Development of the endoderm and gut in medaka, Oryzias latipes

We performed an extensive analysis of endodermal development and gut tube morphogenesis in the medaka embryo by histology and in situ hybridization. The markers used in these analyses included sox17, sox32, foxA2, gata-4, -5, -6 and shh. sox17, sox32, foxA2, and gata-5 and -6 are expressed in the early endoderm to the onset of gut tube formation. Sections of medaka embryos hybridized with foxA2, a pan-endodermal marker during gut morphogenesis, demonstrated that gut tube formation is initiated in the anterior portion and that the anterior and mid/posterior gut undergo distinct morphogenetic processes. Tube formation in the anterior endoderm that is fated to the pharynx and esophagus is much delayed and appears to be independent of gut morphogenesis. The overall aspects of medaka gut development are similar to those of zebrafish, except that zebrafish tube formation initiates at both the anterior and posterior portions. Our results therefore describe both molecular and morphological aspects of medaka digestive system development that will be necessary for the characterization of medaka mutants.     

Expression of the congenital heart disease 5/tryptophan rich basic protein homologue gene during heart development in Medaka fish,Oryzias latipes

The congenital heart disease 5 (CHD5)/tryptophan rich basic protein (WRB) is a protein containing a tryptophan‐rich carboxy‐terminal region, which was discovered in the human fetal heart. In humans, this CHD5/WRB is located between the markers ACTL5‐D21S268 within the Down syndrome (DS) Region‐2 at chromosome 21. Congenital heart disease is commonly linked to DS patients. The functions of this gene product are unknown. To identify the functions of CHD5/WRB in heart formation during embryogenesis, the medaka CHD5 cDNA (mCHD5) was isolated and its gene expression pattern and the localization of its gene product were investigated. The obtained mCHD5 belongs to the CHD5 superfamily, whose members include coiled‐coil proteins. The mCHD5 gene was found to be expressed in the developing heart after stage 28 at which the chamber (ventricle and atrium) differentiation in the heart tube is initiated in the embryo. Its gene product was also detected in the developing heart at embryonic stage 28 and 35. Knocking‐down of mCHD5 function caused severe cardiac disorder, including abnormal chamber differentiation, abnormal looping and ocular abnormality such as Cyclops. Our results provide the mCHD5 gene expression pattern as well as its physiological role during heart formation in a vertebrate model system.     

Human cardiac myosin light chains: Sequence comparisons between myosin LC1 and LC2 from normal and idiopathic dilated cardiomyopathic hearts

The primary structures of light chains isolated from the human myocardium with idiopathic dilated cardiomyopathy (IDC) were determined and compared with the sequence structures of myosin light chains obtained from control human heart myosin. Sequences were determined by chemical analysis and the identity of N-terminal residues established by mass spectrometry. The N-terminal residues in essential (ELC) and regulatory (RLC) light chains were blocked and were identified to be trimethyl alanine. The amino acid sequences of ELC and RLC from control human myosin revealed a high degree of homology with those purified from rat and chicken cardiac myosin. Comparison with a published partial chemical sequence of the human heart myosin light chains revealed significant variations. However, there was very good agreement with published sequences obtained by molecular biological techniques. Sequences of the light chains from cardiomyopathic myosin revealed no difference in the primary structures when compared with control human heart myosin light chains indicating IDC had no influence on, nor was caused by, altered myosin light chain gene expression.     

Spatial-temporal expression of pum1 and pum2 in medaka Oryzias latipes

Two pumilios, pum1 and pum2, were identified in medaka Oryzias latipes. Oryzias latipes pum1 and pum2 are ubiquitous in the adult tissues but with specific expression in the germ cells of gonads, ovary and testis. Pum1 is expressed in the spermatogonia to spermatocytes whilst pum2 presents in spermatocytes of testis only. Oryzias latipes pum1 and pum2 are maternally supplied RNA with ubiquitous expression in the early stages, and embryonic expression of pum1 and pum2 may begin from early gastrula. Both pum1 and pum2 are expressed in the tissues including brain, eye and trunk, and both are expressed in the gonads after hatching. Taken together, Pum1 and Pum2 may play important roles in embryonic and germ cell development of O. latipes.     

