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Cell Rep ; 27(10): 3019-3033.e5, 2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31167145


Homeostatic renewal and stress-related tissue regeneration rely on stem cell activity, which drives the replacement of damaged cells to maintain tissue integrity and function. The Jun N-terminal kinase (JNK) signaling pathway has been established as a critical regulator of tissue homeostasis both in intestinal stem cells (ISCs) and mature enterocytes (ECs), while its chronic activation has been linked to tissue degeneration and aging. Here, we show that JNK signaling requires the stress-inducible transcription factor Ets21c to promote tissue renewal in Drosophila. We demonstrate that Ets21c controls ISC proliferation as well as EC apoptosis through distinct sets of target genes that orchestrate cellular behaviors via intrinsic and non-autonomous signaling mechanisms. While its loss appears dispensable for development and prevents epithelial aging, ISCs and ECs demand Ets21c function to mount cellular responses to oxidative stress. Ets21c thus emerges as a vital regulator of proliferative homeostasis in the midgut and a determinant of the adult healthspan.

Cell Rep ; 24(11): 3072-3086, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30208329


The niche critically controls stem cell behavior, but its regulatory input at the whole-genome level is poorly understood. We elucidated transcriptional programs of the somatic and germline lineages in the Drosophila testis and genome-wide binding profiles of Zfh-1 and Abd-A expressed in somatic support cells and crucial for fate acquisition of both cell lineages. We identified key roles of nucleoporins and V-ATPase proton pumps and demonstrate their importance in controlling germline development from the support side. To make our dataset publicly available, we generated an interactive analysis tool, which uncovered conserved core genes of adult stem cells across species boundaries. We tested the functional relevance of these genes in the Drosophila testis and intestine and found a high frequency of stem cell defects. In summary, our dataset and interactive platform represent versatile tools for identifying gene networks active in diverse stem cell types.

Proteínas de Drosophila/metabolismo , Células-Tronco/metabolismo , Testículo/metabolismo , Animais , Drosophila , Proteínas de Drosophila/genética , Masculino , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
J Vis Exp ; (116)2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27768082


Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

Drosophila melanogaster , Regulação da Expressão Gênica no Desenvolvimento , Discos Imaginais , Animais , Modelos Animais de Doenças , Proteínas de Drosophila , Larva , Microscopia Confocal , Mosaicismo , Neoplasias
Dis Model Mech ; 8(10): 1279-93, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26398940


Cancer initiation and maintenance of the transformed cell state depend on altered cellular signaling and aberrant activities of transcription factors (TFs) that drive pathological gene expression in response to cooperating genetic lesions. Deciphering the roles of interacting TFs is therefore central to understanding carcinogenesis and for designing cancer therapies. Here, we use an unbiased genomic approach to define a TF network that triggers an abnormal gene expression program promoting malignancy of clonal tumors, generated in Drosophila imaginal disc epithelium by gain of oncogenic Ras (Ras(V12)) and loss of the tumor suppressor Scribble (scrib(1)). We show that malignant transformation of the ras(V12)scrib(1) tumors requires TFs of distinct families, namely the bZIP protein Fos, the ETS-domain factor Ets21c and the nuclear receptor Ftz-F1, all acting downstream of Jun-N-terminal kinase (JNK). Depleting any of the three TFs improves viability of tumor-bearing larvae, and this positive effect can be enhanced further by their combined removal. Although both Fos and Ftz-F1 synergistically contribute to ras(V12)scrib(1) tumor invasiveness, only Fos is required for JNK-induced differentiation defects and Matrix metalloprotease (MMP1) upregulation. In contrast, the Fos-dimerizing partner Jun is dispensable for JNK to exert its effects in ras(V12)scrib(1) tumors. Interestingly, Ets21c and Ftz-F1 are transcriptionally induced in these tumors in a JNK- and Fos-dependent manner, thereby demonstrating a hierarchy within the tripartite TF network, with Fos acting as the most upstream JNK effector. Of the three TFs, only Ets21c can efficiently substitute for loss of polarity and cooperate with Ras(V12) in inducing malignant clones that, like ras(V12)scrib(1) tumors, invade other tissues and overexpress MMP1 and the Drosophila insulin-like peptide 8 (Dilp8). While ras(V12)ets21c tumors require JNK for invasiveness, the JNK activity is dispensable for their growth. In conclusion, our study delineates both unique and overlapping functions of distinct TFs that cooperatively promote aberrant expression of target genes, leading to malignant tumor phenotypes.

Carcinogênese/patologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Animais , Carcinogênese/genética , Proliferação de Células , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Modelos Biológicos , Invasividade Neoplásica , Neoplasias/genética , Neoplasias/patologia , Oncogenes , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Supressão Genética , Transcriptoma/genética , Proteínas ras/metabolismo
EMBO J ; 31(15): 3323-33, 2012 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-22781127


Precise gene expression is a fundamental aspect of organismal function and depends on the combinatorial interplay of transcription factors (TFs) with cis-regulatory DNA elements. While much is known about TF function in general, our understanding of their cell type-specific activities is still poor. To address how widely expressed transcriptional regulators modulate downstream gene activity with high cellular specificity, we have identified binding regions for the Hox TF Deformed (Dfd) in the Drosophila genome. Our analysis of architectural features within Hox cis-regulatory response elements (HREs) shows that HRE structure is essential for cell type-specific gene expression. We also find that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact with non-overlapping regions in vivo, despite their similar DNA binding preferences. While Dfd and Ubx HREs exhibit comparable design principles, their motif compositions and motif-pair associations are distinct, explaining the highly selective interaction of these Hox proteins with the regulatory environment. Thus, our results uncover the regulatory code imprinted in Hox enhancers and elucidate the mechanisms underlying functional specificity of TFs in vivo.

Drosophila/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Elementos de Resposta/genética , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação/genética , Drosophila/embriologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Genes Homeobox , Genes de Insetos , Código das Histonas/genética , Código das Histonas/fisiologia , Proteínas de Homeodomínio/metabolismo , Modelos Biológicos , Ligação Proteica , Fatores de Transcrição/fisiologia , Ativação Transcricional