The document summarizes an experiment that aims to identify genes involved in Drosophila hematopoiesis using RNA interference. The experiment crosses flies containing the Hand-Hemolectin-Gal4 construct, which drives GFP expression in blood cells, to flies containing UAS-driven RNAi constructs targeting different genes. Offspring are observed under fluorescence microscopy. Genes are identified as involved in hematopoiesis if RNAi knockdown affects blood cell development as seen by changes in GFP expression patterns compared to controls. Several genes were found to affect hematopoiesis based on this criteria, including genes related to chromosome condensation, cell division, immune function, and protein synthesis.

1. Satkartar Khalsa
Abstract
Hematopoiesis, refering to blood development in an organism, still has many aspects and
mechanisms that we do not know of today. In order to understand hematopoiesis on a more
intimate level, it must be broken down into building blocks. The building blocks that we must
understand are the genes involved in blood development. Through using the UAS-gal4 system,
genes are identified through fluorescent microscopy. By identifying which genes have an effect
on the blood system, including the lymph glands and pericardial nephrocytes, hematopoiesis will
become less of a mystery. After identifying which genes affect the blood system as a whole, it’s
important to see where the genes are specifically needed. Using the hemolectin-dsRNA system,
the cortical zone’s impact on blood development will be tested. If the cortical zone, is affected by
the gene, more information about the location of the genes expression will be obtained. There
were many genes that had an effect on the entire lymph gland and/or the cortical zone, many of
which were involved in signal transduction pathways, and translation.
Introduction
The complex blood formation system present in Drosophila melanogaster still has
elements and aspects unknown to us today. Blood formation in this system is involved in three
different places in the fruit fly: pericardial nephrocytes, lymph gland, and heart. The lymph
gland, which is the main focus of this experiment, is the region in which blood cells, hemocytes,
are formed and maintained. These blood cells are analogous to the vertebrate’s myeloid blood
cells. There are haemolymph in flies and lymph in humans. The main three types of blood cells
present are those that are phagocytes, which are called plasmotocytes, those that assist in clotting
coagulation called lamellocytes, and those that help cytokine secretion, which are known as
crystal cells. The cells that have yet to differentiate into these three types are located in the
medullary zone (MZ). The cells that differentiated into one of the three blood cells are located
on the outskirts of the medullary zone, called the cortical zone (CZ). The niche controlling the
progenitors is known as the Posterior Signaling Center (PSC).
There are important ways in which the progenitors in the medullary zone maintain their
stem-like qualities. The PSC expresses Hedgehog, which is necessary for the upkeep of
progenitors in the MZ. Without both PSC and normal hedgehog expression, the size of the
medullary zone either decreases without the presence of PSC and Hh or increase drastically with
overexpression of Hh and a larger PSC. Hedgehog uses a two-step process in order to signal to
the cells in the medullary zone to sustain their role as progenitors. In the first process, a cleaved
Ci protein enters the nucleus and turns target genes off with the help of a co-repressor. In the
second process, an intact Ci protein enters the nucleus and turns target genes on with the help of
a co-activator. With these two processes, Hh and PSC help maintain the progenitors in the
medullary zone.
The highly complex and conserved process of blood formation still needs to go through
extensive research because of the vague nature of the genes involved in normal hematopoiesis. In
hopes of finding the genes that are required for normal blood formation, the results of this
research must be delved into. To look into what genes are needed for this process, it must be seen
what genes affect the normal and healthy communication between populations of cells, normal
proliferation and differentiation that initiates loss of cells and precursors and also death of cells.
Crossing UAS-gal4 lines with UAS-dsRNA lines will give us larvae to picture using
fluorescence to show the affect of the absence of a certain gene.
Materials and Methods

2. The first step in this research endeavor is getting fly stocks in which to experiment with.
An array of stocks of flies were obtained through the Biomedical Research 10H Lab. The BR
10H Lab acquired TRiP lines through the Drosophila RNAi Screening Center at Harvard
University, VDRC lines through the Vienna Drosophila RNAi Center, and NIG lines through the
Drosophila Genetic Resources Center in Japan. A supplemental table at the end of this paper
reports where each line came from. These lines were selected based on previous research and
knowledge about the genes correlated with the stocks. The genes of each stock were analyzed
using the information regarding the biological and molecular function of the genes in question on
Fly Base. One of the protein sequences was then copied and a BLAST search was performed on
it. The human homologs of the protein were found. Then the conserved domains were identified
as well as their normal function in the protein. Protein function could then be determined based
on the conserved domains functions. Once these stocks were obtained from the corresponding
companies, virgins were collected from hand hemolectin Lineage Tracing (HHLT) line, which
was graciously supplied by the LS Core Office.
