RESUMO
BACKGROUND: During neurogenesis, growing axons must navigate through the complex extracellular environment and make correct synaptic connections for the proper functioning of neural circuits. The mechanisms underlying the formation of functional neural networks are still only partially understood. RESULTS: Here we analyzed the role of a novel gene si:ch73-364h19.1/drish in the neural and vascular development of zebrafish embryos. We show that drish mRNA is expressed broadly and dynamically in multiple cell types including neural, glial, retinal progenitor and vascular endothelial cells throughout the early stages of embryonic development. To study Drish function during embryogenesis, we generated drish genetic mutant using CRISPR/Cas9 genome editing. drish loss-of-function mutant larvae displayed defects in early retinal ganglion cell, optic nerve and the retinal inner nuclear layer formation, as well as ectopic motor axon branching. In addition, drish mutant adults exhibited deficient retinal outer nuclear layer and showed defective light response and locomotory behavior. However, vascular patterning and blood circulation were not significantly affected. CONCLUSIONS: Together, these data demonstrate important roles of zebrafish drish in the retinal ganglion cell, optic nerve and interneuron development and in spinal motor axon branching.
Assuntos
Axônios , Neurogênese , Células Ganglionares da Retina , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/genética , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/fisiologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Axônios/metabolismo , Axônios/fisiologia , Neurogênese/genética , Neurogênese/fisiologia , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Embrião não Mamífero/metabolismo , Retina/metabolismo , Retina/embriologiaRESUMO
The WASF3 gene promotes cancer cell invasion and metastasis, and genetic inactivation leads to suppression of metastasis. To identify small molecules that might interfere with WASF3 function, we performed an in silico docking study to the regulatory pocket of WASF3 using the National Cancer Institute (NCI) diversity set VI small molecule library. Compounds that showed the maximum likelihood of interaction with WASF3 were screened for their effect on cell movement in breast and prostate cancer cells, a well-established predictor of invasion and metastasis. Three hit compounds were identified that affected cell movement, and the same compounds also suppressed cell migration and invasion in vitro in both MDA-MB-231 breast cancer cells and Du145 prostate cancer cells. Using a zebrafish metastasis assay, one of these compounds, NSC670283, showed significant suppression of metastasis in vivo while not affecting cell proliferation. NSC670283 showed a consistent effect on suppression of invasion and metastasis, and cellular temperature shift assays provided support for physical interaction with WASF3. In addition, suppression of cell movement and invasion was accompanied by a decrease in actin filament polymerization. The data in this study suggest that these small molecules inhibit cancer cell invasion and metastasis, and to our knowledge, it is the first identification of a small molecule that can potentially inhibit WASF3-directed metastasis, laying the foundation for medicinal chemistry approaches to enhance the potency of the identified compounds.
RESUMO
(1) Background: Several agencies in the United States play a primary role in ensuring food safety, yet foodborne illnesses result in about 3000 deaths and cost more than USD 15.6 billion each year. The study objectives included analyzing local health departments' (LHDs) level of engagement in food safety and other related services, and LHDs' characteristics associated with those services. (2) Methods: We used data from 1496 LHDs that participated in the 2019 National Profile of Local Health Departments Survey, administered to all 2459 LHDs in the United States. Logistic regression analyses were performed to model multiple dichotomous variables. (3) Results: An estimated 78.9% of LHDs performed food safety inspections, 78.3% provided food safety education, 40.7% provided food processing inspections, and 48.4% engaged in policy and advocacy. The odds for LHDs to directly provide preventive nutrition services were 20 times higher if the LHDs had one or more nutritionists on staff (Adjusted Odds Ratio or AOR = 20.0; Confidence Interval, CI = 12.4−32.2) compared with LHDs with no nutritionists. Other LHD characteristics significantly associated with the provision of nutrition services (p < 0.05) included population size, state governance (rather than local), and LHD having at least one registered, licensed, practical, or vocational nurse. The odds of providing food processing services were lower for locally governed than state-governed LHDs (AOR = 0.5; CI = 0.4−0.7). The odds of performing food safety inspections varied by LHD's population size, whether a nutritionist was on staff, whether it was state-governed (vs. locally), and whether it completed a community health assessment (CHA) within 5 years. (4) Conclusions: LHDs play a critical role in ensuring safe food for Americans, yet variations exist in their performance based on their specific characteristics. Adequate funding and a competent workforce are essential for LHDs to utilize evidence-based practices and engage in policymaking and advocacy concerning food safety.
