METABOLISM AND PHYSIOLOGY
1. Insulin/insulin-like growth factor signaling pathway promotes increased body fat in Drosophila female. Puja Biswas, Colin Miller, Rideout Lab, University of British Columbia.
In many animals, including Drosophila, females store more body fat than males. One gene that
contributes to this sex difference in Drosophila body fat is brummer (bmm), a highly conserved
triglyceride lipase. Normally, bmm mRNA levels are higher in males than in females. This male
bias in bmm mRNA levels contributes to the sex difference in body fat, as loss of bmm largely
eliminates the sex difference in body fat. Yet, it remains unclear which factors regulate the sex
difference in bmm mRNA levels. One widely recognized regulator of bmm mRNA levels is the
insulin/insulin-like growth factor signaling pathway (IIS), where high levels of IIS activity
repress bmm mRNA levels. When we monitored Drosophila insulin-like peptides (Dilps) and IIS
activity in adult flies, females had increased mRNA levels of multiple Dilps than males, and
higher IIS activity. Females also showed higher peripheral insulin sensitivity than males. We
next asked whether higher IIS pathway function in females contributed to their ability to store
more body fat than males. To test this, we used inducible gene expression systems to either
ablate or activate the insulin producing cells (IPCs), a key source of circulating Dilps, in adult
flies shortly after eclosion. Loss of IPCs in females significantly reduced body fat, with no effect
in males. We replicated this female-specific effect on body fat using RNAi to reduce Dilp levels
in the IPCs. Increased IPC activity, on the other hand, augmented body fat in males with no
effect in females. Together, these data suggest that the sex difference in IIS function contributes
to the male-female difference in body fat. While future experiments are needed to test whether
sex-specific IIS regulation contributes to male-biased bmm mRNA levels, and whether the
effects of IIS on body fat are mediated by bmm, our data demonstrate sex differences at multiple
levels of IIS regulation that contribute to a male-female difference in pathway function.
2. Regulation of Neuronal Lipid Droplets. Colin Miller, Rideout Lab, University of British Columbia.
Lipid droplets are specialized lipid storage organelles. They sequester triglycerides and other neutral lipid
species. Lipid droplet formation is tightly associated with nutrient availability and triglyceride metabolism.
They are prominent in adipose tissue but found in almost all cell types. In neurons, lipid droplets are
synthesized from circulating lipids or by de novo synthesis supported by glia. Neuronal lipid droplets are
used to generate energy, maintain membranes, and modify synaptic proteins. Further, neuronal lipid
droplets have been associated with neurodegenerative diseases as a response to cell stressors including
reactive oxygen species. In adipose tissue, the regulation of lipid droplets is well studied; however, the
regulation of neuronal lipid droplets under normal physiological conditions is poorly understood. By
genetically driving a GFP that localizes to lipid droplets we can visualize droplets under a multitude of
conditions. Our work has demonstrated the presence of lipid droplets in neurons of healthy adult flies,
mainly in the mushroom body, optic lobes, and antennal lobes. Over aging, lipid droplets are highest at
eclosion and decease over time. Lastly, manipulating genes associated with triglyceride metabolism effect
the number of lipid droplets in the mushroom body. This suggests triglyceride metabolism has a role in the
regulation of neuronal lipid droplets. In conclusion these finding and future experiments will support our
understanding of neuronal lipid droplet regulation. Additionally, this work will create a foundation for the
study of neuronal lipid droplet function under normal physiological conditions and their role in
neurodegenerative diseases.
3. The role of transformer in sex-dependent body size plasticity. Ferdinand Koranteng. Rideout Lab, University of British Columbia.
Our lab has shown that female, but not male flies, significantly increase their body size when fed a high
protein diet. We have identified a nutrient-responsive axis whereby dietary protein acts through fat body-
derived stunted to promote insulin secretion and growth. Upstream of this mechanism is transformer (tra),
the master regulator of sex determination in flies. While females lose their body size plasticity in the tra
mutant flies, tra overexpression (traF) in males significantly raises their adult weight in the high protein diet,
suggesting a unique role for Tra in the nutrient-body size axis. Currently, we are investigating how Tra
activity is nutrient-dependently modified.
4. Investigating sex differences in the Drosophila fat body. Celena Cherian, Rideout Lab, University of British Columbia.