I crossed two different stocks for each experiment and maintained a control cross
throughout the entire process. For the control of the first half of this experiment I crossed HHLT-
gal4 and 5905. HHLT stands for Hand Hemolectin Lineage Tracing. It contains the UAS-GFP
gene and also produces Gal4. The genetics of this cross is explained in Figure 1 and the actual
mechanism for this cross and how it generates GFP is explained in Figure 2. This means that it
produced the green fluorescent protein, which allowed us to take fluorescent pictures of the
larva. For the second half of the experiment, I crossed Hemolectin-gal4 and 5905, which
exhibited fluorescence only in the cortical zone.
5905 contains mutations that result in the abnormal white color of the eye. However, as
the control for this experiment, it does not include mutations that could have an effect on
hematopoeisis, so it can be assumed that the control cross is what normal healthy blood
development looks like.
In order to understand this control and experimental crosses, it’s important to understand
the mechanism of HHLT-gal4. This stock of flies have two promotors present in blood cell DNA
Figure 1
- A cross between HHLT-
gal4 lines
and UAS-RNAi lines, their
F1 progeny and the F1
progeny’s genetic makeup.
With the combinations of
these two mechanisms,
fluorescence is expressed in
blood cells and a certain
gene is “knocked down” to
test its importance in
hematopoiesis.

3. that directly correlate to this research: hemolectin and hand. Both of these promoters are attached
to the gal4 gene and initiate blood cell expression of Gal4. Hemolectin is only activated while
the blood cells are mature and circulating in the lymph gland. Hand is activated from the late
embryo stage to the first larval instar stage in the cardiac mesoderm such as the heart, lymph
gland, and pericardial tissue. However, the expression in pericardial tissue and heart are decrease
to unreadable levels after this stage. While the organism is not these stages, the gene is turned off
and Gal4 is not created. By using both hand and Hemolectin, HHLT-Gal4 is able to express GFP
through every developmental stage. In order to enhance the image taken of the larvae UAS-FLP
was also utilized. hand-Gal4 was the first of the two promoters to be activated and thus the first
to make gal4. This gal4 then activates another gene UAS-FLP to produce the recombinase FLP.
FLP is able to cut parts of DNA and delete or add DNA into parts of the genomic sequence. In
our case, FLP excises an area of the Actin5C-gal4 gene that inhibits the production of gal4. By
eliminating this part of the Actin5C-gal4 gene, transcription can proceed, which would inevitably
allow the creation of gal4. In all scenarios, when gal4 is present in a cell it binds to the UAS that
is involved in the production EGFP. Lymph gland cells permanently express GFP in all ensuing
stages after the production of gal4 from hand (Mondal, Shim, Evans, & Banerjee, 2014).
The previous description of GFP mechanism and Figure 2 explained the HHLT process
for exhibiting GFP, however the most important concepts of functional genomics and gene
knockdown are still yet to be covered. I crossed HHLT with a variety of stocks that have
different UAS-dsRNA systems. I took males from stocks of various UAS-dsRNA lines and
crossed them with HHLT-gal4. Each individual UAS-dsRNA line corresponded to a specific
gene. I tested these lines and recorded whether the gene “knockdown” had any affect on the
blood development in larva.
The UAS-dsRNA aspect of each line created a loss-of-function phenotype for a specific
gene of interest. I used RNA interference to eliminate the mRNA before it became a protein. The
Figure 2:
- mechanism of the
production of GFP
using gal4 as an
intermediate protein
- as seen through this
figure, UAS-FLP allows
more GFP to be created
in only blood cells

4. upstream activating sequence is not in fact attached to RNAi, as this is just the mechanism. It is
attached to a sequence of DNA that is encoded by an inverted repeat. This means that in the
DNA, there are two sections on the same strand that are homologous. This sequence will cause
the subsequent RNA to invert and become a double stranded piece of RNA that looks like a
hairpin. The actual hairpin is the area in which the sequence is not homologous and therefore
cannot hydrogen bond with itself. These hpRNA’s are then cut up into siRNA’s, also known as
small interfering RNA’s. These short double stranded RNA sequences bind to RISC, RNA-
induced silencing complex, which cleave the double stranded siRNA’s into single strands. RISC
then catalyzes the binding of the target mRNA, where Dicer cleaves both strands into short
fragments. The point of this mechanism is to decrease the concentration of the
target genes mRNA, and therefore decrease the concentration of proteins that
would have been created.
