A Zebrafish Model for a Rare Genetic Disease Reveals a Conserved Role for FBXL3 in the Circadian Clock System

Abstract

The circadian clock, which drives a wide range of bodily rhythms in synchrony with the day–night cycle, is based on a molecular oscillator that ticks with a period of approximately 24 h. Timed proteasomal degradation of clock components is central to the fine-tuning of the oscillator’s period. FBXL3 is a protein that functions as a substrate-recognition factor in the E3 ubiquitin ligase complex, and was originally shown in mice to mediate degradation of CRY proteins and thus contribute to the mammalian circadian clock mechanism. By exome sequencing, we have identified a FBXL3 mutation in patients with syndromic developmental delay accompanied by morphological abnormalities and intellectual disability, albeit with a normal sleep pattern. We have investigated the function of FBXL3 in the zebrafish, an excellent model to study both vertebrate development and circadian clock function and, like humans, a diurnal species. Loss of fbxl3a function in zebrafish led to disruption of circadian rhythms of promoter activity and mRNA expression as well as locomotor activity and sleep–wake cycles. However, unlike humans, no morphological effects were evident. These findings point to an evolutionary conserved role for FBXL3 in the circadian clock system across vertebrates and to the acquisition of developmental roles in humans.

Establishment of cell lines from individual zebrafish embryos

“…Regular veterinary inspections (every three months) confirmed that there were no known pathogens affecting the fish in our facility. The fbxl3 –/– line 14 is registered in the Zebrafish Model Organism Database (ZFIN) as fbxl3a tlv 0 . Eggs were derived from in-crosses of 6–9-month-old fish.…”

“…The genotyping of the 24 hpf embryos was performed via PCR followed by NlaIII restriction enzyme digestion (New England Biolabs) as previously described, 14 with some modifications.…”

“…As proof of principle, here we have used the fbxl3a tlv 08 mutant zebrafish line and its WT sibling line WT tlv 08 . 14 This particular mutant was selected due to the lack of morphological distinguishing features of the homozygous mutant embryos and the availability of a robust molecular genotyping method involving polymerase chain reaction (PCR) and subsequent restriction enzyme digestion. After collecting wild type and mutant embryos, each individual embryo is enzymatically dissociated in parallel, then genotyped via PCR and processed to establish an initial ‘pre-cell line’.…”

Expression of Nerve Growth Factor and Its Receptor TrkA in the Reproductive System of Adult Zebrafish

“…This fascinating model can be utilized in mutagenesis analyses and drug screening tests. It has been used to conduct experiments related to different research fields such as embryology, genetics, cancer, cardiovascular, and organ and tissue regeneration [ 43 , 44 , 45 , 46 ]. In the present report, firstly, in order to identify different cell populations in adult zebrafish testis and ovary, hematoxylin-eosin staining on paraffin sections has been carried out.…”

Zebrafish as a Model for Neurological Disorders

“…In an illustration of the simplicity and power of the zebrafish as a genetic tool, two studies have made use of the relatively new CRISPR/Cas9 technology of gene editing to produce loss-of-function alleles of genes of interest prior to analyzing the induced phenotypes. Starting from a consanguineous family of whom several members presented macrocephaly, facial hypoplasia, hypotonia, and intellectual disability, Confino et al [5] identified the causative mutation in the FBXL3 gene encoding F-box and leucine-rich repeat protein 3 and then generated a loss-of-function allele of fbxl3a, one of the two zebrafish paralogs of this gene. Interestingly, the results suggested that in contrast to the human gene, fbxl3a is not essential for brain development, but is a key component of the circadian clock, as is the case in mice.…”