Cloning and expression analysis of myogenin from flounder (Paralichthys olivaceus) and promoter analysis of muscle-specific expression

Myogenin is a bHLH transcription factor of the MyoD family. It plays a crucial role in myoblast differentiation and maturation. We report here the isolation of flounder myogenin gene and the characterization of its expression patterns. Sequence analysis indicated that flounder myogenin shared a similar structure and the conserved bHLH domain with other vertebrate myogenin genes. Flounder myogenin gene contains 3 exons and 2 introns. Sequence alignment and phylogenetic showed that flounder myogenin was more homologous with halibut (Hippoglossus hippoglossus) myogenin and striped bass (Morone saxatilis) myogenin. Whole-mount embryo in situ hybridization revealed that flounder myogenin was first detected in the medial region of somites that give rise to slow muscles, and expanded later to the lateral region of the somite that become fast muscles. The levels of myogenin transcripts dropped significantly in matured somites at the trunk region. Its expression could only be detected in the caudal somites, which was consistent with the timing of somite maturation. Transient expression analysis showed that the 546 bp flounder myogenin promoter was sufficient to direct muscle-specific GFP expression in zebrafish embryos. (c) 2007 Elsevier Inc. All rights reserved.

Characterization of DYRK2 (dual-specificity tyrosine-phosphorylation-regulated kinase 2) from Zebrafish (Dario rerio)

Proteins of the DYRK (dual-specificity tyrosine-phosphorylation-regulated kinase) family are characterized by the presence of a conserved kinase domain and N-terminal DH box. DYRK2 is involved in regulating key developmental and cellular processes, such as neurogenesis, cell proliferation, cytokinesis, and cellular differentiation. Herein, we report that the ortholog of DYRK2 found in zebrafish shares about 70% identity with that of human, mouse, and chick. RT-PCR showed that DYRK2 is expressed maternally and zygotically. In-situ hybridization results show that DYRK2 is expressed in somite cells that will develop into muscles. Our results provide preliminary evidence for investigating the in-vivo function of DYRK2 in zebrafish muscle development.

Characterization of muscle-regulatory gene, MyoD, from flounder (Paralichthys olivaceus) and analysis of its expression patterns during embryogenesis

Specification and differentiation of skeletal muscle cells are driven by the activity of genes encoding members of the myogenic regulatory factors (MRFs). In vertebrates, the MRF family includes MyoD, Myf5, myogenin, and MRF4. The MRFs are capable of converting a variety of nonmuscle cells into myoblasts and myotubes. To better understand their roles in fish muscle development, we isolated the MyoD gene from flounder (Paralichthys olivaceus) and analyzed its structure and patterns of expression. Sequence analysis showed that flounder MyoD shared a structure similar to that of vertebrate MRFs with three exons and two introns, and its protein contained a highly conserved basic helix-loop-helix domain (bHLH). Comparison of sequences revealed that flounder MyoD was highly conserved with other fish MyoD genes. Sequence alignment and phylogenetic analysis indicated that flounder MyoD, seabream (Sparus aurata) MyoD1, takifugu (Takifugu rubripes) MyoD, and tilapia (Oreochromis aureus) MyoD were more likely to be homologous genes. Flounder MyoD expression was first detected as two rows of presomitic cells in the segmental plate. From somitogenesis, MyoD transcripts were present in the adaxial cells that give rise to slow muscles and the lateral somitic cells that give rise to fast muscles. After 30 somites formed, MyoD expression decreased in the somites except the caudal somites, coincident with somite maturation. In the hatching stage, MyoD was expressed in other muscle cells and caudal somites. It was detected only in muscle in the growing fish.

Fate map of zebrafish left-right organizer

The organizer is a ciliated signalling transient organ, responsible for the patterning of embryo tissues during embryonic development. In higher vertebrates, such as mouse and chick, this organizer (the node and the Hensen’s node, respectively) performs dorsalventral and anteriorposterior axis definition, as well as left-right patterning of the internal organs. In lower vertebrates, such as frog and zebrafish, there is a separate specialized organ for left-right purposes called the Gastrocoel Roof Plate (GRP) and Kupffer’s Vesicle (KV), respectively. It is known that mouse and chick organizer cells give rise to structures like floor plate, notochord, hypochord and somites. Frog GRP originates all these but floor plate. In zebrafish, at 13-14 somite stage (ss) the KV finished its left-right patterning but what happens to this organizer’ cells is still poorly studied. This research attempts to understand the fate and behaviour of the KV cells. We followed the fate of KV cells by live imaging and by tight time-courses with fixed larvae. We assessed in detail their proliferative and death profile, as well as cilia length progression from 9-10 ss until 29-30 ss. We conclude that the KV cells mostly follow the evolutionarily conserved fates described for other organizers. These cells mainly incorporate the notochord and hypochord; few cells incorporate the floor plate and the somites. As a novelty, it is also hypothesized that the hypural cell fate may be among the KV cell fates.

