable. study, the low level of ACTA2 transcripts in just hatched larvae was slightly increased at 1 DPH; however, soon after, Availability of data and materials the expression of ACTA2 transcripts was dramatically in- Not applicable. creased at 2 DPH and kept to be constant until 4 DPH (i.e., Authors’ contributions immediately following the transition to exogenous feeding). SYL carried out the gene expression assays and phylogenetic analysis. JAL This modulation pattern could be explained by the need for performed the molecular cloning and isolation of genes. YKN designed this study, performed the data evaluation, and drafted the manuscript. All the preparation of smooth muscles with the developments authors read and approved the final manuscript. and differentiations of vascular and gastrointestinal organs. Although we did not examine histological ontogenesis of Funding This study was supported by the grant from the Korea Institute of Marine internal organs, initial developments of various smooth Science & Technology Promotion (KIMST; Project #20130369), Ministry of muscle-related organs including intestine might occur in Oceans and Fisheries, Republic of Korea. that period, as scrutinized in a closely related Misgurnus Ethics approval and consent to participate species (M. anguillicaudatus) (Han et al. 2013; Zhang et al. Not applicable. 2016). Subsequently, ACTA2 transcripts were detected in a decreased level (i.e., at 5 DPH and afterwards). Generally, Consent for publication Not applicable. this pattern might be recognized by a stabilization process after the explosive transcriptions in line with the ontogenic Competing interests developments of various smooth muscle-relevant organs. The authors declare that they have no competing interests. However, it may not also be excluded the possibility that ACTA2 transcripts might be diluted in the total RNA tem- Publisher’sNote plates by various other gene transcripts and dominated Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ACTA1 transcripts with the increase of body mass in fry. Collectively, the three α-actin isoforms are dynamically Received: 14 August 2017 Accepted: 3 October 2017 modulated during embryo development and larval on- togeny largely in an isoform-dependent fashion. References Alves-Costa F, Silva MDP, Wasko AP. 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Fisheries Sci. 1997;63:403–6. and we will help you at every step: Venhatesh B, Tay BH, Elgar G, Brenner S. Isolation, characterization and evolution • We accept pre-submission inquiries of nine pufferfish (Fugu rubripes) actin genes. J Mol Biol. 1996;259:655–65. Watabe S. Myogenic regulatory factors and muscle differentiation during � Our selector tool helps you to find the most relevant journal ontogeny in fish. J Fish Biol. 1999;55:1–18. � We provide round the clock customer support Whitesell TR, Kennedy RM, Carter AD, Rollins EL, Georgijevic S, Santoro MM, Childs � Convenient online submission SJ. An α-smooth muscle actin (acta2/αsma) zebrafish transgenic line marking vascular mural cells and visceral smooth muscle cells. PLoS One. 2014;3:e90590. � Thorough peer review Xu Y, He J, Wang X, Lim TM, Gong Z. Asynchronous activation of 10 muscle- � Inclusion in PubMed and all major indexing services specific protein (MSP) genes during zebrafish somitogenesis. Dev Dyn. 2000; � Maximum visibility for your research 219:201–15. Zhang J, Yang R, Yang X, Fan Q, Wei K, Wang W. Ontogeny of the digestive tract Submit your manuscript at in mud loach Misgurnus anguillicaudatus larvae. Aquac Res. 2016;47:1180–90. www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Fisheries and Aquatic Sciences Springer Journals

Molecular characterization of three muscle alpha actin genes in mud loach (Misgurnus mizolepis; Cypriniformes)

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Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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Abstract

Background: Teleosts represent unique features in the regulation of muscle development and growth, with a great deal of myogenic plasticity. Muscle actins are major components to compose muscle fibers, and they play essential roles in cellular mobility and other related functions. In order to understand isoform-dependent roles of muscle actins in the mud loach (Misgurnus mizolepis), this study was aimed to characterize gene structure and expression pattern of the three muscle actin isoforms (α-skeletal, α-cardiac, and α-smooth muscle actins) isolated from this benthic fish species. Results: Mud loach α-actin isoforms (ACTA1, ACTC1, and ACTA2) were fairly conserved in their primary structures and shared a high-sequence identity one another. At genomic level also, all the three isoforms exhibited the same exon-intron organization pattern characterized by eight translated exons. However, in mRNA expression patterns, these three actin isoforms were found to display an apparent isoform-dependency in tissue distribution (i.e., ACTA1 in the skeletal muscles, ACTC1 in the heart, and ACTA2 in the intestines) and developmental regulation (i.e., increased expression of ACTA1 and ACTC1 with the progress of myogenesis, and the significant elevation of ACTA2 during organ development in early larvae). Conclusion: Data from this study suggest that three muscle actin isoforms have undergone certain functional differentiation with regard to their roles in the development and adult musculature, in spite of high structural similarity. Genetic information on muscle actins obtained in this study could provide a useful base to extend our understanding on evolutionary diversification of the myogenic regulation in teleosts. Keywords: Mud loach Misgurnus mizolepis, Alpha actins, Gene structure, mRNA expression Background and amniote vertebrates is the indeterminate growth For the adaptation to aquatic locomotion, teleosts ex- pattern of fish muscles. Most teleosts display a contin- hibit the distinct separation of muscle fiber types (slow- ued growth throughout most of their lifespan, with body twitch fibers to red muscle and fast-twitch