Expression of cardiac myosin light chain 2 during embryonic heart development in medaka fish, Oryzias latipes, and phylogenetic relationship with other myosin light chains

Experimental animals

The orange variety of medaka, Oryzias latipes, was maintained at the University of California Davis medaka facility. Fish were handled according to an approved institutional animal protocol (protocol #06-12291 University of California, Davis). The temperature was controlled at 26°C and a 14:10 h light : dark light cycle regime was used.

For RNA isolation and whole-mount ISH, the fertilized eggs were incubated in 50-mL tubes containing fresh water at 26°C, with gentle shaking to synchronize their development. At specific times embryos were selected, based on when the desired number of somites had developed as determined using bright fields microscopy.

RNA isolation, cDNA cloning and RT–PCR

To obtain the cDNA encoding mcmlc2, we evaluated the expression pattern of available medaka expressed sequence tags (ESTs) (Medaka EST database, Laboratory of Embryology, Graduate School of Science, University of Tokyo, http://medaka.lab.nig.ac.jp/), resembling the myosin light chain gene, which is homologous to that of zebrafish. We found one (MF01FSA042I24) and the specific primers for the rapid amplification of cDNA ends (5′- and 3′-rapid amplification of cDNA ends [RACE])-polymerase chain reaction (PCR) methods, which were designed based on its nucleotide sequence. The medaka stage 25 embryonic cDNA library was constructed using the Marathon cDNA amplification kit (Clontech) from RNA isolated from the whole embryo at stage 25 using the total RNA isolation kit from Stratagene (La Jolla, CA, USA). Both kits were used following the manufacturer's instructions. To clone the cDNA encoding the full length of mcmlc2, a pair of primers specific for the very N-terminal (mcmlc2 F1, 5′-TGTATTTTCCATGTTTGAGCAGTC-3′) and C-terminal (mcmlc2 R1, 5′-TGTGTGATAATGTAGCAGAGTGA-3′) ends of the clone, MF01FSA042I24, coding regions were synthesized. The cDNAs encoding full length of mcmlc2 were cloned from the medaka stage 25 embryonic cDNA library following the 5′- and 3′-RACE-PCR methods with these mcmlc2 specific primers using an Advantage 2 PCR enzyme system (Clontech), or Advantage GC-2 polymerase mix (Clontech), following the instructions of the manufacturing company. The obtained cDNAs encoding 5′- and 3′-terminal regions of mcmlc2 were inserted into a pGem-T Easy vector (Promega, Madison, WI, USA) and sequenced with an ABI 3730 capillary electrophoresis genetic analyzer at the DNA Sequencing Facility in UC Davis. For cardiac myosin heavy chain (MYH), for whole-mount ISH, cDNA encoding MYH_C2 (MF015DA026N10) was obtained from the National Bioresources Project (medaka) (Japan) (http://www.shigen.nig.ac.jp/medaka/) and then the DNA fragment was amplified following the PCR method with specific primers, mMYH_C2 F1 (5′- GAGACT GTGAAA GGTATCCG-3′) and mMYH_C2 R1 (5′-CTTAGCTT ATCGACCTGAGAC-3′). The obtained cDNAs were used for making RNA probes.

Database analysis

Signal sequence cleavage sites were predicted using the PSORT II program through the PSORT WWW Server (http://psort.nibb.ac.jp/). N- and O-linked glycosylation sites were predicted using the websites http://www.cbs.dtu.dk/services/NetNGlyc/ and http://www.cbs.dtu.dk/services/NetOGlyc/, respectively. The predicted amino acids sequences of mcmlc2 were analyzed to find homologous proteins using the National Center for Biotechnology Information (NCBI) protein–protein BLAST database (http://www.ncbi.nlm.nih.gov/BLAST/).

Identification of genomic structure of mcmlc2

The genomic structure of mcmlc2 was identified through the medaka genomic database (http://dolphin.lab.nig.ac.jp/medaka/).

