SINGH K.P. and BRILL J.A. 2014

G3 – Genes – Genomes – Genetics 1
Investigation: Gene Function and Functional Genomics
A Genome-wide Screen to Identify Novel Interactions of
the Phosphatidylinositol Phosphate Signalling Pathway in
Drosophila Spermatogenesis
Singh, Kevinder P. 1, 3, 4
and Brill, Julie A. 1, 2
1
Program in Cell Biology, The Hospital for Sick Children, Toronto ON, Canada; 2
Department of Molecular Genetics, University of
Toronto, Toronto ON, Canada; 3
Department of Human Biology, University of Toronto, Toronto ON, Canada; 4
Department of Cell and
Systems Biology, University of Toronto, Toronto ON, Canada.
Abstract Phosphatidylinositol phosphates (PIPs) are important cell membrane lipids involved in intracellular
signalling and trafficking pathways. In this project, we investigated the role of PIPs in spermatogenesis. We used
Drosophila melanogaster as a model organism due to the conserved nature of genes involved in sperm development.
The objective of our research is to identify novel PIP-pathway interactions. We used transgenic flies expressing a PIP
5-phosphatase, SigD, isolated from Salmonella, in male germ cells. Expression of SigD results in the reduction of
intracellular phosphatidylinositol 4, 5-bisphosphate (PIP2), resulting in male sterility. Infertility is due to defects in
nuclear shaping, impaired axoneme elongation, spermatid cyst bipolarity and defects in F-actin investment cone
polymerization. Females carrying the SigDLow
transgene are fertile and were crossed to males with specific
chromosomal deletions obtained from the Bloomington Stock Centre (refer to materials and methods). Male progeny
from these crosses, which carry the maternal SigDLow
transgene as well as the paternal deletion, were selected for
analysis. Testes were dissected and stained with DAPI to mark nuclei and rhodamine-phalloidin to mark F-actin.
Fluorescence microscopy was used to analyze cells. From a total of 113 deficiency crosses, 20 strongly suppress the
SigDLow
transgene, resulting in a rescue of nuclear shaping defects, axoneme growth, F-actin cone polymerization and
cyst bipolarity, while 8 enhance it. Further analysis at the genetic level using DNArrow©
2.1 has uncovered a gene of
interest, meiosis I arrest (mia). Mia males were crossed to SigDLow females; SigDLow x mia-/+ males displayed a partial
rescue of spermatid cyst bipolarity, nuclear shaping and F-actin investment cone assembly. Immunofluorescence
probing for PIP2 showed a partial upregulation of PIP2 at the nuclear end of spermatid cysts from these flies. Other
genes also revealed from the screen showing partial suppressive interaction were nuclear fallout (nuf) and always
early (aly), both implicated in spermatid development.
Introduction: Spermatogenesis in Drosophila
melanogaster
During spermatogenesis in Drosophila
melanogaster, a germline stem cell divides
producing an undifferentiated stem cell and a
spermatogonium1
. The spermatogonium
subsequently undergoes four rounds of mitotic
divisions to produce 16 mature primary
spermatocytes1
. These 16 primary spermatocytes
then enter meiotic division to produce 64 haploid
spermatids encased within a syncytial cyst in the
testis1
. Spermatid differentiation begins with cells
containing a haploid nucleus (Figure 1A), a
nebenkern composed of two mitochondrial
derivatives and a short axoneme, which will later
develop into the tail of the spermatid1, 2
(Figure
1B). Elongation of the axoneme occurs at the
growing end as polarity is established early
during development2, 3, 4
. The sperm tail or
axoneme is composed of the classical 9+2
microtubule structure (Figure 1C). Elongated
spermatid cysts are unipolar, as all the nuclei are
polarized to one end of the cyst. The spermatid
nuclei undergo differentiation from a round shape
to a characteristic needle-like shape2
(Figure 2).

Singh K P. and Brill J A. 2014
2
Figure 1. Drosophila spermatid development. A.
Early round spermatocytes contain a nebenkern
composed of two mitochondrial derivatives (mt), and
an elongating axoneme (ax). B. Elongation of the
axoneme and the mitochondrial derivative occur
simultaneously. C. Cross-section of the spermatid
axoneme, which is composed of a classical 9+2
microtubule. D. 64 spermatids get encased into a
syncytial cyst; polarity is established as all nuclei face
the one end of the cyst.
Figure 2. Changes in nuclear morphology of a
developing spermatid. Spermatid nuclei begin as
round and develop into a final characteristic needle-
like shape.
Additional research is required to understand the
mechanisms behind the drastic morphological
change of the spermatid nuclei. Once axonemal
elongation is complete, spermatids undergo
individualization, which involves the removal of
unnecessary organelles and cellular components
not required by mature sperm1, 6, 7
. During
individualization, investment cones containing
filamentous actin (F-actin) form around each
nucleus (Figure 3A) and migrate in synchrony
from the nuclear-end of the cyst towards the tail1
.
A cystic bulge or waste bag forms as the F-actin
cones migrate, which is later degraded1
(Figure
3B). Once individualization has occurred the
spermatids are able to enter the seminal vesicles
and are capable of fertilization1, 4
.
Studying spermatogenesis in Drosophila
provides us with a great model for understanding
microtubule assembly as the axoneme can reach
an approximate length of 2 mm, which is 40 times
longer than human sperm1, 3, 4
. In addition, we
also acquire a thorough understanding of F-actin
dynamics and changes in nuclear morphology1, 5
.
It has been previously described that
phosphatidylinositol phosphate signalling is
crucial for regulating the above-mentioned
processes in Drosophila spermatogenesis 3, 4
.
Phosphatidylinositol Phosphate Signalling in
Drosophila Spermatogenesis
Phosphatidylinositol phosphates, or PIPs
henceforth, are important cell membrane lipids
involved in intracellular signalling and trafficking
pathways8
. The goal of our research is to
investigate the role of PIPs in Drosphila
spermatogenesis. The transgenic flies used in our
experiments express varying levels of SigD a PIP
5-phosphatase, isolated from Salmonella3, 4, 14
.
SigD catalyzes the reverse reaction of PIP5-
kinase-1 reducing intracellular membrane bound
phosphatidylinositol 4, 5-bisphosphate or PIP2,

