RESUMO
A major problem in developmental genetics is how HOX transcription factors, like Proboscipedia (PB) and Ultrabithorax (UBX), regulate distinct programs of gene expression to result in a proboscis versus a haltere, respectively, when the DNA-binding homeodomain (HD) of HOX transcription factors recognizes similar DNA-binding sequences. Indeed, the lack of DNA-binding specificity is a problem for all transcription factors (TFs), as the DNA-binding domains generally recognize small targets of five to six bases in length. Although not the initial intent of the study, I found extensive non-specificity of TF function. Multiple TFs including HOX and HD-containing and non-HD-containing TFs induced both wingless and eyeless phenotypes. The TFs Labial (LAB), Deformed (DFD), Fushi tarazu (FTZ), and Squeeze (SQZ) induced ectopic larval thoracic (T) 1 beard formation in T2 and T3. The TF Doublesex male (DSXM) rescued the reduced maxillary palp pb phenotype. These examples of non-specificity of TF function across classes of TFs, combined with previous observations, compromise the implicit, initial assumption often made that an intrinsic mechanism of TF specificity is important for function. Interestingly, the functional complementation of the pb phenotype may suggest a larger role for regulation of expression of TFs in restriction of function as opposed to an intrinsic specificity of TF function. These observations have major ramifications for analysis of functional conservation in evolution and development.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Transcrição/metabolismo , Animais , Feminino , Fatores de Transcrição Fushi Tarazu/metabolismo , Genes Homeobox , Teste de Complementação Genética , Masculino , Mutação , Asas de Animais/metabolismoRESUMO
Observation of how cells divide, grow, migrate and form different parts of a developing organism is crucial for understanding developmental programs. Here, we describe a multicolor imaging tool named Raeppli (after the colorful confetti used at the carnival in Basel). Raeppli allows whole-tissue labeling such that the descendants of the majority of cells in a single organ are labeled and can be followed simultaneously relative to one another. We tested the use of Raeppli in the Drosophila melanogaster wing imaginal disc. Induction of Raeppli during larval stages irreversibly labels >90% of the cells with one of four spectrally separable, bright fluorescent proteins with low bias of selection. To understand the global growth characteristics of imaginal discs better, we induced Raeppli at various stages of development, imaged multiple fixed discs at the end of their larval development and estimated the size of their pouch primordium at those developmental stages. We also imaged the same wing disc through the larval cuticle at different stages of its development; the clones marked by Raeppli provide landmarks that can be correlated between multiple time points. Finally, we used Raeppli for continuous live imaging of prepupal eversion of the wing disc.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Linhagem da Célula , Cor , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Discos Imaginais/citologia , Discos Imaginais/crescimento & desenvolvimento , Proteínas Luminescentes/genética , Proteínas Recombinantes/genética , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimentoRESUMO
The ability to survive and reproduce after cold exposure is important in all kingdoms of life. However, even in a sophisticated genetic model system like Drosophila melanogaster, few genes have been identified as functioning in cold tolerance. The accumulation of the Frost (Fst) gene transcript increases after cold exposure, making it a good candidate for a gene that has a role in cold tolerance. Despite extensive RNAi knockdown analysis, no role in cold tolerance has been assigned to Fst CRISPR is an effective technique for completely knocking down genes, and is less likely to produce off-target effects than GAL4-UAS RNAi systems. We have used CRISPR-mediated homologous recombination to generate Fst-null alleles, and these Fst alleles uncovered a requirement for FST protein in maintaining female fecundity following cold exposure. However, FST does not have a direct role in survival following cold exposure. FST mRNA accumulates in the Malpighian tubules, and the FST protein is a highly disordered protein with a putative signal peptide for export from the cell. Future work is needed to determine whether FST is exported from the Malpighian tubules and directly interacts with female reproductive tissues post-cold exposure, or whether it is required for other repair/recovery functions that indirectly alter energy allocation to reproduction.
