1. Suggesting Potential Candidates for
Future Screens by Reducing the
Expression of Robo and Talin in
Heart Cells of Drosophila
Melanogaster
Written by: Amanda Konash
Student Number: 1145016
Supervisor: Dr. Roger Jacobs
2. 2
Table of Contents
ABSTRACT 3
INTRODUCTION 3
1.1 - Drosophila Melanogaster as Model Organisms 5
1.2 - Drosophila Melanogaster Heart Development 6
1.2.1 - Structure 6
1.2.2 - Development 7
1.2.3 - Function 9
1.3 - Gal 4/UAS System 9
1.4 - The Role of Robo, and Talin 11
1.4.1 - Talin 11
1.4.1.1 - Integrin 12
1.4.2 - Robo 13
METHODS AND MATERIALS 14
2.1 - Control, UAS-RNAi, Gal4, and Balancer Lines 14
2.2 - Fly Maintenance 17
2.3 - Determining Which Chromosome the UAS Lines are on 17
2.4 - Genetic Scheme 18
RESULTS 22
3.1 - UAS-moesin-mCherry 22
3.2 - UAS-Robo 23
3.3 - UAS-Talin 24
DISCUSSION 25
REFERENCES 28
APPENDIX 32
Appendix A - Raw Data From the UAS-RNAi and Gal4 Crosses Conducted 32
Appendix B - Raw Data From Chromosome Testing 33
3. 3
ABSTRACT
Cardiovascular disease is the leading cause of death globally. It costs the United
States approximately $108.9 billion each year for health care services, lost productivity,
and medication. It is also the most common congenital defect in infants affecting three
million fetuses and newborns annually. Drosophila Melanogaster also known as the
common fruit fly has helped immensely in paving the way towards better diagnoses and
treatment of cardiovascular disease due to the great similarities it has with humans. Robo
(a transmembrane receptor) and Talin (a protein coding gene) are very important in the
development of the Drosophila heart. Using Drosophila Melanogaster, a study was
conducted to identify a “sensitized” genotype with reduced viability due to knockdown of
Talin or Robo specifically in the heart. With these genotypes, a future screen will then be
designed to isolate mutations that potentially work with Talin or Robo during heart
development or later function.
INTRODUCTION
Cardiovascular disease is the leading cause of death globally (Bier & Bodmer,
2004). On average 56 million people die per year, with 17.5 million of those deaths
attributed to cardiovascular disease (Bier & Bodmer, 2004). It costs the United States
approximately $108.9 billion each year for health care services, lost productivity, and
medication (Bier & Bodmer, 2004). It is also the most common congenital defect in
infants affecting three million fetuses and newborns annually (Francine et al., 2014).
There have been many medical advances within this field leading to decrease mortality
for example due to an enhanced ability to diagnose and treat these disorders (Sytkowski,
4. 4
1990). To put it in perspective, in the 1950’s cardiovascular disease was responsible for
almost half of all deaths in Canada and is now responsible for less then one-quarter
(Moon, 2008). Also in the 1950’s, fewer than 20% of infants born with complex heart
defects reached adulthood and now more then 90% of infants reach adulthood (Moon,
2008). Despite these accomplishments, there is still a limited understanding of the
developmental and genetic factors involved with cardiovascular disease (Francine et al.,
2014). Further research is also being hindered by the complex and multifactorial nature of
this disease (Tao & Schulz, 2007). Cardiovascular disease is of primary importance to
public health and much research is being done towards the prevention of it (Tao &
Schulz, 2007).
Drosophila Melanogaster also known as the common fruit fly has helped
immensely in paving the way towards better diagnoses and treatment of cardiovascular
disease (Bier & Bodmer, 2004). This is due to the great similarities between Drosophila
and humans. For example, humans contain 1682 identified disease genes, in which 74%
of those are homologs in Drosophila, about 500 of these genes are functionally
equivalent between flies and humans, and 34 of these genes are related to cardiovascular
disease (Bier & Bodmer, 2004). They also have very similar morphologies during the
early stages of heart formation (Bier & Bodmer, 2004). This paper will be focusing on an
in depth look into a study currently being conducted using Drosophila Melanogaster. The
study consists of looking at the effect that reduced signaling of Robo and Talin in the
heart cells has on Drosophila viability. Using these viability results, a suitable sensitized
background can then be chosen, around which a future screen will be designed. The
purpose of the screen is to see how signals from or through Robo and Talin modulate
5. 5
functions of the dorsal vessel such as cardioblast morphogenesis, polarity, and adhesion
dynamics for example. The screen will help increase the understanding of the pathways
involved in heart formation and help identify other proteins or genes, that are involved
along with Robo and Talin. To do this, a UAS-Gal4 system combined with RNA
interference (RNAi) will be used.
