Zebrafish Cacna1fa is required for cone photoreceptor function and synaptic ribbon formation

Abstract

Mutations in the human CACNA1F gene cause incomplete congenital stationary night blindness type 2 (CSNB2), a non-progressive, clinically heterogeneous retinal disorder. However, the molecular mechanisms underlying CSNB2 have not been fully explored. Here, we describe the positional cloning of a blind zebrafish mutant, wait until dark (wud), which encodes a zebrafish homolog of human CACNA1F. We identified two zebrafish cacna1f paralogs and showed that the cacna1fa transcript (the gene mutated in wud) is expressed exclusively in the photoreceptor layer. We demonstrated that Cacna1fa localizes at the photoreceptor synapse and is absent from wud mutants. Electroretinograms revealed abnormal cone photoreceptor responses from wud mutants, indicating a defect in synaptic transmission. Although there are no obvious morphological differences, we found that wud mutants lacked synaptic ribbons and that wud is essential for the development of synaptic ribbons. We found that Ribeye, the most prominent synaptic ribbon protein, was less abundant and mislocalized in adult wud mutants. In addition to cloning wud, we identified synaptojanin 1 (synj1) as the defective gene in slacker (slak), a blind mutant with floating synaptic ribbons. We determined that Cacna1fa was expressed in slak photoreceptors and that Synj1 was initially expressed wud photoreceptors, but was absent by 5 days postfertilization. Collectively, our data demonstrate that Cacna1fa is essential for cone photoreceptor function and synaptic ribbon formation and reveal a previously unknown yet critical role of L-type voltage-dependent calcium channels in the expression and/or distribution of synaptic ribbon proteins, providing a new model to study the clinical variability in human CSNB2 patients.

Congenital stationary night blindness: An analysis and update of genotype–phenotype correlations and pathogenic mechanisms

“…The wud mutants show no other morphological abnormalities than thinner OPLs. The ERG had a small a-wave followed by a delayed and reduced b-wave component (Jia et al, 2014). There is normal visual background adaptation and spontaneous swimming activity, but optokinetic response and optomotor response are absent, indicating that wud mutants are blind.…”

“…Transgene to model Nougaret-pedigree (Dryja et al, 1996) Reduced rod photoreceptor responses (Jia et al, 2014;Muto et al, 2005) c.3430C>T p.Gln1144* Cabp4 NM_144532.2…”

“…Zebrafish models for CACNA1F gene defect. Recently two zebrafish models with Cacna1f mutations have been described by Jia and colleagues (Jia et al, 2014). The so -called "wait until dark (wud)" mutants were identified in a large-scale mutagenesis screen for defects in visual behaviour (Muto et al, 2005).…”

“…The so -called "wait until dark (wud)" mutants were identified in a large-scale mutagenesis screen for defects in visual behaviour (Muto et al, 2005). Genetic mapping localized the gene defects in both to a region harbouring a gene, later named Cacna1fa, which shows strong homology to human CACNA1F (Jia et al, 2014). Subsequent sequencing revealed c.626T>A and c.3430C>T Cacna1f mutations leading to premature stop codons p.Leu209* and p.Gln1144* respectively.…”

Neuronal remodeling in retinal circuit assembly, disassembly, and reassembly

“…For example, loss of ribbon-associated proteins, such as the presynaptic cytomatrix protein bassoon, results in “floating,” unanchored ribbons in the axonal terminals of both cones and rods [39, 40](Figure 3). Floating ribbons are also observed in the cones and rods of mouse [41–43] (Figure 3) and zebrafish [44–46] mutants with neurotransmission defects. However, disruption in ribbon localization may not be due to altered transmitter release per se because ribbons do not float but instead accumulate in abnormal numbers at individual synaptic sites of TeNT-silenced mouse bipolar cells [35].…”