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A Thesis
Entitled
A Visual Screen for Centrosome Mutants in Drosophila melanogaster
by
Sarah E. Hynek
Submitted to the Graduate Faculty as partial fulfillments of the
requirements for the
Master of Science Degree in Biology
Dr. Tomer Avidor-Reiss, Committee Chair
Dr. Deborah Chadee, Committee Member
Dr. Scott Leisner, Committee Member
Dr. John Plenefisch, Committee Member
Dr. Patricia R. Komuniecki, Dean
College of Graduate Studies
The University of Toledo
May 2015
Copyright 2015, Sarah Elizabeth Hynek
This document is copyrighted material. Under copyright law, no parts of this
document may be reproduced without the expressed permission of the author
An Abstract of
A Visual Screen for Centrosome Mutants in Drosophila melanogaster
by
Sarah E. Hynek
Submitted to the Graduate Faculty as partial fulfillment of the
requirements for a Master of Science Degree in Biology
University of Toledo
May 2015
Centrosomes are highly conserved organelles that are composed of two
microtubule-based centrioles surrounded by an amorphous protein cloud of
pericentriolar material (PCM), which is able to nucleate astral microtubules.
They serve as microtubule organizing centers during cell division and are
important for fertilization. During fertilization, upon fusion with the ovum,
the sperm contributes modified centrioles and a haploid set of genetic
material. These modified centrioles recruit maternal PCM proteins and then
form the microtubule sperm aster. These microtubules extend to find the
female pronucleus and facilitate its movement towards, and eventual fusion
with, the male pronucleus. This process creates a complete genome, and
allows the first zygotic cell division to take place.
During spermatogenesis, the centrioles undergo a variety of changes such as
elongation, duplication, and separation, all of which precede centrosome
reduction. This is the process which creates modified centrioles in the mature
sperm to be contributed to the oocyte.
iii
During this phenomenon, the
centrosome loses its astral microtubule nucleating function, PCM, and many
centriolar proteins. By using a forward genetic approach, we have created a
random mutagenesis screen for centriolar mutants in the testes, with the
overall goal of finding those with centrosome reduction defects. We have
chosen ethyl methylsulfonate (EMS) as our mutagen. Using Ana1-GFP or
Asl-GFP, which label the centrioles and PCM respectively, we dissect and
visualize Drosophila testes using fluorescence microscopy. In total we have
examined 1436 mutants, finding many defects including those in testes
morphology, centriole length, spermatid nucleus morphology, and one mutant
of particular interest with Asl-GFP labeling in the mature sperm, indicating
a defect in centrosome reduction.
iv
Acknowledgements
This thesis would not be possible without the unyielding support of my
family, friends, fiancé, advisor, and other dedicated faculty here at UT. No
amount of thank you-s could express how grateful I am for every positive
thought, motivational pep talk, and helpful discussion along the way.
v
Contents
Abstract
iii
Acknowledgements
v
Contents
vi
List of Tables
ix
List of Figures
x
List of Abbreviations
1
xii
Introduction
1
1.1
The Centrosome is Important for Fertility . . . . . . . . . . . . . . . . . 1
1.2
Centrosome Reduction Creates Modified Centrioles
in the Sperm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3
Male Infertility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.4
Spermatogenesis in Drosophila melanogaster . . . . . . . . . . . . . . 5
1.5
Drosophila melanogaster as a model organism . . . . . . . . . . . . . . 7
vi
2
3
Methods and Materials
10
2.1
Fly Stocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.2
EMS Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2.3
Virgin Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.4
Crossing Scheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2.5
Lethality Scoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.6
Heat Shock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.7
Dissections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.8
Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.9
Complementation Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.10
Motility Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.11
Photon Counting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.12
Penetrance Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
A Visual Screen for Centrosome Mutants
16
3.1
Screen Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
3.2
Testes Morphology Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
3.3
Nuclear Morphology Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . 19
vii
3.4
Developmental Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.5
Mutant UT737 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
3.6
Mutant UT1446 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.7
Zuker Collection Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.7.1 Mutant z2984 has Asl-GFP in the Spermatozoa . . . . . . 23
3.7.2 Mutant z2984 is not a Plk4 Mutant . . . . . . . . . . . . . . . . 24
3.7.3 Mutant z2984 is a Complex Genetic Interaction . . . . . . 25
4
Discussion and Future Directions
References
28
31
viii
List of Tables
Table 1
Numerical analysis of the screen . . . . . . . . . . . . . . . . . . . . . . . . 17
Table 2
