RESUMO
Drosophila matrix metalloproteinase 2 (MMP2) is specifically expressed in posterior follicle cells of stage-14 egg chambers (mature follicles) and is crucial for the breakdown of the follicular wall during ovulation, a process that is highly conserved from flies to mammals. The factors that regulate spatiotemporal expression of MMP2 in follicle cells remain unknown. Here, we demonstrate crucial roles for the ETS-family transcriptional activator Pointed (Pnt) and its endogenous repressor Yan in the regulation of MMP2 expression. We found that Pnt is expressed in posterior follicle cells and overlaps with MMP2 expression in mature follicles. Genetic analysis demonstrated that pnt is both required and sufficient for MMP2 expression in follicle cells. In addition, Yan was temporally upregulated in stage-13 follicle cells to fine-tune Pnt activity and MMP2 expression. Furthermore, we identified a 1.1â kb core enhancer that is responsible for the spatiotemporal expression of MMP2 and contains multiple pnt/yan binding motifs. Mutation of pnt/yan binding sites significantly impaired the Mmp2 enhancer activity. Our data reveal a mechanism of transcriptional regulation of Mmp2 expression in Drosophila ovulation, which could be conserved in other biological systems.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Feminino , Drosophila/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Transdução de Sinais/fisiologia , Ovulação/genética , Mamíferos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/genéticaRESUMO
Aminergic signaling is known to play a critical role in regulating female reproductive processes in both mammals and insects. In Drosophila, the ortholog of noradrenaline, octopamine, is required for ovulation as well as several other female reproductive processes. Two octopamine receptors have already been shown to be expressed in the Drosophila reproductive tract and to be required for egg-laying: OAMB and Octß2R. The Drosophila genome contains 4 additional octopamine receptors-Octα2R, Octß1R, Octß3R, and Oct-TyrR-but their cellular patterns of expression in the reproductive tract and potential contribution(s) to egg-laying are not known. In addition, the mechanisms by which OAMB and Octß2R regulate reproduction are incompletely understood. Using a panel of MiMIC Gal4 lines, we show that Octα2R, Octß1R, Octß3R, and Oct-TyrR receptors are not detectable in either epithelium or muscle but are clearly expressed in neurons within the female fly reproductive tract. Optogenetic activation of neurons that express at least 3 types of octopamine receptors stimulates contractions in the lateral oviduct. We also find that octopamine stimulates calcium transients in the sperm storage organs and that its effects in spermathecal, secretory cells, can be blocked by knock-down of OAMB. These data extend our understanding of the pathways by which octopamine regulates egg-laying in Drosophila and raise the possibility that multiple octopamine receptor subtypes could play a role in this process.
Assuntos
Proteínas de Drosophila , Drosophila , Receptores de Amina Biogênica , Animais , Feminino , Masculino , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Octopamina/metabolismo , Sêmen/metabolismo , Proteínas de Drosophila/metabolismo , MamíferosRESUMO
Monoamine neurotransmitters such as noradrenalin are released from both synaptic vesicles (SVs) and large dense-core vesicles (LDCVs), the latter mediating extrasynaptic signaling. The contribution of synaptic versus extrasynaptic signaling to circuit function and behavior remains poorly understood. To address this question, we have previously used transgenes encoding a mutation in the Drosophila Vesicular Monoamine Transporter (dVMAT) that shifts amine release from SVs to LDCVs. To circumvent the use of transgenes with non-endogenous patterns of expression, we have now used CRISPR-Cas9 to generate a trafficking mutant in the endogenous dVMAT gene. To minimize disruption of the dVMAT coding sequence and a nearby RNA splice site, we precisely introduced a point mutation using single-stranded oligonucleotide repair. A predicted decrease in fertility was used as a phenotypic screen to identify founders in lieu of a visible marker. Phenotypic analysis revealed a defect in the ovulation of mature follicles and egg retention in the ovaries. We did not detect defects in the contraction of lateral oviducts following optogenetic stimulation of octopaminergic neurons. Our findings suggest that release of mature eggs from the ovary is disrupted by changing the balance of VMAT trafficking between SVs and LDCVs. Further experiments using this model will help determine the mechanisms that sensitize specific circuits to changes in synaptic versus extrasynaptic signaling.