The role of Wnt signalling in the development of somites and neural crest

The Wnt family of secreted signalling molecules controls a wide range of developmental processes in all metazoans. In this investigation we concentrate on the role that members of this family play during the development of (1) the somites and (2) the neural crest. (3) We also isolate a novel component of the Wnt signalling pathway called Naked cuticle and investigate the role that this protein may play in both of the previously mentioned developmental processes. (1) In higher vertebrates the paraxial mesoderm undergoes a mesenchymal-to-epithelial transformation to form segmentally organised structures called somites. Experiments have shown that signals originating from the ectoderm overlying the somites or from midline structures are required for the formation of the somites, but their identity has yet to be determined. Wnt6 is a good candidate as a somite epithelialisation factor from the ectoderm since it is expressed in this tissue. In this study we show that injection of Wnt6-producing cells beneath the ectoderm at the level of the segmental plate or lateral to the segmental plate leads to the formation of numerous small epithelial somites. We show that Wnts are indeed responsible for the epithelialisation of somites by applying Wnt antagonists which result in the segmental plate being unable to form somites. These results show that Wnt6, the only member of this family to be localised to the chick paraxial ectoderm, is able to regulate the development of epithelial somites and that cellular organisation is pivotal in the execution of the differentiation programmes. (2) The neural crest is a population of multipotent progenitor cells that arise from the neural ectoderm in all vertebrate embryos and form a multitude of derivatives including the peripheral sensory neurons, the enteric nervous system, Schwann cells, pigment cells and parts of the craniofacial skeleton. The induction of the neural crest relies on an ectodermally derived signal, but the identity of the molecule performing this role in amniotes is not known. Here we show that Wnt6, a protein expressed in the ectoderm, induces neural crest production. (3) The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement (Pandur et al. 2002b). Recent work has identified the protein Naked cuticle as an intracellular switch promoting the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked cuticle-1 (cNkd1) and demonstrate that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly our study shows that the expression of cNkd1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

Expression and regulation of Nkd-1, an intracellular component of Wnt signalling pathway in the chick embryo

The Wnt family of secreted signalling molecules control a wide range of developmental processes in all metazoans. The intracellular response to Wnt signalling depends on the choice of signalling cascade activated in the responding cell. Cells can activate either the canonical pathway that modulates gene expression to control cellular differentiation and proliferation, or the non-canonical pathway that controls cell polarity and movement. Recent work has identified the protein Naked Cuticle to act as an intracellular switch to promote the non-canonical pathway at the expense of the canonical pathway. We have cloned chick Naked Cuticle-1 (cNkd-1) and show that it is expressed in a dynamic manner during early embryogenesis. We show that it is expressed in the somites and in particular regions where cells are undergoing movement. Lastly, we show that the expression of cNkd-1 is regulated by Wnt expression originating from the neural tube. This study provides evidence that non-canonical Wnt signalling plays a part in somite development.

Effect of moderate and severe heat stress on avian embryonic Hsp70 gene expression

Stress response is a universal mechanism developed by all organisms to deal with adverse changes in the environment, which lead to the synthesis of heat shock proteins (Hsps). In this study, the effect of moderate (41degreesC) and severe (44degreesC) heat stress on Hsp70 transcript expression pattern was investigated during chicken embryogenesis. Acute exposure to severe heat stress for one hour resulted in a fifteen-fold increase in Hsp70 mRNA levels. The return of stressed embryos to normal incubation temperature resulted in Hsp70 mRNA levels five-fold higher than control after three hours and normal levels after six hours. Moderate heat stress did not induce enhancements on Hsp70 mRNA levels. The spatial expression of Hsp70 transcripts was detected in embryos under normal incubation conditions. Whole-mount in situ hybridization analysis showed that Hsp70 transcripts were constitutively present in somite and in distinct encephalic domains (predominantly in prosencephalon and mesencephalon areas) of the chicken embryo. These results showed that Hsp70 induction is dependent on incubation temperature conditions, suggesting that early chicken embryos may induce a quick emergence response to cope with severe heat stress by increasing Hsp70 mRNA levels.