Phenotypic groups of mutants identified . . . . . . . . . . . . . . . . . . 18
ix
List of Figures
Figure 1
The Centrosome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Figure 2
Sperm Aster Formation in the Zygote . . . . . . . . . . . . . . . . . . . . . 3
Figure 3
Model of Centrosome Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Figure 4
Spermatogenesis in Drosophila melanogaster . . . . . . . . . . . . . . 6
Figure 5
Model of Forward Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Figure 6
Crossing Scheme for the Screen . . . . . . . . . . . . . . . . . . . . . . . . . 12
Figure 7
Testes Morphology Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Figure 8
Sperm Nucleus Morphology Defects . . . . . . . . . . . . . . . . . . . . . 19
Figure 9
Testes Developmental Defects . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Figure 10
Mutant UT737 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Figure 11
Mutant UT1446 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Figure 12
Mutant z2984 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Figure 13
Mutant z2984 Penetrance Quantification . . . . . . . . . . . . . . . . . 24
x
Figure 14
Mutant z2984 Photon Counting Quantification . . . . . . . . . . . . 24
Figure 15
Schematic of a Complementation Test . . . . . . . . . . . . . . . . . . . 25
Figure 16
Genetic Interaction Quantification . . . . . . . . . . . . . . . . . . . . . . 27
xi
List of Abbreviations
Asl . . . . . . . . . . . . . . . . . . Asterless
CyO . . . . . . . . . . . . . . . . . Wings (Marker Mutation/Balancer)
Dr . . . . . . . . . . . . . . . . . . Drop (Eye Marker Mutation)
EMS . . . . . . . . . . . . . . . . . Ethyl Methanesulfonate
Hu . . . . . . . . . . . . . . . . . . Humeral (Bristle Marker Mutation)
MKRS . . . . . . . . . . . . . . . Balancer
PCL . . . . . . . . . . . . . . . . . Proximal Centriole-Like
PCM . . . . . . . . . . . . . . . . . Pericentriolar Material
Tb . . . . . . . . . . . . . . . . . . . Tubby (Pupa Marker Mutation)
TM6B . . . . . . . . . . . . . . . . Balancer
xii
Chapter One
Introduction
1.1 The Centrosome is Important for Fertility
The centrosome is a large organelle in the cell that is conserved throughout
studied animal species, with the known exception of the flatworms,
planarians (Azimzadeh et al., 2012). A pair of microtubule-based centrioles
form the foundation of a typical animal centrosome, however in higher
plants, these are absent (Brown and Lemmon, 2011). These centrioles recruit
and are surrounded by an amorphous matrix of proteins called the
pericentriolar material, or PCM. From the PCM, the centrosome can nucleate
astral microtubules (Azimzadeh and Bornens, 2007) (Figure 1). The
centrosome functions as the major microtubule organizing center of a cell
during cell division, nucleates cilia, and plays a critical role in successful
fertilization (Nigg and Raff, 2009).
The centrosome plays a key role in fertilization by contributing its centrioles
to the zygote (Sutovsky and Schatten, 2000). During oocyte formation in
animals (oogenesis), the centrioles are eliminated, and PCM proteins become
abundant in the cytoplasm (Schatten, 1994). Conversely, during sperm
1
formation in animals (spermatogenesis), the centrosomal components are
eliminated leaving only the centrioles (Manandhar et al., 2005). In all nonrodent animals, when a sperm fuses with an oocyte, its genetic material
(pronucleus) and centrioles, , are contributed to the zygote. The centrioles are
then able to recruit the PCM proteins present in the zygotic cytoplasm and
nucleate astral microtubules (Figure 2) (Callaini and Riparbelli, 1996),
referred to as the sperm aster (Sathananthan et al., 1997; Terada et al.,
2010). The astral microtubules extend throughout the zygote to locate the
female pronucleus, which is then able to move along the astral microtubules
towards the male pronucleus. After fusion of the pronuclei, each centriole
duplicates forming two centrosomes, which allows the first zygotic cell
division to take place (Blachon et al., 2014).
Figure 1 – The centrosome
consists of three components: two
centrioles (green), an amorphous
pericentriolar material cloud
(red), and astral microtubules
(red lines).
2
Figure 2 – The modified
centrioles (green) from the sperm
recruit PCM proteins (red) and
nucleate astral microtubules
(white) to locate the female
pronucleus (pink) and help it
move to fuse with the male
pronucleus (blue).
♀
♂
1.2 Centrosome Reduction Creates Modified Centrioles in the Sperm
During animal spermatogenesis, early spermatids have a typical centrosome
with a pair of centrioles surrounded by PCM that can nucleate astral
microtubules (Manandhar and Schatten, 2000). As spermatids mature
(spermiogenesis), the centrosome loses many of its characteristics via a
process called centrosome reduction. First, the centrosome loses its
microtubule nucleating function; second, the PCM no longer surrounds the
centrioles; and finally, the centrioles degenerate, however, this degeneration
varies between different species (Figure 3) (Manandhar et al., 2000;
Manandhar et al., 1998). For example, mice have completely degenerated
centrioles (Manandhar et al., 1998), monkeys and humans have one intact
and one degenerated centriole (Manandhar and Schatten, 2000), and
Drosophila have one partially degenerated centriole and a partially
degenerated secondary centriolar structure, the PCL (Blachon et al., 2009).