Figure 3. Filamentous actin (F-actin) investment cone
polymerization and migration. Once spermatids have
elongated, they undergo individualization. F-actin
investment cones migrate in synchrony from the
nuclear-end of the spermatid towards the tail,
removing unnecessary organelles and cellular
components not required by mature spermatids. A
cystic bulge or waste bag forms as the cones migrate,
which is degraded. A. DAPI and rhodamine-phalloidin
staining depicting F-actin investment cones associated
with nuclei. B. Migrating F-actin cones and formation
of the cystic bulge as indicated by arrows.
Figure 4. The phosphatidylinositol-phosphate signalling pathway. Phosphoinositol (PI) is phosphorylated by
PI4-kinase producing phosphatidylinositol 4-phosphate (PI4P), which is once again phosphorylated by PIP5-
kinase-1 producing phosphatidylinositol 4, 5-bisphosphate, or PIP2. SigD is a PIP5-phosphatase that catalyzes
the reverse reaction of PIP5-kinase-1, depleting membrane phosphatidylinositol 4, 5-bisphosphate or PIP2.
which is crucial during spermatid development 3, 4
(Figure 4). Phosphatidylinositol phosphate
signalling in Drosophila has been shown to be
involved in cellular growth and differentiation,
establishment of apical-basal polarity, actin
dynamics, as well as regulation of the cell cycle9,
10, 11, 12, 13
.
The Effects of SigD Transgenic Expression
during Drosophila Spermatogenesis
SigD Transgenic Flies have Reduced Levels of
PIP2
SigD, also known as SopB, is a protein
encoded by the Salmonella pathogenicity island 1,
which functions as a virulence factor for bacterial
survival14, 15
. SigD has the capacity to reduce
membrane PIP2, which is extensively associated
with the actin cytoskeleton; reduction causes
membrane ruffling16, 17
. This ruffling of the
membrane allows the pathogen to gain access into
epithelial cells, where it can replicate and escape
host immune responses14
. SigD acts as a
phosphoinositide phosphatase, specifically a PIP
5-phosphatase16
. To study the role of PIP2 in
Drosophila spermatogenesis, the SigD transgene

4
was expressed under the regulation of the
spermatocyte specific β2-tubulin promoter18
. Two
different transgenic lines were used: one with a
high level of SigD expression in the testes
(SigDHigh
) and the second with a lower level of
expression (SigDLow
) 4
. SigDLow
flies carry the
transgene on the X chromosome resulting in
lower expression of the transgene, whereas
SigDHigh
flies carry SigD on the right arm of the
third chromosome, resulting in a higher
expression3, 4
. When the SigD transgene is
introduced into the Drosophila genome, distinct
phenotypes have been observed during
spermatogenesis3, 4
. Males carrying the SigD
transgene show sterility3, 4
.
Partial Reduction in PIP2 Levels During
Spermatogenesis Causes Male Infertility
Previous experiments using
immunofluorescence showed that PIP2 normally
associated with the plasma membrane of
spermatocytes is depleted in SigDLow
expressing
transgenic flies 4
. Spermatids of SigDLow
flies fail
to undergo nuclear elongation; they do not form
F-actin investment cones and consequently do not
undergo individualization3, 4
. Depletion of PIP2 in
SigDHigh
lines causes defects in meiotic
cytokinesis and a complete failure in spermatid
tail elongation3
, a more sever phenotype when
compared to SigDLow
. When an enzymatically
dead, or phosphatase-dead, variant of the SigD
(SigDdead
) was transgenically expressed, no
phenotype was observed confirming catalytic
activity of the functional SigD transgene3
. This
result confirmed that SigD is responsible for the
observed phenotypes as it depletes membrane
PIP2, and ultimately causes male sterility3, 4
.
Reduction in Membrane PIP2 Results in
Spermatid Cyst Bipolarity
Wild type elongating spermatid cysts are
unipolar, as all nuclei localize to one end of the
cyst1, 2, 3, 4
(Figure 5A). It has been previously
reported that a reduction in PIP2 levels results in
spermatid cyst bipolarity4
. Scattered nuclei are
also observed in the SigDLow
and SigDHigh
transgenic flies as a result of PIP2 depletion4
.
Bipolarity of the cyst occurs when nuclei appear
distributed evenly to each end of the cyst and
spermatid tails develop towards the center4
(Figure 5B). Experiments overexpressing sktl
(PIP5-kinase-1) in the SigDLow
transgenic
background resulted in a partial rescue of the
above-described phenotypes4
. Sktl is a PIP5-
kinase, which replenishes membrane PIP2
4
. This
further supports the importance of the PIP2 at the
membrane during spermatid development.
Reduction in Membrane PIP2 Leads to Defective
F-actin Assembly
PIP2 regulates actin dynamics in
association with downstream Rho family
GTPases, considered to be the master regulators
of the cytoskeleton4
. Membrane bound PIP2 is
shown to be involved in the regulation of
downstream effector proteins, such as the N-
WASP and ARP complexes that modulate the
actin cytoskeleton and its interactions7
. It has also
been reported that PIP2 links actin to the plasma
membrane through membrane linker proteins like
spectrin19, 20
. F-actin cone polymerization and
migration are essential for spermatids to undergo
individualization and become mature sperm4
(Figure 5A’). Expression of SigDLow
and SigDHigh
in Drosophila melanogaster testes reduces
membrane PIP2 leading to defective F-actin cone
polymerization and migration4
(Figure 5B’ and
C’). Since F-actin investment cones are required
for spermatid individualization, absence or
reduction of F-actin leads to spermatids incapable
of this process, rendering them non-functional4
.
The objective of our research is to perform
a genetic screen, testing deficiencies on the right
and left arms of the second and third chromosome
of the Drosophila melanogaster genome. We will
cross fertile-virgin females carrying the SigDLow

