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1.
PLoS Genet ; 14(1): e1007164, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29385124

RESUMO

Olfactory sensory neurons choose to express a single odorant receptor (OR) from a large gene repertoire and extend axons to reproducible, OR-specific locations within the olfactory bulb. This developmental process produces a topographically organized map of odorant experience in the brain. The axon guidance mechanisms that generate this pattern of connectivity, as well as those that coordinate OR choice and axonal guidance receptor expression, are incompletely understood. We applied the powerful approach of single-cell RNA-seq on newly born olfactory sensory neurons (OSNs) in young zebrafish larvae to address these issues. Expression profiles were generated for 56 individual Olfactory Marker Protein (OMP) positive sensory neurons by single-cell (SC) RNA-seq. We show that just as in mouse OSNs, mature zebrafish OSNs typically express a single predominant OR transcript. Our previous work suggests that OSN targeting is related to the OR clade from which a sensory neuron chooses to express its odorant receptor. We categorized each of the mature cells based on the clade of their predominantly expressed OR. Transcripts expressed at higher levels in each of three clade-related categories were identified using Penalized Linear Discriminant Analysis (PLDA). A genome-wide approach was used to identify membrane-associated proteins that are most likely to have guidance-related activity. We found that OSNs that choose to express an OR from a particular clade also express specific subsets of potential axon guidance genes and transcription factors. We validated our identification of candidate axon guidance genes for one clade of OSNs using bulk RNA-seq from a subset of transgene-labeled neurons that project to a single protoglomerulus. The differential expression patterns of selected candidate guidance genes were confirmed using fluorescent in situ hybridization. Most importantly, we observed axonal mistargeting in knockouts of three candidate axonal guidance genes identified in this analysis: nrp1a, nrp1b, and robo2. In each case, targeting errors were detected in the subset of axons that normally express these transcripts at high levels, and not in the axons that express them at low levels. Our findings demonstrate that specific, functional, axonal guidance related genes are expressed in subsets of OSNs that that can be categorized by their patterns of OR expression.


Assuntos
Axônios/fisiologia , Genes Controladores do Desenvolvimento , Neurogênese/genética , Neurônios Receptores Olfatórios/fisiologia , Receptores Odorantes/genética , Receptores Odorantes/fisiologia , Animais , Animais Geneticamente Modificados , Diferenciação Celular/genética , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Rede Nervosa/embriologia , Rede Nervosa/fisiologia , Bulbo Olfatório/metabolismo , Proteína de Marcador Olfatório/metabolismo , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Receptores Odorantes/metabolismo , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologia , Transcriptoma , Peixe-Zebra/embriologia , Peixe-Zebra/genética
2.
J Neurosci ; 32(30): 10370-82, 2012 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-22836270

RESUMO

Endocytic trafficking of membrane proteins is essential for neuronal structure and function. We show that Transient Axonal Glycoprotein 1 (TAG1 or CNTN2), a contactin-related adhesion molecule, plays a central role in the differential trafficking of components of the semaphorin3A (Sema3A) receptor complex into distinct endosomal compartments in murine spinal sensory neuron growth cones. The semaphorin3A receptor is composed of Neuropilin1 (NRP1), PlexinA4, and L1, with NRP1 being the ligand-binding component. TAG1 interacts with NRP1, causing a change in its association with L1 in the Sema3A response such that L1 is lost from the complex following Sema3A binding. Initially, however, L1 and NRP1 endocytose together and only become separated intracellularly, with NRP1 becoming associated with endosomes enriched in lipid rafts and colocalizing with TAG1 and PlexinA4. When TAG1 is missing, NRP1 and L1 fail to separate and NRP1 does not become raft associated; colocalization with PlexinA4 is reduced and Plexin signaling is not initiated. These observations identify a novel role for TAG1 in modulating the intracellular sorting of signaling receptor complexes.


Assuntos
Contactina 2/metabolismo , Endocitose/fisiologia , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuropilina-1/metabolismo , Receptores de Superfície Celular/metabolismo , Semaforina-3A/metabolismo , Animais , Cones de Crescimento/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/fisiologia
3.
Neuroscience ; 508: 19-29, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-35940453

RESUMO

Olfactory sensory neurons that express related odorant receptors specifically target large identifiable neuropils called protoglomeruli when they first reach the olfactory bulb in the zebrafish. This crude odorant receptor-related mapping is further refined as odorant receptor-specific glomeruli segregate from protoglomeruli later in development. Netrins are a prominent class of axon guidance molecules whose contribution to olfactory circuit formation is poorly studied. Morpholino knock down experiments have suggested that Netrin/Dcc signaling is involved in normal protoglomerular targeting. Here we extend these findings with more detailed characterization and modeling of netrin expression, and by examining protoglomerular targeting in mutant lines fornetrin1a (ntn1a), netrin1b (ntn1b), and their receptorsunc5b,dcc, andneo1a. We confirm thatntn1a,ntn1b, anddccare required for normal protoglomerular guidance of a subset of olfactory sensory neurons that are labeled with the Tg(or111-7:IRES:Gal4) transgene. We also observe errors in the targeting of these axons inunc5bmutants, but not inneo1a mutants. Our findings are consistent with ntn1a andntn1bacting primarily as attractants for olfactory sensory neurons targeting the central zone protoglomerulus.