1.1 - Drosophila Melanogaster as Model Organisms
Drosophila Melanogaster is the best model organism to use in cardiac research,
due to its dorsal vessel morphology (Jennings, 2011). The dorsal vessel is a highly
ordered and simple linear tube that resembles early stages of development of the
vertebrate heart (Jennings, 2011). In both Drosophila and vertebrates, the cardiac
progenitors come from the lateral mesoderm and are specified through similar cellular
induction pathways and function of transcriptional effectors (Bodmer, 1995). Due to the
simple structure of the dorsal vessel it allows for any morphological changes to be easily
detected (Bodmer, 1995). The dorsal vessel contains two major cell types: the outer non-
myogenic pericardial cells and the inner contractile myocardial cells, which form the
lumen (Bodmer, 1995). As there are only two, these cell types can be easily tracked using
molecular markers in both fixed and living tissues (Bodmer, 1995). Unlike the vertebrate
heart, the dorsal vessel is not involved in the transportation of oxygen as that role has
been adopted by diffusion during embryogenesis and a tracheal system later in
development (Bodmer, 1995). This is advantageous for researchers because cardiac
malfunctions that are common in vertebrate infants and that cause lethality are less
common in Drosophila (Bodmer, 1995). Another advantage of using Drosophila is that
6. 6
the heart tube develops ventral to the ectoderm, directly under the dorsal cuticle (Tao &
Schulz, 2007). This allows for easy imaging of the heart (Tao & Schulz, 2007). Other
reasons that verify Drosophila Melanogaster as an effective model organism is that they
are easy and inexpensive to maintain compared to vertebrate models, they have a short
life cycle, it is very easy to set up genetic crosses, they have a fast generation time, they
proliferate very quickly, and they have a well characterized, completely sequenced, small
genome (Jennings, 2011).
Other organisms have been considered for cardiac research such as mice and
Caenorhabditis elegans, but researchers have identified many disadvantages in using
them. For instance, causing loss of function mutations in human disease-related genes in
mice is very time consuming, costly, and usually results in modest mutant phenotypes
(Moon, 2008). Caenorhabditis elegans, on the other hand, along with every other
invertebrate genetic model lacks a heart, which obviously makes them unsuitable.
1.2 - Drosophila Melanogaster Heart Development
To be able to understand morphological changes occurring in the Drosophila
Melanogaster heart (dorsal vessel), one must understand the structure of the heart, how
the heart develops, and what the main functions are.
1.2.1 - Structure
The structure of the dorsal vessel consists of a linear tube that in its early stages
resembles the vertebrate heart (Tao & Schulz, 2007). As outlined previously, when the
dorsal vessel reaches maturity at the end of embryogenesis it consists of two major cell
7. 7
types that form the inner and outer cell layers (Bodmer, 1995). The dorsal vessel is
segmented and consists of pairs of cardioblasts with a range in size of nuclei due to the
distinct genetic nature of the cells (Tao & Schulz, 2007). It also contains an anterior-
posterior polarity (Tao & Schulz, 2007). The anterior aorta is structured as a narrow
lumen that finishes in the outflow tract of the heart (Tao & Schulz, 2007). The posterior
is structured as a broad lumen that contracts strongly, contains the inflow tracts, and
finishes with four cells that appear to play a major role as the pacemaker of the larval
organ (Tao & Schulz, 2007). An accurate representation of the structure of the dorsal
vessel can be seen in Figure 1.
Figure 1: Structure of the Drosophila adult dorsal vessel (Tao & Schulz, 2007). This
figure displays the different cell types as well as the segmentation pattern present within
the heart.
1.2.2 - Development
Circulatory system development of Drosophila begins when maternal signaling
events specify the ventrally located mesoderm cell layer during embryogenesis shown in
Figure 2 stage 5 (Tao & Schulz, 2007). After specification, the mesoderm cell layer
8. 8
invaginates and spreads laterally (Tao & Schulz, 2007). At stage 9, those laterally
spreading cells start to receive signals from the overlying ectoderm (Tao & Schulz,
2007). These signals cause the cells to become specified as dorsal mesoderm (Tao &
Schulz, 2007). At stage 11, heart precursors are specialized from the dorsal mesoderm
and specify into identifiable clusters of cells (Tao & Schulz, 2007). The heart precursors
are then aligned to form continuous rows of cardioblasts on each side of the embryo (Tao
& Schulz, 2007). From stage 13 to stage 16, the two cardioblast rows start to move
dorsally towards each other (Tao & Schulz, 2007). At stage 16, the cardioblasts rows
encounter each other at the dorsal midline (Tao & Schulz, 2007). The lumen is then
formed from sub-classes of cardioblasts undergoing differentiation programs and the
generation of inflow tracts (Tao & Schulz, 2007). At stage 17, the formation of the dorsal
vessel is complete and synchronized contractions are initiated (Tao & Schulz, 2007).
