Mutations in KEOPS-Complex Genes Cause Nephrotic Syndrome with Primary Microcephaly

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms

Genotyping of CRISPR/Cas9 genome edited Xenopus tropicalis

The targeted nuclease revolution (ZFN, TALEN, and CRISPR/Cas9) has led to a myriad of reports describing genotyping methodologies for genome edited founders (F0-crispants) and their offspring (F1). As such, choosing a specific genotyping methodology for your Xenopus CRISPR/Cas9 experiments can be challenging. In this chapter we will discuss, with emphasis on Xenopus tropicalis (X. tropicalis), different methods for assessing genome editing efficiencies within F0 CRISPR/Cas9 founders and for identification of their hetero-, compound hetero-, and homozygous mutant F1 offspring. For F0 crispants, we will provide the protocols and the respective (dis)advantages of genotyping with heteroduplex mobility assay (HMA), subclone Sanger sequencing, and sequence trace decomposition. Furthermore, we provide a previously unpublished pipe-line for rapid genotyping of F1 offspring-high resolution melting analysis (HRMA) and sequence trace decomposition-procured from breeding with F0 crispants. As such, we report here the current state-of-the-art cost- and time-effective approaches to perform genotyping of CRISPR/Cas9 experiments for the Xenopus tropicalis researcher

The zebrafish histamine H3 receptor modulates aggression, neural activity and forebrain functional connectivity.

AIM: Aggression is a behavioural trait characterized by the intention to harm others for offensive or defensive purposes. Neurotransmitters such as serotonin and dopamine are important mediators of aggression. However, the physiological role of the histaminergic system during this behaviour is currently unclear. Here, we aimed to better understand histaminergic signalling during aggression by characterizing the involvement of the histamine H3 receptor (Hrh3). METHODS: We have generated a novel zebrafish Hrh3 null mutant line using CRISPR-Cas9 genome engineering and investigated behavioural changes and alterations to neural activity using whole brain Ca2+ imaging in zebrafish larvae and ribosomal protein S6 (rpS6) immunohistochemistry in adults. RESULTS: We show that genetic inactivation of the histamine H3 receptor (Hrh3) reduces aggression in zebrafish, an effect that can be reproduced by pharmacological inhibition. In addition, hrh3-/- zebrafish show behavioural impairments consistent with heightened anxiety. Larval in vivo whole brain Ca2+ imaging reveals higher neuronal activity in the forebrain of mutants, but lower activity in specific hindbrain areas and changes in measures of functional connectivity between sub-regions. Adult hrh3-/- zebrafish display brain region-specific neural activity changes in response to aggression of both key regions of the social decision making network, and the areas containing histaminergic neurons in the zebrafish brain. CONCLUSION: These results highlight the importance of zebrafish Hrh3 signalling for aggression and anxiety and uncover the brain areas involved. Targeting this receptor might be a potential novel therapeutic route for human conditions characterized by heightened aggression