Assuntos
Governo Local , Saúde Pública , Inocuidade dos Alimentos , Humanos , Razão de Chances , Estados UnidosRESUMO
The discovery of novel analgesic agents with high potency, low toxicity and low addictive properties remain a priority. This study aims to identify the analgesic potential of quinoline derived α-trifluoromethylated alcohols (QTA) and their mechanism of action. We synthesized and characterized several compounds of QTAs and screened them for antiepileptic and analgesic activity using zebrafish larvae in high thorough-put behavior analyses system. Toxicity and behavioral screening of 9 compounds (C1-C9) identified four candidates (C2, C3, C7 and C9) with antiepileptic properties that induces specific and reversible reduction in photomotor activity. Importantly, compounds C2 and C3 relieved the thermal pain response in zebrafish larvae indicating analgesic property. Further, using novel inâ vivo CoroNa green assay, we show that compounds C2 and C3 block sodium channels and reduce inflammatory sodium signals released by peripheral nerve and tissue damage. Thus, we have identified novel QTA compounds with antiepileptic and analgesic properties which could alleviate neuropathic pain.
Assuntos
Analgésicos/farmacologia , Anticonvulsivantes/farmacologia , Metanol/análogos & derivados , Quinolinas/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismo , Analgésicos/síntese química , Analgésicos/química , Animais , Anticonvulsivantes/síntese química , Anticonvulsivantes/química , Relação Dose-Resposta a Droga , Descoberta de Drogas , Metanol/síntese química , Metanol/química , Metanol/farmacologia , Estrutura Molecular , Quinolinas/síntese química , Quinolinas/química , Bloqueadores dos Canais de Sódio/síntese química , Bloqueadores dos Canais de Sódio/química , Relação Estrutura-Atividade , Peixe-ZebraRESUMO
Precise positioning of neurons resulting from cell division and migration during development is critical for normal brain function. Disruption of neuronal migration can cause a myriad of neurological disorders. To investigate the functional consequences of defective neuronal positioning on circuit function, we studied a zebrafish frizzled3a (fzd3a) loss-of-function mutant off-limits (olt) where the facial branchiomotor (FBM) neurons fail to migrate out of their birthplace. A jaw movement assay, which measures the opening of the zebrafish jaw (gape), showed that the frequency of gape events, but not their amplitude, was decreased in olt mutants. Consistent with this, a larval feeding assay revealed decreased food intake in olt mutants, indicating that the FBM circuit in mutants generates defective functional outputs. We tested various mechanisms that could generate defective functional outputs in mutants. While fzd3a is ubiquitously expressed in neural and non-neural tissues, jaw cartilage and muscle developed normally in olt mutants, and muscle function also appeared to be unaffected. Although FBM neurons were mispositioned in olt mutants, axon pathfinding to jaw muscles was unaffected. Moreover, neuromuscular junctions established by FBM neurons on jaw muscles were similar between wildtype siblings and olt mutants. Interestingly, motor axons innervating the interhyoideus jaw muscle were frequently defasciculated in olt mutants. Furthermore, GCaMP imaging revealed that mutant FBM neurons were less active than their wildtype counterparts. These data show that aberrant positioning of FBM neurons in olt mutants is correlated with subtle defects in fasciculation and neuronal activity, potentially generating defective functional outputs.
