In the inner ear of higher vertebrates, the cupula is the sensory extracellular matrix that sits atop the crista ampullaris and, by coupling fluid motion in the semi-circular canals to the sensory hair cells, it is involved in detecting the rotation of the head and is key for gaze stabilization and proper postural balance. Structural impairment or mechanical detachment of the cupula from the ampulla is associated with a clinical picture of sudden loss of vestibular function. Recently, zona pellucida-like domain containing protein 1 (ZPLD1, also known as Cupulin), has been identified as the major structural component of the cupula in salmon. In mouse, two spontaneous mutations in the Zpld1 gene, “spiral” (sprl) and “circler with hearing” (cwh), but not a predicted loss-of-function deletion (Zpld1−/−), have been reported to cause variably penetrant hyperactive circling behaviour – a phenotypical indicator of a prominent balance dysfunction. Despite the recent findings, little is still known about the role of ZPLD1 in the function and morphology of the cupula. Moreover, a clear phenotypical characterisation of animal models with a complete loss of ZPLD1 protein is still lacking in the literature. In this study, I describe the morphological and behavioural changes in mouse and zebrafish lacking ZPLD1. To characterise the mutant phenotype, animal behaviour was tracked and analysed using a machine-learning approach (DeepLabCut). In addition, mice underwent a short period of light deprivation to determine whether visual cues compensate for loss of ZPLD1. Immunofluorescence microscopy on inner-ear cryosections from fish and mice was used to investigate the morphology of the cupula. Overall, the findings reveal that Zpld1-/- mice exhibit variably penetrant circling behaviour. All mice exhibit increased head-turning activity, and this decreases as the mice mature. A short period of visual deprivation does not cause a significant change in the animal behaviour. Loss of ZPLD1 affects cupular structure, as in its absence the cupula is truncated and fails to reach the roof of the ampulla. In zebrafish ZPLD1 is encoded by two genes, zpld1a and zpld1b. The findings suggest that zpld1a alone is crucial for the expression of ZPLD1, without which the cupula is no longer detected. Moreover, zpld1a homozygous mutants exhibit greater head-turning activity and average speed than zpld1b homozygous mutant fish.</p
