RESUMO
The evolutionarily conserved Roundabout (Robo) family of axon guidance receptors control midline crossing of axons in response to the midline repellant ligand Slit in bilaterian animals including insects, nematodes, and vertebrates. Despite this strong evolutionary conservation, it is unclear whether the signaling mechanism(s) downstream of Robo receptors are similarly conserved. To directly compare midline repulsive signaling in Robo family members from different species, here we use a transgenic approach to express the Robo family receptor SAX-3 from the nematode Caenorhabditis elegans in neurons of the fruit fly, Drosophila melanogaster. We examine SAX-3's ability to repel Drosophila axons from the Slit-expressing midline in gain of function assays, and test SAX-3's ability to substitute for Drosophila Robo1 during fly embryonic development in genetic rescue experiments. We show that C. elegans SAX-3 is properly translated and localized to neuronal axons when expressed in the Drosophila embryonic CNS, and that SAX-3 can signal midline repulsion in Drosophila embryonic neurons, although not as efficiently as Drosophila Robo1. Using a series of Robo1/SAX-3 chimeras, we show that the SAX-3 cytoplasmic domain can signal midline repulsion to the same extent as Robo1 when combined with the Robo1 ectodomain. We show that SAX-3 is not subject to endosomal sorting by the negative regulator Commissureless (Comm) in Drosophila neurons in vivo, and that peri-membrane and ectodomain sequences are both required for Comm sorting of Drosophila Robo1.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Drosophila/genética , Evolução Molecular , Proteínas do Tecido Nervoso/genética , Receptores Imunológicos/genética , Transdução de Sinais , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Endossomos/metabolismo , Teste de Complementação Genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Transporte Proteico , Receptores Imunológicos/química , Receptores Imunológicos/metabolismo , Proteínas RoundaboutRESUMO
lilliputian (lilli), the sole Drosophila member of the FMR2/AF4 (Fragile X Mental Retardation/Acute Lymphoblastic Leukemia) family of transcription factors, is widely expressed with roles in segmentation, cellularization, and gastrulation during early embryogenesis with additional distinct roles at later stages of embryonic and postembryonic development. We identified lilli in a genetic screen based on the suppression of a lethal phenotype that is associated with ectopic expression of the transcription factor encoded by the segmentation gene runt in the blastoderm embryo. In contrast to other factors identified by this screen, lilli appears to have no role in mediating either the establishment or maintenance of engrailed (en) repression by Runt. Instead, we find that Lilli plays a critical role in the Runt-dependent activation of the pair-rule segmentation gene fushi-tarazu (ftz). The requirement for lilli is distinct from and temporally precedes the Runt-dependent activation of ftz that is mediated by the orphan nuclear receptor protein Ftz-F1. We further describe a role for lilli in the activation of Sex-lethal (Sxl), an early target of Runt in the sex determination pathway. However, lilli is not required for all targets that are activated by Runt and appears to have no role in activation of sloppy paired (slp1). Based on these results we suggest that Lilli plays an architectural role in facilitating transcriptional activation that depends both on the target gene and the developmental context.