Semi-quantitative RT–PCR

To identify the gene expression pattern of mcmlc2 and mMHY_C2 during the medaka embryogenesis, semi-quantitative reverse transcription–polymerase chain reaction (sqRT–PCR) following the method described by Marone et al. (2001) using primers specific for mcmlc2, mMYH_C2 and medaka elongation factor α1 (mEFα1) were carried out. The sqRT–PCR was carried out using same kit for RT-PCR described above. A pair of primers described above, mcmlc2 F1 and mcmlc2 R1 for detection of mcmlc2 gene expression and mMYH_C2 F1 and mMYH_C2 R1 for mMYH_C2 gene expression were used. As the control, the primers specific for the mEFα1, mEFα1 810F (5′-CAG GAC GTC TAC AAA ATC GG-3′) and mEFα1160R (5′-AGC TCG TTG AAC TTG CAG GCG-3′) (AB013606) were used. The sqRT–PCR reaction was carried out with one cycle of 50°C for 1 h and 94°C for 300 s, 34 cycles of 94°C for 30 s, 52°C for 30 s, 68°C for 60 s, and 68°C for 6 min for mcmlc2 and for 300 s, 33 cycles of 94°C for 30 s, 53°C for 30 s, 68°C for 60 s, and 68°C for 6 min for mMYH_C2, using a MiniCycler thermal cycler with hot bonnet heat lid (Bio-Rad, Hercules, CA, USA [previously MJ Research]).

Histological detection of mcmlc2 and mMYH_C2 gene expression during embryogenesis

Morpholino injection

To knockdown the function of the mcmlc2 gene product by blocking the translation initiation complex sterically, the antisense oligo (anti-mcmlc2 Morpholino oligo: MO-mcmlc2; 5′-GTCTGGATGACTGCATGAGG ACCTG-3′) were designed and purchased from Gene Tools LLC (Philomath, OR, USA). For injection, the morpholino solutions were diluted to 10–300 µm in a final concentration of 1 × Yamamoto's solution (Yokoi et al. 2006). The same amount of MO-mcmlc2 (200 pL/embryo) and 1 × Yamamoto solution as the control were injected into the one cell stage wild type embryos and then both injected embryos were incubated at 26°C. The embryos were observed at 2 days post-fertilization (2 dpf), 3 dpf, 4 dpf and 5 dpf using a microscope. Their heart contractions were recorded using a CCD camera, and their phenotypic changes were recorded and counted as heart beats per min.

Phylogenetic analysis

Phylogenetical analyses were conducted using MEGA version 4 (Tamura et al. 2007). The deduced amino acid sequences of cDNAs, encoding the MLC2 with high similarity to that of mcmlc2 obtained above, were aligned by using CLUSTAL W (Thompson et al. 1994). The alignment was used to generate a phylogenetic tree by the neighbor-joining methods with a booststrap value from 1000 replicates. The amino acids sequences of MLCs (Table 1) were deduced from cDNA nucleotide sequences retrieved from the NCBI BLAST database (http://www.ncbi.nlm.nih.gov/BLAST/) and Ghost Database (Ciona intestinalis genomic and cDNA resources) (http://ghost.zool.kyoto-u.ac.jp/indexr1.html).

Sequence and cloning analysis of medaka cardiac myosin light chain 2 (mcmlc2)

To understand the molecular structure of mcmlc2, the cDNA encoding mcmlc2 was obtained from the stage 25 embryonic cDNA library. The mcmlc2 cDNA (AB458318) consists of 611 nucleic acids and is predicted to encode the protein of 172 amino acid residues (Fig. 1A). A predicted signal peptide cleavage site was not detected using the methods of von Heijne (1986) using the PSORT II program. The poly (A)⁺ tail addition signal was located at 50 bp (AATAAA) from the 3′-end. The predicted amino acid sequence contained three N-glycosylation modification sites; however, O-glycosylation modification sites were not detected. A conserved EF-hand calcium-binding motif was located in the central to C-terminal portion of the sequence (Fig. 1 A bold letters and Fig. 6). Based on a medaka genomic database search, the genomic structure of mcmlc2 was determined and found to span 1804 nucleotides and include seven exons and six introns (Fig. 1B). The intron-exon structure of the mcmlc2 gene (Fig. 1B) is the same as those of human and mouse, containing seven exons and six introns. By contrast, the fly (Drosophila melanogaster) gene has three exons and the nematode (Caenorhabditis elegans) gene contains four (Doevendans et al. 2000). Based on a BLAST comparison to known proteins (NCBI: http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), medaka cardiac myosin light chain 2 (mcmlc2) was found to belong to the EF-hand (Ca²⁺-binding protein) superfamily. Identity to the myosin light polypeptide 7 (Danio rerio) (NM_131329.2), cardiac myosin light chain 2 (Danio rerio) (AF425743.1), myosin light chain (Xenopus laevis) (NM_001086846.1) and myosin light chain 2a (Homo sapiens) (NM_021223.2) was 85, 84, 79% and 71%, respectively. Homologous amino acid sequences from other species were also used for the phylogenetic analysis below.