HHLT-gal4 and UAS-dsRNA are both homozygous for these genes and therefore when
crossed create heterozygous HHLT-gal4 and UAS-dsRNA lines that have the dual function of
producing gal4 to exhibit the expression of GFP in blood cells, but also carry out post-
transcriptional gene silencing of a target gene. If the progeny exhibits any level of GFP that is
uncharacteristic of the control cross then, the gene can be characterized as a gene associated with
hematopoiesis.
For the second half of this experiment, I used Hml-gal4 to cross with the stocks I
had crossed with HHLT previously. This was done to find the results of whether the stocks
exhibit a phenotype in the cortical zone. I used the same experimental procedure stated above but
will be looking to see if there is a different outcome in the cortical zone. I crossed Hemolectin-
gal4 and the same UAS-dsRNA lines. The only difference with the second part of the experiment
is where the fluorescence is expressed. As stated above, hand and hemolectin are expressed at
Figure 3:
-shows the RNAi
mechanism that
shows how a gene
cane be silenced
in this experiment
- also shows the
result of gene
silencing if the
gene of interest is
important to
hematopoiesis

5. different stages in the larvae’s development. Hemolectin-gal4 exhibits fluorescence in the
cortical zone, where the differentiated cells reside.
Virgin females from HHLT-gal4 or Hml-gal4 were put into a fresh vial conducive for
crossing conditions and were combined with males from UAS-dsRNA lines that were ordered
from the organizations listed above. Approximately 8 males were combined with 12 virgin
females to ensure a successful cross that produced surplus progeny to take pictures of. These
crosses were immediately put into an incubator kept at 29 degrees Celsius to promote the
expression of gal4, which allowed the accurate and amplified fluorescence in the larvae. These
crosses were kept in the incubator for approximately three days and then were transferred to
brand new vials to supply the crosses with fresh food and another vial in which to lay eggs in.
After transferred, both vials were put back into the incubator until larvae crawled up the sides of
the vials.
When the larvae were in third instar larvae stage, using forceps, they were taken out and
washed. They were placed on chilled glass wells and oriented in a uniform manner to be
pictured. They were oriented dorsal side up with the trachea visible and anterior on the top. The
pictures were taken with Auxiocam HRc. Each set of crosses, from week to week, were
compared to the control cross 5905. Each week had a different exposure level and the uniformity
was maintained by comparing the crosses with the control cross.
Results
5905 was used as our control cross because it was known that gal4 would be present in
the blood cells and genes involved in hematopoiesis would not be affected. Therefore
experimental crosses could be compared to this control cross. The control cross was recreated
every week to standardize the pictures taken of the larvae. By measuring the exposure level
needed for each week, the experimental fluorescence could be compared to the uniform 5905’s
level of GFP or RFP.

6. There are four different categories the resulting phenotypes could fit into: phenotype with
less GFP, phenotype with more GFP, outliers, and phenotype consistent with the controlled
cross, which did not have any implications. The RFP phenotypes is attached to the data
organized by the GFP phenotypes. All of the results can be compared to Figure 1A, which
contains the control cross.
Experimental crosses conducted with the HHLT and Hml are included as follows:
The first phenotype discussed exhibits variable decreased function in GFP. In Figure 1
there are seven crosses, 32510, 24354, 34867, 34335, 34483, 103393, and 34711, where there
was significant decrease in GFP.
In 32510, almost all GFP was absent from the progeny. The Hml phenotype exhibited an
increased level of RFP in the secondary lobe of the lymph gland where there had previously been
none as seen in Figure 1B. The gene knock-down associated with this cross is CG7420. CG7420
plays a role in regulating chromosome condensation, which is important in the regulation of gene
expression. The human homolog of this gene is secretion-regulating guanine nucleotide
exchange factor.
In 24354, there is almost no GFP present in the larva as seen in Figure 1C. The Hml
phenotype of the larvae was consistent with the control cross. The gene linked to this cross is
TER94, whose role is protein binding and ATPase activity to facilitate cell division. The human
homolog of this gene is transitional endoplasmic reticulum ATPase.