Isolation of cardiac myofibrils and myosin light chains with in vivo levels of light chain phosphorylation

Conditions are described for the preparation of functional myofibrils and myosin light chains from freeze-clamped beating hearts with the state of light chain phosphorylation chemically ‘frozen’ during the extraction procedure. Myofibrils were shown to be functionally intact by measurement of Ca²⁺ binding and ATPase activity. Highly purified cardiac myosin light chains could be routinely isolated from myofibrillar preparations using ethanol fractionation together with ion-exchange chromotography. Analysis of light chains for covalent phosphate indicated that basal levels of phosphorylation of the 18?20 000 dalton light chain of myosin in rabbit hearts beating in situ or in a perfusion apparatus were 0.3–0.4 mol/mol. Covalent phosphate content of the light chain fraction did not change during perfusion of hearts with 10 μM epinephrine.     

Identification of SNPs,mapping and analysis of allele frequencies in two candidate genes for meat production traits: the porcine myosin heavy chain 2B (MYH4) and the skeletal muscle myosin regulatory light chain 2 (HUMMLC2B)

Myosin is one of the most important skeletal muscle proteins. It is composed of myosin heavy chains and myosin light chains that exist with different isoforms coded by different genes. We studied the porcine myosin heavy chain 2B (MYH4) and the porcine skeletal muscle myosin regulatory light chain 2 (HUMMLC2B) genes. A single nucleotide polymorphism (SNP), identified for each gene, was used for linkage mapping of MYH4 and HUMMLC2B to porcine chromosome (Sscr) 12 and Sscr 3, respectively. The mapping of these two genes was confirmed by using a porcine-rodent radiation hybrid panel, even if for MYH4 the LOD score and the retention fraction were low. Allele frequencies at the two loci were studied in a sample of 307 unrelated pigs belonging to seven different pig breeds. Moreover the distribution of the alleles at these two loci was analysed in groups of pigs with extreme divergent (positive and negative) estimated breeding values (EBV) for four meat production traits that have undergone selection in Italian heavy pigs.     

Structural and functional changes of myosin during development: comparison with adult fast, slow and cardiac myosin.

ATPase (Ca²⁺ and K⁺ activated) activity of myosin prepared from muscles of 3–4 week rabbit embryos (EM) is slighly lower than that of adult fast muscle myosin (FM), but in contrast to the less active adult slow muscle myosin (SM) is stable on exposure to pH 9.2. Studies of the time course, by means of Na dodecyl-SO₄ polyacrylamide gel electrophoresis, of changes in the pattern of polypeptides released by tryptic digestion show that in this regard EM is closest to SM. The light chain complement of EM appears identical with that of FM rather than of SM or cardiac myosin (CM) by the criteria of coelectrophoresis and removal by 5,5′-dithio-2-dinitrobenzoate treatment of LC₂ except that the relative amount of LC₃ is less in EM than in FM. The staining pattern of light meromyosin (EMM) paracrystals prepared from EM is distinct from either the FM, SM or CM LMM staining pattern. These studies suggest that different genes are involved in the coding for embryonic and adult heavy chains.     

Phosphorylation of myosin light chains and C-protein in the myocardium of hibernating ground squirrel Citellus undulatus

A comparative study was made of the extent of phosphorylation of myosin regulatory light chains and C-protein from the left ventricle of the hibernant ground squirrel Citellus undulatus during the periods of hibernation and activity. During hibernation, the light chains were found to be completely dephosphorylated. In active animals, the share of phosphorylated light chains averaged 40–45%. The extent of cardiac C-protein phosphorylation in hibernation was about twice higher than in the active state. Seasonal differences in phosphorylation of the two proteins of ground squirrel myocardium are discussed in the context of adaptation to hibernation.     

