RESUMO
Morphogen-mediated signaling is critical for proper organ development and stem cell function, and well-characterized mechanisms spatiotemporally limit the expression of ligands, receptors, and ligand-binding cell-surface glypicans. Here, we show that in the developing Drosophila ovary, canonical Wnt signaling promotes the formation of somatic escort cells (ECs) and their protrusions, which establish a physical permeability barrier to define morphogen territories for proper germ cell differentiation. The protrusions shield germ cells from Dpp and Wingless morphogens produced by the germline stem cell (GSC) niche and normally only received by GSCs. Genetic disruption of EC protrusions allows GSC progeny to also receive Dpp and Wingless, which subsequently disrupt germ cell differentiation. Our results reveal a role for canonical Wnt signaling in specifying the ovarian somatic cells necessary for germ cell differentiation. Additionally, we demonstrate the morphogen-limiting function of this physical permeability barrier, which may be a common mechanism in other organs across species.
RESUMO
Niches maintain a finite pool of stem cells via restricted space and short-range signals. Stem cells compete for limited niche resources, but the mechanisms regulating competition are poorly understood. Using the Drosophila testis model, we show that germline stem cells (GSCs) lacking the transcription factor Chinmo gain a competitive advantage for niche access. Surprisingly, chinmo-/- GSCs rely on a new mechanism of competition in which they secrete the extracellular matrix protein Perlecan to selectively evict non-mutant GSCs and then upregulate Perlecan-binding proteins to remain in the altered niche. Over time, the GSC pool can be entirely replaced with chinmo-/- cells. As a consequence, the mutant chinmo allele acts as a gene drive element; the majority of offspring inherit the allele despite the heterozygous genotype of the parent. Our results suggest that the influence of GSC competition may extend beyond individual stem cell niche dynamics to population-level allelic drift and evolution.
Assuntos
Células-Tronco Germinativas Adultas/fisiologia , Proteínas de Drosophila/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Germinativas Adultas/metabolismo , Animais , Diferenciação Celular/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Matriz Extracelular/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Germinativas/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Masculino , Proteínas do Tecido Nervoso/genética , Transdução de Sinais/fisiologia , Nicho de Células-Tronco/genética , Nicho de Células-Tronco/fisiologia , Testículo/metabolismo , Fatores de Transcrição/metabolismoRESUMO
In developing organisms, proper tuning of the number of stem cells within a niche is critical for the maintenance of adult tissues; however, the involved mechanisms remain largely unclear. Here, we demonstrate that Thickveins (Tkv), a type I bone morphogenetic protein (BMP) receptor, acts in the Drosophila developing ovarian soma through a Smad-independent pathway to shape the distribution of BMP signal within the niche, impacting germline stem cell (GSC) recruitment and maintenance. Somatic Tkv promotes Egfr signaling to silence transcription of Dally, which localizes BMP signals on the cell surface. In parallel, Tkv promotes Hh signaling, which promotes escort cell cellular protrusions and upregulates expression of the Drosophila BMP homolog, Dpp, forming a positive feedback loop that enhances Tkv signaling and strengthens the niche boundary. Our results reveal a role for non-canonical BMP signaling in the soma during GSC establishment and generally illustrate how complex, cell-specific BMP signaling mediates niche-stem cell interactions.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Células Germinativas/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Animais , Diferenciação Celular , Drosophila/citologia , Drosophila/crescimento & desenvolvimento , Feminino , Células Germinativas/metabolismo , Masculino , Ovário/citologia , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Nicho de Células-TroncoRESUMO
Snail, a zinc-finger transcription factor, controls the process of epithelial-mesenchymal transition, and ectopic expression of this protein may produce cells with stem cell properties. Because the effect of Snail expression in ovarian epithelial cells remains unclear, we generated Drosophila ovarian follicle stem cells (FSCs) with homozygous Scutoid (Sco) mutation. The Sco mutation is a reciprocal transposition that is known to induce ectopic Snail activity. We found that Sco mutant FSCs showed excess proliferation and high competitiveness for niche occupancy, and the descendants of this lineage formed outgrowths that failed to enter the endocycle. Surprisingly, such phenotypes were not rescued by suppressing Snail expression, but were completely restored by supplying Lethal giant larvae (Lgl). The lgl allele is a cell polarity gene that is often mutated in the genome. Importantly, Sco mutants survived in a complementation test with lgl. This result was probably obtained because the Sco-associated lgl allele appears to diminish, but not ablate lgl expression. While our data do not rule out the possibility that the Sco mutation disrupts a regulator of lgl transcription, our results strongly suggest that the phenotypes we found in Sco mutants are more closely associated with the lgl allele than ectopic Snail activity.