In 34867, all but one larvae had significantly decreased GFP. The lymph gland and
pericardial cells were not lit up with GFP, whereas the circulating cells did exhibit diminished
GFP, as seen in Figure 1D. The RFP from the larvae of the cross with Hml had a phenotype that
was consistent with control cross. The gene knocked down in this cross is Contactin and it has
many functions, some of which are receptor plasma membrane protein, and involvement in
immunity as T-cell receptors. The human homolog to this gene is Contactin 3 (plasmacytoma
associated).
In 34335, a third of the progeny did not exhibit GFP. The rest of the larvae had normal
GFP as shown in Figure 1E. The Hml phenotype was consistent with the control. The gene
knocked down in this cross was SLY-1 homologous. Its molecular function is SNARE binding,
which is the selective, non-covalent interaction with a SNARE (soluble N-ethylmaleimide-
sensitive factor attached protein receptor) protein. (Kimura, Mizoguchi, & Ide, 2003) Its human
homolog is sec1 family domain-containing protein 1 isoform a.
Shown in Figure 1F, all of the larvae of 34483 exhibited GFP, however each of them had
varying levels of GFP. Roughly one third of the larvae were normally bright but the rest showed
very dim levels of GFP in the lymph gland and pericardial nephrocytes. Roughly one third of the
larvae had Hml phenotypes consistent with the control. The rest of the larvae had diminished
levels of RFP in the cortical zones. The gene knocked down from these crosses is CG33123. Its
function is anti-codon binding as a leucyl-tRNA synthetase. Its human homolog is leucine--
tRNA ligase, cytoplasmic.
In 103393, there was no GFP present in the lymph glands or any other structures but
there were small areas of concentrated GFP sporadically present throughout the bodies of each
larva as shown in Figure 1G. The Hml larvae exhibited more circulating blood cells as well.
There was also increased expression of RFP in the pericardial nephrocytes. The gene associated
with this stock is crooked neck. Its gene function is RNA processing involved in transcription. Its
human homolog is crooked neck-like protein 1 isoform a.

7. In 34711, there was GFP present in the pericardial nephrocytes, not the lymph glands.
When this stock was crossed with Hml, it had a similar phenotype where the pericardial
nephrocytes were present. The gene that was knocked down affected the cells present in the
lymph gland but not in the pericardial nephrocytes or the circulating blood cells. The knocked
down gene was eIF2B-ε. It plays a role in translation initiation factor activity through the
exchange of GDP for GTP. The human homolog of this gene is eukaryotic translation initiation
factor 2B, subunit 5 epsilon, 82kDa, isoform CRA a.
The second phenotype discussed exhibits variable increased function in GFP but
characteristically similar to the control cross. There are four where there is increased GFP that
looks much like the control cross, which are the crosses 8269, 103383, 31196 and 23659.
In 8269, there was an increase in GFP as seen in Figure 2A. The circulating blood cells
were much more widespread and seemed to be in clumps throughout the entire body compared to
the control cross, where although the circulating cells were present, were not large enough to
pinpoint. The gene knocked down in this cross was head involution defective. It is involved in
protein binding as a topoisomerase, which helps accurate chromosome transmission. It has a
human homolog, which is hCG19253, isoform.
There was a clear increase in both brightness and area in 103383. The area in which the
lymph gland lit up was much larger in size and took up almost half of the body as seen in Figure
2B. The larvae had RFP in the pericardial nephrocytes. The gene knocked down in this cross
was Clathrin heavy chain, which is involved in protein binding, specifically clathrin light chain.
This gene has many human homologs one of which is clathrin heavy chain 1 isoform 1.
In 31196, half of them displayed identical increases in GFP in location and size. The
lymph gland was significantly larger and brighter compared to the controlled cross. As seen in
Figure 2C, the other half of this cross displayed similar amounts of GFP to the controlled cross.
The gene knocked down in this cross was Nucleoporin 93kD-1, which encodes for a protein that
is a component of the nuclear pore complex. It is required for the correct assembly of the nuclear
pore complex. This aspect of the protein assists protein import into the nucleus. Its human
homolog is KIAA0095.
