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Supplementary Figure 1. Patterns of slbo reporter expression. Single-channel images show

GFP driven by the slbo enhancer from the stage 8 egg chambers shown in Figure 1 (a-d here

correspond to images c-f in Fig. 1). Yellow asterisks mark polar cells; pink asterisks indicate non-

GFP-positive gap cells. Scale bar is 10m.

Supplementary Figure 2. Asymmetric arrangement of border cells continues in stage 9. (a)

Lateral view of stage 9 egg chamber from the slbo reporter line, slbo-Gal4, UAS-mCD8-GFP. GFP,

green; Fas III, red; DAPI, blue. Border cells (green) have changed shape to cluster around the polar

cells (red). Scale bar is 20m. (b, d) 3-D reconstructions of optical sections from the anterior

epithelia of a stage 9 egg chamber. Scale bars are 10m. (b) GFP expression shows a gap in

positive cells next to the polar cells (white asterisk). Magenta asterisks mark polar cells. GFP,

green; Fas III, white; DAPI, blue. (c) Graph shows several different patterns of border cell

arrangements with two different border cell markers (slbo or STAT antibody) at stage 9. (d)

Example arrangement of a border cell cluster with 2 gaps. Magenta asterisks mark polar cells; white

asterisks mark gap cells. GFP,green; STAT, red; Arm, white; DAPI, blue.

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Supplementary Figure 3. Domeless expression is continuous across the anterior epithelium.

(a) Lateral view of stage 8 egg chamber from the domeless-LacZ genotype. β-galactosidase, green;

Arm, red; DAPI, blue. Scale bar is 20μm. (b-b’) 3-D reconstructions of optical sections from the

anterior epithelia of a stage 8 egg chamber. (b’) β-gal expression is limited to the nucleus of anterior

epithelial cells. Yellow asterisks mark polar cells. β-gal, green; Slbo, red; Arm, white. Scale bar is

5μm.

Supplementary Figure 4. Patterns of STAT protein expression. Single-channel images show

cells marked with an antibody directed against the STAT protein in the stage 8 egg chambers shown

in Figure 2 (a-c here correspond to images a-c in Fig. 2). Yellow asterisks mark polar cells. Scale

bar is 10µm.

Manning et al., Supplementary Information

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Supplementary Figure 5. Radial border specification pattern adjacent to nurse cell cleft.

(a-c) An individual stage 8 slbo-Gal4 reporter egg chamber imaged from the anterior at different

focal planes (optical sections). slbo-GFP, green; F-actin, phalloidin, red; FASIII, cyan; DAPI, blue.

Scale bar is 10μm. Yellow asterisks indicate polar cells. (a) Anterior-most optical section, which is at

the apical surface of the epithelium. Presumptive border cells, positive for GFP, exhibit radial

organization. (b) A deeper optical section, which is 5 μm past the surface of the most anterior nurse

cell. The cleft is estimated by visualization of cortical F-actin in the nurse cells, and by creating 3-D

reconstructions. (c) Same focal plane as in (a) with the dotted yellow lines outlining the “Y” shaped

cleft that lies below this focal plane. The polar cells are away from the cleft, and sit over a nurse

cell.

Manning et al., Supplementary Information

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Supplementary Figure 6. UPD secretion from only one polar cell does not recapitulate asymmetries observed in vivo. (a, b, c) Representations of the surface of the epithelium spanning 50µm from the center (the polar cells, rectangles). Domain coordinates are set to (0,0,0) at the apical point between the polar cells. A flat, even subapical domain is modeled. For these simulations, Upd is secreted from the left polar cell centered at (-2.5,0) (dark gray), and no source or uptake Upd occurs in the right polar cell. (a’, b’, c’) Magnified view of center of the region, reoriented in a flat representation. (b’, c’) Heatmaps show resultant local Upd concentration (experienced at the follicle cell apical surface) after the simulation is run, similar to Figure 4. Scale bar (right) shows

highest [Upd] in dark red; lowest in blue. (b, b’) The original source value = 45 pM µm s-1 is effectively reduced by half coming from only one polar cell, and is insufficient to produce the above-threshold activation needed for border cell specification. (c, c’) Doubling the source to

= 90 pM µm s-1 yields activated cells nearest to the UPD-secreting polar cell, but none to the other side.

Manning et al., Supplementary Information

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Supplementary Figure 7. Increased UPD secretion yields radial symmetry, as observed in

vivo. (a) A domain with a Y-shaped cleft centered at (0,0,0, approximating three nurse cells at the anterior epithelium, with one variable cleft as in Fig. 4f. This domain is shown from the apical side to illustrate the clefts clearly (the positive directions along the x, y, and z axes are indicated). (b) These

simulations are the same as those from Fig. 4, except the source term is increased to 75 pM µm s-1 to model overexpression of Upd (secreted from the both polar cells). When Upd expression is significantly increased, the model shows radial patterns of cell activation for a variety of cleft dimensions (compare to Fig. 4h), similar to the results of in vivo experiments shown in Fig. 2.