Assuntos
Proteínas de Drosophila/genética , Drosophila/crescimento & desenvolvimento , Regulação da Expressão Gênica , Folículo Ovariano/citologia , Proteínas Supressoras de Tumor/genética , Alelos , Animais , Linhagem da Célula , Proliferação de Células/genética , Senescência Celular , Drosophila/genética , Feminino , Homeostase , Células-Tronco/citologiaRESUMO
Epithelial stem cells undergo constant self-renewal and differentiation to maintain the homeostasis of epithelial tissues that undergo rapid turnover. Recent studies have shown that the epithelial-mesenchymal transition (EMT), which is primarily mediated by Snail via the suppression of E-cadherin, is able to generate cells with stem cell properties. However, the role of Snail in epithelial stem cells remains unclear. Here, we report that Snail directly controls proliferation of follicle stem cells (FSCs) in Drosophila females. Disruption of Snail expression in FSCs compromises their proliferation, but not their maintenance. Conversely, FSCs with excessive Snail expression display increased proliferation and lifespan, which is accompanied by a moderate decrease in the expression of E-cadherin (required for adhesion of FSCs to their niche) at the junction between their adjacent cells, indicating a conserved role of Snail in E-cadherin inhibition, which promote epithelial cell proliferation. Interestingly, a decrease in E-cadherin in snail-knock down FSCs does not restore the decreased proliferation of snail-knock down FSCs, suggesting that adhesion strength of FSCs to their niche is dispensable for Snail-mediated FSC division. Our results demonstrate that Snail controls epithelial stem cell division independently of its known role in the EMT, which contributes to induction of cancer stem cells.
Assuntos
Caderinas/biossíntese , Proteínas de Drosophila/biossíntese , Folículo Ovariano/citologia , Ovário/citologia , Fatores de Transcrição da Família Snail/fisiologia , Células-Tronco/citologia , Animais , Caderinas/genética , Adesão Celular , Divisão Celular , Senescência Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Células Epiteliais/citologia , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Proteínas Luminescentes/análise , Mosaicismo , Fatores de Transcrição da Família Snail/deficiência , Nicho de Células-TroncoRESUMO
Aging influences stem cells, but the processes involved remain unclear. Insulin signaling, which controls cellular nutrient sensing and organismal aging, regulates the G2 phase of Drosophila female germ line stem cell (GSC) division cycle in response to diet; furthermore, this signaling pathway is attenuated with age. The role of insulin signaling in GSCs as organisms age, however, is also unclear. Here, we report that aging results in the accumulation of tumorous GSCs, accompanied by a decline in GSC number and proliferation rate. Intriguingly, GSC loss with age is hastened by either accelerating (through eliminating expression of Myt1, a cell cycle inhibitory regulator) or delaying (through mutation of insulin receptor (dinR) GSC division, implying that disrupted cell cycle progression and insulin signaling contribute to age-dependent GSC loss. As flies age, DNA damage accumulates in GSCs, and the S phase of the GSC cell cycle is prolonged. In addition, GSC tumors (which escape the normal stem cell regulatory microenvironment, known as the niche) still respond to aging in a similar manner to normal GSCs, suggesting that niche signals are not required for GSCs to sense or respond to aging. Finally, we show that GSCs from mated and unmated females behave similarly, indicating that female GSC-male communication does not affect GSCs with age. Our results indicate the differential effects of aging and diet mediated by insulin signaling on the stem cell division cycle, highlight the complexity of the regulation of stem cell aging, and describe a link between ovarian cancer and aging.
Assuntos
Envelhecimento/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Células Germinativas/patologia , Insulina/metabolismo , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais , Animais , Biomarcadores/metabolismo , Contagem de Células , Proliferação de Células , DNA/metabolismo , Dano ao DNA , Feminino , Citometria de Fluxo , Fase G1 , Masculino , Ovário/patologia , Fase S , Nicho de Células-TroncoRESUMO
Stem cells have an innate ability to occupy their stem cell niche, which in turn, is optimized to house stem cells. Organ aging is associated with reduced stem cell occupancy in the niche, but the mechanisms involved are poorly understood. Here, we report that Notch signaling is increased with age in Drosophila female germline stem cells (GSCs), and this results in their removal from the niche. Clonal analysis revealed that GSCs with low levels of Notch signaling exhibit increased adhesiveness to the niche, thereby out-competing their neighbors with higher levels of Notch; adhesiveness is altered through regulation of E-cadherin expression. Experimental enhancement of Notch signaling in GSCs hastens their age-dependent loss from the niche, and such loss is at least partially mediated by Sex lethal. However, disruption of Notch signaling in GSCs does not delay GSC loss during aging, and nor does it affect BMP signaling, which promotes self-renewal of GSCs. Finally, we show that in contrast to GSCs, Notch activation in the niche (which maintains niche integrity, and thus mediates GSC retention) is reduced with age, indicating that Notch signaling regulates GSC niche occupancy both intrinsically and extrinsically. Our findings expose a novel role of Notch signaling in controlling GSC-niche adhesion in response to aging, and are also of relevance to metastatic cancer cells, in which Notch signaling suppresses cell adhesion.