Assuntos
Sistemas CRISPR-Cas , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Expressão Gênica , Animais , Temperatura Baixa , Proteínas de Drosophila/metabolismo , Feminino , Mutação com Perda de Função , Masculino , Túbulos de Malpighi/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , ReproduçãoRESUMO
Key studies led to the idea that transcription factors are composed of defined modular protein motifs or domains, each with separable, unique function. During evolution, the recombination of these modular domains could give rise to transcription factors with new properties, as has been shown using recombinant molecules. This archetypic, modular view of transcription factor organization is based on the analyses of a few transcription factors such as GAL4, which may represent extreme exemplars rather than an archetype or the norm. Recent work with a set of Homeotic selector (HOX) proteins has revealed differential pleiotropy: the observation that highly-conserved HOX protein motifs and domains make small, additive, tissue specific contributions to HOX activity. Many of these differentially pleiotropic HOX motifs may represent plastic sequence elements called short linear motifs (SLiMs). The coupling of differential pleiotropy with SLiMs, suggests that protein sequence changes in HOX transcription factors may have had a greater impact on morphological diversity during evolution than previously believed. Furthermore, differential pleiotropy may be the genetic consequence of an ensemble nature of HOX transcription factor allostery, where HOX proteins exist as an ensemble of states with the capacity to integrate an extensive array of developmental information. Given a new structural model for HOX functional domain organization, the properties of the archetypic TF may require reassessment.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Sítio Alostérico , Processamento Alternativo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Genes Homeobox , Humanos , Dados de Sequência Molecular , Peptídeos/química , Estrutura Terciária de Proteína , Fatores de Transcrição/metabolismoRESUMO
Developmental competence is the response of a cell(s) to information. Determination of adult labial identity in Drosophila requires Proboscipedia (PB) and Sex combs reduced (SCR); however, co-ectopic expression of PB and SCR is not sufficient for induction of ectopic adult labial identity, because the developmental information supplied by PB and SCR is suppressed. The evolutionarily conserved LASCY, DYTQL, NANGE motifs, and the C-terminal domain of SCR are sequence elements that mediate some, or all, of the suppression of ectopic proboscis determination. Therefore, the developmentally competent primordial proboscis cells provide an environment devoid of suppression, allowing PB and SCR to determine proboscis identity. SCR derivatives lacking suppression sequences weakly induce ectopic proboscis transformations independently of PB, suggesting that SCR may be the activity required for induction of adult labial identity, as is the case for larval labial identity. A possible explanation for PB independence of SCR in determination of adult and embryonic labial identity is PB operates as a competence factor that switches SCR from determining T1 identity to labial identity during metamorphosis. Lastly, labial determination is not conserved between SCR and murine HOXA5, suggesting that SCR has acquired this activity during evolution.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Motivos de Aminoácidos , Animais , Antenas de Artrópodes/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Drosophila melanogaster/ultraestrutura , Glândulas Salivares/crescimento & desenvolvimento , Tarso Animal/crescimento & desenvolvimentoRESUMO
The regulation of the initiation of transcription by transcription factors is often assumed to be dependent on specific recognition of DNA-binding sites and nonredundant. However, the redundant induction or rescue of a phenotype by transcription factors, phenotypic nonspecificity, challenges these assumptions. To assess the frequency of phenotypic nonspecificity in the rescue of transcription factor phenotypes, seven transcription factor phenotypes (labial, Deformed, Sex combs reduced, Ultrabithorax, fruitless, doublesex, and apterous) were screened for rescue by the expression of 12, or more, nonresident transcription factors. From 308 assessments of rescue by nonresident transcription factors, 18 rescues were identified across 6 of the 7 transcription factor phenotypes. Seventeen of the 18 rescues were with transcription factors that recognize distinct DNA-binding sites relative to the resident transcription factors. All rescues were nonuniform across pleiotropic transcription factor phenotypes suggesting extensive differential pleiotropy of the rescue. Primarily using RNAi to knockdown expression, and with the exceptions of the requirement of Bric a Brac 1 for female abdominal pigmentation and Myb oncogene-like for wing development, no evidence was found for a role of the other 16 nonresident transcription factor in the transcription factor phenotypes assessed. Therefore, these 16 rescues are likely due to functional complementation and not due to the expression of an epistatic function in the developmental/behavioral pathway. Phenotypic nonspecificity is both differentially pleiotropic and frequent, as on average 1 in 10-20 nonresident transcription factors rescue a phenotype. These observations will be important in future considerations of transcription factors function.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Feminino , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fenótipo , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ciclo Celular/genética , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Proto-Oncogênicas c-myb/metabolismoRESUMO
