RESUMO
The output of the cerebellar cortex is mainly released via cerebellar nuclei which vary in number and complexity among gnathostomes, extant vertebrates with a cerebellum. Cartilaginous fishes, a basal gnathostome lineage, show a conspicuous, well-organized cerebellar nucleus, unlike ray-finned fishes. To gain insight into the evolution and development of the cerebellar nucleus, we analyzed in the shark Scyliorhinus canicula (a chondrichthyan model species) the developmental expression of several genes coding for transcription factors (ScLhx5,ScLhx9,ScTbr1, and ScEn2) and the distribution of the protein calbindin, since all appear to be involved in cerebellar nuclei patterning in other gnathostomes. Three regions (subventricular, medial or central, and lateral or superficial) became recognizable in the cerebellar nucleus of this shark during development. Present genoarchitectonic and neurochemical data in embryos provide insight into the origin of the cerebellar nucleus in chondrichthyans and support a tripartite mediolateral organization of the cerebellar nucleus, as previously described in adult sharks. Furthermore, the expression pattern of ScLhx5,ScLhx9, and ScTbr1 in this shark, together with that of markers of proliferation, migration, and early differentiation of neurons, is compatible with the hypothesis that, as in mammals, different subsets of cerebellar nucleus neurons are originated from progenitors of 2 different sources: the ventricular zone of the cerebellar plate and the rhombic lip. We also present suggestive evidence that Lhx9 expression is involved in cerebellar nuclei patterning early on in gnathostome evolution, rather than representing an evolutionary innovation of the dentate nucleus in mammals, as previously hypothesized.
Assuntos
Evolução Biológica , Calbindinas/metabolismo , Núcleos Cerebelares , Cação (Peixe) , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Animais , Calbindinas/genética , Núcleos Cerebelares/embriologia , Núcleos Cerebelares/metabolismo , Cação (Peixe)/embriologia , Cação (Peixe)/genética , Cação (Peixe)/metabolismo , Proteínas de Peixes/genéticaRESUMO
BACKGROUND: Squaliform sharks represent approximately 27 % of extant shark diversity, comprising more than 130 species with a predominantly deep-dwelling lifestyle. Many Squaliform species are highly specialized, including some that are bioluminescent, a character that is reported exclusively from Squaliform sharks within Chondrichthyes. The interfamiliar relationships within the order are still not satisfactorily resolved. Herein we estimate the phylogenetic interrelationships of a generic level sampling of "squaloid" sharks and closely related taxa using aligned sequences derived from a targeted gene capture approach. The resulting phylogenetic estimate is further used to evaluate the age of first occurrence of bioluminescence in Squaliformes. RESULTS: Our dataset comprised 172 putative ortholog exon sequences. Phylogenetic estimates result in a fully resolved tree supporting a monophyletic lineage of Squaliformes excluding Echinorhinus. Non-luminous Squalidae are inferred to be the sister to a clade comprising all remaining Squaliform families. Our results suggest that the origin of photophores is coincident with an elevated diversification rate and the splitting of families Dalatiidae, Etmopteridae, Oxynotidae and Somniosidae at the transition of the Lower to the Upper Cretaceous. The presence of luminous organs was confirmed for the Sleeper shark genus Zameus. These results indicate that bioluminescence in sharks is not restricted solely to the families Etmopteridae and Dalatiidae as previously believed. CONCLUSIONS: The sister-clade to non-luminous Squalidae comprises five families. The presence of photophores is reported for extant members of three out of these five families based on results of this study, i.e. Lantern sharks (Etmopteridae), Kitefin sharks (Dalatiidae) and Sleeper sharks (Somniosidae). Our results suggest that the origin of luminous organs arose during the rapid diversification event that gave rise to the extant Squaliform families. These inferences are consistent with the idea of diversification of Squaliform sharks being associated with the emergence of new deep-sea habitats in the Lower Cretaceous, which may have been facilitated by the evolution of bioluminescence.