3
This knowledge comes from very few studies, and importantly, the
mechanism and function behind centrosome reduction remains unknown.
1
2
3
Figure 3 – Centrosome reduction takes place in three steps: 1. Loss of
astral microtubules 2. Loss of PCM 3. Loss of centriolar proteins.
1.3 Male Infertility
Approximately 10 – 15% of couples in the United States are infertile (Practice
Committee of American Society for Reproductive Medicine in collaboration
with Society for Reproductive and Infertility, 2008) and it is estimated that in
one-third of these cases, the male is the sole contributor to the problem.
Despite recent advances in the diagnosis of infertility factors, in the male
nearly 50% of all infertility cases have no known cause (Jose-Miller et al.,
2007). Assisted reproductive technology (ART), such as in vitro fertilization
and intracytoplasmic sperm injection (ICSI), is making it possible for those
with male factor infertility to conceive. However, defects in the events after
sperm entry into the oocyte cannot be overcome with this method (Terada,
2007). It is thought that in some of these cases, the centrosome may have
defects that are still causing the infertility (Kovacic and Vlaisavljevic, 2000;
4
Rawe et al., 2000). We hypothesize that one such defect may be in centrosome
reduction.
1.4 Spermatogenesis in Drosophila melanogaster
Spermatogenesis is a highly complex process that must be tightly controlled
to produce correct, functional male gametes. In Drosophila, spermatogenesis
begins at the most apical end of the testes, the tip. Here we find a group of
seven to nine stem cells referred to as the hub (de Cuevas and Matunis,
2011). Each stem cell asymmetrically divides to form one daughter cell, or
spermatagonium, and one stem cell, keeping the hub replenished at all times
(Kiger et al., 2001). The spermatagonium undergoes four mitotic divisions
with incomplete cytokinesis to give a syncytium of sixteen spermatocytes.
During this time, the cells increase to roughly thirty times their original size,
and each spermatocyte has two pairs of centrioles which have elongated
dramatically, and are perpendicular to one another (Gottardo et al., 2013).
These cells are surrounded on either side by two cyst cells, ensuring that each
mass of sixteen cells travel and develop together throughout spermatogenesis
(Figure 4).
After mitosis, the syncytium of spermatocytes, referred to as a bundle,
completes meiosis one and meiosis two. These two cell divisions create a total
of sixty-four haploid cells, each with only one centriole, called the giant
centriole (Fuller, 1993). This group of cells has a round nucleus, and the giant
5
centriole docks to the nucleus while forming the cilium, which serves as the
tail (flagellum) of the sperm cell (Figure 4). As the nucleus begins to change
shape from round to leaf (Fabian and Brill, 2012), the formation of a unique
atypical centriolar structure called the Proximal Centriole Like (PCL) occurs
(Blachon et al., 2009), which serves as the second centriolar structure in the
zygote (Blachon et al., 2014). The nucleus continues to change from leaf to
canoe and then to needle, and in parallel both the major and minor
mitochondria change from round to rod shaped (Fabian and Brill, 2012).
During these changes, centrosome reduction takes place resulting in modified
centrioles in the mature sperm (spermatozoa) which are finally stored in the
seminal vesicle (Figure 4).
Figure 4 – Spermatogenesis in the testes occurs in chronological
order, and begins with stem cells and spermatogonia at the tip (red)
and continues to spermatocytes (blue), then onto spermatids where
the nucleus and mitochondria change shape (orange), and finally to a
mature sperm stored in the seminal vesicle with reduced centriole and
PCL. (purple).
6
1.5 Drosophila melanogaster as a model organism The fruit fly,
Drosophila melanogaster, has been used in basic biological studies for over
100 years, serving as an excellent model for many cellular processes and
human diseases (Roote and Prokop, 2013). Drosophila has a short generation
time of ten days, and can be easily genetically manipulated with an array of
tools and techniques. Using Drosophila to understand an observable
phenotype is advantageous due to its ease of use in forward genetic screens.
The forward genetic approach utilizes the observance of a particular
phenotype followed by random mutagenesis until alteration or elimination of
the phenotype is identified via screening. Forward genetics has five steps: 1)
random mutagenesis, 2) identify a phenotype of interest, 3) identify the gene
responsible, 4) study the function of the protein, and 5) study the ortholog in
other species (Roote and Prokop, 2013) (Figure 5).