In 23659 there was more GFP in some of the larva compared to the control. The lymph
gland GFP was larger and brighter. The lymph gland took up roughly twice as much space than
is characteristic in a normal larvae as Figure 2D illustrates. There were also more circulating
cells that could be seen in clumps, instead of spread throughout the body. These clumps were not
concentrated however, and were roughly transparent but did exhibit GFP. The RFP in the larvae
from the cross with Hml was consistent with the control cross. The gene knocked down in these
crosses was Smg5 and its function is to degrade unusable mRNA and activate telomerase at the
site of polymerization. Its human homolog is KIAA1089 protein.
The third important phenotype were the outliers that did not simply have an
overexpression or under expression of GFP in the lymph glands. These crosses showed a
completely different phenotype that was not consistent with the controlled cross regarding
location of GFP. These crosses exhibited GFP in unique places and in different quantities that
weren’t characteristic of 5905 and were not merely an overproduction or underproduction of
GFP in the lymph gland or pericardial nephrocytes. The crosses that were phenotypically
different from the controlled cross in pattern of GFP were 106240, 25572, 103250, and 110477.

8. With the cross between 106240 and HHLT, there was no clear phenotype shown in
Figure 3A. In a third of the larvae, there seemed to be a distinct area lit up by GFP however, this
area did not correspond to the lymph glands. The centralized region of GFP was located much
lower than the lymph gland, which could be seen in the anterior of the body in the controlled
cross 5905. In the remaining larvae pictured from 106240, the bodies were lit up with GFP,
however there was no concentrated area. The area that was lit up in 106240 could be speculated
as still the lymph gland. When crossed with Hml, the resulting progeny had an increased level of
RFP in the secondary lobe of the lymph gland where there had previously been none. The gene
involved was Ef1α-like factor. Ef1α-like factor’s main biological function is its vital role in
translational termination. The human homolog is eukaryotic peptide chain release factor GTP-
binding subunit ERF3A isoform X2.
In 25572, there were concentrated areas of GFP that were not the lymph gland, illustrated
in Figure 3B. There was a dim lit area that was consistent with the lymph gland. It looked like
the fat body was covering up the brightness of the GFP. However, on top of the fat body there
were abnormal concentrations of GFP. There were layers of GFP spots that were uncharacteristic
of lymph glands. The RFP phenotype of this cross was an increase in brightness in both the
pericardial cells and the secondary lobe of the lymph gland. The gene involved in this cross was
Heparan sulfate 3-O sulfotransferase-A and its main function is [heperan sulfate]-glucosamine 3-
sulfotransferase 1 activity. It helps catalyze chemical reactions. The human homolog of this gene
is Chain A, Human 3-O-Sulfotransferase Isoform 5 With Bound Pap.
Cross 103250 was similar to 25572 in that there were abnormal localizations of GFP in
locations that were clearly not the lymph gland as seem in Figure 3C. Although the lymph gland,
and pericardial nephrocytes lit up with GFP, there were also one or more concentrations on each
larvae that were absent from the control crosses. In general these larvae were brighter than the
control cross because there seemed to be more GFP in random areas. Also, the pericardial cells
were extremely bright and vivid with RFP. The gene knocked down in this cross was Zn finger
homeodomain 1. Its primary function is to create a DNA binding protein that aids in
transcription. Its human homolog is zinc finger E-box-binding homeobox 1 isoform X2.
In 110477, there were unique results in both regular light, and blue light. With regular
light, every larvae had black tumors all over their bodies. They were similar in size and color to a
meconium, a waste accumulation in virgins, however they were present in the larva and in no
orderly manner. Under the blue microscope the melanizations were consistent with the findings
under regular light. However, some of the melanizations seemed to be more developed than
others because covering the GFP were black metastatic growths that looked more rough than the
other melanizations. This phenomenon was not present in all larvae, however showed up in more
than one larva and also in more than one instance on one specific larva. The progeny the resulted
from the cross between this stock and Hml also produced a phenotype. The secondary lobe of the
lymph was lit up with RFP although there were no tumors in this cross. The gene COP9
signalosome subunit 1b has many functions like serving as a DNA damage checkpoint, and
regulation of cell cycle. The human homolog of this gene is unnamed protein product.
Discussion
Throughout the rest of this paper, the genes that produced phenotypes in the genes
mentioned above will be discussed and analyzed by discussing the significance of the
phenotypes, the interrelationship between the Hml and HHLT phenotypes, and any previous
characterization of the known genes. The three phenotypes that are worth discussing are those

9. with diminished GFP, increased GFP, and those that have abnormal or uncharacteristic GFP. The
RFP associated with these GFP phenotypes will also be discussed.
