Supplemental Figure S1. Polarized MTs are essential for cellular localization of axis
determinants in the oocyte. A schematic view of the germarium is shown at the top. Theoocytes, a germline stem cell (GSC) and cystoblasts (CBs) are shown in purple, paleorange and blue, respectively. A schematic diagram of an ovariole is shown in themiddle. The ovariole consists of the germarium at the anterior tip and egg chambers ofincreasing age. Schematic views of egg chambers at stages 1-8 (mid-oogenesis) areshown at the bottom. MT distribution is shown in green. At stages 1-6, MTs extend
along the cortex and fold back slightly at the anterior. The MTOC is detected at theposterior region of the stage 1 oocyte. The oocyte nucleus is encapsulated by MTs. By
stage 7, MTs are rolled up in the shape of a “diaphragm” in the center of the oocyte. In
stages 8-9, as the ‘diaphragm’ has opened, MT bundles project from both the anterior
and the oocyte nucleus in the A-P direction, exhibiting a “horseshoe-like” pattern.

Supplemental Figure S2. (A) Mael is expressed in ovaries and Drosophila S2 cells, anon-gonadal somatic cell line originally established from an embryo (left panel). Maelcan be efficiently knocked down by transfection of double-stranded mael RNA in S2cells (right panel). (B) The Mael signal disappeared from S2 cells after RNAi treatment,confirming that the antibody is Mael-specific. (C) Immunofluorescence analysis ofMael in dividing S2 cells. Mael is highly concentrated at mitotic spindles. (D) The Maelsignal was not detected in mael mutant (maelM391/Df) egg chambers, confirming theantibody specificity. Scale bars indicate 20 µm. (E) Immunoprecipitation wasperformed from ovary lysates using an anti-Mael monoclonal antibody in an
Empigen-based buffer. The immunoprecipitated complexes were stained with silver.
Mael alone was immunoprecipitated, confirming that the antibody does not cross-reactwith proteins other than Mael. (F) The complexes immunoprecipitated from control(maelM391/TM3) and mael mutant (maelM391/Df) ovary lysates using an anti-Maelmonoclonal antibody were immunoblotted using antibodies against Cnn, Msps,
D-TACC and γTub. Mael association with Cnn, Msps, D-TACC and γTub wasconfirmed.

Supplemental Figure S3. (A) The Mael complex immunopurified from ovaries with ananti-Mael antibody was analyzed by immunoblotting (IB) with antibodies for eachmember of the Argonaute family of proteins. The Mael complex does not contain any
members of the Argonaute family. (B) Immunoprecipitation from ovary lysates using
anti-Aub and anti-Piwi monoclonal antibodies. Protein components of MTOC (Cnn andD-TACC) were not detected in the Aub- and Piwi-associated complexes. (C-D) The
Aub- or Piwi-associated complexes purified from ovaries were analyzed byimmunoblotting (IB) with the anti-Mael antibody. Mael was not detected in either
complex. “n.i.” indicates non-immune IgG used as a negative control.

Supplemental Figure S4. Immunohistochemical analyses of the mael mutant ovaries
using anti-Mael (magenta) and anti-Vas (green) antibodies. Vas is a germ cell marker.
Mael was detected both in germline cells and in the Vas-negative somatic follicle cells(surrounded by the dotted-line). Mael was also detected at the mitotic spindles (arrows)
in the dividing cyst cells, which are a Vas-positive cyst. DNA was stained with DAPI(blue).

Supplemental Figure S5. The expression level of αTub in control and mael

(maelM391/Df) ovaries. Western blot analyses of ovary lysates using anti-Mael (upper)
and anti-αTub (middle) antibodies. Ponceau S staining of the blot is shown as a loadingcontrol (lower).

Supplemental Figure S6. Monitoring MT dynamics in living S2 cells using time-lapse
fluorescence microscopy. (A) Live fluorescence microscopy cell analysis of maelRNAi
cells showed delayed mitosis. The time that cells take from metaphase to anaphase wasmeasured (also see C). The mitotic time of maelRNAi cells is longer than that of control
cells. (B) The percentage of mitotic S2 cells in maelRNAi, cnnRNAi, and cdc27RNAi S2 cells.
GFPRNAi was used as a control. The number of mitotic cells of maelRNAi was slightly
increased compared to that of cnnRNAi. cdc27 is known to be an essential cell cycle gene
and was used as a positive control (Goshima et al. 2007) in this study. (C) Time-lapse
fluorescence microscopy imaging of control (top) and maelRNAi (bottom) S2 cells.
AcGFP-αTub and dsRed-Histone 2B were used to detect αTub (green) andchromosomes (red), respectively. Normal bipolar spindle formation during cell divisionappeared normal in maelRNAi S2 cells. However, maelRNAi S2 cells showed, incomparison to the control cells, slightly delayed mitosis (also see A). Time scale is in
min:sec.

Supplemental Figure S7. MT distribution in mael egg chambers containing over 16
germ cells visualized using anti-αTub antibody (white). (A) An egg chamber with twooocyte-like cells was observed in mael ovaries. The accumulation of MTs was notdetected in either oocyte (green arrowheads). Oo indicates Oocyte-like nuclei. (B)Magnified views (bottom) of mael (maelM391/Df) oocytes (indicated with thedotted-line) in (A) are shown. Magnified view of control (maelM391/TM3 shown in Fig.
5C-St 4-5), which shows MT accumulation at the posterior region of the oocyte, is alsoshown. It appears that the MTOC is not formed in the mael egg chambers. DNA wasstained with DAPI (blue).