7
Figure 5 - We utilize the 5 step
forward genetic approach in order
to study centrosome reduction in
the
fruit
fly,
Drosophila
melanogaster.
Ethyl methanesulfonate (EMS), radiation, and transposons are common ways
to induce random mutations (St Johnston, 2002). For the purposes of this
screen, we have chosen to use EMS for three reasons: 1 – EMS generates
single point mutations, and rarely, small deletions (Sega, 1984), often
affecting protein domains, which will generate large numbers of alleles
unlike other techniques that affect the whole protein. 2 – Unlike radiation
and transposons, which have biases towards certain types of genes, EMS has
the potential to mutate all genes. 3 – Identifying the specific mutation is
simple with techniques such as whole genome sequencing.
The original goal of our EMS screen was to identify mutants that do not
undergo normal centrosome reduction.
8
To do this, we utilized a visual
approach, where we dissected the testes of flies whose centrioles were labeled
with Ana1-GFP, a centriolar protein. After dissection, the testes were stained
with hoescht dye to see the nucleus, and observed under a fluorescence
microscope. The phenotype we wanted to identify was flies containing
spermatozoa in the seminal vesicle that still retained Ana1-GFP labeling,
indicating a defect in centrosome reduction had taken place. Finding this
phenotype would represent the first mutant in centrosome reduction,
allowing for both mechanistic and functional insights to be gained.
9
Chapter Two
Methods and Materials
2.1 Fly Stocks
All flies were cultured on standard media at 25°C (Roote and Prokop, 2013).
The HS-Hid-Dr and FRT82 stocks were obtained from the Bloomington Stock
Center (7758 and 2035, respectively). The Asl reduction mutant was obtained
from Dr. Barbara Wakimoto (Wakimoto et al., 2004), who screened through
flies originally from Dr. Charles Zuker (Koundakjian et al., 2004). Transgenic
constructs of Asl-GFP and Ana1-GFP were made in the Avidor-Reiss lab and
sent to BestGene to create fly lines expressing the transgenic protein under
its endogenous promoter.
2.2 EMS treatment
Males 24 – 48 hours old were collected and starved, but not dehydrated, for
approximately 18 hours in a chamber with damp filter paper. After
approximately 18 hours, the males were moved to a chamber with filter paper
dampened with a 25 mM EMS solution for 8 – 10 hours. The EMS solution
was composed of 1% sucrose and green food coloring. Flies were then scored
for green abdomens indicating that the EMS solution was ingested. Flies
10
without green abdomens were disposed of and those with green abdomens
were moved to a fresh food vial overnight to recover before being crossed
(adapted from (Koundakjian et al., 2004).
2.3 Virgin Collection
Females were collected in a vial every 12 hours and considered virgin if no
larva were produced after 3 – 4 days. Those that had a meconium were
separated and labeled as “M+” and were able to be used right away.
2.4 Crossing Scheme
For the first of three crosses, EMS treated males with an isogenized third
chromosome that contained an FRT site (Bloomington stock #2035) were
crossed en masse to virgin females containing the transgene on the second
chromosome, Ana1-GFP, which labels the centrioles. These females also have
the apoptotic gene Hid coupled with a heat inducible Hid promoter on their
third chromosome; this is phenotypically marked with the dominant marker
mutation Drop (Dr).
For the second cross, single males were selected from the first cross’s progeny
that had Ana1-GFP over the balancer CyO, which has the dominant
phenotype of curly wings on the second chromosome. The males had a
mutated third chromosome over the balancer TM6B. Each individual male
was then crossed to 3-4 virgin females as stated above. Virgin collection was
avoided by using a heat shock. Male and female progeny of heat shocked vials
11
were scored for successful heat shock by the absence of flies with the eye
phenotype Dr and then moved to a new vial (cross #3) to mate. After 10 days,
vials were scored for lethality (Figure 6).
Figure 6 – Crossing
scheme used to generate
mutant lines that have
mutations on the third
chromosome.
2.5 Lethality Scoring
Flies in cross 3 contain the balancer TM6B on the third chromosome with the
dominant phenotypic markers Tubby (Tb) for pupae and Humeral (Hu) for
adults. Absence of these marker phenotypes (denoted by a +) indicates fly is
homozygous for any mutations on the third chromosome. The absence of Tb+
pupae in a vial indicated an embryo or larva lethal phenotype when the EMS
induced mutation was homozygous. Tb+ pupae that did not develop enough to
determine the sex were considered pupa lethal. Pupae that developed to
determine the sex but did not have flies exit out of their pupae were
considered pharate adult. Finally, Tb+ pupae with flies that exitd but did not
yield live Humeral plus (Hu+) flies were deemed adult lethal. Vials with Tb+
pupae and Hu+ flies were considered to have viable mutations.
