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Characterization of Drosophila longevity and fecundity regulating genes

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Characterization of Drosophila longevity and fecundity regulating genes

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CHARACTERIZATION OF DROSOPHILA LONGEVITY AND FECUNDITY
REGULATING GENES

by

Yishi Li

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR BIOLOGY)

December 2008

Copyright 2008 Yishi Li

ii

Epigraph

Journeying is hard,
Journeying is hard.
There are many turnings,
Which am I to follow?
I will mount a long wind some day and break the heavy waves.
And set my cloudy sail straight and bridge the deep, deep sea.

-Li Bai

iii
Dedication

To
Sanju Huang and Jianguo Li

iv
Acknowledgements

Foremost, I would like to express my gratitude to all the people whose support made this
dissertation possible. I would like to thank my academic advisor, Dr. John G. Tower. I
would like to thank Dr. Tower for giving me the opportunity to perform this research in
his laboratory. Without his critical suggestions and encouragement, I would not have
been able to achieve any of my goals during my Ph.D. career. I would also like to thank
the other faculty members in my graduate committee, Dr. Steven E. Finkel, Dr. Oscar
M. Aparicio, Dr. Valter D. Longo, and Dr. Lucio Comai. They provided invaluable
suggestions and other support towards my research at all stages. I would like to thank
my former and current my graduate school colleagues, Dr. Morris Waskar, Ji-ping Yuan,
Hongjun Zhang, Junsheng Yang, as each has provided critical support during my career
at USC. I would like to convey my appreciation to my mother Sanju Huang, my father
Jianguo Li, and the rest of my family and friends for their incredible support over these
years. Finally, my special thanks go to the rest of the Molecular and Computational
Biology Program of USC.

v

Table of Contents

Epigraph ii
Dedication iii
Acknowledgements iv
List of Tables vii
List of Figures viii
Abstract x
Chapter 1: Introduction 1
Chapter 2: Identification and characterization of magu and hebe genes 16
in Drosophila Melanogaster
Abstract 16
Introduction 17
Materials and Methods 23
Results 29
Discussion 63
Conclusion 69
Chapter 3: Anti-apoptotic gene dIAP2 lifespan assays 71
Abstract 71
Introduction 71
Materials and Methods 73
Results 73
Discussion 83
Conclusion 84
Chapter 4: Characterization of stem cell driver in Drosophila gut 86
Abstract 86
Introduction 86
Materials and Methods 88
Results 89
Discussion 101

vi
Conclusion 103
Chapter 5: Intra-abdominal injection of Drosophila and RNAi effect 105
Abstract 105
Introduction 105
Materials and Methods 108
Results 111
Discussion 114
Conclusion 115
Chapter 6: Conclusions and future directions 117
Bibliography 125

vii
List of Tables

Table 1: PdL mutant strains and magu/hebe transgenic strains list 41
Table 2: Lifespan assay of mutants and transgenic strains round 1 50
Table 3: Lifespan assay of transgenic strains round 2 52
Table 4: Lifespan assay of D42 driver cross round 1 53
Table 5: Lifespan assay of D42 driver cross round 2 54
Table 6: Lifespan assay of C204 driver cross round 1 55
Table 7: Female fecundity parallel assay of Bam PdL mutant 61
Table 8: Lifespan assay of Bam PdL mutant round 1 61
Table 9: Lifespan assay of Bam PdL mutant round 2 62
Table 10: Lifespan assay of Bam PdL mutant round 3 retest 62
Table 11: Old and new food ingredient comparison 63

viii
List of Figures

Figure 1 Sequence structure of PdL insertion 19
Figure 2 Fecundity of Or-R wild type female flies during aging 21
Figure 3 Cross design for generation of PdL mutants 26
Figure 4 First fecundity assay for homozygous females 30
Figure 5 Magu and hebe PdL insertion maps 38
Figure 6 Structure of USC1.0 with cDNA insertion 40
Figure 7 Northern blotting on mutants and transgenic strains 42
Figure 8 Northern blotting on Or-R wild type strain 45
Figure 9 Lifespan assay of transgenic strains 46
Figure 10 Fecundity test with DOX titration 56
Figure 11 Ban PdL mutant insertion map 59
Figure 12 Parallel fecundity and lifespan assay of ban mutant 59
Figure 13 Lifespan assay of ban mutant on two recipes 60
Figure 14 Ban mutant lifespan assay repeat 60
Figure 15 Original dIAP2 PdL mutant lifespan assay 73
Figure 16 DIAP2 lifespan assay, round 1 76
Figure 17 DIAP2 lifespan assay, round 2 78
Figure 18 DIAP2 lifespan assay, round 3 81

ix
Figure 19 Drosophila gut gene expression by GFP reporter 90
Figure 20 Abdominal RNAi injection station setup 109
Figure 21 Pilot RNAi injection 111
Figure 22 Fecundity test before and after RNAi injection 113

x
Abstract

The regulation of Drosophila melanogaster longevity and fecundity involves many
factors. Longevity is governed by oxidative stress, stem cell loss, dietary restriction, the
insulin/IGF-1 pathway, and other factors. Fecundity is also regulated by multiple tissues
and factors, including the germline stem cells and stem cell niche, the fat body, yolk
proteins, and sex peptides. The fecundity of wild type female Drosophila gradually
declines during aging, suggesting a common pathway regulating longevity and fecundity
machinery. Since both mechanisms involve multiple factors, sorting through the
Gordian’s knot is a formidable task. Using a PdL mutagenesis approach, I screened for a
specific phenotype in thousands of independent mutant strains to examine both
regulatory networks simultaneously. Two novel genes, magu and hebe, were identified
and characterized to regulate longevity and fecundity. While Drosophila lifespan was
extended upon the induction of these genes, fecundity increase requires that the gene
induction be in an ideal range to show the expected phenotypic change. I also performed
several other projects, including studying the lifespan extension effect of dIAP2,
characterization of a Drosophila gut driver strain, and intra-abdominal RNAi injection in
adult Drosophila. These projects provided us insight on longevity, fecundity, anti-
apoptosis, stem cell biology, RNAi and other aspects of Drosophila research. In sum,
Drosophila melanogaster, as a model organism for molecular biology and genetics
study, will continue to contribute to the scientific community.

1
Chapter 1 Introduction

Drosophila aging mechanism
An exhaustive review of Drosophila aging research, including its history, techniques,
and discoveries, would require several volumes to adequately explore the work in this
field. Therefore, for the purpose of the introduction, this sub-section and all the
following sub-sections will only focus on the topics that are of the greatest concern to
my research projects in Dr. Tower’s lab.

Drosophila melanogaster was named after the Greek words “black-bellied dew lover”. It
is commonly known as the fruit fly. Drosophila melanogaster is among the most studied
model organisms in genetics research for a variety of reasons. First, it is very small and
easy to culture to obtain massive numbers of individuals in a short amount of time in the
laboratory. Furthermore, the wild-type fly has relatively short lifespan, which is crucial
for aging research. It has only 4 pairs of chromosomes including one sex chromosome.
The small number of chromosome pairs makes genetic crosses easy to design and
perform. The sex determination by the X and Y chromosomes works by a similar
mechanism to the human sex chromosomes. Interestingly, about 77% of known human
disease genes can be matched to unique Drosophila sequences (Reiter, Potocki et al.
2001). These conserved genes and similarities between the two genomes make
Drosophila a uniquely important tool for molecular biology, genetics and
pharmaceutical research.

2

The Drosophila aging process is affected by an array of factors including, but not
limited to, oxidative and heat-shock stress responses, insulin/IGF-1 signaling pathway,
dietary restriction (DR), and stem cell maintenance.

Oxidative stress response has long been known to be involved in the aging process. As
respiration occurs, oxygen is metabolized, and reactive oxygen species (ROS) are
inevitably released by mitochondria as a by-product. ROS have deleterious effects in
most tissues and are generally suppressed by various endogenous enzymes, including
superoxide dismutase (SOD) and catalase. Over-expression of these enzymes is
sometimes effective to extend Drosophila lifespan (Sun and Tower 1999; Sun, Folk et
al. 2002; Sun, Molitor et al. 2004). Heat-shock proteins have also been shown to be
beneficial for lifespan extension via increased stress resistance (Morrow, Samson et al.
2004; Wang, Benzer et al. 2004). Among the most investigated heat-shock proteins are
Hsp22 and Hsp23, where expression level correlates with life span (Kurapati, Passananti
et al. 2000).

The Insulin/IGF-1 pathway is conserved among various organisms including C.elegans
and Drosophila (Giannakou and Partridge 2007). Functionally conserved components in
this pathway include the insulin receptor (InR), phosphoinositide 3-kinase (PI3K) and
the forkhead transcription factor dFOXO. When certain critical components in the
insulin/IGF-1 pathway are knocked down, such as IGF receptor or the receptor substrate

3
protein, CHICO, the inhibition of dFOXO is lifted, resulting in increased stress
resistance and extended lifespan in both C.elegans (Gems, Sutton et al. 1998) and
Drosophila (Bohni, Riesgo-Escovar et al. 1999; Tatar, Kopelman et al. 2001).

Another conserved factor regulating longevity is DR. It is the only known way to
reliably extend lifespan in the vast majority of organisms without genotypic change.
Some research suggests that manipulating the nutritional intake moderately (for example
by changing the food composition) (Ye, Cui et al. 2007) affects the mortality rate in
Drosophila. This indicates that DR increases Drosophila lifespan through a reduction of
the current risk of death, rather than a slowing down of aging-related damage (Pletcher,
Libert et al. 2005; Giannakou, Partridge et al. 2007). In fact, the stress responses are
modulated by the insulin/IGF-1 pathway and dFOXO. One of the important functions of
dFOXO is to increase the transcription of cell defense genes such as SOD2 and Hsp70
(Lee, Kennedy et al. 2003; Murphy, McCarroll et al. 2003). Over-expression of dFOXO
in the Drosophila fat body correspondingly extends lifespan (Giannakou, Goss et al.
2004; Hwangbo, Gershman et al. 2004). It is also proposed that DR induces a defense
program that provides resistance to an array of stresses (Anderson, Bitterman et al.
2003). DR and IIS appear to crosstalk, but the specific relationship is no yet clear
(Giannakou, Partridge et al. 2008; Min, Tatar et al. 2008).

Drosophila oogenesis and fecundity regulation

4
The process of Drosophila oogenesis has been extensively reviewed in articles and book
chapters (Mahowald 1980; Lin and Spradling 1993). Each female Drosophila has a pair
of ovaries, consisting of 15 to 20 ovarioles each. The ovarioles are the assembly lines of
Drosophila eggs. The anterior part of an ovariole is known as the germarium. The
posterior part is known as the vitellarium (Lin and Spradling 1993). The starting point of
the egg assembly line is the anterior tip of the germarium, where two germline stem cells
reside on average. When these stem cells divide asymmetrically, they generate a new
cystoblast cell and maintain the original stem cell as it was before the division. The
cystoblast cell is enveloped by a single layer of follicle cells, which are generated by
follicle stem cells. The cystoblast cell divides four times to form a 16-cyst-cell cluster.
Only one cell in the cluster will develop into an oocyte, while the others will become
nurse cells. The nurse cells will undergo endoreplication in which they replicate their
DNA without cell division. Then the nurse cells will grow and accumulate yolk protein,
which will later nurture the oocyte. This process is known as “vitellogenesis”. At the late
stage of oogenesis, the nurse cells will undergo apoptosis. The follicle cells will produce
the eggshell, including the respiratory appendages, and then will also undergo apoptosis
(Mahowald 1980; Lin and Spradling 1993). Before the fertilization, the mature eggs are
arrested at metaphase I of meiosis. When mating occurs, sperm cells are ejaculated into
female, along with many different types of accessory gland proteins in the seminal fluid,
to fertilize the egg and release it from the meiosis arrest (Wolfner 2002; Gillott 2003).

5
Female fecundity is greatly affected by many environmental conditions, including
temperature, humidity, and food availability (Alpatov 1932; Kaliss and M.A.Graubard
1936; Siddiqui and C.A.Barlow 1972). Among the most important factors is the influx
of male seminal fluid proteins. A few researchers suggest that male seminal fluid
proteins can increase egg production and decrease female sexual receptivity after mating
has occurred (Wolfner 1997; Chapman 2001; Wolfner 2002; Gillott 2003; Kubli 2003;
Liu and Kubli 2003; Lawniczak and Begun 2004; McGraw, Gibson et al. 2004).
However, the ingredients of seminal fluid are not fully characterized. The fluid contains
the secretions of the male accessory glands and ejaculatory duct (Wolfner 2002; Gillott
2003). Scientists have suggested a list of more than 80 different types of accessory gland
proteins (Swanson, Clark et al. 2001).

A more recent finding suggests that a particular “sex peptide” can increase female egg
production (Chapman, Bangham et al. 2003; Liu and Kubli 2003), and cause release of
juvenile hormone (Moshitzky, Fleischmann et al. 1996). It was previously known that
juvenile hormone may positively affect Drosophila oogenesis and egg production
process (Soller, Bownes et al. 1999; Dubrovsky, Dubrovskaya et al. 2002).

Drosophila PdL mutagenesis
The technique of PdL mutagenesis would not be possible without two crucial steps: P-
element mutagenesis and the tet-on system.

6
After the initial use of Drosophila P-element (Rubin and Spradling 1982; Rubin and
Spradling 1983), hundreds of papers, if not more, described the basic theory and the
application of this transposable element (Ring, Bass et al. 2000; Tower 2000; Landis,
Bhole et al. 2001; Groth, Fish et al. 2004). P-elements attracted so many researchers
because it not only suggests the wide horizontal transfer of transposable elements within
the Drosophila melanogaster species, but it is also a powerful tool to manipulate the
Drosophila genome. P-elements have inverted repeats on both ends (Collins and Rubin
1984), forming the target site for transposase, an enzyme required for P-element
mobility (Rio and Rubin 1988; Mullins, Rio et al. 1989; Rio 1990). When a strain that
bears a P-element is crossed to another strain that bears a transposase coding sequence
(such as “ry
+
2-3”) (Robertson, Preston et al. 1988), the germ line cells of the progeny
will be mutagenized by the transposition of its P-element. The expression level of a gene
located 3’, or, less frequently, 5’, of the new P-element insertion site can be altered,
and/or the gene may be physically disrupted.

It must be emphasized that the above example application alone does not do justice to
even the most fundamental uses of P-element mutagenesis. An in-depth discussion of P-
elements, however, is far beyond the scope of this introduction.

The initial report of a tet-on system in Drosophila can be found in a 1998 research
article (Bieschke, Wheeler et al. 1998). The tet-on system is derived from the binding
mechanism between a protein called the reverse tetracycline repressor (rtR) and its DNA

7
target site, the tetracycline operator (tetO). This binding occurs only in the presence of
tetracycline, or one of its derivatives, such as doxycycline (DOX). When the DNA
binding capability of rtR is coupled to the transcription activation capability of herpes
virus protein VP16, we obtain the transactivator protein rtTA. Previous research
suggested that, in mammals, rtTA turned on transcription of a gene downstream of a
tetO sequence in a DOX-dependent manner. The research demonstrated that tet-on
system also worked efficiently in Drosophila, as indicated by the successful induction of
beta-gal by up to 100 fold (Bieschke, Wheeler et al. 1998).

In later reports, the same group of researchers placed the DOX-inducible promoter, a
combination of the tetO operator and the hsp70 core promoter, within a transposable P-
element, creating a DOX-inducible transposable promoter named PdL (Landis, Bhole et
al. 2001). The promoter, when it is mobilized to a new chromosomal position by the
transposase, is able to induce transcription of a gene downstream of the insertion site in
a DOX-dependent manner. This novel method gave the scientists a powerful tool to
generate many different transgenic Drosophila strains simultaneously with each one
being able to over-express a gene-of-interest specifically in any development stage. The
induction requires only a tiny amount of the DOX chemical in the Drosophila food
(Landis, Bhole et al. 2001). Given enough independent crosses, PdL-based mutagenesis
is able to induce a large fraction of the genes in Drosophila, thus making possible the
investigation of the over-expression phenotypes of a significant fraction of the 14,000
Drosophila genes.

8

Drosophila apoptosis and anti-apoptosis pathways
Programmed cell death, also known as apoptosis, is common among many organisms. It
removes un-needed or damaged cells during development, whether or not caused by
aging itself. The core of the apoptosis machinery is an enzyme named caspase, a
member in protease family (Degterev, Boyce et al. 2003).

When apoptosis is induced, the programmed cell death signal triggers the adaptor Ark,
which activates the initiator caspase Dronc (Quinn, Dorstyn et al. 2000; Yu, Wang et al.
2006). Experiments demonstrated that Ark and Dronc are the central players of caspase-
dependent cell death. When Ark is mutated or knocked down by RNAi, cell death is
either decreased or blocked (Kanuka, Sawamoto et al. 1999; Zimmermann, Ricci et al.
2002; Akdemir, Farkas et al. 2006; Kiessling and Green 2006; Leulier, Ribeiro et al.
2006; Mills, Daish et al. 2006; Srivastava, Scherr et al. 2007). The ectopic expression of
dominant-negative Dronc blocks caspase-dependent cell death in the Drosophila eye
(Hawkins, Yoo et al. 2000; Meier, Silke et al. 2000), and RNAi has the same effect in
the embryo (Quinn, Dorstyn et al. 2000). The Ark/Dronc cascade is regulated by the
inhibitor of apoptosis protein dIAP1 of the IAP family (Yan, Wu et al. 2004; Muro,
Means et al. 2005), which suppresses Dronc and the effector caspase Drice that Dronc
activates (Hawkins, Yoo et al. 2000; Meier, Silke et al. 2000; Chai, Yan et al. 2003).
Taken together, the evidence described above suggests that the cell death signal activates
Dronc in an Ark-dependent manner. Dronc in turn activates Drice, triggering cell death

9
proteins and leading to apoptosis. Their effects can be negatively regulated by the
apoptosis inhibitor IAP proteins.