Assuntos
Evolução Biológica , Cação (Peixe)/classificação , Cação (Peixe)/fisiologia , Animais , Núcleo Celular/genética , Cação (Peixe)/genética , Éxons , Feminino , Fósseis , Filogenia , Alinhamento de SequênciaRESUMO
BACKGROUND: Understanding the evolution of the vertebrate pancreas is key to understanding its functions. The chondrichthyes (cartilaginous fish such as sharks and rays) have often been suggested to possess the most ancient example of a distinct pancreas with both hormonal (endocrine) and digestive (exocrine) roles. The lack of genetic, genomic and transcriptomic data for cartilaginous fish has hindered a more thorough understanding of the molecular-level functions of the chondrichthyan pancreas, particularly with respect to their "unusual" energy metabolism (where ketone bodies and amino acids are the main oxidative fuel source) and their paradoxical ability to both maintain stable blood glucose levels and tolerate extensive periods of hypoglycemia. In order to shed light on some of these processes, we carried out the first large-scale comparative transcriptomic survey of multiple cartilaginous fish tissues: the pancreas, brain and liver of the lesser spotted catshark, Scyliorhinus canicula. RESULTS: We generated a mutli-tissue assembly comprising 86,006 contigs, of which 44,794 were assigned to a particular tissue or combination of tissues based on mapping of sequencing reads. We have characterised transcripts encoding genes involved in insulin regulation, glucose sensing, transcriptional regulation, signaling and digestion, as well as many peptide hormone precursors and their receptors for the first time. Comparisons to mammalian pancreas transcriptomes reveals that mechanisms of glucose sensing and insulin regulation used to establish and maintain a stable internal environment are conserved across jawed vertebrates and likely pre-date the vertebrate radiation. Conservation of pancreatic hormones and genes encoding digestive proteins support the single, early evolution of a distinct pancreatic gland with endocrine and exocrine functions in jawed vertebrates. In addition, we demonstrate that chondrichthyes lack pancreatic polypeptide (PP) and that reports of PP in the literature are likely due cross-reaction with PYY and/or NPY in the pancreas. A three hormone islet organ is therefore the ancestral jawed vertebrate condition, later elaborated upon only in the tetrapod lineage. CONCLUSIONS: The cartilaginous fish are a great untapped resource for the reconstruction of patterns and processes of vertebrate evolution and new approaches such as those described in this paper will greatly facilitate their incorporation into the rank of "model organism".
Assuntos
Encéfalo/metabolismo , Cação (Peixe)/genética , Cação (Peixe)/fisiologia , Perfilação da Expressão Gênica , Fígado/metabolismo , Pâncreas/fisiologia , Sequência de Aminoácidos , Animais , Digestão/genética , Evolução Molecular , Genes Homeobox/genética , Glucose/metabolismo , Insulina/química , Insulina/genética , Insulina/metabolismo , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Especificidade de Órgãos , Pâncreas/citologia , Pâncreas/metabolismo , Receptores de Hormônios Pancreáticos/genética , Transdução de Sinais/genética , Fatores de Transcrição/metabolismoRESUMO
It has been recently shown that the somatostatin gene family was likely composed of at least three paralogous genes in the common ancestor of all extant jawed vertebrates. These three genes, namely SS1, SS2 and SS5, are thought to have been generated through the two rounds of whole-genome duplications (2R) that took place early during the vertebrate evolution. In the present study, we report the cloning of three distinct somatostatin cDNAs from the dogfish Scylorhinus canicula, a member of the group of cartilaginous fish. We decided to call these cDNAs, at least provisionally, SSa, SSb and SSc, respectively. Two of them, SSa and SSb, encode proteins that both contain the same tetradecapeptide sequence at their C-terminal extremity (AGCKNFFWKTFTSC). This putative peptide is identical to that generated by the SS1 gene in other vertebrate species. The last cDNA, SSc, encodes a protein that contains at its C-terminal extremity the same peptide sequence as that generated by the SS2 gene in teleosts (APCKNFFWKTFTSC). Phylogenetic analysis showed that the SSa and SSc genes likely correspond to the dogfish counterparts of the SS1 and SS2 genes, respectively. In contrast, the phylogenetic status of the SSb gene is less clear. Several lines of evidence suggest that it could correspond to the SS5 gene, but this view will need to be confirmed, for example by synteny analysis. Finally, RT-PCR analysis revealed that SSa, SSb and SSc genes are differentially expressed in dogfish tissues, suggesting that the corresponding peptides may exert distinct functions.