Assuntos
Neurônios Motores , Peixe-Zebra , Animais , Axônios , Movimento Celular , Neurogênese , Proteínas de Peixe-Zebra/genéticaRESUMO
CREB (cAMP response element-binding) transcription factors are conserved markers of memory formation in the brain and peripheral circuits. We provide immunohistochemical evidence of CREB phosphorylation in the dwarf cuttlefish, Sepia bandensis, following the inaccessible prey (IP) memory experiment. During the IP experiment, cuttlefish are shown prey enclosed in a transparent tube, and tentacle strikes against the tube decrease over time as the cuttlefish learns the prey is inaccessible. The cues driving IP learning are unclear but may include sensory inputs from arms touching the tube. The neural activity marker, anti-phospho-CREB (anti-pCREB) was used to determine whether IP training stimulated cuttlefish arm sensory neurons. pCREB immunoreactivity occurred along the oral surface of the arms, including the suckers and epithelial folds surrounding the suckers. pCREB increased in the epithelial folds and suckers of trained cuttlefish. We found differential pCREB immunoreactivity along the distal-proximal axis of trained arms, with pCREB concentrated distally. Unequal CREB phosphorylation occurred among the 4 trained arm pairs, with arm pairs 1 and 2 containing more pCREB. The resulting patterns of pCREB in trained arms suggest that the arms obtain cues that may be salient for learning and memory of the IP experiment.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Aprendizagem/fisiologia , Memória/fisiologia , Sepia/fisiologia , Animais , Extremidades/fisiologia , FosforilaçãoRESUMO
This is a proof-of-principle study on the combination of microwaves and multiwalled carbon nanotubes to induce in vivo, localized hyperthermic ablation of cells as a potential methodology for the treatment of localized tumors. Compared to conventional methods, the proposed approach can create higher temperatures in a rapid and localized fashion, under low radiation levels, eliminating some of the unwanted side effects. Following successful ablation of cancer cells in cell culture and zebrafish tumor-xenograft models, it is hypothesized that a cancer treatment can be developed using safe microwave irradiation for selective ablation of tumor cells in vivo using carbon nanotube-Antibody (CNT-Ab) conjugates as a targeting agent. In this study, mice were used as an animal model for the optimization of the proposed microwave treatment strategy. The safe dose of CNT-Ab and microwave radiation levels for mice were determined. Further, CNT-Ab distribution and toxicology in mice were qualitatively determined for a time span of two weeks following microwave hyperthermia. The results indicate no toxicity associated with the CNT-Ab in the absence of microwaves. CNTs are only found in the proximity of the site of injection and have been shown to effectively cause hyperthermia induced necrosis upon exposure to microwaves with no noticeable damage to other tissues that are not in direct contact with the CNT-Ab. To understand the cellular immune response towards CNT-Abs, transgenic zebrafish with fluorescently labeled macrophages and neutrophils were used to assay for their ability to phagocytize CNT-Ab. Our results indicate that macrophages and neutrophils were able to actively phagocytose CNT-Abs shortly after injection. Taken together, this is the first study to show that CNTs can be used in combination with microwaves to cause targeted ablation of cells in mice without any side effects, which would be ideal for cancer therapies.
Assuntos
Técnicas de Ablação/métodos , Micro-Ondas , Nanotubos de Carbono/química , Técnicas de Ablação/efeitos adversos , Animais , Anticorpos/química , Anticorpos/metabolismo , Temperatura Alta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nanotubos de Carbono/toxicidade , Necrose/patologia , Peixe-ZebraRESUMO
Measuring behavior in the form of numerical data is difficult, especially for studies involving complex actions. DanioVision is a closed-chamber system that utilizes subject tracking to comprehensively record behavior, while also mitigating the influence of environmental conditions. We used DanioVision to record activity of juvenile dwarf cuttlefish (Sepia bandensis) during the inaccessible prey (IP) procedure, a memory experiment in which cuttlefish learn to inhibit capture attempts towards inaccessible prey. By quantifying total movement and orientation of the body, we found that cuttlefish show memory by selectively inhibiting tentacle strikes without reducing total movement, or orientation towards the prey. We show that DanioVision can be used to assess multiple components of dynamic responses that are not measurable by direct observation alone and provide new evidence that strike inhibition is the product of learning, and not motor fatigue.