There were seven crosses that exhibited a decrease in GFP in the phenotypes: 32510,
24354, 34867, 34335, 34483, 103393, and 34711. The genes that have proven they play a role in
hematopoiesis by diminishing the amount of blood cells or the expression of blood cells are
relatively CG7420, TER94, Contactin, eIF2B-ε, SLY-1 homologous, CG33123, and crooked
neck.
The interference with CG7420, which produces proteins involved in chromosome
condensation, caused the absence of a blood system. Although there was minimal GFP seen , it is
worth noting that it was still alive. It could speculated that this cross, or any cross with almost no
GFP probably had a heart, but the heart cells are not considered blood cells. The only conclusion
that can be made from the phenotype that is exhibited is the lack of lymph gland, pericardial cells
and circulating blood cells. The knockdown of this vital regulation could have many
consequences due to the widespread need for chromosome condensation. However, the only
known effect of mutations in this gene is fertility. In this cross however, it can be seen that
fertility was not the only possible outcome because hematopoiesis was also affected as seen in
Figure 1B.
In 24354’s cross, by knocking down TER94, the lymph gland and the pericardial
nephrocytes were eliminated. The RFP phenotype didn’t show any abnormalities. TER94’s main
function is ATPase activity to facilitate cell division. Through RNA Interference, all blood cells
were inhibited from dividing, thus the lack of GFP in the progeny as seen in Figure 1C. Some
phenotypes associated with the knock down of this gene are increased mortality in many stages
of growth, flightless, and increased cell death. The link between the Hml cross and HHLT cross
is important because it shows that TER94 is not a vital gene needed in the cortical zone but it is
needed in either the medullary zone or PSC. TER94 has had extensive research done on it and
has been linked to the assembly to the fusome, which is a Drosophila germ cell specific
organelle. The fusome has a direct relationship with the germ cell differentiation into cystoblast.
(León & McKearin, 1999)The human homolog transitional endoplasmic reticulum ATPase, has
been linked to retinal pathology but there has not been extensive research with this gene and its
correlation to hematopoiesis. (Griciuc et al., 2010). Because it has been shown that TER94 has
had a role in cell differentiation, it can be seen that its ability to facilitate cell division has a vital
role in hematopoiesis.
In34867, when contactin was knocked down, almost all GFP was absent from the
progeny indicating that there were no lymph gland or pericardial nephrocyte cells as seen in
Figure 1D. Contactin is involved in cell adhesion with epithelial cells and nerve maturation,
however there is no known link to hematopoiesis yet. Septate junctions (SJ’s) in epthilial cells
and neurons are important for the formation and maintenance of selective barriers and contactin
is vital to the formation of SJ’s. (Faivre-Sarrailh et al., 2004) The closest connection that can be
drawn between this gene and hematopoiesis is that contactin helps establish the glial blood-brain
barrier. There has been similar findings in human diseases such as plasmacytoma. The human
homolog of contactin is Contactin 3 (plasmacytoma associated) and it is normally expressed in
the brain, which consistent with the contactin’s role in establishing blood-brain barrier. (Mock,
Connelly, McBride, Kozak, & Marcu, 1996)
eIF2B-ε’s knockdown showed no GFP in the lymph glands but GFP was still present in
the pericardial nephrocytes in Figure 1H. The gene plays a role in translation initiation factor
activity through the exchange of GDP for GTP. This would mean somewhat eliminating the

10. process preceding formation of the peptide bond between the first two amino acids of a protein.
Without this process, proteins are kept from being made in the lymph gland and this can inhibit
the growth of blood cells in many ways. By dramatically decreasing the efficiency of translation,
proteins, which are the workers of the cell, are not able to carry out individual tasks. It has been
shown that a mutation in this gene can negatively affect white blood cells in humans, which are
the cause of many diseases such as neurodegenerative diseases (Fogli & Boespflug-Tanguy,
2006) and leukoencephalopathy. (Wortham, Martinez, Gordiyenko, Robinson, & Proud,
2014) By knocking down this gene, an important step in creating proteins in the blood cells
was inhibited.
In CG33123’s knockdown, there was no GFP present in the lymph glands or any other
structures but there were small areas of concentrated GFP sporadically present throughout the
bodies of each larva in Figure 1F. There was also increased expression of RFP in the pericardial
nephrocytes. CG33123 functions as an aminoacyl-tRNA synthetase through anticodon binding.
