RESUMO
Multicellular rosettes are transient epithelial structures that serve as intermediates during diverse organ formation. We have identified a unique contributor to rosette formation in zebrafish Kupffer's vesicle (KV) that requires cell division, specifically the final stage of mitosis termed abscission. KV utilizes a rosette as a prerequisite before forming a lumen surrounded by ciliated epithelial cells. Our studies identify that KV-destined cells remain interconnected by cytokinetic bridges that position at the rosette's center. These bridges act as a landmark for directed Rab11 vesicle motility to deliver an essential cargo for lumen formation, CFTR (cystic fibrosis transmembrane conductance regulator). Here we report that premature bridge cleavage through laser ablation or inhibiting abscission using optogenetic clustering of Rab11 result in disrupted lumen formation. We present a model in which KV mitotic cells strategically place their cytokinetic bridges at the rosette center, where Rab11-associated vesicles transport CFTR to aid in lumen establishment.
Assuntos
Divisão Celular/fisiologia , Polaridade Celular/fisiologia , Embrião não Mamífero/fisiologia , Desenvolvimento Embrionário/fisiologia , Células de Kupffer/fisiologia , Organogênese/fisiologia , Peixe-Zebra/embriologia , Animais , Linhagem Celular , Movimento Celular , Cílios/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células de Kupffer/citologia , Mitose , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Myotonic dystrophy type 1 (DM1) is a dominant neuromuscular disorder caused by a trinucleotide (CTG) repeat expansion. Mutant DMPK 3'-untranslated region (3'-UTR) transcripts aggregate in nuclear foci and are thought to impose dominant-negative effects by interacting with RNA binding proteins. We demonstrated previously that the mutant 3'-UTR RNA disrupted C2C12 myoblast differentiation, and that the CUG expansion was necessary for this effect. Several proteins are known to interact with the CUG tract or the region 3' (distal) to it. Here, using a library of transfected C2C12 clones, we show that although transcripts containing a CUG expansion alone or a CUG expansion plus the distal region of the DMPK 3'-UTR accumulate into RNA foci, neither of these RNAs affect C2C12 myogenesis. Thus, RNA foci formation, and perturbation of any RNA binding factors involved in this process, are not sufficient to block myoblast differentiation. Interestingly, we found that transcripts containing expanded CUG tracts can form both nuclear and cytoplasmic RNA foci, demonstrating that factors involved in foci formation are present in the nucleus and cytoplasm. RNA analysis of myogenic markers revealed that the mutant DMPK 3'-UTR mRNA does not affect myoblast determination factors MyoD or Myf5, but significantly impedes upregulation of the differentiation factors myogenin and p21. C2C12 provide a good model to study adult muscle regeneration. Our observations in this system may be relevant to the lack of a regenerative response to continued muscle wasting in DM, and point to defects in early events in the myogenic response to muscle damage.
Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transativadores , Expansão das Repetições de Trinucleotídeos/genética , Regiões 3' não Traduzidas/genética , Animais , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/análise , Citoplasma/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Musculares/análise , Músculo Esquelético/química , Músculo Esquelético/citologia , Mutação , Proteína MyoD/análise , Fator Regulador Miogênico 5 , Miogenina/análise , Miotonina Proteína Quinase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de TempoRESUMO
The mutation causing myotonic dystrophy (DM) has been identified as a CTG expansion in the 3'-untranslated region (3'-UTR) of the DM protein kinase gene ( DMPK ), but the mechanism(s) of pathogenesis remain unknown. Studies using DM patient materials have often produced confusing results. Therefore, to study the effects of the DM mutation in a controlled environment, we have established a cell culture model system using C2C12 mouse myoblasts. By expressing chimeric reporter constructs containing a reporter gene fused to a human DMPK 3'-UTR, we identified both cis and trans effects that are mediated by the DM mutation. Our data show that a mutant DMPK 3'-UTR, with as few as 57 CTGs, had a negative cis effect on protein expression and resulted in the aggregation of reporter transcripts into discrete nuclear foci. We determined by deletion analysis that an expanded (CTG) (n) tract alone was sufficient to mediate these cis effects. Furthermore, in contrast to the normal DMPK 3'-UTR mRNA, a mutant DMPK 3'-UTR mRNA with (CUG)(200)selectively inhibited myogenic differentiation of C2C12 myoblasts. Genetic analysis and the Cre- loxP system were used to clearly demonstrate that the myoblast fusion defect could be rescued by eliminating the expression of the mutant DMPK 3'-UTR transcript. Characterization of spontaneous deletion events mapped the inhibitory effect to the (CTG) (n) expansion and/or the 3' end of the DMPK 3'-UTR. These results provide evidence that the DM mutation acts in cis to reduce protein production (consistent with DMPK haploinsufficiency) and in trans as a 'riboregulator' to inhibit myogenesis.