Figure 5. Effects of SigDLow
and SigDHigh
transgenic expression on spermatid development. A. Wild type
nuclei stained with DAPI, 64 nuclei are polarized to one end of the cyst. A’ Micrograph showing 64 F-actin
investment cones associated with nuclei. A’’ Merged micrograph showing long thin unipolar cysts. B.
Expression of SigDLow
causes defects in nuclear morphology, as well as bipolarity within the cysts, as
indicated by yellow arrows. B’ Lack of F-actin investment cones when SigDLow
is expressed. B’’ Merged
micrograph depicting truncated bipolar spermatid cysts. C. Expression of SigDHigh
leads to complete arrest
in axoneme elongation and nuclear morphology. C’ Complete lack of F-actin investment cones. F-actin is
detected in ring canals formed during incomplete cellular division. C’’ Merged image depicting a loss of
polarity within spermatid cysts.
transgene to Bloomington deficiency males and
select progeny carrying the maternal SigDLow
transgene and the paternal deficiency. We are
looking for dominant suppressers or enhancers of
nuclear shaping, spermatid cyst polarity, F-actin
cone polymerization and axoneme elongation. We
are interested in finding potential genes that
interact with the phosphatidylinositol phosphate
pathway. Performing a genetic screen has proven
in the past to be a reliable methodology in
understanding signal transduction pathways and
their interactions such as the sevenless and ras
signalling pathways, both involved in vital
intracellular signalling transduction processes24,

6
25
.
Materials and Methods
Fly Stocks and Husbandry
Transgenic flies, SigDLow
and SigDHigh
,
were created by microinjecting w1118
(wild type)
embryos with constructs containing the SigD
transgene3, 18
. Due to the sterility of SigD
expressing males, to maintain stocks we select
females carrying the transgene (w+
- red coloured
eyes) and crossed them to w1118
males. All flies
are maintained on standard molasses agar
medium18
. The Bloomington Deficiency Stock
used in our experiments consist of viable flies
carrying large deletions of their genome generated
through the use of transposable elements and
maintained over rearranged wild type
chromosomes, known as balancers. Available:
http://flystocks.bio.indiana.edu/. All SigD, w1118
flies and deficiency crosses were incubated at
25°C to speed larval development. The
Bloomington Deficiency stocks and mutant flies
(mia/aly/nuf/nht/comr/sa) were maintained at
room temperature18
. Mia flies were generated
using ethyl methanesulfonate mutagenesis (EMS)
by Lin et al., 1996. To perform genetic crosses,
we select virgin females carrying the SigDLow
transgene, which are fertile, and cross them with
balanced Bloomington deficiency flies. Male
progeny from these crosses that carry the
maternal SigDLow
transgene as well as the
paternal deficiency were selected for further
analysis (Figure 6).
Chemical Reagents, Micro-dissections and
Fluorescence Staining
Flies were anesthetized with a constant
flow of carbon dioxide. Testes from males 1 to 3
days old were dissected in testis isolation buffer
(TIB: 183mM KCl, 47mM NaCl, 10mM Tris
buffer, pH 6.8 in 100mL double-distilled water)
using a Leica© MZ6 stereomicroscope (Leica
Microsystems, ON). Five testes pairs per cross
were placed in a drop of TIB on a polylysine-
coated slide. A coverslip was placed over the
specimen. Slides were then placed on an inverted
phase contrast microscope where the coverslip
was squashed against the specimen and slide by
removing excess TIB from under the coverslip
using Kimwipes©
. Slides were then immersed into
liquid nitrogen. Once frozen, the coverslip was
removed and slides were placed into a coplin jar
containing anhydrous ethyl alcohol inside a dry
ice incubator. Testes were fixed using 4%
paraformaldehyde (1mL 16% paraformaldehyde
in 3mL phosphate buffered saline). Phosphate
buffered saline (PBS) was made from tablets
(Amresco, OH USA, 1 tablet per 100mL double-
distilled water). Spermatid cysts were
permeabalized using PBT-DOC (0.3% TritonX-
100, 0.3% sodium deoxycholate in 1L PBS).
Rinses were performed using PBT (PBS and 0.1%
TritonX-100). Rhodamine-phalloidin (Molecular
Probes, NY USA) was prepared using 0.5μL per
slide aliquoted into a 1.5mL Eppendorf tube and
dried at 65°C for 10 minutes. Once dried, 2μL of
anhydrous ethyl alcohol was added, followed by
100μL of PTB per slide, and 0.1μL per slide
DAPI (1:1000, Molecular Probes) was added.
Contents were mixed using a vortex for 30
seconds and stored at room temperature. The
Eppendorf tube was covered with aluminum foil
to prevent fluorescent dyes from exposure to
light. Once specimens were stained and washed,
they were mounted with a coverslip using 15μL
9:1 90% glycerol: p-Phenylenediamine (PPD).
Slides were stored at 4°C and analyzed within 2
days.
Immunostaining
Immunostaining was conducted to observe
relative levels of PIP2 in SigDLow
x mia-/+
testes
and associated controls (w1118
, mia-/+
, SigDLow
and
SigDHigh
) Staining procedures are the same as
above (Fluorescence Staining). Except,
specimens were blocked using PBT with bovine
serum albumin (BSA) to block non-specific
antibody-protein interactions (PBTB: 5% BSA in
PBT). Primary antibody was 1:100 mouse IgM
anti-PIP2 (Echelon Bioscience, UT USA)