Assuntos
Neurônios Receptores Olfatórios , Receptores Odorantes , Animais , Bulbo Olfatório/metabolismo , Peixe-Zebra/metabolismo , Receptores Odorantes/metabolismo , Receptores de Netrina/metabolismo , Netrinas/metabolismo , Neurônios Receptores Olfatórios/fisiologia , Axônios/metabolismo , Netrina-1/metabolismo
4.
Neural Dev ; 17(1): 1, 2022 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-34980234

RESUMO

BACKGROUND: Olfactory Sensory Neuron (OSN) axons project from the zebrafish olfactory epithelium to reproducible intermediate target locations in the olfactory bulb called protoglomeruli at early stages in development. Two classes of OSNs expressing either OMP or TRPC2 exclusively target distinct, complementary protoglomeruli. Using RNAseq, we identified axon guidance receptors nrp2a and nrp2b, and their ligand sema3fa, as potential guidance factors that are differentially expressed between these two classes of OSNs. METHODS: To investigate their role in OSN axon guidance, we assessed the protoglomerular targeting fidelity of OSNs labeled by OMP:RFP and TRPC2:Venus transgenes in nrp2a, nrp2b, or sema3fa mutants. We used double mutant and genetic interaction experiments to interrogate the relationship between the three genes. We used live time-lapse imaging to compare the dynamic behaviors of OSN growth cones during protoglomerular targeting in heterozygous and mutant larvae. RESULTS: The fidelity of protoglomerular targeting of TRPC2-class OSNs is degraded in nrp2a, nrp2b, or sema3fa mutants, as axons misproject into OMP-specific protoglomeruli and other ectopic locations in the bulb. These misprojections are further enhanced in nrp2a;nrp2b double mutants suggesting that nrp2s work at least partially in parallel in the same guidance process. Results from genetic interaction experiments are consistent with sema3fa acting in the same biological pathway as both nrp2a and nrp2b. Live time-lapse imaging was used to examine the dynamic behavior of TRPC2-class growth cones in nrp2a mutants compared to heterozygous siblings. Some TRPC2-class growth cones ectopically enter the dorsal-medial region of the bulb in both groups, but in fully mutant embryos, they are less likely to correct the error through retraction. The same result was observed when TRPC2-class growth cone behavior was compared between sema3fa heterozygous and sema3fa mutant larvae. CONCLUSIONS: Our results suggest that nrp2a and nrp2b expressed in TRPC2-class OSNs help prevent their mixing with axon projections in OMP-specific protoglomeruli, and further, that sema3fa helps to exclude TRPC2-class axons by repulsion from the dorsal-medial bulb.


Assuntos
Neurônios Receptores Olfatórios , Peixe-Zebra , Animais , Axônios , Bulbo Olfatório , Mucosa Olfatória , Condutos Olfatórios
5.
Methods Mol Biol ; 1493: 299-309, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27787860

RESUMO

The study of receptor endocytosis is important to our understanding of the signal transduction events initiated by axon guidance cues in growth cones. Fab fragments of antibodies to guidance receptors and endocytic cargoes like transferrin and cholera toxin-B are the tools of choice for studying the dynamics of receptor internalization and intracellular trafficking by different pathways. We describe a method where in vitro cultures of growth cones are incubated with these ligands in the presence or absence of Sema3A, followed by stripping of remaining ligand on cell-surface and analysis by immunofluorescence techniques. These techniques can be employed for studying the endocytosis of any axon guidance receptor in response to attractive or repulsive guidance cues and, in particular, to allow the differential trafficking of specific receptor components to be revealed.


Assuntos
Transporte Biológico , Endocitose , Cones de Crescimento/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Receptores de Superfície Celular/metabolismo , Semaforina-3A/metabolismo , Imunofluorescência , Gânglios Espinais/citologia , Células HEK293 , Humanos , Técnicas In Vitro , Ligantes
6.
Neural Dev ; 12(1): 18, 2017 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-29020985

RESUMO

BACKGROUND: The axons of Olfactory Sensory Neurons (OSNs) project to reproducible target locations within the Olfactory Bulb (OB), converting odorant experience into a spatial map of neural activity. We characterized the initial targeting of OSN axons in the zebrafish, a model system suitable for studying axonal targeting early in development. In this system the initial targets of OSN axons are a small number of distinct, individually identifiable neuropilar regions called protoglomeruli. Previously, Olfactory Marker Protein-expressing and TRPC2-expressing classes of OSNs were shown to project to specific, non-overlapping sets of protoglomeruli, indicating that particular subsets of OSNs project to specific protoglomerular targets. We set out to map the relationship between the classical Odorant Receptor (OR) an OSN chooses to express and the protoglomerulus its axon targets. METHODS: A panel of BACs were recombineered so that the axons of OSNs choosing to express modified ORs were fluorescently labeled. Axon projections were followed into the olfactory bulb to determine the protoglomeruli in which they terminated. RESULTS: RNA-seq demonstrates that OSNs express a surprisingly wide variety of ORs and Trace Amine Associated Receptors (TAARs) very early when sensory axons are arriving in the bulb. Only a single OR is expressed in any given OSN even at these early developmental times. We used a BAC expression technique to map the trajectories of OSNs expressing specific odorant receptors. ORs can be divided into three clades based upon their sequence similarities. OSNs expressing ORs from two of these clades project to the CZ protoglomerulus, while OSNs expressing ORs from the third clade project to the DZ protoglomerulus. In contrast, OSNs expressing a particular TAAR project to multiple protoglomeruli. Neither OR choice nor axonal targeting are related to the position an OSN occupies within the olfactory pit. CONCLUSIONS: Our results demonstrate that it is not the choice of a particular OR, but of one from a category of ORs, that is related to initial OSN target location within the olfactory bulb. These choices are not related to OSN position within the olfactory epithelium.


Assuntos
Axônios , Neurogênese/fisiologia , Bulbo Olfatório/embriologia , Neurônios Receptores Olfatórios/citologia , Animais , Animais Geneticamente Modificados , Peixe-Zebra
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