Manning et al., Supplementary Information

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Supplementary Figure 8. halfpint (hfp) mutant ovaries have grossly normal follicle cell

development. (a) Ovariole from a hfp13/ hfp9 mutant female, stained with antibodies directed

against SLBO (green) to mark border and centripetal cells, and Armadillo (ARM, red, a’) to reveal β -

catenin, which is enriched in border cells, and stained with DAPI (blue, a’’) to show nuclei. Scale bar

is 50μm (a’) ARM expression is normal in younger stage follicle cells and border cells, which have

migrated (arrow). (a’’) Normal development of stalk cells is apparent (arrowheads) when all follicle

cell nuclei are marked.

Supplementary Figure 9. Stage 8 anterior follicle cells lack active Caspase 3. (a-a’) Lateral

view of a stage 5 egg chamber from a slbo-reporter female. A supernumerary polar cell is marked by

the expression of active Caspase 3 (GFP,green; Casp3 antibody, red; DAPI, blue. Scale bar is

20μm. (b-b’) Lateral view of stage 8 egg chamber from a slbo-reporter fly. There is no detectable

active Caspase 3 expression, and nuclear DNA looks normal, not punctate. GFP, green; Casp3, red;

DAPI, blue. Scale bar is 20μm.

Manning et al., Supplementary Information

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Supplementary Table 1: Lateral cleft measurements for stage 8 egg chambers.

Sub-apical clefts were measured by lateral imaging of phalloidin-stained egg chambers, using optical sections

and 3-D projections as needed (see Methods). Only egg chambers with two visible nurse cells at the anterior

were analyzed for this table; only clefts juxtaposed against presumptive border cells were included in analysis.

Error is given as Standard Error of the Mean (SEM), and multiple measurements per egg chamber were used

to determine average sizes in mutants. For slbo-reporter, total two-dimensional area was approximated as a

triangle representing the domain created by two nurse cells against the epithelium. For hfp mutants, since

nurse cells did not fill the space, a straight-edged crescent shape (see Fig. 5b) was measured, and the area

was approximated as a rectangular domain; the triangular cleft domain was relatively small and was not

included.

Genotype Stage 8 egg chamber

Cleft Depth (μm)

Cleft width (μm)

Cleft area (μm2)

slbo-Gal4; UAS-mCD8GFP

1 2.91 6.77 9.85

2 3.51 8.83 15.50

3 3.99 6.8 13.57

4 5.87 4.31 12.65

5 6.28 4.15 13.03

6 3.95 9.8 19.36

7 3.24 8.48 13.74

8 3.68 3.61 6.64

9 1.97 2.73 2.69

10 2.04 3.28 3.35

11 3.98 6.29 12.51

12 2.59 4.28 5.54

AVG 3.67±0.4 5.78±0.7 10.70 (triangular)

hfp13, st, cu, e, ca/ TM2, Ubx

1 2.75 14.74 40.54

2 2.13 8.75 18.64

3 2.0 17.06 32.12

4 2.63 11.75 30.90

AVG 2.38±0.2

13.07±1.8

30.55 (rectangular)

hfp38, st, cu, e, rec2/ hfp13, st, cu, e, ca

1 3.2 15.00 48

2 2.0 14.44 28.88

3 2.0 10.7 21.4

4 2.1 19.60 41.16

AVG 2.33±0.3 14.94±1.8 34.86 (rectangular)

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Supplementary Table 2: Presumptive border cell cluster arrangements relative to the

nurse cell cleft when polar cells juxtaposed one nurse cell.

Presumptive border cell (BC) numbers were scored by upright imaging of egg chambers from slbo-Gal4;UAS-

mCD8GFP females. BC arrangement was determined as described in the text. Total number of border cells

was determined by GFP and Slbo protein expression by antibody staining; Fas III expression was used as a

marker for the Polar Cells (PC), and DAPI DNA dye revealed all follicle cells (FC) in the domain. In these

cases, three or four nurse cells lay adjacent to the epithelium, creating a Y or H shaped cleft. For cases in

which all BC sat over the nurse cell itself, not the cleft, a radial activation pattern was observed.

BC arrangement # of BC (slbo +)

Shape of Cleft

Position of BC cluster Position of gap if present

radial 5 "Y" (H) over nurse cell nuclei no gap

radial 10 "Y' over nurse cell nuclei no gap

radial 8 "Y" over nurse cell nuclei no gap

radial 9 "Y" over nurse cell nuclei no gap

1 gap 10 "Y" over nurse cell nuclei, 3 cells over one section of “Y”

gap above nc

1 gap 6 "Y" over one section of "Y" gap above divot

1 gap 9 "Y" over one section of "Y" gap above nc