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1 Supporting Information Ho et al /pnas SI Materials and Methods In immunostaining, antibodies from Developmental Studies Hybridoma ank were -FasII (mouse, 1:200), - (mouse, 1:100), and -Elav (rat, 1:100). Other primary antibodies used were - (rabbit, 1:200; Upstate), -as-3 Asp-175 (rabbit, 1:500; ell Signaling), - -gal (rabbit, 1:200; IN Pharmaceuticals), - (rat, 1:500; provided by M. hat), and -Eagle (rabbit, 1:1000; provided by J. Urban). All secondary antibodies coupled to y3 or y5 were from Jackson ImmunoResearch (1:500). S2 cell transfection was performed using 0.3 g ofmt-gal4 and 0.25 g of each UAS construct. pha-agowd, pha- G9144, and pha-ub were constructed using the Gateway system (Invitrogen). Generation of Flag-Gcm was done by overlapping PR using the following forward and reverse oligonucleotide primers (the wild-type terminus of gcm was removed from puast-gcm upon SacII digestion and the Flagtagged PR product digested by SacII was inserted): 5 -TTGAATGGTGTTGGAAATAGGTT-3 and 5 -GGGTATTTATGTATGTTTTGTAGT- TTTATGTATGTTTTGTAGTGAATAGATGGG- ATGTGTGTTGA-3. In Western blot, primary antibodies used were -Flag and -HA (1:5,000, Sigma) and secondary antibodies used were -mouse-hrp and -rabbit-hrp (1:5,000, Jackson). Developing EL reagents were from Perkin-Elmer. Immunoprecipitations of the Flag proteins were done using Flag M2 beads (Sigma). HA proteins were immunoprecipitated by HA antibodies conjugated to Protein A beads (GE Healthcare). In RNA-mediated interference, 30 g of dsrnas was used to treat S2 cells for 2 days before the transfection of plasmids for 3 days. dsrnas for slimb and ago were generated using PR with primers mh205/mh206 and ago5 /ago3. After amplifications, PR products of the targeted region ( 500 bp) were cloned into TOPO vector (Invitrogen) and plasmids of forward and reverse orientations were both selected. dsrnas were prepared using the Promega T7 RiboMaxTM express system. Sequences for the primers used are as follows: mh205, 5 -GAAGGTTAAAATATG-3 ; mh206, 5 -GATAGTGTGGATTAGGGT-3 ; ago5, 5 -TAATAGATATATAGGGAGAGAA- TGAGTTAGGAGA-3 ; and ago3, 5 -TAATAGATATATAGGGAGAA- GATTGTGTGAAGGAAG-3. Q-PR was performed using mrna isolated from late stage embryos. Primers were designed using Primer Express software (Applied iosystems). cdnas were made from fly embryonic RNAs of designated genotypes by a High apacity Reverse Transcription Kit (Applied iosystems). cdnas were incubated with Syber Green dye (Applied iosystems) for detection of the RNA products. Results were recorded and analyzed by the Ai 7500 system. Sequences for the primers used are as follows: repo, 5 -AAGAAGGATGAGATGTGTT-3 and 5 -AGTGGAGGAATATTG-3 ; ttk, 5 -ATTAAGAAAAGTAAGTTAA-3 and 5 -GGGAAATTGGATGT-3 ; yc, 5 -AATGGTGGTTTG-3 and 5 -AGTATGGTTGTGAA-3 ; and yc E, 5 -GATGTGTTTGTGATAGAGAAT-3 and 5 -GTTTAGTTTATTGATTGG-3. The software Metamorph was used for measurements of immunofluorescence intensity and the number of colocalized cells. For all measurements, mutant samples and their corresponding heterozygous controls were stained in the same reaction tube; therefore the staining conditions are identical for the comparison. All images were taken with the same exposure/gain and a threshold was used so that 15% of the total signal was eliminated as background. Abdominal segments of the embryos were used for the measurements. 1of10

2 A pflag-gcm + + pslimb-ha + - pha-slimb WD - + Slimb Slimb WD Flag-Gcm IP: Flag I: HA pmt-gal pha-ub(ko) pha-ub pflag-gcm dsrna slimb dsrna ago dsrna slimb+ago dsrnas Slimb Slimb WD Input IP: Flag I: HA Ub-Gcm α-tubulin 72 Flag-Gcm lane % dsrna ago slimbn β-tubulin Fig. S1. iochemical analysis of Gcm. (A) Gcm interacts with Slimb through the WD domain. S2 cells transfected with pflag-gcm and pslimb-ha (lane 1) or pha-slimb WD (lane 2) were analyzed by coimmunoprecipitation by Flag antibody and Western blot by HA antibody. Results in A show that only full-length Slimb protein interacts with Gcm, not Slimb WD. () Ubiquitinated Gcm was detected as smears when both pflag-gcm and pha-ub were transfected (lane 3). Immunoprecipitation was done by Flag antibody and Western blot by HA antibody. Ub-Gcm species were reduced when both slimb and ago dsrnas were added to S2 cells (21%, lane 9). Single knockdown of slimb or ago resulted in some reduction (70%, lane 7) or no reduction (106%, lane 8). The average of reduction in Ub-Gcm was obtained from 3 independent experiments. () RT-PR analysis of slimb and ago mrna levels in S2 cells treated with respective dsrnas. -Tubulin is shown as a control. 2of10