incubated overnight at 4°C. Secondary antibody
was 1:1000 anti-mouse IgM conjugated to Alexa
488-Green fluorochromes and incubated for 1
hour at room temperature. In addition, specimens
were fluorescently counterstained with DAPI
(1:1000, Molecular Probes) to observe nuclei.
Fluorescence Microscopy
Flourescently labelled specimens were
analysed using an upright epiflourescence
microscope (Zeiss: Axioplan 2) equipped with an
AxioCam wide range camera. Axiovision Rel 4.8
software (Carl Zeiss) was employed to analyze
and configure micrographs. An X-cite 120 LED
wide-field fluorescence microscopic light source
was used for illumination. Micrographs were
processed using Microsoft Office Profesional
Photo Editor (Microsoft, CA USA).
DNArrow©
Version 2.1 (Formerly DNArrow©
2.0)
To analyze the results produced by the
genetic screen we employed DNArrow©
2.1
software (Papadopoli A. and Brill J. A.,
unpublished), which takes results generated from
the genetic screen and narrows down regions of
the genome which are of interest or regions that
have not been tested. Data from each cross is
input using webhosting site
https://www.x10hosting.com/ (web-login
credentials available in Appendix A) where data
is loaded and stored and will sync to DNArrow©
2.1 available at
http://dnarrow.x10.mx/dnarrow.html (web-login
credentials available in Appendix A). To operate
DNArrow©
2.1, we selected which chromosome
arm we wanted to analyze. Data loaded into the
active colum represents intervals that have been
tested and are true suppressors or true enhancers.
Data loaded into the inactive column are intervals
that have been tested and show no interaction.
The collapse column takes overlapping
interacting and non-interacting regions and
subtracts them, essentially narrowing in on
regions of the genome that show strong activity
while avoiding the non-active areas. Interval
output from the remaining section of the program
indicates regions of the genome that have not
been tested. Information from this program is
used to test new Bloomington Deficiencies which
span regions of the genome described at positive
hits or areas that have not been tested. Data entry
before a web-based hosting site (x10hosting), was
google.doc. Both x10hosting and google.doc were
updated to ensure accurate comparison and to
have a backup of the data. A complete guide on
data input and output regarding x10hosting and
DNArrow©
2.1 are available in Appendix C.
Figure 6. SigDLow
x Deficiency genetic cross. Virgin
females carrying the SigDLow
transgene on the X
chromosome are crossed with Bloomington deficiency
flies, which contained large autosomal deletion, which
are balanced. From the F1, male progeny carrying the
maternal SigDLow
transgene and paternal deficiency
are selected for dissection and florescence staining.
Results
A total of 113 Bloomington deficiencies
crosses were screened, from which 20 resulted in
very strong suppression and 8 resulted in an
enhancement of the SigDLow
phenotype. There
were varying degrees of interaction; some strong
suppressors or enhancers of one or multiple of the
following: nuclear shaping defects, spermatid cyst
bipolarity, F-actin investment cone
polymerization and axoneme elongation.
Df(3R)24971 strongly suppresses SigD associated
phenotypes

Figure 7. Example of a strong suppressor of F-actin cone polymerization, spermatid cyst bipolarity and axoneme
length. A. SigDLow
expressing virgin females were crossed to Df(3R)24971 males, progeny carrying the maternal
transgene and the paternal deficiency were selected for analysis. B. Phase micrograph showing length of
spermatid cysts, notice the length and size resemble wild type. C. DAPI fluorescence staining showing partial
rescue of bipolarity, nuclei are polarized to the leading edge of the cyst. D. Rhodamine-phalloidin fluorescence
staining showing completely formed F-actin investment cones. E. Merged micrograph. F. Flybase image
depicting the deleted region that the deficiency spans, the blue, red and white arrows represent genes,
highlighted in yellow is the deficiency tested.
An example of a strong suppressor is
Df(3R)24971, which consists of a deleted segment
from the right arm of the third chromosome
(deleted breakpoints, 3R:[83F1—84B2]). SigDLow
virgin females were crossed to Df(3R)24971
males, F1 progeny carrying the maternal SigDLow
transgene and paternal Df(3R)24971 were
selected for analysis (Figure 7A). DAPI
fluorescence staining marking nuclei showed a
partial rescue of bipolarity and nuclear shaping
defects (Figure 7C). A complete rescue of F-actin
cone polymerization was also observed (Figure
7D). Furthermore, the spermatid cysts displayed a
wild type characteristic length (Figure 7B/E).
Df(3R)24971 contained many deleted genes, in
order to precisely predict which gene or group of
genes is responsible for the suppression event we
have to narrow down this large interval by testing
smaller deficiencies that span the larger deletion
(Figure 7F).
Df(2R)24931 strongly enhances SigD associated
phenotypes
An example of an enhancer of nuclear
shaping and spreading was Df(2R)24931, a
deletion on the right arm of the second
chromosome (Deleted breakpoints, 2R: [52A10—
52D2]). Again, we crossed SigDLow
virgin
females

Figure 8. Example of an enhancer of nuclear shaping and spreading. A. SigDLow
expressing virgin females with
Bloomington deficiency stock Df(2R)24931 males, progeny expressing the maternal SigDLow
transgene and
paternal deficiency selected for analysis. B. Phase micrograph depicting irregular spermatid cyst formation
defective elongation. C. DAPI fluorescent staining showing nuclear spreading, and rounded shaped nuclei. D.
Rhodamine-phalloidin fluorescently stained micrograph depicting a lack of F-actin investment cones. E. Merged
image. F. Flybase image depicting genes deleted in deficiency, highlighted in yellow is the deficiency tested.
to Df(2R)24931 males and selected F1 progeny
that carried the maternal SigDLow
transgene and
the paternal Df(2R)24931 deletion (Figure 8A).
We observed a truncation of the spermatid cysts
(Figure 8B/E) and spreading of round nuclei
throughout the cysts (Figure 8C). There was no F-
actin investment cone polymerization (Figure
8D). This deficiency was a large deletion,
spanning several genes. Narrowing of the interval
by testing smaller spanning deficiencies is needed
to hypothesize which gene or genes is or are
responsible for the enhancement.
DNArrow©
2.1 and genetic interval narrowing
Once a deficiency has been crossed to
SigDLow
, dissected and analyzed and results
showed a suppression, enhancement or no
interaction, we employed an online genomic
database called Flybase GBrowse
(http://flybase.org/cgi-bin/gbrowse/dmelabs/),
which describes the details of the deletion, as well
as a complete description of the all the genes that
span these deleted segments. In order to ensure
accuracy, testing smaller spanning deficiencies
within the larger region allows us to narrow large
deficiencies in which many genes have been
deleted. DNArrow©
2.1 was employed to narrow
in on intervals considered to be regions of high
interaction. An example of a positive hit that

10
Figure 9. DNArrow©
2.1 narrowing of interval
Df(3R)BSC10257 reveals mia and pcmt as genes of
interest. By testing smaller deficiencies, which span
the larger deletion allows us to narrow in on genes on
interest. Df(3R)BSC10257 resulted in suppression,
followed by Df(3R)179, which also resulted in
suppression. Finally, Df(3R)LT was tested and showed
strong suppression of SigDLow
. Df(3R)LT spanned two
genes, mia and pcmt, mia-/+
flies were crossed with
SigDLow
. Results shown in Figure 10.
Figure 10. DNArrow©
2.1 reveals mia as gene of interest in strong suppression of SigDLow
. A. Df(3R)LT
deficiency males were crosses with SigDLow
resulting in a strong suppression of SigDLow
phenotypes. B. Mia -/+
heterozygous mutant were crossed against SigDLow
virgin females, F1 male progeny that carried the maternal
SigDLow
transgene and paternal mia-/+
knockout resulted in a strong suppression of SigDLow
. C. Phase contrast
micrograph depicting leading edge of spermatid cyst. D. DAPI fluorescence staining showing a rescue of nuclear
morphology and partial rescue of cyst polarity. E. Rhodamine-phalloidin fluorescence staining showing a rescue
of F-actin investment cones. F. Merged micrograph depicting F-actin investment cone co-localization with
nuclei at leading edge of spermatid cyst.

