RESUMO
BACKGROUND: Urodele amphibians are capable of regenerating their organs after severe damage. During such regeneration, participating cells are given differentiation instructions by the surrounding cells. Limb regeneration has been investigated as a representative phenomenon of organ regeneration. Cells known as blastema cells are induced after limb amputation. In this process, dermal fibroblasts are dedifferentiated and become undifferentiated similar to limb bud cells. Just like limb bud cells, the induced blastema cells are positioned along the three limb developmental axes: the dorsoventral, the anteroposterior, and the proximodistal. The accurate developmental axes are essential for reforming the structures correctly. Despite the importance of the developmental axes, the relationship between the newly establishing developmental axes and existing limb axes was not well described with molecular markers. RESULTS: In this study, we grafted skin from GFP-transgenic axolotls and traced the cell lineage with position-specific gene expressions in order to investigate the correlation of the newly established axes and cellular origin. Shh- and Lmx1b-expressing cells emerged from the posterior skin and dorsal skin, respectively, even though the skin was transplanted to an inconsistent position. Shox2, a posterior marker gene, could be activated in cells derived from distal skin. CONCLUSIONS: Our results suggest that the location memories on anteroposterior and dorsoventral axes are relatively stable in a regenerating blastema though cellular differentiation is reprogrammed.
Assuntos
Ambystoma mexicanum/embriologia , Ambystoma mexicanum/fisiologia , Animais , Diferenciação Celular/fisiologia , Extremidades/embriologia , Extremidades/fisiologia , Imunofluorescência , Hibridização In Situ , Regeneração/fisiologiaRESUMO
Axolotl (Ambystoma mexicanum) is a critically endangered salamander species and a model organism for regenerative and developmental biology. Despite life-long neoteny in nature and in captive-bred colonies, metamorphosis of these animals can be experimentally induced by administering Thyroid hormones (THs). However, microbiological consequences of this experimental procedure, such as host microbiota response, remain largely unknown. Here, we systematically compared host bacterial microbiota associated with skin, stomach, gut tissues and fecal samples, between neotenic and metamorphic axolotls based on 16S rRNA gene sequences. Our results show that distinct bacterial communities inhabit individual organs of axolotl and undergo substantial restructuring through metamorphosis. Skin microbiota among others, shifted sharply, as highlighted by a major transition from Firmicutes-enriched to Proteobacteria-enriched relative abundance and precipitously decreased diversity. Fecal microbiota of neotenic and metamorphic axolotl shared relatively higher similarity, suggesting that diet continues to shape microbiota despite fundamental transformations in the host digestive organs. We also reproduced the previous finding on reduction in regenerative capacity in limbs of axolotl following metamorphosis, highlighting the need to investigate whether shifts in microbiota is causally linked to regenerative capacity of axolotl. The initial results on axolotl microbiota provide novel insights into microbiological aspects of axolotl metamorphosis and will establish a baseline for future in-depth studies.
