RESUMO
Class I Basic Helix-Loop-Helix (bHLH) transcription factors form homodimers or heterodimers with class II bHLH proteins. While bHLH heterodimers are known to have diverse roles, little is known about the role of class I homodimers. In this manuscript, we show that a linked dimer of Daughterless (Da), the only Drosophila class I bHLH protein, activates Atonal (Ato) expression and retinal neuron differentiation synergistically with the retinal determination factor Eyeless (Ey). The HLH protein Extramacrocheate (Emc), which forms heterodimer with Da, antagonizes the synergistic activation from Da but not the Da-Da linked dimer with Ey. We show that Da directly interacts with Ey and promotes Ey binding to the Ey binding site in the Ato 3׳ enhancer. Interestingly, the Ey binding site in the Ato 3׳ enhancer contains an embedded E-box that is also required for the synergistic activation by Ey and Da. Finally we show that mammalian homologs of Ey and Da can functionally replace their Drosophila counterparts to synergistically activate the Ato enhancer, suggesting that the observed function is evolutionary conserved.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Modelos Moleculares , Proteínas do Tecido Nervoso/metabolismo , Neurônios Retinianos/fisiologia , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Imunoprecipitação da Cromatina , Primers do DNA/genética , Proteínas de Ligação a DNA/química , Dimerização , Proteínas de Drosophila/química , Discos Imaginais/embriologia , Discos Imaginais/metabolismo , Imuno-Histoquímica , Microscopia de Fluorescência , Neurônios Retinianos/metabolismoRESUMO
Inactivation of the retinoblastoma gene Rb leads to defects in cell proliferation, differentiation, or apoptosis, depending on specific cell or tissue types. To gain insights into the genes that can modulate the consequences of Rb inactivation, we carried out a genetic screen in Drosophila to identify mutations that affected apoptosis induced by inactivation of the Retinoblastoma-family protein (rbf) and identified a mutation that blocked apoptosis induced by rbf. We found this mutation to be a new allele of head involution defective (hid) and showed that hid expression is deregulated in rbf mutant cells in larval imaginal discs. We identified an enhancer that regulates hid expression in response to developmental cues as well as to radiation and demonstrated that this hid enhancer is directly repressed by RBF through an E2F binding site. These observations indicate that apoptosis of rbf mutant cells is mediated by an upregulation of hid. Finally, we showed that bantam, a miRNA that regulates hid translation, is expressed in the interommatidial cells in the larval eye discs and modulates the survival of rbf mutant cells.
Assuntos
Apoptose/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteína do Retinoblastoma/genética , Fatores de Transcrição/genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Elementos Facilitadores Genéticos , Genes Reporter , MicroRNAs/genética , MicroRNAs/metabolismo , Dados de Sequência Molecular , Mutação , Fenótipo , Células Fotorreceptoras de Invertebrados/citologia , Células Fotorreceptoras de Invertebrados/fisiologia , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismoRESUMO
The determination of neuronal identity in Drosophila cells depends on the accurate expression of proneural genes. The proneural gene atonal (ato) encodes a basic-HLH protein required for photoreceptor and chordotonal organ formation. The initial expression of ato in imaginal discs is regulated by sequences that lie 3' to its open reading frame. In this report, we show that the initial ato transcription in different imaginal discs is regulated by distinct 3' cis-regulatory sequences. The eye-specific ato 3' cis-regulatory sequence consists of two distinct elements we term 2.8 PB and 3.6 BP that regulate ato transcription during different stages of eye development. The 2.8 PB enhancer contains a highly conserved consensus binding site for the retinal determination (RD) factor Sine oculis (So). Mutation of this So binding site abolishes 2.8 PB enhancer activity. Furthermore the RD factors So and Eyes absent (Eya) are required for 2.8 PB enhancer activity and can induce ectopic 2.8 PB reporter expression. In contrast, ectopic Dpp signaling is not sufficient to induce ato 3' enhancer activation but can induce increased levels of RD factor Dachshund (Dac) and synergize with So and Eya to increase ato 3' enhancer activity. These results demonstrate a direct mechanism by which the RD factors regulate ato expression and suggest an important role of Dpp in the activation of ato 3' enhancer is to regulate the levels of RD factors.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Drosophila/genética , Olho/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Proteínas do Tecido Nervoso/metabolismo , Organogênese , Retina/metabolismo , Animais , Animais Geneticamente Modificados , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Drosophila/embriologia , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero , Elementos Facilitadores Genéticos , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Elementos Reguladores de Transcrição , Retina/embriologia , Análise de Sequência de DNA , Transcrição GênicaRESUMO
The ability to generate spiral ganglion neurons (SGNs) from stem cells is a necessary prerequisite for development of cell-replacement therapies for sensorineural hearing loss. We present a protocol that directs human embryonic stem cells (hESCs) toward a purified population of otic neuronal progenitors (ONPs) and SGN-like cells. Between 82% and 95% of these cells express SGN molecular markers, they preferentially extend neurites to the cochlear nucleus rather than nonauditory nuclei, and they generate action potentials. The protocol follows an in vitro stepwise recapitulation of developmental events inherent to normal differentiation of hESCs into SGNs, resulting in efficient sequential generation of nonneuronal ectoderm, preplacodal ectoderm, early prosensory ONPs, late ONPs, and cells with cellular and molecular characteristics of human SGNs. We thus describe the sequential signaling pathways that generate the early and later lineage species in the human SGN lineage, thereby better describing key developmental processes. The results indicate that our protocol generates cells that closely replicate the phenotypic characteristics of human SGNs, advancing the process of guiding hESCs to states serving inner-ear cell-replacement therapies and possible next-generation hybrid auditory prostheses. © Stem Cells Translational Medicine 2017;6:923-936.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Humanas/citologia , Células Receptoras Sensoriais/citologia , Gânglio Espiral da Cóclea/citologia , Animais , Tronco Encefálico/citologia , Linhagem Celular , Linhagem da Célula , Movimento Celular , Sobrevivência Celular , Técnicas de Cocultura , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos , Células-Tronco Neurais/citologia , RatosRESUMO
Connection of tubules into larger networks is the key process for the development of circulatory systems. In Drosophila development, tip cells of the tracheal system lead the migration of each branch and connect tubules by adhering to each other and simultaneously changing into a torus-shape. We show that as adhesion sites form between fusion cells, myosin and microtubules form polarized bundles that connect the new adhesion site to the cells' microtubule-organizing centres, and that E-cadherin and retrograde recycling endosomes are preferentially deposited at the new adhesion site. We demonstrate that microtubules help balancing tip cell contraction, which is driven by myosin, and is required for adhesion and tube fusion. We also show that retrograde recycling and directed secretion of a specific matrix protein into the fusion-cell interface promote fusion. We propose that microtubule bundles connecting these cell-cell interfaces coordinate cell contractility and apical secretion to facilitate tube fusion.