Decreased phosphatase activity, increased Ca2+ sensitivity, and myosin light chain phosphorylation in urinary bladder smooth muscle of newborn mice

Developmental changes in the regulation of smooth muscle contraction were examined in urinary bladder smooth muscle from mice. Maximal active stress was lower in newborn tissue compared with adult, and it was correlated with a lower content of actin and myosin. Sensitivity to extracellular Ca2+ during high-K+ contraction, was higher in newborn compared with 3-wk-old and adult bladder strips. Concentrations at half maximal tension (EC50) were 0.57 +/- 0.01, 1.14 +/- 0.12, and 1.31 +/- 0.08 mM. Force of the newborn tissue was inhibited by approximately 45% by the nonmuscle myosin inhibitor Blebbistatin, whereas adult tissue was not affected. The calcium sensitivity in newborn tissue was not affected by Blebbistatin, suggesting that nonmuscle myosin is not a primary cause for increased calcium sensitivity. The relation between intracellular [Ca2+] and force was shifted toward lower [Ca2+] in the newborn bladders. This increased Ca2+ sensitivity was also found in permeabilized muscles (EC50: 6.10 +/- 0.07, 5.77 +/- 0.08, and 5.55 +/- 0.02 pCa units, in newborn, 3-wk-old, and adult tissues). It was associated with an increased myosin light chain phosphorylation and a decreased rate of dephosphorylation. No difference was observed in the myosin light chain phosphorylation rate, whereas the rate of myosin light chain phosphatase-induced relaxation was about twofold slower in the newborn tissue. The decreased rate was associated with a lower expression of the phosphatase regulatory subunit MYPT-1 in newborn tissue. The results show that myosin light chain phosphatase activity can be developmentally regulated in mammalian urinary bladders. The resultant alterations in Ca2+ sensitivity may be of importance for the nervous and myogenic control of the newborn bladders.     

Expression of proto-oncogenes and muscle specific genes during cardiac hypertrophy and development in rats and humans

A regulatory interdependence of expression of proto-oncogenes and muscle specific genes observed in smooth muscle was examined in cardiac muscle during normal development and hypertrophy both in rats and humans. During normal development in rats, myosin light chain 2 expression is very low at prenatal stages, while c-fos expression starts from the early stages of embryonic development. In aorta constricted rats c-fos induction occurs within 30 min whereas myosin light chain 2 expression is sufficiently high only after 3 or 4 days of post operative period. In the case of humans, the expression of myosin light chain 2 as well as c-fos occurs at high levels during embryonic development. Similar results were obtained with tissue samples obtained from patients with cardiac abnormalities. Induction of the c-fos gene in cultured myocytes by 12-O-tetradeeanoylphorbol 13-acetate has no influence on the expression of myosin light chain 2. These studies were extended with studies on c-myc and Β-myosin heavy chain gene expression which revealed a similar pattern of expression as that of c-fos and myosin light chain 2. These results have indicated that the expression of proto-oncogenes in cardiac muscle may be independently regulated from the expression of muscle specific genes.     

Gonadal sex differentiation and expression of Sox9a2, Dmrt1, and Foxl2 in Oryzias luzonensis

Oryzias luzonensis is closely related to the medaka, O. latipes. The sex of both species is determined by an XX‐XY system. However, the testis determining gene (DMY/Dmrt1bY) found in O. latipes does not exist in O. luzonensis. Instead, a different gene is thought to act as a testis determining gene. In this study, we focused the gonadal sex differentiation process in O. luzonensis under different testis determining gene. First, we observed the gonadal development of O. luzonensis histologically. We then analyzed the expression of Sox9a2/Sox9b, Dmrt1, and Foxl2 during early development. Our results suggest that the sexual differentiation of germ cells in O. luzonensis is initiated later than in O. latipes. However, the timing of the sexual differentiation of the supporting cell linage is similar between the species. genesis 47:289–299, 2009. © 2009 Wiley‐Liss, Inc.     