Its human homolog, leucine-tRNA ligase, cytoplasmic makes protein and without it, there’s a
breakdown in transcription because it is not capable of sensing the levels of leucine and it cannot
activate other proteins for amino acid signaling. (Han et al., 2012)
When the gene SLY-1 homologous was knocked down, a third of the progeny did not
exhibit GFP as seen in Figure 1E. The phenotype resulting from the cross of this stock with Hml
was consistent with the control. There has been little research conducted on either SLY-1
homologous or the human homolog, sec1 family domain-containing protein 1 isoform a.
However, based on the data collected from this experiment and the knowledge obtained about
the role of SLY-1 homologous through conserved domains, we can make an assumption of how
this gene affects hematopoiesis. Its molecular function is SNARE binding, which is the selective,
non-covalent interaction with a SNARE protein, which means that it is involved in phagocytosis
and intracellular transportation. The knock-down of this gene understandably has implications
with blood formation in this model organism because phagocytosis is often the immune’s
response to foreign objects. Without this mechanism, the immune system is compromised and
the blood development cannot happen normally.
When crooked neck was knocked down, no GFP was present in the lymph glands or any
other structures but there are small areas of concentrated GFP sporadically present throughout
the bodies of each larva. There is no characteristic placement of the circulating blood cells but
they are nonetheless present. The larvae pictured from the Hml cross with this stock exhibited
more circulating blood cells as well. There was also increased expression of RFP in the
pericardial nephrocytes, shown in Figure 1G. Crooked neck’s main role is as a RNA processing
protein, which helps with transcription. As stated previously, transcription and translation are
extremely important for the creation of a normal cell and thus a normal blood system. Without
this important protein, it is easy to understand why the GFP would be diminished. In a study, it
suggests that Hedgehog, an important gene in the regulation of progenitors in the blood system,
regulates both Ci and Su(fu) levels through crooked neck. (Liu et al., 2014). Without progenitors
in the lymph gland, the entire system falls apart, which explains why GFP was diminished.
The second phenotype discussed with HHLT was the increase in GFP in the phenotype
produced. There are four crosses in which this occurred: 103383, 8269, 31196, and 23659. Their
genes respectively are Clathrin heavy chain, head involution defective, Nucleoporin 93kD-1 and
Smg5.

11. Clathrin heavy chain produced an increase in GFP in regards to the lymph glands
brightness and area, whown in Figure 2B. The RFP was in the cortical zone and pericardial
nephrocytes. Clathrin heavy chain’s function is protein binding. As a coat-protein, it binds to
clathrin light chain and is used to build small vesicles in order to transport molecules within
cells. When clathrin heavy chain is not present, there is an overduplication of centrosome and
therefore an increase in replication. (Olszewski, Chandris, Park, Eisenberg, & Greene, 2014)
This explains the increase in GFP. Because the RFP in the cortical zone was normal, it can be
seen that this overduplication is mainly present in the medullary zone with the progenitors. The
progenitors are most affected by the increase in division of cells.
Head involution defective is involved in protein binding as a topoisomerase, which helps
accurate chromosome transmission. When this gene was knocked down, there was an increase in
GFP in the circulating blood cells and the RFP in the cortical zone was normal, seen in Figure
2A. Although its molecular function serves as a topoisomerase, head involution defective’s
biological function is an activator of cell death. (Zhou et al., 1997) When this gene is knocked
down, there are less apoptic signals in the cell and therefore, less cells die. This explains why
there was an increase in GFP. Because this gene maintained a normal RFP phenotype, it suggests
that the gene is mainly expressed by progenitors instead of the differentiated cells in the cortical
zone.
Nucleoporin 93kD-1 is a nuclear pore assembler and as such determines the selectivity of
the pore. Half of the progeny displayed increases in GFP in location and size, while the other
half remained at a level consistent with the control cross as seen in Figure 2C. The increase in
GFP can be explained through Nup93-1’s function. Without a selective barrier between the
nucleus and cytoplasm, things can enter and exit without much difficulty. (Mansfeld et al., 2006)
Without the barrier, inhibitors that maintain the cellular division are no longer able to work
effectively. Therefore cell division continues without inhibition, creating more cells and
therefore more GFP.
Smg5 degrades unusable mRNA and activates telomerase at the site of polymerization.
