|
Fig. 3 bette davis (bdav) Nicotine response mutation is linked to a GBT insertion in zebrafish chaperone containing protein 8 (cct8). (A) The founder F1 animal (Fig. 2C) harbored three independently expressed GBTs. Upon out-crossing and further analysis, animals with altered nicotine response profile (A) harboring only a single GFP locus were isolated and propagated for six generations formally isolating the bdav nicotine response locus. Nicotine was administered at the standard dose of 10 μM. (B) bdav heterozygous fish show no difference in response to allyl isothiocyanate or extreme temperatures when compared to their wild-type siblings. (C) The single expressed GFP locus is encoded by a GBT integration in predicted intron two of zebrafish chaperone containing protein 8 (cct8). (D) RT-PCR analysis of cct8 transcript in wild-type, heterozygous, and homozygous bdav embryos. Primers in exons 1 and 5 were used to detect wild-type transcript of the cct8 gene. Primers in exon 5 and the GFP portion of the transposon were used to determine expression of transposon mRNA. RT-PCR using primers in β-actin were performed as an internal control. (E) Sagittal imaging of a whole mount in situ hybridization for cct8 at 1 day post fertilization (pf) (top) through 5 days pf (bottom) shows high levels of neural expression. (F) Phylogenetic analysis (Ensembl) indicates cct8 is an evolutionarily ancient gene and is encoded by a single ortholog in humans and other mammals. *, P < 0.05 when comparing to control or acute. **, P < 0.05 when comparing to corresponding treatment group.