RESUMEN
Hippo-Yorkie (Hpo-Yki) signaling is central to diverse developmental processes. Although its redeployment has been amply demonstrated, its context-specific regulation remains poorly understood. The Drosophila eye disc is a continuous epithelium folded into two layers, the peripodial epithelium (PE) and the retinal progenitor epithelium. Here, Yki acts in the PE, first to promote PE identity by suppressing retina fate, and subsequently to maintain proper disc morphology. In the latter process, loss of Yki results in the displacement of a portion of the differentiating retinal epithelium onto the PE side. We show that Protein Phosphatase 2A (PP2A) complexes comprising different substrate-specificity B-type subunits govern the Hpo-Yki axis in this context. These include holoenzymes containing the Bâ´ subunit Cka and those containing the B' subunits Wdb or Wrd. Whereas PP2A(Cka), as part of the STRIPAK complex, is known to regulate Hpo directly, PP2A(Wdb) acts genetically upstream of the antagonistic activities of the Hpo regulators Sav and Rassf. These in vivo data provide the first evidence of PP2A(B') heterotrimer function in Hpo pathway regulation and reveal pathway diversification at distinct developmental times in the same tissue.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismoRESUMEN
Hearing in infants is essential for brain development, acquisition of verbal language skills, and development of social interactions. Therefore, it is important to diagnose hearing loss soon after birth so that interventions can be provided as early as possible. Most newborns in the United States are screened for hearing deficits and commercially available next-generation sequencing hearing loss panels often can identify the causative gene, which may also identify congenital defects in other organs. One of the most prevalent autosomal dominant congenital hearing loss syndromes is branchio-oto-renal syndrome (BOR), which also presents with defects in craniofacial structures and the kidney. Currently, mutations in three genes, SIX1, SIX5, and EYA1, are known to be causative in about half of the BOR patients that have been tested. To uncover new candidate genes that could be added to congenital hearing loss genetic screens, we have combined the power of Drosophila mutants and protein biochemical assays with the embryological advantages of Xenopus, a key aquatic animal model with a high level of genomic similarity to human, to identify potential Six1 transcriptional targets and interacting proteins that play a role during otic development. We review our transcriptomic, yeast 2-hybrid, and proteomic approaches that have revealed a large number of new candidates. We also discuss how we have begun to identify how Six1 and co-factors interact to direct developmental events necessary for normal otic development.
RESUMEN
The specification of organs, tissues and cell types results from cell fate restrictions enacted by nuclear transcription factors under the control of conserved signaling pathways. The progenitor epithelium of the Drosophila compound eye, the eye imaginal disc, is a premier model for the study of such processes. Early in development, apposing cells of the eye disc are established as either retinal progenitors or support cells of the peripodial epithelium (PE), in a process whose genetic and mechanistic determinants are poorly understood. We have identified protein phosphatase 2A (PP2A), and specifically a STRIPAK-PP2A complex that includes the scaffolding and substrate-specificity components Cka, Strip and SLMAP, as a critical player in the retina-PE fate choice. We show that these factors suppress ectopic retina formation in the presumptive PE and do so via the Hippo signaling axis. STRIPAK-PP2A negatively regulates Hippo kinase, and consequently its substrate Warts, to release the transcriptional co-activator Yorkie into the nucleus. Thus, a modular higher-order PP2A complex refines the activity of this general phosphatase to act in a precise specification of cell fate.
