RESUMO
In the present study, we have analyzed in detail the functions of Htt during growth and development of the protist, Dictyostelium discoideum by creating mutants (both overexpressor and knockout). The mRNA was present at all stages of growth and development. Overexpression of htt did not show any major anomaly, while deletion resulted in delayed aggregation territory formation and showed asynchronous development especially after slug stage. The slugs formed by htt - cells showed aberration in anterior-posterior boundary, showing increased prestalk region. DdHtt regulates STAT transcription factors in the tip organizer region that help maintain patterning and culmination. In chimeras with the wild-type, htt - cells preferentially localized to the tip of the slug and basal disc regions of the fruiting body showing prestalk/stalk bias, while the overexpressing cells majorly populated the prespore/spore region showing spore bias. These differences could be attributed to protein kinase A (PKA)-regulated cyclic adenosine monophosphate (cAMP) signaling.
Assuntos
AMP Cíclico/metabolismo , Dictyostelium/metabolismo , Deleção de Genes , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Linhagem da Célula , Proliferação de Células/genética , Dictyostelium/citologia , Dictyostelium/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Agregados Proteicos , Proteínas de Protozoários/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição STAT/metabolismo , Esporos/citologia , Esporos/metabolismoRESUMO
We have earlier classified the homeobox containing genes from Dictyostelium discoideum and identified 5 genes belonging to the TALE class. Here, we have characterized hbx9, a member of TALE class. In situ hybridization results show it to be preferentially expressed in the prestalkA (pstA) cells but not in the prestalkO (pstO) cells. Disruption of the hbx9 gene in Ax2 cells delay initiation of development and also slows cell proliferation. There was decreased cadA expression during the development of hbx9- cells, which results in inappropriate cell contacts, altered cell motility and patterning properties. ecmA and ecmB were mis-expressed in the pstO and pstA regions resulting in an effectively smaller pstO region. Surprisingly, the total ecmAO expression in pstAO cells was comparable to the wild type, suggesting that in hbx9- cells, ecmA was expressed in the pstO region. In hbx9-, proportioning and patterning was altered in favour of pstA cell-type. Moreover, precocious expression of ecmB and pspA was also observed in the vegetative cells. Expression profiling of components of cAMP signalling suggest that Hbx9 controls cell proportioning of prestalk cells by modulating cAMP signalling during growth-to-development transition.
Assuntos
Diferenciação Celular/genética , Dictyostelium/genética , Proteínas de Homeodomínio/genética , Morfogênese/genética , Proteínas de Protozoários/genética , Animais , Dictyostelium/crescimento & desenvolvimento , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Dictyostelium discoideum encodes a single Rheb protein showing sequence similarity to human homologues of Rheb. The DdRheb protein shares 52 percent identity and 100 percent similarity with the human Rheb1 protein. Fluorescence of Rheb yellow fluorescent protein fusion was detected in the D. discoideum cytoplasm. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization analyses showed that rheb is expressed at all stages of development and in prestalk cells in the multicellular structures developed. When the expression of rheb as a fusion with lacZ was driven under its own promoter, the beta-galactosidase activity was seen in the prestalk cells. D. discoideum overexpressing Rheb shows an increase in the size of the cell. Treatment of the overexpressing Rheb cells with rapamycin confirms its involvement in the TOR signalling pathway.