Dickkopf-1 (Dkk1) is a secreted protein that negatively modulates the Wnt/␤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/␤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 ␤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 ␤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/␤catenin and Wnt/PCP pathways.[Keywords: Dickkopf-1; HSPG; Wnt/PCP; gastrulation movements] Supplemental material is available at http://www.genesdev.org.

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.

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.

LRRK2 mutations are a major cause of Parkinson's disease. Pathogenicity of LRRK2 loss-of-function is controversial, as knockout in rodents induces no brain-specific effects and knockdown studies in zebrafish are conflicting. Here we show that deletion of the ~60-kbp-long zebrafish lrrk2 locus elicits a pleomorphic, albeit transient brain phenotype in maternal-zygotic mutants (mzLrrk2). Intriguingly, 11-month-old mzLrrk2 adults display increased amine catabolism. Additionally, we find decreased mitosis in the larval brain and reduced stab injury-induced neuronal regeneration in the adult telencephalon. Finally, hypokinesia associates with loss of lrrk2 in larvae. Our results demonstrate that lrrk2 knockout has an early neurodevelopmental effect. We report for the first time perturbed amine catabolism in a LRRK2 knockout. We propose mzLrrk2 zebrafish as a valuable tool to study LRRK2 loss-of-function in vivo, and provide a link between LRRK2 and the control of basal cell proliferation in the brain, potentially critical upon challenges like brain injury.Recently, a third paper rekindled the initial claims, describing a lrrk2 MO-induced phenotype with macroscopic developmental abnormalities (24). These discrepancies revived concerns over the consistency of MO-induced knockdown to assess gene loss-of-function due to the variability and transiency of induced changes and the risk of off-target effects (25). Although the analysis of MO-induced phenotypes may still provide useful information, their validation would inevitably entail the generation of reliable null alleles (26).Using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome-editing tool, we report here the deletion of the ~60-kbp-long zebrafish lrrk2 locus containing the entire open reading frame (ORF), resulting in an unambiguous null allele. This is the first report describing LRRK2 deficiency in a vertebrate in vivo model where no residual or truncated protein is produced.We characterized the phenotype of the brain, as the organ of possibly highest relevance for PD. We find that maternal-zygotic lrrk2 mutants display a pleomorphic, but transient neurodevelopmental phenotype, including increased apoptosis, delayed myelination, reduced and morphologically abnormal microglia/leukocytes, and reduced catecholaminergic neurons. We also find a correlation between hypokinesia and loss of lrrk2 in larvae. Importantly, for the first time in a LRRK2 knockout model, we report perturbed amine catabolism in older animals. Finally, we observe decreased mitosis in the larval brain and impaired neuronal regeneration after stabbing the adult telencephalon. Our results suggest a link between zebrafish Lrrk2 and the control of cell proliferation in the brain, with crucial implications for the self-healing capacity upon lesion.