Fig. 3: rbfox1 LoF disrupts zebrafish larvae HPI axis, bdnf/trkb2 pathway and pac1a expression. TRKB modulation restore HPI gene expression and behaviour.

A Expression levels of bdnf and trkb2 full-length (TK+) and truncated (TK-) in 5 days post fertilisation (dpf) larvae (rbfox1^+/+ and rbfox1^19del/19del). trkb2 expression is shown in a stacked bar format, where the directly measured TK+ (long form) is shown as the lower portion and the inferred TK- (short form) is shown on top. B Expression levels of HPI axis genes crhb, nr3c2, nr3c1, crhr1, crhr2, mc1r, pomca and kf19 in 5dpf larvae (rbfox1^+/+ and rbfox1^19del/19del). C Whole-body cortisol measurement of 5dpf larvae (left panel) and adult fish (right panel) in resting physiological conditions. Cortisol values are normalised per whole-body homogenate (g) (rbfox1^+/+ and rbfox1^19del/19del). D, E Expression levels of crhb and nr3c2 in presence or absence of the TRKB selective D antagonist ANA-12 and E agonist 7,8-DHF in 5dpf larvae (rbfox1^+/+ and rbfox1^19del/19del). F–I Behavioural assays in presence or absence of the TRKB agonist 7,8-DHF in rbfox1 larvae (rbfox1^+/+ and rbfox1^19del/19del): F mean distance travelled, G average speed, H startle response to white light stimulus and I proportion of responders over time in the response and habituation to acoustic startle assay. J–N Whole mount in situ hybridisation (ISH) for trkb2 anti-sense riboprobe in J rbfox1^+/+ and K rbfox1^19del/19del, and L trkb2 sense riboprobe in rbfox1^+/+ 5dpf larvae (lateral view in the main boxes and dorsal view in the smaller boxes on the top right corner). M, N Sagittal cryosection of trkb2 ISH in M rbfox1^+/+ and N rbfox1^19del/19del 5 dpf larvae. Black boxes in M-a and N-a represent the region of the brain showed in higher magnification panels in M-a’ and N-a’. Scale bars: 200 µm in J), K) and L); 100 µm in M-a) and N-a); 50 µm in M-a’) and N-a’). O Schematic depiction (sagittal) of zebrafish larval brain indicating position of levels illustrated by ISH on sagittal cryosections. P trkb2 ISH intensity mean in the hypothalamus of 5dpf zebrafish larvae, rbfox1^+/+ versus rbfox1^19del/19del. N = 4 larvae x genotype. Q Expression levels of pac1-hop and pac1-short in rbfox1^+/+ and rbfox1^19del/19del 5 dpf larvae. For qPCR experiments, reference genes were actin – β 2 (actb2) and ribosomal protein L13a (rpl13a). Each green dot/pink triangle in A-D, K-L represents a pool of 15 larval heads (eyes and jaw removed), while yellow ones represent larvae exposed to TRKB drugs. Where indicated, we used Log₁₀ transformation to normalise the data facilitating a clearer visualisation of trends within the dataset. All larvae employed were progeny of rbfox1^+/19del in-cross and were genotyped after experiments and prior to data analysis. For cortisol measurement (G), each dot/triangle represent a pool of 12 larvae in the left panel (larvae) and a single whole zebrafish in the right panel (adult fish). In all graphs: bars represent standard error of the mean (SEM); * p < 0.05; ** p < 0.01; **** p < 0.0001.