Figure 11. Immunostaining of SigDLow
x mia-/+
flies shows a partial upregulation of PIP2 at the membrane. A.
Wild type spermatid cysts counterstained with DAPI to mark nuclei at leading edge. A’. PIP2 immunostain
shows PIP2 localized at the leading edge of the spermatid cysts with nuclei. A’’ Merged image showing nuclei
and PIP2 co-localization. B. SigDLow
spermatid cysts counterstained with DAPI showing bipolarity as well as
nuclear spreading. B’. SigDLow
spermatid cysts immuno stained with anti-PIP2 antibody show no PIP2. B’’.
Merged image SigDLow
and PIP2 note the absence of PIP2. C. SigDLow
x mia-/+
spermatid cysts counterstained
with DAPI showing nuclei at one end of several cysts. C’. Anti-PIP2 antibody immunostaining shows a partial
upregulation of PIP2 associated with the nuclei. C’’. Merged image of spermatid cysts shows PIP2 localization at
the leading edge.
came from DNArrow©
2.1 analysis was meiosis I
arrest (mia). Df(3R)BSC10257 was tested and
resulted in a strong suppression. The genomic
deletion that deficiency spanned had many genes.
A smaller deficiency within the larger deletion
(Df(3R)BSC179) resulted in another suppression.
Once more a smaller deficiency within the larger
deficiency was tested (Df(3R)LT) and resulted in
a strong suppression of SigDLow
associated
phenotypes. Df(3R)LT deficiency covers only two
genes: mia and pcmt, we tested mia-/+
flies with
SigDLow
and this resulted in a strong suppression
in SigD phenotypes (Figure 9).
Mia-/+
Heterozygous Mutants Result in
Suppression of SigDLow
Associated Phenotypes
Using the data from the genetic screen and
investigating regions of strong activity, we were
able to narrow in on a single gene of interest.

Table 1. DNArrow©
2.1 collapsed interval output. Regions on chromosomes 2 (L/R) and 3 (L/R) that show
interaction, only suppressing intervals are depicted. These intervals consist of regions where multiple
deficiencies have been tested and showed interaction; non-interacting deficiencies are subtracted from
interacting regions effectively narrowing down the search for interacting genes.
Collapsed
Coordinate (Kbps)
Size
(Kbps)
Chromosome
Arm
Interaction Genes Description
1119134.. 1151484 32 350 2L SUP Pino
CG4552
Iris
CG4577
CR45717
MFS3
CG4749
Tfb4
Vps29
4031318.. 4197800 166 484 2L SUP Ed
CR44984
Sr-CIII
Sr-CI
CG2955
Or24a
CR44057
CG31961
Lectin-240b
CG3714
Lectin-240b
cell surface
protein,
involved in cell
to cell
communication
11006679.. 11067029 60 350 2L SUP Nup154
Art8
dUTPase
Samuel
CG14913
CG18666
11358603.. 11445762 87 159 2L SUP Salr
CR43681
Salm
Salr (spalt-
related) shows
high testes
expression
13669537.. 13680154 10 617 2L SUP CG18507
CG31814
15338532.. 15349955 11 423 2R SUP betaTub56D
CG7744
par-1
mei-W68
betaTub56D is
a structural
constituent of
the

cytoskeleton
par-1 is
involved in
cellular
component
organization
and biogenesis
18401650.. 18480501 78 851 2R SUP px
CG6018
CG11362
Dnr1
5601375.. 5684102 82 727 3L SUP Eaf6
Blimp-1
Lin-28
Sse
Sif
Sif is predicted
to have
RacGEF
activity and
also contains a
PH domain,
where PIPs
and Rac/Rho
family
GTPases
interact
271425.. 327733 56 308 3L SUP Miple2
CG32845
Ttm2
RhoGEF3
Fwd
CG32344
Atac3
RhoGEF3 and
Fwd involved
in the PIP
pathway
11997642.. 12074922 77 280 3L SUP CG6828
Rols
CR43994
CG43993
CG6793
CG6793
CG43638
10743982.. 10920164 176 182 3R SUP Sdr
CR45598
CR45599
CG14861
RpL10Aa

14
CR44944
Dpr9
CG6974
CG6966
Table 2. Results from heterozygous knockouts mutants tested with SigDLow
Meiosis I arrest, or mia, was observed as one of
two genes deleted in a deficiency (Df(3R)LT) that
was crossed with SigDLow
(Figure 10A/B). F1
testes once dissected and stained showed a strong
suppression of SigDLow
phenotypes (Figure 10C-
E). There was a partial rescue of bipolarity
(Figure 10D), nuclear shaping and F-actin cone
polymerization (Figure 10E). Mia encodes for a
transcription factor, which is involved in the
progression of the male cell cycle during
spermatogenesis21, 22
; it is also implicated in
cellular differentiation after meiosis23
. We go on
to further investigate the observed suppression of
SigDLow
, and to classify the mechanism of
interaction.
SigDLow
x mia-/+
flies show a partial up-regulation
of PIP2 at the membrane
Allele (-/+)
x SigDLow
Fluorescence Microscopic Analysis Results
nuf
TM3
(nuclear fallout)
-Partial rescue of bipolarity
-Partial rescue of F-actin investment
cones
-Cysts length resembles SigDLow
Weak
Suppressor
mia1 st e
TM6b
(meiosis I arrest)
-Rescue of bipolarity
-Rescue of nuclear morphology
-Rescue of F-actin investment cones
-Axoneme elongation resembles w1118
(wild type)
-Testis seem larger then wild type flies
Strong
Suppressor
(Results Figures
10/11)
aly5 red
TM6c
(always early)
-Some F-actin cones are seen through
the cysts
-Partial rescue of nuclear elongation
V. Weak
Suppressor
comr, cnbw
CyO
(cookie monster)
No Interaction No Interaction
sa’red
TM3
(spermatocyte arrest)
w; nht cnbw
SM6a
(no hitter)