RESUMO
Adrenergic signaling is known to play a critical role in regulating female reproductive processes in both mammals and insects. In Drosophila , the ortholog of noradrenaline, octopamine (Oa), is required for ovulation as well as several other female reproductive processes. Loss of function studies using mutant alleles of receptors, transporters, and biosynthetic enzymes for Oa have led to a model in which disruption of octopaminergic pathways reduces egg laying. However, neither the complete expression pattern in the reproductive tract nor the role of most octopamine receptors in oviposition is known. We show that all six known Oa receptors are expressed in peripheral neurons at multiple sites within in the female fly reproductive tract as well as in non-neuronal cells within the sperm storage organs. The complex pattern of Oa receptor expression in the reproductive tract suggests the potential for influencing multiple regulatory pathways, including those known to inhibit egg-laying in unmated flies. Indeed, activation of some neurons that express Oa receptors inhibits oviposition, and neurons that express different subtypes of Oa receptor can affect different stages of egg laying. Stimulation of some Oa receptor expressing neurons (OaRNs) also induces contractions in lateral oviduct muscle and activation of non-neuronal cells in the sperm storage organs by Oa generates OAMB-dependent intracellular calcium release. Our results are consistent with a model in which adrenergic pathways play a variety of complex roles in the fly reproductive tract that includes both the stimulation and inhibition of oviposition.
RESUMO
Across species, ovulation is a process induced by a myriad of signaling cascades that ultimately leads to the release of encapsulated oocytes from follicles. Follicles first need to mature and gain ovulatory competency before ovulation; however, the signaling pathways regulating follicle maturation are incompletely understood in Drosophila and other species. Our previous work has shown that the bHLH-PAS transcription factor Single-minded (Sim) plays important roles in follicle maturation downstream of the nuclear receptor Ftz-f1 in Drosophila. Here, we demonstrate that Tango (Tgo), another bHLH-PAS protein, acts as a co-factor of Sim to promote follicle cell differentiation from stages 10 to 12. In addition, we discover that re-upregulation of Sim in stage-14 follicle cells is also essential to promote ovulatory competency by upregulating octopamine receptor in mushroom body (OAMB), matrix metalloproteinase 2 (Mmp2) and NADPH oxidase (NOX), either independently of or in conjunction with the zinc-finger protein Hindsight (Hnt). All these factors are crucial for successful ovulation. Together, our work indicates that the transcriptional complex Sim:Tgo plays multiple roles in late-stage follicle cells to promote follicle maturation and ovulation.
Assuntos
Proteínas de Drosophila , Metaloproteinase 2 da Matriz , Animais , Feminino , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Oogênese/genética , Ovulação/genéticaRESUMO
The Serotonin Transporter (SERT) regulates extracellular serotonin levels and is the target of most current drugs used to treat depression. The mechanisms by which inhibition of SERT activity influences behavior are poorly understood. To address this question in the model organism Drosophila melanogaster, we developed new loss of function mutations in Drosophila SERT (dSERT). Previous studies in both flies and mammals have implicated serotonin as an important neuromodulator of sleep, and our newly generated dSERT mutants show an increase in total sleep and altered sleep architecture that is mimicked by feeding the SSRI citalopram. Differences in daytime versus nighttime sleep architecture as well as genetic rescue experiments unexpectedly suggest that distinct serotonergic circuits may modulate daytime versus nighttime sleep. dSERT mutants also show defects in copulation and food intake, akin to the clinical side effects of SSRIs and consistent with the pleomorphic influence of serotonin on the behavior of D. melanogaster. Starvation did not overcome the sleep drive in the mutants and in male dSERT mutants, the drive to mate also failed to overcome sleep drive. dSERT may be used to further explore the mechanisms by which serotonin regulates sleep and its interplay with other complex behaviors.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Masculino , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Serotonina , Corte , Drosophila/metabolismo , Sono/genética , Mutação , Comportamento Alimentar , Mamíferos/metabolismoRESUMO