Assuntos
Ambystoma mexicanum/embriologia , Metamorfose Biológica , Microbiota , Animais , Dieta , Espécies em Perigo de Extinção , Microbiota/genética , RegeneraçãoAssuntos
Ambystoma mexicanum , Animais Selvagens , Conservação dos Recursos Naturais , Extinção Biológica , Adulto , Ambystoma mexicanum/embriologia , Ambystoma mexicanum/genética , Ambystoma mexicanum/crescimento & desenvolvimento , Ambystoma mexicanum/fisiologia , Animais , Animais de Laboratório/genética , Animais de Laboratório/fisiologia , Animais Selvagens/genética , Animais Selvagens/fisiologia , Conservação dos Recursos Naturais/economia , Conservação dos Recursos Naturais/tendências , Ecossistema , Pool Gênico , Genômica , História do Século XVI , História do Século XIX , História do Século XX , História do Século XXI , Humanos , Endogamia , Lagos , México , Camundongos , Dinâmica Populacional , Regeneração/fisiologia , Medicina Regenerativa , Disrafismo Espinal , Hormônios Tireóideos/história , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: The endogenous ability to dedifferentiate, re-pattern, and re-differentiate adult cells to repair or replace damaged or missing structures is exclusive to only a few tetrapod species. The Mexican axolotl is one example of these species, having the capacity to regenerate multiple adult structures including their limbs by generating a group of progenitor cells, known as the blastema, which acquire pattern and differentiate into the missing tissues. The formation of a limb regenerate is dependent on cells in the connective tissues that retain memory of their original position in the limb, and use this information to generate the pattern of the missing structure. Observations from recent and historic studies suggest that blastema cells vary in their potential to pattern distal structures during the regeneration process; some cells are plastic and can be reprogrammed to obtain new positional information while others are stable. Our previous studies showed that positional information has temporal and spatial components of variation; early bud (EB) and apical late bud (LB) blastema cells are plastic while basal-LB cells are stable. To identify the potential cellular and molecular basis of this variation, we compared these three cell populations using histological and transcriptional approaches. RESULTS: Histologically, the basal-LB sample showed greater tissue organization than the EB and apical-LB samples. We also observed that cell proliferation was more abundant in EB and apical-LB tissue when compared to basal-LB and mature stump tissue. Lastly, we found that genes associated with cellular differentiation were expressed more highly in the basal-LB samples. CONCLUSIONS: Our results characterize histological and transcriptional differences between EB and apical-LB tissue compared to basal-LB tissue. Combined with our results from a previous study, we hypothesize that the stability of positional information is associated with tissue organization, cell proliferation, and pathways of cellular differentiation.
Assuntos
Ambystoma mexicanum/embriologia , Plasticidade Celular/genética , Extremidades/embriologia , Botões de Extremidades/embriologia , Regeneração/genética , Ambystoma mexicanum/genética , Animais , Diferenciação Celular/genética , Plasticidade Celular/fisiologia , Proliferação de Células/genética , Botões de Extremidades/fisiologia , Regeneração/fisiologia , Transdução de Sinais/genéticaRESUMO
The impressive morphological diversification of vertebrates was achieved in part by innovation and modification of the pharyngeal skeleton. Extensive fate mapping in amniote models has revealed a primarily cranial neural crest derivation of the pharyngeal skeleton. Although comparable fate maps of amphibians produced over several decades have failed to document a neural crest derivation of ventromedial elements in these vertebrates, a recent report provides evidence of a mesodermal origin of one of these elements, basibranchial 2, in the axolotl. We used a transgenic labeling protocol and grafts of labeled cells between GFP+ and white embryos to derive a fate map that describes contributions of both cranial neural crest and mesoderm to the axolotl pharyngeal skeleton, and we conducted additional experiments that probe the mechanisms that underlie mesodermal patterning. Our fate map confirms a dual embryonic origin of the pharyngeal skeleton in urodeles, including derivation of basibranchial 2 from mesoderm closely associated with the second heart field. Additionally, heterotopic transplantation experiments reveal lineage restriction of mesodermal cells that contribute to pharyngeal cartilage. The mesoderm-derived component of the pharyngeal skeleton appears to be particularly sensitive to retinoic acid (RA): administration of exogenous RA leads to loss of the second basibranchial, but not the first. Neural crest was undoubtedly critical in the evolution of the vertebrate pharyngeal skeleton, but mesoderm may have played a central role in forming ventromedial elements, in particular. When and how many times during vertebrate phylogeny a mesodermal contribution to the pharyngeal skeleton evolved remain to be resolved.
