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Developmental Cell, Volume 23
Supplemental Information
let-7-Complex MicroRNAs Regulate
the Temporal Identity of Drosophila
Mushroom Body Neurons via chinmo
Yen-Chi Wu, Ching-Huan Chen, Adam Mercer, and Nicholas S. Sokol
INVENTORY OF SUPPLEMENTAL INFORMATION
1. Supplemental Figure 1. let-7-C expression in the CNS.
Figure provides additional data regarding let-7-C miRNA expression in the
CNS, including northern blots of miR-100 and miR-125, schematic of
transcriptional reporters used in this study, and analysis of one of those
(let-7-C3miR::optGal4) in the CNS.
2. Supplemental Figure 2. Additional characterization of let-7-C MB phenotypes
Figure provides additional characterization of let-7-C mutant mushroom
body phenotypes, as well as verification that strains used to assay let-7-C
dosage effects contain differing levels of let-7.
3. Supplemental Figure 3. Additional analysis of chinmo 3UTR extension
Figure provides schematic of chinmo 3UTR, verification of UAS-miRNA
constructs used to assay chinmo 3UTR luciferase reporters in Figure 3G,
and pictures of fly eyes whose pigment was quantified in Figure 3H.
4. Supplemental Figure 4. let-7 and miR-125 microRNAs regulate MB temporal
identity and chinmo expression
Figure presents ectopic expression of miR-100, let-7, and miR-125
individually.
5. Supplemental Experimental Procedures
6. Supplemental References
Figure S1. let-7-C expression in the CNS.
(A,B) miR-100 (A) and miR-125 (B) are expressed in a similar expression
profiles as let-7, shown in Figure 1B. Total RNA was extracted from CNS tissues
dissected from staged animals and probed for miR-100, miR-125 and U6 snRNA.
(C) Schematics of let-7-C locus and transcriptional reporters used to assay let-7-C
expression. The let-7-C locus spans ~18kb and encodes a ~2.4kb primary let-7-C
transcript that contains miR-100, let-7 and miR-125. The let-7-C::Gal4
transcriptional reporter is the result of a Gal4 knock-in into the endogenous let-7-
C locus, and is described in Sokol, 2008. The let-7-Cp12.5kb::lacZ transcriptional
reporter contains 12.5kb of the first let-7-C intron upstream of lacZ, and is
described in (Chawla and Sokol, 2012). The let-7-C3miR::optGal4 transcriptional
reporter contains a modified version of the full-length let-7-C locus in which all
three microRNAs were deleted upstream of a Drosophila codon optimized gal4
cDNA (Pfeiffer et al., 2010). (D-F) let-7-C3miR::optGal4 transcriptional reporter
driving UAS-GFP::lacZ::nls (white in left panels, green in right panels) is not
detected in (D) white prepupae (0h) stained for Dac (magenta, bottom panels)
but is detected in increasing numbers of MB neurons in (E) 12-h pupae (12h),
and (F) 24-h pupae (24h). Arrowheads mark cells expressing let-7-
C3miR::optGal4 in 12 h pupae. Scale bar: D, E, F, 30 m.
Figure S2. Additional characterization of let-7-C MB phenotypes.
(A-D) Single cell clones of wild-type MB neurons (green), generated at various
selected stages, showing the unique and distinguishable morphologies of (A) ,
(B) /, (C) p. /, and (D) / neuronal subtypes. (A) The lobe (weakly
labeled with anti-FasII antibodies in magenta) is derived from MB neurons born
before the mid-third instar stage, and these neurons project axons medially. In
contrast, /, p. /, and / subtypes project axons dorsally as well as
medially. (B) / neurons are identified based on the absence of FasII
expression and are born in mid-to-late L3. (C, D) The p. /, and / subtypes
are both strongly labeled with anti-FasII antibodies. They can be distinguished
from one another since the medial axons of p. / neurons terminate prior to
those of / subtypes (arrow in C), and p. / neurons project only one primary
dendrite into the calyx (arrowhead in C) whereas / neurons project more than
one primary dendrite (arrowhead in D) (Zhu et al., 2003; Zhu et al., 2006). (E)
Percentages of different subtypes of MB neurons (y-axis) among single and two-
cell clones that were induced in wildtype animals at different times relative to
puparium formation (x-axis), showing the normal timing of transitions between
/, p. /, and / subtypes. This data is from a duplicate experiment as those
presented in Figure 2A. (F) Percentages of different subtypes of MB neurons (y-
axis) among single and two-cell clones that were induced in wildtype and let-7-C
mutant animals at different timepoints relative to 2nd-to-3rd larval ecdysis (x-
axis), and indicates that the / transition is not delayed in let-7-C mutants.