Since the discovery of the IAP protein family in early 90’s in baculoviruses (Crook,
Clem et al. 1993; Birnbaum, Clem et al. 1994; Clem and Miller 1994), the Drosophila
homologs have subsequently been researched by different research groups (Hay,
Wassarman et al. 1995; Vernooy, Copeland et al. 2000). IAP family proteins are
identified by multiple repeats of a 70-amino-acid motif named Baculovirus IAP repeat
(BIR) on the N-terminus and a RING ubiquitin ligase domain on the C-terminus (Hay,
Wassarman et al. 1995). There are 3 BIR-bearing proteins in Drosophila, including
dIAP1 (Hay, Wassarman et al. 1995), dIAP2 (Hay, Wassarman et al. 1995; Duckett,
Nava et al. 1996; Liston, Roy et al. 1996; Uren, Pakusch et al. 1996), and dBruse
(Vernooy, Chow et al. 2002). The functions and pathways involving dIAP1 and dBruse
have been extensively reviewed in the literature. Since our longevity strain bears the tet-
on-dIAP2 sequence and over-expresses dIAP2, among the 3 BIR-bearing proteins,
dIAP2 will be the focus of the following introduction section.

The most investigated member in IAP protein family is dIAP1, which is responsible for
the survival and maintenance of different types of cells by inhibiting active caspases
(Hay, Huh et al. 2004; Hay and Guo 2006). Unlike its sibling dIAP1, dIAP2 is not
primarily known for its inhibition of apoptosis effect. Rather than anti-apoptosis
functions, dIAP2 was found to be related to innate immune responses to Gram-negative

10
bacterial infections (Gesellchen, Kuttenkeuler et al. 2005; Kleino, Valanne et al. 2005).
Even though there may not be an immediately obvious relation between anti-apoptosis
and the innate immune response, different groups of researchers have suggested that
cells under apoptotic stress could be the target of the immune response in autoimmunity.
This finding provided the scientists a unique point of view about the potential links
between anti-apoptosis and innate immune response on molecular level (Takatsu,
Nakamura et al. 2000; Georgel, Naitza et al. 2001; Mukae, Yokoyama et al. 2002;
Mahoney and Rosen 2005). A more recent finding suggested that dIAP2 regulates the
immune response in a dose-dependent manner (Huh, Foe et al. 2007). Taken together,
dIAP2 molecular functions could provide an interesting model as the cross-point of
immune response, apoptosis and aging.

Drosophila stem cells
Researchers have long been aware of the existence of germline stem cells and somatic
stem cells in the anterior part of the ovarioles in the Drosophila ovary (Lin and
Spradling 1993). It is no wonder that the asymmetrical division of these germline stem
cells starts the whole oogenesis process. Readers also should not be surprised to find that
the somatic cells that surround the germline stem cells consitutute a stem cell “niche” to
support them with molecular signaling. These signals are crucial for germline stem cell
maintenance, division and differentiation (Lin and Spradling 1993; Bhat and Schedl
1997; King and Lin 1999).

11
When a stem cell generates a new daughter cell, it divides in an asymmetrical manner.
For example, when a germline stem cell in the germarium generates a new cystoblast
cell, the division results in a new cystoblast cell in the ovariole, whereas the original
germline stem cell keeps its “stemness” and remains functionally identical to how it was
before the division. This self-renewal process guarantees the continuous production of
new eggs (Lin and Spradling 1993).

Stem cells are unable to keep their stemness without the support the microenvironment
they reside in, which is named a “niche”. The niche not only produces maintenance,
growth and differentiation factors for stem cells (Watt and Hogan 2000; Spradling,
Drummond-Barbosa et al. 2001; Szakmary, Cox et al. 2005), but, perhaps more
importantly, also ensures that the stem cells that reside in it occasinally divide in a new,
symmetrical manner to replenish a lost or damaged stem cell (Xie and Spradling 2000).

In the human and mouse digestive systems, the intestinal cells are continuously
replenished by stem cells (Playford 1998; Bischoff, Sellge et al. 1999; Lorentz and
Bischoff 2001; Sellge, Lorentz et al. 2004; Micchelli and Perrimon 2006; Ohlstein and
Spradling 2006). Surprisingly, until recently, little was known about the possible
existence of stem cells in Drosophila intestine. Drosophila intestine cells were
previously described to be relatively stable. Later, in 2006, two separate research groups
reported the discovery of Drosophila intestine stem cells in Drosophila midgut
(Micchelli and Perrimon 2006; Ohlstein and Spradling 2006). As with many other stem

12
cells, the Notch signaling pathway regulates the differentiation of these multipotent stem
cells (Ohlstein and Spradling 2006). A more recent finding related to intestine stem cell
differentiation suggests that Notch signaling and Notch ligand Delta level determine the
differentiation fate of daughter cells of intestine stem cells (Ohlstein and Spradling
2007). A high Delta level with strong Notch signaling results in enterocyte cells, while
weak Notch signaling results in enteroendocrine cells (Ohlstein and Spradling 2007).
Germline stem cells, intestine stem cells, the niches, and signal pathway that support and
regulate the stem cells provides us an invaluable model system for our longevity and
other studies.

Drosophila RNAi
RNA interference (RNAi) is a relatively new technique. However, because it is easy to
carry out and it is able to precisely knock down any gene in a sequence-specific manner,
in the last a few years, RNAi has become the predominant choice for decreasing the
expression level of a gene of interest in most model organisms.

When dsRNA segments are introduced into eukaryotic cells, if their sequence is
complementary to that of an endogenous gene, the expression of the endogenous gene
will be inhibited by a mechanism known as RNA interference (RNAi) (Fire, Xu et al.
1998; Fire 1999; Hunter 1999; Bosher and Labouesse 2000). The evolutionary origin of
RNAi remains unclear, but most researchers agree that it is most likely a defense
mechanism that protects the host organism from virus invasions or restricts the motility

13
of transposable elements (Montgomery and Fire 1998; Cogoni and Macino 1999;
Ketting, Haverkamp et al. 1999; Tabara, Sarkissian et al. 1999; Zambon, Vakharia et al.
2006).

After dsRNA is introduced into the cell, the long strand of dsRNA is processed into 21-
25 nucleotide long sequences called small interfering RNA (siRNA) by the protein
complex Dicer (Hamilton and Baulcombe 1999; Zamore, Tuschl et al. 2000; Bernstein,
Caudy et al. 2001; Ketting, Fischer et al. 2001). The resulting siRNA is further
incorporated into an RNA-protein complex called RNA-induced silencing complex
(RISC) (Hammond, Bernstein et al. 2000). Along with siRNA, RISC recognizes the
complementary mRNA of the siRNA and targets it for destruction (Martinez,
Patkaniowska et al. 2002).

Because this targeted degradation has such a high sequence specificity, RNAi has
rapidly become the preferred tool for knocking down genes when performing functional
studies in both molecular biology labs and in a clinical research environment. Genome-
wide screening with RNAi in Drosophila cells and adults performed by many
independent research groups provide us a very efficient and useful method to investigate
various gene functions with high throughput (Wheeler, Bailey et al. 2004; Dorner, Lum
et al. 2006; Flockhart, Booker et al. 2006; Mathey-Prevot and Perrimon 2006; Derre,
Pypaert et al. 2007; Dietzl, Chen et al. 2007; Moffat, Reiling et al. 2007; Perrimon,
Friedman et al. 2007; Ramadan, Flockhart et al. 2007).

14

Not long after the discovery of RNAi itself, scientists started to try using it to knock
down Drosophila genes in a conditional and inheritable manner (Kennerdell and
Carthew 2000). The pioneer scientist and some of the followers shared a similar
approach to achieve RNAi in Drosophila (Kennerdell and Carthew 2000; Hammond,
Caudy et al. 2001; Adams and Sekelsky 2002; Allikian, Deckert-Cruz et al. 2002). They
constructed an inverted repeat of the target gene sequence and cloned it into a vector that
is able to transcribe the repeat conditionally. The whole construct was injected into the
early stage developing Drosophila embryo by microinjection to generate a transgenic
strain. The inverted repeat structure will fold back upon itself after transcription,
essentially becoming a dsRNA and targeting the gene of interest. The same Dicer and
RISC mechanism will process the dsRNA, and then eventually achieve RNAi. By taking
advantage of the conditional promoter upstream of the inverted repeat, researchers are
able to knock down genes during any stage of development in any tissue. This creative
RNAi design resulted in very effective interference. With only a few improvements
(Adams and Sekelsky 2002; Lee and Carthew 2003), the conditional transcription of
inverted repeats is still the major weapon in scientists’ RNAi arsenal (Allikian, Deckert-
Cruz et al. 2002; Dzitoyeva, Dimitrijevic et al. 2003; Lum, Yao et al. 2003; Dasgupta
and Perrimon 2004; Baeg, Zhou et al. 2005; Gwack, Sharma et al. 2006; Perrimon,
Friedman et al. 2007; Chen, Shi et al. 2008).

15
However, as with all other molecular biology techniques, RNAi by transcription of
inverted repeat has its own disadvantages.. Researchers often have to inject hundreds of
embryos, hoping to obtain one successful transformant. It also takes weeks or even
months to balance the successful transformant into a homozygous strain before it can be
used to knock down their favorite gene in a live fly. Another research group tried an
alternative approach, inducing RNAi by simply injecting dsRNA into the abdomen of
anesthetized adult Drosophila (Dzitoyeva, Dimitrijevic et al. 2001). The same research
group used this method to characterize the molecular functions of a few genes in the
central nervous system (Dzitoyeva, Dimitrijevic et al. 2001; Dzitoyeva, Dimitrijevic et
al. 2003). This new method is able to knock down a gene of interest with reasonable
efficacy in just a few days, compared to weeks or months for the traditional technique.
However, perhaps because the intra-abdominal dsRNA injection on adult flies requires
mastery of fly injection techniques, until now, other than this original research group,
reports of successful adult RNAi injection in Drosophila are still very rare.

16
Chapter 2
Identification and characterization of magu and hebe genes
in Drosophila melanogaster

Abstract
During Drosophila aging, the mortality rate increases exponentially and progeny
production per animal declines dramatically, correlating with decreased count and cell
division of somatic and germ-line stem cells in the gonads. To search for genes that
might promote both longevity and fecundity, a P element transposon (PdL), containing
an outwardly-directed, doxycycline-inducible promoter was used to generate conditional
overexpression mutations. Mutant females were screened for increased fecundity at late
ages in the presence of doxycycline. Two genes were identified, named hebe (CG1623)
and magu (CG2264), that when over-expressed in adult flies could increase lifespan by
~5-30% in both sexes and increase female fecundity at late ages. Transcripts of magu are
enriched in Drosophila stem cells, and magu encodes a protein related to the human
SMOC2 regulator of angiogenesis. While moderate over-expression of magu in adult
females increased fecundity at late ages, high-level over-expression of magu was
maternal-effect lethal. The data demonstrate that adult-specific over-expression of magu
and hebe genes can increase lifespan and modulate female fecundity. The ability of
magu over-expression to increase both lifespan and female fecundity provides evidence
against a necessary trade-off between reproduction and longevity.

17
Introduction
The size of the Drosophila melanogaster genome is around 180 Mb, containing
approximately 14,000 genes (Celniker and Rubin 2003). There is no doubt that the
Drosophila genome project provided the scientific community with an invaluable asset,
a solid foundation from which to begin genomic and proteomic studies. But taking
advantange of a database of this size is always a challenge for genomics researchers. Of
the 14,000 genes, some have of course already been extensively characterized. To
research the molecular functions of the remaining genes, a large scale method would be
the only practical way to put so many candidates under the microscope at the same time.
Many research groups have applied more than one type of method to try to achieve this
goal. Microarrays are probably the easiest method to investigate the functions of
multiple genes simultaneously, but it provides the least amount of information (Gupta
and Oliver 2003; Shin, Zhao et al. 2005; Lai, Parnell et al. 2007). This is inevitable due
to the unique approach of microarrays themselves. When the organism shows a certain
phenotype in a particular life stage (Arbeitman, Furlong et al. 2002), microarrays only
answer the questions about what gene is up- or down-regulated. By studying the
correlations between these expression patterns, one can obtain basic clues about what
genes may or may not work together in a regulatory pathway or interact with each other.
However, gene expression patterns alone do not provide further information about
specific functions, especially for novel genes.

18
One can also rely on large scale RNAi projects to predict the molecular functions of new
genes. A few reports already demonstrate the successful application of this novel but
powerful approach on Drosophila on a large scale (Mathey-Prevot and Perrimon 2006;
Dietzl, Chen et al. 2007). By knocking down the expression of many different genes
individually and culturing the flies into adulthood, one can directly observe the
corresponding phenotypes in any life stage. With easy access to mutant flies, researchers
can also design genetic crosses between different knock-down mutant strains to
investigate the relations between the genes in the same or different regulation pathways.
However, as with all other molecular biology techniques, RNAi has disadvantages.
RNAi simulates a loss-of-function mutation. It does not allow the over-expression of the
target gene. Therefore it is difficult to obtain the gain-of-function mutant phenotype by
only performing RNAi mutations.

Our lab devised a novel approach to generate a large quantity of random mutants that
can over-express many genes individually. With this system, gain-of-function mutation
phenotypes can be readily generated and observed (Landis, Bhole et al. 2001). The
system takes advantage of P-element mutagenesis and the tet-on system to create the
concept of PdL mutagenesis. By using transposase to excise and insert the PdL
conditional promoter into random locations in the genome (Figure 1), a random gene can
be over-expressed, and its gain-of-function phenotype can be studied. Given enough
mutants, one can cover almost the whole genome to study the gain-of-function mutant

19
phenotype of any gene. In this research project, I used this technique to identify genes
related to fecundity and longevity.

Figure 1: The sequence structure of PdL insertion. 5’ and 3’ are labeled by –P and –OH.
7-Tet-O operator, polylinker site, and Hsp70 core promoter are labeled. Pry1 and IR
annealing sites are labeled. Their 5’ to 3’ orientation is labeled by arrows. The two Taq1
sites in the polylinker and downstream sequence are labeled.

Each female fly has one pair of ovaries, consisting of 15-20 ovarioles each. The
ovarioles is anatomically divided into an anterior part named the germarium, and a
posterior part named the vitellarium (Kirilly and Xie 2007). On average, two germline
stem cells are located at the anterior tip of the germarium in each ovariole. They
maintain their stemness using asymmetrical division with self-renewal (the standard
stem cell model) to produce a daughter stem cell and a new cystoblast in each division
round (Deng and Lin 2001; Kirilly and Xie 2007). The new cystoblast cell then
undergoes 4 rounds of mitosis to become a 16-cyst-cell-cluster. One of the 16 cells will
develop into an oocyte while the others will become nurse cells (Deng and Lin 2001).
This newly produced oocyte is surrounded by a single layer of follicle cells that are
produced by somatic stem cells located at the anterior tip of germarium (Margolis and

20
Spradling 1995). The whole structure moves towards the posterior end to enter
vitellarium. The oocyte undergoes 14 development stages. During the middle and late
stages, the nurse cells provide nutrition to the developing oocyte, and later undergo
apoptosis. In the late stages, follicle cells secrete eggshell proteins (?) endochorion and
exochorion and eventually also undergo apoptosis, leaving only the egg shell
surrounding the mature egg (Mahowald 1980; Cavaliere, Taddei et al. 1998; Waring
2000; Trougakos, Papassideri et al. 2001; Nezis, Stravopodis et al. 2002).

Oogenesis is a complex process involving numerous molecular signals. Any mistake in
the egg assembly line would affect the female fecundity, resulting in altered egg
production behavior. As confirmed by previous experiments, the fecundity of the female
flies gradually declines after they pass the young age. (Figure 2) The females lose the
majority of their fecundity at about 70 days of age. Since the generation of new oocyte
results from the asymmetrical division of germline stem cells, it can be postulated that
the reduced fecundity in the late age of female flies is caused by the aging process in
those stem cells. Ovary germline stem cells are supported by niches surrounding them
(Kirilly and Xie 2007; Nystul and Spradling 2007; Song, Call et al. 2007). The
maintenance mechanism of the niches is mediated by a Jak/Stat signaling pathway
(Lopez-Onieva, Fernandez-Minan et al. 2008). It has been reported that removal of the
Jak/Stat pathway results in the differentiation of the germline stem cells, destroying their
stem-ness (Lopez-Onieva, Fernandez-Minan et al. 2008). It is also demonstrated that the
Dpp and Notch signaling pathways, regulated by Jak/Stat, affects the niche size and

21
ensure the undifferentiated state of the germline stem cells enveloped by the niches
(Song, Call et al. 2007; Lopez-Onieva, Fernandez-Minan et al. 2008). A recent report
described that the decreased BMP signaling in the late age niche cells resulted in loss of
germline stem cells (Pan, Chen et al. 2007). Another source of stem cell aging is E-
cadherin. The same report demonstrated that a decrease of E-cadherin in the niches in
late age flies resulted in germline stem cell aging (Pan, Chen et al. 2007). Interestingly,
stem cell aging could be counteracted by the over-expression of the enzyme superoxide
dismutase (SOD). Over expression of SOD in either germline stem cells or in niche cells
is sufficient to prolong germ line stem cell lifespan and induce their proliferation (Pan,
Chen et al. 2007). This finding is consistent with the sufficiency of SOD over-expression
to extend Drosophila lifespan (Sun and Tower 1999; Sun, Folk et al. 2002).

