Extended Data Fig. 6: SPARC3-Out-GAL80 reveals morphologies of individual Trpγ+ neurons.
From: A discrete neuronal population coordinates brain-wide developmental activity
a. Schematic of the SPARC3-Out-GAL80 cassette. PhiC31 recombines one of two competing attP target sequences with one attB target sequence. Rxn 1 leads to loss of the GAL80 ORF, disinhibiting GAL4-driven effector expression. Rxn 2 preserves Tubulin promoter driven GAL80 expression, maintaining GAL4 inhibition. Three progressively truncated variants for the first attP sequence were designed (25) to bias the recombination in favor of Rxn 2, resulting in frequent (Dense), sporadic (Intermediate), or rare (Sparse) loss of GAL80 and disinhibition of GAL4>UAS expression. b. Map of pHD-3xP3-DsRed-ΔattP (a CRISPR-HDR-donor precursor) showing multiple cloning sites for homology arm insertion (right). c. Map of pHD-3xP3-DsRed-ΔattP-CRISPR-donor (example includes homology arms targeting the Su(Hw)AttP5 region of the Drosophila genome). d. Assembled SPARC3-Out-GAL80 cassette; see Materials and Methods for details. MCS, multiple cloning site. gRNA, guide RNA. HDVR, hepatitis delta virus ribozyme sequence. e, g. Single Trpγ+ neuron (orange, manually segmented) in the context of others (cyan) labeled using SPARC. Neurons expressing myr::SM-V5. Reference marker (magenta), Ncad. Image MIP of stitched confocal stacks of 72 hAPF brain. Scale bar, 100µm. f. Trpγ+ visual processing neurons identified in 72 hAPF brains using SPARC. We observed a given neuron up to three times in 30 sparsely labeled brains.