Asunto(s)
Proteínas de Drosophila , Drosophila , Proteínas Adaptadoras Transductoras de Señales , Animales , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Epitelio/metabolismo , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Retina/metabolismoRESUMEN
BACKGROUND: BMP signaling is involved in myriad metazoan developmental processes, and study of this pathway in Drosophila has contributed greatly to our understanding of its molecular and genetic mechanisms. These studies have benefited not only from Drosophila's advanced genetic tools, but from complimentary in vitro culture systems. However, the commonly-used S2 cell line is not intrinsically sensitive to the major BMP ligand Dpp and must therefore be augmented with exogenous pathway components for most experiments. RESULTS: Herein we identify and characterize the responses of Drosophila ML-DmD17-c3 cells, which are sensitive to Dpp stimulation and exhibit characteristic regulation of BMP target genes including Dad and brk. Dpp signaling in ML-DmD17-c3 cells is primarily mediated by the receptors Put and Tkv, with additional contributions from Wit and Sax. Furthermore, we report complex regulatory feedback on core pathway genes in this system. CONCLUSIONS: Native ML-DmD17-c3 cells exhibit robust transcriptional responses to BMP pathway induction. We propose that ML-DmD17-c3 cells are well-suited for future BMP pathway analyses.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Receptores de Activinas Tipo II/metabolismo , Animales , Línea Celular , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Receptores de Superficie Celular/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Transcripción Genética/genéticaRESUMEN
Drosophila has three types of visual organs, the larval eyes or Bolwig's organs (BO), the ocelli (OC) and the compound eyes (CE). In all, the bHLH protein Atonal (Ato) functions as the proneural factor for photoreceptors and effects the transition from progenitor cells to differentiating neurons. In this work, we investigate the regulation of ato expression in the BO primordium (BOP). Surprisingly, we find that ato transcription in the BOP is entirely independent of the shared regulatory DNA for the developing CE and OC. The core enhancer for BOP expression, atoBO, lies ~6kb upstream of the ato gene, in contrast to the downstream location of CE and OC regulatory elements. Moreover, maintenance of ato expression in the neuronal precursors through autoregulation-a common and ancient feature of ato expression that is well-documented in eyes, ocelli and chordotonal organs-does not occur in the BO. We also show that the atoBO enhancer contains two binding sites for the transcription factor Sine oculis (So), a core component of the progenitor specification network in all three visual organs. These binding sites function in vivo and are specifically bound by So in vitro. Taken together, our findings reveal that the control of ato transcription in the evolutionarily derived BO has diverged considerably from ato regulation in the more ancestral compound eyes and ocelli, to the extent of acquiring what appears to be a distinct and evolutionarily novel cis-regulatory module.
Asunto(s)
Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/genética , Elementos de Facilitación Genéticos , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Homeostasis/genética , Región de Flanqueo 5'/genética , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión/genética , ADN/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/metabolismo , Proteínas de Homeodominio/metabolismo , Larva/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Especificidad de Órganos , Transcripción Genética , Activación Transcripcional/genéticaRESUMEN
Limb development membrane protein-1 (LMBR1)/lipocalin-interacting membrane receptor (LIMR)-type proteins are putative nine-transmembrane receptors that are evolutionarily conserved across metazoans. However, their biological function is unknown. Here, we show that the fly family member Lilipod (Lili) is required for germ-line stem cell (GSC) self-renewal in the Drosophila ovary where it enhances bone morphogenetic protein (BMP) signaling. lili mutant GSCs are lost through differentiation, and display reduced levels of the Dpp transducer pMad and precocious activation of the master differentiation factor bam. Conversely, overexpressed Lili induces supernumerary pMad-positive bamP-GFP-negative GSCs. Interestingly, differentiation of lili mutant GSCs is bam-dependent; however, its effect on pMad is not. Thus, although it promotes stem cell self-renewal by repressing a bam-dependent process, Lilipod enhances transduction of the Dpp signal independently of its suppression of differentiation. In addition, because Lili is still required by a ligand-independent BMP receptor, its function likely occurs between receptor activation and pMad phosphorylation within the signaling cascade. This first, to our knowledge, in vivo characterization of a LMBR1/LIMR-type protein in a genetic model reveals an important role in modulating BMP signaling during the asymmetric division of an adult stem cell population and in other BMP signaling contexts.