Figure 2: Female fecundity of Or-R wild type flies. Number of progeny per female per
day is plotted against age in days. Error bars: standard error of the mean.

Other molecular signals also affect female reproductive behavior. Females require sex
peptide receptor to respond to sex peptides from the male accessory gland proteins. The

22
influx of sex peptides dramatically increases egg production following mating (Kubli
2003; Liu and Kubli 2003; Swanson 2003; Yapici, Kim et al. 2008). Other factors for
Drosophila reproductive behavior include the regulation of juvenile hormone (JH) and
ecdysteroids. These stimulate the uptake of yolk proteins in developing oocytes
(Bownes, Ronaldson et al. 1993; Kelly 1994). The yolk protein is mainly produced in
the fat body, which potentially could affect the female reproductive behavior as well
(Gilbert, Serafin et al. 1998).

Since oogenesis is such a complex and highly regulated process, this Gordian Knot
poses great challenges to researchers in genetics, stem cell biology and development
biology. The advantages of our conditional over-expression system make it relatively
easy to study the impact of a specific gene over-expression at any life stages. Other
conditional over-expression systems work in a similar fashion, but use a different trigger
to over-express the gene of interest. For example, the FLP-out system (REF?) triggers
gene over-expression by heat shock, which is known to affect fly longevity and
reproductive behavior by itself, and therefore is not the best choice for these studies.
Both the tet-on and gene switch system use a chemical-based triggering mechanism and
do not affect normal fly physiology (Tower 2000; Landis, Bhole et al. 2001; Roman,
Endo et al. 2001; Stebbins, Urlinger et al. 2001; Allikian, Deckert-Cruz et al. 2002;
McGuire, Roman et al. 2004; Ford, Hoe et al. 2007). Therefore, they are ideal options
for the identification and characterization of fecundity and longevity regulating genes. In
this research, I used PdL mutagenesis to screen for altered fecundity and longevity

23
phenotypes. The traditional tet-on system was constructed to confirm these phenotypes
on transgenic lines when the re-tests were performed.

Materials and Methods
Drosophila strains and culture

Drosophila strains and culture conditions are as previously described (Ford, Hoe et al.
2007). Experiments were preformed at 25
o
C using a standard cornmeal/agar media
(Ren, Webster et al. 2007). To cause conditional gene expression using the Tet-on
system, flies were cultured on media containing doxycycline and ampicillin each at a
final concentration of 64ug/ml (“+DOX”). For the control groups (“-DOX”), flies were
cultured on media containing ampicillin only.

Generation of wild type female fecundity curve

A cohort of 40 Oregon-R virgin female flies was collected over a period of 48 hours and
designated as 1 day old. They were separated into 10 vials with 4 virgins in each vial
together with 4 young Oregon-R males and kept at 25
o
C. Flies in each vial were
transferred to fresh food vials every second day and the number of dead flies was
recorded, and the old vials were kept at 25
o
C for subsequent progeny counts. Each
month 4 young Oregon-R males were added to each vial to replenish the old males, and

24
the experiment was performed until all the females were dead. The number of pupae in
each vial was counted 10 days after each transfer. For each vial, the number of pupae
was divided by the number of females in that specific period to yield progeny per
female. The average pupae number per female +/-SEM was calculated across the 10
replicate vials. A preliminary version of these data was presented in a review article
(Waskar, Li et al. 2005), and the data are included here with further statistical analysis
for comparison purposes.

Genetic screen for new PdL mutations

Virgin flies from strain PdL45C1 were crossed to males from strain delta2-3. The male
progeny containing both the PdL45C1 insertion and delta2-3 were selected by their
phenotypic markers and crossed to virgins of strain rtTA(3)E2. The virgin progeny flies
containing a new PdL insertion and rtTA(3)E2 have the ability to over-express the gene
immediately downstream of the PdL insertion, and ~8,000 of these mutants were
generated and analyzed. (See figure 3 for cross design detail.) Each individual mutant
female was combined with 3 young Oregon-R male flies and cultured on -DOX food for
the first 35 days of their lifespan. During this process the flies were transferred to fresh
food vials every two days. At the end of the 35 day period, the flies were then cultured
on –DOX food for 4 days (“Time Period 1”). The flies were then cultured on +DOX
food for 6 days to allow any gene over-expression and phenotypes to become apparent.
The flies were then cultured on +DOX food for an additional 4 days (“Time Period 2”).

25
During these steps the flies were always transferred to fresh food vials every other day.
Pupal number was counted in vials 10 days after transfer. Because control Oregon-R
wild type flies showed significant decrease in fecundity between Time Period 1 and
Time Period 2, any mutants that did not show a significant decrease in fecundity in this
interval were considered potential positives and were further characterized. The site of
insertion of PdL was determined using inverse PCR, sequencing and comparison
between the PdL flanking sequence and fly genomic DNA databases using NCBI-
BLAST, as previously described (Landis, Bhole et al. 2001).

26

Figure 3: The cross design for generation of PdL mutants. Male and virgin females are
labeled by their corresponding symbols. Balancers on different chromosomes are labeled
by their corresponding phenotypic markers. A brief diagram of the screening procedure
that followed the cross is included.

Creation of constructs and transgenic flies

The cDNA of gene of interest was ordered from BACPAC (http://bacpac.chori.org/)
(BACPAC Resources Center at Children's Hospital Oakland Research Institute in
Oakland, California). The cDNA was amplified from its clone vector POT1. For magu
the forward primer was 5’-TGACGAATTCGAACTGCTAAG-3’; for hebe the forward
primer was 5’-TTCAAAGGCAGACAGACATGG-3’; the reverse primer anneals to the
POT1 vector and was 5’-CGTTAGAACGCGGCTACAATT-3’. The amplified cDNAs

27
were ligated into the USC1.0 vector that contains the mini-white
+
marker gene (Allikian,
Deckert-Cruz et al. 2002) to create p{magu} and p{hebe} transformation constructs,
respectively. Multiple independent transgenic strains were generated for each construct
by embryo microinjection using standard protocols (Rubin and Spradling 1982) and the
y-ac-w recipient strain (Patton, Gomes et al. 1992).

Northern blot

Northern blot assay was performed essentially as previously described (Landis, Bhole et
al. 2003). Briefly, strains bearing a PdL insertion or a transgene insertion were crossed
with rtTA(3)E2 flies. The male and female progeny were collected separately and
cultured on +DOX and –DOX food for 10 days. Total RNA was extracted from 30 flies
of each group using Trizol kit (Invitrogen), resolved on 2% agarose gels, and then
transferred to Genescreen membrane (Dupont/NEN). Restriction fragments from the
cDNA clones of the magu and hebe genes were radiolabelled and used as gene-specific
probes. Ribosomal protein 49 (Rp49) probe was used as loading control. Transcript size
was determined by comparison with 1 Kb RNA ladder (Gibco-BRL) according to the
manufacturer’s instructions.

Female fecundity assays

28
Female fecundity was assayed in a longitudinal protocol analogous to the screening
procedure described above, except that multiple flies (four vials each containing 4
females) were used to provide replication and statistical inference (Figure 4). Female
fecundity was also assayed in a parallel protocol, which involved four replicate vials,
each containing four females and four wild-type males, for each of +DOX and –DOX
conditions. Flies were transferred to fresh vials every other day. The progeny numbers
(pupae) were recorded in the old vials in the time intervals between day 10 to 20 and
between day 30 to 40. The average progeny per female for each time interval was
calculated, and +DOX values were compared to –DOX values using unpaired, two-sided
t-tests. For certain experiments, the final concentration of DOX in the food was diluted
1:5 and 1:25 as indicated.

Lifespan assays

Lifespan assays were performed at 25
o
C with passage to fresh food vials every other day
as previously described (Ford, Hoe et al. 2007). Each cohort consisted of ~125 flies at
~25 flies per vial, and median lifespans of +DOX samples were compared to –DOX
controls using log-rank tests.

29
Protein motif analyses

The protein motif search was performed using four different databases: InterPro
(http://www.ebi.ac.uk/interpro/), GenomeNet (http://www.genome.jp/), ScanProsite
(http://expasy.org/cgi-bin/scanprosite), and Sanger Institute Pfam
(http://pfam.sanger.ac.uk/).

Results
Screening for gene over-expression mutations that increase late-age female fecundity
using PdL

In Drosophila, female fecundity peaks in the first two weeks, and then declines
dramatically with advancing age (Figure 2) (Waskar, Li et al. 2005; Zhao, Xuan et al.
2008). To identify genes that might positively regulate both fecundity and lifespan, PdL
mutagenesis was employed to create gene over-expression mutations. The PdL P
element contains an outwardly-directed, DOX-regulated promoter that will cause over-
expression of genes located downstream of the 3’ end of the element. The rtTA(3)E2
transgenic strain contains a construct in which the powerful, tissue-general promoter
from the cytoplasmic actin gene (actin5C) drives expression of the artificial transcription
factor rtTA. The rtTA transcription factor is activated upon interaction with DOX, and
then binds to specific sites (Tet-operator sites) in the PdL promoter, thereby mediating
DOX-inducible transcription. In this way, feeding DOX to a fly containing both PdL

30
and rtTA(3)E2 can cause robust over-expression of a gene located 3’ to PdL in all of the
somatic tissues of the fly.

Figure 4: The first fecundity test after balancing to homozygosity. Male flies from each
strain was crossed to rtTA(3)E2 virgin females. The progeny females were kept with
young Or-R males for continued mating. The fecundity was assayed with the method
described in methods section. X-axis represents the early and late time period. Y-axis
represents progeny per female per day. Y-error bar represents standard deviation. A t-
test comparing the early and late time period fecundity was performed, and the p-value
is shown under each curve.

Strain PdL[45C1] contains an insert of PdL on the second chromosome, which is also
marked with the dominant mutation Sternopleural (Sp). Virgins of this strain were
crossed to males from strain delta2-3, which expresses the P element transposase. The
male progeny containing both the PdL[45C1] insertion and delta2-3 were selected by
their phenotypic markers. In these males the transposase will cause transposition of PdL
to new sites in the genome in the germ line cells, and therefore these males are called
“dysgenic”. The dysgenic males were crossed to virgins of strain rtTA(3)E2 to produce
mutant female progeny bearing a new insertion of PdL on either the first, second or third
chromosomes (indicated by asterisk), as well as the rtTA(3)E2 driver. The new

31
insertions were identified by the presence of the mini-white+ marker gene in PdL, and
the absence of the Sp mutation marking the chromosome bearing the starting PdL
insertion. Approximately 8,000 of these mutant females were generated and analyzed.
The mutant females were combined with Oregon-R male flies and cultured on -DOX
food for the first 35 days of their lifespan. During this process the flies were transferred
to fresh food vials every two days. At the end of the 35 day period, the female flies were
then individually cultured in the presence of 3 Oregon-R wild type males on –DOX food
for 4 days (“time period 1”), to allow measurement of progeny production. The flies
were then cultured on +DOX food for 6 days to allow any gene over-expression and
phenotypes to become apparent. The flies were then cultured on +DOX food for an
additional 4 days (“time period 2”) to again allow measurement of progeny production.
During these steps the flies were always being transferred to fresh food vials every other
day. The number of progeny (pupae) produced by the mutant females was counted in
each vial 10 days after transfer. Because control Oregon-R wild type flies showed
significant decrease in fecundity between time period 1 and time period 2 (Figure 4), any
mutant females that did not show a significant decrease in fecundity in this interval were
considered potential positives and were further characterized. Eight mutant female
strains were identified that showed little or no decrease in fecundity between time period
1 and time period 2. These eight promising new PdL insertions were balanced, and re-
tested for fecundity effects using replicate females (Figure 4), and further tested for
effects on lifespan (described below). Two mutants consistently gave positive results

32
and were further pursued, hereafter referred to as P{PdL}hebe and P{PdL}magu, named
after immortal deities from Chinese mythology.

Conditional over-expression of hebe and magu cDNAs

Inverse PCR was used to clone the genomic DNA immediately adjacent to the 3’ end of
the PdL insertions in lines P{PdL}hebe and P{PdL}magu. Comparison of the flanking
sequences to the GenBank database revealed that P{PdL}hebe was inserted 6,721bp
upstream of the translation start site for gene CG1663 (hebe), while P{PdL}magu was
located in the first intron of gene CG2264 (magu), 4,646bp upstream of the translation
start site. In both cases the 3’ end of PdL was oriented towards the open reading frame,
suggesting that DOX would cause over-expression of functional, full-length proteins.
cDNA clones for both hebe and magu were obtained from a commercial source
(BACPAC Resources Center at Children's Hospital Oakland Research Institute).
Northern blot analysis confirmed the DOX-dependent over-expression of both the hebe
and magu genes in the P{PdL}hebe and P{PdL}magu mutant strains, in both male and
female adult flies (Figure 7A, F).

To confirm the phenotypic effects of hebe and magu over-expression, the corresponding
cDNAs were cloned into the USC1.0 vector, to allow for DOX-dependent over-
expression in an otherwise wild-type genetic background (Figure 6). Multiple
independent lines were generated for each over-expression construct (Table 1), and

33
Northern blot analysis confirmed DOX-dependent over-expression of the transgenes in
both male and female adult flies (Figure 7B-E, G-H).

Ubiquitous over-expression of hebe and magu in adults increases male and female
lifespan

The rtTA(3)E2 ubiquitous driver was used to cause DOX-dependent over-expression of
hebe and magu genes in adult flies. The original P{PdL}hebe and P{PdL}magu
insertions were assayed, along with multiple independent cDNA over-expression
construct insertions for each gene, in replicate experiments. Representative survival
curves are presented (Figure 9), and the statistical analysis is presented for each strain
and replicate experiment (Table 2). Control genotypes were generated by crossing the
Oregon-R wild-type and w[1118] injection strains to the rtTA(3)E2 ubiquitous driver
strain, to generate progeny expressing rtTA but containing no target transgene. In these
controls DOX treatment caused no significant change in lifespan of either male or
female flies (Figure 9A, B; Table 2). In males, the P{PdL}hebe mutation resulted in
increases of 31% in median and 24% in mean lifespan. In females, the mutation yielded
a 2.2% increase in median and 11% increase in mean lifespan. The three independent
hebe cDNA over-expression strains yielded increases in median lifespan in males
ranging from 7% to 26% percent, while increases in females ranged from 6% to 23%.
Similar but somewhat more variable results were obtained upon ubiquitous over-
expression of magu. The P{PdL}magu mutation yielded 9.3% percent increase in

34
median lifespan and a 5.3% increase in mean lifespan in males. In females this mutation
yielded a 2.2% increase in median lifespan and a 5.3% increase in mean lifespan. Five
independent magu cDNA over-expression construct insertions were assayed in a first
experiment, and three of these (P{magu}102, P{magu}67-1, and P{magu}39) yielded
increases in median lifespan in both males and females ranging from 2-18%. In
contrast, line P{magu}103 yielded 14% increase in median lifespan in males but no
increase in females, while line P{magu}67-2 gave no increase in either sex. In a second
experiment, with the ubiquitous rtTA(3)E2 driver, the two magu cDNA lines tested
(P{magu}102 and P{magu}67-1) each gave increases in median lifespan in both males
and females, ranging from 2.6-21%. The specific chromosomal site of insertion of P
element constructs can affect expression levels in various tissues, and we hypothesize
that this may be why results varied for the five independent magu cDNA strains.
Nevertheless, two of the magu cDNA strains, P{magu}102 and P{magu}67-1, yielded
significant increases for both males and females in replicate experiments. Therefore, we
conclude that although lifespan effects varied considerably across strains and
experiments, ubiquitous over-expression of both hebe and magu can increase lifespan in
both male and female adult flies.

