Journal: bioRxiv

Article Title: Unilateral gene expression via maternally controlled zygotic recombination in Drosophila

doi: 10.64898/2026.10.14.759140

Figure Lengend Snippet: (A) Embryonic smg-ФC31 -mediated SPARC recombination (i.e., MCZR-SPARC) in the adult brain of 3 example flies. Top: schematic representations of expression patterns, as in Extended Data Fig. 1. Bottom: confocal MIPs of immunostained adult brains. Brp counterstain shows neuropil extent. Scale bar: 100 μm. (B) Top: frequency of asymmetric GFP+ patches by patch size and temperature at the time of fertilization. Each fly is categorized by the size of its asymmetric GFP+ patch(es). Complete patches cover one entire brain hemisphere, large patches cover at least a quarter of the central brain, and small patches cover less than a quarter of the central brain. White numbers inside bars indicate the number flies in each group. p = 0.021 for large vs. small patch rate (Boschloo’s Exact). Bottom: frequency of asymmetric GFP+ patches by patch size and maternal age at the time of fertilization. Large patches: Spearman’s ρ = -0.94, p = 0.005; small patches: Spearman’s ρ = 0.71, p = 0.111. Group sizes for 3, 7, 12, 22, 27, and 28 days are 8, 23, 17, 12, 10, and 5, respectively. See also Extended Data Fig. 4 and Supplementary Table 1. (C) MCZR-SPARC recombination for an individual cell type (s-LNv). Left: an example full brain MIP. RFP is driven constitutively, while GFP depends on maternal smg-ФC31 activity. Teal box indicates area of magnification. Scale bar: 100 μm. Right: magnified area for three different brains, one with symmetric GFP expression in s-LNv neurons, one with no expression, and one showing complete asymmetric expression. Asymmetric example brain is the same as shown on the left (teal arrow). Dashed yellow lines indicate the midlines in each image, and yellow arrowhead highlights missing GFP signal. Scale bar: 20 μm. (D) Examples of “complete” (top row) and “partial” (middle and bottom rows) asymmetry in different cell types. Each image is a MIP through the indicated brain regions. For Tm9 (bottom row), the left and right optic lobes of a single fly are shown as separate images. Scale bars: 50 μm. See also Supplementary Table 2. (E) K -means clustering of example cell types based on complete (colored bars) and partial (gray bars) asymmetry rates. At k = 3, cell types group approximately according to the neuroblast type from which they derive (yellow: Optic Lobe, red: Central Brain Type II, blue: Antennal and Central Brain Type I). N is shown at each leaf position in the dendrogram. (F) Complete and partial asymmetry rates by neuroblast type. While the overall rate of any asymmetry is consistent across neuroblast types ( χ = 2.4, p = 0.48), complete asymmetry rates differ significantly ( χ = 16.3, p < 0.001). Colors from (E). (G) Conceptual model of the scaling efficiency for different methods used to generate asymmetric mosaics in the fly brain. Pink band represents the observed floor (large patch rate, 28%) to theoretical ceiling (50%) of complete asymmetry rates for MCZR-SPARC across target population sizes. Darkest part of the band is the mean asymmetry rate across all tested cell types (33%). Lines represent population size scaling efficiency of post-mitotic ФC31 driving SPARC-D (black), post-mitotic hs-Flp driving FlpOut (blue), and the gynandromorph method (gray). Dots are real data points from other studies that used the post-mitotic SPARC and FlpOut methods , and the corresponding lines are exponential extrapolations from those points.

Article Snippet: CaSpeR4-SGS plasmid ( smg5’UTR-GFP-smg3’UTR ; kind gift of Howard Lipshitz) containing a GFP open reading frame flanked by smg UTRs was verified for sequence integrity via NotI-HF and NheI-HF restriction analysis (New England Biolabs, #R3189S and #R3131S).

Techniques: Expressing, Confocal, Activity Assay