fibers to size and muscle mass increasing, albeit at a slow rate white muscle) into discrete layers in their striated mus- until mortality or senescence occurs (Johnston et al. cles, which has not been seen in other amniote verte- 2011). Muscle growth in teleosts is signified by the com- brates (Watabe 1999; Kiessling et al. 2006). White bined contribution of a long-lasting hyperplasia (increase muscle (glycolytic, anaerobic metabolism; for fast-start, in fiber number) as well as the hypertrophy (increase in burst swimming) and red muscle (oxidative metabolism; fiber size), which is hardly observable in many other for slow, sustained movement) occupy distinct axial re- higher vertebrates (Kiessling et al. 2006; Johnston et al. gions (Kiessling et al. 2006; Chong et al. 2009). Another 2011). Furthermore, metabolic and contractile character- striking difference in a skeletal musculature between fish istics of teleost muscles also represent significant plasti- city and flexibility with regard to not only environmental conditions (e.g., temperature (Hall et al. 2003; Johnston * Correspondence: yoonknam@pknu.ac.kr et al. 2008; Stickland et al. 1988; Johnston 2006)) but Department of Marine Bio-Materials and Aquaculture, Pukyong National University, Busan 48513, Korea © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 2 of 11 also reproductive status (e.g., gonad maturation development stages and adult tissues (Perrin and Ervasti (Johnston et al. 2011; Mathana et al. 2012)). 2010; Bertola et al. 2008). However, in contrast to rich Taken together, teleosts have developed unique or spe- information on mammalian counterparts, the compara- cialized features in the architecture and physiology of tive expression profiles of the muscle actin isoforms in a muscles during their evolutionary history, and such adap- given single species have remained to be further ex- tive processes undoubtedly have resulted in modifications plored in teleosts. of the genetic pathways modulating muscle growth and Mud loach (Misgurnus mizolepis Günther; Cyprini- functions in teleosts, as compared to terrestrial vertebrates formes) is an aquaculture-relevant food fish in Korea (Kim (Morita 2000; Mudalige et al. 2007). For this reason, iden- et al. 1994). This species has various advantageous merits tification of genetic determinants for muscle-specific as a candidate model system for muscle research in teleosts. genes from teleosts may be an important basis to study As a model fish, it possesses general merits such as easiness the evolutionary diversification of the musculature in the of laboratory maintenance, transparent embryonic develop- vertebrate lineage. Also, from an aquaculture viewpoint, ment, and relatively short generation time (Nam et al. myogenic genes are potentially of biotechnological interest 2000). Besides, mud loach undergoes characteristic changes from the perspective of growth regulation and quality con- in locomotory behavior during early larval development, trol of the flesh product. Understanding these key genes suggesting that this species is a good subject to visualize the could provide the base in order to develop future pro- interrelationships between the differentiation of different grams involving genetic manipulation and selection of de- muscle types and larval swimming behavior. Furthermore, sired growth trait(s) in commercially important fish with this species, dramatically induced muscle hyperplasia species (Johnston et al. 2008). has been demonstrated by a GH-transgenesis (Nam et al. Actins are one of major components of muscle tissues 2001; Nam et al. 2008), which can provide useful platform to compose the myofibrils of musculature. As an evolu- to examine differential regulation of genetic pathways asso- tionary conserved protein, actins play essential roles in ciated with muscle growth in hyperplasia/hypertrophy-ac- maintaining cytoskeletal structure, cellular mobility, cell celerated fishes. Taken together, our long-term goal is to list division and differentiation, intracellular movement, and this species as a research model for myogenesis of teleosts. contractile processes, which are associated with a wide This study, as an initial step toward our goal, was aimed to spectrum of physiological aspects in vertebrates (Perrin isolate genetic determinants for three representative muscle and Ervasti 2010). The actin multigene family has been actins (skeletal, cardiac, and smooth muscle types) from known to be evolved through duplication and divergence mud loach and to examine the isoform-dependent from a common ancestral gene, leading diverse isoforms expression patterns in adult tissues and developing embryos (and subisoforms) in the extant actin genes (Miwa et al. and larvae. 1991). Although actin isoforms share remarkably high sequence identity one another, each isoform exhibits dis- Methods tinct regulation pattern for its spatial and temporal ex- Molecular cloning of α-actin isoform cDNAs and genomic pression (Perrin and Ervasti 2010). Further, teleosts have genes been known to retain multiple paralog copies of actin From next-generation sequencing (NGS) database of mud isoform genes depending on taxonomic lineages as a re- loach whole fry transcriptome (unpublished data), expressed sult of a whole-genome duplication (WGD) event (Kim sequence tag clones showing significant homology to previ- and Nam 2009; Glasauer and Neuhauss 2014). It poten- ously known vertebrate muscle actins were collected. The tially implies that certain subfunctionalization and/or fragment sequences were assembled into contigs using neofunctionalization might have occurred in teleostean Sequencher software (Gene Codes, Ann Arbor, MI, USA) actin isoform groups (Venhatesh et al. 1996). Muscle ac- and subjected to NCBI GenBank BLASTx search to verify tins (alpha actins) in teleosts can be broadly classified their potential annotations as muscle alpha actin members. into three primary types, α-skeletal, α-cardiac, and α- From this pilot analysis, three distinct actin isoforms were smooth muscle actins. Alpha-skeletal (ACTA1) and α- identified