Over-expression of hebe and magu preferentially in adult motor-neurons and gut
increases female lifespan

35
We have recently described a system consisting of three transgenic constructs that
allows for tissue-specific, DOX-dependent transgene expression that can be modulated
over 3 orders of magnitude (Ford, Hoe et al. 2007). The D42 driver is a well-
characterized GAL4/UAS system driver that yields expression of the yeast GAL4
transcription factor throughout the developing nervous system and preferentially in
motor neuron tissue in adult flies (Parkes, Elia et al. 1998). The UAS-rtTAm2alt
insertion (Stebbins, Urlinger et al. 2001) is a “bridge construct” that is regulated by
GAL4 to yield expression of rtTA only in those same tissues. Finally, as discussed
above, the Tet-on promoter in PdL (or USC1.0 for the cDNA constructs) will be
activated by rtTA only the presence of DOX. Therefore the combination of these three
constructs yields DOX-inducible expression of transgenes in adult flies, preferentially in
motor neuron tissue (Ford, Hoe et al. 2007). By the similar mechanism, C204 driver
over-express the gene of interest in Drosophila. We examined the life span extension
effect using these two driver strains. (Figure 9A and 9B)

The control genotype for the motor neuron expression experiments contained the motor
neuron driver and the bridge construct, but no target construct, and in these flies there
was no significant change in lifespan in either males or females in replicate experiments
(Table 3). Two independent hebe cDNA lines were tested, P{hebe}DX2 and
P{hebe}AX, and in a first experiment these lines yielded increases in median lifespan in
females of 22% and 38%, respectively; no significant effect was obtained in males. Two
independent magu cDNA over-expression strains were also tested, P{magu}102 and

36
P{magu}67-1, and both lines yielded increases in median lifespan in females of 28%,
but neither showed a significant alteration in lifespan in males. Therefore in a first
experiment utilizing the D42 motor neuron driver, both hebe and magu over-expression
increased lifespan in female flies but not male flies. In contrast, in a replicate
experiment, both the hebe and magu cDNA over-expression constructs yielded increases
in median lifespan in both males and females, ranging from 9-37%. In summary, while
the lifespan increases varied considerably across strains and experiments, the conditional
over-expression of hebe and magu in adult motor neuron tissue consistently increased
lifespan in females, with less consistent increases observed in males.

Ubiquitous over-expression of hebe and magu in adult females modulates fecundity.

To confirm the effects of hebe and magu over-expression on adult female fecundity, the
original PdL mutations, P{PdL}hebe and P{PdL}magu, as well as two independent
cDNA expression lines for each gene, were ubiquitously over-expressed in adult
females, using varying amounts of DOX to vary the degree of over-expression. Adult
females were cultured on –DOX media and media supplemented with varying
concentrations of DOX, and female fecundity was assayed at two time periods: from day
10 to day 20 (time period 1), and from day 30 to day 40 (time period 2) (Figure 10). The
control genotype contained the rtTA(3)E2 driver and no target construct, and in these
control flies DOX had no significant effect on female fecundity at either time point
(Figure 10A). In contrast the P{PdL}magu insertion increased female fecundity at late

37
ages (Figure 10B), consistent with the screening procedure by which it was identified
(above). The magu cDNA over-expression line p{magu}67-1 also increased female
fecundity at late ages, but in this case there was an apparent trade-off, with reduced
female fecundity at the earlier time point (Figure 10D). Interestingly, the magu cDNA
line P{magu}102 caused dose-dependent decreases in female fecundity at both time
points (Figure 10C). Similar reductions were obtained with line p{magu}103, and these
reductions in fecundity had a significant maternal-effect lethal component, as evidenced
by the failure to hatch of a large fraction of laid eggs (data not shown). The reductions
in female fecundity observed with lines P{magu}102 and P{magu}103 may be due to
especially high and perhaps toxic levels of cDNA over-expression in adult females in
these lines; in contrast the P{PdL}magu and P{magu}67-1 lines that produced increased
female fecundity were associated with modest increases in magu expression (Figure 7B
and C, and data not shown). For the original P{PdL}hebe mutation, the change in late-
age female fecundity did not reach statistical significance in subsequent experiments. On
the other hand, the cDNA line P{hebe}DX2 did result in increased fecundity at late ages
(Figure 10C), and each hebe over-expression line was associated with decreased
fecundity at the earlier time point, demonstrating that hebe over-expression can
modulate female fecundity.

38
Figure 5: The sequence map for magu and hebe PdL mutant strains. PdL insertion is
labeled by an inverted triangle. The length of the chromosome is labeled by 5kb major
tick marks and 1kb minor tick marks. The genes on either strand of the chromosome are
labeled by wide gray arrows pointing 5’ to 3’. At the bottom of each diagram, each
transcript is labeled by long gray arrows, with blocks representing exons and lines
representing introns.
A: Sequence map of the magu PdL insertion.
B: Motif prediction in the Magu protein gene product.
C: Sequence map of the hebe PdL insertion.

39

40

Figure 6: The structure of USC1.0 with gene of interest cDNA insertion.
A: The target sequence of 2-3 transposase. The inverted repeat target sequences are
labeled by the triangles pointing outward. Different structural components are labeled by
boxes filled by different textures.
B: Magu and hebe cDNA sequence cloned into USC1.0 between PstI and EcoRI sites.
The 15-25 bp upstream of the ATG start codon were also cloned into USC1.0 to ensure
successful transcription.

41

Construct Line name Chromosome
magu 2
nd
PdL
hebe 2
nd
39 2
nd
40 3
rd

102 3
rd
103 2
nd
67-1 3
rd
P{magu}
67-2 3
rd
DX1 2
nd
DX2 2
nd
AX 3
rd

28-1 3
rd
28-2 3
rd
8 3
rd
P{hebe}
4 2
nd

Table1: The list of strains includes the positive PdL mutants generated by PdL
mutagenesis and the magu and hebe transgenic strains established by microinjection.

42
Figure 7: Northern blots confirming the expression of ether magu or hebe transcripts in
PdL mutant strains and transgenic lines. The tested line males were crossed to
rtTA(3)E2 virgin females. The progeny were fed with DOX for 10 days before assaying
expression. The loading control is Ribosome protein 49 (RP49). The transcripts of
interest are labeled.
A: P{PdL}magu PdL mutant; B: P{magu}103 magu transgenic line; C: P{magu}67-1
magu transgenic line; D: P{magu}102 magu transgenic line; E: P{magu}67-2 magu
transgenic line; F: P{PdL}hebe PdL mutant; G: P{hebe}DX2 hebe transgenic line; H:
P{hebe}DX1 hebe transgenic line.

43

44

45

Figure 8: Northern blot verifying the expression of magu transcripts in different tissues
of Or-R wild type female flies. The P{PdL}magu males were crossed to rtTA(3)E2
virgin females. The progeny were fed with DOX for 10 days before assaying. Ribosome
protein 49 (RP49) is used as loading control. The transcript of interest is labeled.

46
Figure 9A: Lifespan assay of transgenic strains established from magu and hebe cDNA
transgenes. The male flies from each test strain were crossed to either rtTA(3)E2
ubiquitous driver or D42 motor neuron driver female flies. The male and female progeny
were collected and treated with +/-DOX during aging. The x-axis represents fly age in
days. The y-axis represents the percent of the experimental group surviving to that age.
+DOX and –DOX treatments are labeled with empty diamond dots and solid square
dots, respectively.
A: W1118 strain with rtTA(3)E2 ubiquitous driver, male progeny lifespan;
B: W1118 strain with rtTA(3)E2 ubiquitous driver, female progeny lifespan,;
C: P{magu}67-1 magu transgenic strain with rtTA(3)E2 driver, male progeny lifespan;
D: P{magu}67-1 magu transgenic strain with rtTA(3)E2 driver, female progeny
lifespan;
E: P{magu}102 magu transgenic strain with rtTA(3)E2 driver, male progeny lifespan;
F: P{magu}102 magu transgenic strain with rtTA(3)E2 driver, female progeny lifespan;
G: P{hebe}DX-2 hebe transgenic strain with rtTA(3)E2 driver, male progeny lifespan;
H: P{hebe}DX-2 hebe transgenic strain with rtTA(3)E2 driver, female progeny lifespan;
I: W1118 strain, with D42 motor neuron driver, female progeny lifespan;
J: P{magu}102 magu transgenic strain, with D42 driver, female progeny lifespan.

47

48
Figure 9B: Lifespan assay of transgenic strains established from magu and hebe cDNA
transgenes. The male flies from each test strain were crossed to C204 gut driver female
flies. The male and female progeny were collected and treated with +/-DOX during
aging. The x-axis represents fly age in days. The y-axis represents the percent of the
experimental group surviving to that age. +DOX and –DOX treatments are labeled with
empty diamond dots and solid square dots, respectively.
A: W1118 strain with C204 driver, male progeny lifespan;
B: W1118 strain with C204driver, female progeny lifespan,;
C: P{magu}102 magu transgenic strain with C204 driver, male progeny lifespan;
D: P{magu}102 magu transgenic strain with C204 driver, female progeny lifespan;
E: P{magu}67-1 magu transgenic strain with C204 driver, male progeny lifespan;
F: P{magu}67-1 magu transgenic strain with C204 driver, female progeny lifespan;
G: P{hebe}DX-2 hebe transgenic strain with C204 driver, male progeny lifespan;
H: P{hebe}DX-2 hebe transgenic strain with C204 driver, female progeny lifespan;
I: P{hebe}AX hebe transgenic strain with C204 driver, male progeny lifespan;
J: P{hebe}AX hebe transgenic strain with C204 driver, female progeny lifespan;.

49

50

Genotype Treat Sex MD MA S.D. ΔMD ΔMA P
W1118 +DOX M 92 87.2 14.5 0 0.12% 0.448
W1118 -DOX M 92 87.0 14.9 N/A N/A N/A
W1118 +DOX F 76 80.1 14.2 0.00% 0.21% 0.672
W1118 -DOX F 76 79.9 14.3 N/A N/A N/A
OR-R +DOX M 92 85.7 16.8 2.22% -0.69% 0.678
OR-R -DOX M 90 86.3 13.8 N/A N/A N/A
OR-R +DOX F 76 78.2 13.4 0.00% -2.04% 0.977
OR-R -DOX F 76 79.9 12.3 N/A N/A N/A
P{PdL}magu +DOX M 94 88.1 15.8 9.30% 8.59% 4.50E-06
P{PdL}magu -DOX M 86 81.1 15.2 N/A N/A N/A
P{PdL}magu +DOX F 92 90. 7 8.7 2.22% 5.28% 4.85E-05
P{PdL}magu -DOX F 90 86.1 9.0 N/A N/A N/A
P{PdL}hebe +DOX M 92 88.8 15.6 31.43% 24.21% 9.10E-15
P{PdL}hebe -DOX M 70 71.5 18.5 N/A N/A N/A
P{PdL}hebe +DOX F 92 90.7 10.9 2.22% 11.25% 1.02E-07
P{PdL}hebe -DOX F 90 81.6 14.7 N/A N/A N/A
P{magu}102 +DOX M 98 96.0 17.7 8.89% 23.77% 1.11E-16
P{magu}102 -DOX M 90 77.5 22.8 N/A N/A N/A
P{magu}102 +DOX F 90 86.8 11.0 7.14% 9.34% 0.00176
P{magu}102 -DOX F 84 79.4 15.9 N/A N/A N/A
P{magu}103 +DOX M 96 88.2 18.1 14.29% 20.13% 5.47E-05
P{magu}103 -DOX M 84 73.4 26.9 N/A N/A N/A
P{magu}103 +DOX F 72 76.7 14.3 0.00% 4.66% 0.652
P{magu}103 -DOX F 72 73.36 19.9 N/A N/A N/A
P{magu}67-1 +DOX M 96 93.6 17.8 2.13% 11.64% 6.79E-07
P{magu}67-1 -DOX M 94 83.8 20.7 N/A N/A N/A
P{magu}67-1 +DOX F 90 86.9 13.1 18.42% 13.85% 8.91E-08
P{magu}67-1 -DOX F 76 76.3 15.4 N/A N/A N/A
P{magu}67-2 +DOX M 86 84.6 15.8 -4.44% -3.91% 0.439
P{magu}67-2 -DOX M 90 88.0 7.9 N/A N/A N/A
P{magu}67-2 +DOX F 90 81.3 23.4 0.00% 0.12% 0.688
P{magu}67-2 -DOX F 90 81.2 18.8 N/A N/A N/A
P{magu}39 +DOX M 86 85.0 20.1 7.50% -3.98% 0.762
P{magu}39 -DOX M 80 88.5 18.1 N/A N/A N/A
P{magu}39 +DOX F 94 88.7 16.7 4.44% 0.67% 0.0405
P{magu}39 -DOX F 90 88.1 11.9 N/A N/A N/A

Table 2: Lifespan assay on magu and hebe mutants and transgenic strains, using Or-R
and W1118 as control, crossed to the E2 driver; round 1 of 2. Abbreviations: M: male;
F: female; MD: Median; MA: Mean; S.D.: Standard Deviation; ΔMD: Percent change of
Median; ΔMA: Percent change of Mean; P: P-values obtained from a log-rank test.

51

Genotype Treat Sex MD MA S.D. ΔMD ΔMA P
P{PdL}hebe +DOX M 92 88.8 15.6 31.43% 24.21% 9.10E-15
P{PdL}hebe -DOX M 70 71.5 18.5 N/A N/A N/A
P{PdL}hebe +DOX F 92 90.7 10.9 2.22% 11.25% 1.02E-07
P{PdL}hebe -DOX F 90 81.6 14.7 N/A N/A N/A
P{hebe}DX1 +DOX M 90 83.5 21.5 7.14% 9.14% 0.00028
P{hebe}DX1 -DOX M 84 76.5 20.9 N/A N/A N/A
P{hebe}DX1 +DOX F 72 77.8 13.6 5.88% 9.16% 0.000506
P{hebe}DX1 -DOX F 68 71.2 14.8 N/A N/A N/A
P{hebe}DX2 +DOX M 96 85.5 23.4 26.32% 15.62% 2.34E-10
P{hebe}DX2 -DOX M 76 74.0 21.3 N/A N/A N/A
P{hebe}DX2 +DOX F 84 81.3 14.1 20.00% 15.65% 1.10E-09
P{hebe}DX2 -DOX F 70 70.3 13.5 N/A N/A N/A
P{hebe}AX +DOX M 92 88.6 15.3 21.05% 12.60% 3.24E-08
P{hebe}AX -DOX M 76 78.6 15.1 N/A N/A N/A
P{hebe}AX +DOX F 86 83.1 14.6 22.86% 11.80% 6.16E-08
P{hebe}AX -DOX F 70 74.3 14.7 N/A N/A N/A

Table 2, continued: Lifespan assay on magu and hebe mutants and transgenic strains,
using Or-R and W1118 as control, crossed to E2 driver; round 1 of 2. Abbreviations: M:
male; F: female; MD: Median; MA: Mean; S.D.: Standard Deviation; ΔMD: Percent
change of Median; ΔMA: Percent change of Mean; P: P-values obtained by a log-rank
test.

52

Genotype Treat Sex MD MA S.D. ΔMD ΔMA P
W1118 +DOX M 72 68.0 14.2 2.86% 1.61% 0.76
W1118 -DOX M 70 66.9 15.3 N/A N/A N/A
W1118 +DOX F 72 68.2 14.1 2.86% -0.52% 0.384
W1118 -DOX F 70 68.6 13.7 N/A N/A N/A
P{magu}102 +DOX M 82 75.3 18.1 20.59% 20.72% 1.06E-12
P{magu}102 -DOX M 68 62.4 20.0 N/A N/A N/A
P{magu}102 +DOX F 80 77.0 14.2 2.56% 2.63% 0.000502
P{magu}102 -DOX F 78 75.0 10.7 N/A N/A N/A
P{magu}67-1 +DOX M 72 72.7 12.4 9.09% 18.39% 1.73E-07
P{magu}67-1 -DOX M 66 61.4 17.3 N/A N/A N/A
P{magu}67-1 +DOX F 76 73.1 14.3 8.57% 4.60% 3.64E-05
P{magu}67-1 -DOX F 70 69.8 9.8 N/A N/A N/A
P{hebe}DX-2 +DOX M 69 66.8 18.6 11.29% 9.54% 2.39E-05
P{hebe}DX-2 -DOX M 62 61.0 14.0 N/A N/A N/A
P{hebe}DX-2 +DOX F 78 75.1 14.8 14.71% 14.07% 4.50E-13
P{hebe}DX-2 -DOX F 68 65.8 10.1 N/A N/A N/A
P{hebe}AX +DOX M 70 65.7 19.7 12.90% 7.04% 2.09E-05
P{hebe}AX -DOX M 62 61.4 13.0 N/A N/A N/A
P{hebe}AX +DOX F 74 72.2 13.0 8.82% 8.57% 4.70E-07
P{hebe}AX -DOX F 68 66.5 11.1 N/A N/A N/A

Table 3: Lifespan Assay on magu and hebe transgenic lines, using W1118 as control,
crossed to E2 driver; round 2 of 2. Abbreviations: M: male; F: female; MD: Median;
MA: Mean; S.D.: Standard Deviation; ΔMD: Percent change of Median; ΔMA: Percent
change of Mean; P: P-values obtained by a log-rank test.