12
2.6 Heat Shock
The heat shock was performed two times during cross two (refer to Figure 6).
Parent flies were moved to new vials 24 hours after the creation of cross two,
and the empty vials containing food and embryos were placed in a 37°C water
bath for one hour to activate the heat inducible Hid promoter to kill the
embryos. After another 24 hours, parents flies were disposed of, and the
empty vials containing food and embryos were again placed in a 37°C water
bath for one hour. This resulted in two copies of each cross two fly line. After
10 - 12 days, progeny flies were scored for Dr to confirm the success of the
heat shock. If Dr flies were found, the flies and their vial were discarded
because they were undesired progeny of cross two.
2.7 Dissections
All dissections were performed on very late pupae or adults 1 – 3 days old as
described in (Basiri et al., 2013) with the following changes: the testes were
cut into 2 pieces for higher quality staining, and incubated in the stain
(1:1000 µg DAPI or Hoescht) for 10 minutes.
2.8 Microscopy
Screening of the mutant testes was done with a Leica SP5 fluorescent
microscope using a 100x objective. Centriolar mutations were imaged with a
Leica SP8 confocal microscope using a 63x objective. Confocal images were
13
taken as Z-stacks and formatted with maximum projection for image
generation purposes.
2.9 Complementation Tests
Complementation tests were performed if a mutant phenocopied a known
mutation on the third chromosome. Heterozygote flies of the known gene and
the phenocopying gene were crossed, and the pupae with one copy of the
known mutation and one copy of the unknown mutation were dissected.
Testes that maintained the mutant phenotype were considered to not
complement, and those that exhibited a wild type phenotype were considered
to complement. Mutants that did not complement were suggested to be new
alleles of known genes. Mutants that complemented were suggested to
represent a new gene.
2.10 Motility Assays
Sperm motility was determined using males 24 – 48 hours old. Their testes
were dissected and placed in 3µL of 0.5% NaCl and crushed with a glass
coverslip. Slides were analyzed using a Leica SP5 microscope and phase
contrast light with a 100x objective for spermatozoa with motile tails.
2.11 Photon Counting
Testes were dissected as stated above in “Dissections,” with the following
changes: the seminal vesicle was punctured before staining to release the
14
spermatozoa, and slides were not fixed. We used three different stages of
sperm development to determine photon levels of Asl-GFP in the centrosome:
round spermatid (onion stage), late spermatid, and spermatozoan. Five sperm
at each stage from five different testes (25 measurements total) were
collected using the Leica SP8 Confocal Microscope. The Asl-GFP images were
taken as Z-stacks using the counting mode and then analyzed for number of
photons at maximum projection. Statistics were generated using a two-tailed
Student’s T test.
2.12 Asl-GFP in the Spermatozoa Quantification
Testes were dissected as stated above in “Dissections,” with the following
change: the seminal vesicle was punctured before fixing and staining. Testes
from 5 different flies were analyzed, and 20 sperm from each were counted as
either having or not having Asl-GFP in their spermatozoa. Statistics were
generated using a two-tailed Student’s T test.
15
Chapter Three
A Visual Screen for Centrosome Mutants
3.1 Screen Statistics
In total, 7466 mutant lines have been attempted, and of those 2361 survived.
The surviving lines were scored for lethality, and 887 lines were embryo or
larva lethal, 38 were pupa lethal, and 199 were pharate adult/adult lethal.
Mutations that were embryo, larva, or pupa lethal were discarded, and those
that were pharate adult/adult lethal were retained because they still
produced pupae that were screened. Pharate adults develop fully but do not
exit from the pupa. In total, 1124 lines out of the established 2361 lines were
lethal, a 48% lethality rate. Therefore, a total of 1436 lines were screened
(Table 1).
We targeted the third chromosome in this screen due to the availability of
genetic tools in our lab. The third chromosome contains around 6000 genes
with 4300 of them representing genes estimated to be essential (Koundakjian
et al., 2004). By assuming a Poisson distribution (Pollock and Larkin, 2004),
we calculated that we have induced 2.86 mutations per chromosome,
resulting in approximately 4107 mutations visually screened for centrosome
16
and testes defects. Poisson distribution takes advantage of knowing the
number of total genes on the targeted chromosome, and of those, how many
are essential. By comparing the lethality rate of the EMS-induced mutations
with the number of predicted essential genes, we are able to estimate the
number of mutations per chromosome the EMS has caused.