Study of reciprocal effects of cardiac myosin and tropomyosin isoforms on actin-myosin interaction with in vitro motility assay

Interaction of myosin with actin in striated muscle is controlled by Ca²⁺ via thin filament associated proteins: troponin and tropomyosin. In cardiac muscle there is a whole pattern of myosin and tropomyosin isoforms. The aim of the current work is to study regulatory effect of tropomyosin on sliding velocity of actin filaments in the in vitro motility assay over cardiac isomyosins. It was found that tropomyosins of different content of α- and β-chains being added to actin filament effects the sliding velocity of filaments in different ways. On the other hand the velocity of filaments with the same tropomyosins depends on both heavy and light chains isoforms of cardiac myosin.     

Truncation of vertebrate striated muscle myosin light chains disturbs calcium-induced structural transitions in synthetic myosin filaments

Electron microscopy and negative staining techniques have been used to show that the proteolytic removal of 13 amino acids from the N-terminus of essential light chain 1 and 19 amino acids from the N-terminus of the regulatory light chain of rabbit skeletal and cardiac muscle myosins destroys Ca(2+)-induced reversible movement of subfragment-2 (S2) with heads (S1) away from the backbone of synthetic myosin filaments observed for control assemblies of the myosin under near physiological conditions. This is the direct demonstration of the contribution of the S2 movement to the Ca(2+)-sensitive structural behavior of rabbit cardiac and skeletal myosin filaments and of the necessity of intact light chains for this movement. In muscle, such a mobility might play an important role in proper functioning of the myosin filaments. The impairment of the Ca(2+)-dependent structural behavior of S2 with S1 on the surface of the synthetic myosin filaments observed by us may be of direct relevance to some cardiomyopathies, which are accompanied by proteolytic breakdown or dissociation of myosin light chains.     

Structures of smooth muscle myosin and heavy meromyosin in the folded, shutdown state

Remodelling the contractile apparatus within smooth muscle cells allows effective contractile activity over a wide range of cell lengths. Thick filaments may be redistributed via depolymerisation into inactive myosin monomers that have been detected in vitro, in which the long tail has a folded conformation. Using negative stain electron microscopy of individual folded myosin molecules from turkey gizzard smooth muscle, we show that they are more compact than previously described, with heads and the three segments of the folded tail closely packed. Heavy meromyosin (HMM), which lacks two-thirds of the tail, closely resembles the equivalent parts of whole myosin. Image processing reveals a characteristic head region morphology for both HMM and myosin, with features identifiable by comparison with less compact molecules. The two heads associate asymmetrically: the tip of one motor domain touches the base of the other, resembling the blocked and free heads of this HMM when it forms 2D crystals on lipid monolayers. The tail of HMM lies between the heads, contacting the blocked motor domain, unlike in the 2D crystal. The tail of whole myosin is bent sharply and consistently close to residues 1175 and 1535. The first bend position correlates with a skip in the coiled coil sequence, the second does not. Tail segments 2 and 3 associate only with the blocked head, such that the second bend is near the C-lobe of the blocked head regulatory light chain. Quantitative analysis of tail flexibility shows that the single coiled coil of HMM has an apparent Young's modulus of about 0.5 GPa. The folded tail of the whole myosin is less flexible, indicating interactions between the segments. The folded tail does not modify the compact head arrangement but stabilises it, indicating a structural mechanism for the very low ATPase activity of the folded molecule.     

The nonmuscle myosin regulatory light chain gene mlc-4 is required for cytokinesis, anterior-posterior polarity, and body morphology during Caenorhabditis elegans embryogenesis.

Using RNA-mediated genetic interference in a phenotypic screen, we identified a conserved nonmuscle myosin II regulatory light chain gene in Caenorhabditis elegans, which we name mlc-4. Maternally supplied mlc-4 function is required for cytokinesis during both meiosis and mitosis and for establishment of anterior-posterior (a-p) asymmetries after fertilization. Reducing the function of mlc-4 or nmy-2, a nonmuscle myosin II gene, also leads to a loss of polarized cytoplasmic flow in the C. elegans zygote, supporting models in which cytoplasmic flow may be required to establish a-p differences. Germline P granule localization at the time of cytoplasmic flow is also lost in these embryos, although P granules do become localized to the posterior pole after the first mitosis. This result suggests that a mechanism other than cytoplasmic flow or mlc-4/nmy-2 activity can generate some a-p asymmetries in the C. elegans zygote. By isolating a deletion allele, we show that removing zygotic mlc-4 function results in an elongation phenotype during embryogenesis. An mlc-4/green fluorescent protein transgene is expressed in lateral rows of hypodermal cells and these cells fail to properly change shape in mlc-4 mutant animals during elongation.     