53

Genotype Treat Sex MD MA S.D. ΔMD ΔMA P
W1118 +DOX M 38 35.8 11.1 11.76% 0.00% 0.809
W1118 -DOX M 34 35.8 11.1 N/A N/A N/A
W1118 +DOX F 34 34.1 6.1 6.25% 3.65% 0.315
W1118 -DOX F 32 32.9 7.3 N/A N/A N/A
P{magu}102 +DOX M 38 37.2 14.5 11.76% -0.53% 0.884
P{magu}102 -DOX M 34 37.4 13 N/A N/A N/A
P{magu}102 +DOX F 46 46.7 14.7 27.78% 36.55% 0.0009
P{magu}102 -DOX F 36 34.2 13.2 N/A N/A N/A
P{magu}67-1 +DOX M 44 41.9 15.7 0.00% -3.68% 0.547
P{magu}67-1 -DOX M 44 43.5 11.1 N/A N/A N/A
P{magu}67-1 +DOX F 46 45.3 13.5 27.78% 23.43% 2.14E-06
P{magu}67-1 -DOX F 36 36.7 13.7 N/A N/A N/A
P{hebe}DX2 +DOX M 38 39.4 12.9 11.76% 10.36% 0.127
P{hebe}DX2 -DOX M 34 35.7 13.9 N/A N/A N/A
P{hebe}DX2 +DOX F 44 42.3 13.9 22.22% 17.83% 0.00121
P{hebe}DX2 -DOX F 36 35.9 10.9 N/A N/A N/A
P{hebe}AX +DOX M 28 32.7 14.1 -22.2% -8.91% 0.187
P{hebe}AX -DOX M 36 35.9 15 N/A N/A N/A
P{hebe}AX +DOX F 44 39.6 16 37.50% 24.53% 0.00887
P{hebe}AX -DOX F 32 31.8 14.4 N/A N/A N/A

Table 4: Lifespan Assay on magu and hebe transgenic lines using W1118 as control,
crossed to D42 motor neuron driver; round 1 of 2. Abbreviations: M: male; F: female;
MD: Median; MA: Mean; S.D.: Standard Deviation; ΔMD: Percent change of Median;
ΔMA: Percent change of Mean; P: P-values obtained by a log-rank test.

54

Genotype Treat Sex MD MA S.D. ΔMD ΔMA P
W1118 +DOX M 50 48.4 10.5 0 -1% 0.948
W1118 -DOX M 50 48.9 8.8 N/A N/A N/A
W1118 +DOX F 50 51.7 7.6 -3.8% 5.5% 0.583
W1118 -DOX F 52 49.0 11.5 N/A N/A N/A
P{magu}102 +DOX M 52 45.9 17.6 36.8% 32.7% 1.34E-07
P{magu}102 -DOX M 38 34.6 16.4 N/A N/A N/A
P{magu}102 +DOX F 48 45.3 16.3 9.1% 13.0% 4.29E-07
P{magu}102 -DOX F 44 40.1 11.2 N/A N/A N/A
P{magu}67-1 +DOX M 50 44.8 18.4 31.6% 29.5% 7.16E-08
P{magu}67-1 -DOX M 38 34.6 17.3 N/A N/A N/A
P{magu}67-1 +DOX F 46 41.3 15.6 21.0% 15.0% 5.83E-07
P{magu}67-1 -DOX F 38 35.9 12.0 N/A N/A N/A
P{hebe}DX2 +DOX M 50 48.4 12.4 19.0% 17.8% 2.46E-09
P{hebe}DX2 -DOX M 42 41.1 10.0 N/A N/A N/A
P{hebe}DX2 +DOX F 54 48..4 16.6 17.4% 15.2% 1.75E-05
P{hebe}DX2 -DOX F 46 42 16.2 N/A N/A N/A
P{hebe}AX +DOX M 46 45.8 12.0 9.5% 13.6% 2.45E-06
P{hebe}AX -DOX M 42 40.3 9.8 N/A N/A N/A
P{hebe}AX +DOX F 52 44.9 17.6 18.2% 13.7% 0.000207
P{hebe}AX -DOX F 44 39.5 16.5 N/A N/A N/A

Table 5: Lifespan assay on magu and hebe transgenic lines using W1118 as control,
crossed to D42 motor neuron driver; round 2 of 2. Abbreviations: M: male; F: female;
MD: Median; MA: Mean; S.D.: Standard Deviation; ΔMD: Percent change of Median;
ΔMA: Percent change of Mean; P: P-values obtained by a log-rank test.

55

Genotype Treat Sex MD MA S.D. ΔMD ΔMA P
W1118 +DOX M 60 59.9 11.1 0.00% -1.64% 0.115
W1118 -DOX M 60 60.9 14
W1118 +DOX F 60 58.3 12.3 3.45% 4.86% 0.967
W1118 -DOX F 58 55.6 15.5
P{hebe}AX +DOX M 68 64 15.5 17.24% 18.30% 1.46E-06
P{hebe}AX -DOX M 58 54.1 16.2
P{hebe}AX +DOX F 68 64.2 17.8 21.43% 28.40% 5.45E-09
P{hebe}AX -DOX F 56 50 19.7
P{hebe}DX2 +DOX M 68 64.1 15.6 13.33% 11.67% 0.00204
P{hebe}DX2 -DOX M 60 57.4 15.6
P{hebe}DX2 +DOX F 68 63.3 17.1 17.24% 19.21% 1.45E-07
P{hebe}DX2 -DOX F 58 53.1 17.1
P{magu}102 +DOX M 70 65.9 16.6 12.90% 10.02% 0.000292
P{magu}102 -DOX M 62 59.9 13
P{magu}102 +DOX F 62 59.1 20.4 -3.13% 8.64% 0.00661
P{magu}102 -DOX F 64 54.4 18.1
P{magu}67-1 +DOX M 68 68.4 12.5 0.00% 3.64% 0.0331
P{magu}67-1 -DOX M 68 66 11.7
P{magu}67-1 +DOX F 70 70 15.2 9.38% 17.45% 7.20E-08
P{magu}67-1 -DOX F 64 59.6 17

Table 6: Lifespan assay on magu and hebe transgenic lines using W1118 as control,
crossed to C204 gut driver; round 1 of 1. Abbreviations: M: male; F: female; MD:
Median; MA: Mean; S.D.: Standard Deviation; ΔMD: Percent change of Median; ΔMA:
Percent change of Mean; P: P-values obtained by a log-rank test.

56
Figure 10: The DOX-titration fecundity assay. Male flies from each strain were crossed
to rtTA(3)E2 virgin females. The progeny females were kept with fresh Or-R males for
continuous mating. The experiment was performed as described in the methods section.
The early and late time periods are plotted on the X-axis. Time period 1 contains ages
10-20 days. Time period 2 contains ages 30-40 days. Time period 3 contains ages 50-60
days (if there were still live females at that age.). Different DOX titration groups are
labeled by grayscale. The Y-axis shows progeny per female per day. Error bars represent
standard deviation. A t-test comparing the early and late time period fecundity was
performed for each strain. A: W1118; B: P{PdL}magu PdL mutant; C: P{magu}102
magu transgenic line; D: P{magu}67-1 magu transgenic line; E: P{PdL}hebe PdL
mutant; F: P{hebe}DX2 hebe transgenic line; G: P{hebe}AX hebe transgenic line;

57

58
During the screening procedure, my colleague Ji-Ping Yuan performed another
screening assay with a slightly different approach. In this screen, the mutant female flies
were treated with +DOX from childhood. Their fecundity and lifespan was compared to
that of Or-R wild type flies. After being balanced to homozygosity, the strains that were
positively characterized in the screening round were assayed for fecundity and longevity
phenotypes, again on separate +/-DOX treatment groups. One PdL mutant was thus
generated with increased fecundity and lifespan upon DOX induction. The PdL insertion
of this mutant was mapped. The insertion was 14,026bp 5’ upstream of gene bantam
(See figure 11 for the PdL insertion map of the mutant.). The fecundity of the PdL
mutant was assayed throughout of the fecund life of the tested female (Figure 12: A, C.).
During the fecundity assay, it became evident that the PdL mutant showed significantly
increased fecundity upon the induction of DOX. The lifespan extension effect of the PdL
mutant was assayed twice. In these two rounds of lifespan assays, only female progeny
were examined. The first round of the lifespan assay was performed on our previous
food recipe; the second round was performed on both our previous recipe and our new
recipe (See table 10 for the comparison between the old and new recipes). During both
rounds of the assays, the female progeny showed increased lifespan. (See figures 12, 13
for lifespan assay curves.) However, the lifespan assay was repeated for a third round
with the same assay protocol on the new food. In this round, the lifespan extension effect
was not observed (Figure 14). The expression of the gene bantam in these PdL mutant
progeny was checked by Northern blotting with the methods described above. However,
I had some technical difficulty with the blots. The Northern blotting was unsuccessful;

59
therefore it did not show the over-expression of gene bantam upon DOX induction (Data
not shown).

Figure 11: The PdL insertion sequence map for screen 2 Ban PdL mutant strain. PdL
insertion is labeled by the inverted triangle. The length of the chromosome is labeled by
5kb major tick marks and 1kb minor tick marks. The genes on either strand of the
chromosome are labeled by wide gray arrows pointing 5’ to 3’.

Figure 12: The parallel fecundity and lifespan test result curves for screen 2 Ban PdL
mutant strain. The fecundity and lifespan assays were performed as previously
described. The fecundity and lifespan curves are labeled as above. Only female progeny
were assayed for lifespan in this round. A: Or-R fecundity curve; B: Or-R lifespan
curve; C: Ban PdL mutant fecundity curve; D: Ban PdL mutant lifespan curve

60

Figure 13: The lifespan assay of Ban PdL mutant performed on two different food
recipes. The lifespan assay was performed as described above. The lifespan curves are
labeled as above. Different food recipes with different +/-DOX group are labeled by
different colors and dots. For food recipe comparison, see table 10. New food recipe
with +DOX is labeled red. New food recipe with –DOX is labeled blue. Old food recipe
with +DOX is labeled purple. Old food recipe with –DOX is labeled green. Only female
progeny were assayed for lifespan in this round. A: Or-R lifespan curve; B: Ban PdL
mutant lifespan curve

Figure 14: The repeat round of the lifespan assay of Ban PdL mutant. The lifespan assay
was performed as described above. The lifespan curves are labeled as above. A: W1118
male; B: P{PdL}ban PdL mutant male; C: W1118 female; D: P{PdL}ban PdL mutant
female

61

genotype Treat MA S.D. ΔMA P
Or-R +DOX 1.6 1.1 -10.1% 0.731
Or-R -DOX 1.7 1.3 N/A N/A
P{PdL}bam +DOX 1.5 1.2 273% 0.006
P{PdL}bam -DOX 0.4 1.9 N/A N/A

Table 7: Female fecundity parallel assay on original bam PdL mutant. Abbreviations:
MA: mean of egg production number per female per day; S.D.: standard deviation of the
mean egg production number per female per day within the same cohort; ΔMA: change
of mean egg production number when comparing the +DOX and –DOX cohorts; P: P-
value given by the unpaired, two-sided t-test

Genotype Treat Sex MD MA S.D. ΔMD ΔMA P
Or-R +DOX F 78 67.2 14.7 0 -7.81% 0.171
Or-R -DOX F 78 72.9 13.3 N/A N/A N/A
P{PdL}bam +DOX F 68 64.2 17.8 21.43% 28.40% 0.00594
P{PdL}bam -DOX F 56 50 19.7 N/A N/A N/A

Table 8: Lifespan assay on bam mutant, using Or-R as control, crossed to E2 driver;
round 1 of 2. Abbreviations: M: male; F: female; MD: Median; MA: Mean; S.D.:
Standard Deviation; ΔMD: Percent change of Median; ΔMA: Percent change of Mean;
P: P-value given by the log-rank test

62

Genotype Treat MD MA S.D. ΔMD ΔMA P
Or-R +DOX(NF) 40 38.4 9.2 0.00% -0.52% 0.868
Or-R - DOX(NF) 40 38.6 8.0 N/A N/A N/A
Or-R +DOX 43 37.8 4.1 7.5% 9.45% 1.16E-03
Or-R -DOX 40 39.6 4.7 N/A N/A N/A
P{PdL}bam +DOX(NF) 90 87.3 6.1 12.5% 14.50% 7.24E-08
P{PdL}bam - DOX(NF) 80 76.3 4.7 N/A N/A N/A
P{PdL}bam +DOX 82 77.1 5.3 10.81% 12.23% 2.33E-09
P{PdL}bam -DOX 74 68.7 4.9 N/A N/A N/A

Table 9: Lifespan assay on bam mutant, using Or-R as control, crossed to E2 driver;
round 2 of 2. In this round, two different food recipes were used. See table 10 for food
recipe comparison. Abbreviations: NF: New Food; M: male; F: female; MD: Median;
MA: Mean; S.D.: Standard Deviation; ΔMD: Percent change of Median; ΔMA: Percent
change of Mean; P: P-value given by the log-rank test

Genotype Treat Sex MD MA S.D. ΔMD ΔMA P
W1118 +DOX M 62 61.8 15.6 3.33% 4.92% 0.0718
W1118 -DOX M 60 58.9 13.1 N/A N/A N/A
W1118 +DOX F 58 57.8 12.6 0.00% 2.12% 0.095
W1118 -DOX F 58 56.6 10.1 N/A N/A N/A
P{PdL}bam +DOX M 70 68.1 11.0 12.9% 11.82% 7.04E-08
P{PdL}bam -DOX M 62 60.9 8.5 N/A N/A N/A
P{PdL}bam +DOX F 70 67.7 9.0 6.06% 4.31% 0.2
P{PdL}bam -DOX F 66 64.9 11.0 N/A N/A N/A

Table 10: Lifespan assay retest on bam mutant, using W1118 as control, crossed to E2
driver. This time, only the new food recipe was used (identical to the new food recipe in
the last round lifespan assay). Abbreviations: M: male; F: female; MD: Median; MA:
Mean; S.D.: Standard Deviation; ΔMD: Percent change of Median; ΔMA: Percent
change of Mean; P: P-value given by the log-rank test

63

For One Liter Old Food New Food
Water (L) 1 1
Sucrose (g) 0 0
Dextrose (g) 0 105
Molasses (ml) 100 0
Agar (g) 9 8
Yeast (g) 41 26
Cornmeal (g) 100 50
Tegosept (g) 2.5 1.7
95% Ethanol (ml) 22.5 8.6
Propionic Acid (ml) 8 1.9
phosphoric acid 0 0

Table 11: This table compares the difference between old and new food recipes.

Discussion
Fly female fecundity declines during the majority of the fly lifespan, as manifested by
progressively decreasing egg production as the flies age. The fecundity of 80-day old
females is on average less than 10% of 10-day olds. There must be potential correlations
between the age and the fecundity of those females at the genetic level. It is also
plausible that when fly lifespan is extended by genotypic change, fecundity would also
be affected positively. The correlations between aging and fecundity can be partially
explained by the stem cell decay theory (Kirilly and Xie 2007; Pan, Chen et al. 2007;
Song, Call et al. 2007; Lopez-Onieva, Fernandez-Minan et al. 2008). In old animals, the
germline stem cells stop division and become differentiated, or are even lost altogether.
This process greatly reduces the potential for oogenesis in female flies. One of the

64
original questions that I was trying to answer when I embarked on this project was, can
we determine which aspects of the aging process affect female fecundity changes? By
setting up specific criteria for genetic screening, one can score virtually any phenotype
that one is interested in. Therefore, two PdL mutants were selected not only for their
fecundity increase phenotype, but also for their lifespan extension phenotype. The
lifespan and fecundity assays that followed verified that, as was observed in the
screening rounds, lifespan extension is more straightforward to attain, while the
fecundity assay required DOX titrations to adjust the gene over-expression level.

The initial screening cross design was a typical example of establishing a conditional
over-expression mutant library that could handle any number of mutant strains. Given
enough time, it is able to score any phenotype that interests the researcher, and can cover
the entire 180 Mb Drosophila genome containing 14,000 genes (Celniker and Rubin
2003). Our screening took 3 full-time and 3 part-time researchers 4 months. In the
process, we harvested roughly 20,000 independent random mutants from 9 rounds of
crosses. By statistical estimates, this design still had not covered the whole genome.

The initial screen scored for mutants that showed fecundity increase during fly aging.
After the establishment of transgenic lines, both the original PdL mutant strains and
transgenic lines were re-tested for the lifespan extension phenotype. There was no
significant difference between the +DOX group and the –DOX group in either sex in the
Or-R wild type and W1118 control strains, as is expected since these lines contain no

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rtTA inducible promoters. Lifespan increased significantly in both PdL mutant stains as
well as in the transgenic strains (Figure 9). This result is consistent with the data from
the preceding screening round. Lifespan also increased significantly in the transgenic
lines and in both sexes, and more so than in the PdL lines. This additional increase is not
unexpected because the transgenic lines bear an extra copy of the transgene, which can
result in higher over-expression when the flies are fed with DOX. The positive lifespan
change was also confirmed by another cross with different driver lines. The initial
screening and lifespan retests described above were performed by using rtTA(3)E2
driver strain, which expresses the transgene ubiquitously. The tissue specific driver line
D42 expresses the gene of interest in motor neurons. From the lifespan curves, the
lifespan extension effect on the females is obvious relative to the W1118 control strain.

The fecundity assay results were less straightforward, with initial tests of the transgenic
strains showing decreased fecundity in late life, opposite to the PdL results. Our
hypothesis was that extreme over-expression of the transgene has a deleterious effect in
this phenotype, with just moderate over-expression resulting in a positive effect on
fecundity. Following reports that decreased DOX concentration in the fly food leads to a
decrease in the level of over-expression of the transgene (Ford, Hoe et al. 2007), we
tested this hypothesis by using DOX at 1/5 and 1/25 of the original concentration.