Lines Attempted
7466
Total Surviving Lines
2361
Early Lethal (No Tb+ pupa)
887
Pupa Lethal (unable to tell male/female)
38
Pharate Adult/Adult Lethal (No Hu+ flies)
199
Total Lethal
1124
Viable
1237
Lines Able to be Screened (Viable + Pharate
Adult)
1436
Screened
1436
Mutations per chromosome
2.86 (48% lethality)
Total mutation studied in the screen
2.86 x 1436 = 4107
Total Centriole Mutants
34 (2%)
Table 1 – Numerical analysis of the screen to date, including lines
created, lethal phenotypes scored, induced mutations based on Poisson
Distribution, and total centriolar mutants found.
17
Phenotype
Number of Mutants
No spermatozoa in seminal vesicle
358
Testes Deformity
54
Developmental Defects
21
Nuclei Defects
16
Long Giant Centriole
29
No PCL
1
Short Giant Centriole
3
Centrosome Reduction
0
3.2 Testes Morphology Mutants
We have identified 54 mutants with testes deformities (Table 2). These
deformities represent a range of phenotypes. Many of the mutants have a
large bulge in the tip of the testes, leaving the base very small and skinny
(Figure 7). Other variations of a testes deformities include small testes and
round, ball-shaped testes (Not shown).
18
Figure 7 – Mutant 20 has a
massive bulb testes tip and
mutant 5402 has a less severe
defect with a large tip. Testes
tips denoted with an arrow.
3.3 Nuclear Morphology Mutants
Mutants we identified that have defects in their nuclear morphology exhibit a
large range of phenotypes. In the screen, we identified 16 mutants with some
sort of nuclear defect. Of these, 8 had curved nuclei, and 10 showed a
developmental arrest (Table 2). The most common is an arrest between leaf
and canoe stage which gives an amorphous shape that never progresses to
the needle like shape of a normal mature sperm. In these mutants, the
nucleus often elongates but is unable to morphologically change shape. We
have also found mutants that showed a curved (Figure 8) or abnormal
nucleus shape as a spermatozoa, and mutants arrest at the round spermatid
stage, referred to in the mouse and human system as globozoospermia.
Figure 8 – Mutant with
spermatozoa nuclei (blue) that
are curved (right) instead of
needle shaped (left).
19
3.4 Developmental Mutants
We have found a subset of mutations that affect the location of various stages
in spermatogenesis, 21 in total exhibit this phenotype (Table 2). Normally in
the testes, certain developmental stages are confined to specific areas of the
testes, for example, stem cells and spermatocytes are always found at the tip
of the testes. Along the outer edge of the testes before spiraling begins is
where meiotic cells can be found (Figure 9). In mutants with developmental
defects, we see spermatocytes and meiotic cells present very late, where we
normally find bundles of maturing sperm that are canoe to needle shaped.
We have also identified a mutant that appears to have too many stem cells in
its hub (not shown).
Figure 9 – Control testes (left)
with spermatocytes in their proper
position. Defective testes (mutant
850; right) with spermatocytes
much later towards the base of the
testes where typically more
mature sperm are located.
20
3.5 Mutant UT737
Mutant UT737 exhibits a short giant centriole (Figure 10), and this
phenocopies a known mutation in Bld10, a centriolar protein. We performed a
complementation test between mutant UT737 and a Bld10 mutant, and
found that the phenotype failed to complement, suggesting that this mutation
is an allele of Bld10. Interestingly, we find that this mutant has motile
sperm, but the known Bld10 mutant does not have motile sperm. This
suggests that it may be a mutation in a different protein domain or a more
mild mutation than the known allele. Flies with one copy of UT737 and one
copy of the known Bld10 allele do not have motile sperm, but do exhibit a
short giant centriole.
Figure 10 – Mutant UT737
(right) has a short giant
centriole compared to control
(left). Centriole and PCL
labeled by Ana1-GFP, nucleus
stained with DAPI (blue).
3.6 Mutant UT1446
Ana1-GFP labels both the giant centriole and the PCL in the spermatids
(Blachon et al., 2009). Mutant UT1446 is devoid of Ana1-GFP labeling of the
21
PCL in the spermatids (Figure 11). This mutant phenocopies a mutation in
the know centriolar protein Poc1. A complementation test between the Poc1
allele k245 and mutant UT1446 failed to complement, suggesting that this
mutant is an allele of Poc1. Flies with one copy of the k245 allele and one
copy of the UT1446 allele show a normal giant centriole length, yet still does
not have Ana1-GFP labeling of the PCL. Sequencing of the mutant confirmed
that indeed, it is a mutation in Poc1. There are 3 other known alleles of Poc1,
each exhibiting a PCL without Ana1 labeling and having a short giant
centriole. Mutant UT1446 has the PCL phenotype, but has a normal length
giant centriole, suggesting that its mutation is PCL specific.
Figure 11 – Ana1-GFP labels the
giant centriole and PCL in wild
type spermatids (left), however in
the UT1446 mutant (Poc1, right)
there is no Ana1-GFP labeling of
the PCL.