Sexual dimorphism has fascinated biologists for centuries and differences in external appearances,
behaviour and reproduction have been well documented across many species. However, there is a lack of
knowledge surrounding sex differences in developmental and physiological processes. One such
physiological aspect is systemic growth. Males and females of many species across the animal kingdom
differ in body size and studies using Drosophila have begun to unravel the mechanisms behind this sexual
size dimorphism. In 2015, our lab published a paper showing that the sex of the larval fat body, an
endocrine organ equivalent to mammalian adipose tissue and liver, contributes to the male-female
difference in body size. Yet, we lack detailed knowledge of male-female differences in the fat body that
would explain why the sexual identity of this cell type regulates body size. Clues into how the fat body might
differ between the sexes to influence body size emerge from more than 25 years of studies on larval
growth. These studies show that the correct regulation of mRNA translation and mitochondrial function are
essential for flies to achieve a normal body size. Unfortunately, none of these studies included both sexes.
This study aims to understand whether sex differences exist in mRNA translation and mitochondrial
function, and whether these differences contribute to male-female differences in body size.
CELL BIOLOGY AND DEVELOPMENT
5. Understanding the role of translational control in oocyte function. Kaicheng Ma, Greenblatt Lab, University of British Columbia.
The control of protein translation in both time and space is essential for animal development and for the
continual function of cells such as neurons and germ cells. Mutations in the (fragile X messenger
ribonucleoprotein 1 (FMR1) gene, encoding the RNA binding protein FMRP, lead to fragile X syndrome
(FXS) and fragile X primary ovarian insufficiency (FXPOI). Our lab found that in the Drosophila female
germline, FMRP acts at the level of translation initiation to boost protein production from mRNAs encoding
unusually large proteins, many of which are encoded by the fly orthologs of human autism-associated
genes. The goal of my research project is to gain fundamental insights into translational control
mechanisms by understanding the mechanism by which FMRP promotes translation of its targets. To gain
mechanistic insights into FMRP's function, I took advantage of a novel assay which allows us to visualize
FMRP activity in vivo. FMRP-dependent translation drives the formation of microscopically visible particles
of one of its targets, a ~590 kDa ubiquitin ligase Poe in developing Drosophila nurse cells. By screening for
the loss of Poe particles using single-molecule fluorescence in situ hybridization (smFISH), I discovered a
potential requirement for two RNA binding proteins, lingerer (lig) and ovarian tumour (otu), which also
contain domains that either bind to or remove ubiquitin groups. Based on my preliminary data, we
hypothesize that FMRP functions as part of a larger complex of RNA-binding proteins that bind to and/or
modify the ubiquitination status of core translation regulatory factors.
6. Characterizing Robinow Syndrome DVL1 mutations in Drosophila melanogaster. Gamze Akarsu, Verheyen Lab, Simon Fraser University.
Insights into how development is regulated by sophisticated and interconnected signaling networks can be
gained from studies on developmental diseases. Deciphering the effects of mutations that result in
abnormal development through these networks can be difficult due to their complexity. Since flies have little
genetic redundancy and are substantially easier, cheaper, and faster to grow than other vertebrate models,
using Drosophila melanogaster can help solve the challenge of investigating such human disorders. In this
study we are focusing on characterizing the altering effects on signaling of 3 Dishevelled 1 (DVL1) variants
obtained from Robinow Syndrome (RS) patients. RS is a rare developmental disorder characterized by
short-limb dwarfism and fetal facies like broad nasal tips and widely spaced eyes phenotypes. RS has been
linked to certain mutations in different genes which are components of the non-canonical/Planar Cell
Polarity (PCP) pathway of Wnt signaling. Wnt signaling plays a role in both homeostasis and embryonic
development. DVL is a relay molecule for both canonical and non-canonical/PCP Wnt signaling cascades.
