Goldstone, J. V. | bioRxiv Science

bioRxiv2021

Orphan cytochrome P450 20A1 CRISPR/Cas 9 mutants and neurobehavioral phenotypes in zebrafish

Orphan cytochrome P450 (CYP) enzymes are those for which biological substrates and function(s) are unknown. Cytochrome P450 20A1 (CYP20A1) is the last human orphan P450 enzyme, and orthologs occur as single genes in every vertebrate genome sequenced to date. The occurrence of high levels of CYP20A1 transcripts in human substantia nigra and hippocampus and abundant maternal transcripts in zebrafish eggs strongly suggest roles both in the brain and during early embryonic development. Patients with chromosome 2 microdeletions including CYP20A1 show hyperactivity and bouts of anxiety, among other conditions. Here, we created zebrafish CYP20A1 mutants using CRISPR/Cas9, providing vertebrate models with which to study the role of CYP20A1 in behavior and other neurodevelopmental functions. The homozygous cyp20a1 null mutants exhibited significant behavioral differences from wild-type zebrafish, both in larval and adult animals. Larval cyp20a1-/- mutants exhibited a strong increase in light-simulated movement (i.e., light-dark assay), which was interpreted as hyperactivity. Further, the larvae exhibited mild hypoactivity during the adaptation period of the optomotor assays. Adult cyp20a1 null fish showed a pronounced delay in adapting to new environments, which is consistent with an anxiety paradigm. Taken together with our earlier morpholino cyp20a1 knockdown results, the results described herein suggest that the orphan CYP20A1 has a neurophysiological role.

pharmacology and toxicology↗

bioRxiv2019

CRISPR-Cas9 mutated pregnane x receptor (pxr) retains pregnenolone-induced expression of cytochrome p450 family 3, subfamily A, polypeptide 65 (cyp3a65) in zebrafish (Danio rerio) larvae.

Pregnane x receptor (PXR) is a nuclear receptor that regulates transcriptional responses to drug or xenobiotic exposure in many vertebrate species. One key response is the induction of cytochrome P450 3A (CYP3A) transcription. PXR is a promiscuous receptor activated by a wide range of ligands that can differ across species, making functional studies on its role in the chemical defensome, most relevant when approached in a species-specific manner. Genetic knockout studies in mammals have shown a requirement for PXR in ligand-dependent activation of CYP3A expression or reporter gene activity. Transient knockdown in zebrafish revealed a similar requirement, however it is not known what the effect of a genetic knockout would be in the zebrafish model. Here, we report on generation of two zebrafish lines each carrying a genetic deletion in the pxr coding region, predicted to result in loss of a functional gene product. To our surprise zebrafish larvae, homozygous for either of the pxr mutant alleles, retain their ability to induce cyp3a65 mRNA expression following exposure to the established zebrafish Pxr ligand, pregnenolone (PN). Thus, zebrafish carrying pxr alleles with sizable deletions in either the DNA binding or the ligand binding domains do not yield a loss-of-function phenotype, suggesting that a compensatory mechanism is responsible for cyp3a65 induction. Two alternative possibilities are that Pxr is not required for the effect or that truncated yet functional mutant Pxr is sufficient for the effect.

pharmacology and toxicology↗