Materials and Methods
dcr-1d102 results in a Q1948X mutation and therefore truncates the second RNAIII domain in Dcr-1.
Generation of Transgenic Lines
Full-length
Dacapo
PCR
products
were
GCTCTAGATTAGTTGTTGTGGCGCGGCCGCTT
synthesized
and
using
the
the
forward
reverse
primer
primer
GCTTCAGAATGGTCAGTGCCCGAGTCCTG (Invitrogen) from the template pSK+ dap (a gift
from Christian Lehner). PCR products were then digested with XbaI and cloned into p-hsCaSpeR and
pUAS-P vectors (a gift from Pernille Rorth). This XbaI fragment contained only the coding region;
the 5’ and 3’ untranslated regions were not included. Transformant lines were generated by germline
transformation in a w1118 background. Four independent transformant lines were obtained for UASDap (UAS-Dap-18 was analysed in Fig4d, other independent insertions gave similar results) and one
transformant line for hs-Dap (hs-Dap-7).
Dacapo Overexpression
Ectopic expression of Dacapo was achieved by two different ways: 1) heat-shock-induced
expression by administering 3x50 min heat-shocks in 36 hours and dissected 3-6 days after the last
heat-shock; and 2) Gal4/UAS-dependent overexpression with the germline driver nanos (nos Gal4
VP16).
Generation of clones and kinetic- and statistical-analysis
Drosophila melanogaster stocks were raised on standard cornmeal-yeast-agar-medium at 25oC.
Clones were induced using the FLP-FRT system for mitotic recombination (Supplementary Ref1). To
obtain imaginal disc clones, we heat-shocked second-instar larvae for 120 minutes at 37 C and
dissected them after 2 days. Transient cyst clones were induced by a single 50 minute heat-shock and
the animals were dissected after 5 days at 21C; cysts were counted only if they had not yet reached
the 16-cell stage and if no stem cell clone was observed in the germarium.
Division frequencies were measured using germaria containing one GFP+ GSC and one
GFP- GSC. The total number of cysts from a GSC that are produced in a given time window
provides a measurement of GSC division frequency. In our case, the time window spanned from the
first heat-shock treatment to the time of harvesting the adults. Therefore, we limited our counts to the
region of the germarium that was anterior to the easily-identifiable GFP+/+ cyst. This cyst developed
from the first daughter cell of the clonal GSC (GFP-) after heat-shock induced mitotic recombination.
Since dcr-1 GSCs could have been induced by either of the two heat-shocks that were given, the total
number of GSC progeny (cysts) found in the relevant region could vary (Fig3b). However, in all
cases, the number of cysts produced from the dcr-1 GSCs was lower than for control GSCs regardless
of time. Cyst production from homozygous clonal GSCs was divided by the cyst production from
heterozygous non-clonal GSCs in the same germarium to obtain the division index (Fig3c).
The student T-test was used to determine the statistical significance in Fig4c. The Dap 3’UTRs
from D. melanogaster and D. pseudoobscura were analyzed for potential miRNA-binding sites. Four
algorithms were independently applied: the program miRanda24, an EMBL program27, TargetScan28
and a program devised by Nakahara and Carthew (Supplementary Ref3).
Staining Procedures
Antibody stainings, BrdU-labeling (90 minute pulse) and confocal microscopy was performed
as described previously17-18 (Supplementary Ref2). A two-photon laser-scanning microscope (Leica
TCS SP/MP) was used in this study.
Supplementary References
1. K. G. Golic, Science 252, 958 (Jun 17, 1991).
2. M. Keller Larkin et al., Dev Genes Evol 209, 301 (May, 1999).
3. K.Nakahara and R.Carthew, unpublished.
Supplementary Table 1. Dcr-1 mutant GSCs are blocked in G1/S cell cycle
transition
n = number of GSCs counted
% GSCs that show positive staining for cell cycle
markers
CycE
Dap
CycA
CycB
BrdU
PH3
43.2%
46.0%
67.1%
48.4%
49.2%
11%
n=88
n=50
n=76
n=64
n=131
n=134
hsFlp; FRT82B dcr-1Q1147X/FRT82B dcr-1Q1147X
85.0%
68.5%
58.3%
48.4%
39.3%
2.9%
(8 days post induction)
n=40
n=35
n=24
n=31
n=87
n=35
96%
96%
33.0%
17.0%
23.8%
0.0%
n=25
n=24
n=24
n=47
n=61
n=10
Genotype
Control dcr-1/+ (GFP+)
hsFlp; FRT82B dcr-1Q1147X/FRT82B Ubi-GFP
Mutant dcr-1/dcr-1 (GFP-)
(12 days post induction)
Supplementary Figure 1 Legend
The dcr-1d102 allele yields a phenotype identical to, albeit slightly milder than the
dcr-1Q1147X allele. (a) dcr-1d102 germaria are smaller then wild-type and exhibit a marked
decrease in germline cyst number. (b) dcr-1d102 allele shows an increase in the frequency
of CycE expression whereas GSCs that are homozygous for the parental chromosome
express CycE with frequencies similar to what is observed for wild-type GSCs. No
significant effect on CycE in dcr-2 mutant clones was observed. Days indicate the
timepoint after clonal induction.
Supplementary Figure 2 Legend
The dcr1-related reduction in cell division rate and cell cycle delay are
germline stem cell-specific. (a-a’’) dcr-1 (dcr-1Q1147X) clones in a leg imaginal disc.
Clones are marked by dotted line in panels a’ and a’’. Legend refers to top clone and its
sister clone, which are in the best orientation. The number of cells in the dcr-1 clone (a’,
black) is approximately equal to the number of cells in the adjacent sister clone (a, bright
green). a’’ Only a small percentage of cells within the dcr-1 clone are cyclin E-positive
(while 96% of dcr-1 GSCs are CycE positive; Fig3e). (b) The average number of cells in
dcr-1 imaginal disc clones is approximately equal to the number of cells in the sister (WT
aka GFP+/+) clones. Data is shown for 21 individual imaginal disc clones (n=21). (c) Loss
of dcr-1Q1147X does not cause transient germline cysts to express CycE with the high
frequency observed in dcr-1 GSCs. (d) CycE is expressed in transient dcr-1Q1147X
germline cysts with a frequency similar to that observed for wild-type transient cysts.
Supplementary Figure 3 Legend
Analysis of Dacapo expression in GSCs.
(a) Dap protein is detected with high
frequency in dcr-1Q1147X GSCs (no GFP, arrow). (b) CycE and Dap-5gm co-expressed in
a wild type GSC. (c) In dcr-1 GSCs CycE expression is more frequent than in wild type
(85% and 40% respectively). However, Dap-5gm frequency is similar in wild type and
dcr-1 GSCs. (d) Over-expression of Dacapo lacking the 3’UTR partially phenocopies dcr1 (37%, n=85). Quantitations with these assays are shown in Fig4b-d.
Supplementary Figure 4 Legend
Computational prediction of miRNA binding sites in the dap 3’ UTR. (a) The
complete 3’ UTR of D. melanogaster dap mRNA with the predicted microRNA target sites
(shown in blue type) and their corresponding microRNAs. The miR-7 is additionally
underlined since it partially overlaps with one of the miR-289 sites. (b) These specific
sites were each predicted by at least two of four independent algorithms listed
Supplementary Ref3
26,27,28,
to be targets of the indicated miRNAs. Each algorithm only scored a site as
positive if it was also found in the 3’UTR sequence of the D. pseudoobscura dap gene.
Thus the sites are conserved in dap between different Drosophila species. The position
of the 5’ nucleotide of each target site in the dap 3’UTR sequence is indicated.