Assuntos
Ambystoma mexicanum/embriologia , Evolução Biológica , Padronização Corporal , Osso e Ossos/embriologia , Faringe/embriologia , Ambystoma mexicanum/genética , Animais , Embrião não Mamífero/metabolismo , Mesoderma/embriologia , Crista Neural/embriologia , Tretinoína/metabolismoRESUMO
The segmental series of somites in the vertebrate embryo gives rise to the axial skeleton. In amniote models, single vertebrae are derived from the sclerotome of two adjacent somites. This process, known as resegmentation, is well-studied using the quail-chick chimeric system, but the presumed generality of resegmentation across vertebrates remains poorly evaluated. Resegmentation has been questioned in anamniotes, given that the sclerotome is much smaller and lacks obvious differentiation between cranial and caudal portions. Here, we provide the first experimental evidence that resegmentation does occur in a species of amphibian. Fate mapping of individual somites in the Mexican axolotl (Ambystoma mexicanum) revealed that individual vertebrae receive cells from two adjacent somites as in the chicken. These findings suggest that large size and segmentation of the sclerotome into distinct cranial and caudal portions are not requirements for resegmentation. Our results, in addition to those for zebrafish, indicate that resegmentation is a general process in building the vertebral column in vertebrates, although it may be achieved in different ways in different groups.
Assuntos
Ambystoma mexicanum/embriologia , Somitos/embriologia , Somitos/ultraestrutura , Coluna Vertebral/embriologia , Animais , Padronização Corporal , Diferenciação Celular , Somitos/transplanteRESUMO
There is still confusion about the homology of several cranial muscles in salamanders with those of other vertebrates. This is true, in part, because of the fact that many muscles present in early ontogeny of amphibians disappear during development and specifically during metamorphosis. Resolving this confusion is important for the understanding of the comparative and evolutionary morphology of vertebrates and tetrapods because amphibians are the phylogenetically most plesiomorphic tetrapods, concerning for example their myology, and include two often used model organisms, Xenopus laevis (anuran) and Ambystoma mexicanum (urodele). Here we provide the first detailed report of the cranial muscle development in axolotl from early ontogenetic stages to the adult stage. We describe different and complementary types of general muscle morphogenetic gradients in the head: from anterior to posterior, from lateral to medial, and from origin to insertion. Furthermore, even during the development of neotenic salamanders such as axolotls, various larval muscles become indistinct, contradicting the commonly accepted view that during ontogeny the tendency is mostly toward the differentiation of muscles. We provide an updated comparison between these muscles and the muscles of other vertebrates, a discussion of the homologies and evolution, and show that the order in which the muscles appear during axolotl ontogeny is in general similar to their appearance in phylogeny (e.g. differentiation of adductor mandibulae muscles from one anlage to four muscles), with only a few remarkable exceptions, as for example the dilatator laryngis that appears evolutionary later but in the development before the intermandibularis.
Assuntos
Ambystoma mexicanum/crescimento & desenvolvimento , Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , Filogenia , Ambystoma mexicanum/embriologia , Animais , Larva/crescimento & desenvolvimento , Metamorfose Biológica , Morfogênese , Músculo Esquelético/embriologiaRESUMO
Traditionally, the cartilaginous viscerocranium of vertebrates is considered as neural crest (NC)-derived. Morphological work carried out on amphibian embryos in the first half of the XX century suggested potentially mesodermal origin for some hyobranchial elements. Since then, the embryonic sources of the hyobranchial apparatus in amphibians has not been investigated due to lack of an appropriate long-term labelling system. We performed homotopic transplantations of neural folds along with the majority of cells of the presumptive NC, and/or fragments of the head lateral plate mesoderm (LPM) from transgenic GFP+ into white embryos. In these experiments, the NC-derived GFP+ cells contributed to all hyobranchial elements, except for basibranchial 2, whereas the grafting of GFP+ head mesoderm led to a reverse labelling result. The grafting of only the most ventral part of the head LPM resulted in marking of the basibranchial 2 and the heart myocardium, implying their origin from a common mesodermal region. This is the first evidence of contribution of LPM of the head to cranial elements in any vertebrate. If compared to fish, birds, and mammals, in which all branchial skeletal elements are NC-derived, the axolotl (probably this is true for all amphibians) demonstrates an evolutionary deviation, in which the head LPM replaces NC cells in a hyobranchial element. This implies that cells of different embryonic origin may have the same developmental program, leading to the formation of identical (homologous) elements of the skeleton.
