Fig. 1: Neuronal activity modulates synapse formation in the developing PDE axon.
From: An activity-regulated transcriptional program directly drives synaptogenesis
a, Schematic representation and line scan of the PDE axon and its synapses labeled using a combinatorial approach. Endogenous FLP-on (FRT) GFP::ELKS-1 labels active zones and endogenous FLP-on (FRT3) mScarlet::TBA-1 labels microtubules (for axon morphology) when combined with a transgene expressing a dopaminergic-specific flippase (dat-1p::FLP). b, Top: schematic representation of PDE-silencing experiments. Transgenic animals expressing dat-1p::HisCl1 were placed on media containing histamine at the L2 stage and imaged at the L4 stage. Bottom: schematic representation of PDE-excitation experiments. Transgenic animals expressing dat-1p::ChR2 were subjected to blue light at the L2 or L4 stage and imaged either 2 h or 48 h post-treatment. This schematic was created with BioRender.com. c, Line scans of ELKS-1 in the PDE axon of animals carrying wyEx8629(dat-1p::HisCl), treated with 0 mM or 10 mM histamine. d, Quantification of ELKS-1 in PDE of animals shown in c (n = 14 for both conditions). e, Line scans of ELKS-1 in the PDE axon of animals carrying wyEx10629(dat-1p::ChR2), treated with or without blue light. f, Quantification of ELKS-1 of animals shown in e (n = 19 for both conditions). g, Transgenic animals carrying wyEx8629(dat-1p::HisCl) were imaged for their endogenous FOS-1::GFP signal in PVD and PDE neuronal nuclei (white dashed circles) in the presence or absence of 10 mM histamine. h, Quantification of FOS-1::GFP in animals shown in f (n = 11 for both conditions). i, Normalized expression of PDE FOS-1::GFP presented in h, compared to each paired PVD’s (sister cell of PDE) expression (n = 11 for both conditions). j, Transgenic animals carrying wyEx10630(dat-1p::ChR2) were treated at L3 with or without blue light for ten cycles (15 s on and 60 s off) and allowed to recover for 2 h. FOS-1::GFP was imaged in PVD and PDE nuclei (white dashed circles). k, Quantification of FOS-1::GFP in animals shown in i (n = 15 for both conditions). l, Normalized expression of PDE FOS-1::GFP presented in k compared to each paired PVD’s expression (n = 15 for both conditions). For images with line scans, scale bar = 20 μm; for images of neuronal nuclei, scale bar = 10 μm. For all graphs, medians are represented in thick dashed lines and quartiles are represented in thin dashed lines. P values presented were calculated using two-tailed unpaired Student’s t tests. MGV, mean gray value.