Expression of mcmlc2 and mMYH_C2 during embryogenesis

To determine the relationship between the timing of both mcmlc2 and mMYH_C2 gene expression and the timing of cardiovascular system development during embryogenesis, sqRT–PCR and whole-mount ISH were carried out following the materials and methods described earlier. As shown in Figure 2, expression of mcmlc2 was detected in the mRNA obtained from stage 20 embryos. At this stage, the cardiac mesoderm was initiated to migrate from the neural crest to become the bi-lateral cardiac mesoderm (Fig. 3A–G). Furthermore, the expression of mMHY_C2 was detected first in stage 18 embryos in this experiment. After stage 24, the expression of mMYH_C2 seemed to be increased.

Following the whole-mount ISH analysis, the specific timing and location of mcmlc2 gene expression compared with that of mMYH_C2, was determined in the embryos at certain embryonic stages (Fig. 3). The expression of mcmlc2 was first detected at stage 21 (Fig. 3A,B). Its expression was found in bilateral stripes of cells located at the posterior region of the head. Using microscopy, and viewing the dorsal side, the cardiomyocyte precursors expressing mcmlc2 were seen to separate into bilateral mesodermal tubes (Fig. 3A,B). As the two sheet-structures migrate and make contact at the midline, they were found to form a bridge of cells that fuse at the midline on the ventral side. The posterior and anterior edges of these tube-structures then bent towards the middle becoming more circular in shape, and eventually forming a cone. In stage 22 embryos, heart tube formation is initiated. We discovered that the heart cone (tube) elongates from the dorsal area of the body cavity towards the ventral area of the cavity (3, 4). A difference is noted in zebrafish, as the heart cone (tube) elongates from the ventral side to the dorsal side, but with no mention of a relation to the body cavity (Stainier et al. 1993). During this stage, the migration of the cardiomyocyte precursors was completed and the heart tubular structure (heart cone) could be clearly observed (Fig. 3C–F). Following the growth of the embryo's body around the circumference of the yolk, the heart cone began growth towards the body cavity (Fig. 3E,F). By the end of this stage, the cardiomyocyte precursor cells expressing mcmlc2 could be detected underneath the head and extending from the posterior end of the mid-brain to the anterior end of the hindbrain. The one-layer cone structure expressing mcmlc2 then became more tubular and grew straight from the ventral surface of the embryo toward the inner layer of the body cavity, whose wall lay against the yolk mass (Fig. 3F,G). During this stage, following the embryo formation, the body cavity extends further toward the posterior end of the eye vesicles. Expression of mMYH_C2 was also observed in the elongating heart tube in stage 23 embryos; however, the expression pattern was not the same as that of mcmlc2. In the developing heart tube, some cells expressed the mMYH_C2 (Fig. 3H–J); however, its expression could not be detected in all areas of the entire tube as observed with the mcmlc2 (Fig. 3G). The heart tube subsequently continued to straighten and became thicker. At stage 24 (the 16-somite stage), the heart tube had initiated a rhythmic beat of approximately 33–64 beats per min.

By the end of stage 25, the heart tube (cone) expressing mcmlc2 was thicker than that observed in previous stages. It extended to the anterior end of the forebrain. The expression of mMYH_C2 also continued at the same elongating heart tube at stage 25 (data not shown). At stage 25 (18–19-somite stage), a heartbeat of 70 to 80 beats per min could be detected, and contractions could be observed moving towards the anterior cardinal vein and the root of the dorsal aorta. This contraction was observed in the whole developing tube synchronously. By this stage, chamber differentiation had been initiated, and the heart beat could be seen expanding and contracting in a direction toward the right, lateral side. The anterior portion of the heart tube will differentiate into the atrium and the posterior portion into the ventricle; however, at this stage the patterns of expression of both mcmlc2 and mMYH_C2 were the same as they were at the earlier stages.