Assuntos
Ambystoma mexicanum/embriologia , Ambystoma mexicanum/fisiologia , Animais , Animais Geneticamente Modificados , Aves , Osso e Ossos/embriologia , Cartilagem/embriologia , Peixes , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Cabeça/embriologia , Coração/embriologia , Mesoderma/embriologia , Mesoderma/metabolismo , Mesoderma/fisiologia , Miocárdio/patologia , Crista Neural/metabolismo , Crista Neural/patologiaRESUMO
Muscle differentiation has been widely described in zebrafish and Xenopus, but nothing is known about this process in amphibian urodeles. Both anatomical features and locomotor activity in urodeles are known to show intermediate features between fish and anurans. Therefore, a better understanding of myogenesis in urodeles could be useful to clarify the evolutionary changes that led to the formation of skeletal muscle in the trunk of land vertebrates. We report here a detailed morphological and molecular investigation on several embryonic stages of Ambystoma mexicanum and show that the first differentiating muscle fibers are the slow ones, originating from a myoblast population initially localized close to the notochord that forms a superficial layer on the somitic surface afterwards. Subsequently, fast fibers differentiation ensues. We also identified and cloned A. mexicanum Myf5 as a muscle-specific transcriptional factor likely involved in urodele muscle differentiation.
Assuntos
Ambystoma mexicanum/embriologia , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular , Ambystoma mexicanum/anatomia & histologia , Ambystoma mexicanum/genética , Animais , Padronização Corporal , Clonagem Molecular , Embrião não Mamífero/embriologia , Embrião não Mamífero/ultraestrutura , Desenvolvimento Embrionário , Ensaios Enzimáticos , Imuno-Histoquímica , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Rápida/ultraestrutura , Fibras Musculares de Contração Lenta/metabolismo , Fibras Musculares de Contração Lenta/ultraestrutura , Músculo Esquelético/embriologia , Músculo Esquelético/ultraestrutura , Mioblastos Esqueléticos/metabolismo , Fator Regulador Miogênico 5/genética , Fator Regulador Miogênico 5/metabolismo , Miosinas/genética , Miosinas/metabolismo , Notocorda/embriologia , Notocorda/ultraestrutura , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: A major step during the evolution of tetrapods was their transition from water to land. This process involved the reduction or complete loss of the dermal bones that made up connections to the skull and a concomitant enlargement of the endochondral shoulder girdle. In the mouse the latter is derived from three separate embryonic sources: lateral plate mesoderm, somites, and neural crest. The neural crest was suggested to sustain the muscle attachments. How this complex composition of the endochondral shoulder girdle arose during evolution and whether it is shared by all tetrapods is unknown. Salamanders that lack dermal bone within their shoulder girdle were of special interest for a possible contribution of the neural crest to the endochondral elements and muscle attachment sites, and we therefore studied them in this context. RESULTS: We grafted neural crest from GFP+ fluorescent transgenic axolotl (Ambystoma mexicanum) donor embryos into white (d/d) axolotl hosts and followed the presence of neural crest cells within the cartilage of the shoulder girdle and the connective tissue of muscle attachment sites of the neck-shoulder region. Strikingly, neural crest cells did not contribute to any part of the endochondral shoulder girdle or to the connective tissue at muscle attachment sites in axolotl. CONCLUSIONS: Our results in axolotl suggest that neural crest does not serve a general function in vertebrate shoulder muscle attachment sites as predicted by the "muscle scaffold theory," and that it is not necessary to maintain connectivity of the endochondral shoulder girdle to the skull. Our data support the possibility that the contribution of the neural crest to the endochondral shoulder girdle, which is observed in the mouse, arose de novo in mammals as a developmental basis for their skeletal synapomorphies. This further supports the hypothesis of an increased neural crest diversification during vertebrate evolution.
