(A) MATLAB reconstruction of distal gonad. Top row, DIC image is one Z-plane; all others are Z-projections. Far left, DIC was used to determine gonadal outline; middle-left, DAPI reveals nuclei (blue); middle-right, signal from sygl-1 exon probes shows sygl-1 cytoplasmic mRNAs and nuclear active transcription sites (ATS) (magenta); far right, signal from sygl-1 intron probes show only ATS (yellow). Bottom row, outputs from custom MATLAB code (Source code 1). Far left, gonadal outline (red dashed line), middle-left, germ cell nuclei false-colored in a depth gradient (yellow-blue) according to Z-position and with DTC (cyan) excluded; middle-right, cytoplasmic mRNAs (magenta) with nuclear ATS excluded computationally; far right, ATS (red) with smFISH signals scaled according to intensity. Scale bar: 5 μm. (B) Signal intensities of cytoplasmic dots from sygl-1 exon probes. A total of 222,260 spots were analyzed from 78 gonads. Raw values from Z-planes were normalized to background levels in the same plane and the mean intensity value set to 10 arbitrary units (a.u.) for each gonad. CV: coefficient of variation (CV <1, significantly narrow distribution). (C,D) Signal intensities of sygl-1 ATS. A total of 2627 spots were analyzed from 78 gonads. (C) Intensities of sygl-1 ATS using exon probes. We first normalized raw values to background levels in the same Z-plane and then normalized to mean intensity of sygl-1 cytoplasmic spots seen with the same probe in the same gonad. Mean intensity of sygl-1 ATS was 172.0 a.u., or roughly 17-fold more than the mean intensity of sygl-1 individual mRNAs. (D) Intensities of sygl-1 ATS using intron probes. Raw values were normalized to background levels in the same Z-plane. (E) Number of sygl-1 ATS per nucleus in 7018 nuclei (78 gonads). Error bars: standard deviation. (F) Pair-wise comparisons of sygl-1 ATS intensities within one nucleus (78 gonads), using normalized values from exon probe. Each black dot represents one pairing. Grey dashed line indicates a perfect correlation (Pearson’s correlation coefficient r = 1); r indicates the correlation coefficient from data in the graph. (G-J) sygl-1 transcriptional activity is independent of cell cycle stage. The cell cycle stage was monitored for DNA content (summed DAPI signal) or nuclear size, which are correlated (Figure 2—figure supplement 1A), in all nuclei located 0–30 µm (1–7 gcd) from the distal end (n = 6979 nuclei total); n.s.: not significant (p>0.05) by t-test. The cell cycle is also independent from nuclear location in the gonad (Figure 2—figure supplement 1B,C). (G,H) DNA content (G) or nuclear size (H) was compared between ATS-positive and ATS-negative cells. For all box-and-whisker plots in this study, the bold line in the box shows the median; top and bottom of box are the third and first quartiles, respectively; whiskers, maximum and minimum of data points; circles, outliers (value greater than 1.5X first or third quartile from the median). (I, J) Summed sygl-1 ATS intensity was estimated by pooling all ATS signal intensities (a.u.) within the same nucleus. Each black dot represents a single nucleus.