(G) Average number of neurons as detected by the H24 Gal4 driver in wildtype
(wt) or let-7-CKO1 and let-7-CKO2 transheterozgyotes. (H) Average number of /
neurons as detected by the c739 Gal4 driver in wildtype (wt) or let-7-CKO1 and let-
7-CKO2 transheterozgyotes. (I) Average number of cells in OK107-Gal4 labeled
wildtype or let-7-CKO2 adult MB clones generated in newly hatched larvae. Figures
in parentheses represent the total number of clones (E, F, I) or brains (G and H)
analyzed for each genotype. (J-M) let-7 expression correlates with let-7-C copy
number. (J) Northern blot analysis of total RNA extracted from age-matched
adults carrying four copies (4X), two copies (2X), or zero copies (0X) of let-7-C
probed with let-7 as well as two loading controls, U6 and miR-1. (K)
Quantification of northern blot results in J indicating the fold of let-7 expression
in 2x and ox samples relative to 4x sample, normalized to either U6 or miR-1. (L)
Northern blot analysis of total RNA extracted from age-matched adults carrying
two copies (2X), one copy (1X), or zero copies (0X) of let-7-C probed with let-7 as
well as two loading controls, U6 and miR-1. (M) Quantification of northern blot
results in L indicating the fold of let-7 expression in 1x and ox samples relative to
2x sample, normalized to either U6 or miR-1. (N-Q) Characterization of let-7-C
mutant midline crossing phenotype. Adult single-two cell MARCM clones of let-
7-C MB neurons (green in N, P) were generated at 0-6 and 18-24 hr after pupal
formation, respectively. lobe neuron axons usually stop at the midline (dotted
line in N, P) whereas some p. / and / neurons cross the midline in let-7-C
mutant brains (asterisks in N, P). Although let-7-C mutant p. / neurons cannot
be identified based on their premature termination, they can still be reliably
identified based on the primary dendrite number (arrowheads). (O, Q) Average
number of p. / (O) and / (Q) neurons that display midline crossing
phenotype. Genotype for N-Q: UAS-mCD8::GFP, hsFLP /+; let-7-CKO1/KO2;
FRT3L2A/FRT3L 2A, tubP-GAL80; GAL4-OK107/+. Scale bar in N and P is 50 m.
Values are presented as mean SEM.
Figure S3. Additional analysis of chinmo 3UTR extension
(A) Schematic of 3end of the chinmo locus showing genomic organization of two
annotated chinmo transcripts (RF and RG) that encode distinct protein isoforms
(PA and PB, respectively). ORFs are indicated in black and UTR sequence is in
white (top). A Gbrowse screenshot of developmental RNA-seq data taken from
Flybase (middle). Note that the signal extending beyond the 3end of chinmo-RF
and chinmo-RG represents the previously unannotated ~6kb extension of the
chinmo 3UTR. Schematic of chinmo 3UTR (bottom). A indicates the location of
the stop codon of the Chinmo-PA isoform, whereas B indicates the location of the
stop codon of the Chinmo-PB isoform. ProxAn indicates location of the chinmo-
RF and RG putative polyadenylation sites, whereas distAn indicates location of
polyadenylation site based on RNA-seq data. The 1.4kb fragment indicates
sequence containing a cluster of predicted let-7 (orange) and miR-125 (blue)
binding sites. Sequences of and predicted base-pairing between let-7-C miRNAs
and chinmo 3UTR are shown. Numbering is relative to first nucleotide of chinmo
3UTR. Red boxes indicate DNA deleted in chinmo1.4kbmut construct. (B-G) let-7-C
miRNAs are expressed in KC-167 cells and repress luciferase controls (B, C, D)
Northern blot analysis of total RNA extracted from KC-167 cells transfected with
UAS-let-7-C, UAS-let-7-Clet-7, miR-125, UAS-let-7-CmiR-100, miR-125, UAS-let-7-CmiR-
100, let-7, UAS-let-7-C3miR, or no DNA (-) and probed for miR-100 (B), let-7 (C), or
miR-125 (D). Blots were also probed with U6 as a loading control. (E) Fold
repression of luciferase reporters containing synthetic miR-100 sites followed by
the white in cell cultures in which UAS-let-7-C, UAS-let-7-Clet-7, miR-125, or UAS-
let-7-C3miR were transfected. P < 0.0001 comparing UAS-let-7-C and UAS-let-7-
C3miR. P = 0.0003 comparing UAS-let-7-Clet-7, miR-125 and UAS-let-7-C3miR. (F)
Fold repression of luciferase reporters containing synthetic let-7 sites followed by
the white in cell cultures in which UAS-let-7-C, UAS-let-7- CmiR-100, miR-125, or
UAS-let-7-C3miR were transfected. P = 0.0006 comparing UAS-let-7-C and UAS-
let-7-C3miR. P < 0.0001 comparing UAS-let-7-CmiR-100, miR-125 and UAS-let-7-
C3miR. (G) Fold repression of luciferase reporters containing synthetic miR-125
sites followed by the white in cell cultures in which UAS-let