Assuntos
Adesão Celular , Proteínas de Drosophila/fisiologia , Receptores Notch/fisiologia , Nicho de Células-Tronco , Células-Tronco/fisiologia , Envelhecimento , Animais , Proteínas Morfogenéticas Ósseas/fisiologia , Proteínas Cdh1/metabolismo , Proliferação de Células , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Feminino , Proteínas de Ligação a RNA/fisiologia , Transdução de SinaisRESUMO
The stem cell niche houses and regulates stem cells by providing both physical contact and local factors that regulate stem cell identity. The stem cell niche also plays a role in integrating niche-local and systemic signals, thereby ensuring that the balance of stem cells meets the needs of the organism. However, it is not clear how these signals are merged within the niche. Nutrient-sensing insulin/FOXO signaling has been previously shown to directly control Notch activation in the Drosophila female germline stem cell (GSC) niche, which maintains the niche and GSC identity. Here, we demonstrate that FOXO directly activates transcription of fringe, a gene encoding a glycosyltransferase that modulates Notch glycosylation. Fringe facilitates Notch inactivation in the GSC niche when insulin signaling is low. We also show that the Notch ligand predominantly involved is GSC niche-derived Delta. These results reveal that FOXO-mediated regulation of fringe links the insulin and Notch signaling pathways in the GSC niche in response to nutrition, and emphasize that stem cells are regulated by complex interactions between niche-local and systemic signals.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Células Germinativas/citologia , Células Germinativas/metabolismo , Insulina/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Nicho de Células-Tronco , Animais , Sequência de Bases , Contagem de Células , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Feminino , Glicosilação , Modelos Biológicos , Dados de Sequência Molecular , Mutação/genética , N-Acetilglucosaminiltransferases/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica , Receptor de Insulina/metabolismo , Transdução de Sinais , Transcrição Gênica , Peixe-ZebraRESUMO
The cyclin-dependent protein kinase family regulates a wide range of cellular functions such as cell cycle progression, differentiation, and apoptosis. In this study, we identified a zebrafish cyclin-dependent protein kinase-like 1 protein called zebrafish cdkl1 (zcdkl1), which shared a high degree of homology and conserved synteny with mammalian orthologs. zcdkl1 exhibited abilities for phosphorylation of myelin basic protein and histone H1. RT-PCR analysis revealed that zcdkl1 was expressed starting from fertilization and continuing thereafter. In adult tissues, zcdkl1 was predominantly detected in brain, ovary, and testis, and was expressed at low levels in other tissues. At 50% epiboly stage, zcdkl1 was widely expressed. At 12 to 48 h post-fertilization, zcdkl1 was predominantly expressed in the hypochord, the medial and lateral floor plate, and the pronephric duct. Interference of zcdkl1 expression resulted in abnormalities, such as brain and eye malformation, pericardial edema, and body axis curvature. Disruption of zcdkl1 reduced neurogenin-1 in the brain and sonic hedgehog expression in the floor plate region. These deformities were apparently rescued by co-injection of zcdkl1 mRNA. Findings of this study indicate that zcdkl1 plays an essential role in zebrafish development.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/classificação , Desenvolvimento Embrionário , Células HEK293 , Proteínas Hedgehog/metabolismo , Histonas/metabolismo , Humanos , Dados de Sequência Molecular , Morfogênese , Proteína Básica da Mielina/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Proteínas de Peixe-Zebra/genéticaRESUMO
Calcium/calmodulin-dependent protein kinase II (CaM-KII) plays a critical role in neuronal functions. In this report, we demonstrate the expression patterns, functional analysis, and development role of the two zebrafish CaM-KII inhibitors, cam-kiin1 and cam-kiin2. Both of these genes were detected in the 5-somite stage and are persistently expressed thereafter. The RNA transcripts of cam-kiin1 were prominently expressed in the forebrain and hindbrain regions, especially in the telencephalon, while cam-kiin2 was detected in the anterior brain region and neurons of the hindbrain. Through GST-pull down, co-immunoprecipitation, and kinase assay, cam-kii inhibitors can bind to and reduce cam-kiiα activity. However, no overt alternation of brain marker such as ngn1, otx2, and pax2.1 was observed in morphants received each one or combined MO. Our results suggest that the two cam-kii inhibitors exhibited distinct expression pattern and may play a minor role in zebrafish brain development.