(A–E) Assessment of synaptic oxytocin content in robo2 mutant was performed as described in Figure 1F. Graph showing the number and size of neurohypophyseal synapses (NS) (A,B) and colocalizing OXT puncta (C,D) and the number of neurohypophyseal projecting axons (E) in 8 days post-fertilization (dpf) robo2+/+ (n = 13) vs robo2-/- (n = 17) larvae (*p<0.05 Student’s t-test with Cohen's d = 0.82; ns denotes not significant). (F–J) Assessment of synaptic oxytocin content upon slit3 knock-down. Transgenic (oxt:EGFP) embryos were injected with injection buffer or injection buffer with 0.85 ng of morpholino targeted to the translational start site of slit3. Graph showing the number and size of NS (F,G) and colocalizing OXT puncta (H,I) and the number of neurohypophyseal projecting axons (J) in 8 days post-fertilization (dpf) control (n = 15) vs slit3 morpholino injected (n = 14) larvae (**p<0.05 with Cohen’s d = 1.23; ns denotes not significant, Student’s t-test). (K) Hypophyseal POMC cells are localized near OXT synapses. Confocal Z-stacks maximum intensity projection of hypophysis region of 5 day old triple transgenic (pomc:EGFP; oxt:Gal4; UAS:NTR-mCherry). The hypophysis area showing OXT NS adjacent to anterior and posterior clusters of POMC cells are shown. Scale: 10 μM. (L–R) Local overexpression in mosaic hypophyseal POMC clones located. Transgenic embryos expressing the pomc:Gal4 driver were injected with transposon-based transgenic vectors containing either control UAS:tRFP (M,P) or UAS: Slit3-EmGFP (N,Q). The injected 5-dpf larvae were subjected to immunostaining with anti-OXT protein and anti-GFP. OXT-puncta (arrows) within a distance of 2 μM (yellow dashed ellipse) from the clone surface were quantified. Scale bar: 2 µm. Bar graphs showing the mean volume of OXT puncta upon expression of tRFP vs Slit3 in hypophyseal POMC anterior (n = 15 and 18, respectively) or posterior clone (n = 10 and 10, respectively) position (*p<0.05 Student’s t-test with Cohen’s d = 1.14). Error bars indicate SEM in (A-J, O and R).
Experimental data for Figure 5: Neurohypophyseal synaptic parameters upon gain- or loss-of function of slit3 or robo2.
The excel sheet includes values for number and volume of neurohypophyseal synapses and OXT puncta from confocal imaging of larval neurohypophysis of multiple experiments namely: 1) robo2 +/+ vs robo2 -/- 2) Mock vs morpholino-injected embryos 3) Mosaic transgenesis experiments by hypophyseal expression of tRFP vs slit3.
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