Assuntos
Ambystoma mexicanum/embriologia , Pescoço/embriologia , Crista Neural/anatomia & histologia , Ombro/embriologia , AnimaisRESUMO
In this chapter we provide a set of different protocols for the ultrastructural analysis of amphibian (Xenopus, axolotl) tissues, mostly of embryonic origin. For Xenopus these methods include: (1) embedding gastrulae and tailbud embryos into Spurr's resin for TEM, (2) post-embedding labeling of methacrylate (K4M) and cryosections through adult and embryonic epithelia for correlative LM and TEM, and (3) pre-embedding labeling of embryonic tissues with silver-enhanced nanogold. For the axolotl (Ambystoma mexicanum) we present the following methods: (1) SEM of migrating neural crest (NC) cells; (2) SEM and TEM of extracellular matrix (ECM) material; (3) Cryo-SEM of extracellular matrix (ECM) material after cryoimmobilization; and (4) TEM analysis of hyaluronan using high-pressure freezing and HABP labeling. These methods provide exemplary approaches for a variety of questions in the field of amphibian development and regeneration, and focus on cell biological issues that can only be answered with fine structural imaging methods, such as electron microscopy.
Assuntos
Ambystoma mexicanum/anatomia & histologia , Microscopia Eletrônica/métodos , Xenopus laevis/anatomia & histologia , Ambystoma mexicanum/embriologia , Animais , Embrião não Mamífero/ultraestrutura , Substituição ao Congelamento/métodos , Imuno-Histoquímica/métodos , Microscopia Eletrônica/instrumentação , Coloração e Rotulagem/métodos , Fixação de Tecidos/métodos , Xenopus laevis/embriologiaRESUMO
Nkx2.5, a homeodomain-containing transcription factor, is known to be necessary for normal heart development in vertebrates. It is one of the earliest lineage-restricted genes expressed in cardiovascular progenitor cells and knowledge of its expression patterns has important therapeutic implications for damaged cardiomyocytes. Mexican axolotl is a unique system to study heart development for two reasons: the presence of a mutant phenotype lacking organized myofibrils due to sarcomeric tropomyosin deficiency and the ability to induce metamorphosis by administration of exogenous thyroid hormone. In this study, we cloned and sequenced the as yet uncharacterized Nkx2.5 cDNA from normal and cardiac mutant axolotl heart RNA. Comparison of cDNA sequences of Nkx2.5 from normal and mutant axolotl hearts did not show differences suggesting that loss of function mutation in Nkx2.5 is not responsible for the mutant phenotype. However, quantitative studies show higher expression of Nkx2.5 in mutant hearts raising the possibility that increased expression of Nkx2.5 may contribute to the mutant phenotype. We also evaluated quantitative changes in expression of Nkx2.5 in axolotl hearts during embryonic and postembryonic heart development induced by exogenous thyroid hormone. There is an apparent increase in Nkx2.5 transcript levels in metamorphosed hearts.
Assuntos
Ambystoma mexicanum/embriologia , Coração/embriologia , Proteínas de Homeodomínio/metabolismo , Mutação , Miocárdio/metabolismo , Tiroxina/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Metamorfose Biológica , Dados de Sequência Molecular , Fenótipo , Análise de Sequência de DNA , Fatores de Transcrição/genéticaRESUMO
Ambystoma mexicanum is an endemic salamander of Xochimilco, a wetland of the basin of Mexico valley. Nowadays, axolotl populations are decreasing due environmental stressors. Particularly, studies about organophosphorus pesticides (OPPs; i.e. chlorpyrifos and malathion) toxicity are of great importance due to their intensive use in agricultural activities in Xochimilco. Thus, the aim of this study was to evaluate under controlled conditions the toxicity of chlorpyrifos (CPF) and malathion (MLT) on embryos and larvae (stage 44 and 54) of A. mexicanum. Embryos and larvae were exposure 96h from 0.5 to 3mg CPFL(-1) and from 10 to 30mg MLTL(-1) in independent tests. Embryos at the end of this period were maintained 9d without pesticide in order to identify possible recuperation. Differences obtained in mortality, hatching success, development, morphological abnormalities, behaviour and activity, suggest that toxicity of CPF and MLT differs in embryos and larval stages. Embryos were less sensitive to OPPs acute exposure than axolotl larvae; additionally, toxicity of CPF in larval stages was greater than MLT. On the other hand, data obtained in axolotl embryos during the period of recuperation to CPF in particular as delay and inhibition of development, malformations and success of hatching, indicated that these responses turned out more sensitive than mortality. This study allowed to identify the toxicological potential of OPPs on early developmental stages of A. mexicanum and it is a valuable contribution for the future management of the axolotl wild population.