The expression patterns of mcmlc2 and mMYH_C2 in embryos at stages 28 to 39 are shown in Figure 3(K,M,N) (mcmlc2) and Figure 3(L,O,P) (mMYH_C2). In stage 28–30 embryos, the shape of the heart tube appears differentiated into a segmental (chamber) structure expressed by both mcmlc2 and mMYH_C2 (Fig. 3K,L). At stage 36, heart looping had been initiated and expression of mcmlc2 was found in both the atrium and the ventricle (Fig. 3M). In stages 38 to 39, the expression of mcmlc2 was detected in both differentiated ventricle and atrium (Fig. 3N); however, the mMYH_C2 gene expression was detected only in the differentiated ventricle (Fig. 3O,P).

Abnormal heart formation in MO-mcmlc2 injected embryos

To understand the physiological function of mcmlc2 during embryogenesis, a morpholino designed to knockdown the expression of mcmlc2 gene products was injected into one cell stage embryos. MO-mcmlc2 injected embryos were examined under the microscope every 24 h after injection for phenotypic abnormalities. As a control, an identical amount of 1 × Yamamoto solution was injected into the embryo; however, no phenotypic abnormalities were observed.

Cardiac abnormalities in MO-mcmlc2 injected embryos were found to vary in a dose-dependent manner. The survival ratio to hatching of MO-mcmlc2 embryos at various doses was 20/20 (10 µm MO-mcmlc2), 11/11 (30 µm MO-mcmlc2), 14/24 (100 µm MO-mcmlc2) and 0/23 (300 µm MO-mcmlc2). Of interest, all three test embryos given a MO-mcmlc2 injected dose exhibited delays in their development and growth during embryogenesis in the injected dose dependent manner. The effect was less apparent in the 10 µm MO-mcmlc2 embryos, but becomes more apparent in the 30 µm MO-mcmlc2 and the 100 µm MO-mcmlc2 injected embryos. We also observed a dose-dependent reduction in the numbers of erythrocytes in the blood vessels of embryos injected with 30 µm or more of MO-mcmlc2. Furthermore, the observed erythrocytes were light red in color, suggesting lower hemoglobin content. Most abnormal phenotypes were observed in the developing heart. Figure 4 shows the typical abnormalities of a MO-mcmlc2 injected embryo at 9 dpf. In 9 dpf embryos, the cardiac phenotypes in most of embryos injected 10 µm MO-mcmlc2 were normal; however, in some embryos, as shown in Figure 4(C,D), the atria became longer than those of the control (Fig. 4A,B). At the 30 µm concentration (Fig. 4E,F), the ventricles were found to be smaller than that of normal embryos, and were not aligned normally with the atria. In these embryos, the atria were longer than those of normal embryos, and their sinus venosus were larger than those of normal embryos (Fig. 4A,B,E,F). At the 100 µm concentration, most of these embryos developed a greatly elongated atrium, a small ventricle and a large sinus venosus. The size of the differentiated ventricle was less than two thirds of that found in normal embryos and most of the hearts did not function correctly. This was because, during heart contraction, the blood capacity of the pumping ventricle was small; and therefore inadequate for pumping the blood to the blood vessels (Fig. 4G,H). In some embryos, the chambers were not correctly differentiated, and appeared only as largely elongated tube structures (data not shown).

Figure 5 shows the cardiac abnormalities in the 300 µm MO-mcmlc2 injected embryos. Most embryos (98%) had a larger and elongated atrium, and a large, abnormal sinus venosus (Fig. 5B–D,G–I). The abnormal thickening and enlargement in these heart tubes were first observed in 2 dpf embryos (Fig. 5A,F). In normal embryos, the heart tube reached the margin of the head and had visibly differentiated chambers by 3 dpf (Fig. 3M). However, in these morphants of the same age, the ventricles were very small as described above, the atria were more elongated in their extension from the head region than that observed in normal embryos, and the heart's looping process was absent (Fig. 5B,G). In 300 µm MO-mcmlc2 injected embryos at 4 dpf, we observed slightly enlarged atria and large sinuses venosus (Fig. 5C,H). In the 5 dpf morphants, the atria and sinuses venosus appeared larger (Fig. 5D,I), and all embryos died before hatching.