When the larval transitions into its adult form, the dorsal vessel will then experience
substantial cellular remodelling (Wolf & Rockman, 2011).
Figure 2: Stages of Drosophila heart development (Tao & Schulz, 2007). Stages 5 to 11
present lateral views, where as stages 12 to 17 present dorsal views.
9. 9
1.2.3 - Function
The dorsal vessel is an organ used for hemolymph circulation (Tao & Schulz,
2007). Hemolymph is a fluid consisting of free-floating cells (haemocytes) that are
analogous to the fluids and cells that make up the blood and lymph in vertebrates (Tao &
Schulz, 2007). Within the heart there are ostial cells that create inflow tracts facilitating
the movement of hemolymph into the circulatory system (Molina & Cripps, 2001). This
flow of hemolymph is responsible for transporting immune cells, nutrients, and
molecules necessary to maintain homeostasis (Wolf & Rockman, 2011). The dorsal
vessel is not responsible for oxygen transport like in mammals (Wolf & Rockman, 2011).
Oxygen transport in flies occurs through other systems outlined earlier.
1.3 - Gal 4/UAS System
One system that can be used to target specific genes within the heart of
Drosophila Melanogaster is the UAS-Gal4 system. The Upstream Activating Sequence
(UAS) is an enhancer that Gal4 (a yeast regulatory protein consisting of a DNA-binding
domain and an activation domain) specifically binds to to activate transcription. This
system works by using two distinct transgenic lines that are initially separated (Bier &
Bodmer, 2004). In the case of Drosophila, one strain of flies will contain the Gal4 line
and in another strain of flies there will be a target gene in the form of a cDNA transgene
placed downstream of the UAS (Bier & Bodmer, 2004). When the Gal4 line and UAS
line are separate the target gene is inactive and the UAS line will be viable (Bier &
Bodmer, 2004). Once crossed, the progeny that inherit both the Gal4 line and the UAS
10. 10
line will have the target gene activated (Bier & Bodmer, 2004). The progeny can then be
inspected for any functional or morphological defects or lethality (Bier & Bodmer, 2004).
Not only can this system be used to express target genes, but if coupled with
transgenic RNA interference (RNAi), then the RNAi constructs can be used to turn down
or silence the genes of interest in temporal- or tissue-specific manners (Haley et al.,
2003). RNA interference (RNAi) is the silencing of gene expression through cleavage,
degradation, or blocked translation of a target gene’s mRNA (Yuan et al., 2002). In
Drosophila, the expression of transgenic RNAi begins after a cross between UAS and a
Gal4 driver has been achieved (Ni et al., 2008). The Gal4-UAS system controls the
expression of a gene fragment that is dimerized to produce a double-stranded RNA
(dsRNA) hairpin structure, which then triggers an RNAi response (Ni et al., 2008). This
RNAi response begins with the dsRNA being cut by a type III RNase enzyme called
Dicer into short 21 to 25 base pair molecules called small interfering RNAs (siRNAs)
(Haley et al., 2003). These siRNAs then bind to several proteins creating a RNA-induced
silencing complex (RISC). Energy in the form of adenosine triphosphate (ATP) is then
used to unzip the double stranded siRNA effectively creating single stranded RNA
(ssRNA) (Haley et al., 2003). This step activates the RISC (Haley et al., 2003). Once the
RISC is activated, it can recognize and bind to the target mRNA (Haley et al., 2003).
Once bound, the subunits of RISC begin cleaving the mRNA preventing protein
production (Haley et al., 2003). Once the gene of interest is no longer functional, the
intrinsic dynamics within the progeny will change and these changes can be studied
(Haley et al., 2003). Figure 3 supplies a visual representation of RNAi within Drosophila.
11. 11
Figure 3: Mechanism of RNA interference in Drosophila (D’Adamo, 2006).
1.4 - The Role of Talin, and Robo
There have been many studies conducted on Drosophila heart tubulogenesis that
have identified many essential cell surface factors that have direct implications in human
disease. Robo, and Talin are just two of those factors that play major roles in the
formation and functioning of the Drosophila heart. These two factors will be of primary
focus in the study being conducted.
1.4.1 - Talin
Talin is a signaling molecule located downstream of Integrin (Brown et al., 2002).
Talin (an adaptor protein) is unique for its requirement in both inside and outside integrin
function (Brown et al., 2002). Outside the cell, when Talin binds to the beta subunit on
the integrin tail it activates the integrin, increasing the affinity of extracellular matrix
12. 12
ligands (Brown et al., 2002). On the inside of the cell, Talin works to indirectly and
directly link integrins to the actin cytoskeleton (Brown et al., 2002). In general, Talin
serves as a scaffolding protein to allow for the formation of integrin adhesion complexes
(IAC) at integrin-actin junctions (Brown et al., 2002). It also regulates the organization of
the actin filament network as well as the composition at focal adhesions (Brown et al.,
2002). The structure of Talin is unique in that it is very large (~250kDa protein)
comprised of two domains, a small N-terminal head, and an extended C-terminal rod
(Brown et al., 2002). The N-terminal is used for interacting with membrane proteins.