Supplemental Figure S8. Distribution of DE-cadherin in mael mutant ovaries. (A) The
expression pattern of DE-cadherin in the germarial region of the control ovary. Highconcentrations of DE-cadherin are found in both anterior and posterior follicle cells(arrows). Oocytes, judged by Orb staining (A’), lie adjacent to DE-cadherin-positive
follicle cells (A’’). (B) The expression pattern of DE-cadherin in the germarial region ofthe mael mutant ovary. DE-cadherin in both anterior and posterior follicle cells was notdetected in mael ovaries. DE-cadherin was detected in the mael oocyte (indicated withan arrow, compare with A). In addition, DE-cadherin was localized in a disorderly
fashion in some egg chambers (indicated with horizontal square bracket). (C) The
expression pattern of DE-cadherin in the mael mutant egg chamber with over 16 germ
cells. DE-cadherin accumulated not only in the posterior- but also in theanterior-localized oocyte. DNA was visualized using DAPI. Scale bars indicate 20 µm.

Supplemental Figure S9. Possible models for mechanisms giving rise to the egg
chambers containing over 16 germ cells, based on Huynh (Huynh 2006). In Drosophila,
the cystoblast divides precisely four times with incomplete cytokinesis to form a 16-cell
cyst. Normal egg chambers contain 15 nurse cells, an oocyte, and 15 ring canals(Normal). When two egg chambers juxtaposed to each other are fused, one egg chamber
contains 30 nurse cells, two oocytes and 30 ring canals (Fusion). Cysts with insufficientrounds of cell division generates less than 15 nurse cells and ring canals (mitoticdefects: less than 4 rounds of cell division). A cyst with an extra round of cell division
generates 31 nurse cells and ring canals, and an oocyte (mitotic defects: 5 rounds of cell
division). In the case of fusion, the number (r) of ring canals is n-2, where n is thenumber of germ cells (r = n-2), whereas in the case of mitotic defects, the number ofring canals is n-1 (r = n-1). White circles indicate germ cells, blue circles indicate
oocytes, and red lines indicate ring canals.

Sato_supplemental Fig.S1
Germarium

Region 3(St. 1)

Region 2b
Region 2a
Region 1

GSCs
CBs
Centrosome migration Posterior positioning(Oocyte determination) of Oocyte

MitosisGermarium 24689101114

OocyteMTOC

St. 1-6

St. 7

St. 8

Mael

62-

Mael

47.547.5-

αTub

Mael αTub
Mael DAPIDAPICinterphase

αTub

metaphase

anaphase

EkDa
220-+ + -F

Control

mael

IPIP

Input
n.i.
anti-Mael
Input
n.i.
anti-Mael62-

maelM391/Df: IP: Lysate
Msps

160IB

83-

anti-Mael83-

maelM391/TM3

anti-Mael

175-

D
Mael

175-

mael RNAi

Mael Vas
αTub DAPIkDa

GFP RNAi

n.i.

B
Mael αTub
Mael DAPI
DAPI

kDa

mael RNAi

S2

S2

A

Ovary

Sato_supplemental Fig.S2

D-TACC
Cnn

120-

γTub

10090-

Mael

807060-

50-

-Mael

- MaelAub

Mael
D-TACC

Ago3
Cnn

Aub

Piwi

Piwi
Mael

anti-Piwi

Piwi
Aub-MaelMael

anti-Aub

IP

n.i.

C
anti-Mael

n.i.

Input

anti-Piwi

IP

Input

Ago2
anti-AubB

IB

IB

Ago1
n.i.

IP

Input

anti-Mael

n.i.

Input

AIB

IB

Sato_supplemental Fig.S3

DIP

Sato_supplemental Fig.S4

Mael

Vas

Mael
Vas

Mael
Vas
DAPI

Mael
Tub

Ponceau S

maelControl

Sato_supplemental Fig.S5

Sato_supplemental Fig.S6

A

B

CControl

Prophase

Metaphase

Anaphase

Telophase

maelRNAi

Metaphase

Anaphase

Telophase

Sato_supplemental Fig.S7

A

maelM391/Df

7 8 9 Oo
5 6 1326
4 1112
3 10 16 18 25
15 17 24
2
1 14 2223
Oo 1920 21

DAPI

B

maelM391/TM3

Tub

Tub DAPI

maelM391/Df

Sato_supplemental Fig.S8
A maelM391/TM3

B maelM391/Df

DE-cadB’A’

Orb
B’’A’’

DE-cad Orb
B’’’

A’’’

merge
C maelM391/Df

C’

C’’

C’’’

DE-cadOrb

DE-cad Orb

mergeSato_supplemental Fig.S9
Mitotic defect1(less than 4 rounds of cell division)
less than 15 Nurse cells
less than 15 Ring canals

12

12

3

4

5

6

1

2

3

4

7

8

13

5

6

14

9

1

2

10

11

3

4

1215

78

16

13

5

6

14

13

5

6

14

9

1

2

10

9

1

2

10

11

3

4

12

11

3

4

12

15

7

8

16

15

78

16

Normal(4 rounds of cell division)
15 Nurse cells
15 Ring canals
1 Oocyte

2925 2630

13 17

910 18 14215126222373482415 19 11 12 20 16
31

27 28

32Fusion
30 Nurse cells
30 Ring canals
2 Oocyte

Mitotic defect(5 rounds of cell division)
31 Nurse cells?
31 Ring canals
1 Oocyte?