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Proteínas Morfogenéticas Óseas/metabolismo , Proliferación Celular/fisiología , Proteínas de Drosophila/fisiología , Transducción de Señal/fisiología , Células Madre/citología , Animales , DrosophilaRESUMEN
The fruit fly Drosophila melanogaster has two types of external visual organs, a pair of compound eyes and a group of three ocelli. At the time of neurogenesis, the proneural transcription factor Atonal mediates the transition from progenitor cells to differentiating photoreceptor neurons in both organs. In the developing compound eye, atonal (ato) expression is directly induced by transcriptional regulators that confer retinal identity, the Retinal Determination (RD) factors. Little is known, however, about control of ato transcription in the ocelli. Here we show that a 2kb genomic DNA fragment contains distinct and common regulatory elements necessary for ato induction in compound eyes and ocelli. The three binding sites that mediate direct regulation by the RD factors Sine oculis and Eyeless in the compound eye are also required in the ocelli. However, in the latter, these sites mediate control by Sine oculis and the other Pax6 factor of Drosophila, Twin of eyeless, which can bind the Pax6 sites in vitro. Moreover, the three sites are differentially utilized in the ocelli: all three are similarly essential for atonal induction in the posterior ocelli, but show considerable redundancy in the anterior ocellus. Strikingly, this difference parallels the distinct control of ato transcription in the posterior and anterior progenitors of the developing compound eyes. From a comparative perspective, our findings suggest that the ocelli of arthropods may have originated through spatial partitioning from the dorsal edge of an ancestral compound eye.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ojo Compuesto de los Artrópodos/embriología , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Proteínas del Tejido Nervioso/genética , Neurogénesis/genética , Células Fotorreceptoras de Invertebrados/citología , Activación Transcripcional/genética , Animales , Animales Modificados Genéticamente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Sitios de Unión , Ojo Compuesto de los Artrópodos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción PAX6/genética , Transactivadores/genéticaRESUMEN
The v-ATPase is a fundamental eukaryotic enzyme that is central to cellular homeostasis. Although its impact on key metabolic regulators such as TORC1 is well documented, our knowledge of mechanisms that regulate v-ATPase activity is limited. Here, we report that the Drosophila transcription factor Mitf is a master regulator of this holoenzyme. Mitf directly controls transcription of all 15 v-ATPase components through M-box cis-sites and this coordinated regulation affects holoenzyme activity in vivo. In addition, through the v-ATPase, Mitf promotes the activity of TORC1, which in turn negatively regulates Mitf. We provide evidence that Mitf, v-ATPase and TORC1 form a negative regulatory loop that maintains each of these important metabolic regulators in relative balance. Interestingly, direct regulation of v-ATPase genes by human MITF also occurs in cells of the melanocytic lineage, showing mechanistic conservation in the regulation of the v-ATPase by MITF family proteins in fly and mammals. Collectively, this evidence points to an ancient module comprising Mitf, v-ATPase and TORC1 that serves as a dynamic modulator of metabolism for cellular homeostasis.
Asunto(s)
Proteínas de Drosophila/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Factores de Transcripción/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , Animales , Línea Celular Tumoral , Membrana Celular/metabolismo , Drosophila , Activación Enzimática , Homeostasis/fisiología , Humanos , Melanocitos/metabolismo , Melanoma/genética , ATPasas de Translocación de Protón Mitocondriales/genética , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño , Transcripción Genética/genética , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
A host of classical and molecular genetic tools make Drosophila a tremendous model for the dissection of gene activity. In particular, the FLP-FRT technique for mitotic recombination has greatly enhanced gene loss-of-function analysis. This technique efficiently induces formation of homozygous mutant clones in tissues of heterozygous organisms. However, the dependence of the FLP-FRT method on cell division, and other constraints, also impose limits on its effectiveness. We describe here the generation and testing of tools for Mutant Analysis by Rescue Gene Excision (MARGE), an approach whereby mutant cells are formed by loss of a rescue transgene in a homozygous mutant organism. Rescue-transgene loss can be induced in any tissue or cell-type and at any time during development or in the adult using available heat-shock-induced or tissue-specific flippases, or combinations of UAS-FLP with Gal4 and Gal80ts reagents. The simultaneous loss of a constitutive fluorescence marker (GFP or RFP) identifies the mutant cells. We demonstrate the efficacy of the MARGE technique by flip-out (clonal and disc-wide) of a Ubi-GFP-carrying construct in imaginal discs, and by inducing a known yki mutant phenotype in the Drosophila ovary.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Mosaicismo , Proteínas Nucleares/genética , Recombinación Genética , Transactivadores/genética , Animales , Análisis Mutacional de ADN , Femenino , Homocigoto , Discos Imaginales/crecimiento & desarrollo , Discos Imaginales/metabolismo , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Fenotipo , Proteínas Señalizadoras YAPRESUMEN