Assuntos
Sepia , Animais , Decapodiformes , Aprendizagem , Memória , Comportamento PredatórioAssuntos
Infecções por Coronavirus/epidemiologia , Equidade em Saúde/organização & administração , Pneumonia Viral/epidemiologia , Saúde Pública , Determinantes Sociais da Saúde/estatística & dados numéricos , Fatores Etários , Betacoronavirus , COVID-19 , Comorbidade , Infecções por Coronavirus/etnologia , Abastecimento de Alimentos/estatística & dados numéricos , Hospitalização/estatística & dados numéricos , Humanos , Pandemias , Pneumonia Viral/etnologia , Características de Residência , SARS-CoV-2 , Fatores Socioeconômicos , Estados UnidosRESUMO
Contactin2 (Cntn2)/Transient Axonal Glycoprotein 1 (Tag1), a neural cell adhesion molecule, has established roles in neuronal migration and axon fasciculation in chick and mouse. In zebrafish, antisense morpholino-based studies have indicated roles for cntn2 in the migration of facial branchiomotor (FBM) neurons, the guidance of the axons of the nucleus of the medial longitudinal fascicle (nucMLF), and the outgrowth of Rohon-Beard (RB) central axons. To study functions of Cntn2 in later stages of neuronal development, we generated cntn2 mutant zebrafish using CRISPR-Cas9. Using a null mutant allele, we detected genetic interactions between cntn2 and the planar cell polarity gene vangl2, as shown previously with cntn2 morphants, demonstrating a function for cntn2 during FBM neuron migration in a sensitized background of reduced planar cell polarity signaling. In addition, maternal-zygotic (MZ) cntn2 mutant larvae exhibited aberrant touch responses and swimming, suggestive of defects in sensorimotor circuits, consistent with studies in mice. However, the nucMLF axon convergence, FBM neuron migration, and RB outgrowth defects seen in morphants were not seen in the mutants, and we show here that they are likely off-target effects of morpholinos. However, MLF axons exhibited local defasciculation in MZcntn2 mutants, consistent with a role for Cntn2 in axon fasciculation. These data demonstrate distinct roles for zebrafish cntn2 in neuronal migration and axon fasciculation, and in the function of sensorimotor circuits.
Assuntos
Adesão Celular/genética , Contactina 2/genética , Neurogênese/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Axônios/metabolismo , Sistemas CRISPR-Cas , Movimento Celular/genética , Polaridade Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Morfolinos/genética , Morfolinos/metabolismo , Neurônios Motores/metabolismo , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
In this study the rational design, synthesis, and anticancer activity of quinoline-derived trifluoromethyl alcohols were evaluated. Members of this novel class of trifluoromethyl alcohols were identified as potent growth inhibitors in a zebrafish embryo model. Synthesis of these compounds was carried out with an sp(3) -C-H functionalization strategy of methyl quinolines with trifluoromethyl ketones. A zebrafish embryo model was also used to explore the toxicity of ethyl 4,4,4-trifluoro-3-hydroxy-3-(quinolin-2-ylmethyl)butanoate (1), 2-benzyl-1,1,1-trifluoro-3-(quinolin-2-yl)propan-2-ol (2), and trifluoro-3-(isoquinolin-1-yl)-2-(thiophen-2-yl)propan-2-ol (3). Compounds 2 and 3 were found to be more toxic than compound 1; apoptotic staining assays indicated that compound 3 causes increased cell death. In vitro cell proliferation assays showed that compound 2, with an LC50 value of 14.14â µm, has more potent anticancer activity than cisplatin. This novel class of inhibitors provides a new direction in the discovery of effective anticancer agents.
Assuntos
Álcoois/farmacologia , Antineoplásicos/farmacologia , Descoberta de Drogas , Hidrocarbonetos Fluorados/farmacologia , Quinolinas/farmacologia , Quinolinas/toxicidade , Peixe-Zebra/embriologia , Álcoois/síntese química , Álcoois/química , Álcoois/toxicidade , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/toxicidade , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Hidrocarbonetos Fluorados/síntese química , Hidrocarbonetos Fluorados/química , Hidrocarbonetos Fluorados/toxicidade , Modelos Animais , Estrutura Molecular , Quinolinas/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Herbarin A and B were isolated from the fungal strains of Cladosporium herbarum found in marine sponges Aplysina aerophoba and Callyspongia aerizusa. Total synthesis of Herbarin A and B was achieved by carrying out a multi-step synthesis approach, and the antioxidant properties were evaluated using FRAP assay. Toxicity of these compounds was determined using a zebrafish embryo model.
