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