Shared and unique consequences of Joubert Syndrome gene dysfunction on the zebrafish central nervous system

Abstract

Joubert Syndrome (JBTS) is a neurodevelopmental ciliopathy defined by a highly specific midbrain-hindbrain malformation, variably associated with additional neurological features. JBTS displays prominent genetic heterogeneity with >40 causative genes that encode proteins localising to the primary cilium, a sensory organelle that is essential for transduction of signalling pathways during neurodevelopment, among other vital functions. JBTS proteins localise to distinct ciliary subcompartments, suggesting diverse functions in cilium biology. Currently, there is no unifying pathomechanism to explain how dysfunction of such diverse primary cilia-related proteins results in such a highly specific brain abnormality. In order to identify the shared consequence of JBTS gene dysfunction, we carried out transcriptomic analysis using zebrafish mutants for the JBTS-causative genes cc2d2aw38, cep290fh297, inpp5ezh506, talpid3i264 and togaram1zh510 and the Bardet-Biedl syndrome-causative gene bbs1k742. We identified no commonly dysregulated signalling pathways in these mutants and yet all mutants displayed an enrichment of altered gene sets related to central nervous system function. We found that JBTS mutants have altered primary cilia throughout the brain, however do not display abnormal brain morphology. Nonetheless, behavioural analyses revealed reduced locomotion and loss of postural control which, together with the transcriptomic results, hint at underlying abnormalities in neuronal activity and/or neuronal circuit function. These zebrafish models therefore offer the unique opportunity to study the role of primary cilia in neuronal function beyond early patterning, proliferation and differentiation.

Ciliogenesis defects after neurulation impact brain development and neuronal activity in larval zebrafish

“… 59 , 60 , 86 , 87 , 88 , 89 Of note, cerebellar defects have only been described in a single zebrafish mutant so far, carrying a loss of function allele in the Joubert gene arl13b . 71 , 86 , 90 Notably, our results in the elipsa mutants are similar to the ones reported for the Joubert gene arl13b mutant zebrafish. 90 By characterizing cerebellar malformation in the elipsa mutant, our findings are therefore opening new avenues for understanding cilia-related control of cerebellar development in Joubert’s syndrome using zebrafish as model system.…”

“…We next aimed to identify whether specific cell populations were particularly affected in the mutants, considering that the cerebellum is composed of both glutamatergic excitatory and GABAergic inhibitory neurons. 69 , 70 , 71 We first assessed the excitatory glutamatergic neurons, which consist of mainly eurydendroid cells in addition to some granule cells, 70 ( Figures 3 E and 3F) using the transgenic line Tg(vglut2a:dsRed) , 72 but did not observe a major difference in the location of excitatory glutamatergic neurons despite the abnormal cerebellar morphology. In contrast, the number of parvalbumin-expressing inhibitory Purkinje cells ( Figures 3 G and 3H) was significantly reduced in the mutant animals ( Figure 3 H).…”

“…We could unfortunately not record data from older animals since mutant zebrafish with ciliogenesis defects, including elipsa mutant, typically display a curled tail and do not survive to adulthood. Recent work revealed that the Joubert syndrome gene cc2d2a mutant zebrafish displayed locomotory and postural defects, 71 hinting at underlying abnormalities in neuronal activity and circuit function in this mutant. Future work using cell-specific manipulation of cilia will be critical to pinpoint the precise function of cilia in different cell types of the brain.…”

“…The staining protocol for acetylated tubulin and SV2 staining was performed as per. 71 The larvae were first euthanized and fixed in 2% trichloroacetic acid (TCA) for 3 hours at RT. After incubation, the larvae were washed 4 x 5 mins in PBS.…”