Analysis of the origin and development of hatching gland cells by transplantation of the embryonic shield in the fish, Oryzias latipes

Hatching gland cells of the medaka, Oryzias latipes, have been observed to differentiate from the anterior end of the hypoblast, which seems to first involute at the onset of gastrulation. These results suggest that the hatching gland cells of medaka originate from the embryonic shield, the putative organizer of this fish. The present study investigated whether hatching gland cells really originate from the embryonic shield in the medaka. Transplantation experiments with embryonic shield and in situ hybridization detection of hatching enzyme gene expression as a sign of terminal differentiation of the gland cells were carried out. The analysis was performed according to the following processes. First, identification and functional characterization of the embryonic shield region were made by determining the expression of medaka goosecoid gene and its organizer activity. Second, it was confirmed that the embryonic shield had an organizer activity, inducing a secondary embryo, and that the developmental patterns of hatching gland cells in primary and secondary embryos were identical. Finally, the hatching gland cells as identified by hatching enzyme gene expression were found to coincide with the dye-labeled progeny cells of the transplanted embryonic shield. In conclusion, it was determined that hatching gland cells were derived from the embryonic shield that functioned as the organizer in medaka.     

Expression of murine early embryonic antigens, SSEA-1 and antigenic determinant of EMA-1, in embryos and ovarian follicles of a teleost medaka (Oryzias latipes)

Stage-specific embryonic antigen-1 (SSEA-1) and the antigenic determinant of monoclonal antibody EMA-1 are expressed in a stage-specific manner in mouse early embryos. To study whether these antigens generally exist in fish, expression of the antigens was examined in embryos, ovarian follicles, and adult tissues of a teleost medaka (Oryzias latipes), using immunohistochemical techniques. In 1-cell-stage embryos, these carbohydrate antigens were found in numerous cytoplasmic granules in the blastodisc and the cortical cytoplasm. These granules gradually decreased in number as the embryos developed. In 4-cell-stage embryos, the antigens appeared on the cleavage planes and were located on the cleavage planes within the blastoderm in the following cleavage stages. In blastula-stage embryos, the expression was ubiquitously found on the cell surface of blastomeres. At the mid-gastrula stage, the antigens were restricted to the enveloping layer, yolk syncytial layer, and cortical cytoplasm, but were rarely found in deep cells that contribute to formation of the embryonic body. In later-stage embryos and adult fish, the antigens were located in various tissues. In ovarian follicles, the antigens were found in granules of oocytes and granulosa cells. These observations were basically consistent with those in mice; however, expression in 1-cell-stage embryos and ovarian follicles has not been observed in mice. This unexpected finding suggests that the antigens are produced in granulosa cells and transferred to 1-cell-stage embryos via oocytes, and that the antigens involved in the early developmental process are maternally prepared in teleosts.     

Requirements of myocyte-specific enhancer factor 2A in zebrafish cardiac contractility

Myocyte-specific enhancer factor 2A (MEF2A) regulates a broad range of fundamental cellular processes including cell division, differentiation and death. Here, we tested the hypothesis that MEF2A is required in cardiac contractility employing zebrafish as a model organism. MEF2A is highly expressed in heart as well as somites during zebrafish embryogenesis. Knock-down of MEF2A in zebrafish impaires the cardiac contractility and results in sarcomere assembly defects. Dysregulation of cardiac genes in MEF2A morphants suggests that sarcomere assembly disturbances account for the cardiac contractile deficiency. Our studies suggested that MEF2A is essential in cardiac contractility.