Both the N- and C-terminal are binding sites for beta integrin (Brown et al., 2002).
1.4.1.1 - Integrins
As mentioned, Talin play a huge role in integrin expression. Integrins are non-
covalent heterodimeric transmembrane receptors that facilitate communication between
the extracellular matrix and the cytoplasmic signaling adaptors to the cells cytoskeleton
(Narasimha & Brown, 2000). Integrins consist of a large extracellular domain, a
transmembrane domain and a short cytoplasmic tail (Narasimha & Brown, 2000). Each
integrin also contains one alpha (α) and one beta (β) subunit. When an integrin receptor
becomes bound to an extra cellular matrix (ECM) ligand, a conformational change is
triggered that exposes binding sites within the integrin cytoplasmic tails (Morse &
Brahme, 2014). These sits mediate specific protein-protein interactions between the
integrin subunits and numerous cytoskeletal adaptors or signaling proteins (Morse &
Brahme, 2014). This enables a physical connection from the cells interior to the outside
of the cell and bidirectional signaling across the plasma membrane (Morse & Brahme,
13. 13
2014). Inside the cell, Integrins act as a signaling hub mediating cell locomotion,
adhesion, polarization, survival, differentiation, cytoskeleton dynamics, and cell
migration (Narasimha & Brown, 2000). One of its more specific functions is its
involvement in maintaining both cellular and extracellular matrix structural identity by
anchoring cells to the ECM, which confers a sense of tissue stability and improves
structural integrity of the cell (Narasimha & Brown, 2000). Drosophila is comprised of
five alpha integrin subunits labeled as αPS1-5 and two beta integrin subunits labeled as
βPS and βv (Narasimha & Brown, 2000).
1.4.2 - Robo
Robo (roundabout) is a transmembrane receptor that plays a major role in dorsal
formation. There are three genes associated with roundabout, Robo, Robo 2 and Robo 3
(Medioni et al., 2010). These three genes are receptors for Slit and are part of a slit-Robo
pathway in Drosophila (Medioni et al., 2010). This pathway serves many functions in
dorsal development such as adhesion, guiding cardioblast alignment, controlling cardiac
cell polarization during alignment, the maintenance of two cell populations during dorsal
migration, and ultimately the formation of the lumen (Medioni et al., 2010). There have
been many studies conducted on detecting the expression locations of the Robo receptors
during development (Santiago-Martínez et al., 2006). The cardioblasts express the single
Robo receptor, and further away from the dorsal midline, the pericardial cells express
both Robo and Robo 2 (Santiago-Martínez et al., 2006). Through experimentation,
researchers have been able to see what happens to development when organisms
experience loss of function mutations of their different Robo receptors (Santiago-
14. 14
Martínez et al., 2006). These loss of function mutations in Robo and Robo 2
simultaneously can lead to a twisted heart tube, parts of the heart tube missing,
aggregations of the cardioblasts, defects in cell adhesion resulting in gaps in the rows of
cardioblasts and pericardial cells, and severe defects in the alignment of the two cell
types (Santiago-Martínez et al., 2006). When the receptors are ablated separately, milder
defects are observed (Santiago-Martínez et al., 2006). With Robo mutants, there is an
occasional misposition of a cardioblast cell (Santiago-Martínez et al., 2006). With Robo 2
mutants, often one or more pericardial cells will migrate too far dorsally and be found
close to the dorsal midline in front of the rows of cardioblasts (Santiago-Martínez et al.,
2006). When Robo 3 is ablated, there are no changes in development, which leads
researchers to believe that it is unlikely involved in cardiac development (Qian et al.,
2005). Further research needs to be conducted.
METHODS AND MATERIALS
2.1 - Control, UAS-RNAi, Gal4, and Balancer Lines
There were various fly lines used to conduct this experiment (Table 1). The
controls that were used are y1
w1118
(yw, BL 6598) and UAS-moesin-mCherry (UAS-
moe-mCherry, Millard and Martin 2008). The UAS-RNAi lines used include UAS-Talin-
RNAi (Vienna Drosophila RNAi Center (VRDC)), and UAS-Robo (Roundabout,
VRDC). The Gal4 lines used include, Tin∆C-Gal4 (Tinman, Lo and Frasch 2001), Svp-
Gal4 (Seven up, R.Cripps), dMEF-Gal4 (myocyte enhancing factor 2, Ranganayakulu et
al. 1996), Hand-Gal4 (A. Pauluat 22), and B2-3-20-Gal4 (Heart, Jacobs' lab central
stocks). Heart was created via a transposon exchange using the B2-3-20 enhancer trap.
