Fig. S1
.
PPE and
brk
reporter expression in mid
-stage egg chambers.
Large
brk
reporter expression
in mid
-stage egg chambers from wildtype
brkNFgfp
(A) or when
PPEdist
(B) or
PPEprox
(C) is deleted.
White arrows indicate polar cell (PC)/border cell (BC) expression that persists in
brkNFgfp-ΔPPEdist
.
Direct fusion reporter expression for full length (2kb)
PPE
(D),
PPEdist
(E) and
PPEprox
(F) in mid
-stage
egg chambers. Yellow arrowhead indicates germline expression that is associated with
PPEdist-
mCherry
only. In all images, anterior is left and scale bar = 20 μm.
Development: doi:10.1242/dev.199890: Supplementary information
Development • Supplementary information
Fig
.
S2
.
dpp
and
brk
are not co-e
xpressed in
mid-
to late-s
tage egg chambers.
Expression
pattern
of
brk
and
dpp
as visualized by HCR and co-stained with Lam, Spec in WT (
yw
: maximum projections,
A-A
” and sections B-B
”),
ΔPPEdist
(C-C
”) and
ΔPPEprox
(D-D”)
shows that
dpp
and
brk
domains do
not
overlap
in mid-late stage egg chambers.
Germline
brk
expression indicated with white arrow and is only
associated w
ith
ΔPPEprox
mutant background. Sc
ale bar = 20 μm.
Development: doi:10.1242/dev.199890: Supplementary information
Development • Supplementary information
Development: doi:10.1242/dev.199890: Supplementary information
Development • Supplementary information
Fig. S3. Tissue-s
pecific perturbation of
brk
expression with additional drivers supports a role for
brk
in the anterior ECs
and the germline for maintaining germ
cell homeostasis.
Schematics
showing expression patterns of additional GAL4 drivers used to affect
brk
expression in somatic and
germline sub-populations and the resulting quantification of pMad
+
niche cells: (A) anterior
germarium follicle cells, except cap cells (
c587-G
AL4
), (B) a subset of region 1 and 2a escort cells
(
GMR25A11-G
AL4
), (C) follicle stem cells and their progeny (
109-3
0-G
AL4
), (D) terminal filament and
cap cells (
bab1-G
AL4
), and (E) the
cystoblast region of the germline (
bam-G
AL4
).
The
number
of
pMad+ cells in the niche region was quantified for each genotype and plotted; green bar signifies
“normal” range of 2-3
pMad+ cells
per
germarium. One-way ANOVA was used for statistical
comparison of each dataset to its respective control genotype (see Methods). n = number of germaria,
error bars represent mean±s.d. (see Table S2).
Development: doi:10.1242/dev.199890: Supplementary information
Development • Supplementary information
Fig. S4. Loss of
brk
in follicle cells leads to mid
-late stage follicle phenotypes that
are separable
from
pMad+ cell
regulatory role.
Representative germaria resulting from broad FC
brk
knockdown driven
by either
tj-GAL4
(constrained to adult using
GAL80
TS
; A) or
c587-
GAL4
(constrained to adult using
GAL80
TS
, B) have different effects on
pMad+ cell
number (as visualized by pMad+ staining in the niche;
indicated by yellow arrowheads - see also Fig. S3); while both lead to loss of mid-stage egg chambers
(late stage egg chambers abutting germaria indicated with white arrows) as well as perturbed or absent
fusomes (indicated by yellow bracket). These drivers differ in strength, as indicated by signal from UAS-
mCD8
-GFP (E, F), in a trend that corresponds to the severity of their effects on
pMad+ cell
number. Drivers
active in subsets of follicle cells:
109-
30-GAL4
is expressed in FSCs and shows a loss of mid-stage egg
chambers similar to that of
tj-GAL4
-
and
c587
-
GAL4
-driven
brk
RNAi (C). Whereas
GMR25A11
which is
active in anterior ECs has a moderate effect on
pMad+ cell
number but mid to late-stage egg chambers
appear normal (D). In all images, anterior is to the left and scale bars = 20μm. (G) Number of egg chambers
(including the germarium) per ovariole was quantified for follicle cell
brk
knockdown and one-way ANOVA
was used for statistical comparison of each dataset to the control genotype (
yw
; see Methods). n = number
of ovarioles, error bars represent mean±s.d. (see Table S2
).
Development: doi:10.1242/dev.199890: Supplementary information
Development • Supplementary information
Fig. S5.
dpp
RNAi demonstrates that increase in pMad+
cell number associated with
Δ
PPEprox
mutant rel
ates to
dpp
.
RNAi against
dpp
was performed using either
tj-GAL4
or
GMR25A11
-GAL4
in
wildtype and
ΔPPEprox
mutants (without
GAL80
TS
).
ΔPPEprox, tj-
GAL4
control has significantly more
pMad+ cells than wildtype but
dpp
RNAi with either
tj-GAL4
or
GMR25A11
-GAL4
in the mutant background
mirror the knockdown in a wildtype background, indicating a dominant effect of
dpp
in the EC.
One
-way
ANOVA was used for statistical comparison of each dataset to control genotype (
tj-GAL4
; see Methods).
n = number of
germaria, error bars represent mean±s.d. (see Table S2).
Development: doi:10.1242/dev.199890: Supplementary information
Development • Supplementary information
Fig. S6. Expression
of additional
cap cell markers
in ECs
suggest
these
cells
have
altered
fate
when
brk
is upregulated.
Staining for cap cell markers En and Cad in wt (A,A’) compared
to mutant
conditions shows enrichment of Cad at ECs where En is upregulated (see also, Fig. 6K-P
) in
ΔPPEprox
(B,B’), and upon
brk
overexpression driven by either
c587-G
AL4
(constrained to adult with
GAL80
TS
;
C,C’)
or
tj-G
AL4
(D,D’), but not when driven by
nos-G
AL4
(E,E’) . Inset area indicated with yellow dashed
box. In all images anterior is left, scale bars = 20 μm.
Table S1.
Drosophila
fly stocks
Click here to download Table S1
Table S
2
.
Detailed statistics
Click here to download Table S
2
Table S
3
.
Primers and genomic sequences
Click here to download Table S
3
Development: doi:10.1242/dev.199890: Supplementary information
Development • Supplementary information