SigDLow
x mia-/+
flies were dissected and
immunostained with primary mouse anti-PIP2
IgM antibody and then with a secondary anti-IgM
conjugated to Alexa 488-green fluorochrome, to
observe the relative amounts of PIP2 within the
cells. In wild type spermatid cysts, PIP2 is
concentrated to the leading edge of the cysts,
where all 64 nuclei polarize (Figure 11A and A’).
In SigDLow
there is a marked reduction in PIP2 at
the leading edge of the cysts. As bipolarity is
established there is very little to no PIP2 observed
(Figure 11B and B’). SigDLow
x mia-/+
flies show
partial rescue of bipolarity as well as rescue of
PIP2 to the leading edge of the spermatid cysts
(Figure 11C and C’). These results suggest that
mia-/+
is replenishing PIP2 at the membrane in the
presence of SigDLow
resulting in the rescue of
bipolarity defects. An explanation for this
observation is described in the discussion section.
DNArrow©
2.1 intervals of interest
We employed DNArrow©
2.1 to narrow
down regions of the genome that show strong
interaction or regions of the genome that we have
not tested. Table 1 describes several collapsed
intervals or regions of the genome that show
strong suppression. Several of these regions
contain genes of interest; some of these genes
have testes specific expression patterns or are
involved in intracellular signalling processes.
Many PIP pathway and SigD transgene regulators
appear, which support the effectiveness of the
screen and sensitivity to detect controls. For
example in suppressor region 2R:
15338532..15349955, betaTub56D appears as a
gene of interest, BetaTub56D is a structural
constituent of the cells cytoskeleton and a major
constituent of microtubules, which form the
spermatid tail. Another example is sif, from
suppressor interval 3L: 5601375..5684102, has
been predicted to have RacGEF activity. Sif also
contains a pleckstrin homology (PH) domain that
facilitates RacGEF binding to phosphoinositols. It
has also been implicated in regulating actin
filament based structures within columnar
epithelial cells26
. In suppressor interval 3L:
271425..327733 RhoGEF3 was also picked up,
Rho family GTPases bind to PIPs in the
membrane and facilitate downstream F-actin
polymerization through the N-WASP and ARP
complexes. Fwd was also revealed within the
same interval of chromosome 3L as RhoGEF3,
fwd encodes a PI 4-kinase, which performs the
first phosphorylation event in the
phosphatidylinositol phosphate signalling
pathway and regulates cytokinesis during male
meiosis10
. Appearing as a suppressor of SigD
phenotypes may suggest a regulatory role of fwd
in regulating PIP2.
Nuf-/+
and aly-/+
display weak suppression of
SigDLow
Individual alleles of heterozygous mutants
were crossed with SigDLow
. Nuf-/+
showed a weak
suppression of nuclear shaping and F-actin cone
polymerization (Table 2). Nuf has been shown to
be a downstream effector of Rab11, which is
implicated in endosomal trafficking and actin
cytoskeleton arrangements during cytokinesis28
.
Further investigation is required to associate nuf
involvement in the PIP pathway. Always early or
aly-/+
was also tested and showed a weak
suppression of F-actin cone polymerization
(Table 2). Aly is a transcription factor involved in
the regulation of the RNA polymerase II promoter
during spermatid development27
. Further research
is required to associate these genes with the PIP
pathway.
Discussion
Our research has shown that by using a
genetic screen we can successfully discover
dominant suppressors and enhancers of a
particular phenotype. By testing numerous
deficiencies we were able to narrow in on genes,
which have a large potential of regulating the
phosphatidylinositol phosphate signalling
pathway. To screen for novel interactions of the
PIP pathway, we used the phosphatase SigD, to

16
deplete membrane PIP2. By crossing flies, which
express SigDLow
with deficiency flies, it was
possible to detect which genes are vital in the
regulation of PIP2 at the membrane.
Further exploring Df(3R)24971 which
showed a strong suppression of SigD, we want to
know which gene or genes is or are resulting in
the suppression event. Most noticeable of the
suppression was the rescue of F-actin investment
cones, which were well shaped, as well as the
length of the cysts, which indicates complete
elongation of the axoneme. This perhaps indicates
that when these genes are present, they result in
down-regulation of PIP2 at the membrane, or an
alternate hypothesis is that they are altering the
function of our transgene.
When we analyze the results from
Df(2R)24931 which showed an enhancement of
the nuclear shaping defects, as the nuclei arrest in
a round shape as well as being scattered
throughout the cysts; and showed non-classical
cyst formation. This indicates to us that there is a
gene or group of genes that, under wild type
conditions are regulating PIP2 at the membrane.
Deletion of these genes is resulting in a worse
phenotype, which perhaps indicates that these
genes normally function to regulate PIP2 at the
membrane.
We observed a strong suppression of
SigDLow
when we crossed SigDLow
carrying virgin
female flies to heterozygous mutant knockouts of
mia. Results from SigDLow
x mia-/+
flies can act as
a control for the basis of the transgenic expression
of SigD. Both SigD and mia (and upstream
regulators boule and twine) are under the
regulation of the spermatocyte specific β2-tubulin
promoter29
, when the mia-/+
is knocked-out we
observed a strong suppression of SigDLow
phenotypes. This evidence confirms the
effectiveness of the SigD transgene in producing
the phenotypes previously described, as well as
the sensitivity of the screen, as a promoter control
was detected. As described in the results, there are
many other genes of great interest, which will be
tested in the future. We hope to find regulators of
the PIP pathway such as interactions that can
replenish membrane PIP2.
The results from the screen give us new
insight in genes involved in sperm development
in Drosophila. Due to the shared homology of
genes between Drosophila and humans, much of
the genetic and cellular process can be applied to
both models. The results from our screen present
new regulators, which affect the PIP pathway. It
is important to note that not only is the PIP
pathway involved in spermatogenesis in
Drosophila, rather the molecular properties of
cells, apical-basal polarity, actin dynamics and
changes in nuclear morphology are involved in
many cell based pathologies such as cancer. Many
variants of epithelial cancers are due to the loss of
apical-basal polarity, which is influenced by the
distribution of membrane bound
phosphatidylinositol 4, 5-bisphosphate (PIP2) and
phosphatidylinositol 3, 4, 5-trisphosphate (PIP3)
within the cell. Down-regulation of these vital
lipoproteins results in loss of cell-cell/cell-basal
lamina adhesion and activation of a
hypersensitive migratory behavior. Implications
from this screen may reveal novel players in
regulating cellular metastasis and migration of
cells.
Conclusion
Studying the phosphatidylinositol
phosphate signalling pathway in Drosophila
provides us with an excellent model of
understanding microtubule dynamics, F-actin
polymerization, cellular polarity and nuclear
morphology. Our results show the effectiveness
of performing a screen to detect novel interaction
of the phosphatidylinositol phosphate pathway.
The implications from this research will help us to
understand the vast network of intracellular
signalling transduction pathways that regulate
cellular homeostasis. We are better able to
understand the role of phosphatidylinositol
phosphates during spermatid development and
gain further knowledge of its interactions.

