RESUMO
Germline stem cells in the Drosophila ovary are maintained by a somatic niche. The niche is structurally and functionally complex and contains four cell types, the escort, cap, and terminal filament cells and the newly identified transition cell. We find that the large Maf transcription factor Traffic jam (Tj) is essential for determining niche cell fates and architecture, enabling each niche in the ovary to support a normal complement of 2-3 germline stem cells. In particular, we focused on the question of how cap cells form. Cap cells express Tj and are considered the key component of a mature germline stem cell niche. We conclude that Tj controls the specification of cap cells, as the complete loss of Tj function caused the development of additional terminal filament cells at the expense of cap cells, and terminal filament cells developed cap cell characteristics when induced to express Tj. Further, we propose that Tj controls the morphogenetic behavior of cap cells as they adopted the shape and spatial organization of terminal filament cells but otherwise appeared to retain their fate when Tj expression was only partially reduced. Our data indicate that Tj contributes to the establishment of germline stem cells by promoting the cap cell fate, and controls the stem cell-carrying capacity of the niche by regulating niche architecture. Analysis of the interactions between Tj and the Notch (N) pathway indicates that Tj and N have distinct functions in the cap cell specification program. We propose that formation of cap cells depends on the combined activities of Tj and the N pathway, with Tj promoting the cap cell fate by blocking the terminal filament cell fate, and N supporting cap cells by preventing the escort cell fate and/or controlling the number of cap cell precursors.
Assuntos
Proteínas de Drosophila/genética , Fatores de Transcrição Maf Maior/genética , Ovário/citologia , Proteínas Proto-Oncogênicas/genética , Nicho de Células-Tronco , Animais , Drosophila/citologia , Drosophila/genética , Proteínas de Drosophila/metabolismo , Feminino , Fatores de Transcrição Maf Maior/metabolismo , Oogônios/citologia , Oogônios/metabolismo , Ovário/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismoRESUMO
We have characterized the gene emperor's thumb (et) and showed that it is required for the regulation of apoptosis in Drosophila. Loss-of-function mutations in et result in apoptosis associated with a decrease in the concentration of DIAP1. Overexpression of one form of et inhibits apoptosis, consistent with et having an anti-apoptotic function; however, overexpression of a second form of et induces apoptosis, indicating that the two forms of et may have competing functions. et encodes a protein deubiquitinase, suggesting it regulates apoptosis by controlling the stability of apoptotic regulatory proteins.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Apoptose/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/metabolismo , Endopeptidases/fisiologia , Genes de Insetos , Sequência de Aminoácidos , Animais , Drosophila/genética , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Filogenia , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Drosophila photoreceptors undergo marked changes in their morphology during pupal development. These changes include a five-fold elongation of the retinal cell body and the morphogenesis of the rhabdomere, the light sensing structure of the cell. Here we show that twinstar (tsr), which encodes Drosophila cofilin/ADF (actin-depolymerizing factor), is required for both of these processes. In tsr mutants, the retina is shorter than normal, the result of a lack of retinal elongation. In addition, in a strong tsr mutant, the rhabdomere structure is disorganized and the microvilli are short and occasionally unraveled. In an intermediate tsr mutant, the rhabdomeres are not disorganized but have a wider than normal structure. The adherens junctions connecting photoreceptor cells to each other are also found to be wider than normal. We propose, and provide data supporting, that these wide rhabdomeres and adherens junctions are secondary events caused by the inhibition of retinal elongation. These results provide insight into the functions of the actin cytoskeleton during morphogenesis of the Drosophila eye.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas dos Microfilamentos/metabolismo , Morfogênese , Fatores de Despolimerização de Actina/genética , Junções Aderentes/metabolismo , Animais , Citoesqueleto/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/embriologia , Feminino , Masculino , Proteínas dos Microfilamentos/genética , Mutação , Fenótipo , Células Fotorreceptoras de Invertebrados/anatomia & histologia , Células Fotorreceptoras de Invertebrados/embriologia , Células Fotorreceptoras de Invertebrados/metabolismo , Retina/citologia , Retina/embriologiaRESUMO
Learning assistant (LA) programs have been implemented at a range of institutions, usually as part of a comprehensive curricular transformation accompanied by a pedagogical switch to active learning. While this shift in pedagogy has led to increased student learning gains, the positive effect of LAs has not yet been distinguished from that of active learning. To determine the effect that LAs would have beyond a student-centered instructional modality that integrated active learning, we introduced an LA program into a large-enrollment introductory molecular biology course that had already undergone a pedagogical transformation to a highly structured, flipped (HSF) format. We used questions from a concept test (CT) and exams to compare student performance in LA-supported HSF courses with student performance in courses without LAs. Students in the LA-supported course did perform better on exam questions common to both HSF course modalities but not on the CT. In particular, LA-supported students' scores were higher on common exam questions requiring higher-order cognitive skills, which LAs were trained to foster. Additionally, underrepresented minority (URM) students particularly benefited from LA implementation. These findings suggest that LAs may provide additional learning benefits to students beyond the use of active learning, especially for URM students.