Assuntos
Cação (Peixe)/genética , Somatostatina/genética , Animais , Clonagem Molecular , DNA Complementar , Evolução Molecular , Neuropeptídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The currently recognised dystrophin protein family comprises the archetype, dystrophin, its close relative, utrophin or dystrophin-related protein (DRP), and a distantly related protein known as the 87K tyrosine kinase substrate. During the course of a phylogenetic study of sequences encoding the characteristic C-terminal domains of dystrophin-related proteins, we identified an unexpected novel class of vertebrate dystrophin-related sequences. We term this class dystrophin-related protein 2 (DRP2), and suggest that utrophin/DRP be renamed DRP1 to simplify future nomenclature. DRP2 is a relatively small protein, encoded in man by a 45 kb gene localized to Xq22. It is expressed principally in the brain and spinal cord, and is similar in overall structure to the Dp116 dystrophin isoform. The discovery of a novel relative of dystrophin substantially broadens the scope for study of this interesting group of proteins and their associated glycoprotein complexes.
Assuntos
Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Distrofina/química , Distrofina/genética , Proteínas de Membrana , Proteínas Musculares , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Proteínas do Citoesqueleto/classificação , Cação (Peixe)/genética , Distrofina/biossíntese , Peixes/genética , Humanos , Camundongos , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Distribuição Tecidual , Utrofina , Cromossomo XRESUMO
Pax6 is involved in the control of neuronal specification, migration, and differentiation in the olfactory epithelium and in the generation of different interneuron subtypes in the olfactory bulb. Whether these roles are conserved during evolution is not known. Cartilaginous fish are extremely useful models for assessing the ancestral condition of brain organization because of their phylogenetic position. To shed light on the evolution of development of the olfactory system in vertebrates and on the involvement of Pax6 in this process, we analyzed by in situ hybridization and immunohistochemistry the expression pattern of Pax6 in the developing olfactory system in a basal vertebrate, the lesser spotted dogfish Scyliorhinus canicula. This small shark is becoming an important fish model in studies of vertebrate development. We report Pax6 expression in cells of the olfactory epithelium and olfactory bulb, and present the first evidence in vertebrates of strings of Pax6-expressing cells extending along the developing olfactory nerve. The results indicate the olfactory epithelium as the origin of these cells. These data are compatible with a role for Pax6 in the development of the olfactory epithelium and fibers, and provide a basis for future investigations into the mechanisms that regulate development of the olfactory system throughout evolution.
Assuntos
Cação (Peixe)/genética , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Nervo Olfatório/metabolismo , Fatores de Transcrição Box Pareados/genética , Proteínas Repressoras/genética , Animais , Cação (Peixe)/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Fator de Transcrição PAX6RESUMO
The transport mechanisms for water, ammonia and urea in elasmobranch gill, kidney and gastrointestinal tract remain to be fully elucidated. Aquaporin 8 (AQP8) is a known water, ammonia and urea channel that is expressed in the kidney and respiratory and gastrointestinal tracts of mammals and teleost fish. However, at the initiation of this study in late 2019, there was no copy of an elasmobranch aquaporin 8 gene identified in the genebank even for closely related holocephalon species such as elephant fish (Callorhinchus milii) or for the elasmobranch little skate (Leucoraja erinacea). A transcriptomic study in spiny dogfish (Squalus acanthias) also failed to identify a copy. Hence this study has remedied this and identified the AQP8 cDNA sequence using degenerate PCR. Agarose electrophoresis of degenerate PCR reactions from dogfish tissues showed a strong band from brain cDNA and faint bands of a similar size in gill and liver. 5' and 3' RACE was used to complete the AQP8 cDNA sequence. Primers were then designed for further PCR reactions to determine the distribution of AQP8 mRNA expression in dogfish tissues. This showed that AQP8 is only expressed in dogfish brain and AQP8 therefore clearly can play no role in water, ammonia and urea transport in the gill, kidney or gastrointestinal tract. The role of AQP8 in dogfish brain remains to be determined.