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Antioxidantes/síntese química , Ácidos Graxos Insaturados/síntese química , Pironas/síntese química , Animais , Antioxidantes/farmacologia , Antioxidantes/toxicidade , Cladosporium/química , Cladosporium/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Ácidos Graxos Insaturados/química , Ácidos Graxos Insaturados/toxicidade , Modelos Animais , Pironas/química , Pironas/toxicidade , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Van gogh-like 2 (Vangl2), a core component of the Wnt/planar cell polarity (PCP) signaling pathway, is a four-pass transmembrane protein with N-terminal and C-terminal domains located in the cytosol, and is structurally conserved from flies to mammals. In vertebrates, Vangl2 plays an essential role in convergence and extension (CE) movements during gastrulation and in facial branchiomotor (FBM) neuron migration in the hindbrain. However, the roles of specific Vangl2 domains, of membrane association, and of specific extracellular and intracellular motifs have not been examined, especially in the context of FBM neuron migration. Through heat shock-inducible expression of various Vangl2 transgenes, we found that membrane associated functions of the N-terminal and C-terminal domains of Vangl2 are involved in regulating FBM neuron migration. Importantly, through temperature shift experiments, we found that the critical period for Vangl2 function coincides with the initial stages of FBM neuron migration out of rhombomere 4. Intriguingly, we have also uncovered a putative nuclear localization motif in the C-terminal domain that may play a role in regulating CE movements.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Neurônios Motores/metabolismo , Peixe-Zebra/crescimento & desenvolvimento , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Movimento Celular/genética , Nervo Facial/metabolismo , Gastrulação/genética , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Estrutura Terciária de Proteína , Rombencéfalo/crescimento & desenvolvimento , Rombencéfalo/metabolismo , Peixe-Zebra/metabolismoRESUMO
Vangl2, a core component of the Planar Cell Polarity pathway, is necessary for the caudal migration of Facial Branchiomotor (FBM) neurons in the vertebrate hindbrain. Studies in zebrafish suggest that vangl2 functions largely non-cell autonomously to regulate FBM neuron migration out of rhombomere 4 (r4), but the cell-type within which it acts is not known. Here, we demonstrate that vangl2 functions largely in floor plate cells to regulate caudal neuronal migration. Furthermore, FBM neurons fail to migrate caudally in the mouse Gli2 mutant that lacks the floor plate, suggesting an evolutionarily conserved role for this cell type in neuronal migration. Although hindbrain floor plate cilia are disorganized in vangl2 mutant embryos, cilia appear to be dispensable for neuronal migration. Notably, Vangl2 is enriched in the basolateral, but not apical, membranes of floor plate cells. Taken together, our data suggest strongly that Vangl2 regulates FBM neuron migration by acting in floor plate cells, independently of cilia function.
Assuntos
Cílios/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Membrana/genética , Neurônios Motores/metabolismo , Rombencéfalo/metabolismo , Proteínas de Peixe-Zebra/genética , Animais , Movimento Celular , Polaridade Celular , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Neurogênese , Rombencéfalo/citologia , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Proteína Gli2 com Dedos de ZincoRESUMO
During development, facial branchiomotor (FBM) neurons, which innervate muscles in the vertebrate head, migrate caudally and radially within the brainstem to form a motor nucleus at the pial surface. Several components of the Wnt/planar cell polarity (PCP) pathway, including the transmembrane protein Vangl2, regulate caudal migration of FBM neurons in zebrafish, but their roles in neuronal migration in mouse have not been investigated in detail. Therefore, we analyzed FBM neuron migration in mouse looptail (Lp) mutants, in which Vangl2 is inactivated. In Vangl2(Lp/+) and Vangl2(Lp/Lp) embryos, FBM neurons failed to migrate caudally from rhombomere (r) 4 into r6. Although caudal migration was largely blocked, many FBM neurons underwent normal radial migration to the pial surface of the neural tube. In addition, hindbrain patterning and FBM progenitor specification were intact, and FBM neurons did not transfate into other non-migratory neuron types, indicating a specific effect on caudal migration. Since loss-of-function in some zebrafish Wnt/PCP genes does not affect caudal migration of FBM neurons, we tested whether this was also the case in mouse. Embryos null for Ptk7, a regulator of PCP signaling, had severe defects in caudal migration of FBM neurons. However, FBM neurons migrated normally in Dishevelled (Dvl) 1/2 double mutants, and in zebrafish embryos with disrupted Dvl signaling, suggesting that Dvl function is essentially dispensable for FBM neuron caudal migration. Consistent with this, loss of Dvl2 function in Vangl2(Lp/+) embryos did not exacerbate the Vangl2(Lp/+) neuronal migration phenotype. These data indicate that caudal migration of FBM neurons is regulated by multiple components of the Wnt/PCP pathway, but, importantly, may not require Dishevelled function. Interestingly, genetic-interaction experiments suggest that rostral FBM neuron migration, which is normally suppressed, depends upon Dvl function.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Neurônios Motores/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Fosfoproteínas/fisiologia , Animais , Diferenciação Celular , Movimento Celular , Polaridade Celular , Proteínas Desgrenhadas , Camundongos , Camundongos Knockout , Camundongos Mutantes , Camundongos Transgênicos , Modelos Neurológicos , Neurônios Motores/citologia , Rede Nervosa/citologia , Rede Nervosa/embriologia , Rede Nervosa/fisiologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Neurogênese/genética , Neurogênese/fisiologia , Receptores Proteína Tirosina Quinases/deficiência , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/fisiologia , Rombencéfalo/citologia , Rombencéfalo/embriologia , Via de Sinalização Wnt , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologiaRESUMO
The amino acid sequence across the DNA-binding homeodomain of Gbx2 is highly conserved across multiple species. In mice, Gbx2 is essential for establishment of the midbrain-hindbrain boundary (MHB), and in development of anterior hindbrain structures, rhombomeres (r) 1-r3, and the r2/r3-derived cranial nerve V. In contrast, studies in zebrafish have implicated gbx1 in establishment of the MHB. Therefore, we tested potential roles for gbx2 in anterior hindbrain development in zebrafish. gbx2 knockdown with antisense morpholino results in increased cell death in r2, r3, and r5 and a truncation of the anterior hindbrain, similar to the defect in Gbx2(-/-) mice. Moreover, there is abnormal clustering of cranial nerve V cell bodies in r2 and r3 indicative of defects in aspects of anterior hindbrain patterning. These phenotypes can be rescued by expression of the mouse GBX2 protein. These results suggest that gbx2/Gbx2 has an evolutionarily conserved role in anterior hindbrain development.
Assuntos
Evolução Biológica , Proteínas de Homeodomínio/fisiologia , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Morte Celular/genética , Morte Celular/fisiologia , Proliferação de Células , Sequência Conservada/fisiologia , Embrião de Mamíferos , Embrião não Mamífero , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Rombencéfalo/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Polycystic kidney disease (PKD) is one of the leading causes of end-stage renal disease in humans and is characterized by progressive cyst formation, renal enlargement, and abnormal tubular development. Currently, there is no cure for PKD. Although a number of PKD genes have been identified, their precise role in cystogenesis remains unclear. In the jcpk mouse model of PKD, mutations in the bicaudal C gene (Bicc1) are responsible for the cystic phenotype; however, the function of Bicc1 is unknown. In this study, we establish an alternative, nonmammalian zebrafish model to study the role of Bicc1 in PKD pathogenesis. Antisense morpholinos were used to evaluate loss of Bicc1 function in zebrafish. The resulting morphants were examined histologically for kidney cysts and structural abnormalities. Immunostaining and fluorescent dye injection were used to evaluate pronephric cilia and kidney morphogenesis. Knockdown of zebrafish Bicc1 expression resulted in the formation of kidney cysts; however, defects in kidney structure or pronephric cilia were not observed. Importantly, expression of mouse Bicc1 rescues the cystic phenotype of the morphants. These results demonstrate that the function of Bicc1 in the kidney is evolutionarily conserved, thus supporting the use of zebrafish as an alternative in vivo model to study the role of mammalian Bicc1 in renal cyst formation.
Assuntos
Modelos Animais de Doenças , Doenças Renais Policísticas , Proteínas de Ligação a RNA/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra , Sequência de Aminoácidos , Animais , Embrião não Mamífero/anatomia & histologia , Embrião não Mamífero/patologia , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Doenças Renais Císticas/metabolismo , Doenças Renais Císticas/patologia , Camundongos , Dados de Sequência Molecular , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fenótipo , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia , Proteínas de Ligação a RNA/genética , Alinhamento de Sequência , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The transmembrane protein Van gogh-like 2 (Vangl2) is a component of the noncanonical Wnt/Planar Cell Polarity (PCP) signaling pathway, and is required for tangential migration of facial branchiomotor neurons (FBMNs) from rhombomere 4 (r4) to r5-r7 in the vertebrate hindbrain. Since vangl2 is expressed throughout the zebrafish hindbrain, it might also regulate motor neuron migration in other rhombomeres. We tested this hypothesis by examining whether migration of motor neurons out of r2 following ectopic hoxb1b expression was affected in vangl2(-) (trilobite) mutants. Hoxb1b specifies r4 identity, and when ectopically expressed transforms r2 to an "r4-like" compartment. Using time-lapse imaging, we show that GFP-expressing motor neurons in the r2/r3 region of a hoxb1b-overexpressing wild-type embryo migrate along the anterior-posterior (AP) axis. Furthermore, these cells express prickle1b (pk1b), a Wnt/PCP gene that is specifically expressed in FBMNs and is essential for their migration. Importantly, GFP-expressing motor neurons in the r2/r3 region of hoxb1b-overexpressing trilobite mutants and pk1b morphants often migrate, even though FBMNs in r4 of the same embryos fail to migrate longitudinally (tangentially) into r6 and r7. These observations suggest that tangentially migrating motor neurons in the anterior hindbrain (r1-r3) can use mechanisms that are independent of vangl2 and pk1b functions. Interestingly, analysis of tri; val double mutants also suggests a role for vangl2-independent factors in neuronal migration, since the valentino mutation partially suppresses the trilobite mutant migration defect. Together, the hoxb1b and val experiments suggest that multiple mechanisms regulate motor neuron migration along the AP axis of the zebrafish hindbrain.