3.7 Zuker Collection Mutants
A small collection of male sterile mutants originally from the Zuker
Collection (Koundakjian et al., 2004) is available in our lab. Mutations in
these flies were induced by EMS treatment. The flies have Asl-GFP, a PCM
protein, instead of Ana1-GFP. In parallel to the Ana1-GFP screen, we also
screened these Asl-GFP mutants. Surprisingly, we found that mutant z2984
22
as a heterozygote has Asl-GFP labeling in the spermatozoa (Figure 12),
indicating a dominant mutation in which centrosome reduction did not go to
completion.
Figure 12 – Spermatozoa from control
(left) and mutant z2884 (right) flies.
Mutant z2984 retains Asl-GFP labeling
of the centrosome (arrow), while the
control does not, indicating a defect in
centrosome reduction.
3.7.1 Mutant z2984 has Asl-GFP in the Spermatozoa
In heterozygotes with the z2984 mutation, 89%(±8) of their mature
spermatozoa have Asl-GFP labeling compared to 0% (±0) in control flies (n=5
testes, p<0.0001) (Figure 13). Photon counting quantification of this
phenotype reveals that the mutant has a statistically significant increase in
the total amount of Asl present in the spermatozoa compared to the control
(Figure 14), confirming that this mutant does indeed have a defect in
centrosome reduction of Asl.
23
Figure 13 – Analysis of
spermatozoa in both wildtype
and
z2984
for
Asl-GFP
labeling. The z2984 mutant has
a significant increase in the
amount of spermatozoa with
Asl-GFP labeling, n = 5,
p<0.001
Percent Spermatozoa with
Asl-GFP labeling
***
100
80
60
% Szoa with
Asl-GFP
40
20
0
Wild type
z2984
Photon Counting Asl-GFP
100,000
10,000
1,000
*
100
z2984
10
control
1
Figure 14 – Photon counting
at round, intermediate, and
mature stage sperm shows a
significant increase in the
amount of Asl-GFP in the
mature sperm, n = 5, p <
0.001. Graph shown in a
logarithmic scale.
3.7.2 Mutant z2984 is not a Plk4 Mutant
Mutant z2984 phenocopies a known, dominant mutation in Plk4, which does
not undergo complete reduction of Asl in the spermatozoa. Plk4 is on the
third chromosome, and mutant z2984 came from an EMS screen for the third
chromosome. A complementation test was performed with Plk4 and z2984
heterozygotes, with both mutations over the balancer, TM6B (Figure 15).
This balancer has two dominant markers, Tb for pupae and Hu for flies,
24
allowing for those with plk4 and z2984 to be identified as Tb+ pupae or Hu+
adults. Because both mutations are dominant, the presence of Asl-GFP in the
mature spermatozoa could not be used as a criteria for complementation,
therefore the plk4 homozygote phenotype of uncoordination and meiotic
defects was used. Upon analysis of flies containing plk4 and z2984, we found
that they were coordinated, and had no meiotic defects in the testes,
suggesting that mutation z2984 was not a Plk4 mutation.
Figure 15 – Schematic representation of a complementation test between
plk4 (blue) and z2984 (green).
3.8.3 Mutant z2984 is a Complex Genetic Interaction
Because the z2984 mutation is a dominant mutation, it is not guaranteed
that it resides on the third chromosome. We next needed to map the mutation
to a chromosome using genetic crosses. Using a z2984 mutant male crossed to
a wild type female, we negatively controlled for the X chromosome, and
25
positively controlled for the 2nd and 3rd chromosome as fathers do not pass
their X chromosome onto their sons. Our results showed that males with a
wild type X chromosome and wild type 2nd and 3rd chromosomes over the
balancer CyO (2nd) or TM6B (3rd) had Asl-GFP in 0% of their spermatozoa (n
= 5 testes). Males with a wild type X chromosome and the z2984 mutation on
the 3rd chromosome over the balancer TM6B had Asl-GFP labeling in about
2% of their mature sperm (n = 5 testes), significantly less than the original
phenotype of 89%. Lastly, we found that males with a wild type X
chromosome and z2984 mutation on the 3rd chromosome over the balancer
MKRS had Asl-GFP labeling in 0% of their spermatozoa (n = 5 testes).
Statistical analysis of the percent of spermatozoa with Asl-GFP labeling
shows no significant difference between the three groups. Together these
data suggest that the mutation is located on the X chromosome.
Using z2984 mutant females, we positively controlled for the X chromosome.
Our results show unexpectedly that progeny males with a predicted mutated
X chromosome and a wild type 3rd chromosome over the balancer TM6B have
Asl-GFP labeling in only 1% of their mature sperm on average (n = 5 testes).