Assuntos
Aquaporinas , Rajidae , Squalus acanthias , Amônia/metabolismo , Animais , Aquaporinas/genética , Encéfalo/metabolismo , DNA Complementar/metabolismo , Cação (Peixe)/genética , Cação (Peixe)/metabolismo , Peixes/metabolismo , Brânquias/metabolismo , Intestinos , Rim/metabolismo , Mamíferos/metabolismo , Rajidae/metabolismo , Squalus acanthias/genética , Squalus acanthias/metabolismo , Ureia/metabolismo , Água/metabolismoRESUMO
BACKGROUND: Teeth and tooth-like structures, together named odontodes, are repeated organs thought to share a common evolutionary origin. These structures can be found in gnathostomes at different locations along the body: oral teeth in the jaws, teeth and denticles in the oral-pharyngeal cavity, and dermal denticles on elasmobranch skin. We, and other colleagues, had previously shown that teeth in any location were serially homologous because: i) pharyngeal and oral teeth develop through a common developmental module; and ii) the expression patterns of the Dlx genes during odontogenesis were highly divergent between species but almost identical between oral and pharyngeal dentitions within the same species. Here we examine Dlx gene expression in oral teeth and dermal denticles in order to test the hypothesis of serial homology between these odontodes. RESULTS: We present a detailed comparison of the first developing teeth and dermal denticles (caudal primary scales) of the dogfish (Scyliorhinus canicula) and show that both odontodes develop through identical stages that correspond to the common stages of oral and pharyngeal odontogenesis. We identified six Dlx paralogs in the dogfish and found that three showed strong transcription in teeth and dermal denticles (Dlx3, Dlx4 and Dlx5) whereas a weak expression was detected for Dlx1 in dermal denticles and teeth, and for Dlx2 in dermal denticles. Very few differences in Dlx expression patterns could be detected between tooth and dermal denticle development, except for the absence of Dlx2 expression in teeth. CONCLUSIONS: Taken together, our histological and expression data strongly suggest that teeth and dermal denticles develop from the same developmental module and under the control of the same set of Dlx genes. Teeth and dermal denticles should therefore be considered as serial homologs developing through the initiation of a common gene regulatory network (GRN) at several body locations. This mechanism of heterotopy supports the 'inside and out' model that has been recently proposed for odontode evolution.
Assuntos
Cação (Peixe)/embriologia , Cação (Peixe)/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Dente/embriologia , Fatores de Transcrição/genética , Animais , Evolução Biológica , Cação (Peixe)/anatomia & histologia , Odontogênese , Dente/anatomia & histologia , Dente/metabolismoRESUMO
The skeleton is one of the most important features for the reconstruction of vertebrate phylogeny but few data are available to understand its molecular origin. In mammals the Runt genes are central regulators of skeletogenesis. Runx2 was shown to be essential for osteoblast differentiation, tooth development, and bone formation. Both Runx2 and Runx3 are essential for chondrocyte maturation. Furthermore, Runx2 directly regulates Indian hedgehog expression, a master coordinator of skeletal development. To clarify the correlation of Runt gene evolution and the emergence of cartilage and bone in vertebrates, we cloned the Runt genes from hagfish as representative of jawless fish (MgRunxA, MgRunxB) and from dogfish as representative of jawed cartilaginous fish (ScRunx1-3). According to our phylogenetic reconstruction the stem species of chordates harboured a single Runt gene and thereafter Runt locus duplications occurred during early vertebrate evolution. All newly isolated Runt genes were expressed in cartilage according to quantitative PCR. In situ hybridisation confirmed high MgRunxA expression in hard cartilage of hagfish. In dogfish ScRunx2 and ScRunx3 were expressed in embryonal cartilage whereas all three Runt genes were detected in teeth and placoid scales. In cephalochordates (lancelets) Runt, Hedgehog and SoxE were strongly expressed in the gill bars and expression of Runt and Hedgehog was found in endo- as well as ectodermal cells. Furthermore we demonstrate that the lancelet Runt protein binds to Runt binding sites in the lancelet Hedgehog promoter and regulates its activity. Together, these results suggest that Runt and Hedgehog were part of a core gene network for cartilage formation, which was already active in the gill bars of the common ancestor of cephalochordates and vertebrates and diversified after Runt duplications had occurred during vertebrate evolution. The similarities in expression patterns of Runt genes support the view that teeth and placoid scales evolved from a homologous developmental module.