Assuntos
Ambystoma mexicanum/embriologia , Clorpirifos/toxicidade , Malation/toxicidade , Praguicidas/toxicidade , Animais , Embrião não Mamífero/metabolismo , Compostos Organofosforados/toxicidadeAssuntos
Ambystoma mexicanum/embriologia , Embrião não Mamífero/transplante , Extremidades/transplante , Ambystoma mexicanum/genética , Animais , Animais Geneticamente Modificados , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Transgenes/genética , Transplante HomólogoRESUMO
The discovery of the naturally occurring cardiac non-function (c) animal strain in Ambystoma mexicanum (axolotl) provides a valuable animal model to study cardiomyocyte differentiation. In homozygous mutant animals (c/c), rhythmic contractions of the embryonic heart are absent due to a lack of organized myofibrils. We have previously cloned a partial sequence of a peptide cDNA (N1) from an anterior-endoderm-conditioned-medium RNA library that had been shown to be able to rescue the mutant phenotype. In the current studies we have fully cloned the N1 full length cDNA sequence from the library. N1 protein has been detected in both adult heart and skeletal muscle but not in any other adult tissues. GFP-tagged expression of the N1 protein has revealed localization of the N1 protein in the endoplasmic reticulum (ER). Results from in situ hybridization experiments have confirmed the dramatic decrease of expression of N1 mRNA in mutant (c/c) embryos indicating that the N1 gene is involved in heart development.
Assuntos
Ambystoma mexicanum/embriologia , Proteínas de Anfíbios/metabolismo , Retículo Endoplasmático/metabolismo , Coração/embriologia , Proteínas Musculares/metabolismo , Ambystoma mexicanum/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , Regulação para Baixo , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Músculo Estriado/metabolismo , MutaçãoRESUMO
The fate of single somites has not been analyzed from a comparative perspective with modern cell-marking methods. Most of what we know is based on work using quail-chick chimeras. Consequently, to what degree cell fate has been conserved despite the anatomical differences among vertebrates is unknown. We have analyzed the cell fate of the cranialmost somites, with the focus on somite two, in the Mexican axolotl (Ambystoma mexicanum). Somite cells were marked by injection of dextran-fluorescein and detected using immunofluorescence after 2 months of development in paraffin sections. Our data confirm and extend earlier studies based on classical histology in salamanders. We show that somite two contributes to different muscles, skeletal elements, and connective tissues of the head and cranial trunk region. Cells from somites two and three migrate latero-ventrally and contribute to the hypobranchial muscles mm. geniohyoideus and rectus cervicis. We provide evidence that the specific formation of the hypobranchial musculature from ventral processes of the somites might be variable in different classes of vertebrates. We further demonstrate that mm. cucullaris and dilatator laryngis, which were earlier thought to have a branchial origin, arise from somitic material in a manner very similar to the findings in quail-chick chimeras. Our findings indicate that the pattern of somitic derivatives is highly conserved within tetrapods.
