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Fig. 6 S1
Single-cell labelling and projection tracing of OXT neurons.
(A) A scheme describing the single-cell labeling method used to trace the projections made by each OXT-ergic neuron. Transgenic Tg(oxt:egfp) embryos were co-microinjected with OXT-specific Gal4 driver, (oxt:Gal4) construct (Blechman et al., 2011) together with constructs harbouring membrane localized caax-tRFP under the control of a multimerized Gal4 upstream activation sequence (10xUAS). Injected embryos were fixed at five dpf, stained for tRFP and EGFP and imaged by a confocal microscope. Injection was calibrated to drives the mosaic expression of caax-tRFP at low efficiency, resulting in single-cell genetic labeling in OXT-ergic neurons. (B) An example of single-cell labeling of an hypothalamo-neurohypophyseal OXT projecting neuron to the posterior pituitary/neurohypophysis. (C) Labeled cells were traced from the 3D confocal Z-Stack using the Fiji image processing and analysis package for 3D tracing and reconstitution with the Simple Neurite Tracer plugin.