Assuntos
Desenvolvimento Ósseo/genética , Cordados/crescimento & desenvolvimento , Cordados/genética , Evolução Molecular , Animais , Sequência de Bases , Galinhas/genética , Galinhas/crescimento & desenvolvimento , Condrogênese/genética , Cordados não Vertebrados/genética , Cordados não Vertebrados/crescimento & desenvolvimento , Subunidades alfa de Fatores de Ligação ao Core/genética , Primers do DNA/genética , Cação (Peixe)/genética , Cação (Peixe)/crescimento & desenvolvimento , Duplicação Gênica , Regulação da Expressão Gênica no Desenvolvimento , Feiticeiras (Peixe)/genética , Feiticeiras (Peixe)/crescimento & desenvolvimento , Proteínas Hedgehog/genética , Humanos , Modelos Genéticos , Odontogênese/genética , Osteogênese/genética , Filogenia , Urocordados/genética , Urocordados/crescimento & desenvolvimentoRESUMO
Shark immunoglobulin new antigen receptor (IgNAR, also referred to as NAR) variable domains (Vs) are single-domain antibody (sdAb) fragments containing only two hypervariable loop structures forming 3D topologies for a wide range of antigen recognition and binding. Their small size ( approximately 12kDa) and high solubility, thermostability and binding specificity make IgNARs an exceptional alternative source of engineered antibodies for sensor applications. Here, two new shark NAR V display libraries containing >10(7) unique clones from non-immunized (naïve) adult spiny dogfish (Squalus acanthias) and smooth dogfish (Mustelus canis) sharks were constructed. The most conserved consensus sequences derived from random clone sequence were compared with published nurse shark (Ginglymostoma cirratum) sequences. Cholera toxin (CT) was chosen for panning one of the naïve display libraries due to its severe pathogenicity and commercial availability. Three very similar CT binders were selected and purified soluble monomeric anti-CT sdAbs were characterized using Luminex(100) and traditional ELISA assays. These novel anti-CT sdAbs selected from our newly constructed shark NAR V sdAb library specifically bound to soluble antigen, without cross reacting with other irrelevant antigens. They also showed superior heat stability, exhibiting slow loss of activity over the course of one hour at high temperature (95 degrees C), while conventional antibodies lost all activity in the first 5-10min. The successful isolation of target specific sdAbs from one of our non-biased NAR libraries, demonstrate their ability to provide binders against an unacquainted antigen of interest.
Assuntos
Toxina da Cólera/imunologia , Cação (Peixe)/imunologia , Imunoglobulinas/isolamento & purificação , Receptores de Antígenos/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos/isolamento & purificação , Sequência Conservada , Cação (Peixe)/genética , Temperatura Alta , Imunoglobulinas/química , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Dados de Sequência Molecular , Biblioteca de Peptídeos , Estrutura Terciária de Proteína , Receptores de Antígenos/química , Análise de Sequência de ProteínaRESUMO
Because the cerebellum emerged at the agnathan-gnathostome transition and cartilaginous fishes are at the base of the gnathostome lineage, this group is crucial to determine the basic developmental pattern of the cerebellum and to gain insights into its origin. We have systematically analyzed key events in the development of cerebellum and cerebellum-related structures of the shark Scyliorhinus canicula. Three developmental periods are distinguished based on anatomical observations combined with molecular analysis. We present neurochemical and genoarchitectonic evidence on the onset of cerebellar development, the rostral and caudal cerebellar boundaries, the compartmentalization of the cerebellum, and correspondence of cerebellar domains to rhombomeric segmentation of the rostral hindbrain. Our observations, mainly based on the expression pattern of ScHoxA2, support the origin of both the upper and lower auricular leaves from r1 and exclude any cerebellar origin from r2. Correlation between subrhombomeres r1a/r1b and cerebellar domains is proposed based on the ScEn2 expression. The ScEn2 and ScOtx2 expression patterns revealed an antero-posterior cerebellar compartmentalization similar to that of mammals, and supported certain fissures (commonly used to define cerebellar domains) as reliable anatomical landmarks. At difference from mammals, the expression of ScEn2 along the cerebellar median-lateral axis does not reveal a multiple-banded pattern. The present study provides an atlas of cerebellar development in one of the most basal extant gnathostome lineages and emphasizes the importance of combining classic descriptive with modern molecular studies to gain knowledge on the ancestral condition of cerebellar developmental processes and the origins and evolution of the cerebellum.