Assuntos
Movimento Celular/fisiologia , Neurônios Motores/fisiologia , Rombencéfalo/embriologia , Rombencéfalo/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/metabolismo , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Proteínas com Domínio LIM , Fator de Transcrição MafB/genética , Fator de Transcrição MafB/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Confocal , Modelos Neurológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo , Rombencéfalo/citologia , Transdução de Sinais , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Interactions between a neuron and its environment play a major role in neuronal migration. We show here that the cell adhesion molecule Transient Axonal Glycoprotein (Tag1) is necessary for the migration of the facial branchiomotor neurons (FBMNs) in the zebrafish hindbrain. In tag1 morphant embryos, FBMN migration is specifically blocked, with no effect on organization or patterning of other hindbrain neurons. Furthermore, using suboptimal morpholino doses and genetic mutants, we found that tag1, lamininalpha1 (lama1) and stbm, which encodes a transmembrane protein Vangl2, exhibit pairwise genetic interactions for FBMN migration. Using time-lapse analyses, we found that FBMNs are affected similarly in all three single morphant embryos, with an inability to extend protrusions in a specific direction, and resulting in the failure of caudal migration. These data suggest that tag1, lama1 and vangl2 participate in a common mechanism that integrates signaling between the FBMN and its environment to regulate migration.
Assuntos
Moléculas de Adesão Celular Neuronais/fisiologia , Laminina/fisiologia , Proteínas de Membrana/fisiologia , Neurônios Motores/fisiologia , Rombencéfalo/embriologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/embriologia , Animais , Adesão Celular , Movimento Celular , Contactina 2 , Rombencéfalo/fisiologia , Transdução de Sinais , Peixe-Zebra/fisiologiaRESUMO
BACKGROUND: How axon guidance signals regulate growth cone behavior and guidance decisions in the complex in vivo environment of the central nervous system is not well understood. We have taken advantage of the unique features of the zebrafish embryo to visualize dynamic growth cone behaviors and analyze guidance mechanisms of axons emerging from a central brain nucleus in vivo. RESULTS: We investigated axons of the nucleus of the medial longitudinal fascicle (nucMLF), which are the first axons to extend in the zebrafish midbrain. Using in vivo time-lapse imaging, we show that both positive axon-axon interactions and guidance by surrounding tissue control initial nucMLF axon guidance. We further show that two guidance molecules, transient axonal glycoprotein-1 (TAG-1) and laminin-alpha1, are essential for the initial directional extension of nucMLF axons and their subsequent convergence into a tight fascicle. Fixed tissue analysis shows that TAG-1 knockdown causes errors in nucMLF axon pathfinding similar to those seen in a laminin-alpha1 mutant. However, in vivo time-lapse imaging reveals that while some defects in dynamic growth cone behavior are similar, there are also defects unique to the loss of each gene. Loss of either TAG-1 or laminin-alpha1 causes nucMLF axons to extend into surrounding tissue in incorrect directions and reduces axonal growth rate, resulting in stunted nucMLF axons that fail to extend beyond the hindbrain. However, defects in axon-axon interactions were found only after TAG-1 knockdown, while defects in initial nucMLF axon polarity and excessive branching of nucMLF axons occurred only in laminin-alpha1 mutants. CONCLUSION: These results demonstrate how two guidance cues, TAG-1 and laminin-alpha1, influence the behavior of growth cones during axon pathfinding in vivo. Our data suggest that TAG-1 functions to allow growth cones to sense environmental cues and mediates positive axon-axon interactions. Laminin-alpha1 does not regulate axon-axon interactions, but does influence neuronal polarity and directional guidance.