The Gal4/UAS system is one of the most powerful tools for the study of cellular and developmental processes in Drosophila. Gal4 drivers can be used to induce targeted expression of dominant-negative and dominant-active proteins, histological markers, activity sensors, gene-specific dsRNAs, modulators of cell survival or proliferation, and other reagents. Here, we describe novel atonal-Gal4 lines that contain regions of the regulatory DNA of atonal, the proneural gene for photoreceptors, stretch receptors, auditory organ, and some olfactory sensilla. During neurogenesis, the atonal gene is expressed at a critical juncture, a time of transition from progenitor cell to developing neuron. Thus, these lines are particularly well suited for the study of the transcription factors and signaling molecules orchestrating this critical transition. To demonstrate their usefulness, we focus on two visual organs, the eye and the Bolwig. We demonstrate the induction of predicted eye phenotypes when expressing the dominant-negative EGF receptor or a dsRNA against Notch in the developing eye disc. In another example, we show the deletion of the Bolwig's organ using the proapoptotic factor Hid. Finally, we investigate the function of the eye specification factor Eyes absent or Eya in late retinal progenitors, shortly before they begin morphogenesis. We show that Eya is still required in these late progenitors to promote eye formation, and show failure to induce the target gene atonal and consequent lack of neuron formation.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Drosophila/genética , Proteínas del Ojo/genética , Ojo/metabolismo , Proteínas del Tejido Nervioso/genética , Células Madre/metabolismo , Animales , Animales Modificados Genéticamente , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Ojo/citología , Ojo/crecimiento & desarrollo , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hibridación in Situ , Morfogénesis/genética , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/genética , Neuronas/metabolismo , Interferencia de ARN , Receptores Notch/genética , Receptores Notch/metabolismo , Retina/citología , Retina/crecimiento & desarrollo , Retina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Proneural transcription factors drive the generation of specialized neurons during nervous system development, and their dynamic expression pattern is critical to their function. The activation of the proneural gene atonal (ato) in the Drosophila eye disc epithelium represents a critical step in the transition from retinal progenitor cell to developing photoreceptor neuron. We show here that the onset of ato transcription depends on two distant enhancers that function differently in subsets of retinal progenitor cells. A detailed analysis of the crosstalk between these enhancers identifies a critical role for three binding sites for the Retinal Determination factors Eyeless (Ey) and Sine oculis (So). We show how these sites interact to induce ato expression in distinct regions of the eye field and confirm them to be occupied by endogenous Ey and So proteins in vivo. Our study suggests that Ey and So operate differently through the same 3' cis-regulatory sites in distinct populations of retinal progenitors.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Drosophila/embriología , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factores de Transcripción Paired Box/metabolismo , Proteínas Represoras/metabolismo , Retina/embriología , Células Madre/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Sitios de Unión , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Elementos de Facilitación Genéticos , Proteínas del Ojo/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/genética , Hibridación in Situ , Proteínas del Tejido Nervioso/fisiología , Sistema Nervioso/embriología , Neuronas/metabolismo , Neuronas/fisiología , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/genética , Proteínas Represoras/genética , Transcripción GenéticaRESUMEN
The Microphthalmia-associated Transcription Factor (MITF) governs numerous cellular and developmental processes. In mice, it promotes specification and differentiation of the retinal pigmented epithelium (RPE), and in humans, some mutations in MITF induce congenital eye malformations. Herein, we explore the function and regulation of Mitf in Drosophila eye development and uncover two roles. We find that knockdown of Mitf results in retinal displacement (RDis), a phenotype associated with abnormal eye formation. Mitf functions in the peripodial epithelium (PE), a retinal support tissue akin to the RPE, to suppress RDis, via the Hippo pathway effector Yorkie (Yki). Yki physically interacts with Mitf and can modify its transcriptional activity in vitro. Severe loss of Mitf, instead, results in the de-repression of retinogenesis in the PE, precluding its development. This activity of Mitf requires the protein phosphatase 2â¯A holoenzyme STRIPAK-PP2A, but not Yki; Mitf transcriptional activity is potentiated by STRIPAK-PP2A in vitro and in vivo. Knockdown of STRIPAK-PP2A results in cytoplasmic retention of Mitf in vivo and in its decreased stability in vitro, highlighting two potential mechanisms for the control of Mitf function by STRIPAK-PP2A. Thus, Mitf functions in a context-dependent manner as a key determinant of form and fate in the Drosophila eye progenitor epithelium.