Structural analysis of the Pimelodus maculatus (Lacépède, 1803) embryogenesis (Siluriformes: Pimelodidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fetotoxicity caused by the interaction between zinc and arsenic in mice

Arsenic is an environmental pollutant that induces congenital malformations in experimental models and can contribute to human birth defects. The environmental exposure to arsenic is relatively small when compared with the doses required to cause teratogenicity in mice and other laboratory animals. In order to study the action of zinc in the arsenic-induced teratogenicity, in the present work mice were either pretreated with zinc and later with arsenic or were treated simultaneously with zinc and arsenic in vivo and in vitro. Following administration of arsenate on gestation day 8, pregnant females were killed on the 17th day of gestation; maternal and fetal data were collected by laparotomy and used to calculate reproductive parameters. Fetuses were analyzed for the presence of external malformation and, after the appropriate processing, visceral and skeletal analyses were accomplished. Conceptuses were exposed in whole embryo culture to arsenicals on gestation day 8 (3-6 somite stage). After a 26 h culture period, morphological development was assessed. Neither pretreatment with zinc nor simultaneous administration of zinc prevented arsenic teratogenicity in these experimental models. (C) 2002 Wiley-Liss, Inc.

Larval stages of Stenocionops furcatus (Olivier, 1791) (Decapoda : Brachyura : Majoidea) and a reappraisal of larval morphological characters for Mithracidae

Stenocionops furcatus is a spider crab found in the western Atlantic, from Georgia, USA to Rio Grande do Sul, Brazil, on sand, coral, rocks or mud bottoms from the intertidal zone to 180 m. We describe all laboratory-reared larval stages of S. furcatus obtained from the northern coast of São Paulo State, Brazil, and compare our data with existing larval descriptions for the genus and other mithracids. The larval development of S. furcatus consists of two zoeal stages and one megalopa. The durations of the first and second zoeal stage were similar to4 and 5 days respectively, the megalopa appearing 10-18 days after hatching. Our results show that the zoeae of S. furcatus differ from those of other Mithracidae by possessing four setae on the proximal lobe of the coxal endite of the maxilla, instead of five, and by the presence of mid-dorsal setae on the third abdominal somite in the second zoeal stage, which are lacking in other mithracids. Larval descriptions for Stenocionops in two previous publications were attributed to the subspecies S. furcatus coelatus from the Caribbean. Larvae from Brazilian waters closely resemble one of these accounts, suggesting that this taxon extends beyond the West Indies and that the other description represents larvae of S. furcatus furcatus. Additional morphological details, not available previously, are provided.

A morphometric study of the mud crab, Panopeus austrobesus Williams, 1983 (Decapoda, Brachyura) from a subtropical mangrove in South America

The relative growth of the xanthid crab, Panopeus austrobesus was investigated by means of the allometric method. Crabs were obtained in the mangrove formed by the estuary of the rivers Comprido and Escuro (23degrees29'24S 45degrees10'12W), Ubatuba, São Paulo State, Brazil. All crabs were measured to obtain their carapace width (CW) and length (CL), abdomen width (AW) at the basis of the 5(th) somite, and major cheliped propodus length (PL) and height (PH). Males were also measured for their gonopod length (GL). The size of crabs based on CW ranged from 4.0 to 44.8 mm for males and 3.1 to 34.5 mm for females. The relative growth equation (Y = aX(b)) based on the relationship between GL and CW suggested that males reach their sexual maturity near 14.6 mm CW. Such relationship shows a positive allometry during the juvenile phase and an isometric growth in adult life. In females, the estimated size at 50% maturity is 13.0 mm CW, based on the relationship AW vs. CW. Males reach larger sizes than females, which probably provides them better conditions to protect females during courtship. Concerning cheliped size, approximately 73% of the crabs analysed (N = 209), disregarding sex, have the right PL larger than the left. The PL growth shows that specimens with a left major cheliped (26%) have a higher allometric coefficient, despite being smaller considering their CW. Such a difference may compensate the smaller size of the crab during defense or prey capture.