15. 15
The balancer chromosomes used include CyO and TM3,Sb, which were both obtained
from Jacobs' lab central stocks (Table 2).
The Gal4 lines used in this project are all expressed in the heart, however they
differ from one another in their specific expression pattern. Tin-Gal4 for instance is one
of these lines. The tinman gene is expressed early in embryogenesis in two major cell
types in the Drosophila heart, the cardial and pericardial cells (Evans et al., 1995). Tin-
Gal4 is also expressed in these cells, but more specifically in the tinman cells of the
embryonic heart (Wolf & Rockman, 2011). Each side of the embryonic dorsal vessel is
arranged in sets of four tin positive cardioblasts that form the contractile cells of the heart
and are separated by pairs of Svp-expressing ostial cells (Wolf & Rockman, 2011).
Tinman has the function of determining the formation of visceral mesoderm, heart
progenitors, specific somatic muscle precursors and glia-like mesodermal cells (Evans et
al., 1995). When a loss of function occurs, the visceral mesoderm and the heart
primordial fail to develop, and the fusion of the anterior and posterior endoderm is
impaired (Bodmer, 1993). Another Gal4 line that was used is Hand-Gal4. In Drosophila,
the hand gene is expressed at a late stage in embryogenesis in the cardioblasts, pericardial
nephrocytes, and the lymph gland hematopoietic pogenitors (Lo et al., 2007). The hand
gene functions as a transcriptional activator and without it the heart and lymph gland fail
to develop. The lack of function of the hand gene can also result in hypoplastic
myocardium and a deficiency of pericardial and lymph gland hematopoietic cells,
accompanied by cardiac apoptosis (Lo et al., 2007). Svp-Gal4 is another Gal4 line that
was used. This orphan nuclear receptor gene, svp is primarily expressed early in
embryogenesis in the myocardial cells in the posterior aorta (Shah et al., 2011). Svp-Gal4
16. 16
is also expressed here, but more specifically in the seven-up cells in the embryonic heart
(Wolf & Rockman, 2011). This is opposite of the expression pattern found with Tin-Gal4
(Wolf & Rockman, 2011). They form muscular ostia, which permit hemolymph to enter
the heart for circulation, and they form the wall of the dorsal vessel (Molina & Cripps,
2001). Another Gal4 line that was used is Dmef-Gal4. The dmef gene in Drosophila is a
transcription factor required for muscle development (Iklé et al., 2008). It is expressed
early in embryogenesis, accumulates in the embryonic muscles and activates target genes
throughout the mesoderm (Iklé et al., 2008). Without this gene the fly is unable to form
cardiac, visceral, and skeletal muscles (Iklé et al., 2008). The final Gal4 line used is
Heart-Gal4, which has an uncharacterized pattern thought to be in the dorsal vessel
(personal communication, J. Vanderploeg).
Table 1: A list of stocks used within this experiment
Stock Full Name Source (Stock #)
yw y1
w1118
Bloomington (#6598)
UAS-moe-mCherry Moesin-mCherry Millard and Martin 2008
UAS-RNAi Lines
UAS-Talin-RNAi Talin Vienna Drosophila RNAi
Center (VRDC) (#40399)
UAS-Robo Roundabout VDRC (100624KK)
GAL4 Lines
Tin∆C-Gal4 Tinman Lo and Frasch 2001
Svp-Gal4 Seven up R. Cripps
dMEF-Gal4 Myocyte enhancer factor 2 Ranganayakulu et al. 1996
Hand-Gal4 Hand A. Paululat 22
B2-3-20-Gal4 Heart Jacobs' lab central stocks
Table 2: A list of the balancers used within this experiment
Name Source (Stock #)
Chromosome 2: Sco/CyO Jacobs' lab central stocks
Chromosome 3: D/TM3 Jacobs' lab central stocks
17. 17
2.2 - Fly Maintenance
Part of this experiment involves collecting virgin females that will be used in
setting up crosses. Before these females can be collected though, the control, UAS, Gal4,
and balancer stocks needed to be built up. The stocks were kept in polystyrene vials
containing a sucrose, yeast, and agar medium, and stored at 25°C. To ensure the
maximum number of eggs were being laid, twice a week (Monday and Thursday) the
adult flies were transferred to new vials containing fresh food.
