Figure Legends
Figure 1. Cell type-specific biosynthetic RNA tagging reveals limited rRNA synthesis in neurons. A. Fluorescent RNA signal (green) in a single brain lobe following 24 hours of EC-tagging targeted to neural progenitors (insc > CD:UPRT ) or neurons (nSyb > CD:UPRT ). Scale bar is 10 µm.B. Relative precursor rRNA (pre-rRNA) and Syt1transcript abundance following 24 hours of EC-tagging in neurons (nSyb -tag) or neural progenitors (insc -tag), as measured by RT-qPCR. Fold-change in relative abundance (nSyb -tag /insc -tag), after normalization to a RNA polymerase II subunit, is shown. Data are the mean and standard deviation from two biological replicates. C. The fluorescent signal is predominately ribosomal RNA. Dissected brains were soaked in EU alone (left panel), EU plus the mRNA synthesis inhibitor triptolide (middle panel), or EU plus the mRNA and rRNA synthesis inhibitor actinomycin D (right panel). An equivalent region of central brain is shown in each confocal stack. Scale bar is 10 µm. D. The EU-RNA signal localizes to the nucleolus of neuroblasts. EU-RNA signal alone (left panel), overlay of EU-RNA and the nucleolus marker Udd (middle panel), overlay of EU-RNA and the proliferating cell marker PCNA (right panel). All panels are from the same confocal stack. The large PCNA positive cells are neuroblasts. Scale bar is 10 µm.
Figure 2. Biosynthetically tagged rRNA is only detected in newly born neurons. A. Fluorescent RNA signal (green) in a single brain lobe following 4-hours of EU feeding. Wor-gal4 driven expression ofUAS-mtd-Tomato (wor > mtd-Tomato ) results in Tomato signal in the plasma membrane of neuroblasts and their progeny, including neurons made before the 4-hour EU feeding (mtd-Tomato in magenta, left panel). Antibody stain for the neuron-specific protein Elav is shown in magenta in the right panel (the two panels are from the same single confocal image). In the left panel, a single EU-positive neuroblast is indicated by an arrow and associated EU-positive GMCs are indicated by asterisks. There are no EU-positive neurons in this image. Scale bar is 10 µm. B. Plot showing the relationship between EU feeding time and the number of EU-positive neurons. Data are the mean and standard deviation for multiple brain lobes (4-hour feeding n = 8, 6-hour feeding n = 6, 24-hour feeding n = 6). C. Fluorescent RNA signal (green) in progenitors and neurons following 24 hours of EU feeding. A single confocal image is shown, with Pros and Elav signal separated. Pros is expressed in INPs, GMCs and neurons while Elav is only expressed in neurons. Multiple EU-positive neurons are visible and all are located adjacent to progenitors (progenitors are Pros-positive, Elav-negative). Scale bar is 10 µm. D. Fluorescent RNA signal (green) in a cross section of larval central brain following 24 hours of EU feeding. Recently born neurons are located near the periphery while older neurons are located deeper, near the neuropil (asterisk). Scale bar is 10 µm.
Figure 3. Progenitor rRNA associates with mitotic chromosomes and is passed to progeny cells. A. Fluorescent RNA signal (green) following a 16-hour EU feeding, co-stained with antibodies for PHH3 (mitotic chromosomes) and Miranda (cortex of mitotic neuroblasts, INPs, and newly formed GMCs). A single confocal image is shown, with one neuroblast (outlined by white line) repositioned for clarity. Two mitotic neuroblasts with a basal Miranda crescent and chromosomes at the metaphase plate are shown. Scale bar is 10 µm. B. Same experimental conditions as part A. Two adjacent neuroblasts and recently produced GMCs (indicated by arrows) are shown. Scale bar is 10 µm.C. Fluorescent RNA signal (green) following a 6-hour EU feeding (pulse) and a 6-hour EU feeding followed by 18 hours of feeding in the absence of EU and an excess of unmodified uridine (chase). Top images show dorsal brain lobe regions following the pulse (left panel) and chase (right panel). Bottom images show deeper brain lobe regions following the pulse (left panel) and chase (right panel). Neurons were identified by antibody staining for Elav (magenta). Scale bar is 10 µm.
Figure 4. Inhibition of rRNA inheritance causes neurodevelopment and protein synthesis defects. A. Summary of rRNA sources in RNA Polymerase I knockdown experiments (progenitor-derived = green, neuron-derived = blue). B. Eclosion failure in control (UAS-Polr1B{RNAi} with no Gal4 activation) and Polr1Bknockdown flies. C. HPG-based imaging of protein synthesis in control and Polr1B knockdown flies. HPG-tagged proteins are labeled green, Elav is labeled magenta. Scale bar is 10 µm. D.Quantification of protein synthesis: fluorescence intensity (arbitrary units (a.u.)) was measured in a fixed-size area of neurons (Elav-positive) across all three genotypes. Three regions were measured per brain lobe and six brain lobes were analyzed per genotype. ** = p-value < 1 x 10-5, Student’s t-test compared to wildtype.