RESUMO
The extracellular matrix protein Tgfbi has been shown to localise to myotendinous junctions in mouse and postulated to interact with the transmembrane protein Integrin alpha7beta1, which, in parallel with the Dystrophin-associated protein complex, is critical for linkage between the extracellular matrix and the cytoskeleton of muscle fibres. Here we use a GFP-tagged form of Tgfbi to analyse its distribution in the developing skeletal muscle of the zebrafish embryos and antisense morpholino oligonucleotides to investigate the function of the endogenous protein. We find that although tagged Tgfbi accumulates at the myosepta, the attachment of muscle fibres to the myosepta is established and maintained normally in morphant embryos; however, the fibres show a marked reduction in their growth and a disruption of their myofibril bundles.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Miofibrilas/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra , Animais , Proteínas da Matriz Extracelular/genética , Humanos , Camundongos , Fibras Musculares Esqueléticas/citologia , Miofibrilas/ultraestrutura , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fator de Crescimento Transformador beta/genética , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genéticaRESUMO
MicroRNAs (miRNAs) regulate gene expression by inhibiting translation of target mRNAs through pairing with miRNA recognition elements (MREs), usually in 3' UTRs. Because pairing is imperfect, identification of bona fide mRNA targets presents a challenge. Most target recognition algorithms strongly emphasize pairing between nucleotides 2-8 of the miRNA (the 'seed' sequence) and the mRNA but adjacent sequences and the local context of the 3' UTR also affect targeting. Here, we show that dispatched 2 is a target of miR-214. In zebrafish, dispatched 2 is expressed in the telencephalon and ventral hindbrain and is essential for normal zebrafish development. Regulation of dispatched 2 by miR-214 is via pairing with three, noncanonical, weak MREs. By comparing the repression capacity of GFP reporters containing different dispatched 2 sequences, we found that a combination of weak sites, which lack canonical seed pairing, can effectively target an mRNA for silencing. This finding underscores the challenge that prediction algorithms face and emphasizes the need to experimentally validate predicted MREs.
Assuntos
Proteínas de Membrana/genética , MicroRNAs/metabolismo , Interferência de RNA , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Regiões 3' não Traduzidas/química , Animais , Pareamento de Bases , Embrião não Mamífero/anatomia & histologia , MicroRNAs/química , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologiaRESUMO
Muscle cells are surrounded by extracellular matrix, the components of which play an important role in signalling mechanisms involved in their development. In mice, loss of collagen XV, a component of basement membranes expressed primarily in skeletal muscles, results in a mild skeletal myopathy. We have determined the complete zebrafish collagen XV primary sequence and analysed its expression and function in embryogenesis. During the segmentation period, expression of the Col15a1 gene is mainly found in the notochord and its protein product is deposited exclusively in the peri-notochordal basement membrane. Morpholino mediated knock-down of Col15a1 causes defects in notochord differentiation and in fast and slow muscle formation as shown by persistence of axial mesodermal marker gene expression, disorganization of the peri-notochodal basement membrane and myofibrils, and a U-shape myotome. In addition, the number of medial fast-twitch muscle fibers was substantially increased, suggesting that the signalling by notochord derived Hh proteins is enhanced by loss of collagen XV. Consistent with this, there is a concomitant expansion of patched-1 expression in the myotome of morphant embryos. Together, these results indicate that collagen XV is required for notochord differentiation and muscle development in the zebrafish embryo and that it interplays with Shh signalling.
Assuntos
Colágeno/metabolismo , Desenvolvimento Muscular , Notocorda/embriologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Membrana Basal/embriologia , Membrana Basal/metabolismo , Membrana Basal/ultraestrutura , Padronização Corporal/genética , Clonagem Molecular , Colágeno/antagonistas & inibidores , Colágeno/genética , Proteínas Hedgehog/metabolismo , Dados de Sequência Molecular , Neurônios Motores/fisiologia , Desenvolvimento Muscular/genética , Notocorda/química , Notocorda/metabolismo , Transdução de Sinais , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genéticaRESUMO
Signalling by members of the Hedgehog family of secreted proteins plays a central role in the development of many animal species. In the zebrafish embryo, the specification of myoblast fates is controlled by Hedgehog signals emanating from axial midline structures. Distinct muscle cell identities are induced by varying levels of signalling activity. The SET domain transcription factor, Blimp1, is a key target of Hedgehog signalling in this process.