In these DOX titration fecundity assays (see figure 10), DOX concentration had no
effect on the late life fecundity of control W1118 flies. This consistency allows the fair

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comparison of fecundity measurements in either the PdL mutants or the transgenic
strains. In the magu PdL mutant, early-life fecundity was increased by +DOX. The
increase was not as strong when the DOX concentration was reduced, and almost
disappeared at the lowest DOX concentration. In late life, this trend is even more
noticeable. The original DOX levels triggered a significant fecundity increase relative to
the –DOX group, and this increase was reduced by lower DOX concentrations.
However, even in the lowest DOX concentration group, fecundity was still higher than
in the –DOX group. When looking at P{magu}102, one of the magu transgenic strains,
the highest DOX concentration resulted in the lowest fecundity, and the fecundity
increased as the DOX concentration was lowered, in both early and late life. This
phenomenon is consistent with our hypothesis, and it is confirmed in another magu
transgenic line, P{magu}67-1 (See figure 10D.). When looking at the hebe PdL mutant
and transgenic lines, it is clear that in late life, fecundity can be increased by over-
expressing the hebe gene. In the hebe PdL mutant, the fecundity increase was seen on
the lowest DOX concentration group during both early and late life. This result implies
that in order to increase fecundity, there is an optimal expression range that gives the
largest fecundity benefit. Expression levels outside of this range will instead be
detrimental to fecundity. This hypothesis is also confirmed by two hebe transgenic lines.
Their fecundity was greatly increased by +DOX over-expression, but lowering the DOX
concentration to 1/5 yielded an even larger fecundity benefit.

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Although both novel genes, magu and hebe, showed consistent lifespan extension and
fecundity increase, not too much is known about their molecular function. The magu
gene protein product has been reported to interact with Slam (Giot, Bader et al. 2003).
This interaction was confirmed by a high-throughput yeast two hybrid project. The
interaction has not been confirmed to occur in vivo. However, Slam regulates polarized
membrane growth in oogenesis (Lecuit, Samanta et al. 2002), which might implicate
magu itself in oogenesis. Endogenous magu expression also correlates with the Dorsal
and Toll genes, both of which are regulators of embryo axis formation during oogenesis
(Stathopoulos, Van Drenth et al. 2002). These findings suggest that magu could indeed
play a role in oogenesis, consistent with its role regulating fecundity demonstrated in our
experiments. Magu might also be involved in spermatogenesis, as its transcript has been
reported to be enriched more than 20-fold in the germline stem cells at the very tip of the
testes (Terry, Tulina et al. 2006). These findings suggest that some genes, including
magu, could play highly related roles in both spermatogenesis and oogenesis. The
molecular functions of magu homologs also offer some clues. The Magu ortholog in rat
is known as SMOC2, and it is highly expressed in the early embryo and in the ovary of
mice (Vannahme, Gosling et al. 2003; Mager, Schultz et al. 2006). The human Magu
ortholog is called Smap2, and in humans the expression level is highest in the aorta. Its
expression is upregulated during neointima formation, which suggests a role in
angiogenesis (Nishimoto, Hamajima et al. 2002; Rocnik, Liu et al. 2006).

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Unfortunately, hebe is even less well documented than magu. A search for protein
motifs in the hebe coding region surprisingly yielded no results (Kanehisa, Goto et al.
2002; Vannahme, Gosling et al. 2003; de Castro, Sigrist et al. 2006; Finn, Mistry et al.
2006; Rocnik, Liu et al. 2006; Mulder, Apweiler et al. 2007). The only previous report
relating to hebe function showed that a P-element-induced hebe (CG1623) mutation
resulted in a 62% decrease in the male aggression behavior index (Edwards, Rollmann et
al. 2006). Whether or not male aggression behavior is related to our hebe lifespan
extension and fecundity increase effect still deserves further investigation.

The PdL mutant from another screen also showed lifespan increase on two different food
recipes. (See table 10 for food recipe comparison) The mapping of the PdL insertion
suggests that the insertion occurred about 14 kb upstream of the miRNA gene bantam
(ban). Two early research articles suggested that ban is involved in the regulation of
Drosophila cell growth (Hipfner, Weigmann et al. 2002; Brennecke, Hipfner et al.
2003). Ectopic expression of ban microRNA promotes cell proliferation and inhibits
apoptosis (Brennecke, Hipfner et al. 2003). A recent article also suggested an anti-
apoptosis role for ban: ionizing radiation induces expression of ban, which in turn
represses hid to limit radiation-induced apoptosis pathways (Jaklevic, Uyetake et al.
2008). It will be interesting to see whether bantam over-expression was indeed behind
the lifespan extension effect of this PdL mutant strain.

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Conclusion
Our findings on the two novel genes provide further evidence that the aging process can
be altered by the expression of one or two key players. Magu and hebe PdL mutants
from our screen yielded significant lifespan increase when expressed either ubiquitously
or specificifically in motor neuron tissue. This lifespan increase was consistent across
our 3 rounds of lifespan assay tests. It was also confirmed by the transgenic lines for
both novel genes.

Gene magu shared Kazal type serine protease inhibitor and SPARC extracellular Ca
2+
binding domains with its homologs. Previous reports of these homologs suggest
potential roles for magu in oogenesis, embryo development, spermatogenesis, and
angiogenesis. For hebe, the lack of conserved domains in this protein prevented us from
predicting potential functions other than the lifespan extension and fecundity regulation
effects found in our experiments.

Both the original PdL mutants and the corresponding transgenic strains showed lifespan
extension effects, and the fecundity regulation effect was also verified by our titration
DOX fecundity assays. By lowering the DOX concentration, the magu transgene
expression level was brought down to an ideal level. The transgenic lines showed a
beneficial effect in female fecundity. These findings suggest that there is an optimal
range of expression level for these genes to show their largest beneficial effects. Female
oogenesis is an extremely complicated process that involves stem cell maintenance and

70
decay, niche cell aging, sex peptide receptors, and yolk protein production, among
others. For this highly regulated and sophisticated machinery, one should not be
surprised to see that pouring too much or too little lubricant in the mechanism of a Swiss
watch could both be detrimental to the overall efficiency.

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Chapter 3
Anti-apoptotic gene dIAP2 lifespan assays

Abstract
Anti-apoptosis pathways are among the most important regulation mechanisms for
longevity research in various model organisms. The role of anti-apoptotic genes in
Drosophila aging is still under extensive research. The Tower lab stock contains a PdL
mutant strain that able to over express the Drosophila IAP2 (dIAP2) gene. I aimed to
test whether the over expression of dIAP2 could bring a longevity benefit to the animal.
After a few test rounds of lifespan assays on the dIAP2 over-expressing flies, I found
that dIAP2 overexpression did not result in lifespan extension. Differences in the
expression level of the gene between the first and second rounds could account for our
failure to observe an effect. However, the third-round lifespan assay suggested that
dIAP2 could be beneficial to adult fly longevity, if the gene is over-expressed only in
childhood; nevertheless, a few more rounds of the assay should be performed to confirm
such an extension effect. The potential role of dIAP2 in Drosophila longevity
mechanism largely remains to be determined.

Introduction
The protein products of Drosophila apoptosis-inducing genes such as Reaper, HID and
GRIM can physically interact with members of the inhibitors of apoptosis (IAP) protein
family (Vucic, Kaiser et al. 1998). Through these interactions, IAPs block apoptosis.

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Knocking out dIAP1 function results in early embryonic cell death and caspase activity
(Hawkins, Wang et al. 1999; Wang, Hawkins et al. 1999). It was also found that dIAP1
is capable of suppressing cell dealth caused by Drosophila caspase drICE activity
(Kaiser, Vucic et al. 1998). The above discoveries suggest that the Drosophila IAP, like
its human counterpart, family may play important roles in apoptosis and aging
processes.

During a pilot screening for extended lifespan strains in Tower lab fly stocks, my
colleague Gary Landis found that a candidate PdL insertion strain exhibits an unusually
long lifespan. (Figure 15) When I took over this research project, I mapped the location
of the PdL insertion. The sequencing data indicates that the insertion is located 5’
upstream of Drosophila IAP2 gene, a member of Drosophila IAP family. By crossing
this PdL strain with either ubiquitous or tissue-specific driver strains, I was able to over-
express the dIAP2 gene. I hypothesized that when overexpressed, dIAP2 may extend
Drosophila lifespan by inhibiting apoptosis pathways during aging.

73

Figure 15: The original dIAP2 PdL mutant lifespan assay. This assay only compared the
+DOX lifespan of Or-R and dIAP2 PdL mutant with rtTA(3)E2 cross. Only males were
assayed in this round. The lifespan assay was performed as described above. The
lifespan curves are labeled as above.

Materials and Methods
Drosophila strains, culture conditions, and lifespan assays have been described
previously (Landis, Bhole et al. 2003; Landis, Abdueva et al. 2004; Sun, Molitor et al.
2004; Ford, Hoe et al. 2007). The Or-R wild type and w1118 strains were used as
control lines (Lott, Kreitman et al. 2007). The PdL-dIAP2 strain was used as the
experimental line. Multiple driver lines were used (Ford, Hoe et al. 2007), including the
Dy42-901 motor neuron driver, Elav-GAL4-901 muscle driver, Actin-rtTA965
ubiquitous driver, Actin-GAL4-901 ubiquitous driver, Tubulin-GAL4-901 ubiquitous
driver, and Actin-rtTA(3)E2 ubiquitous driver. PdL insertion mapping was performed as
previously described (Landis, Bhole et al. 2003).

Results

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In the first round lifespan assay (Figure 16), only male progeny were used. It can be
noticed that the progeny flies from almost all the drivers that are crossed to PdL-dIAP2
strain showed significantly extended lifespan. Among them, the progeny from the Actin-
GAL4-901 driver cross, which expresses dIAP2 ubiquitously, showed the least lifespan
extension. The progeny from Tubulin-GAL4-901 showed the largest lifespan extension.
Tubulin is also a ubiquitous driver strain. The other tissues-specific drivers also showed
moderate lifespan extension.

In the second round lifespan assay (Figure 17), both male and female flies were used and
assayed separately. In this experiment, in the male progeny, lifespan extension can still
be observed in the Dy42 cross, but the extension percentage was much less than in the
previous round. The extension almost completely disappeared in the Elav-GAL4-901,
Actin-rtTA965, and Actin-GAL4-901 crosses. The most dramatic change occurred in the
Tubulin-GAL4-901 cross. In the previous round, this cross gave the largest lifespan
extension. In this round, however, the +DOX group lived even less than the –DOX
group. Interestingly, in the female group of the second round, Tubulin-GAL4-901
became the only group that did give lifespan extension. All other groups did not result in
any lifespan extension at all. Therefore, the preliminary conclusion from this round is
completely different from the first round. This round also lacks Or-R or W1118 as
control lines.

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The third round lifespan assay was a novel approach (Figure 18), during which +DOX
and –DOX food was provided only during larval development. When the flies grew into
adults, they were only provided regular food without any DOX added, regardless of
group of origin. Also, in this round, only one driver strain was used, the rtTA(3)E2
ubiquitous driver. The male and female adults were again both assayed for lifespan
extension separately. This round included Or-R wild type and W1118 as control strains.
Interestingly, in both male and female group, Or-R and W1118 showed decreased
lifespan, although the effect was less severe in males than in females. In both male and
female groups, dIAP2 and rtTA(3)E2 cross progeny flies showed lifespan increase.
Females demonstrated a larger increase than males.

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Figure 16: The first round of the dIAP2 lifespan assay. The males of dIAP2 PdL mutant
were crossed with each of five different driver strain virgin females. The male progeny
were assayed for lifespan. Only males were assayed in this round. The lifespan curves
were labeled in the same way as above lifespan assays. A: Dy42 driver; B: Elav driver;
C: Actin-rtTA965 driver; D: Actin-GAL4-901 driver; E: Tubulin driver;

77

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Figure 17: The second round of dIAP2 lifespan assay. The dIAP2 PdL mutant strain was
crossed with each different driver strain virgin females. The progeny were assayed for
lifespan. The lifespan curves were labeled in the same way as above lifespan assays. A:
Dy42 driver, male; B: Elav driver, male; C: Actin-rtTA965 driver, male; D: Actin-
GAL4-901 driver, male; E: Tubulin driver, male; F: Dy42 driver, female; G: Elav driver,
female; H: Actin-rtTA965 driver, female; I: Actin-GAL4-901 driver, female; J: Tubulin
driver, female

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80

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Figure 18: The third round of the dIAP2 lifespan assay. Males of each strain were
crossed with rtTA(3)E2 driver virgin females. Both sexes of the progeny were assayed
for lifespan. The lifespan curves were labeled in the same way as above lifespan assays.
A: Or-R cross, male; B: Or-R cross, female; C: W1118 cross, male; D: W1118 cross,
female; E: dIAP2 cross, male; F: dIAP2 cross, female

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83

Discussion
The first two rounds of experiments showed somewhat contradicting results. In the first
round, although only male flies were assayed for lifespan, almost all the crosses
suggested lifespan extension. On the other hand, in the second round lifespan assays, the
male group lifespan assay was essentially the repeat of the first round, but all the
lifespan extension effects were diminished except the Dy42 motor neuron driver cross.
The best lifespan extension in the first round, given by the Tubulin driver, turned into a
negative effect in the second round. In the female group, lifespan extension was only
observed in Tubulin driver cross, but not any other group. These contradicting results
were especially difficult to analyze because of the lack of wild type control groups. But
by taking a closer look at the comparison between the two rounds of experiments, some
general trends can still be elucidated. The lifespan extension effects in the second round
were generally diminished relative to the first round. The extension from Dy42 in the
second round was obviously less than in the first round. Rather than the positive effect
that was shown in the first round, the Tubulin cross resulted in a negative effect in the
second round. Although we cannot be certain, the overall decaying trend could be due to
a decreased expression of rtTA transcription activator across all the driver lines. Because
DOX is a light-sensitive reagent, leaving the DOX stock solution in room temperature or
exposing it to strong sunlight for an extended period of time could result in the
degradation of the reagent. A degraded DOX stock solution could lower the efficiency

84
of the rtTA transcription activation machinery, reducing trans-activation of the transgene
and thereby reducing the lifespan extension effects that were observed in the first round.

The design of the third round experiment was a completely novel approach. By adding
DOX during the larval development stage, the experiment took full advantage of the
Tet-on system, which is able to express the gene of interest at any desired time point.
The expression of dIAP2 in the larvae could provide an anti-apoptotic effect and in turn
be beneficial to Drosophila longevity even if the overexpression is not continued into
adulthood. This hypothesis was confirmed by the increased lifespan of both male and
female groups in this round. The decreased lifespan effect in both male and female
groups and in both control strains (Or-R wild type and W1118) may suggest a
deleterious effect of DOX on the developing larvae. Although there are no previous
reports of such an effect (Bieschke, Wheeler et al. 1998; Landis, Bhole et al. 2001; Ford,
Hoe et al. 2007), the significantly decreased lifespan of those adults could be concerning
to Drosophila researchers looking to apply DOX on developing larvae for longevity
research.

Conclusion
The first two rounds of lifespan assays in the dIAP2 project gave a complicated and
somewhat intriguing result, consisting of a successful lifespan extension phenotype in
the first round that disappeared in the second round. The vanishing of the lifespan
extension could be due to the degradation of DOX reagents by abnormal environment

85
temperature or sunlight exposure, although this is only an educated guess. It would be
very useful to have a wild type control group when analyzing the first round lifespan
assay data, which is a powerful lesson to me and to all other future Drosophila
researchers when we design our lifespan experiments.

In the third round experiment, the positive effect on adult lifespan could be due to a
long-lasting benefit caused by dIAP2 over-expression in developing larvae. DIAP2 has
not been reported as an anti-apoptosis regulator, unlike its IAP family sibling dIAP1
(Wang, Hawkins et al. 1999; Meier, Silke et al. 2000; Chai, Yan et al. 2003; Yan, Wu et
al. 2004; Muro, Means et al. 2005). However, previous reports suggest its involvement
in the innate immune response in flies (Leulier, Ribeiro et al. 2006; Huh, Foe et al.
2007). An increased innate immune response could result in a stronger tolerance to
environmental stress in the larvae, which in turn could benefit their adult lifespan. The
lifespan extension seen in the third round assay suggests an interesting hypothesis that a
greater innate immune response could be beneficial for lifespan extension.

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Chapter 4
Characterization of stem cell drivers in Drosophila gut

Abstract
Stem cells are un-differentiated cells found in various tissues in Drosophila. Their
ability to differentiate into different types of somatic cells, combined with their ability to
self-replicate without limit make them a unique model system for molecular biologists.
The germline stem cells located in the Drosophila ovary and testes have been
extensively investigated in the last decade. However, it was believed that the intestine
somatic cells in Drosophila gut were relatively stable and thus do not require somatic
stem cells to replenish damaged or lost cells. Then, two research group independently
reported the existence of Drosophila midgut stem cells. I selected a pool of genes that
are expressed in Drosophila ovary and characterized their expression in the Drosophila
midgut. By a GFP illumination reporter, the experiment suggested that 3 of the genes
could have abundant expression in Drosophila midgut. These positive candidate genes
provided an opportunity to construct tissue-specific driver strains that are able to over-
express the gene of interest in Drosophila midgut tissue. This tool could then be used to
investigate the molecular and biological functions of these genes in the midgut.