Acknowledgments
I would like to thank all members of the
Brill lab, firstly, Dr. Julie A. Brill for this
incredible opportunity, Dr. Lacramioara Fabian
for being wonderful mentor and role model
throughout this research project, Gordon
Polevoy, Lilit Antonyan, Dayag Sheikhkarimli,
Jonathan Ma for his biochemistry expertise in
helping me with my protein gel and Andrew
Papadopoli for his contributions in developing
DNArrow©
2.0 and 2.1 which allowed us narrow
down large genetic intervals and investigate
regions of interest. I would also like to thank Dr.
Helen White-Cooper for mia and aly flies used in
my experiments. I am grateful to those institutions
whose funding made my research possible. Words
cannot express what a career changing
experience this has been!
References
1. Fuller MT. (1993). Spermatogenesis. Cold
Spring Harbor, New York: Cold Spring
Harbor Press.
2. Fabian L and Brill JA. (2012). Drosophila
spermiogenesis: Big things come from
little packages. Spermatogenesis; 2:3,
p197-212.
3. Wei HC, Rollins J, Fabian L, Hayes M,
Polevoy G, Bazinet C. (2008). Depletion
of plasma membrane PtdIns(4,5)P2
reveals essential roles for
phosphoinositides in flagellar biogenesis.
J Cell Sci; 121:1076- 84.
4. Fabian L, Wei HC, Rollins J, Noguchi T,
Blankenship JT, Bellamkonda K. (2010).
Phosphatidylinositol 4,5-bisphosphate
directs spermatid cell polarity and exocyst
localization in Drosophila. Mol Biol Cell;
21:1546-55.
5. Tates AD. (1971). Cytodifferentiation
during spermatogenesis in Drosophila
melanogaster: An electron microscope
study. Leiden: Rijksuniversiteit.
6. Tokuyasu KT, Peacock WJ, Hardy RW.
(1972). Dynamics of spermiogenesis in
Drosophila melanogaster. I.
Individualization process. Z Zellforsch
Mikrosk Anat 124:479-506.
7. Yins HL and Janmey PA. (2003).
Phosphoinositide regulation of the actin
cytoskeleton. Annu. Rev. Physiol. 65, 761–
789.
8. Roth MG. (2004). Phosphoinositides in
constitutive membrane traffic. Physiol.
Rev. 84, 699-730.
9. Leevers SJ, Weinkove D, MacDougall
LK, Hafen E and Waterfield MD. (1996).
The Drosophila phosphoinositide 3-kinase
Dp110 promotes cell growth. EMBOJ. 15,
6584-6594.
10. Brill JA, Hime GR, Scharer-Schuksz M.
and Fuller MT. (2000). A phospholipid
kinase regulates actin organization and
intercellular bridge formation during
germline cytokinesis. Development 127,
3855-3864.
11. Bateman JM. and McNeill H. (2004).
Temporal control of differentiation by the
insulin receptor/tor pathway in
Drosophila. Cell 119, 87-96.
12. Pilot F, Philippe JM, Lemmers C and
Lecuit T. (2006). Spatial control of actin
organization at adherens junctions by a
synaptotagmin-like protein Btsz. Nature
442, 580-584.
13. Pinal N, Goberdhan DC, Collinson L,
Fujita Y, Cox IM, Wilson C. and Pichaud
F. (2006). Regulated and polarized
PtdIns(3,4,5)P3 accumulation is essential
for apical membrane morphogenesis in
photoreceptor epithelial cells. Curr. Biol.
16, 140-149.