The NR5A-family nuclear receptors are highly conserved and function within the somatic follicle cells of the ovary to regulate folliculogenesis and ovulation in mammals; however, their roles in Drosophila ovaries are largely unknown. Here, we discover that Ftz-f1, one of the NR5A nuclear receptors in Drosophila, is transiently induced in follicle cells in late stages of oogenesis via ecdysteroid signaling. Genetic disruption of Ftz-f1 expression prevents follicle cell differentiation into the final maturation stage, which leads to anovulation. In addition, we demonstrate that the bHLH/PAS transcription factor Single-minded (Sim) acts as a direct target of Ftz-f1 to promote follicle cell differentiation/maturation and that Ftz-f1's role in regulating Sim expression and follicle cell differentiation can be replaced by its mouse homolog steroidogenic factor 1 (mSF-1). Our work provides new insight into the regulation of follicle maturation in Drosophila and the conserved role of NR5A nuclear receptors in regulating folliculogenesis and ovulation.
When animals reproduce, females release eggs from their ovaries which then get fertilized by sperm from males. Each egg needs to properly mature within a collection of cells known as follicle cells before it can be let go. As the egg matures, so do the follicle cells surrounding it, until both are primed and ready to discharge the egg from the ovary. Mammals rely on a protein called SF-1 to mature their follicle cells, but it is unclear how this process works. Most animals from humans to fruit flies release their eggs in a very similar way, using many of the same proteins and genes. For example, the gene for SF-1 in mammals is similar to a gene in fruit flies which codes for another protein called Ftz-f1. Since it is more straightforward to study ovaries in fruit flies than in humans and other mammals, investigating this protein could shed light on how follicle cells mature. However, it remained unclear whether Ftz-f1 plays a similar role to its mammalian counterpart. Here, Knapp et al. show that Ftz-f1 is present in the follicle cells of fruit flies and is required for them to properly mature. Ftz-f1 controlled this process by regulating the activity of another protein called Sim. Further experiments found that the gene that codes for the SF-1 protein in mice was able to compensate for the loss of Ftz-f1 and drive follicle cells to mature. Studying how ovaries release eggs is an essential part of understanding female fertility. This work highlights the similarities between these processes in mammals and fruit flies and may help us understand how ovaries work in humans and other mammals. In the future, the findings of Knapp et al. may lead to new therapies for infertility in females and other disorders that affect ovaries.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Proteínas Nucleares/genética , Folículo Ovariano/fisiologia , Ovulação , Fatores de Transcrição/genética , Animais , Drosophila melanogaster/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Transcrição GênicaRESUMO
Proper development and maturation of a follicle is essential for successful ovulation and reproduction; however, the molecular mechanisms for follicle maturation, particularly for somatic follicle cell differentiation, are poorly understood. During Drosophila oogenesis, the somatic follicle cells encasing oocytes undergo two distinct well-established transitions: the mitotic to endocycle switch at stage 6/7 and the endocycle to gene amplification switch at stage10A/10B. Here, we identify a novel third follicle cell transition that occurs in the final stages of oogenesis (stage 13/14). This late follicle cell transition is characterized by upregulation of the transcription factor Hindsight (Hnt), and downregulation of the homeodomain transcription factor Cut and the zinc-finger transcription factor Tramtrack-69 (Ttk69). We demonstrate that inducing expression of Cut in stage 14 follicle cells is sufficient to inhibit follicle rupture and ovulation through its negative regulation of Hnt and promotion of Ttk69 expression. Our work illustrates the importance of the stage13/14 transition for follicle maturation and demonstrates the complex regulation required for somatic follicle cells to differentiate into a state primed for follicle rupture and ovulation.