Male progeny that had a predicted mutated X chromosome and the z2984
mutation on the 3rd chromosome over the balancer TM6B showed Asl-GFP in
the spermatozoa in 80% of their mature sperm (n = 5 testes). Even more
surprising was that when males had a predicted mutated X and the z2984
mutation over the balancer MKRS we saw Asl-GFP labeling in 0% of the
26
spermatozoa (n = 5 testes) (Figure 16). Statistical analysis of these data
show a significant difference between the percent spermatozoa with Asl-GFP
in mutant x, z2984 on the third, and the balancer TM6B compared to all
other male progeny. These data suggest a complex genetic interaction
between a mutation on the X chromosome, the z2984 mutation, and the
balancer TM6B. Due to the complex nature of this interaction, we have
chosen not to pursue further studies on it. However, determining the genes
responsible would be possible with whole genome sequencing. In order to do
this, we would need to obtain the original fly line that the mutant was
created from and compare its sequence to mutant z2984.
z2984 Males x Wild type Females
Wild type Males x z2984 Females
***
Figure 16 – Percent of spermatozoa with Asl-GFP labeling in z2984 males
crossed with wild type females (left) as positive control for the 2 nd and 3rd
chromosomes and negative control for the X chromosome. Percent of
spermatozoa with Asl-GFP labeling in z2984 females crossed with wild type
males (right) as positive control for the X chromosome and negative control
for the 2nd and 3rd chromosomes. N = 5 testes, p<0.0001 for x*; 3*/TM6B
compared to all other progeny scored.
27
Chapter Four
Discussion and Future Directions
Even though we did not succeed in our original goal to find a centrosome
reduction mutant in the centriolar protein Ana1, the screen was still
successful in many ways. Here we have shown that the visual approach to
screening in the Drosophila testes is an effective way to find mutations in
both the centrosome and in the many areas of spermatogenesis. Previously,
centrosome mutations were found using a behavioral screen that looked for
uncoordinated flies. Overall in this screen, we were able to identify 34
centrosome mutations, about 2% of the total mutants found. This represents
one of the largest collections of centrosome mutations, which can be further
investigated by our lab and by other labs in the field.
We are currently following up with 2 mutants from this screen, the Bld10
(UT737) mutant and the Poc1 (UT1446) mutation. UT737 is being sequenced
to find its specific mutation. We are interested in this mutant because it has
motile sperm unlike any other Bld10 mutants. The UT1446 mutant is
currently being examined by multiple members of the lab, both biochemically
28
and genetically. A paper is in progress regarding this mutant and its impact
on male fertility.
The third chromosome of Drosophila contains roughly 6000 genes
(Koundakjian et al., 2004). In our EMS screen, we generated an estimated
4107 mutations, potentially covering 68% of the genes on the third
chromosome. This leaves nearly 2000 genes that were potentially not hit
based on our estimations. We also did not find more than one allele for any
known gene that we could score. This screen was done over 2 years, so in
order to generate enough mutations to potentially mutate every gene on the
third chromosome once, the screen would need to continue for at least 1 more
year.
Instead of extending the EMS screen, we have chosen to pursue an RNAi
screen in the lab. The RNAi screen will be targeting all known kinases and
phosphatases in the Drosophila genome due to our lab’s recent finding that
the kinase, Plk4, regulates the centrosome reduction of the PCM protein Asl.
RNAi lines for all of the 251 kinases and 86 phosphatases are available in the
Vienna Drosophila RNAi Center and the Transgenic RNAi Center (Dietzl et
al., 2007). The advantage of doing an RNAi-based screen is that the gene
causing the phenotype is already known, and that the RNAi can be
specifically expressed in the germline cells, minimizing its impact on fly
viability.
29
Our hypothesis regarding centrosome reduction was that it is essential for
male fertility, and recent work in the lab supports this. The plk4 mutant in
our lab has a defect in the centrosome reduction of the PCM protein Asl.
When Asl is labeled with GFP in this mutant, fluorescence is still observed in
the mature sperm, just like the z2984 mutant (Figure 12) Embryos that are
fathered by sperm with this defect have an arrest or a delay in development,
resulting in a decrease in fertility by about 20%. We would anticipate that
having multiple mutations that affect the reduction of various centrosomal
proteins would lead to a greater reduction in the fertility of these sperm.
One important future goal of the screen is to create a resource of these
mutations for other labs that have an interest in any of the phenotypes we
identified. For example, a group in Pennsylvania we met at the recent
Drosophila conference was very interested in our testes deformity mutants
that have large bulb tips. The Drosophila community greatly benefits from
mutant collections of EMS screens that are made available to the public. We
anticipate that further screening and mutant line creation will lead to a large
library of testes specific mutants beneficial to many labs.
30
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34