The Drosophila Hox gene, Sex combs reduced (Scr), is required for patterning the larval and adult, labial and prothoracic segments. Fifteen Scr alleles were sequenced and the phenotypes analyzed in detail. Six null alleles were nonsense mutations (Scr(2), Scr(4), Scr(11), Scr(13), Scr(13A), and Scr(16)) and one was an intragenic deletion (Scr(17)). Five hypomorphic alleles were missense mutations (Scr(1), Scr(3), Scr(5), Scr(6), and Scr(8)) and one was a small protein deletion (Scr(15)). Protein sequence changes were found in four of the five highly conserved domains of SCR: the DYTQL motif (Scr(15)), YPWM motif (Scr(3)), Homeodomain (Scr(1)), and C-terminal domain (CTD) (Scr(6)), indicating importance for SCR function. Analysis of the pleiotropy of viable Scr alleles for the formation of pseudotracheae suggests that the DYTQL motif and the CTD mediate a genetic interaction with proboscipedia. One allele Scr(14), a missense allele in the conserved octapeptide, was an antimorphic allele that exhibited three interesting genetic properties. First, Scr(14)/Df had the same phenotype as Scr(+)/Df. Second, the ability of the Scr(14) allele to interact intragenetically with Scr alleles mapped to the first 82 amino acids of SCR, which contains the octapeptide motif. Third, Scr(6), which has two missense changes in the CTD, did not interact genetically with Scr(14).
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Homeodomínio/metabolismo , Larva/metabolismo , Fatores de Transcrição/metabolismo , Alelos , Animais , Western Blotting , Códon sem Sentido/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Larva/genética , Larva/crescimento & desenvolvimento , Mutação de Sentido Incorreto/genética , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência , Fatores de Transcrição/genéticaRESUMO
Homeotic selector (HOX) transcription factors (TFs) regulate gene expression that determines the identity of Drosophila segments along the anterior-posterior (A-P) axis. The current challenge with HOX proteins is understanding how they achieve their functional specificity while sharing a highly conserved homeodomain (HD) that recognize the same DNA binding sites. One mechanism proposed to regulate HOX activity is differential post-translational modification (PTM). As a first step in investigating this hypothesis, the sites of PTM on a Sex combs reduced protein fused to a triple tag (SCRTT) extracted from developing embryos were identified by Tandem Mass Spectrometry (MS/MS). The PTMs identified include phosphorylation at S185, S201, T315, S316, T317 and T324, acetylation at K218, S223, S227, K309, K434 and K439, formylation at K218, K309, K325, K341, K369, K434 and K439, methylation at S19, S166, K168 and T364, carboxylation at D108, K298, W307, K309, E323, K325 and K369, and hydroxylation at P22, Y87, P107, D108, D111, P269, P306, R310, N321, K325, Y334, R366, P392 and Y398. Of the 44 modifications, 18 map to functionally important regions of SCR. Besides a highly conserved DNA-binding HD, HOX proteins also have functionally important, evolutionarily conserved small motifs, which may be Short Linear Motifs (SLiMs). SLiMs are proposed to be preferential sites of phosphorylation. Although 6 of 7 phosphosites map to regions of predicted SLiMs, we find no support for the hypothesis that the individual S, T and Y residues of predicted SLiMs are phosphorylated more frequently than S, T and Y residues outside of predicted SLiMs.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Proteínas de Homeodomínio/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Homeodomínio/genéticaRESUMO
Drosophila transcription factor (TF) function is phenotypically nonspecific. Phenotypic nonspecificity is defined as one phenotype being induced or rescued by multiple TFs. To explain this unexpected result, a hypothetical world of limited specificity is explored where all TFs have unique random distributions along the genome due to low information content of DNA sequence recognition and somewhat promiscuous cooperative interactions with other TFs. Transcription is an emergent property of these two conditions. From this model, explicit predictions are made. First, many more cases of TF nonspecificity are expected when examined. Second, the genetic analysis of regulatory sequences should uncover cis-element bypass and, third, genetic analysis of TF function should generally uncover differential pleiotropy. In addition, limited specificity provides evolutionary opportunity and explains the inefficiency of expression analysis in identifying genes required for biological processes.