Assuntos
Regulação para Baixo/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Proteínas de Homeodomínio/genética , Proteínas Nucleares/genética , Folículo Ovariano/crescimento & desenvolvimento , Ovulação , Fatores de Transcrição/genética , Animais , Proteínas de Drosophila/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Oogênese/genética , Folículo Ovariano/citologia , Ovulação/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Regulação para Cima/genéticaRESUMO
BACKGROUND: Contractile films that smooth the surface of skin upon drying are popular among consumers due to their "instant" effect and perceivable smoothing benefits. The objective of our study was to correlate an in vitro measurement of contractile force with in vivo smoothing performance, thereby enabling rapid screening of film-forming technologies for impactful cosmetic results. METHODS: We introduce and characterize an in vitro method to measure drying stress of film-containing formulations. This method is used to measure the drying stresses of seven different cosmetic film formulations. We then evaluate these formulas in a blinded clinical study, measuring their effect on under-eye and Crow's Feet area smoothing through bioinstrumentation (3D PRIMOS imaging) and blinded expert grading of images. RESULTS: The in vitro drying stress measurement was found to be repeatable and sensitive enough to detect differences between formulations with typical amounts of film-forming agents. Significant correlation was found between the in vitro drying stress measurements and under-eye smoothing measured by 3D imaging (R2 = 0.71). Expert grading confirmed that film formulas deliver perceivable smoothing in the under-eye and Crow's Feet regions 15 minutes after application. CONCLUSION: The in vitro method described here can be used to predict the efficacy of formulations that deliver smoothing benefits to consumers. For consumer use, the esthetic properties of a formula should be balanced with film performance, guided by this model which predicts skin smoothing efficacy.
Assuntos
Cosméticos/farmacologia , Fármacos Dermatológicos/farmacologia , Envelhecimento da Pele/efeitos dos fármacos , Administração Cutânea , Cosméticos/administração & dosagem , Face , Humanos , Técnicas In Vitro , Soroalbumina Bovina/farmacologia , Higiene da Pele , Estresse Fisiológico/fisiologiaRESUMO
Ovulation, the process of releasing a mature oocyte from the ovary, is crucial for animal reproduction. In order for the process of ovulation to occur, a follicle must be fully matured and signaled to rupture from the ovary. During follicle rupture in both mammals and Drosophila, somatic follicle cells are enzymatically degraded to allow the oocyte to be liberated from the follicle. Here, we describe a detailed protocol of our newly developed ex vivo follicle rupture assay in Drosophila, which represents a first assay allowing direct quantification of follicles' capacity to respond to ovulation stimuli and rupture. This assay can be modified to stimulate rupture with other reagents (for example, ionomycin) or to query enzymatic activity (in situ zymography). In addition, this assay allows genetic or pharmacological screens to identify genes or small molecules regulating follicle rupture in Drosophila.
RESUMO
Although ecdysteroid signaling regulates multiple steps in oogenesis, it is not known whether it regulates Drosophila ovulation, a process involving a matrix metalloproteinase-dependent follicle rupture. In this study, we demonstrated that ecdysteroid signaling is operating in mature follicle cells to control ovulation. Moreover, knocking down shade (shd), encoding the monooxygenase that converts ecdysone (E) to the more active 20-hydroxyecdysone (20E), specifically in mature follicle cells, blocked follicle rupture, which was rescued by ectopic expression of shd or exogenous 20E. In addition, disruption of the Ecdysone receptor (EcR) in mature follicle cells mimicked shd-knockdown defects, which were reversed by ectopic expression of EcR.B2 but not by EcR.A or EcR.B1 isoforms. Furthermore, we showed that ecdysteroid signaling is essential for the proper activation of matrix metalloproteinase 2 (Mmp2) for follicle rupture. Our data strongly suggest that 20E produced in follicle cells before ovulation activates EcR.B2 to prime mature follicles to be responsive to neuronal ovulatory stimuli, thus providing mechanistic insights into steroid signaling in Drosophila ovulation.
Assuntos
Drosophila/metabolismo , Ovulação/metabolismo , Transdução de Sinais/fisiologia , Esteroides/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Drosophila/fisiologia , Proteínas de Drosophila/metabolismo , Ecdisona/metabolismo , Ecdisterona/metabolismo , Feminino , Genes de Insetos/fisiologia , Metaloproteinase 2 da Matriz/metabolismo , Oogênese/fisiologia , Folículo Ovariano/metabolismo , Folículo Ovariano/fisiologia , Ovulação/fisiologia , Isoformas de Proteínas/metabolismo , Receptores de Esteroides/metabolismoRESUMO
Rodent models are being extensively used to investigate the effects of traumatic injury and develop and assess the mechanisms of repair and regeneration. We present quantitative assessment of two-dimensional (2D) kinematics of overground walking and for the first time three-dimensional (3D) joint angle kinematics of all four limbs during treadmill walking in intact adult female Long-Evans rats. Gait cycle with subphases and intralimb and interlimb cyclograms are presented. Phase relationships between joint angles on a cycle-by-cycle basis and interlimb footfalls are assessed using a simple technique. Electromyogram (EMG) data from major flexor and extensor muscles for each of the hindlimb joints and elbow extensor muscles of the forelimbs synchronized to the 3D kinematics are also obtained. Overground walking kinematics, provides information on base of support, stride length, and hindfoot rotation. Treadmill walking kinematics indicate primarily monophasic angle trajectories for the hip and shoulder joints, weak double peak patterns for the knee and elbow joints, and a prominent double peak pattern for the ankle joints. Maximum flexion of the knee during swing precedes that of the ankle, which precedes that of the hip. A mild exercise regimen over 8 weeks does not alter the kinematics. EMG activity indicates specific relationships of the neural activity to joint angle kinematics. We find that the ankle flexors as well as the hip and elbow extensors maintain constant burst duration with changing cycle duration. Data and techniques described here are likely to be useful for quantitative assessment of altered gait and neural control mechanisms after neurotrauma.
Assuntos
Marcha/fisiologia , Locomoção/fisiologia , Músculo Esquelético/fisiologia , Fenômenos Fisiológicos do Sistema Nervoso , Animais , Fenômenos Biomecânicos , Eletromiografia , Teste de Esforço , Extremidades/inervação , Extremidades/fisiologia , Feminino , Articulações/inervação , Articulações/fisiologia , Modelos Animais , Contração Muscular/fisiologia , Músculo Esquelético/inervação , Condicionamento Físico Animal/fisiologia , Amplitude de Movimento Articular/fisiologia , Ratos , Ratos Long-EvansRESUMO
Recent research has shown that after spinal cord injury, the nervous system reorganizes. Nevertheless, little is known of the effects of neural reorganization, or plasticity, on motor skills. In this work, we present a method that utilizes kinetic and kinematic analysis, for investigating balance control in a rodent model of incomplete spinal cord injury. In this setup, the animals sit unconstrained on their hindlimbs on a platform while they eat a Fruitloop. In this posture, the animal is supporting all the body weight on its hindquarters removing the need for the animal to support itself on its hindlimb or maintain appropriate forelimb-hindlimb coordination for functional gait. The platform is bolted to a force transducer to provide measurements of shear forces in orthogonal directions in the horizontal plane. Reflective markers on the hip and tail of the animal indicate sway of the animals body. Using this method the effect of extensive 12 week long treadmill locomotor training on balance control in rodents with incomplete thoracic spinal cord contusion injury (iSCI) was assessed. One iSCI rat did not undergo training, while a sham injured and 3 other iSCI rats underwent training. The shear forces and sway produced by the injured untrained rat were much larger than for the sham or the injured but trained rats. Stance width assessed from overground walking in a separate study was also larger in the injured untrained rat. These data suggest that balance control may be improved by a locomotor training paradigm. This simple method for assessing balance control could thus be utilized in longitudinal studies to evaluate the effectiveness of pharmacological and locomotor therapies for repair and recovery after spinal cord injury.