2.3 - Determining Which Chromosome the UAS Lines are on
Before the experiment could be conducted, the chromosomes that Robo and
Moesin-mCherry were on had to be identified, as it was previously unknown. This is
important because this information is used in determining what balancers need to be
crossed with the UAS-RNAi lines. If the UAS-RNAi lines are crossed to incorrect
balancers then the results will be inaccurate. To do this virgin females from both
transgenes were first crossed with males from the D/TM3 stock and Sco/CyO stock
(Figure 4 Cross #1). Once the progeny eclosed, the ones from the UAS line crossed with
D/TM3 with the phenotype wild type wings and stubble and the ones from the UAS line
crossed with Sco/CyO with the phenotype curly wings and wild type hairs were kept.
From these two sets of progeny, males were collected from each and crossed with female
virgins from the yw stock (Figure 4 Cross #2). Once the progeny emerged after ~12 days,
they were split into groups based on their phenotypes and then counted. In relation to
Figure 4, if all four phenotypes were present in the progeny for example, then the
location of the transgene, UAS-moesin-mCherry on chromosome 3 would be incorrect. If
18. 18
only genotypes two and four were to appear though, then this transgene would in fact be
on chromosome 3. This rational also applies to the UAS line of interest being crossed
with Sco/Cyo, but instead of looking at chromosome 3, chromosome 2 is being looked at.
Figure 4: A sample cross used in determining what chromosome the UAS-moesin-
mCherry transgene is on.
2.4 - Genetic Scheme
Once the stocks were built up, ten female virgins were collected from each UAS-
RNAi line and crossed with ten males from their associated balancer stocks. For example,
the Talin transgene is on chromosome 3 and so its associated balancer that it was crossed
with is D/TM3,Sb, which is also located on chromosome 3 (Figure 5). Before the flies
were crossed, the females and males were kept separate in glass pairwise tubes containing
a sucrose, yeast, and agar medium, until enough were collected. Five flies were kept in
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19. 19
each pairwise tube. Once all 20 flies were collected five females and five males from the
separate tubes were crossed together to create a total or two pairwise tubes with ten flies
each. After the cross was set up it took about 12 days before progeny begin emerging.
During these 12 days the adults were flipped into new pairwise tubes every Monday and
Thursday. One week after the cross was started, 30 female virgins from each of the Gal4
stocks began to be collected and separated into three pairwise tubes with ten females
each. Once progeny began emerging, if working with a transgene on chromosome 3, the
progeny that had stubble and wild type wings were kept, while the progeny with wild
type hairs and dichaete wings were discarded. If working with a transgene on
chromosome 2, the progeny that had curly wings and wild type hairs were kept, and the
progeny with wild type wings and no bristles on the edge of their thorax were discarded.
From the progeny that were kept, 15 males were transferred and separated into three
pairwise tubes. These males were then crossed with the females from a Gal4 line as
shown in Figure 5 cross #2. This created a total of three pairwise tubes each containing
five males and ten virgin females. Since females do not lay very well the first few days,
after about three days the flies from each of these pairwise tubes were flipped into three
new pairwise tubes and the old tubes were thrown out. At this point the females should
now be laying eggs very well. After another two days, the flies were then flipped again
into new pairwise tubes. The three tubes now containing the flies were kept at room
temperature to continue laying more eggs, while the three old tubes were labelled with
either RT (room temperature), 18°C, or 29°C and transferred to areas with those
temperatures. This last step was repeated two more times until there were nine pairwise
tubes total with three sitting at RT, three at 18°C, and three at 29°C. Once all nine were
20. 20
set, the next step was to wait until the first progeny eclosed. Since metabolism in flies
increases as temperature increases, the flies sitting at 29°C developed and eclosed the
fastest, followed by the ones at room temperature and then the ones at 18°C. Once the
flies began emerging, they were separated by phenotype and recorded under their
associated genotypic and temperature specific column as shown in Appendix A. The flies
in the 29°C pairwise tubes were only counted for seven days after first emergence, where
as the flies in the RT and 18°C pairwise tubes were counted for ten. Counting time was
less for the 29°C flies because of their greater metabolism creating a faster life cycle.
This experiment required only first generation progeny and so counting ceased before the
second generation had the chance to emerge which generally took seven to ten days
depending on the temperature the flies were kept at. The flies were counted every
morning. After all the counting was completed, percent viability was calculated using the
equation in Figure 6.
21. 21
Figure 5: A sample cross of female virgins from a UAS-Talin stock, that is located on
chromosome 3 being crossed with males with the phenotype of dichaete wings over
stubble. The male progeny from this first cross that had the phenotype of wild type wings
and stubble were then crossed with female virgins from a Gal4 line. The resulting
progeny from this second cross are shown.
% 𝑉𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 =
𝐸𝑥𝑝𝑒𝑟𝑖𝑚𝑒𝑛𝑡𝑎𝑙
𝐶𝑜𝑛𝑡𝑟𝑜𝑙
∗ 100
% 𝑉𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 =
113
100
∗ 100
% 𝑉𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 = 113%
Figure 6: A sample calculation of percent viability from the UAS-Talin and TinGal4
cross at room temperature. Experimental refers to the progeny that had both the Gal4 and
♀
!(!)