Asunto(s)
Proteínas de Drosophila , Factor de Transcripción Asociado a Microftalmía , Proteínas Señalizadoras YAP , Animales , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Factor de Transcripción Asociado a Microftalmía/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Proteínas Señalizadoras YAP/metabolismo , Proteínas Señalizadoras YAP/genética , Transactivadores/metabolismo , Transactivadores/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Ojo/metabolismo , Ojo/crecimiento & desarrollo , Proteína Fosfatasa 2/metabolismo , Proteína Fosfatasa 2/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Epitelio/metabolismo , Diferenciación Celular , Proteínas de HomeodominioRESUMEN
The Drosophila eye develops from the larval eye disc, a flattened vesicle comprised of continuous retinal and peripodial epithelia (PE). The PE is an epithelium that plays a supporting role in retinal neurogenesis, but gives rise to cuticle in the adult. We report here that the PE is also necessary to preserve the morphology of the retinal epithelium. Depletion of the adherens junction (AJ) components ß-Catenin (ß-Cat), DE-Cadherin or α-Catenin from the PE leads to altered disc morphology, characterized by retinal displacement (RDis); so too does loss of the Ajuba protein Jub, an AJ-associated regulator of the transcriptional coactivator Yorkie (Yki). Restoring AJs or overexpressing Yki in ß-Cat deficient PE results in suppression of RDis. Additional suppressors of AJ-dependent RDis include knockdown of Rho kinase (Rok) and Dystrophin (Dys). Furthermore, knockdown of ßPS integrin (Mys) from the PE results in RDis, while overexpression of Mys can suppress RDis induced by the loss of ß-Cat. We thus propose that AJ-Jub-Yki signaling in PE cells regulates PE cell contractile properties and/or attachment to the extracellular matrix to promote normal eye disc morphology.
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Uniones Adherentes , Proteínas de Drosophila , Animales , Uniones Adherentes/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Transactivadores/metabolismo , Transducción de Señal , Epitelio/metabolismo , Drosophila/metabolismoRESUMEN
We report here the construction of Tubby-RFP balancers for the X, 2nd and 3rd chromosomes of Drosophila melanogaster. The insertion of a 2xTb-RFP transgene on the FM7c, CyO, and TM3 balancer chromosomes introduces two easily scorable, dominant, developmental markers. The strong Tb phenotype is visible to the naked eye at the larval L2, L3, and pupal stages. The RFP associated with the cuticle is easily detected at all stages from late embryo to adult with the use of a fluorescence stereomicroscope. The FM7c Bar 2xTb-RFP, CyO Cy 2xTb-RFP, and TM3 Sb 2xTb-RFP balancers will greatly facilitate the analysis of lethals and other developmental mutants in L2/L3 larvae and pupae, but also provide coverage of other stages beginning in late embryogenesis through to the adult.