3 A Elav w 1118 slimb P1943 ago 3 D E F G Elav w 1118 slimb P1943 ago 3 H I Eagle /twi- Fig. S2. Neural phenotypes in slimb and ago mutants. (A ) Single slimb or ago mutant exhibits wild-type NS glial patterns. Embryos were stained for (green) and Elav (magenta) to recognize glial and neuronal cells. The following genotypes are shown: (A) w 1118,() slimb P1493, and () ago 3.(D G) Normal glial numbers in PNS of double mutants. Analysis of PNS is shown for w 1118, slimb P1493, ago 3, and embryos stained for (green) and Elav (magenta). ircles indicate the LH-associated glia, and arrows indicate the D supporting glia. (H and I) Lineage composition in neuroglial 6 4T remains normal in mutant embryos. Eagle (green), the marker for the progenies of 6 4T, was used together with (magenta) to analyze the lineage components in 6 4T. Note that in mutants, no neuron glia cell conversion is observed, and vice versa. (Scale bars: 20 m.) 3of10

4 A gcm N7-4 ; gcm N7-4 /+; Fig. S3. gcm suppresses the mutant phenotype. -stained NS is shown for (A), gcm N7 4 ; (), and gcm N7 4 / ; (). (Scale bars: 20 m.) 4of10

5 S15 S13 S11 S10 A A A D D D E E E S12 Gcm Gcm gcm 26 Fig. S4. Gcm expression patterns in wild-type embryos. (A D) Wild-type embryos stained for Gcm are shown for stage 10 (A) with arrows indicating Gcm (magenta) and (green) double-positive LG and arrowheads indicating Gcm-positive and -negative cells adjacent to the LG. ( and ) A subset of Gcm-positive precursor cells in S11 () and S13 () express (arrows and insets). (D) y S15, Gcm is detected as scattered punta in the nuclei of -positive cells (arrows and insets). (E) Ingcm 26 embryos, no Gcm expression is detected in S15, except for a midline pattern. (Scale bar: 10 m.) 5of10

6 gcm-gal4 A D sca>gcm gcm>gcm E Number of glia/ hemisegment (#) 40 ** ** gcm-gal4 gcm>gcm repo>gcm repo>gcm Fig. S5. Gcm overexpression triggered by gcm-gal4 mimics the glial phenotype. staining shows S15 NS glial patterns of gcm-gal4 (A), sca gcm (), gcm gcm (), and repo gcm (D). (Scale bars: 20 m.) (E) Quantification of glial cell numbers for gcm-gal4 (n 60), gcm gcm (n 70), and repo gcm (n 64). 6of10

7 /twi- A D as-3 E E E E F G G Hoechst G G gcm> gcm> gcm>+gcm F F F Hoechst gcm>+gcm H H H H I I I I Hoechst Hoechst Hoechst Fig. S6. Enhanced proliferation in and gcm gcm mutants. (A D) The increase of mitotic cells in double mutants. The /twi- (A and ) and ( and D) NSs were stained for (A and ) and as-3 ( and D) to detect proliferative and apoptotic cells, respectively. (Scale bar: 20 m.) (E H) Gcm promotes glial proliferation. Embryos of gcm (E and G) and gcm gcm (F and H) were marked by md8 (E H), (E H ), Hoechst (E and F ), and (G and H ). E H are merged images. (Scale bars: 10 m.) (I I ) An enlarged view of two -positive cells (red) labeled with (blue) and the nuclear marker Hoechst (green). 7of10

8 /twi- A S12, svp-lacz A /twi- Gcm LacZ D D Gcm yc LacZ yc Fig. S7. Enhanced Gcm and yc expression in the 5 6A lineage of mutants. (A D) 5 6A progenies marked by svp-lacz (green) show expressions of Gcm (magenta in A and or white in A and ) or yc (magenta in and D or white in and D ) in/twi- embryos (A and ) and mutants ( and D) in S12. (Scale bars: 10 m.) 8of10

9 A 2210 /twi- D E gcm> F gcm>+gcm Slit Slit G /twi- H I J /twi- gcm> gcm> +gcm Fig. S8. Axonal and midline structures of and gcm gcm embryos remain intact. (A D) Axons labeled by 2210 appear normal in /twi- (A), (), gcm (), and gcm gcm (D) embryos. The midline structures marked by Slit (green in E and F and white in G J) also appear similar in /twi- (E and G), (F and H), gcm (I), and gcm gcm (J) embryos. (Scale bars: 20 m for A F and 10 m for G J.) 9of10

10 J29; /twi- J29; A E LacZ D gcm> gcm>; Mz97> F HRP Mz97>; Fig. S9. affects glial cell processes and migration. (A and ) /twi- and embryos carrying the enhancer trap J29 were stained to identify the four peripheral glia. In the mutant, ectopic LacZ-positive peripheral glia (in green) were often clustered near the NS/PNS exit zone (arrows in ) whereas in the /twi-, regular numbers of glia were migrating into the stereotyped position (arrow in A). Moreover, the dorsal-most glia that originated in the PNS (arrowhead in A) was not seen in the mutant anymore. ( and D) Glial cell processes in the control and NSs were labeled with gcm md8.inthe NS, glial processes were not well extended (arrow in D) and they were abnormally protruded as compared to the control (arrow in ). (E and F) Processes of peripheral glia were marked with Mz97 md8. Glial nuclei were labeled with (blue) and axons were labeled with HRP (red). In the wild-type embryo, glial processes often extended to where the oenocytes were (circle in E) whereas processes were shorter and improperly extended in the mutants (arrows in F). (Scale bars: 20 m for A D and 10 m for E and F.) 10 of 10