|
Fig. 6
Modulation of Stress Response by Alternative Splicing of PAC1 (A) Quantitative PCR analysis of the two pac1 splice isoforms in mice that were subjected to a foot shock challenge. PVN punches were harvested at different time points following stress initiation. The analyzed pac1 cDNA fragments corresponding to the short and long (hop) splice isoform are schematically shown. The hop cassette is encoded by exon 14 (E14). -p < 0.05, n = 11. (B) Calculated ratio between the amounts of the long and short pac1 isoforms shown in (A) as a function of time after the initiation of the foot shock stressor showing a positive linear correlation. R2 = 0.9, n = 11. (C) The top part shows a scheme depicting pac1a gene structure including the respective binding sites for two antisense MO oligonucleotides designed either to block expression of both PAC1 isoforms (pac1a-ATG MO) or prevent the inclusion of the hop exon by alternative splicing (pac1a-hop MO). The bottom part shows a RT analysis of 6-day-old larvae showing the effect of the pac1a-hop MO. The long (hop) and short splice variants (arrows) are visualized in the control larvae, whereas only the short isoform is present in pac1a-hop MO-injected animals. Inclusion of adjacent constitutive exons is not affected by the pac1a-hop MO. (D–F) Quantitative PCR analyses of crh mRNA following a physical stress challenge of 6-day-old larva. The amount of crh mRNA was measured in individual fish larvae at different time points of recovery. (D) Stress challenge was applied to either mock-treated 6-day-old larvae (control, n = 14) or their siblings that were injected with antisense pac1a-ATG MO (n = 12), which prevents the formation of both PAC1 isoforms. Alternatively, transgenic otp:Gal4 embryos expressing Gal4 in Otp+ cells were coinjected with pac1a-ATG MO together with transposon-based vector constructs harboring PAC1′s short (pac1a-ATG MO+PAC1-short, n = 4) and long (pac1a-ATG MO+PAC1-long, n = 4) isoforms under the control of ten UAS elements (UAS:PAC1-short or UAS:PAC1-long, respectively). -p d 0.01. (E) Stress challenge was applied to either mock-treated 6-day-old transgenic otp:Gal4 larvae (control, n = 11) or their siblings, which were injected with a pac1a-hop MO (n = 12) or pac1a-ATG MO together with UAS:PAC1-long construct (pac1a-ATG MO+PAC1-long, n = 6). -p d 0.05. (F) Stress challenge was applied to either a otp:Gal4 transgenic line larvae (control, n = 10) or their siblings, which were injected with constructs containing the short (n = 7) or long (n = 6) PAC1 isoform under the control of ten UAS elements (otp:Gal4 >> UAS:PAC1-short or otp:Gal4 >> UAS:PAC1-long, respectively). The amount of crh mRNA was measured as described above. -p < 0.05. (G) Analysis of cortisol content following a physical stress challenge applied to either mock-treated 6-day-old larvae (control) or their siblings, which were injected with pac1a-hop MO. Whole-body cortisol levels were measured at different time points of recovery in tissue extracts derived from pools of ten larvae. -p < 0.05, n = 3. The following abbreviations are used: AS, alternative spliced exon; MO, morpholino; ns, not significant; RQ, relative quantity.