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Cromosomas de Insectos/genética , Drosophila melanogaster/genética , Animales , Cromosomas de Insectos/metabolismo , Clonación Molecular , Cruzamientos Genéticos , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Larva/genética , Larva/crecimiento & desarrollo , Masculino , Mutación , Fenotipo , Pupa/genética , Pupa/crecimiento & desarrollo , TransgenesRESUMEN
The proneural genes are fundamental regulators of neuronal development in all metazoans. A critical role of the fly proneural factor Atonal (Ato(Dm)) is to induce photoreceptor neuron formation in Drosophila, whereas its murine homolog, Atonal7(Mm) (aka Ath5) is essential for the development of the ganglion cells of the vertebrate eye. Here, we identify the Bombyx mori ato homolog (ato(Bm) ). In a pattern strikingly reminiscent of ato(Dm), the ato(Bm) mRNA is expressed as a stripe in the silkworm eye disc. Its DNA-binding and protein-protein interaction domain is highly homologous to the Ato(Dm) bHLH. Targeted expression of Ato(Bm) in the endogenous ato(Dm) pattern rescues the eyeless phenotype of the fly ato(1) mutant and its ectopic expression induces similar gain-of-function phenotypes as Ato(Dm). Rescue experiments with chimeric proteins show that the non-bHLH portion of Ato(Bm) (N-region) can effectively substitute for the corresponding region of the fly transcription factor, even though no apparent conservation can be found at the amino acid level. On the contrary, the highly similar bHLH domain of Ato(Bm) cannot similarly substitute for the corresponding region of Ato(Dm). Thus, the bHLH(Bm) domain requires the Ato(Bm) N-region to function effectively, whereas the bHLH(Dm) domain can operate well with either N-region. These findings suggest a role for the non-bHLH portion of Ato proteins in modulating the function of the bHLH domain in eye neurogenesis and implicate specific aa residues of the bHLH in this process.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Bombyx/embriología , Drosophila/embriología , Ojo/embriología , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Bombyx/metabolismo , ADN , Drosophila/metabolismo , Proteínas de Drosophila , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Homología de Secuencia de AminoácidoRESUMEN
Six family transcription factors play important roles in craniofacial development. Their transcriptional activity can be modified by cofactor proteins. Two Six genes and one cofactor gene (Eya1) are involved in the human Branchio-otic (BO) and Branchio-otic-renal (BOR) syndromes. However, mutations in Six and Eya genes only account for approximately half of these patients. To discover potential new causative genes, we searched the Xenopus genome for orthologues of Drosophila cofactor proteins that interact with the fly Six-related factor, SO. We identified 33 Xenopus genes with high sequence identity to 20 of the 25 fly SO-interacting proteins. We provide the developmental expression patterns of the Xenopus orthologues for 11 of the fly genes, and demonstrate that all are expressed in developing craniofacial tissues with at least partial overlap with Six1/Six2. We speculate that these genes may function as Six-interacting partners with important roles in vertebrate craniofacial development and perhaps congenital syndromes.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Animales , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Biología Computacional , Embrión no Mamífero/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hibridación in Situ , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Cresta Neural/embriología , Factores de Transcripción/genética , Proteínas de Xenopus/genéticaRESUMEN
Homeobox transcription factors of the vertebrate CRX/OTX family play critical roles in photoreceptor neurons, the rostral brain and circadian processes. In mouse, the three related proteins, CRX, OTX1, and OTX2, fulfill these functions. In Drosophila, the single founding member of this gene family, called orthodenticle (otd), is required during embryonic brain and photoreceptor neuron development. We have used global gene expression analysis in late pupal heads to better characterize the post-embryonic functions of Otd in Drosophila. We have identified 61 genes that are differentially expressed between wild type and a viable eye-specific otd mutant allele. Among them, about one-third represent potentially direct targets of Otd based on their association with evolutionarily conserved Otd-binding sequences. The spectrum of biological functions associated with these gene targets establishes Otd as a critical regulator of photoreceptor morphology and phototransduction, as well as suggests its involvement in circadian processes. Together with the well-documented role of otd in embryonic patterning, this evidence shows that vertebrate and fly genes contribute to analogous biological processes, notwithstanding the significant divergence of the underlying genetic pathways. Our findings underscore the common evolutionary history of photoperception-based functions in vertebrates and invertebrates and support the view that a complex nervous system was already present in the last common ancestor of all bilateria.
