Loss of Atoh8 Affects Neurocranial and Axial Skeleton Development in Zebrafish.

Background: The basic helix-loop-helix (bHLH) transcription factor atonal homologue 8 (Atoh8) has been implicated in various developmental and physiological processes by means of transient knockdown and conditional knockout approaches in zebrafish, chick and mouse. Despite its demonstrated involvement in multiple tissues, the role of Atoh8 remains elusive in zebrafish. A recent permanent knockout study in zebrafish investigated the role of Atoh8 on the background of previous morpholino studies which demonstrated various developmental defects but could not find any of the morpholino-based effects in the mutant. In mice, a knockout study demonstrated involvement of the transcription factor in skeletal development, showing that disruption of the gene results in reduction of skeletal size. We investigated a mutant fish line generated using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9)-technology for possible phenotypic effects on zebrafish skeletogenesis.

Methods: Here, we present a CRISPR/Cas9-generated permanent zebrafish mutant and investigate the phenotypic effects of the knockout on the developing zebrafish craniofacial and axial skeleton. We investigated the expression pattern of the gene in wildtype and conducted detailed morphometric analysis for a variety of bone and cartilage elements of the developing skeleton at 12 days post fertilisation (dpf) in zebrafish siblings from a heterozygous mating using detailed morphometric measurements and statistical analysis of the results.

Results: Homozygous mutants are viable into late adulthood and show no overt morphological phenotype. Despite the prominent appearance of signal in various embryonic and larval craniofacial and axial skeletal structures, detailed morphometric analysis revealed only subtle phenotypic effects of the mutation on skeletal development in zebrafish. We found the formation of the orbital cartilages of the developing neurocranium and the progress of chordacentra mineralisation to be negatively affected by loss of the transcription factor.

Conclusions: Despite the very subtle phenotypic effect of our mutation, we were able to show involvement of in the skeletal development of zebrafish. We attribute the mild phenotype to a compensatory mechanism induced by nonsense-mediated degradation of messenger ribonucleic acid (mRNA) as suggested in the recent literature. The effect of -disruption on zebrafish skeletal development suggests that the loss of cannot be compensated for at interfaces where more than one embryonic cell lineage contributes to bone and cartilage formation.