However, there are still many things that should be investigated in PCP signaling, which regulate
cytoskeletal processes and directs cell polarity within the epithelial plane. In vertebrates there are 3 DVL
proteins while Drosophila has a common ortholog, Dsh. The 3 DVL1 variants we study have distinct
frameshift mutations that cause the C-terminus to be replaced by the same new peptide sequence with no
known homology. The GAL4-UAS system is used to express human wildtype DVL1, and the variants found
in patients. Our studies have demonstrated that these DVL1 patient variations ectopically stimulate
PCP/JNK signaling, trigger apoptosis, and interfere with the stability of Armadillo/b-catenin, thus inhibiting
canonical Wnt signaling. Additionally, the mutations cause a number of novel phenotypes in wing tissue,
including anomalies in the anterior cross vein, ectopic bristles, and vein thickening. These suggest new
roles of mutant DVL1 in other conserved signaling pathways. We gain insight into the underlying
mechanisms of non-canonical Wnt signaling and, more broadly, how development is affected in people with
RS by knowing how conserved signaling pathways are disrupted by these DVL1 variations. Future
therapies for RS patients may be influenced by the data provided by our studies.
7. The Influence of Basigin on Focal Adhesion Complexes at The Perineurial Glial Membrane. Sophie Roth, Auld Lab, University of British Columbia.
In Drosophila melanogaster, glial cells play an important role in ensheathing and protecting nerves from
damage. The perineurial glia are a conserved class of glia that ensure proper ensheathment of nerves
through interactions with the extracellular matrix (ECM). Glia-ECM interactions occur through focal
adhesion complexes (FACs) comprised of Integrins and intracellular adapter proteins including Talin. Loss
of FACs leads to the loss of the glial sheath and disruption of nervous system function. Integrins also
associate with the transmembrane protein Basigin (aka Neuroplastin) in the glia. Loss of Basigin leads to
deformation of the glial membrane, cytoskeleton and the overlying ECM. Since the loss of Basigin
phenotype can be rescued by loss of Integrin, we hypothesize that Basigin negatively regulates Integrin
activation in order to modulate focal adhesion strength across the glial membrane. This leads to the
questions: 1. Does the increase in Integrin activation with Basigin knockdown lead to changes in tension
across the membrane? 2. What is the role of Basigin in glial FACs? To investigate these questions, a FRET
(fluorescence resonance energy transfer) sensor in the protein Talin will be used to visualize and quantify
changes in Integrin activation when Basigin is knocked down. The sensor includes donor and acceptor
fluorophores connected by a flexible linker peptide. Increased Integrin activation will lead to a decrease in
FRET as the two fluorophores become separated. FRET acceptor photobleaching will be performed in the
nerves of live larvae to compare controls with Basigin knockdown. This study will help elucidate the role of
Basigin in glial FACs and provide insight into the nature of the interaction between Basigin and Integrin.
Overall, investigation into focal adhesion complex maintenance in glia is an important concept in all animals
because of their role in establishing and maintaining glial sheath formation and protecting nervous system
function.
8. Exploring human CDK19 variants and the effects of N-acetylcystine amide using Drosophila as a model organism. Katie Sew, Verheyen Lab, Simon Fraser University.
Cyclin-dependent kinase 8 (Cdk8) is a serine/threonine kinase that has been well characterized as a
mediator in the regulation of gene transcription. We aim to investigate Cdk8’s potential mediator-
independent functions, along with the functions of it’s human ortholog, CDK19 in collaboration with the
Bellen Lab. We found that expression of Cdk8 can modulate mitochondrial morphology under physiological
conditions. In muscles, expression of Cdk8 leads to a fragmented mitochondrial phenotype, whereas
depletion of Cdk8 leads to an elongated phenotype. A kinase dead version of Cdk8 further enhances the
elongated phenotype, which suggests the change in mitochondrial morphology is likely a kinase-dependent
event. The Bellen Lab identified in their previous publication two de novo mutations in CDK19 that are
responsible for patients’ epilepsy as well as neurodevelopmental delays. Their preliminary data showed that
the wild-type CDK19 (CDK19WT) was able to rescue the neuronal defects caused by the depletion of Cdk8,
whereas two identified de novo mutations further enhanced the defective phenotypes. To further validate
their findings, we examined the mitochondrial morphology using the same lines, and found that CDK19WT
resulted in fragmented mitochondria whereas we found elongated mitochondria with expression of the
mutant CDK19 variants. Interestingly, the elongated mitochondrial phenotype is also seen in fibroblast
samples taken from a patient with a de novo CDK19 mutation, which is consistent with what is modeled in
Drosophila. In addition to the mitochondrial phenotypes, we observed that knocking down Cdk8 caused
defects in climbing ability. Using the drug, N-acetylcysteine amide (NACA), that has been previously proven
to be protective against mitochondrial and neuronal defects, we wanted to see if NACA could revert the
defective phenotypes caused by depletion of Cdk8. Our preliminary data has shown that Cdk8 knock-down
flies reared on food supplemented with NACA have improved climbing ability compared to the ones reared
on normal food. By exploring the roles of Cdk8, CDK19, and the effects of NACA, we hope to provide
therapeutic insights to patients with CDK19 mutations.
