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. 2024 Jan 5;12:RP90133. doi: 10.7554/eLife.90133

Figure 5. Bsh:DamID reveals Bsh direct binding to L4 identity genes and pan-neuronal genes.

(A) Schematic for TaDa method. (B) Both Dam and Bsh:Dam show high Pearson correlation coefficients among their three biological replicates, with lower Pearson correlation coefficients between Dam and Bsh:Dam. A heatmap is generated using the multiBamSummary and plotCorrelation functions of deepTools. (C) Bsh:Dam peaks in L4 (46–76 hr after pupa formation [APF]) called by find_peaks and L4 scRNAseq data (48 hr APF; 60 hr APF) (Jain et al., 2022) are combined (see Materials and methods). (D) Bsh:Dam shows strong signals in L4 identity genes and pan-neuronal genes. The Dam alone signal indicates the open chromatin region in L4 neurons. The y axes of Bsh:Dam signal represent log2 (Bsh:Dam/Dam) scores. Bsh peaks in L4 neurons were generated using find_peaks (FDR<0.01; min_quant = 0.9) and peaks2genes. (E) Summary.

Figure 5.

Figure 5—figure supplement 1. UAS-Bsh:Dam expressed by 31C06-Gal4 is functional and specifically expressed in L4 neurons; Bsh:Dam shows a binding peak in ap locus but not pdm3 or zfh1 in L4 neurons.

Figure 5—figure supplement 1.

(A) Schematic of the system used to restrict Bsh:Dam expression spatially to L4 (using the 31C06-Gal4 driver) and temporally to 46–76 hr after pupa formation (APF) (using the temperature-sensitive Gal4 inhibitor, Gal80ts; red T-bar). Note the high expression of GFP from the first ORF (thick green arrow), and low expression of Bsh or Bsh:Dam following the readthrough of the stop codons prior to the second ORF (thin green arrow). (B–B’’) 31C06-Gal4, Gal80ts, UAS-Bsh:Dam is expressed in Ap+ L4 neurons when conditionally expressed from 46 to 76 hr after pupa formation (APF). Scale bar, 10 µm. (C–E) Bsh:Dam retains Bsh function and can partially rescue Ap and Pdm3 expressions in Bsh-null mutant flies (Bsh-null mutant; R27G05-Gal4>UAS-myrGFP-Bsh:Dam). Note that Bsh:Dam expression from the second ORF is extremely low to avoid toxicity and is not detected by immunostaining. The white circle outlines the cell body area of lamina neurons. (E) Quantification of the number of Ap+ or Pdm3+ or Bsh+ cell bodies within z-stack of 2.88 µm in control (C–C’’) and Bsh:Dam (D–D’’). Scale bar, 10 µm. Data are presented as mean ± SEM. Each dot represents a brain. Control: n=5 brains; Bsh:Dam: n=4 brains. ns = not significant, **p<0.001, unpaired t-test. (F) Bsh:Dam shows strong signals in ap gene but not pdm3 or zfh1 gene in L4 neurons during the synapse formation window. The Dam alone signal indicates the open chromatin region in L4 neurons. The y axes of Bsh:Dam signal represent log2 (Bsh:Dam/Dam) scores. Bsh peaks in L4 neurons were generated using find_peaks (FDR<0.01; min_quant = 0.9) and peaks2genes.