RESUMO
The Drosophila maxillary palpus that develops during metamorphosis is composed of two elements: the proximal maxillary socket and distal maxillary palp. The HOX protein, Proboscipedia (PB), was required for development of the proximal maxillary socket and distal maxillary palp. For growth and differentiation of the distal maxillary palp, PB was required in the cells of, or close to, the maxillary socket, as well as the cells of the distal maxillary palp. Therefore, PB is required in cells outside the distal maxillary palp for the expression, by some mechanism, of a growth factor or factors that promote the growth of the distal maxillary palp. Both wingless (wg) and hedgehog (hh) genes were expressed in cells outside the distal maxillary palp in the lancinia and maxillary socket, respectively. Both wg and hh were required for distal maxillary palp growth, and hh was required noncell autonomously for distal maxillary palp growth. However, expression of wg-GAL4 and hh-GAL4 during maxillary palp differentiation did not require PB, ruling out a direct role for PB in the regulation of transcription of these growth factors.
RESUMO
For honey bee and other social insect colonies the 'queen substance' regulates colony reproduction rendering workers functionally sterile. The evolution of worker reproductive altruism is explained by inclusive fitness theory, but little is known of the genes involved or how they regulate the phenotypic expression of altruism. We previously showed that application of honeybee queen pheromone to virgin fruit flies suppresses fecundity. Here we exploit this finding to identify genes associated with the perception of an ovary-inhibiting social pheromone. Mutational and RNAi approaches in Drosophila reveal that the olfactory co-factor Orco together with receptors Or49b, Or56a and Or98a are potentially involved in the perception of queen pheromone and the suppression of fecundity. One of these, Or98a, is known to mediate female fly mating behaviour, and its predicted ligand is structurally similar to a methyl component of the queen pheromone. Our novel approach to finding genes associated with pheromone-induced sterility implies conserved reproductive regulation between social and pre-social orders, and further helps to identify candidate orthologues from the pheromone-responsive pathway that may regulate honeybee worker sterility.
Assuntos
Drosophila/genética , Drosophila/fisiologia , Genes de Insetos , Testes Genéticos/métodos , Feromônios/metabolismo , Animais , Fertilidade , Infertilidade , Comportamento Sexual Animal , OlfatoRESUMO
The protein encoded by the Drosophila pair-rule gene fushi tarazu (ftz) is required for the formation of the even-numbered parasegments. Here we analyze the phenotypes of ectopic expression of FTZ and FTZ protein deletions from the Tubulin alpha1 (Tubalpha1) promoter. Fusion of ftz to the Tubalpha1 promoter resulted in low-level ectopic expression of FTZ relative to FTZ expressed from the endogenous ftz gene. The effects of ectopic expression of four FTZ proteins, FTZ(1-413) (full length wild-type FTZ), FTZ(delta257-316) (a complete deletion of the HD), FTZ(delta101-150) (a deletion that includes the major FTZ-F1 binding site) and FTZ(delta151-209) were determined. Ectopic expression of FTZ(1-413), FTZ(delta257-316) and FTZ(delta101-151) did not result in an anti-ftz phenotype; however, ectopic expression of FTZ(1-413), and FTZ(delta257-316) did result in a ftz(Ual/Rpl)-like phenotype. In addition, low-level ectopic expression of FTZ(1-413) and FTZ(delta257-316) rescued ftz phenotypes. This was an important observation because the even-numbered parasegment pattern of FTZ expression is considered important for normal segmentation. Therefore, the rescue of ftz phenotypes by low-level FTZ expression in all cells of the embryo suggests that the even-numbered parasegment expression pattern of FTZ is not the sole factor restricting FTZ action. Low-level ectopic expression of FTZ(delta151-209) resulted in the anti-ftz phenotype and rescued hypomorphic ftz-f1 phenotypes indicating that FTZ(delta151-209) is a hyperactive FTZ molecule. Therefore, the region encompassing amino acids 151-209 of FTZ is required in some manner for repression of FTZ activity. These results are discussed in relation to the current understanding of the mechanism of FTZ action.