Asunto(s)
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Vertebrados/genética , Vertebrados/metabolismo , Animales , Animales Modificados Genéticamente , Ritmo Circadiano/genética , Drosophila/crecimiento & desarrollo , Evolución Molecular , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Operón Lac , Fototransducción/genética , Masculino , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Fotorreceptoras de Invertebrados/crecimiento & desarrollo , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Especificidad de la Especie , Vertebrados/crecimiento & desarrolloRESUMEN
The Decapentaplegic and Notch signaling pathways are thought to direct regional specification in the Drosophila eye-antennal epithelium by controlling the expression of selector genes for the eye (Eyeless/Pax6, Eyes absent) and/or antenna (Distal-less). Here, we investigate the function of these signaling pathways in this process. We find that organ primordia formation is indeed controlled at the level of Decapentaplegic expression but critical steps in regional specification occur earlier than previously proposed. Contrary to previous findings, Notch does not specify eye field identity by promoting Eyeless expression but it influences eye primordium formation through its control of proliferation. Our analysis of Notch function reveals an important connection between proliferation, field size, and regional specification. We propose that field size modulates the interaction between the Decapentaplegic and Wingless pathways, thereby linking proliferation and patterning in eye primordium development.
Asunto(s)
Proteínas de Drosophila/fisiología , Ojo/embriología , Proteínas de la Membrana/fisiología , Regiones Promotoras Genéticas/fisiología , Transducción de Señal/fisiología , Animales , Tipificación del Cuerpo , Diferenciación Celular , Drosophila/embriología , Proteínas de Drosophila/metabolismo , Epitelio/embriología , Epitelio/fisiología , Regulación del Desarrollo de la Expresión Génica , Cabeza/anatomía & histología , Cabeza/embriología , Proteínas de Homeodominio , Inmunohistoquímica/métodos , Proteínas de Insectos/fisiología , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Mutación , Receptores Notch , Transducción de Señal/genética , Factores de Tiempo , Factores de TranscripciónRESUMEN
The MITF protein is a member of the MYC family of basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factors and is most closely related to the TFE3, TFEC, and TFEB proteins. In the mouse, MITF is required for the development of several different cell types, including the retinal pigment epithelial (RPE) cells of the eye. In Mitf mutant mice, the presumptive RPE cells hyperproliferate, abnormally express the retinal transcriptional regulator Pax6, and form an ectopic neural retina. Here we report the structure of the Mitf gene in Drosophila and demonstrate expression during embryonic development and in the eye-antennal imaginal disc. In vitro, transcriptional regulation by Drosophila Mitf, like its mouse counterpart, is modified by the Eyeless (Drosophila Pax6) transcription factor. In vivo, targeted expression of wild-type or dominant-negative Drosophila Mitf results in developmental abnormalities reminiscent of Mitf function in mouse eye development. Our results suggest that the Mitf gene is the original member of the Mitf-Tfe subfamily of bHLH-Zip proteins and that its developmental function is at least partially conserved between vertebrates and invertebrates. These findings further support the common origin of the vertebrate and invertebrate eyes.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Drosophila melanogaster/genética , Ojo/embriología , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción/química , Factores de Transcripción/fisiología , Alelos , Secuencia de Aminoácidos , Animales , Secuencia Conservada , ADN Complementario/metabolismo , Evolución Molecular , Genes Dominantes , Genoma , Ratones , Factor de Transcripción Asociado a Microftalmía , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Epitelio Pigmentado Ocular/embriología , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transcripción Genética , Activación Transcripcional , TransfecciónRESUMEN
Congenital hearing loss is an important clinical problem because, without early intervention, affected children do not properly acquire language and consequently have difficulties developing social skills. Although most newborns in the US are screened for hearing deficits, even earlier diagnosis can be made with prenatal genetic screening. Genetic screening that identifies the relevant mutated gene can also warn about potential congenital defects in organs not related to hearing. We will discuss efforts to identify new candidate genes that underlie the Branchiootorenal spectrum disorders in which affected children have hearing deficits and are also at risk for kidney defects. Mutations in two genes, SIX1 and EYA1, have been identified in about half of the patients tested. To uncover new candidate genes, we have used the aquatic animal model, Xenopus laevis, to identify genes that are part of the developmental genetic pathway of Six1 during otic and kidney development. We have already identified a large number of potential Six1 transcriptional targets and candidate co-factor proteins that are expressed at the right time and in the correct tissues to interact with Six1 during development. We discuss the advantages of using this system for gene discovery in a human congenital hearing loss syndrome.