Sexual maturity of the speckled swimming crab Arenaeus cribrarius (Lamarck, 1818) (Decapoda, Brachyura, Portunidae), in the Ubatuba Littoral, São Paulo state, Brazil

The sizes at morphological and physiological maturity of male and female Arenaeus cribrarius were estimated to determine if both events are synchronous. Animals were captured with otto-trawls at Ubatuba, Brazil. A total of 2356 specimens, 977 males and 1379 females, were obtained. The major carapace width without spines (CW), the propodus length of the major cheliped (PL) and the width of the 5th abdominal somite (AW) were measured with vernier calipers. Allometric relationships and gonadal development were analyzed to determine the maturity in both sexes. The size at the onset of male morphological maturity was estimated at CW 52 mm, smaller than the CW 63.4 mm physiological maturity size observed. For females, these events are synchronous since both estimates converged at CW 59.7 mm. The onset of functional sexual maturity in A. cribrarius at CW 63.4 and 59.7 mm in males and females, respectively, would indicate a minimum size of CW 64 mm for fishing purposes. Differences between allometric and gonadal estimates indicate the importance of considering both methods. A comparison of the present results with other available data in portunid crabs is provided.

The first zoeal stage of the genus Cronius (Brachyura, Portunidae) from the Brazilian coast, hatched in a laboratory

This study describes the first zoeal stage of Cronius ruber and Cronius tumidulus. The zoeae can be distinguished using the setation of the cephalic appendages and the lateral process on the abdominal somite.

Larval development of Apiomithrax violaceus (A. Milne Edwards, 1868) (Decapoda : Brachyura : Majoidea : Pisidae) reared in laboratory conditions, and a review of larval characters of Pisidae

Apiomithrax violaceus (A. Milne Edwards, 1868 ) is a pisid majoid crab occurring in tropical and subtropical coastal waters of the eastern and western South Atlantic. Larval development consists of two zoeal stages and a megalopa. Beginning with the first zoea, the duration of each larval stage at 24degreesC was 3-8 (5+/-1), 3-5 (4+/-0.5) and 9-15 (11+/-2) days, the megalopa and first crab instar appearing 9-11 (10+/-1) and 20-27 (23+/-2) days after hatching, respectively. Larval characters agree with those proposed for the Majoidea, in having nine or more setae on the scaphognathite in the first zoea and well-developed pleopods in the second zoea. However, larvae of A . violaceus do not fit larval pisid features. Zoeal stages differ from most other Pisidae in having lateral spines, a long rostral spine extending beyond the antenna, two spines per telson fork and a dorsolateral process on the third abdominal somite. The megalopa differs in having a spine dorsally on the carapace and on the basial segment of the second pereiopod. Two characters that are potentially unique to Apiomithrax include a zoeal antenna with an exopod that is much longer than the protopod, and a rostral spine that is longer than the dorsal spine. These characters should facilitate the identification of this taxon and could also be useful for phylogenetic studies. A review of larvae of 28 species among 14 genera indicated that there is no apparent single larval character that differentiates the Pisidae, with more limited phylogenetic analyses suggesting that this is a paraphyletic group. Apiomithrax , Eurynolambrus , Pisoides , Rochinia and Scyra have the most divergent morphological characters within the family. The analysis and inclusion of additional taxa is likely to shed more light on the sister-group relationships of the Pisidae. However, based on the extent of morphological interspecific variability of known larvae it is likely that the group, as presently defined by adult morphology, is not monophyletic.

On the morphology of the worker immatures of the leafcutter ant Atta sexdens linnaeus (Hymenoptera: Formicidae)

Leafcutter ants of the genus Atta Fabricius are serious agricultural pests. Morphological studies of immature stages within this group are few, and the data provided for species of considerable importance are usually incomplete. In this study, the immatures of Atta sexdens Linnaeus are described and compared using light and scanning electron microscopy. Only specimens from founding stage colonies (i.e., lacking adult workers) were used. The existence of four larval instars was estimated by a frequency plot of maximum head widths, and the larvae of different instars differed from each other mainly by their bodily dimensions. Worker larvae belonged to two distinct morphological castes: (1) gardeners and nurses and (2) within-nest generalists. The worker larvae described in this study differed from a previous description of the same species by the following traits: the existence of a genal lobe, the number of clypeal hairs, the presence of two hairs on the ninth abdominal somite, the presence of hairs on the anterior surface of the labrum, and the shape of the maxillary palpus. This study provides a comparative analysis of immature stages of A. sexdens that may be relevant to future morphological and biological studies of the Attini. Microsc. Res. Tech. 75:10591065, 2012. (c) 2012 Wiley Periodicals, Inc.