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Fig. S2
Expression of Otp and CRH in the Mouse PVN and Levels of Zebrafish crh in Response to Osmotic Challenge, Related to Figure 1
(A and B) Expression of Otp and CRH in the mouse PVN. Coronal sections through the adult mouse hypothalamus showing colocalization of Otp and crh in the mouse paraventricular nucleus.
(A) Floating brain sections were subjected to in situ hybridization with a crh- directed probe followed by immuno-staining with an anti-Otp antibody. High-magnification of the crh+;Otp+ area (black rectangle) is shown in panel A′.
(B) Double immuno-fluorescence staining with guinea pig anti-CRH antibogy (Bachem California, Torrance, USA) together with rabbit anti-Otp antibody, which was generated in our lab.
(C and D) Attenuated osmotic stress response in otpam866 mutant. Osmotic stress challenge was applied to 6 day-old progenies of a otpam866-/+ cross by incubation in 50% artificial seawater for 4 min (See details in the Supporting Material). The amounts of crh (C, n=6) and a2bp1 (D, n=5) mRNAs were measured in control and stressed fish larvae using quantitative PCR (qPCR). Larvae were genotyped by sequencing and crh mRNA levels of mutant (-/-) and heterozygous (-/+) animals were plotted accordingly. *p<0.05.
(E) Representative examples of otp:Gal4 transgenic line that was co-injected with transposon-based (Tol2) vector harboring EGFP under the control of UAS elements together with transposase mRNA. EGFP expression is readily visible in 6-day old larvae.