Assuntos
Drosophila melanogaster/citologia , Células Epiteliais/citologia , Matriz Extracelular/metabolismo , Microtúbulos/metabolismo , Actinas/metabolismo , Animais , Fenômenos Biomecânicos , Caderinas/metabolismo , Fusão Celular , Movimento Celular , Drosophila melanogaster/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Junções Intercelulares/metabolismo , Mutação/genética , Miosinas/metabolismo , Fenótipo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Traqueia/citologiaRESUMO
Pax6 and its Drosophila homolog Eyeless (Ey) play essential roles during eye development. Ey/Pax6 contains two distinct DNA binding domains, a Paired domain (PD) and a Homeodomain (HD). While Ey/Pax6 PD is required for the expression of key regulators of retinal development, relatively little is known about the HD-dependent Ey function. In this study, we used the UAS/GAL4 system to determine the functions of different Ey domains on cell growth and on retinal development. We showed that Ey can promote cell growth, which requires the HD but not the PD. In contrast, the ability of Ey to activate Ato expression and induce ectopic eye formation requires the PD but not the HD. Interestingly, deletion of the HD enhanced Ey-dependent ectopic eye induction while overexpression of the HD only Ey forms antagonizes ectopic eye induction. These studies revealed a novel function of Ey HD on cell growth and a novel antagonistic effect of Ey HD on Ey PD-dependent eye induction. We further show the third helix of the Ey HD can directly interact with the RED subdomain in Ey PD and that deletion of the HD increased the binding of Ey PD to its target. These results suggest that the direct interaction between the HD and the PD potentially mediates their antagonistic effects. Since different Ey splicing forms are expressed in overlapping regions during normal development, we speculate that the expression ratios of the different Ey splice forms potentially contribute to the regulation of growth and differentiation of these tissues.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Retina/metabolismo , Animais , Animais Geneticamente Modificados/metabolismo , Sítios de Ligação , Diferenciação Celular , Proliferação de Células , Drosophila/metabolismo , Proteínas de Drosophila/antagonistas & inibidores , Elementos Facilitadores Genéticos , Proteínas do Olho/antagonistas & inibidores , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/antagonistas & inibidores , Fatores de Transcrição Box Pareados/metabolismo , Estrutura Terciária de Proteína , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Retina/citologia , Asas de Animais/crescimento & desenvolvimentoRESUMO
Cell proliferation and cell type specification are coordinately regulated during normal development. Cyclin E, a key G1/S cell cycle regulator, is regulated by multiple tissue-specific enhancers resulting in dynamic expression during Drosophila development. Here, we further characterized the enhancer that regulates cyclin E expression in the developing peripheral nervous system (PNS) and show that multiple sequence elements are required for the full cyclin E PNS enhancer activity. We further show that Wg signaling is important for the expression of cyclin E in the sensory organ precursor (SOP) cells through two conserved TCF binding sites. Blocking Wg signaling does not completely block SOP cell formation but does completely block SOP cell proliferation as well as the subsequent differentiation.
Assuntos
Ciclina E/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica no Desenvolvimento , Sistema Nervoso Periférico/embriologia , Transdução de Sinais , Proteína Wnt1/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular , Proliferação de Células , Sequência Conservada , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Células-Tronco Embrionárias/metabolismo , Elementos Facilitadores Genéticos/genética , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Sistema Nervoso Periférico/citologia , Sistema Nervoso Periférico/metabolismo , Deleção de Sequência , Fatores de Transcrição TCF/metabolismoRESUMO
The shattered1 (shtd1) mutation disrupts Drosophila compound eye structure. In this report, we show that the shtd1 eye defects are due to a failure to establish and maintain G1 arrest in the morphogenetic furrow (MF) and a defect in progression through mitosis. The observed cell cycle defects were correlated with an accumulation of cyclin A (CycA) and String (Stg) proteins near the MF. Interestingly, the failure to maintain G1 arrest in the MF led to the specification of R8 photoreceptor cells that undergo mitosis, generating R8 doublets in shtd1 mutant eye discs. We demonstrate that shtd encodes Apc1, the largest subunit of the anaphase-promoting complex/cyclosome (APC/C). Furthermore, we show that reducing the dosage of either CycA or stg suppressed the shtd1 phenotype. While reducing the dosage of CycA is more effective in suppressing the premature S phase entry in the MF, reducing the dosage of stg is more effective in suppressing the progression through mitosis defect. These results indicate the importance of not only G1 arrest in the MF but also appropriate progression through mitosis for normal eye development during photoreceptor differentiation.