9. Investigating the Role of Cdk8 in a Drosophila Model of Parkinson Disease. Claire Shih, Verheyen Lab, Simon Fraser University.
Cyclin-dependent kinases 8 (Cdk8) are serine-threonine kinases that function as transcriptional regulators,
acting as part of a conserved kinase module in association with the Mediator complex. While its mediator
function as gene regulator has been well-characterized, potential mediator-independent functions of Cdk8
have not been fully investigated. When Cdk8 is ubiquitously knocked down in flies, progeny with desired
genotype have phenotypic effects including held-up and droopy wing postures, reduced life span, and
defects in both flight and climbing abilities. Interestingly, these phenotypic effects are characteristic of flies
with mutations in either PTEN-induced putative kinase 1 (Pink1) or Parkin, two well-established players
associated with Parkinson’s disease (PD). Pink1 and Parkin normally regulate the homeostasis of
mitochondria in a process known as mitophagy. Impaired or dysfunctional mitochondria will be recognized
by Pink1 and Parkin and targeted for degradation by autophagy. Since tissue specific knock down of Cdk8
also resulted in impaired climbing ability, we hypothesized that Cdk8 functions in a common pathway with
Pink1 and Parkin in mitophagy. Muscle-specific expression of Cdk8 was able to suppress defects in
climbing ability and thorax indentation caused by the loss of function Pink1 allele, pink1B9; relative to
pink1B9 mutants. Furthermore, mitochondrial and muscle fiber morphologies were restored when Cdk8 was
overexpressed in the pink1B9 mutant background. Having determined that Cdk8 in muscle can suppress
Pink1B9 mutant phenotypes, we next asked how the modulation of Cdk8 expression might influence the
CNS. Neuron-specific knockdown of Cdk8 in flies caused a significant decrease in the climbing ability and
longevity compared to a white RNAi control; with a particularly strong effect on males, suggesting potential
sex-specific effects for future studies. Altogether, we show that Cdk8 has mediator-independent roles in
muscle and neuronal tissue and is able to revert the defective phenotypes caused by pink1B9 mutant allele.
NEUROSCIENCE
10. How to wrap a nerve: glia-ECM communication. Katie Clayworth, Auld Lab, University of British Columbia.
Peripheral nervous system (PNS) health is largely dependent on proper glial cell functioning during
development. Myelinating and non-myelinating Schwann cells (MSCs and NMSCs, respectively) are glial
cells in the PNS that ensheathe and protect axons. Communication between Schwann cells and the
extracellular matrix (ECM) is essential for PNS development. The ECM protein laminin is important for MSC
development, however very little is known about the mechanisms underlying the role of laminin in NMSC
development. We use developing Drosophila wrapping glia (WG), which ensheathe axons similarly to
NMSCs, as a model to study the role of laminin in NMSC development. We found that laminin appears to
be the only ECM protein expressed around WG, as perlecan, viking (collagen), and nidogen are not
expressed in this region of the nerve. We found strong expression of LanA (one of two laminin alpha
subunits in Drosophila), around WG. Wing blister, the other laminin alpha subunit, is not strongly expressed
the peripheral nerve. Knockdown of LanA in WG eliminated LanA expression around WG and caused WG
swellings, suggesting that LanA is expressed by WG. Preliminary data suggests that LanA is most often
found at the adaxonal WG membrane (between WG and axons), rather than the abaxonal WG membrane
(between WG and its adjacent glial layer, subperineurial glia). These results potentially indicate a form of
WG polarization, a feature that has not been well understood in WG thus far. Finally, further preliminary
data suggests LanA is localized preferentially around motor axons rather than sensory axons, potentially
reflecting a role of WG-derived laminin in animal movement. Due to the highly conserved nature of laminins,
our results have implications for NMSC development—thus improving our understanding of the factors
underlying glia-ECM communication in all animals.
11. Targeted RNA sequencing to investigate the neurobiological and molecular basis of Aedes aegypti smell and taste. Leisl Brewster, Matthews Lab, University of British Columbia.