Assuntos
Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Alelos , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Fatores de Transcrição Fushi Tarazu , Teste de Complementação Genética , Proteínas de Homeodomínio/química , Especificidade de Órgãos , Fenótipo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Deleção de Sequência/genética , Tubulina (Proteína)/genéticaRESUMO
In 1932, Müller first used the term "antimorphic" to describe mutant alleles that have an effect that is antagonistic to that of the wild-type allele from which they were derived. In a previous characterization of mutant alleles of the Drosophila melanogaster Hox gene, Sex combs reduced (Scr), we identified the missense, antimorphic allele Scr(14), which is a Ser10-to-Leu change in the N-terminally located, bilateran-specific octapeptide motif. Here we propose that the cause of Scr(14) antimorphy is the acquisition of a leucine zipper oligomerization motif spanning the octapeptide motif and adjacently located protostome-specific LASCY motif. Analysis of the primary and predicted secondary structures of the SCR N-terminus suggests that while the SCR(+) encodes a short, α-helical region containing one putative heptad repeat, the same region in SCR(14) encodes a longer, α-helical region containing two putative heptad repeats. In addition, in vitro cross-linking assays demonstrated strong oligomerization of SCR(14) but not SCR(+). For in vivo sex comb formation, we observed reciprocal inhibition of endogenous SCR(+) and SCR(14) activity by ectopic expression of truncated SCR(14) and SCR(+) peptides, respectively. The acquisition of an oligomerization domain in SCR(14) presents a novel mechanism of antimorphy relative to the dominant negative mechanism, which maintains oligomerization between the wild-type and mutant protein subunits.
Assuntos
Alelos , Proteínas de Drosophila/genética , Drosophila/genética , Genes Homeobox , Zíper de Leucina/genética , Fatores de Transcrição/genética , Motivos de Aminoácidos , Animais , Animais Geneticamente Modificados , Bases de Dados de Proteínas , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Fenótipo , Análise de Sequência de DNA , Fatores de Transcrição/químicaRESUMO
NIP/DuoxA, originally cloned as a protein capable of binding to the cell fate determinant Numb in Drosophila, was recently identified as a modulator of reactive oxygen species (ROS) production in mammalian systems. Despite biochemical and cellular studies that link NIP/DuoxA to the generation of ROS through the dual oxidase (Duox) enzyme, the in vivo function of NIP/DuoxA has not been characterized to date. Here we report a genetic and functional characterization of nip in Drosophila melanogaster. We show that nip is essential for Drosophila development as nip null mutants die at the 1(st) larval instar. Expression of UAS-nip, but not UAS-Duox, rescued the lethality. To understand the function of nip beyond the early larval stage, we generated GAL4 inducible UAS-RNAi transgenes. da(G32)-GAL4 driven, ubiquitous RNAi-mediated silencing of nip led to profound abnormality in pre-adult development, crinkled wing and markedly reduced lifespan at 29 degrees C. Compared to wild type flies, da-GAL4 induced nip-RNAi transgenic flies exhibited significantly reduced ability to survive under oxidative stress and displayed impaired mitochondrial aconitase function. Our work provides in vivo evidence for a critical role for nip in the development and oxidative stress response in Drosophila.