18
14. Hapfelmeier S, Ehrbar K, Stecher B,
Barthel M, Kremer M, and Hardt WD.
(2004). Role of the Salmonella
Pathogenicity Island 1 Effector Proteins
SipA, SopB, SopE, and SopE2 in
Salmonella enterica Subspecies 1 Serovar
Typhimurium Colitis in Streptomycin-
Pretreated Mice. Infection and Immunity,
72:2 p. 795–809.
15. Norris FA, Wilson MP, Wallis TS, Galyov
EE, and Majerus PW. (1998). SopB, a
protein required for virulence of
Salmonella dublin, is an inositol
phosphate phosphatase. Proc. Natl. Acad.
Sci. USA 95, 14057-14059.
16. Marcus SL, Wenk MR, Steele-Mortimer
O. and Finlay BB. (2001). A synaptojanin-
homologous region of Salmonella
typhimurium SigD is essential for
inositolphosphatase activity and Akt
activation. FEBS Lett. 494, 201-207.
17. Terebiznik MR, Vieira OV, Marcus SL,
Slade A, Yip CM, Trimble WS, Meyer T,
Finlay BB and Grinstein S. (2002).
Elimination of host cell PtdIns (4,5) P2 by
bacterial SigD promotes membrane fission
during invasion by Salmonella. Nat. Cell
Biol. 4, 766-773.
18. Wong R, Hadjiyanni I, Wei HC, Polevoy
G, McBride R, Sem KP and Brill JA.
(2005). PIP2 hydrolysis and calcium
release are required for cytokinesis in
Drosophila spermatocytes. Curr. Biol. 15,
1401-1406.
19. Niggli V, Andreoli C, Roy C, and
Mangeat P. (1995). Identification of a
phosphatidylinositol-4, 5-bisphosphate-
binding domain in the N-terminal region
of ezrin. FEBS Lett. 376, 172–176.
20. Hirao M, Sato N, Kondo T, Yonemura S,
Monden M, Sasaki T, Takai Y and Tsukita
S. (1996). Regulation mechanism of ERM
(ezrin/radixin/ moesin) protein/plasma
membrane association: possible
involvement of phosphatidylinositol
turnover and Rho-dependent signaling
pathway. J. Cell Biol. 135, 37–51.
21. Fuller MT. (1998). Genetic control of cell
proliferation and differentiation in
Drosophila spermatogenesis. Semin. Cell
Dev. Biol. 9(4): 433--444.
22. Lin TY, Viswanathan S, Wood C, Wilson
PG, Wolf N, Fuller MT. (1996).
Coordinate developmental control of the
meiotic cell cycle and spermatid
differentiation in Drosophila
males. Development 122(4): 1331--1341.
23. White-Cooper H, Schafer MA, Alphey
LS, Fuller MT. (1998). Transcriptional
and post-transcriptional control
mechanisms coordinate the onset of
spermatid differentiation with meiosis I in
Drosophila. Development 125(1): 125—
134.
24. Simon MA, Bowtell DD, Dodson GS,
Laverty TR, Rubin GM. (1991). Ras1 and
a putative guanine nucleotide exchange
factor perform crucial steps in signaling
by the sevenless protein tyrosine kinase.
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25. Therrien M, Morrison DK, Wong AM,
Rubin GM. (2000). A genetic screen for
modifiers of a kinase suppressor of Ras-
dependent rough eye phenotype in
Drosophila. Genetics 156: 1231–1242.
26. Georgiou M, Baum B. (2010). Polarity
proteins and Rho GTPases cooperate to
spatially organize epithelial actin-based
protrusions. J. Cell Sci. 123(7): 1089--
1098.

27. Jiang J, White-Cooper H. (2003).
Transcriptional activation in Drosophila
spermatogenesis involves the mutually
dependent function of aly and a novel
meiotic arrest gene cookie monster.
Development 130(3): 563--573.
28. Riggs B, Fasulo B, Royou A, Mische S,
Cao J, Hays TS, Sullivan W. (2007). The
concentration of Nuf, a Rab11 effector, at
the microtubule-organizing center is cell
cycle regulated, dynein-dependent, and
coincides with furrow formation. Mol Biol
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29. Lin TY, Viswanathan S, Wood C, Wilson
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differentiation in Drosophila males.
Development 122.

Appendix.
Part A: Data Input: DNArrow© Version 2.1 (Papadopoli and Brill, unpublished)
Singh, Kevinder P. March 2014.
Provided below is a detailed, step-by-step guide to inputting data into DNArrow©
Version 2.1. For
information pertaining to the operations of program pleases contact: papadopoli.andrew@gmail.com;
singhkevin.singh@mail.utoronto.ca
*Disclaimer: The following screenshot images were taken March of 2014, updates to the web-hosting
site may change the format/organization of components on the site. Please input accordingly.
1. Visit site: https://www.x10hosting.com/

2. Add credentials: Username: dnarrowx Password: fwdgenebrill
3. Wait to be redirected to the homepage.

22
4. Click: Databases.
5. Click: PHPMyAdmin.

6. Click: _dmelres in the left side panel.
7. Click: dmel_data.

24
8. Click: Insert.

9. Data entry. In stk, add the Bloomington Stock Number of the deficiency. In sym, add the symbol of
the deficiency, arm for chromosome arm (ex. 3-Left), lft/rgt for the left and right breakpoints of the
deficiency, available through Flybase. Res_type for results: SUP for suppressor and ENH for enhancer,
true implies a strong interaction (strong suppressor or strong enhancer), false means no interaction or
weak interactions (Remember to focus on the strong interactions, but don’t dismiss weak interactions,
in the comments sections mention your observations). Add your initials in Ins and comments.

26
Part B: DNArrow©
2.1 Data loading and results output
1. Visit: http://dnarrow.x10.mx Add credentials, Username: jbrill Password: fwdgenebrill
2. Select chromosome arm (2L/R, 3L/R), then select enhancer (E) or suppressor (S) for
appropriate analysis.

3. Click the plus (+) symbol beside the E and S to load data, select current arm for data, which is
specific for a single chromosome arm.
4. Data load, if suppressor for 2L is selected data in the suppressors column will load (if enhancer
is selected, column will now load enhancers under same column, column title will then change
to enhancers). Non-interacting regions of the chromosome arm will also load under the inactive
column.

28
5. Click the play arrow at the bottom of the collapse column, this will output intervals where
inactive and active regions have combine, and in cancelled out areas where there is no
interaction and outputs intervals where a potentially interacting gene is located.

6. By clicking the play arrow under the remaining column, the outputs are intervals of the current
chromosome arm that have not been tested by any deficiency. These data indicate either no
deficiency available in the area, or areas that where there was no deficiency available.
7. Take the intervals generated by the collapse column and input them into the GBrowse section
of Flybase. This will allow you to see what genes and or deficiencies span this area.

30
8. Smaller interval. Intervals where there are less then 8-10 genes are good indicators for further
analysis, larger intervals with many genes need to be narrowed down further, all deficiencies
that span this area should be looked into.
9. To see what deficiencies cover this region, you can view the spanning aberrations by clicking
the spanning aberrations line.
10. Load all intervals into an excel file and list genes/deficiencies available, as well as comments
regarding genes (alleles available, type of allele – RNAi or transposable elements),
molecular/biological function, expression patters (testis specific expression patterns).
11. To order Bloomington Deficiency Flies please contact Dr. Lacramioara Fabian
(lala.fabian@utoronto.ca) or Gordon Polevoy (gordon.polevoy@sickkids.ca).

SINGH K.P. and BRILL J.A. 2014

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