Assuntos
Evolução Biológica , Cerebelo/embriologia , Cação (Peixe)/embriologia , Morfogênese , Animais , Cerebelo/metabolismo , Cação (Peixe)/genética , Cação (Peixe)/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Especificidade da EspécieRESUMO
The cerebellum is present in all extant gnathostomes or jawed vertebrates, of which cartilaginous fishes represent the most ancient radiation. Since the isthmic organizer induces the formation of the cerebellum, comparative genoarchitectonic analysis on the meso-isthmo-cerebellar region of cartilaginous fishes with respect to that of jawless vertebrates could reveal why the isthmic organizer acquires the ability to induce the formation of the cerebellum in gnathostomes. In the present work we analyzed the expression pattern of a variety of genes related to the cerebellar formation and patterning (ScOtx2, ScGbx2, ScFgf8, ScLmx1b, ScIrx1, ScIrx3, ScEn2, ScPax6 and ScLhx9) by in situ hybridization, and the distribution of Pax6 protein in the developing hindbrain of the shark Scyliorhinus canicula. The genoarchitectonic code in this species revealed high degree of conservation with respect to that of other gnathostomes. This resemblance may reveal the features of the ancestral condition of the gene network operating for specification of the rostral hindbrain patterning. Accordingly, the main subdivisions of the rostral hindbrain of S. canicula could be recognized. Our results support the existence of a rhombomere 0, identified as the ScFgf8/ScGbx2/ScEn2-positive and mainly negative ScIrx3 domain just caudal to the midbrain ScIrx1/ScOtx2/ScLmx1b-positive domain. The differential ScEn2 and Pax6 expression in the rhombomere 1 revealed anterior and posterior subdivisions. Interestingly, dissimilarities between S. canicula and lampreys (jawless vertebrates) were noted in the expression of Irx, Lhx and Pax genes, which could be part of significant gene network changes through evolution that caused the emergence of the cerebellum.
Assuntos
Cação (Peixe)/embriologia , Cação (Peixe)/genética , Regulação da Expressão Gênica no Desenvolvimento , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Animais , Evolução Biológica , Cerebelo/embriologia , Cerebelo/metabolismoRESUMO
We have determined the complete nucleotide sequence of the mitochondrial DNA (mtDNA) of the dogfish, Scyliorhinus canicula. The 16,697-bp-long mtDNA possesses a gene organization identical to that of the Osteichthyes, but different from that of the sea lamprey Petromyzon marinus. The main features of the mtDNA of osteichthyans were thus established in the common ancestor to chondrichthyans and osteichthyans. The phylogenetic analysis confirms that the Chondrichthyes are the sister group of the Osteichthyes.
Assuntos
DNA Mitocondrial/genética , Cação (Peixe)/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Grupo dos Citocromos b/genética , Região de Controle de Locus Gênico , Dados de Sequência Molecular , Filogenia , RNA de Transferência/genéticaRESUMO
Na(+)/H(+) Exchanger (NHE) proteins mediate cellular and systemic homeostasis of sodium and acid and may be the major sodium uptake method for fishes. We cloned and sequenced NHE2 and NHE3 from the gill of the North Pacific Spiny Dogfish shark Squalus suckleyi and expressed them in functional form in NHE-deficient (AP-1) cell lines. Estimated IC50 for inhibition of NHE activity by amiloride and EIPA were 55 µmol l(-1) and 4.8 µmol l(-1), respectively, for NHE2 and 9 µmol l(-1) and 24 µmol l(-1), respectively, for NHE3. Phenamil at 100 µmol l(-1) caused less than 16% inhibition of activity for each isoform. Although the IC50 are similar for the two isoforms, dfNHE2 is less sensitive than human NHE2 to inhibition by amiloride and EIPA, while dfNHE3 is more sensitive than human NHE3. These IC50 estimates should be considered when selecting inhibitor doses for fishes and for reinterpretation of previous studies that use these pharmacological agents.
Assuntos
Transporte Biológico Ativo/efeitos dos fármacos , Cação (Peixe)/genética , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Proteínas de Peixes/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Linhagem Celular , Clonagem Molecular/métodos , Cricetinae , Proteínas de Peixes/genética , Brânquias/metabolismo , Concentração Inibidora 50 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
Different approaches can be used to elucidate the unsolved questions concerning taxonomic evolution in cartilaginous fish. The study of the karyological characteristics of these vertebrates by combining molecular and traditional techniques of chromosome preparation and banding has been demonstrated to be a very effective method. In this paper we studied the localization and the composition of the constitutive heterochromatin by using C- and restriction endonuclease-banding in four selachian species, belonging to two of the four superorders. We also characterized two different types of repetitive genomic sequences in these species: satellite DNA and (TTAGGG)(n) telomeric sequences. Finally, we analysed the nuclear ribosomal gene to determine the number of the nucleolar organizers and their position on chromosomes by using silver staining, chromomycin A(3), and FISH (fluorescent in situ hybridization). The results showed a prevailingly telomeric localization of constitutive heterochromatin in the Galeomorphii, the presence of additional nucleolar organizer sites in Raja asterias, an exclusively telomeric localization of the (TTAGGG)(n) sequences in Scyliorhinus stellaris and both telomeric and interstitial in Taeniura lymma. These data, together with those concerning the conservation of the satellite DNA, seem to support the hypothesis that Chondrichthyes have an evolutionary history leading them to the acquisition of large genomes rich in highly repeated sequences and subjected to some selective pressures favoring the conservation of this DNA fraction.