Assuntos
Drosophila melanogaster , Envelhecimento/genética , Animais , Animais Geneticamente Modificados/metabolismo , Drosophila/genética , Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/fisiologia , Desenvolvimento Embrionário/genética , Larva/genética , Larva/metabolismo , Estresse Oxidativo/genética , Proteínas/genética , Proteínas/metabolismo , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Transgenes , Asas de Animais/metabolismoRESUMO
The Drosophila HOX transcription factor, Sex combs reduced (SCR), is required for determining labial and the first thoracic segmental identity. A Protein Phosphatase 2A holoenzyme assembled with the PP2A-B' regulatory subunit is proposed to specifically interact with, and dephosphorylate, the SCR homeodomain activating SCR protein activity. To test this hypothesis further, a null mutation was created in the PP2A-B' gene, PP2A-B'(Delta), using Flip-mediated, site-specific recombination. The number of sex comb bristles, salivary gland nuclei and pseudotracheal rows are SCR-dependent and were counted as a measure of SCR activity in vivo. Adults and larvae homozygous for PP2A-B'(Delta) showed no decrease in SCR activity. In addition, no evidence of functional redundancy of PP2A-B' with other regulatory subunits, Twins (TWS) and Widerborst (WDB), for dephosphorylation and activation of SCR activity was observed. In conclusion, a PP2A holoenzyme containing the PP2A-B' regulatory subunit has no role in the dephosphorylation and activation of SCR, and analysis of functional redundancy of PP2A regulatory subunits uncovered no evidence supporting a role of PP2A activity in dephosphorylation and activation of SCR.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteína Fosfatase 2/química , Fatores de Transcrição/metabolismo , Animais , Núcleo Celular/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Homozigoto , Masculino , Modelos Genéticos , Mutação , Fenótipo , Fosforilação , Estrutura Terciária de Proteína , Glândulas Salivares/metabolismoRESUMO
Arista versus tarsus determination is well investigated in Drosophila, yet it remains unresolved whether Antennapedia (ANTP) cell autonomously or noncell autonomously determines tarsus identity and whether Sex combs reduced (SCR) is the HOX protein required for normal tarsus determination. Three observations rule out a cell autonomous role for ANTP in tarsus determination. (i) Clonal ectopic overexpression of ANTP did not repress the expression of the arista determining protein Homothorax (HTH) in early 3rd stadium antennal imaginal discs. (ii) Clonal ectopic expression of ANTP did not transform the arista to a tarsus. (iii) Ectopic overexpression of ANTP, Labial (LAB), Deformed (DFD), SCR, Ultrabithorax (UBX), Abdominal-A (ABD-A), or Abdominal-B (ABD-B), using the dppGAL4 driver, resulted in arista-to-tarsus transformations, and repressed HTH/Extradenticle (EXD) activity noncell autonomously in early 3rd stadium antennal imaginal discs. SCR may not be the HOX protein required for normal tarsus determination, because co-ectopic expression of Proboscipedia (PB) inhibited the arista-to-tarsus transformations induced by ectopic expression of DFD, SCR, ANTP, UBX, ABD-A, and ABD-B. The proposal that SCR is the HOX protein required for normal tarsus determination is dependent on SCR being the sole target of PB suppression, which is not the case. Therefore, the possibility exists that normal tarsus determination is HOX independent.
Assuntos
Drosophila melanogaster/genética , Tarso Animal/metabolismo , Animais , Proteína do Homeodomínio de Antennapedia/genética , Proteína do Homeodomínio de Antennapedia/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Varredura , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tarso Animal/crescimento & desenvolvimento , Tarso Animal/ultraestrutura , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Numb, an evolutionarily conserved cell fate-determining factor, plays a pivotal role in the development of Drosophila and vertebrate nervous systems. Despite lacking a transmembrane segment, Numb is associated with the cell membrane during the asymmetric cell division of Drosophila neural precursor cells and is selectively partitioned to one of the two progeny cells from a binary cell division. Numb contains an N-terminal phosphotyrosine-binding (PTB) domain that is essential for both the asymmetric localization and the fate specification function of Numb. We report here the isolation and characterization of a novel PTB domain-binding protein, NIP (Numb-interacting protein). NIP is a multipass transmembrane protein that contains two PTB domain-binding, NXXF motifs required for the interaction with Numb. In dividing Drosophila neuroblasts, NIP is colocalized to the cell membrane with Numb in a basal cortical crescent. Expression of NIP in Cos-7 cells recruited Numb from the cytosol to the plasma membrane. This recruitment of Numb to membrane by NIP was dependent on the presence of at least one NXXF site. In Drosophila Schneider 2 cells, NIP and Numb were colocalized at the plasma membrane. Inhibition of NIP expression by RNA interference released Numb to the cytosol. These results suggest that a direct protein-protein interaction between NIP and Numb is necessary and sufficient for the recruitment of Numb to the plasma membrane. Recruitment of Numb to a basal cortical crescent in a dividing neuroblast is essential for Numb to function as an intrinsic cell fate determinant.