The yellow fever mosquito, Aedes aegypti, is a vector for viruses that have devastating impacts on global
health and economies. Mosquitoes rely on their senses of smell and taste to execute behaviours such as
host seeking or finding oviposition sites. Dozens to hundreds of chemoreceptors expressed in sensory
tissues such as the antennae, legs and proboscis bind to ligands in the environment, thereby activating
these sensory neurons; sending a signal to the brain and driving behaviour. However, it is not clear how
these chemoreceptors are expressed within and organized across sensory neurons in order to encode
complex chemical stimuli and drive behaviour. To address this, we are developing a targeted RNA
sequencing protocol in Ae. aegypti to profile gene expression in genetically identified populations of sensory
neurons. This will be done using the Q-binary expression system to express a nuclear-targeted green
fluorescent protein (GFP) that can be used to isolate labeled neurons from whole tissues using dounce
homogenization. This will be followed by fluorescence-activated cell sorting (FACS). We will generate
sequencing libraries from pooled nuclei to obtain “cell-type specific transcriptomes” from neuronal
populations of interest, as well as carry out single nucleus RNA sequencing on these populations.
Characterizing chemoreceptor expression within and across sensory neurons will help to elucidate the
mechanisms by which a limited receptor repertoire can encode the inordinate diversity of the chemical
world; shedding light on the evolution of sensory systems across insects and informing future mosquito
control strategies.
12. Taste coding and processing in Drosophila taste projection neurons. Jinfang Li, Gordon Lab, University of British Columbia.
Taste is an important survival cue which informs animals about the safety and nutritional qualities of food.
Therefore, the coding of taste in the nervous system needs to be robust and unambiguous. There is
currently a controversy on whether taste coding in the brain follows a labelled line or combinatorial model.
Here we investigate the Drosophila melanogaster taste system, which shares many commonalities with
mammalian taste, to gain more insights into its coding strategies. We focus on the taste projection neurons
(TPNs) that connect gustatory receptor neurons (GRNs) with higher order processing centres such as the
mushroom bodies (MB). We identified three distinct types of TPNs, each of which encodes a single taste
modality: sweet, high salt, or bitter. We found that each type of TPNs is more narrowly tuned compared to
GRNs. Moreover, behavioural and EM circuit tracing results showed that each TPN type is not only
modality specific, but also functionally specific by selectively synapsing with subpopulations of higher order
neurons that are known to play important roles in reinforcement learning and other complex behaviours. We
concluded that while the coding of some taste modalities in GRNs is combinatorial, the coding in the
identified TPNs mostly adheres to a labelled line model. This strategy allows parallel processing, which
facilitates decoding of complex taste information in the brain.
13. Investigating the function of the autophagy-related protein ATG4D using a Drosophila model. Emily McMann, Gorski Lab, Simon Fraser University.
Macroautophagy (hereafter “autophagy”) is a highly conserved intracellular recycling process that occurs at
basal levels in all eukaryotic cells. Autophagy serves to maintain cellular homeostasis by engulfing excess,
old, and damaged organelles and proteins in double-membrane structures (termed autophagosomes) and
delivers them to the lysosome for degradation. Among the 30+ proteins that regulate the process of
autophagy are the ATG4 cysteine proteases. In mammals, there are four ATG4 homologues (ATG4A-D)
that function to prime Atg8-family members for conjugation to phosphatidylethanolamine in the
autophagosomal membranes, as well as remove them from the outer membrane after autophagosome
formation. The ATG4 homologue ATG4D has been the least well-studied of the ATG4 family members as
initial investigations found its priming capabilities to be weak and limited to a single Atg8-family protein,
leading to the belief that ATG4D was redundant to the other ATG4 homologues. However, apparent loss of
function mutations in ATG4D have since been implicated in a canine neurodegenerative disease, termed
neurodegenerative vacuolar storage disease (NVSD), which is characterized by motor coordination defects,
progressive cerebellar ataxia and Purkinje cell loss, and vacuolization of neuronal and fibroblast cells. More
recently, NVSD-like phenotypes have been identified in human patients with mutations in ATG4D. It has yet
to be determined how exactly ATG4D loss of function contributes to the phenotypes observed in NVSD, or
even if the phenotypes are the result of a defect in autophagy or some other process. Drosophila provide an
excellent model to study human disease and protein function, and we aim to gain insights into the function
of ATG4D via the knockdown and knockout of the Drosophila homologue Atg4b. Additionally, we will utilize
the Gal4-UAS system to express human pathogenic variants of ATG4D in an Atg4b knockout background
to create a disease model for the in vivo study of NVSD.