Assuntos
Elasmobrânquios/genética , Genoma , Animais , Sequência de Bases , DNA/genética , DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , DNA Satélite/genética , Cação (Peixe)/genética , Heterocromatina/genética , Hibridização in Situ Fluorescente , Cariotipagem , Tubarões/genética , Rajidae/genética , Telômero/genéticaRESUMO
The amino acid, either a glutamine (Q) or an arginine (R), at the Q/R site of the pore-lining segment (M2) of a vertebrate AMPA receptor subunit critically influences the properties of the receptor. The R codon of the mammalian AMPA receptor subunit 2 (GRIA2) transcript is not coded by the chromosomal sequence, but is created by posttranscriptional RNA editing activities. On the other hand, the R codons of some teleost GRIA2 homologs are coded by chromosomal sequences. To elucidate the evolution of the utilization of Q/R RNA editing in modifying vertebrate GRIA2 transcripts, the GRIA2 genes of five fish species and an amphibian were studied. The putative hagfish GRIA2 homolog (hfGRIA2) encodes an R codon, whereas shark and bullfrog GRIA2 genes specify a Q codon at the genomic Q/R site. All gnathostoma GRIA2 genes possess an intron splitting the coding regions of M2 and the third hydrophobic region (M3). The intronic components required for Q/R RNA editing are preserved in all the Q-coding vertebrate GRIA2 genes but are absent from the R-coding GRIA2 genes. Interestingly, the hfGRIA2 is intronless, suggesting that hfGRIA2 is unlikely evolved from a Q/R editing-competent gene. Results of this study suggest that modification of GRIA2 transcripts by Q/R editing is most likely acquired after the separation of the Agnatha and Gnathostome.
Assuntos
Arginina/genética , Evolução Molecular , Glutamina/genética , Edição de RNA , Receptores de AMPA/genética , Animais , Cação (Peixe)/genética , Feiticeiras (Peixe)/genética , Conformação de Ácido Nucleico , Subunidades Proteicas , Rana catesbeiana/genética , Tetraodontiformes/genética , Tilápia/genética , Peixe-Zebra/genéticaRESUMO
Recent studies have shown that vertebrates, including teleostean fishes, amphibians, birds and mammals, contain two distinct insulin-like growth factor (IGF) genes. In contrast agnathans, represented by hagfish, apparently have only one IGF that has features characteristic of both IGF-I and IGF-II. Between these groups the elasmobranchs occupy a critical position in terms of the phylogeny of IGFs. We sought to determine if gene duplication and divergence of IGF-I and IGF-II occurred before or after divergence of elasmobranchs from other vertebrates by cloning IGF-like molecules from Squalus acanthias. Our analysis shows that Squalus liver produces two distinct IGF-like molecules. One has greater sequence identity to, and conserved features characteristic of, known IGF-I molecules, while the other is more IGF-II like. These results suggest that the prototypical IGF molecule duplicated and diverged in an ancestor of the extant gnathostomes.
Assuntos
Cação (Peixe)/genética , Genes/genética , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like I/genética , Filogenia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada/genética , Fígado/química , Dados de Sequência Molecular , Família Multigênica , RNA/análise , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The co-existence of insulin-like growth factor 1 (IGF-1) with the classical islet hormones insulin (INS), glucagon (GLUC), somatostatin (SOM) and pancreatic polypeptide (PP) in the endocrine pancreas of representative species of cyclostomes (Myxine glutinosa), cartilaginous fish (Raja clavata, Squalus acanthias) and bony fish (Cottus scorpius, Carassius auratus, Cyprinus carpio, Anguilla anguilla) was studied by the use of monoclonal and polyclonal antisera and the double immunofluorescence technique. In all species investigated, IGF-1-like-immunoreactive cells were found in the endocrine pancreas, however, in varying localization. In Myxine glutinosa, all INS-immunoreactive cells and some of the SOM-immunoreactive cells contained IGF-1-like-immunoreactivity. In Raja and Squalus, only a minority of the INS-immunoreactive cells also displayed IGF-1-like-immunoreactivity. The majority of the IGF-1-like-immunoreactivity was observed in SOM- and in GLUC-immunoreactive cells. Different results were obtained in bony fish. In Cottus, in the Brockmann bodies and the small islets IGF-1-like- and INS-immunoreactivities co-existed to 100%. In contrast, in the other bony fish studied IGF-1-like-immunoreactivity was not observed in INS-immunoreactive cells: in Cyprinus, IGF-1-like-immunoreactivity was found in GLUC-, PP- and SOM-immunoreactive cells and in Carassius and Anguilla, in SOM-immunoreactive cells only. Thus, in all bony fish species with the exception of Cottus, IGF-1 and insulin display a distinct cellular distribution, similar to that of mammals. The present results, thus, may indicate that the branching of IGF-1 and insulin has occurred at the phylogenetic level of bony fish.