To evaluate the possible role of mcmlc2 in heart function, we measured heart rates and visually examined heart contraction in both experimental and control embryos. No statistically significant difference was found between the heart rates of the two groups, but there were important differences in the intensity and coordination of contractions. In some MO-mcmlc2 injected embryos, the amplitude of the heart contraction was small. In other embryos, the contractile pattern was similar to that observed in the heart tube before the initiation of chamber differentiation, with a wave of contraction moving back and forth along the long axis, but not with a synchronous rhythm observed in normal embryos. The rhythm observed in these embryos was asynchronous.

Phylogenetic analysis of myosin light chains

Using the deduced amino acid sequence of mcmlc2 reported in this paper, we constructed a phylogenetic tree to understand its relationship to other MLCs using the neighbor joining method. Figure 6 shows the phylogenetic analysis of MLC-2, including medaka cardiac myosin light chain 2 (mcmlc2). The analysis identified four major clades (Fig. 6A–D). Monophyletic origin of clades A, C and D is indicated by their high bootstrap probability (94–90%), but that of clade B is poorly supported (bootstrap probability = 35%). The genes included in clade A encode proteins characterized as fast skeletal MLC-2 (Fig. 6A). This clade was separated into two sub-clades: the ray-finned fish type and the tetrapod type. All members of clade A are only expressed in skeletal muscle (Fig. 6A1 and 6A2). The MLC-2s in clade B were characterized as the skeletal/light polypeptide 2/ventricular/cardiac muscle isoform. In Ornithorhynchus anatinus (platypus), the gene encoding MLC-2 (XM_001506105) and belonging to clade B is expressed in the liver, lung, brain, and spleen (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db = nuccore&id = 149408856). This may suggest that clade B has evolved many additional functions beyond those of clade A. All genes belonging to clade C encoded MLC polypeptide7 and/or atrial MLC-2. Finally, the proteins in clade D (Fig. 6D) have been characterized as the ‘superfast myosin regulatory light chain 2 and/or myosin regulatory light chain 5’. Currently, the genes encoding MLC-2 in clade D (Fig. 5D) have only been found among the vertebrates in mammals and reptiles. There is little information about which tissues express the MLC-2s in clade D; however, one report for Felis catus (domestic cat) (P41691) finds that the gene is expressed in the jaw-closing muscle (Qin et al. 1994). The classification of MLC-2s based on their expression patterns matched the phylogenetic tree obtained from this study. The medaka cardiac myosin light chain 2 (mcmlc2) reported here was found to belong to clade C (Fig. 6C). The expression of several members of this clade has been analyzed and shown to be restricted to the heart. Our analysis of mcmlc2 expression demonstrates that it is also heart-specific. This conclusion is supported by our whole-mount ISH analysis, which shows heart-specific expression (Fig. 3). We hypothesize that the divergence of MLC-2s during evolution may be related to the change in physiological requirements of the molecule being expressed in each tissue. The amino acid sequence of MLC-2 includes at least two conserved domains: the DC binding domain, which is homologous to the divalent cation-binding domain for both Ca²⁺ and Mg²⁺ in the human cardiac regulatory light chain (Szczesna-Cordary et al. 2004) (1, 7), and the EF-hand motif (Gifford et al. 2007) (Fig. 1). The mutations in the DC binding domains of human ventricular myosin light chain 2 (accession# P10916), N47 K (N47 = essential amino acid for calcium binding 1, or EACB1) and R58Q (R58 = EACB2) corresponds to N/A/D/E/G/S/P/Q115 in the DC binding domain and R/K/Q126 in Figure 7; and each is caused by the loss of calcium binding activity in the molecule (Szczesna-Cordary et al. 2004). Based on our hypothesis, we focused on the Ca²⁺ binding domains, particularly on the amino acid change in the DC binding domain of the MLC-2s molecule. To identify possible key differences in amino acid sequence in and surrounding the DC domains of MCL-2s in all of the clades described in Figure 6, an alignment of the predicted amino acid sequence including the DC binding region was made. It was based on the 78 predicted amino acid sequences spanning the DC domain from the other species using CLUSTAL W as seen in Figure 8 and Table 1 (Thompson et al. 1994). The alignment data from Figure 7 were also used to draw a phylogenetic tree (Fig. 8). The polypeptide sequence of Ciona myosin light chain in Figure 6 was selected as a standard polypeptide sequence from which to compare members of different clades. Table 1 summarizes the sequence similarities present in Figure 7. As shown in Table 1, 24, 8, 25, 4, 10 and 6 polypeptide amino acid sequences were included in A1, A2, B, C, and D clades (Fig. 6, Table 1), respectively.