Introduction
Stem cells in Drosophila, like those in other organisms, renew themselves and produce
new somatic cells. Their “stemness” status largely depends on a biological and chemical

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signal maintained by their surrounding stem cell niche, such as Jak/Stat signaling
pathways (Spradling, Drummond-Barbosa et al. 2001). Scientists has long known that
Drosophila stem cells of many types play important roles in oogenesis (Drummond-
Barbosa and Spradling 2001), brain development (Bello, Izergina et al. 2008) and
spermatogenesis (Tulina 2003). Different types of stem cells are also found in many
tissues and organs of Drosophila, including ovary (King and Lin 1999; Zhang and
Kalderon 2001), testes (Tran, Brenner et al. 2000), and central nervous system (CNS)
(Toriya, Tokunaga et al. 2006). However, because the Drosophila intestine cells were
considered to be relatively stable, scientists did not describe the stem cells in Drosophila
digestive system until late 2005. (Ohlstein and Spradling 2006) Various groups of
researchers discovered that there is a group of stem cells in the Drosophila posterior
midgut (Ohlstein and Spradling 2006) and midgut epithelium (Micchelli and Perrimon
2006). Their approach inspired us to discover potential candidate genes that would be
specifically expressed in Drosophila gut stem cells.

Meanwhile, my colleague Morris Waskar gathered a number of Drosophila stock strains
bearing the coding sequence of GAL4 driven by ovary stem cell promoters. Starting
from the research described above, I aimed to perform a screen to see whether any ovary
stem cell genes are expressed in the Drosophila gut as well.

In aging and other Drosophila studies, researchers are often required to express their
favorite genes in different tissues. By artificially creating different tissue expression

88
patterns, researchers can determine different phenotypes based on how the whole
organism, a specific tissue, or a specific organs is affected. For example, if the main
cellular function of a novel gene is in Drosophila fat body, the researcher expects to see
a larger phenotypic change when the gene is over-expressed in fat body than when it is
over-expressed in a reproductive organ, in which the gene supposedly plays no role.
Therefore, different tissue-specific driver strains can provide scientists with much
insight when investigating various gene functions (Ford, Hoe et al. 2007). From the
screen described above, if a candidate gene can be characterized to be abundantly
expressed in Drosophila gut, it could become a powerful tool to investigate the gut stem
cell functions and establish the driver strain to over-express any gene of interest in
Drosophila gut.

Materials and Methods
Drosophila culture conditions were as previously described (Landis, Bhole et al. 2003;
Landis, Abdueva et al. 2004; Sun, Molitor et al. 2004; Ford, Hoe et al. 2007). The
following Drosophila strains were used: Or-R wild type, Actin5C-GAL4, CG13377-
GAL4, Kap3-GAL4, dawdle-GAL4, Hexo2-GAL4, CrebA-GAL4, C355-GAL4,
CG31305-GAL4, lola-GAL4, UAS-eGFP. When establishing the crosses, the UAS-
eGFP strain virgin flies were crossed with the male flies from the other strains. The
progeny male and female flies that bear both GAL4 and eGFP construct were collected
and cultured in a 25
o
C incubator throughout the fly lifespan. At designated time points,
flies were dissected in 1X PBS solution. The midgut from the dissection was then

89
immediately observed under GFP microscope. A photograph was taken by a SPOT
camera and SPOT image capture software (Diagnostic Instruments Inc., Sterling
Heights, MI) with the following parameters: gamma 1.50; exposure 1000ms; gain 4. For
each fly cross, dissection pictures of both male and female were taken at the 10, 30, and
50 day time points.

Results
It can be seen that on the Or-R flies, which are used as the negative control, no
fluorescence was observable at any time point. (See figure 19 for pictures.) The
dissected midgut of Actin5C-GAL4 flies (positive control) is fluorescent at the 10 day
time point. The fluorescence decreased at later time points. CG13377-GAL4, dawdle-
GAL4, and lola-GAL4 did not fluoresce at any time point. Kap3-GAL4, Hexo2-GAL4
showed relatively strong fluorescence at the 10 day and 30 day (early) time points, but it
was significantly reduced by the 50 day time point.

CrebA-GAL4, C355-GAL4, and CG31305-GAL4, all showed strong fluorescence
throughout all 3 time points. The fluorescence signal was not reduced in 50 day old flies
relative to 10 day and 30 day young flies.

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Figure 19: The GFP illumination pictures of the dissected gut. Males from each strain
were crossed with UAS-eGFP virgin females. Both sexes of the progeny were
maintained in 25
o
C continuously. For each of the 10, 30, and 50 day time points, both
sexes of the progeny flies were dissected in 1X PBS solution. The picture of the midgut
was taken under the conditions and parameters described in methods section. For each
picture, the time point and sex are labeled. In some of the pictures, the Drosophila gut is
not clearly visible because of the lack of fluorescence. In those cases, a red arrow is
pointing to dark colored gut to indicate the location of the gut. A: Or-R; B: Actin5C-
GAL4; C: CG13377-GAL4; D: Kap3-GAL4; E: dawdle-GAL4; F: Hexo2-GAL4; G:
CrebA-GAL4; H: C355-GAL4; I: CG31305-GAL4; J: lola-GAL4

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94

95

96

97

98

99

100

101
Discussion
The experiment was performed successfully, as demonstrated by the negative and
positive control groups. The negative Or-R flies did not fluoresce at all in any of the
time points (Figure 19A). The lack of GFP meant that GAL4 was not produced in the
Or-R cross progeny flies, as was expected. Actin5C is a ubiquitous promoter that is
extensively used when high expression levels of a gene of interest are required (Ford,
Hoe et al. 2007). As can be seen in the 10 day and 30 day time point pictures, the
fluorescence was relatively strong at 10 days (Figure 19B), but disappeared in later time
points. This could suggest that the expression of Actin5C in the fly gut decreases during
fly aging. Many researchers have used this promoter as a fairly standard method to
express genes of interest ubiquitously in all tissues (Bieschke, Wheeler et al. 1998; Sun
and Tower 1999; Ford, Hoe et al. 2007). From the results of this experiment, it is still
unclear whether or not the expression level of Actin5C specifically in fly gut remains
stable throughout fly lifespan. This issue deserves further investigation for anyone who
would like to express their gene of interest specifically in Drosophila gut.

3 out of 10 crosses showed no fluorescence signal in any of the time points, including
CG13377-GAL4, dawdle-GAL4, and lola-GAL4 (Figure 19C, E, J.). The lack of GFP
signal suggests that although these genes may express abundantly in Drosophila ovary
stem cells (Morris Waskar unpublished data), their expression in Drosophila gut remains
low throughout the fly lifespan.

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In 2 out of 10 crosses, progeny showed relatively strong illumination signals during
early life stages. These were Kap3-GAL4, Hexo2-GAL4 (Figure 19D, F.). Both showed
strong signal in fly midgut when the flies are 10 days old. The signal, however,
diminished when the flies became older. This result is quite understandable and
expected. As the animals age, the nurturing cells in the stem cell niches decay, and the
loss of somatic hub cells results in slower division of germline stem cells, and eventually
the reduction of germline stem cell number (Wallenfang, Nayak et al. 2006; Boyle,
Wong et al. 2007; Pan, Chen et al. 2007).

3 crosses (CrebA-GAL4, C355-GAL4, and CG31305-GAL4), especially CrebA-GAL4,
showed a strong GFP illumination signal at all time points (Figure 19G, H. I.). This high
expression of the candidate genes is surprising. Because of the reasons described above,
it was expected that the genes’ expression would be decreased as the flies age. One
hypothesis to explain this is that the reduction of germline stem cells described above
does not apply to gut stem cells, or that the rate of reduction is much slower than
predicted by extrapolating from the case of germline stem cells. Another explanation is
that the replenishing rate of gut stem cells is higher than in other tissues due to the high
turnover of intestinal somatic cells (Micchelli and Perrimon 2006; Ohlstein and
Spradling 2006). In this case, we would predict gut stem cells to be much more tolerant
to niche cell decay and and thus to reduction of their molecular signals. Another possible
reason is that although the somatic hub cells in niches decay and may result in reduction
of gut stem cells, the average per-cell expression level of these 3 genes increases as the

103
flies age. Tissue-specific expression measurements or tissue in-situ hybridization will be
required to discriminated between these hypotheses.

I must point out that the above conclusion is based on the assumption that the cells that
showed illumination signal was indeed gut stem cells, as indicated by other research
groups that intestinal stem cells widely exist in Drosophila midgut (Micchelli and
Perrimon 2006; Ohlstein and Spradling 2006). These candidate genes were also shown
to express in ovary stem cells rather than somatic cells (Morris Waskar, unpublished
data). In order to verify the identity of these stem cells, a staining with GFP antibody to
dissected midgut and stem cell mosaic analysis (Ohlstein and Spradling 2006) can be
performed to check if the GFP illumination signal indeed originates from intestinal stem
cell themselves.

Conclusion
The project was successfully performed, according to the expected results from both
positive and negative control strain crosses and all the remaining experimental strains.
Three strains (CG13377-GAL4, dawdle-GAL4, and lola-GAL4) did not show any
detectable GFP fluorescence, indicating a low or nonexistent level of expression in the
fly midgut. More importantly, another three strains showed expected decreasing GFP
signal throughout the fly lifespan. This decreasing expression pattern was consistent
with previous reports showing somatic hub cell decay and reduction of stem cell

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numbers, although these previous studies were performed on Drosophila ovary, not gut
(Wallenfang, Nayak et al. 2006; Boyle, Wong et al. 2007; Pan, Chen et al. 2007).

The most exciting finding comes from the 3 crosses that gave the highest GFP
fluorescence intensity at all the 3 time points, especially the CrebA-GAL4 cross. The
extremely high expression of these genes provided us an excellent candidate pool to
score for gut expression driver strains. As described above, Drosophila researchers
would always like to express their genes of interest within different tissues. By
expressing the favorite gene in certain tissues, researchers can investigate potential
molecular functions of the gene in those tissues. Because the CrebA-GAL4 strain cross
showed extraordinarily high expression from 10 day to 50 day old flies, it became a
great tool to establish a gut expression driver strain. My colleague Daniel Ford has
established and characterized such a gut driver line based on this strain. I was also able
to use this strain to express two of my favorite genes in Drosophila gut and obtained
some interesting results, as described in Chapter 3 of this dissertation.

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Chapter 5
Intra-abdominal injection of Drosophila and RNAi effect

Abstract
RNAi has become the tool of choice when researchers need to knock down a gene of
interest. In Drosophila, RNAi was achieved mainly in cells or in adults by expressing
the hairpin structure of a target gene. This approach has the disadvantage of being time-
consuming and labor-intensive. Intra-abdominal injection methods have been reported to
knock down target genes successfully. I tried to repeat their approach by injecting the
siRNA targeting two genes related to oogenesis, ORC2 and chiffon. The expected RNAi
effect was not achieved, as indicated by the non-significant change of female Drosophila
fecundity after the injection and the comparison between injected and un-injected
groups. This unexpected result could be attributable to a few reasons. The injected
siRNA was not chemically modified or protected. The original RNAi finding was
targeting genes in the central nervous system (CNS), while my target gene is involved in
oogenesis. This gives the injected siRNA an extra burden, as it has to penetrate the ovary
membrane and ovarioles sheath cells to reach the developing oocyte. The siRNA
injection approach can still be valuable, given that the proper troubleshooting steps are
performed.

Introduction

106
RNAi interference (RNAi) is a mechanism that knocks down gene expression in variety
of organisms (Nishikura 2001). When a long dsRNA is introduced into the organism, its
double-stranded structure is recognized by a ribonuclease III enzyme named Dicer,
which subsequently cleaves it into smaller fragments named small interfering RNAs
(siRNA). In turn, siRNA directs the cleavage of homologous messenger RNA after
siRNA is incorporated into the RNA-induced silencing complex (RISC) (Nishikura
2001; Hannon 2002). The cleavage of messenger RNA then results in a substantial
decrease in expression of that gene in the organism.

Since its discovery a decade ago, RNAi has become an important and powerful research
tool to investigate gene function in an array of organisms, both in vivo and in vitro. At
the same time, RNAi has also been considered as an promising candidate tool in a
therapeutic research environment for the treatment of diseases (Kim and Rossi 2008).
Recently, researchers developed a large scale RNAi injection method for Drosophila
embryos to identify and characterize the molecular functions of the 14,000 Drosophila
genes (Cornell, Fisher et al. 2008). For molecular biology and genetics research, clinical
and therapeutic applications, RNAi is becoming an indispensable tool for the
advancement of academic and commercial research.

It has been a long time since the first scientist injected DNA into a developing
Drosophila embryo, trying to create a Drosophila strain bearing his gene of interest.
(Rubin and Spradling 1982) More recently, scientists have been injecting dsRNAs into

107
Drosophila embryos to knock down genes of interest, and to characterize their function
(Zappe, Fish et al. 2006). This technique involves an inverted repeat of a sequence
specific to the target gene, and clones the inverted repeat structure into a vector that
contains a conditional promoter (Allikian, Deckert-Cruz et al. 2002). The whole
construct is then injected into developing fly embryo to establish the transgenic lines that
bear the inverted repeat sequence, which can in turn be conditionally transcribed. By
using a conditional promoter to control transcription, when desired, the inverted repeat
will be transcribed, and the transcript will fold back on itself and become a dsRNA. The
dsRNA will then be recognized and processed by the Dicer and RISC machinery
described above, triggering the RNAi mechanism to knock down the target gene.

We believe that the technique of RNAi injection into the developing embryo has its
value. However, after the injection, it takes more than 10 days for the embryo to fully
develop into adult. It will also take another 10 days or so, depending on the genetic cross
design, to manifest the phenotype. When screening large number of genes, the workload
could be relatively heavy. And the project could thus become time-consuming.

Because of the inherent disadvantage of RNAi by embryo injection, some researchers
started to think of alternative routes for Drosophila RNAi. By taking advantage of
bacteria that express dsRNA, C.elegans researchers are able to feed them with dsRNA-
expressing bacteria to achieve RNAi easily (Fraser, Kamath et al. 2000). In 2001, the
Drosophila researchers started to inject dsRNA into adult Drosophila abdomen, trying

108
to trigger RNAi of their favorite gene (Dzitoyeva, Dimitrijevic et al. 2001). Inspired by
their success, I aimed to try this novel RNAi approach. I hypothesized that the target
gene expression level would be decreased due to the RNAi effect by injection of dsRNA
into adult Drosophila abdomen.

Materials and Methods
Drosophila culturing conditions were previously described (Landis, Bhole et al. 2003;
Landis, Abdueva et al. 2004; Sun, Molitor et al. 2004; Ford, Hoe et al. 2007). Or-R wild
type female flies were used for injection in all experiments. Injection siRNA and buffer
were ordered from Dharmacon Inc (Chicago, IL). The sequences of siRNA are as
follows: Chiffon: 5’-AACGCAAAGGAGAGCGAT-3’; ORC2: 5’-
AGCCATGCTTCGCAATGTGAA-3’. The siRNA are all dissolved in standard siRNA
buffer provided by Dharmacon Inc. in the concentration of 0.8ug/ul. The injection
apparatus was fixed on a metal rod (See figure 20 for injection station setup.). The
injection glass tubing was ordered from FHC Inc. (Bowdoin, ME 04287, Alsil tubing
with fiber, 1.0ODX0.75ID). The optimal injection volume can be adjusted by a screw on
the apparatus. Green food dye was used during pilot experiment to demonstrate
successful abdomen injection in adult flies.

109
Figure 20: The setup of the abdominal injection station. A: Injection apparatus including
the micromanipulator to adjust the positioning and angles of the injection needle, the
injection pump substituted by large syringe. (Syringe can be replaced by Eppendorf
FemtoJet injection pump to give much better performance.) B: Injection needles are
pulled from FHC Alsil capillary tubing. C: Narishige needle puller for pulling the needle
from the capillary tubing. D: prepared needles. E: overall setup of the injection station
including the apparatus and dissection scope. F: Close-up look of the injection station
showing the anesthetized flies and injection needle.

110

111

Figure 21: The pilot injection with green food dye. Injection aimed at the lower
abdomen of the female fly shown. The injected abdomen is labeled by red arrow
indicating the green stain. A and B: The anesthetized female fly before the injection. C
and D: The same anesthetized female fly immediately after the injection.

Results
In the pilot experiment, green food dye was used as the substitute to the siRNA
solutions. (See figure 21 for injected fly pictures.) As indicated by the faint green color

112
in the abdomen of female flies, the injection delivered the injection buffer into the
abdomen. From the ventral and side view of the female fly before and after the injection
(Same female fly was used to take the picture shown in the figures.), the injected food
dye permeated and diffused around female ovary and midgut. After the fly recovered
from anesthesia, the fly was able to crawl and fly normally. The death rate of injected
flies was not significantly different from the untreated control flies (data not shown).

However, multiple rounds of injection of chiffon or ORC2 siRNA did not result in
decreased fly fecundity, contrary to my hypothesis. Comparing the fecundity curves
after multiple injection rounds of Chiffon and ORC2 injections to those of GFP and
buffer control injections, no obvious decrease of female fecundity could be observed.
From the fecundity curve, it is evident that on the day following the injection, the
fecundity was decreased significantly across all injection groups, including buffer and
GFP siRNA control groups, as well as Chiffon and ORC experimental groups. This is
probably due to a disruption to normal fly physiology by the injection procedure itself.
After two days of recovery, however, the fecundity of all groups returned to normal
levels. The fecundity between control and experimental groups was not significantly
different throughout the remaining experiment period. This pattern was consistent across
all 3 rounds of the injection experiment. (See figure 22 for fecundity curves.)