!(!)
;
!"#!!"#$%
!"#!!"#$%
𝑥
!(!)
!(!)
;
!
!"!,!"
♂
♂
!(!)
!(!)
;
!"#!!"#$%
!"!,!"
𝑥
!(!)
!(!)
;
!"#$%&!
!"#$%&!
♀
+(𝟐)
+(𝟐)
;
𝑻𝒊𝒏𝑮𝒂𝒍𝟒
𝑻𝑴𝟑, 𝑺𝒃
+(𝟐)
+(𝟐)
;
𝑻𝒊𝒏𝑮𝒂𝒍𝟒
𝑼𝑨𝑺 − 𝑻𝒂𝒍𝒊𝒏
Progeny
Cross
#1
Cross
#2
22. 22
the RNAi, while the Control only has the Gal4. The raw data can be found in Appendix
A.
RESULTS
The goal was to cross each UAS-RNAi line to each Gal4 line in order to reduce
expression of the UAS lines in the heart cells. The ones that were completed include
UAS-Moesin-mCherry crossed with Tin-Gal4, UAS-Talin crossed with Tin-Gal4 and
Svp-Gal4, and UAS-Robo crossed with Tin-Gal4, Svp-Gal4, Hand-Gal4, Dmef-Gal4, and
Heart-Gal4. A sample cross showing how each of the crosses were conducted is located
in Figure 5 and the results of these crosses are located below.
3.1 - UAS-Moesin-mCherry
The first step was in determining which chromosome this transgene was on. By
looking at the results located in Appendix B, it is apparent that UAS-moesin-mCherry is
in fact located on chromosome 2 and not on chromosome 3 as previously thought. It is
good to note that since this transgene was believed to be on chromosome 3 before it was
tested, the experiment was conducted as so.
As shown in Figure 7, percent viabilities were greater than 100% for each
temperature. This means that in all cases there was a greater number of progeny with the
UAS-moesin-mCherry and Tin-Gal4 lines, than there was with just the Gal4 line.
23. 23
Figure 7: The percent viability of the control, UAS-Moesin-mCherry when crossed with
TinGal4. The number on the bottom of each bar represents the sample size.
3.2 - UAS-Robo
The experiment began by determining which chromosome this transgene was
located on. By looking at the results located in Appendix B, it is apparent that UAS-Robo
is located on chromosome 2. After this information was obtained there were five crosses
conducted. As shown in Figure 8, each cross at each temperature obtained a percent
viability greater then 100%. The data is not available for Robo knockdown with Svp-
Gal4 and Hand-Gal4 as there was not enough time to collect that data.
129
194
217
0
20
40
60
80
100
120
140
160
TinGal4
%
Viability
Genotype
18
degrees
Celcius
Room
Temperature
29
degress
Celcius
24. 24
Figure 8: The percent viability of UAS-Robo when crossed with TinGal4, DmefGal4,
HeartGal4, SvpGal4, and HandGal4. The numbers on the bottom of each bar represents
the sample size.
3.3 - UAS-Talin
As shown in Figure 9, percent viabilities varied greatly between the two crosses
that were conduced with UAS-Talin. Talin knockdown with Tin-Gal4 showed a percent
viability of 93% at 18°C, over 100% at RT, and 0% at 29°C. Talin knockdown with Svp-
Gal4 on the other hand showed percent viabilities great then 100% at all temperatures.
The latter results are not a good representation though as sample sizes were very low,
indicating a weak cross. For more accurate results, this cross needs to be done again.
137
41
114
N/A
N/A
233
112
111
158
154
189
78
67
176
42
0
20
40
60
80
100
120
140
160
180
200
220
Tin>
RoboRNAi
dMEF>
RoboRNAi
Heart>
RoboRNAi
Svp>
RoboRNAi
Hand>
RoboRNAi
%
Viability
Genotype
18
degrees
Celcius
Room
Temperature
29
degress
Celcius
25. 25
Figure 9: The percent viability of UAS-Talin when crossed with TinGal4 and SvpGal4.
The numbers on the bottom of each bar represents the sample size.
DISCUSSION
In the future, a screen will be designed to isolate mutations that potentially work
with Talin or Robo during heart development or later function. Therefore the goal is to
identify a “sensitized” genotype with reduced viability due to knockdown of Talin or
Robo specifically in the heart. The UAS-Gal4 lines that would be the best are the ones
that produce a percent viability less than a 100% or greater then 0%. This will show that
the associated UAS-Gal4 pathways used to produce a fully functional heart have been
hindered in some way. These percentages are required due to the fact that the screen that
will be performed will be isolation mutations that weaken the stocks even further. At 0%
84
8
213
72
65
9
0
20
40
60
80
100
120
140
160
180
200
220
Tin>TalinRNAi
Svp>TalinRNAi
%
Viability
Genotype
18
degrees
Celcius
Room
Temperature
29
degress
Celcius
26. 26
viability, there is no way to weaken a stock further and at 100% viability, the stocks are
at full health and so any mutations used will not be helpful as it will not be known if that
mutation is weakening the pathway of interest or another pathway.