There was a slight increase in GFP as seen in Figure 2D compared to the control cross in the
lymph gland and with the circulating cells. The RFP in the progeny was consistent with the
control cross. When this gene is knocked down, there are less proteins to degrade nonsense
mRNA, which can have an effect on adipogenesis. (Cho, Han, Park, & Kim, 2013) With a lower
cell differentiation and less regulation of the progenitors, there would be an increase in the lymph
glands, but not specifically in the cortical zone, where the differentiated cells exist.
The last phenotype that has shown genes that affect normal hematopoiesis are the
abnormal, unique phenotypes. The four crosses in this category are 106240, 25572, 103250, and
110477. The respective genes are Ef1α-like factor, Heparan sulfate 3-O sulfotransferase-A, Zn
finger homeodomain 1, and COP9 signalosome subunit 1b.
Ef1α-like factor did not have a clear phenotype, seen in Figure 3A. There were
centralized region of GFP is located much lower than the lymph gland, which could be
speculated as the lymph gland but in a different location. In the other larvae, there is no area of
concentrated GFP. Ef1α-like factor’s function is translational termination. When this gene is
knocked down, it affects translational termination, which henceforth dramatically changes other
proteins sequence, shape and function. With other proteins having different functions or no
function, there’s no conclusive way to explain how the abnormal phenotype arose.

12. Heparan sulfate 3-O sulfotransferase-A’s main function is [heperan sulfate]-glucosamine
3-sulfotransferase 1 activity. The phenotype was atypical because it had the regular lymph gland
GFP present but also have very bright concentrations of GFP that were random in placement and
size, shown in Figure 3B. It helps catalyze chemical reactions and has had the phenotypic results
of increased mortality and fertility. There is a possibility that the reaction it catalyzed inhibited
the formation of blood cells anywhere but the lymph gland, circulating cells, and the pericardial
nephrocytes. When the catalyst was knocked down, the inhibitory role the gene played would be
null, which would explain the random occurrences of GFP.
Zn finger homeodomain 1’s biological function was DNA binding aiding in transcription.
It encodes for a protein that aids in RNA polymerase DNA binding transcription factor activity
which regulates eukaryotic transcription processes. The GFP that resulted from the knockdown
of this gene was extremely sporadic and unpredictable as seen in Figure 3C and the phenotypic
results for the obstruction of this gene is increased mortality and lethality. Like previous crosses,
when transcription or translation are interfered with, a variety of different results can occur. In
one case, this gene inhibited lmd pathway. (Myllymäki & Rämet, 2013) By the loss of
downregulating proteins and pathways, hematopoiesis becomes abnormal and unpredictable,
especially when the change is capable of affecting many different pathways and proteins.
COP9 signalosome subunit 1b had phenotypes of sporadic and random GFP
concentrations, but the peculiar part of the phenotype was the presence of melanizations as seen
in Figure 3D. COP9 signalosome subunit 1b has many functions, one of which is mitotic G2
DNA damage checkpoint. It does not allow cells with mutations in the DNA at certain
checkpoints to divide. Another function of this gene is lateral inhibition, where the gene creates
proteins that signal to equivalent cells surrounding it to differentiate into a cell with a different
fate. There are other roles this gene has like assisting with structural integrity, the ATP-
dependent degradation of ubiquitinated proteins, regulation of cell cycle and female germ-line
stem cell maintenance but the ones explained are some functions that could explain the
extremely interesting results of the progeny of this cross. Possible phenotypes only include
lethality and increased mortality. This cross had melanizations on all of the larvae. The human
homolog of this gene has been linked to carcinogenic results. (Luo, Yang, Takihara, Knoetgen,
& Kessel, 2004) It showed that this unnamed protein product may mediate the cell cycle and
therefore inhibit cell proliferation.
Although this research has shed an enormous amount of light on the blood development
in not only Drosophila but also humans. There can still be more information gained from this
type of experiment. It shouldn’t stop at whether only the cortical zone expresses the gene. This
research should expand into the medullary zone and the posterior signaling center. (Grigorian,
Mandal, & Hartenstein, 2011) It’s important to know what genes the progenitor cells express,
because the stem-cell nature of these cells could provide a lot of information regarding many
illnesses like acute myeloid leukemia. (Crozatier & Vincent, 2011) By using the same UAS-gal4
drive and crossing it with UAS-dsRNA but using the driver in different locations, we could
increase the specificity of our knowledge about blood development and the diseases and genes
associated with the complex process.

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