Assuntos
Músculos/embriologia , Crânio/embriologia , Somitos/embriologia , Ambystoma mexicanum/embriologia , Animais , Embrião não Mamífero/embriologia , Embrião não Mamífero/ultraestrutura , Somitos/ultraestruturaRESUMO
The Mexican axolotl, Ambystoma mexicanum, has been a useful animal model to study heart development and cardiac myofibrillogenesis. A naturally-occurring recessive mutant, gene "c", for cardiac non-function in the Mexican axolotl causes a failure of myofibrillogenesis due to a lack of tropomyosin expression in homozygous mutant (c/c) embryonic hearts. Myofibril-inducing RNA (MIR) rescues mutant hearts in vitro by promoting tropomyosin expression and myofibril formation thereafter. We have studied the effect of MIR on the expression of various isoforms of cardiac troponin T (cTnT), a component of the thin filament that binds with tropomyosin. Four alternatively spliced cTnT isoforms have been characterized from developing axolotl heart. The expression of various cTnT isoforms in normal, mutant, and mutant hearts corrected with MIR, is evaluated by real-time RT-PCR using isoform specific primer pairs; MIR affects the total transcription as well as the splicing of the cTnT in axolotl heart.
Assuntos
Ambystoma mexicanum/embriologia , Coração/embriologia , Miocárdio/metabolismo , Miofibrilas/fisiologia , RNA/metabolismo , Troponina T/genética , Troponina T/metabolismo , Ambystoma mexicanum/genética , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Coração/fisiologia , RNA/genética , Relação Estrutura-AtividadeRESUMO
The Mexican axolotl, Ambystoma mexicanum, is an excellent animal model for studying heart development because it carries a naturally occurring recessive genetic mutation, designated gene c, for cardiac nonfunction. The double recessive mutants (c/c) fail to form organized myofibrils in the cardiac myoblasts resulting in hearts that fail to beat. Tropomyosin expression patterns have been studied in detail and show dramatically decreased expression in the hearts of homozygous mutant embryos. Because of the direct interaction between tropomyosin and troponin T (TnT), and the crucial functions of TnT in the regulation of striated muscle contraction, we have expanded our studies on this animal model to characterize the expression of the TnT gene in cardiac muscle throughout normal axolotl development as well as in mutant axolotls. In addition, we have succeeded in cloning the full-length cardiac troponin T (cTnT) cDNA from axolotl hearts. Confocal microscopy has shown a substantial, but reduced, expression of TnT protein in the mutant hearts when compared to normal during embryonic development.
Assuntos
Ambystoma mexicanum/metabolismo , Miocárdio/metabolismo , Troponina T/metabolismo , Ambystoma mexicanum/embriologia , Ambystoma mexicanum/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião não Mamífero/metabolismo , Imunoquímica , Dados de Sequência Molecular , Contração Muscular , Mutação , Miocárdio/citologia , Ligação Proteica , Homologia de Sequência de Aminoácidos , Tropomiosina/metabolismo , Troponina T/genéticaRESUMO
The Mexican axolotl, Ambystoma mexicanum, serves as an intriguing model to investigate myofibril organization and heart development in vertebrates. The axolotl has a homozygous recessive cardiac lethal gene "c" which causes a failure of ventricular myofibril formation and contraction. However, the conus of the heart beats, and has organized myofibrils. Tropomyosin (TM), an essential component of the thin filament, has three known striated muscle isoforms (TPM1alpha, TPM1kappa, and TPM4alpha) in axolotl hearts. However, it is not known whether there are differential expression patterns of these tropomyosin isoforms in various segments of the heart. Also, it is not understood whether these isoforms contribute to myofibril formation in a segment-specific manner. In this study, we have utilized anti-sense oligonucleotides to separately knockdown post-transcriptional expression of TPM1alpha and TPM4alpha. We then evaluated the organization of myofibrils in the conus and ventricle of normal and cardiac mutant hearts using immunohistochemical techniques. We determined that the TPM1alpha isoform, a product of the TPM1 gene, was essential for myofibrillogenesis in the conus, whereas TPM4alpha, the striated muscle isoform of the TPM4 gene, was essential for myofibrillogenesis in the ventricle. Our results support the segmental theory of vertebrate heart development.