113

Figure 22:. The fecundity assay result of four rounds of abdomen RNAi injection. The
fecundity curves are labeled in the same way as other fecundity curves described above.
Groups with different injected reagents were labeled with different color lines and dots.
Blue line: no injection. Purple line: siRNA buffer solution only. Green line: GFP control
siRNA dissolved in buffer solution. Brown line: Chiffon siRNA dissolved in buffer
solution. Dark purple line: ORC2 siRNA dissolved in buffer solution. A, B, C, D show
round 1, 2, 3, and 4 of the injection, respectively.

114
Discussion
The purpose of the pilot experiment was to test whether injection into the fly abdomen
was able to deliver the injection solution to the abdomen without affecting the normal
physiology of female flies. According to the comparison picture for the same female fly
before and after green food dye injection, the food dye was successfully delivered to the
area around ovary and midgut. Although the abdomen of the injected fly was a little
inflated, it went back to normal appearance after just two days. The death rate of the
injected flies identical to that of control flies suggests that the normal physiological
function of adult female flies was not affected.

Because Chiffon (Landis and Tower 1999) and ORC2 (Loupart, Krause et al. 2000;
Okudaira, Ohno et al. 2005) play important roles in Drosophila chorion (eggshell)
formation in the oogenesis process, we expected that knocking down these two genes by
siRNA injection would decrease female fecundity significantly. The unaffected female
fecundity rate indicates that the oogenesis mechanism and chorion formation process
remained intact. A few details of the experimental design could account for the failure of
abdomen injection RNAi in this context, explained in the following paragraphs.

It is possible that RNase is readily available in the abdominal cavity of female flies. The
injected siRNA was not protected or chemically modified to prevent RNase degradation.
Therefore, it is possible that the siRNA was degraded by RNase in the fly abdomen,
before reaching target cells and triggering the RNAi machinery. However, in the original

115
report of intra-abdominal injection RNAi, the researchers did not describe any type of
chemical modification either (Dzitoyeva, Dimitrijevic et al. 2001). Since they
successfully achieved RNAi under the described conditions, it is difficult to imagine the
RNA degradation would be a major concern in my experiments, suggesting an
explanation specific to the genes I wanted to knock down.

It is also possible that after the injection, the siRNA solution was restricted to the
abdominal cavity. If siRNA was not able to permeate the ovariole sheath cells, it could
not directly take effect inside the follicle cells responsible for the formation of chorion.
To ensure the penetration of siRNA through the ovariole sheath cell, the best strategy
would be to perform microinjection directly on the ovariole. However, without killing
the fly, it would be extremely difficult to perform such a microinjection. This obstacle is
due to limited research tools and to the experimental design itself.

Conclusion
Intra-abdominal injection was not able to achieve the expected RNAi effect, although
the pilot experiment was an excellent training for the intra-abdominal injection
technique. From the pilot experiment, I designed the structure of the injection station
apparatus from scratch and later added the injection pump machine (Eppendorf
FemtoJet), which makes the combination a great tool to perform this type of injection.
My colleague, Chunli Ren, also benefited from the injection station. She was able to
inject up to 3,000 flies within two days after extensive practice.

116

A few reasons could explain why the siRNA injection was not able to achieve the
expected result. Although the unmodified siRNA appears unlikely to be the cause, the
method would surely benefit if some type of chemical modification was performed to
the siRNA before the injection to make it more robust to possible RNase degradation.
The injection technique could also be improved to ensure more accurate injection into
certain abdomen organs. If a different gene that works in other cells that are more
readily permeable from the abdominal cavity could be selected, a different siRNA that
targets the gene could be designed. By changing the target gene strategy, the expected
RNAi effect by intra-abdominal injection may be achieved.

117
Chapter 6
Conclusions and future directions

My research was focused on strategies and regulation mechanisms that can extend the
lifespan of Drosophila melanogaster. My Ph.D. research projects include: screening,
identification, and characterization of novel Drosophila genes magu and hebe, their
functional analysis, verification of the lifespan extension effect of gene dIAP2,
characterization of Drosophila gut stem cell driver strains, and intra-abdominal injection
RNAi approaches.

Drosophila is a great model organism to study. Its tiny body is very easy to manipulate
when it is anaesthetized by carbon dioxide (CO
2
). The culturing condition only requires
a standard cornmeal food that contains sugar, yeast, and cornmeal. It does not require
aseptic culturing conditions in most cases. Drosophila is also not vulnerable to common
infectious disease, unlike some mammalian lab animals. The genome of Drosophila only
contains 4 pairs of chromosomes, which makes it easy to design and perform genetic
crosses to score for required phenotypes. After a few decades of Drosophila research,
the pioneers have paved the road and given us many powerful research tools.
Mutagenesis can be performed by ionizing and UV radiation (Ashburner, Golic et al.
1989), chemical mutagens (Obe, Sperling et al. 1971), P-element transformation, PdL
mutagenesis (Bieschke, Wheeler et al. 1998), etc. The flies that bear different genotypes
can often be scored by the different genetic markers on different chromosomes

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(Ashburner, Golic et al. 1989). With all these advantages being considered, Drosophila
is probably the most important model organism for molecular biology and genetics
research. Although I haven’t worked on any other model organisms in my Ph.D. career,
I am quite confident that Drosophila will continue to be the favorite lab animal that
biologists love to work with.

The genetic design of PdL mutagenesis is relative simple and straightforward. However,
in order to obtain massive amount of mutants, a very large scale genetic cross must be
set up. It took more than 4 months for 3 full-time and 3 part-time researchers to obtain
around 20,000 mutants. After multiple rounds of screening, tests, and retests, the two
most promising genes were selected for further research. Gene “magu” (originally
named CG2264) was able to extend Drosophila lifespan when induced by the ubiquitous
driver rtTA(3)E2 or by the motor neuron specific driver D42, or the gut driver C204. It
has been reported that the magu protein interacts with the protein Slam (Giot, Bader et
al. 2003), which gives us some insight about potential molecular functions of this novel
gene. Slam is a developmental regulator that affects polarized membrane growth during
oogenesis, or the cleavage of the fly embryo, to be more specific (Lecuit, Samanta et al.
2002). Another report, based on microarray data, suggests that magu expression
correlates with Dorsal and Toll, the two major transcription factors involved in dorso-
ventral axis formation in the Drosophila embryo (Stathopoulos, Van Drenth et al. 2002).
These data and interactions may imply that magu does regulate the oogenesis process.
Another research article suggests that magu is enriched in the very tip of the testes.

119
Relative to expression in somatic stem cells, magu is enriched 20.5-fold in germ line
stem cells (Terry, Tulina et al. 2006). Could this mean that magu is also involved in the
spermatogenesis process? According to an NCBI BLAST search (McGinnis and
Madden 2004), magu has many orthologs in various species including worm, fruit fly,
dog, rat, and human. The rat and human orthologs are named SMOC2 (SPARC related
modular calcium binding 2) and Smap2, respectively. A group of researchers reported
that the expression level of SMOC2 was high in the early embryo (Vannahme, Gosling
et al. 2003; Mager, Schultz et al. 2006) and ovary (Vannahme, Gosling et al. 2003) of
mice, consistent with the expression level data of magu and its potential role in early
embryo development. Smap2, the human ortholog of magu, is reported to be more
highly expressed in aorta than in any other tissue. It is also expressed in muscle tissue,
reproductive tissues, digestive tissues, and secretory tissues (Nishimoto, Hamajima et al.
2002). These data also indicated that that Smap2 mRNA was up-regulated during
neointima formation in a rat carotid endarterectomy model, suggesting smap2’s potential
role in the progression of atherosclerosis in the aorta. Another study reported that
SMOC2 is a novel angiogenic factor that can stimulate endothelial cell proliferation,
migration, and potentiates angiogenic effects of growth factors, which leads to the
formation of blood vessels (Rocnik, Liu et al. 2006). The orthologs in these species
share the following conserved domains: the Kazal type serine protease inhibitor,
Thyroglobulin type I repeats, and the SPARC extracellular Ca2+ binding domain. These
domains are also found on magu, according to protein motif searches (Kanehisa, Goto et

120
al. 2002; Vannahme, Gosling et al. 2003; de Castro, Sigrist et al. 2006; Finn, Mistry et
al. 2006; Rocnik, Liu et al. 2006; Mulder, Apweiler et al. 2007).

The original find of the magu mutant was based on increased fecundity in late age flies.
However, the fecundity tests on transgenic magu strains showed complicated results.
High expression levels of magu in transgenic strains resulted in decreased fecundity
instead of increased. This may indicate that the over-accumulation of potentially positive
effectors sometimes results in negative effect. This hypothesis is partially verified by the
titration DOX fecundity assay. Overall, magu is a very interesting gene that may be
involved in oogenesis, spermatogenesis, and angiogenic processes.

In the multiple rounds of lifespan assays, the hebe PdL mutant strain and its transgenic
strains clearly extend lifespan when the transgene is over-expressed ubiquitously and at
least in two different tissue types. The fecundity assays of hebe PdL mutants and
transgenic lines showed intriguing results similar to those in the magu lines. The over-
accumulation hypothesis was verified by the titration DOX fecundity assay for this gene
as well. As to molecular function prediction and protein-interaction relations, we were
surprised to find that the hebe sequence did not match any interaction partner proteins,
nor did it contain any known protein motifs, according to various protein databases
(Kanehisa, Goto et al. 2002; Vannahme, Gosling et al. 2003; de Castro, Sigrist et al.
2006; Finn, Mistry et al. 2006; Rocnik, Liu et al. 2006; Mulder, Apweiler et al. 2007).
So far the only report about the potential molecular functions of hebe was based on the

121
observation that a P-element insertion mutant in hebe (CG1623) resulted in a 62%
decrease in male aggression behavior index (Edwards, Rollmann et al. 2006). However,
no other papers exist related to this gene’s function, protein interaction partners or
molecular pathways.

Through the screening round, my previous colleague Jiping Yuan successfully scored a
PdL mutant that extends lifespan. The lifespan extension effect was confirmed by Jiping
on both the old and new food recipes. The mapping of PdL indicates that the insertion
landed 5’ upstream of the miRNA gene bantam. It was reported recently that bantam is
among the apoptosis-limiting factors after ionizing radiation (Jaklevic, Uyetake et al.
2008). Normal bantam function is also required to sustain cell proliferation (Hipfner,
Weigmann et al. 2002; Brennecke, Hipfner et al. 2003; Thompson and Cohen 2006).
The mapping shows that the distance between PdL insertion and bantam gene is a little
over 14kb. Whether or not the PdL insertion is able to over-express the gene bantam
over the 14kb gap is still questionable. Northern blot did not confirm the induction of
bantam by tet-on cross and +DOX feeding. A repeat of lifespan assay on bantam PdL
mutant was performed by crossing it to rtTA(3)E2 ubiquitous driver strain, and the cross
did not show a significant increase in lifespan, contrary to the previous two rounds of
lifespan assays. It is possible that there is an unknown and unpredicted gene in the 14kb
gap between the PdL insertion and bantam gene. With a few questions remaining
unanswered, this PdL mutant strain still offers interesting challenges awaiting the
researchers to discover.

122

The IAP protein family consists of dIAP1, dIAP2 and dBruse. They share the unique
structural characteristic, the 70-amino-acid motif Baculovirus IAP repeat (BIR) on the
N-terminal and RING ubiquitin ligase domain on the C-terminal. It was reported that
dIAP1 strongly regulates anti-apoptosis pathways (Meier, Silke et al. 2000; Chai, Yan et
al. 2003; Yan, Wu et al. 2004; Muro, Means et al. 2005). However, reports of dIAP2
have been focused on innate immune responses to bacterial infections, rather than anti-
apoptosis pathways (Huh, Foe et al. 2007). So far the studies on dIAP2 have not been
nearly as extensive as those on dIAP1. The lifespan extension effect of dIAP2 on
Drosophila has not captured the attention of fly researchers. According to our lifespan
data on the dIAP2 crosses, the dIAP2 lifespan extension effect on adult flies was
complicated and could be contradictory between different test rounds. One round of the
lifespan assay suggested that dIAP2 may bring lifespan benefit to the adult flies if they
have the induced dIAP2 expression in developing larvae. This finding still needs to be
confirmed by a few more rounds of retests before a definitive conclusion can be drawn.

Stem cell research has been a hot topic not only in the field of molecular biology, but
also in pharmaceutical and clinical researches. Its unique feature of differentiation and
almost unlimited capability of division brought researcher enormous interest.
Drosophila germline stem cells have correspondingly been extensively investigated in
the last decade. However, Drosophila intestinal cells were believed to be relatively
stable, and it was thus inferred that somatic stem cells would exist in the intestine. In

123
2006, two independent research labs identified midgut stem cells for the first time
(Micchelli and Perrimon 2006; Ohlstein and Spradling 2006). These findings motivated
the others to look for more types of stem cells in other Drosophila tissues (Egger, Boone
et al. 2007; McClure and Schubiger 2007; Bello, Izergina et al. 2008). Whenever
possible, scientists want to express their favorite genes in different tissues and seek
phenotypic changes to investigate potential functions of those genes. By confirming the
large quantity expression of CrebA, C355, and CG31305 in the gut, we made it possible
to construct a stem cell driver strain that can induce a gene of interest in Drosophila gut.
Indeed, my colleague Daniel Ford has constructed and characterized such a driver strain
based on this finding. With this combined effort from both of us, the newly constructed
gut driver would be helpful for other fly researchers to perform gene over-expression in
Drosophila gut tissue.

RNAi is another example of an extensively investigated field in biology, especially in
the last five years. Performing RNAi in Drosophila S2 cells and adults has been quite
successful (Cherry 2008; Rogers and Rogers 2008). Achieving RNAi in adult flies by
expressing the hairpin structure has been a standard approach among fly researchers
(Giordano, Rendina et al. 2002). The main disadvantage of hairpin structure expression
from DNA is the long period of time it usually takes to generate an RNAi trangenic
strain and the extensive amount of lab works this technique requires. With the adult
abdomen injection technique, it should be possible to achieve the RNAi effect within
just a few days. The first publication detailing this new approach appeared in 2001

124
(Dzitoyeva, Dimitrijevic et al. 2001). However, reports from other research groups
showing success with this approach have been rare. My own RNAi injection experiment
did not achieve the expected result. And it could be due to a few reasons. The first
explanation is the lack of chemically-modified RNA oligos. The more plausible
explanation, however, is a limited permeating capability of siRNA oligos through ovary
membrane and ovarioles sheath cells. The siRNA injection approach may require more
precise mastery of injection techniques than embryo injection, too. With all the above
obstacles removed, RNAi by abdominal injection could be full of potential for anyone
who would like to perform RNAi on adult flies with a quick and easy approach.

In sum, Drosophila melanogaster was a great model organism for molecular biology and
genetics research. I screened about 8,000 mutants to obtain two most interesting genes.
Magu and Hebe gene extend lifespan when over-expressed. magu has orthologs across
many species, which gives us some insights about its potential functions in oogenesis,
male spermatogenesis, and blood vessel formation. The Drosophila stem cell driver
strain that Daniel Ford and I characterized will be a useful tool for expressing a gene of
interest in the Drosophila gut. RNAi triggered by intra-abdominal injection technique is
still under development. There are a few roadblocks that need to be removed before any
widespread application of this technique. As an indispensable research tool, Drosophila
will remain probably the favorite model organism for molecular biologists and
geneticists.

125
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Abstract (if available)

Abstract The regulation of Drosophila melanogaster longevity and fecundity involves many factors. Longevity is governed by oxidative stress, stem cell loss, dietary restriction, the insulin/IGF-1 pathway, and other factors. Fecundity is also regulated by multiple tissues and factors, including the germline stem cells and stem cell niche, the fat body, yolk proteins, and sex peptides. The fecundity of wild type female Drosophila gradually declines during aging, suggesting a common pathway regulating longevity and fecundity machinery. Since both mechanisms involve multiple factors, sorting through the Gordian’s knot is a formidable task. Using a PdL mutagenesis approach, I screened for a specific phenotype in thousands of independent mutant strains to examine both regulatory networks simultaneously. Two novel genes, magu and hebe, were identified and characterized to regulate longevity and fecundity. While Drosophila lifespan was extended upon the induction of these genes, fecundity increase requires that the gene induction be in an ideal range to show the expected phenotypic change. I also performed several other projects, including studying the lifespan extension effect of dIAP2, characterization of a Drosophila gut driver strain, and intra-abdominal RNAi injection in adult Drosophila. These projects provided us insight on longevity, fecundity, anti-apoptosis, stem cell biology, RNAi and other aspects of Drosophila research. In sum, Drosophila melanogaster, as a model organism for molecular biology and genetics study, will continue to contribute to the scientific community.

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Asset Metadata

Creator Li, Yishi (author)

Core Title Characterization of Drosophila longevity and fecundity regulating genes

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Molecular Biology

Degree Conferral Date 2008-12

Publication Date 10/31/2008

Defense Date 08/19/2008

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag Drosophila,fecundity,longevity,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tower, John (committee chair), Aparicio, Oscar Martin (committee member), Comai, Lucio (committee member), Finkel, Steven E. (committee member), Longo, Valter D (committee member)

Creator Email yishili@gmail.com,yishili@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1735

Unique identifier UC1416831

Identifier etd-Li-2382 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-119373 (legacy record id),usctheses-m1735 (legacy record id)

Legacy Identifier etd-Li-2382.pdf

Dmrecord 119373

Document Type Dissertation

Rights Li, Yishi

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu