RESUMEN
Internal sensory neurons innervate body organs and provide information about internal state to the CNS to maintain physiological homeostasis. Despite their conservation across species, the anatomy, circuitry, and development of internal sensory systems are still relatively poorly understood. A largely unstudied population of larval Drosophila sensory neurons, termed tracheal dendrite (td) neurons, innervate internal respiratory organs and may serve as a model for understanding the sensing of internal states. Here, we characterize the peripheral anatomy, central axon projection, and diversity of td sensory neurons. We provide evidence for prominent expression of specific gustatory receptor genes in distinct populations of td neurons, suggesting novel chemosensory functions. We identify two anatomically distinct classes of td neurons. The axons of one class project to the subesophageal zone (SEZ) in the brain, whereas the other terminates in the ventral nerve cord (VNC). We identify expression and a developmental role of the POU-homeodomain transcription factor Pdm3 in regulating the axon extension and terminal targeting of SEZ-projecting td neurons. Remarkably, ectopic Pdm3 expression is alone sufficient to switch VNC-targeting axons to SEZ targets, and to induce the formation of putative synapses in these ectopic target zones. Our data thus define distinct classes of td neurons, and identify a molecular factor that contributes to diversification of axon targeting. These results introduce a tractable model to elucidate molecular and circuit mechanisms underlying sensory processing of internal body status and physiological homeostasis.SIGNIFICANCE STATEMENT How interoceptive sensory circuits develop, including how sensory neurons diversify and target distinct central regions, is still poorly understood, despite the importance of these sensory systems for maintaining physiological homeostasis. Here, we characterize classes of Drosophila internal sensory neurons (td neurons) and uncover diverse axonal projections and expression of chemosensory receptor genes. We categorize td neurons into two classes based on dichotomous axon target regions, and identify the expression and role of the transcription factor Pdm3 in mediating td axon targeting to one of these target regions. Our results provide an entry point into studying internal sensory circuit development and function, and establish Pdm3 as a regulator of interoceptive axon targeting.
Asunto(s)
Axones/ultraestructura , Proteínas de Drosophila/metabolismo , Neurogénesis/fisiología , Factores del Dominio POU/metabolismo , Células Receptoras Sensoriales/citología , Animales , Axones/metabolismo , Tipificación del Cuerpo/fisiología , Drosophila , Células Receptoras Sensoriales/metabolismo , Tráquea/inervaciónRESUMEN
To better understand how organisms make decisions on the basis of temporally varying multi-sensory input, we identified computations made by Drosophila larvae responding to visual and optogenetically induced fictive olfactory stimuli. We modeled the larva's navigational decision to initiate turns as the output of a Linear-Nonlinear-Poisson cascade. We used reverse-correlation to fit parameters to this model; the parameterized model predicted larvae's responses to novel stimulus patterns. For multi-modal inputs, we found that larvae linearly combine olfactory and visual signals upstream of the decision to turn. We verified this prediction by measuring larvae's responses to coordinated changes in odor and light. We studied other navigational decisions and found that larvae integrated odor and light according to the same rule in all cases. These results suggest that photo-taxis and odor-taxis are mediated by a shared computational pathway.
Asunto(s)
Quimiotaxis/fisiología , Drosophila/fisiología , Luz , Modelos Biológicos , Actividad Motora/fisiología , Odorantes , Navegación Espacial/fisiología , Animales , Optogenética/métodos , Estimulación Luminosa , Estimulación QuímicaRESUMEN
BACKGROUND: The tumor suppressors p14(ARF) (ARF) and p16(INK4A) (p16) are encoded by overlapping reading frames at the CDKN2A/INK4A locus on chromosome 9p21. In human melanoma, the accumulated evidence has suggested that the predominant tumor suppressor at 9p21 is p16, not ARF. However, recent observations from melanoma-prone families and murine melanoma models suggest a p16-independent tumor suppressor role for ARF. We analyzed a group of melanoma metastases and cell lines to investigate directly whether somatic alterations to the ARF gene support its role as a p16-independent tumor suppressor in human melanoma, assuming that two alterations (genetic and/or epigenetic) would be required to inactivate a gene. METHODS: We examined the p16/ARF locus in 60 melanoma metastases from 58 patients and in 9 human melanoma cell lines using multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction (PCR) to detect deletions, methylation-specific PCR to detect promoter methylation, direct sequencing to detect mutations affecting ARF and p16, and, in a subset of 20 tumors, immunohistochemistry to determine the effect of these alterations on p16 protein expression. All statistical tests were two-sided. RESULTS: We observed two or more alterations to the ARF gene in 26/60 (43%) metastases. The p16 gene sustained two or more alterations in 13/60 (22%) metastases (P = .03). Inactivation of ARF in the presence of wild-type p16 was seen in 18/60 (30%) metastases. CONCLUSION: Genetic and epigenetic analyses of the human 9p21 locus indicate that modifications of ARF occur independently of p16 inactivation in human melanoma and suggest that ARF is more frequently inactivated than p16.
Asunto(s)
Silenciador del Gen , Genes p16 , Melanoma/genética , Melanoma/patología , Proteína p14ARF Supresora de Tumor/genética , Animales , Línea Celular Tumoral , Cromosomas Humanos Par 9 , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Metilación de ADN , Eliminación de Gen , Humanos , Immunoblotting , Inmunohistoquímica , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Repeticiones de Microsatélite , Mutación , Proteínas de Neoplasias/genética , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras GenéticasRESUMEN
Glia mediate a vast array of cellular processes and are critical for nervous system development and function. Despite their immense importance in neurobiology, glia remain understudied and the molecular mechanisms that direct their differentiation are poorly understood. Rap/Fzr is the Drosophila homolog of the mammalian Cdh1, a regulatory subunit of the anaphase-promoting complex/cyclosome (APC/C). APC/C is an E3 ubiquitin ligase complex well characterized for its role in cell cycle progression. In this study, we have uncovered a novel cellular role for Rap/Fzr. Loss of rap/fzr function leads to a marked increase in the number of glia in the nervous system of third instar larvae. Conversely, ectopic expression of UAS-rap/fzr, driven by repo-GAL4, results in the drastic reduction of glia. Data from clonal analyses using the MARCM technique show that Rap/Fzr regulates the differentiation of surface glia in the developing larval nervous system. Our genetic and biochemical data further indicate that Rap/Fzr regulates glial differentiation through its interaction with Loco, a regulator of G-protein signaling (RGS) protein and a known effector of glia specification. We propose that Rap/Fzr targets Loco for ubiquitination, thereby regulating glial differentiation in the developing nervous system.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/citología , Proteínas RGS/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Animales , Apoptosis , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Proteínas Cdh1 , Recuento de Células , Diferenciación Celular , Linaje de la Célula , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Larva/crecimiento & desarrollo , Larva/metabolismo , Modelos Biológicos , Neuronas/citología , Unión Proteica , Transporte de ProteínasRESUMEN
In the developing Drosophila eye, Rap/Fzr plays a critical role in neural patterning by regulating the timely exit of precursor cells. Rap/Fzr (Retina aberrant in pattern/Fizzy related) is an activator of the E3 Ubiquitin ligase, the APC (Anaphase Promoting Complex-cyclosome) that facilitates the stage specific proteolytic destruction of mitotic regulators, such as cyclins and cyclin-dependent kinases. To identify novel functional roles of Rap/Fzr, we conducted an F(1) genetic modifier screen to identify genes which interact with the partial-loss-function mutations in rap/fzr. We screened 2741 single P-element, lethal insertion lines and piggyBac lines on the second and third chromosome for dominant enhancers and suppressors of the rough eye phenotype of rap/fzr. From this screen, we have identified 40 genes that exhibit dosage-sensitive interactions with rap/fzr; of these, 31 have previously characterized cellular functions. Seven of the modifiers identified in this study are regulators of cell cycle progression with previously known interactions with rap/fzr. Among the remaining modifiers, 27 encode proteins involved in other cellular functions not directly related to cell-cycle progression. The newly identified variants fall into at least three groups based on their previously known cellular functions: transcriptional regulation, regulated proteolysis, and signal transduction. These results suggest that, in addition to cell cycle regulation, rap/fzr regulates ubiquitin-ligase-mediated protein degradation in the developing nervous system as well as in other tissues.