Assuntos
Peixes/genética , Fator de Crescimento Insulin-Like I/análise , Insulina/análise , Ilhotas Pancreáticas/citologia , Filogenia , Anguilla/genética , Animais , Carpas/genética , Cação (Peixe)/genética , Imunofluorescência , Carpa Dourada/genética , Imuno-Histoquímica , Insulina/genética , Fator de Crescimento Insulin-Like I/genética , Rajidae/genéticaRESUMO
Relaxin is a 6-kd polypeptide that exerts important hormonal effects in many female mammals. Relaxin is produced by the ovary, placenta, or uterus in many mammalian species. The functions of relaxin in the male mammal are not yet firmly established, but there is some evidence suggesting an exocrine effect on sperm motility and fertilizability. In the male mammals that have been studied, relaxin is produced by the prostate gland (human) or seminal vesicles (boar). However, in the bird, the testis is the likely source of relaxin. Among the elasmobranchs, ovaries obtained from dogfish sharks have been shown to contain a polypeptide hormone that is structurally, biologically, and immunologically similar to mammalian relaxins, but the male reproductive tract of this species has not previously been investigated as a potential source of relaxin. Extracts of testes obtained from mature dogfish sharks have now been tested by a specific relaxin bioassay and by a homologous porcine radioimmunoassay for the presence of relaxin. Both crude and partially purified testicular extracts contained unmistakable guinea pig pubic symphysis-"relaxing" activity and relaxin-like immunoactivity. Following immunoaffinity purification, the shark testis polypeptide had an apparent specific activity of 88 microg porcine relaxin equivalents per milligram in the radioimmunoassay, which is similar to the immunoactivity of pure shark ovarian hormones. These data, therefore, strongly support the view that in dogfish sharks, the male as well as the female gonad produces relaxin. Furthermore, as the dogfish shark has existed as a species for about 200 million years, the data suggest that testicular relaxin appeared early in vertebrate evolution.
Assuntos
Relaxina/isolamento & purificação , Testículo/química , Animais , Evolução Biológica , Cromatografia por Troca Iônica , Cação (Peixe)/genética , Feminino , Cobaias , Masculino , Radioimunoensaio , Relaxina/química , Relaxina/genéticaRESUMO
cDNA fragments of both the alpha- and beta-subunits of the Na, K-ATPase and a cDNA fragment of the secretory form of Na-K-Cl cotransporter from the European dogfish (Scyliorhinus canicula) were amplified and cloned using degenerate primers in RT-PCR. These clones were used along with a sCFTR cDNA from the related dogfish shark, Squalus acanthias to characterise the expression of mRNAs for these ion transporters in the dogfish rectal gland subsequent to an acute feeding episode. Following a single feeding event where starved dogfish were fed squid portions (20 g squid/kg fish), there was a delayed and transient 40-fold increase in the activity of Na, K-ATPase in crude rectal gland homogenates. Increases in enzyme activity were apparent 3 h after the feeding event and peaked at 9 h before returning to control values within 24 h. These increases in activity were accompanied by small and transient decreases in plasma sodium and chloride concentrations lasting up to 3 days. Significant increases in the expression of mRNAs for alpha- and beta-subunits of the Na, K-ATPase, the Na-K-Cl cotransporter and CFTR chloride channel were detected but not until 1-2 days after the feeding event. It is concluded that the transient increase in Na, K-ATPase activity is not attributable to increases in the abundance of alpha- and beta-subunit mRNAs but must be associated with some, as yet unknown, post-transcriptional activation mechanism.