Out of all the crosses conducted there was only one that ended up with a percent
viability less than 100% and greater than 0%. This was UAS-Talin knockdown with
TinGal4 at 18°C. This would suggest that UAS-Talin knockdown with TinGal4 would be
the best to use in generating a future screen, but just like many of the results that are
appearing way over 100%, this result could appear slightly under due to high amounts of
statistical error present in the results, and thus it could actually be 100%. This is
especially true since the UAS-Gal4 system is temperature dependent and thus becomes
more efficient at higher temperatures (Duffy, 2002). Therefore, one would expect a
greater amount of reduced viability as the temperature increases, as shown in the RT and
29°C results, not decreases as shown in the 18°C results (Duffy, 2002). This means that
there is still the potential for this cross to be used at a higher temperature. Since the RT
percent viability is over 100% and the 29°C percent viability is 0%, it is hypothesized
that by instead testing this cross at a temperature between 26°C and 28°C, that percent
viabilities lower then 100% and greater then 0% could be achieved.
With UAS-moesin-mCherry, even though it was crossed using the incorrect
balancer, the data is still useful. As a control, it is expected that it should have no effect
on heart development and thus viability should not be reduced. This means that all
phenotypes should be seen at a 1:1 ratio, which is roughly what was seen.
Before the experiment was conducted, it was expected that the UAS-Gal4 system
would produce reduced viability in all Robo and Talin crosses as this system should be
27. 27
reducing their expression in the heart cells. Instead the majority of the crosses except two
(mentioned above) at each temperature showed no reduced viability. There are many
reasons why this could be the case. The first reason could be that even though the RNAi’s
are reducing expression levels, this could still be enough for the flies to be healthy.
Another reason could be that the RNAi’s are just not working due to low responsiveness
of the Gal4 to the UAS line for example. Other lines that could be tested include Integrin,
3Robo-RNAi, or Slit-RNAi.
During the experiment, there were a series of Gal4’s that were used. The purpose
of using different Gal4’s is because they all have different expression patterns. This
means that with each cross set up, when the UAS-Gal4 system becomes active, there will
be reduced expression in different pathways at different stages in Drosophila
development each time. This can ultimately show us which pathways have the greatest or
least impact on viability. Another Gal4 that could be used is actin-Gal4.
In conclusion, the results suggest that none of the lines should be used in a future
screen without further experimentation first. The results were not what was expected and
there were very high amounts of statistical error. These need to be corrected before this
study can proceed.
28. 28
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1738(94)00032-Q
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Andrew, A. (2010). Expression of Slit and Robo Genes in the Developing Mouse
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Molina, M. R., & Cripps, R. M. (2001). Ostia, the inflow tracts of the Drosophila heart,
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32. 32
APPENDIX A: Raw Data From the Control, UAS-RNAi and Gal4 Crosses
For the following tables, the red represents the results at 18°C, the blue represents
results at RT, and the green represents results at 29°C.
Table 1: Results From UAS-Moesin-mCherry
Line Control # of Control
Progeny
Experimental # of
Experimental
Progeny
%
Viability
Sample
Size
Tin
TinGal4>+ 63 TinGal4>UAS-
Moesin-
McherryRNAi
66 105 129
77 117 152 194
83 116 143 217
Table 2: Results From UAS-Talin
Line Control # of Control
Progeny
Experimental # of
Experimental
Progeny
%
Viability
Sample
Size
Tin
TinGal4>+ 44 TinGal4>UAS-
TalinRNAi
41 93 85
100 113 113 213
65 0 0 65
Svp
SvpGal4>+ 4 SvpGal4>UAS
-TalinRNAi
4 100 8
29 43 148 72
3 6 200 9
Table 3: Results From UAS-Robo
Line Control # of Control
Progeny
Experimental # of
Experimental
Progeny
%
Viability
Sample
Size
Tin TinGal4>+ 56 TinGal4>UAS-
RoboRNAi
81 145 136
116 117 101 233
94 95 101 189
Dmef DmefGal4>
+
13 DmefGal4>UA
S-RoboRNAi
28 215 41
54 58 107 112
38 40 105 78
Heart HeartGal4>
+
40 HeartGal4>UAS
-RoboRNAi
74 185 114
50 61 122 111
22 45 204 67
Svp SvpGal4>+ N/A SvpGal4>UAS-
RoboRNAi
N/A N/A N/A
60 98 163 158
83 93 112 176