to be potentially designated α-skeletal, α-cardiac, cardiac (ACTC1) actins are expressed in striated and α-smooth muscle actins, respectively. muscles, importantly involved in locomotory action of In order to get full-length complementary DNA (cDNA) skeletal muscles and cardiac contractility, respectively. for each isoform, vectorette polymerase chain reaction On the other hand, α-smooth muscle actin (ACTA2)is (PCR) was conducted to both 5′-and 3′-directions using expressed in vascular and visceral smooth muscles (Kim the excised phagemid stock of the mud loach fry whole and Nam 2009; Venhatesh et al. 1996). Mostly from body cDNA library (Agilent, Santa Clara CA, USA) as a mammalian studies, the expression of these three actin template for PCR amplification. Oligonucleotides used in isoforms has been known to be regulated through both this study were listed in Additional file 1: Table S1. PCR- isoform-specific and cooperative ways depending on isolated fragments of each isoform were cloned into Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 3 of 11 pGEM-T easy vector (Promega, Madison, WI, USA), se- [BW] = 30.1 ± 3.5 g) were selected. From each individual, quenced and assembled into contigs. Based on the contig the brain, eye, fin (caudal), gill, heart, intestine, kidney, liver, sequence for each actin isoform, full-length continuous muscle (skeletal muscle), spleen, skin, and gonad (ovary or fragment of each isoform was re-isolated from mud loach testis) were surgically removed. Upon removal, tissues were total RNA (mixed total RNA from muscles and internal or- immediately frozen on dry ice and stored at − 80 °C deep gans) by reverse transcription-PCR (RT-PCR). Amplified freezer until used for RNA extraction. RT-PCR products were purified and sequenced directly in In order to prepare embryo and larvae samples, artificial order to determine the representative cDNA sequence for induced spawning was performed according to the each α-actin isoform. methods described previously (Kim et al. 1994; Nam et al. Based on the cDNA sequence, genomic gene of each iso- 2001). Briefly, carp pituitary extract (Sigma-Aldrich, St. form was PCR-amplified from a mud loach genomic DNA Louis, MO, USA) was delivered to six female and six male template prepared with caudal fin. Amplified fragment(s) broodfish via intraperitoneal injection at the dose levels of for each isoform gene was directly sequenced at both direc- 10 μg/g BW and 2 μg/g BW, respectively. Pooled eggs tions by primer walking method. From the genomic gene (from three females based on egg quality) were divided sequence, the genome walking to 5′-upstream region was into three egg batches. Each egg batch was inseminated by carried out in order to identify putative non-translated sperm (saline-diluted) from each of three individual males exon. Genome walking was conducted using Universal in order to prepare three fertilized egg groups. Insemin- Genome Walker Kit (Clontech Laboratories Inc., Mountain ation was done using wet method. Fertilized eggs were View, CA, USA) according to the manufacturer’sinstruc- rinsed with clean tap water (1-μm-filtered) and placed on tions. Depending on isoform genes, two to five rounds of an incubator at 25 ± 1 °C with a constant aeration to ad- genome walking were carried out. Assembled 5′-upstream just the dissolved oxygen level to be 7 ± 1 ppm through- region for each isoform was re-isolated together with its out the experiment. Fertilization rates and hatching downstream coding region in a continuous fragment, and success of the each replicate group were higher than 98 finally, the representative sequence of each isoform gene and 85%, respectively. During the embryonic develop- was determined by direct sequencing. ment, approximately 300 embryos were randomly sam- pled at 0 (just fertilized), 2, 4, 6, 8, 12, 16, 20, 24, and 28 Bioinformatic sequence analysis (hatch-out) hours post fertilization (HPF). Information on Open reading frame (ORF) in each isoform cDNA was pre- the embryogenesis of mud loach can be referred to previ- dicted using the ORF finder tool in NCBI page (https:// ous report (Kim et al. 1987). After hatching, the hatched www.ncbi.nlm.nih.gov/orffinder/), and the ORF sequence larvae from each fertilization group were transferred to was deduced into amino acid sequence using the same tool. one of three larval incubation tanks (each 60-L rectangu- Homology of each isoform to previously known orthologs lar plastic container) equipped with thermostat-assisted was examined by BLAST searches against NCBI GenBank heater and aeration apparatus. Larvae were reared at the database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple same temperature as above. After yolk sac absorption sequence alignments of nucleotide and amino acid se- (around 60 h after hatch), larvae were fed with 100-μm quences were carried out using the ClustalW tool (http:// commercial diet powder for flounder larvae (Woosung www.genome.jp/tools-bin/clustalw). Physicochemical pa- Feed Cor., Korea) and live Artemia nauplii (INVE Aqua- rameters of protein (with deduced amino acid sequence) culture Inc., Salt Lake City, UT, USA). During the larval were computed using ExPASy ProtParam tool (http:// rearing period, about 100 larvae from each replicate group web.expasy.org/protparam/). Phylogenetic analysis of actin were randomly sampled every day until 7 days post- isoforms was conducted by MEGA 7 (ver. 7.0.26) software hatching (DPH). Sampled larvae were frozen on dry ice using neighbor-joining algorithm (http://www.megasoftwar- and kept in deep freezer until used for RNA extraction. e.net/). In order to compare genomic structure of teleost ac- tins, actin genes were searched against Gene database in RT-qPCR assay NCBI (https://www.ncbi.nlm.nih.gov/gene/), and genomic Total RNA was extracted using TriPure Reagent (Roche context of each output was filed for further comparison. Applied Science, Mannheim, Germany) and RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to manufac- Preparation of tissue and developmental samples turers’ recommendations. Quality and quantity of total Mud loach specimens used in this study were laboratory- RNA extracted from tissues were verified by MOPS- bred strains that have been maintained in Experimental formaldehyde agarose gel electrophoresis and NanoDrop Fish Culture Station, Pukyong National University, Busan, Microvolume spectrophotometer (Thermo Fisher Scientific, South Korea. In order to examine the expression pattern of Waltham, MA, USA). Two microgram of total RNA from actin isoforms in adult tissues, 12 normally grown healthy each sample was reverse transcribed into cDNA by using individuals (six females and six males; average body weight Omniscript® Reverse Transcription Kit (Qiagen, Germany) Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 4 of 11 according to the manufacturer’s protocol. During reverse Three mud loach α-actin isoforms represented highly transcription (RT) reaction, a mud loach 18S rRNA reverse similar calculated molecular masses (41,959–41,995 Da) primer was included in order to prepare the normalization and theoretical isoelectric points (pI = 5.22–5.23). Se- control for quantitative reverse transcription PCR (RT- quence identity among them at the amino acid level was qPCR) assay (Nam et al. 2011). The synthesized cDNAs at least more than 98%, in which there were only eight were fourfold diluted with sterile distilled water, and 2 μL non-conservative amino acid substitutions out of 377 of diluted cDNA was used for each quantitative PCR residues (Fig. 1). Thereby, our sequence data consistently (qPCR) amplification. PCR was run with LightCyler480® II confirm the known appraisal on the divergence of actin Real-Time PCR System (Roche Applied Science, Germany) isoform genes from a common ancestral form through and LightCycler® DNA Master SYBR Green I (Roche Ap- duplication events (Miwa et al. 1991; Venhatesh et al. plied Science, Germany). Primer pairs for each actin iso- 1996). In addition, from the multiple sequence align- form and 18S rRNA normalization control were confirmed ments of each isoform with its representative orthologs to amplify the specific band based on the melting curve also showed a considerably high degree of amino acid analysis after the thermal cycling. PCR efficiency of each sequence identity ranging from 97 to 100% among spe- primer pair was validated to be higher than at least 92% cies (data not shown). based on the standard curve for each gene. Quantitative A closer look into the sequence alignment among the PCR assay of each cDNA sample was carried out in tripli- three mud loach α-actin isoforms indicates that substitution cates. After each thermal cycle, amplification signals were patterns in several non-conserved residues are similar with analyzed with LightCyler480® II Software (ver. 1.5; Roche those observed in other teleost actin isoforms. For example, Applied Science, Germany). Relative mRNA expression the alanine/serine substitution found at position 157 (Ala/ levels of each actin isoform in each tissue and developmen- Ser-157) is also found (1) between pufferfish α-skeletal actin tal stages were estimated by ΔCt method based on the isoform 1 and isoform 2 (Venhatesh et al. 1996), (2) be- normalization against its own level of 18S rRNA control tween rattail fish (Coryphaenoides acrolepis and C. cinereus) (Schmittgen and Livak 2008). Significantly different expres- α-skeletal actin isoforms (Morita 2000), and also (3) be- sion levels among tissues and developmental stages were tween the two types of Leporinus macrocephalus skeletal α- addressed by one-way ANOVA followed by Duncan’smul- actin isoforms (Alves-Costa et al. 2015). In particular, the tiple ranged test at the level of P =0.05. substitution in L. macrocephalus α-actin isoforms was ob- served between actin isoforms isolated respectively from Results and discussion white muscle (Ser-157) and red (Ala-157) muscle (Alves- Characteristics of cDNA and deduced amino acid Costa et al. 2015). This amino acid substitution occurs in sequences the vicinity of actin ATP-binding site and has been pro- The three α-actin isoforms isolated from this species posed to influence the actin’s affinity to ATP (also to diva- were designated, ACTA1 [1268 bp excluding the poly(A lent cation) based on the assumption that Ser has a higher +) tail], ACTC1 (1273 bp), and ACTA2 (1607 bp) based hydrogen bonding capacity than Ala (Morita 2000; Alves- on the homology search and molecular phylogeny (see Costa et al. 2015). Affinity of actin to ATP has also been re- below). The three isoform cDNAs showed the same stop ported to affect the thermal stability of fish actins (more codon (TAA). Canonical putative polyadenylation signal stable in ATP-bound forms) extracted from myofibril pow- (AATAAA) was predicted in the three isoform, 23– der (Torigai and Konno 1997). Similarly, the substitutions 26 bp prior to the poly(A+) tail. However, ACTA2 among mud loach α-actin isoforms Thr/Ser-91, Val/Ile-167, showed an additional polyadenylation signal (same and Ala/Thr-280 are observed among subisoforms of puf- AATAAA sequence) in the vicinity of the poly(A+) tail ferfish α-skeletal actin (Venhatesh et al. 1996). (10 bp prior to the tail). The 5′- and 3′-untranslated re- gions (UTRs) of ACTA2 are relatively longer than that of Molecular phylogeny ACTA1, similarly reported in other cypriniform fish In order to designate the nomenclature of three α-actin (Kim and Nam 2009). All of the three isoforms revealed isoforms cloned in mud loach, a molecular phylogenetic a single ORF (each 1131 bp excluding stop codon) to en- analysis was carried out with the taxa sampled from rep- code the same number of amino acids (377 amino acids, resentative teleost and amniote species (Fig. 2). Molecu- including the first two Met-Cys residues known to be lar phylogenetic analysis resulted in a monophyletic processed after translation). Sequences of the three iso- status of three α-actin isoforms (skeletal, cardiac, and forms are available in GenBank under the accession smooth muscle forms), although the topology was not numbers KX347544 (ACTA1), KX347545 (ACTC1), supported by the high bootstrap value. Teleost α-skeletal and KX347546 (ACTA2). Summarized information on actins and α-smooth muscle actins formed the two the sequence characteristics of mud loach α-actin iso- monophyletic clades, where mud loach ACTA1 and forms is also provided in Additional file 2: Table S2. ACTA2 branched off, respectively, in each of the two Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 5 of 11 Fig. 1 Primary polypeptide structures (a) and genomic organization (b) of mud loach α-actin isoforms ACTA1 (skeletal muscle actin), ACTC1 (cardiac muscle actin), and ACTA2 (smooth muscle actin). In the multiple sequence alignment (a), non-identical amino acid residues are indicated by boldface, red letters, and the positions for seven introns are noted by arrowheads. In the schematic representation for genomic architecture (b), all the three actin isoforms possess a non-translated exon 1 (NTE1) and eight translated exons (E2–E9). Translation initiation codon (ATG) in the first translated exon is indicated by arrow in ACTA2. Lengths of translated regions in exons (vertical boxes) are identical for three isoforms, while size of introns (horizontal line) is variable among isoforms clusters (skeletal and cardiac). Mud loach ACTA1 iso- Inconsistency between phylogenetic affiliation and form showed the closest phylogenetic affiliation to previously known taxonomic appraisal of the species Cyprinus carpio ACTA1, while mud loach ACTA2 dis- has been reported in previous attempt on the recon- played the closest relationship with Hemibarbus mylo- struction of phylogenetic tree of teleost muscle actins, don and Danio rerio ACTA2 orthologs. However, in the in which complex pattern of phylogenetic relationships present analysis, teleost α-cardiac actins did not form a would be apparent due to significant gene duplications monophyletic clade. Rather, they were divided into two and diversifications of actin paralogs in teleost genomes subclades, in which the C. carpio and D. rerio ACTC1s (Kim and Nam 2009). Gene duplications following formed a small, their own subclade, although the node differential subfunctionalization(s) seemed to have did not receive the statistical support. Furthermore, the occurred in an isoform- and species (or lineage)- mud loach ACTC1 was not closely affiliated with ortho- dependent fashion, as particularly noticed in Salmoni- logs from the two cypriniform species (C. carpio and D. formes, Gadiformes, Cypriniformes, and Tetraodonti- rerio); instead, it is closely related with α-cardiac ortho- formes (Johnston et al. 2008; Kim and Nam 2009). logs from distantly related species such as Takifugu Furthermore, certain muscle actin isoforms observed in rubripes and Oreochromis niloticus. Because we did not these taxa exhibited no clear affiliation to currently sample all the presumed α-cardiac actins from the tele- established orthology in vertebrate actin multigene osts according to our primary objective of molecular family, making the phylogenetic explanation more com- phylogeny (i.e., type identification of three mud loach plicated (Kim and Nam 2009; Venhatesh et al. 1996). isoforms for their proper nomenclatures), extensive re- Whatever the evolutionary mechanism(s), teleostean constructions of the tree with much more complete se- muscle actins have experienced different evolutionary quence sampling might also be needed in future. Also, history in comparison to amniote counterparts, and di- the further mining of additional paralog actin isoforms versified paralogs could be the potential source of from the mud loach genome might be helpful to explain muscle plasticity to interact with their environmental the reason behind this unexpected finding. changes or variations. Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 6 of 11 Fig. 2 Phylogenetic neighbor-joining tree showing the genetic affiliations of mud loach actin isoforms with either teleost α-skeletal (ACTA1), α- cardiac (ACTC1), or α-smooth muscle (ACTA2) actins. Tree was drawn with amino acid sequences of selected muscle α-actin isoforms using MEGA7 software (ver. 7.0.26), including amniote γ-cytoplasmic actins as outgroups. Each node was tested by bootstrap method (1000 replicates), and only bootstrap values of 50% or above were shown here Gene structure and organization translated exon (E2–E9) was perfectly identical among the From the genomic cloning, the coding sequence of each three isoform genes (129, 129, 111, 85, 162, 192, 182, and isoform gene was clearly matched to its corresponding 144 bp, respectively for E2 to E9). On the other hand, cDNA counterpart, and the GT/AG exon-intron splicing lengths of non-coding, intronic lengths are variable among rule was well conserved at each boundary region. Genomic the three isoform genes (Fig. 1; Additional file 2: Table S2). architectures (exon-intron organization) of the three actin Actins are one of the most conserved protein groups, isoforms were found to be identical (eight translated exons), and extremely high sequence similarly of actin proteins again strongly suggesting that they have derived from a are commonly found among almost eukaryotic organ- common ancestral gene that had gone through duplications isms. However, in contrast to such a homogenous fea- and conversions. Based on the alignment of cDNA and ture at protein level, genomic exon-intron organization genomic sequences, each of three mud loach α-actin iso- of actin genes (i.e., number of exons) has been known to forms was proven to possess a non-translated exon 1 be variable among species and/or taxonomic lineages (NTE1). Lengths of NTE1s found in ACTA1 (KX347541), (Bertola et al. 2008; Kusakabe et al. 1999). For example, ACTC1 (KX347542), and ACTA2 (KX347543)were24, most mammalian α-skeletal and α-cardiac actin genes 22, and 33 bp, respectively. Although the presence of NTE1 reveal six translated exons with the conserved intron po- has not been extensively characterized in fish α-actin genes, sitions, while α-vascular and γ-enteric smooth muscle our bioinformatic survey has shown that similar NTE1s actin genes exhibit eight translated exons in mammals. exist in many other teleost α-actin genes (data not shown) Ascidian muscle actins show four or five exons depend- as reported in human actin genes (Miwa et al. 1991). Be- ing on larval and adult muscle types. Many insect spe- sides the NTE1, three α-actin isoform genes from mud cies lost all introns in their α-skeletal actin genes. loach represented equally eight translated exons (exon E2 Caenorhabditis elegans (nematode) have three or four to exon E9) and the length of coding sequence in each exons in its actin genes (Bertola et al. 2008; Kusakabe et Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 7 of 11 al. 1999). Further, teleost species, which have experi- mud loach tissues (Fig. 3). Mud loach ACTA1 transcripts enced WGD in their evolutionary history, are known to were detectable in all the tissues examined. As expected, retain more diverse paralogs in actin multigene family. skeletal muscle exhibited the most robust expression of With this context, the gene architecture (exon-intron ACTA1 among tissues (P < 0.05). Besides the muscular organization) of teleost actins is thought to be relatively expression, the intestine, skin, gill, and eye revealed the more diversified, compared to ones established in other moderate expression of ACTA1, whereas other vertebrates including mammals. However, barring only a remaining tissues displayed weak expression levels few examples, the comparative information on genomic (P < 0.05). The lowest expression of ACTA1 was found organization patterns of teleost actin genes has not been in the ovary (P < 0.05). On the other hand, ACTC1 tran- sufficiently described. scripts were not clearly detectable in several tissues in- As mentioned above, mud loach displays a uniform cluding the brain, fin, kidney, liver, and gonads under pattern of genomic organization (eight translated exons the present RT-qPCR conditions. The heart expressed interrupted by seven introns) for all the three actin para- the highest amount of ACTC1 transcripts. Although logs. However, our bioinformatic survey to other teleost skeletal muscle showed the second highest expression actin loci strongly indicates that such a uniform pattern level of ACTC1, the expressed amount in skeletal muscle observed in mud loach may not be conserved in all was significantly lower than that in the heart (P < 0.05). other teleostean genomes. Rather, the exon-intron Gill and spleen exhibited quite a low expression of organization is found to be highly species- and isoform- ACTC1 in this species, while the eye, intestine, and skin dependent in the teleost lineage (Additional file 3: Table showed a modest expression of ACTC1 (P < 0.05). S3). For ACTA1 (skeletal actin), the gene organization Meanwhile, the expression pattern of ACTA2 was also with six translated exons is the most frequently found ubiquitously observable in all the tissues. The highest form, which is corresponding to the organization pattern expression was found in the intestine, followed by the of all known mammalian α-skeletal actin genes. ACTA1s spleen (P < 0.05). Interestingly, testis revealed an active with six translated exons are exemplified in channel cat- transcription of ACTA2, of which expression level corre- fish Ictalurus punctatus (GenBank Gene ID = sponded to the third highest among all the tissues exam- 108263396), pufferfish Takifugu rubripes (Gene ID = ined. The heart also expressed a moderate level of 101069447), large yellow croaker Larimichthys crocea ACTA2 transcripts while the other remaining tissues (Gene ID = 104920157), zebrafish Danio rerio (Gene ID were found to show a weak or minute expression of = 550445), and tilapia Oreochromis niloticus (Gene ID = ACTA2 (P < 0.05). 100534413). On the other hand, Japanese flounder Para- Overall, tissue distribution patterns of the three actin lichthys olivaceus and common carp Cyprinus carpio isoform mRNAs observed in the present study are well represent different numbers (six to eight) of translated in agreement with their annotated nomenclatures ad- exons depending upon paralog copies of ACTA1. Mean- dressed by phylogenetic analysis, as the highest or pre- while, many fish species examined in the present study dominant expression levels of skeletal (ACTA1)-, cardiac reveal various paralogs (subisoforms) with differential (ACTC1)-, and smooth muscle (ACTA2)-actin genes in exon numbers (six to eight) within the ACTC1 isoform the myotomal muscles, heart, and intestine, respectively. (cardiac type). Exceptional species is zebrafish, which Such an isoform-dependent expression pattern indicates highlights only the eight-exon-structured ACTC1 cur- a certain subfunctionalization of these three actin iso- rently. Unlike striated muscle actins (ACTA1 and forms with regard to their tissue-specific roles in the ACTC1), all the ACTA2 (smooth muscle type) isoforms musculature of adult mud loach (Perrin and Ervasti are comprised of eight translated exons, suggesting tele- 2010; Bertola et al. 2008). Within the isoform ACTA1, osts have retained the conserved gene organization pat- one notable finding is its active transcription in the in- tern for ACTA2 with mammalian ACTA2s (vascular, testine, which is not generally congruent with the ex- aorta actins). Taken together, the number of translated pected pattern of the skeletal muscle actins, since the exons of actin genes in teleosts is species- and isoform- intestine is a known smooth muscle tissue. Previously, a dependent, in which the organization pattern could also histological analysis has reported that loach would pos- be further diversified among paralog copies within a sess the proximal intestine consisting of intermingled given isoform. Collectively, the present analysis is striated and smooth muscle cells (Hara et al. 1989). One strongly suggestive of the complex evolutionary history plausible, but untested, assumption on this finding (i.e., of actin multigene family in the teleost lineage. the considerably active expression of ACTA1 in the in- testine) is that this species uses its intestine as an Tissue distribution and expression levels accessory air-breathing organ (Luo et al. 2016), and Based on the RT-qPCR assay, mRNA expression pat- thereby mud loach may require a unique contractility in terns of three actin isoforms were examined in adult order to eject air bubbles from its intestine. Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 8 of 11 a a’ b b’ c c’ Fig. 3 RT-qPCR analysis of mud loach ACTA1 (a), ACTC1 (b), and ACTA2 (c) transcripts in the brain (Br), eye (Ey), fin (Fi), gill (Gi), heart (He), intestine (In), kidney (Ki), liver (Li), skeletal muscle (Mu), spleen (Sp), skin (Sk), ovary (Ov), and testis (Te). Expression levels of each isoform were normalized against 18S rRNA control. Histograms excluding the muscle (for ACTA1), heart (for ACTC1), and intestine (for ACTA2) are also provided in a′, b′, and c′, respectively, for better resolution of expressed levels in other tissues. Within each isoform, means (± s.d.) with different letters indicate statistical difference based on one-way ANOVA followed by Duncan’s multiple ranged tests (P < 0.05). In b′, tissues showing undetectable mRNA expression levels of ACTC1 are indicated by nd As a main isoform playing essential roles in cardiac suggests certain putative roles of ACTA2 in the develop- contractility, the predominant expression of ACTC1 in ment of testis, which is also similar with previous identi- the mud loach heart is not surprising. Similar or redun- fication of a testis-specific anomalous actin isoform in dant expression pattern of ACTC1 with ACTA1 (i.e., ac- the pufferfish (Venhatesh et al. 1996). tive expression in skeletal muscle as well as the modest expression in the eye, intestine, and skin) has also been Developmental and ontogenic expression patterns examined previously (Bertola et al. 2008). On the other Three actin isoforms are differentially modulated during hand, the vigorous expression of ACTA2 in the intestine the period of embryonic development and larval early on- is in agreement with its functional involvements mainly togeny in mud loach (Fig. 4). During embryonic develop- in smooth muscle-contained tissues. In mammals, two ment, the onset expression of ACTA1 mRNAs was faintly differentiated isoforms of smooth muscle actins, ACTA2 detected by RT-qPCR at late gastrula stage (8 HPF; char- (vascular/aorta type) and ACTG2 (gastrointestinal type), acterized by 70% epiboly cover). The expression was grad- have been reported (Miwa et al. 1991). However, our ually increased with the progress of somitogenesis until 24 peer-review has been suggestive of no clearly character- HPF (corresponding to pre-hatching stage exhibiting more ized ACTG2 in teleosts. Rather, teleosts potentially rep- than 30 somites), suggesting the essential roles of ACTA1 resented novel and/or anomalous subisoforms/isoforms in embryonic myogenesis in mud loach embryos (Hall et with little orthology to mammalian actins (Kim and al. 2003; Xu et al. 2000; Moutou et al. 2001). Afterward, Nam 2009; Venhatesh et al. 1996). A noticeable finding the transcript level of ACTA1 in mud loach embryos was in the expression pattern of mud loach ACTA2 is the sharply increased until hatch-out and further rigorously prominently high expression in the testis, which is not until 2 DPH. Then, the expression of ACTA1 was rapidly seen with other two isoforms. The present finding dropped to the level observed at just hatching (28 HPF) Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 9 of 11 earlier initiation of myogenesis in fish has been proposed to reflect the requirement to generate swimming propul- sion at earlier life stages imposed by external fertilization (Johnston et al. 2011). Sharp increase of the expression from 24 DPH might be in relation with embryos’ prepar- ation to develop skeletal muscles for hatching event. After hatch, the mud loach yolk sac larvae rarely swim with the settlement at the bottom. However, in this period also, they seem to continue active transcriptions of skeletal muscle genes including ACTA1 in order to make them fully ready for the start-up of active swimming immedi- ately after yolk sac absorption (i.e., the first time full-scale swimming in their lives) (Fujimoto et al. 2006; Gap et al. 2014). Once larvae are fulfilled with transcripts, ACTA1 transcription is downregulated in a certain degree and then stabilized in subsequent phases. Transition to ex- ogenous feeding after yolk sac absorption usually occur at the period from 2 DPH to 3 DPH at 25 °C in mud loach, which is in accordance with our explanation on the larval ACTA1 gene expression pattern. On the other hand, the ACTC1 expression began to be apparent at 12 HPF (4-somite stage), continuously stimu- lated with the progressive development of embryonic myotomes until 24 HPF and quickly upregulated to reach a sharp peak at 28 HPF (just hatching). Redundant expres- sion of ACTA1 and ACTC1 in the development of striated muscle during embryogenesis is a known phenomenon in vertebrates (Bertola et al. 2008). Although ACTC1 eventu- ally becomes the main actin isoform in the heart in adult- hood, it has also been reported to be largely associated with early muscle development (Bertola et al. 2008). A steep increase of ACTC1 expression from the 24 DPH in mud loach embryo is congruent with the initial heart beat- ing and circulation. Afterward, the transcription of Fig. 4 Developmental regulations of mud loach ACTA1, ACTC1,and ACTC1 was rapidly downregulated until 5 DPH, although ACTA2 transcripts, as addressed by RT-qPCR assay. Expression levels of there was a lag period of downregulation from 1 DPH to 2 each isoform were normalized against the expression of 18S rRNA DPH. This is also similar with previous findings, in which control in each sample. Abbreviations for developmental stages are early the expression of α-cardiac actin would be downregulated blastula (EB), early gastrula (EG), late gastrula (LG), four-somite stage and α-skeletal actin becomes the dominant isoform in (4-SM), twelve- smote stage (12-SM), twenty-somite stage (20-SM), pre-hatching stage (PH), just hatching (JH) and yolk sac absorption (YSA). later developmental stages, eventually accounting for al- Based on ANOVA, statistically different means are indicated by different most proportion of the total striated muscle actins in letters (a–j for ACTA1 and ACTC1,and a–d for ACTA2 shown under the grown-up vertebrate animals (Sassoon et al. 1988). X-axis labels). To avoid complicated viewing, T-bars for standard Unlike the above two actin isoforms, mRNA expression deviations are omitted from the final illustration of ACTA2 was not clearly projected during embryonic de- velopment stages, and the initiation of its expression was and kept to be relatively constant in subsequent ages. observed at hatching (at 28 HPF) in a very low amount, Myogenesis has been known to initiate at an earlier stage which differs the previous demonstration that α-smooth of development in teleost embryos than in amniotes; the muscle actin would be also expressed during early cardiac myogenic precursor cells for slow and fast muscle in fish and skeletal muscle development in various myofibroblast- embryos have been reported to be spatially segregated be- like cells in mammalian embryogenesis (Bertola et al. 2008). fore somite formation (i.e., as early as gastrulation) Currently, it is not clear whether other paralog subiso- (Hirsinger et al. 2004). Hence, the onset expression of form(s) of mud loach ACTA2 may participate in the devel- ACTA1 transcripts at late gastrula stage in this study is opment of early embryonic muscles or not. However, our broadly in agreement with the previous observation. The finding on the absence of ACTA2 transcription in Lee et al. Fisheries and Aquatic Sciences (2017) 20:27 Page 10 of 11 embryogenesis is, at least in part, accordant with the previ- Additional files ous study on zebrafish ACTA2 to show that mural cells Additional file 1: Table S1. Oligonucleotide primers used for cloning (main expression site of zebrafish ACTA2)would develop and expression assay in this study. (PDF 69 kb) relatively late as compared to ones in mammals (Whitesell Additional file 2: Table S2. Summarized information on the mud loach et al. 2014). The authors have hypothesized that the slow α-actin isoform cDNAs and genomic gene. (PDF 75 kb) development of vascular mural cells might be a reflection Additional file 3: Table S3. Genomic exon-intron organization of muscle of the small body size of the zebrafish, and thus, there actin genes in teleost species examined in this study. (PDF 102 kb) would be little need to develop contractile smooth muscle at an early embryo stage (Whitesell et al. 2014). In this Acknowledgements Not applicable. study, the low level of ACTA2 transcripts in just hatched larvae was slightly increased at 1 DPH; however, soon after, Availability of data and materials the expression of ACTA2 transcripts was dramatically in- Not applicable. creased at 2 DPH and kept to be constant until 4 DPH (i.e., Authors’ contributions immediately following the transition to exogenous feeding). SYL carried out the gene expression assays and phylogenetic analysis. JAL This modulation pattern could be explained by the need for performed the molecular cloning and isolation of genes. YKN designed this study, performed the data evaluation, and drafted the manuscript. All the preparation of smooth muscles with the developments authors read and approved the final manuscript. and differentiations of vascular and gastrointestinal organs. Although we did not examine histological ontogenesis of Funding This study was supported by the grant from the Korea Institute of Marine internal organs, initial developments of various smooth Science & Technology Promotion (KIMST; Project #20130369), Ministry of muscle-related organs including intestine might occur in Oceans and Fisheries, Republic of Korea. that period, as scrutinized in a closely related Misgurnus Ethics approval and consent to participate species (M. anguillicaudatus) (Han et al. 2013; Zhang et al. Not applicable. 2016). Subsequently, ACTA2 transcripts were detected in a decreased level (i.e., at 5 DPH and afterwards). Generally, Consent for publication Not applicable. this pattern might be recognized by a stabilization process after the explosive transcriptions in line with the ontogenic Competing interests developments of various smooth muscle-relevant organs. The authors declare that they have no competing interests. However, it may not also be excluded the possibility that ACTA2 transcripts might be diluted in the total RNA tem- Publisher’sNote plates by various other gene transcripts and dominated Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. ACTA1 transcripts with the increase of body mass in fry. Collectively, the three α-actin isoforms are dynamically Received: 14 August 2017 Accepted: 3 October 2017 modulated during embryo development and larval on- togeny largely in an isoform-dependent fashion. References Alves-Costa F, Silva MDP, Wasko AP. 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Fisheries Sci. 1997;63:403–6. and we will help you at every step: Venhatesh B, Tay BH, Elgar G, Brenner S. Isolation, characterization and evolution • We accept pre-submission inquiries of nine pufferfish (Fugu rubripes) actin genes. J Mol Biol. 1996;259:655–65. Watabe S. Myogenic regulatory factors and muscle differentiation during � Our selector tool helps you to find the most relevant journal ontogeny in fish. J Fish Biol. 1999;55:1–18. � We provide round the clock customer support Whitesell TR, Kennedy RM, Carter AD, Rollins EL, Georgijevic S, Santoro MM, Childs � Convenient online submission SJ. An α-smooth muscle actin (acta2/αsma) zebrafish transgenic line marking vascular mural cells and visceral smooth muscle cells. PLoS One. 2014;3:e90590. � Thorough peer review Xu Y, He J, Wang X, Lim TM, Gong Z. Asynchronous activation of 10 muscle- � Inclusion in PubMed and all major indexing services specific protein (MSP) genes during zebrafish somitogenesis. 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Fisheries and Aquatic SciencesSpringer Journals

Published: Oct 30, 2017

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