As shown in Figure 7, among the genes homologous to mcmlc2 obtained through public databases, compared with the amino acid residue at EACB1 in Ciona, those which have not changed the EACB1 belonged only to clade D (type 25, 26), clade B (type 8) and clade A (type 4). In other molecules, EACB1 appears to have changed from E to D, N, A, G, S, Q, or P. Of note, EACB1 in all molecules belonging to clade C has been changed from E to S/Q/P/A. In the case of EACB2, the deduced amino acid sequences in types 3, 4, 5–13, 15, 21 and 22 (Fig. 7, Table 1) were not different from R in Ciona, and in other types, the amino acid at the position of EACB2 has been changed from R to Q/K. Of interest, types 11–13 and 15 in clade B, as described in Figure 7, are identical to both EACB1 (N) and EACB2 (R) in the DC binding domains of human ventricular myosin light chain 2 (accession# P10916), which enables the MLC molecule to bind with Ca²⁺. Types 11–13 and 15 in clade B have all been characterized as skeletal/light polypeptide 2/ventricular/cardiac muscle isoforms in our phylogenetic tree (Fig. 6). This may suggest that MLC-2s with this amino acid haplotype physiologically binds to Ca²⁺ in muscle. We hypothesize that compared with the sequence in Ciona (Fig. 7, type 27), if Ciona is considered as the ancestral stage, then the ancestor MLC-2 did not have any, or at least, a very weak binding activity with Ca²⁺ via EABC1 and EABC2. Also, the N at EABC1 in the clade B was obtained later by acquiring the Ca²⁺ binding activity in this molecule during evolution.

In clade C, characterized as the atrial MCL-2, the amino acid at the position of EACB1 was S/Q/P/A, not E or N. However, if EABC1 were ‘N’, the Ca²⁺ may bind to this molecule through this site. At the position of EACB2, all deduced amino acid sequences in clade C, except for types 21 and 22, also appear different at EABC2 in human ventricular myosin light chain 2 (accession# P10916), R58, which is another important amino acid residue for Ca²⁺ binding activity in this molecule.

This suggests that the DC domains in the atrial MLC-2s may not have binding activity with Ca²⁺ through EACB1, or very weak binding in the molecule belonging to type 21 and 22 via EACB2. Because of this, during molecular evolution, the Ca²⁺ binding activity contributed by EACB1 was acquired later because of mutational change of these two residues, and the character of the atrial MLC-2s may be different from those of other cardiac MLCs in the ventricle, as evolved in clade B.

To identify which domains have contributed to the molecular divergence of MLC-2s, we carried out a phylogenetic analysis on several subdomains. They included the N-terminal region, the domain homologous to the DC binding domain in HCRLC (Fig. 7), the domain homologous to the EF-hand calcium binding motif (Olsson & Sjölin 2001), the region between the two homologous Ca²⁺-binding domains, and the C-terminal region. Figure 8 shows the neighbor-joining tree for the DC binding domain homologous regions based on data in shown in Figure 7. Interestingly, the tree topology is very similar to the one shown in Figure 6, with high statistical probability at the separation point between A B and C (83%). However, the phylogenetic trees based on the other polypeptide sequences did not produce the same topology. The topology of the phylogenetic trees in 6, 8 are similar. Differences are the sequences used in Figure 8 as they represent a shorter amino acid sequence with a tighter focus on the DC domain. Also, the bootstrap values for each node generally became lower in the phylogenetic tree constructed with the amino acid sequences from which the polypeptide sequence shown in Figure 7 were removed. These results demonstrated that the hypothetical accuracy of the tree shown in Figure 6 may be accurate. This suggests the separation of each clade from the ancestor molecule may be due primarily to mutational changes of amino acid residues in the DC domain and its vicinity.