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.