Assuntos
Desempenho Acadêmico , Avaliação Educacional , Aprendizagem , Estudantes , Demografia , Feminino , Humanos , Masculino , Biologia Molecular/educação , Aprendizagem Baseada em Problemas , Inquéritos e QuestionáriosRESUMO
The bric à brac (bab) locus is composed of two paralogous genes, bab1 and bab2, in Drosophila melanogaster. Bab1 and Bab2 are nuclear proteins that contain a broad complex, tramtrack, bric à brac/poxviruses and zinc-finger (BTB/POZ) domain. Many BTB/POZ proteins are transcriptional regulators of which the majority contain C(2)H(2) zinc-finger motifs. There is no detectable zinc-finger motif in either Bab protein. However, they share the Bab conserved domain (BabCD) that is highly conserved between Bab1 and Bab2, and the Bab proteins of several other species, e.g. Anopheles gambiae, Apis mellifera and Drosophila virilis. Here we show that Bab2 binds to several discrete sites on polytene chromosomes including the bab locus, and that the BabCD of both Bab1 and Bab2 binds in vitro to the cis-regulatory regions of bab1 and bab2. Our results indicate that the BabCD binds to A/T-rich regions and that its optimum binding sites contain TA or TAA repeats. The BabCD is a composite DNA binding domain with a psq motif and an AT-Hook motif; both motifs are required for DNA binding activity. Structural similarities suggest that the BabCD may bind to DNA in a similar manner as some prokaryotic recombinases.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Drosophila/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Anopheles/genética , Abelhas/genética , Sítios de Ligação/genética , Cromossomos/genética , Cromossomos/metabolismo , Sequência Conservada/genética , DNA/genética , Pegada de DNA/métodos , Proteínas de Ligação a DNA/genética , Desoxirribonuclease I/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Ensaio de Desvio de Mobilidade Eletroforética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genéticaRESUMO
Cre/loxP recombination enables cellular specificity and, in the case of inducible systems, temporal control of genomic deletions. Here we used a SM22α tamoxifen-inducible Cre line to inactivate ß1 integrin in adult smooth muscle. Interestingly, analysis of two distinct ß1 loxP transgenic mice revealed vastly different outcomes after ß1 integrin deletion. Lethality occurred 4 weeks postinduction in one Cre/loxP line, while no apparent phenotype was seen in the other line. Genetic analysis revealed appropriate DNA excision in both cases; however, differences were found in the degree of protein loss with absolutely no change in protein levels in the model that lacked a phenotype. Seeking to understand protein persistence despite appropriate recombination, we first validated the flox allele using a constitutive Cre line and demonstrated its ability to mediate effective protein inactivation. We then examined the possibility of heterozygous cell selection, protein turnover, and deletion efficiency with no success for explaining the phenotype. Finally, we documented the presence of the Cre-recombination episomal product, which persisted in tissue samples with no protein loss. The product was only noted in cells with low proliferative capacity. These findings highlight the potential for protein expression from the products of Cre-recombinase excised genes, particularly when deletion occurs in low turnover populations.
Assuntos
Integrases/metabolismo , Integrina beta1/metabolismo , Recombinação Genética , Alelos , Animais , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Heterozigoto , Homozigoto , Cinética , Camundongos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Plasmídeos/genéticaRESUMO
Planar cell polarity (PCP) is a level of tissue organization in which cells adopt a uniform orientation within the plane of an epithelium. The process of tissue polarization is likely to be initiated by an extracellular gradient. Thus, determining how cells decode and convert this graded information into subcellular asymmetries is key to determining how cells direct the reorganization of the cytoskeleton to produce uniformly oriented structures. Twinstar (Tsr), the Drosophila homolog of Cofilin/ADF (actin depolymerization factor), is a component of the cytoskeleton that regulates actin dynamics. We show here that various alleles of tsr produce PCP defects in the wing, eye and several other epithelia. In wings mutant for tsr, Frizzled (Fz) and Flamingo (Fmi) proteins do not properly localize to the proximodistal boundaries of cells. The correct asymmetric localization of these proteins instructs the actin cytoskeleton to produce one actin-rich wing hair at the distal-most vertex of each cell. These results argue that actin remodeling is not only required in the manufacture of wing hairs, but also in the PCP read-out that directs where a wing hair will be secreted.
Assuntos
Fatores de Despolimerização de Actina/genética , Polaridade Celular , Proteínas de Drosophila/fisiologia , Drosophila/citologia , Drosophila/crescimento & desenvolvimento , Proteínas dos Microfilamentos/fisiologia , Actinas/metabolismo , Alelos , Sequência de Aminoácidos , Animais , Caderinas/metabolismo , Sequência Conservada , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Receptores Frizzled/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Receptores Acoplados a Proteínas G/metabolismo , Distribuição Tecidual , TransgenesRESUMO
The bric à brac (bab) locus acts as a homeotic and morphogenetic regulator in the development of ovaries, appendages and the abdomen. It consists of two structurally and functionally related genes, bab1 and bab2, each of which encodes a single nuclear protein. Bab1 and Bab2 have two conserved domains in common, a BTB/POZ domain and a Psq domain, a motif that characterizes a subfamily of BTB/POZ domain proteins in Drosophila. The tissue distribution of Bab1 and Bab2 overlaps, with Bab1 being expressed in a subpattern of Bab2. Analysis of a series of mutations indicates that the two bab genes have synergistic, distinct and redundant functions during imaginal development. Interestingly, several reproduction-related traits that are sexually dimorphic or show diversity among Drosophila species are highly sensitive to changes in the bab gene dose, suggesting that alterations in bab activity may contribute to evolutionary modification of sex-related morphology.