RESUMEN
LRRK2 gain-of-function is considered a major cause of Parkinson's disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD.
Asunto(s)
Monoaminas Biogénicas/metabolismo , Encéfalo/metabolismo , Eliminación de Gen , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Ansiedad/genética , Encéfalo/embriología , Encéfalo/enzimología , Sistemas CRISPR-Cas , Larva/metabolismo , Monoaminooxidasa/metabolismo , Olfato/genética , Natación , Pez Cebra/embriologíaRESUMEN
Serodiagnostic tests for antibody detection to estimate the immunoprotective status regarding SARS-CoV-2 support diagnostic management. This study aimed to investigate the performance of serological assays for COVID-19 and elaborate on test-specific characteristics. Sequential samples (n = 636) of four panels (acute COVID-19, convalescent COVID-19 (partly vaccinated post-infection), pre-pandemic, and cross-reactive) were tested for IgG by indirect immunofluorescence test (IIFT) and EUROIMMUN EUROLINE Anti-SARS-CoV-2 Profile (IgG). Neutralizing antibodies were determined by a virus neutralization test (VNT) and two surrogate neutralization tests (sVNT, GenScript cPass, and EUROIMMUN SARS-CoV-2 NeutraLISA). Analysis of the acute and convalescent panels revealed high positive (78.3% and 91.6%) and negative (91.6%) agreement between IIFT and Profile IgG. The sVNTs revealed differences in their positive (cPass: 89.4% and 97.0%, NeutraLISA: 71.5% and 72.1%) and negative agreement with VNT (cPass: 92.3% and 50.0%, NeutraLISA: 95.1% and 92.5%) at a diagnostic specificity of 100% for all tests. The cPass showed higher inhibition rates than NeutraLISA at VNT titers below 1:640. Cross-reactivities were only found by cPass (57.1%). Serodiagnostic tests, which showed substantial agreement and fast runtime, could provide alternatives for cell-based assays. The findings of this study suggest that careful interpretation of serodiagnostic results obtained at different times after SARS-CoV-2 antigen exposure is crucial to support decision-making in diagnostic management.
Asunto(s)
COVID-19 , Humanos , COVID-19/diagnóstico , Inmunidad Humoral , SARS-CoV-2 , Pruebas Serológicas , Inmunoglobulina G , Prueba de COVID-19RESUMEN
BACKGROUND: Studies in mouse, Xenopus and chicken have shown that Otx2 and Gbx2 expression domains are fundamental for positioning the midbrain-hindbrain boundary (MHB) organizer. Of the two zebrafish gbx genes, gbx1 is a likely candidate to participate in this event because its early expression is similar to that reported for Gbx2 in other species. Zebrafish gbx2, on the other hand, acts relatively late at the MHB. To investigate the function of zebrafish gbx1 within the early neural plate, we used a combination of gain- and loss-of-function experiments. RESULTS: We found that ectopic gbx1 expression in the anterior neural plate reduces forebrain and midbrain, represses otx2 expression and repositions the MHB to a more anterior position at the new gbx1/otx2 border. In the case of gbx1 loss-of-function, the initially robust otx2 domain shifts slightly posterior at a given stage (70% epiboly), as does MHB marker expression. We further found that ectopic juxtaposition of otx2 and gbx1 leads to ectopic activation of MHB markers fgf8, pax2.1 and eng2. This indicates that, in zebrafish, an interaction between otx2 and gbx1 determines the site of MHB development. Our work also highlights a novel requirement for gbx1 in hindbrain development. Using cell-tracing experiments, gbx1 was found to cell-autonomously transform anterior neural tissue into posterior. Previous studies have shown that gbx1 is a target of Wnt8 graded activity in the early neural plate. Consistent with this, we show that gbx1 can partially restore hindbrain patterning in cases of Wnt8 loss-of-function. We propose that in addition to its role at the MHB, gbx1 acts at the transcriptional level to mediate Wnt8 posteriorizing signals that pattern the developing hindbrain. CONCLUSION: Our results provide evidence that zebrafish gbx1 is involved in positioning the MHB in the early neural plate by refining the otx2 expression domain. In addition to its role in MHB formation, we have shown that gbx1 is a novel mediator of Wnt8 signaling during hindbrain patterning.
Asunto(s)
Tronco Encefálico/embriología , Tronco Encefálico/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/metabolismo , Animales , Tipificación del Cuerpo/genética , Tronco Encefálico/citología , Diferenciación Celular/genética , Linaje de la Célula/genética , Proteínas del Citoesqueleto/genética , Regulación hacia Abajo/genética , Regulación del Desarrollo de la Expresión Génica/genética , Genes Homeobox/genética , Proteínas de Homeodominio/genética , Mesencéfalo/citología , Mesencéfalo/embriología , Mesencéfalo/metabolismo , Biología Molecular/métodos , Mutación/genética , Tubo Neural/citología , Tubo Neural/embriología , Tubo Neural/metabolismo , Factores de Transcripción Otx/genética , Factores de Transcripción Otx/metabolismo , Rombencéfalo/citología , Rombencéfalo/embriología , Rombencéfalo/metabolismo , Transducción de Señal/genética , Activación Transcripcional/genética , Proteínas Wnt/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Dickkopf-1 (Dkk1) is a secreted protein that negatively modulates the Wnt/beta catenin pathway. Lack of Dkk1 function affects head formation in frog and mice, supporting the idea that Dkk1 acts as a "head inducer" during gastrulation. We show here that lack of Dkk1 function accelerates internalization and rostral progression of the mesendoderm and that gain of function slows down both internalization and convergence extension, indicating a novel role for Dkk1 in modulating these movements. The motility phenotype found in the morphants is not observed in embryos in which the Wnt/beta catenin pathway is overactivated, and that dominant-negative Wnt proteins are not able to rescue the gastrulation movement defect induced by absence of Dkk1. These data strongly suggest that Dkk1 is acting in a beta catenin independent fashion when modulating gastrulation movements. We demonstrate that the glypican 4/6 homolog Knypek (Kny) binds to Dkk1 and that they are able to functionally interact in vivo. Moreover, Dkk1 regulation of gastrulation movements is kny dependent. Kny is a component of the Wnt/planar cell polarity (PCP) pathway. We found that indeed Dkk1 is able to activate this pathway in both Xenopus and zebrafish. Furthermore, concomitant alteration of the beta catenin and PCP activities is able to mimic the morphant accelerated cell motility phenotype. Our data therefore indicate that Dkk1 regulates gastrulation movement through interaction with LRP5/6 and Kny and coordinated modulations of Wnt/beta catenin and Wnt/PCP pathways.