1. Insulin/insulin-like growth factor signaling pathway promotes increased body fat in Drosophila female. Puja Biswas, Colin Miller, Rideout Lab, University of British Columbia.
In many animals, including Drosophila, females store more body fat than males. One gene that
contributes to this sex difference in Drosophila body fat is brummer (bmm), a highly conserved
triglyceride lipase. Normally, bmm mRNA levels are higher in males than in females. This male
bias in bmm mRNA levels contributes to the sex difference in body fat, as loss of bmm largely
eliminates the sex difference in body fat. Yet, it remains unclear which factors regulate the sex
difference in bmm mRNA levels. One widely recognized regulator of bmm mRNA levels is the
insulin/insulin-like growth factor signaling pathway (IIS), where high levels of IIS activity
repress bmm mRNA levels. When we monitored Drosophila insulin-like peptides (Dilps) and IIS
activity in adult flies, females had increased mRNA levels of multiple Dilps than males, and
higher IIS activity. Females also showed higher peripheral insulin sensitivity than males. We
next asked whether higher IIS pathway function in females contributed to their ability to store
more body fat than males. To test this, we used inducible gene expression systems to either
ablate or activate the insulin producing cells (IPCs), a key source of circulating Dilps, in adult
flies shortly after eclosion. Loss of IPCs in females significantly reduced body fat, with no effect
in males. We replicated this female-specific effect on body fat using RNAi to reduce Dilp levels
in the IPCs. Increased IPC activity, on the other hand, augmented body fat in males with no
effect in females. Together, these data suggest that the sex difference in IIS function contributes
to the male-female difference in body fat. While future experiments are needed to test whether
sex-specific IIS regulation contributes to male-biased bmm mRNA levels, and whether the
effects of IIS on body fat are mediated by bmm, our data demonstrate sex differences at multiple
levels of IIS regulation that contribute to a male-female difference in pathway function.
2. Regulation of Neuronal Lipid Droplets. Colin Miller, Rideout Lab, University of British Columbia.
Lipid droplets are specialized lipid storage organelles. They sequester triglycerides and other neutral lipid
species. Lipid droplet formation is tightly associated with nutrient availability and triglyceride metabolism.
They are prominent in adipose tissue but found in almost all cell types. In neurons, lipid droplets are
synthesized from circulating lipids or by de novo synthesis supported by glia. Neuronal lipid droplets are
used to generate energy, maintain membranes, and modify synaptic proteins. Further, neuronal lipid
droplets have been associated with neurodegenerative diseases as a response to cell stressors including
reactive oxygen species. In adipose tissue, the regulation of lipid droplets is well studied; however, the
regulation of neuronal lipid droplets under normal physiological conditions is poorly understood. By
genetically driving a GFP that localizes to lipid droplets we can visualize droplets under a multitude of
conditions. Our work has demonstrated the presence of lipid droplets in neurons of healthy adult flies,
mainly in the mushroom body, optic lobes, and antennal lobes. Over aging, lipid droplets are highest at
eclosion and decease over time. Lastly, manipulating genes associated with triglyceride metabolism effect
the number of lipid droplets in the mushroom body. This suggests triglyceride metabolism has a role in the
regulation of neuronal lipid droplets. In conclusion these finding and future experiments will support our
understanding of neuronal lipid droplet regulation. Additionally, this work will create a foundation for the
study of neuronal lipid droplet function under normal physiological conditions and their role in
neurodegenerative diseases.
3. The role of transformer in sex-dependent body size plasticity. Ferdinand Koranteng. Rideout Lab, University of British Columbia.
Our lab has shown that female, but not male flies, significantly increase their body size when fed a high
protein diet. We have identified a nutrient-responsive axis whereby dietary protein acts through fat body-
derived stunted to promote insulin secretion and growth. Upstream of this mechanism is transformer (tra),
the master regulator of sex determination in flies. While females lose their body size plasticity in the tra
mutant flies, tra overexpression (traF) in males significantly raises their adult weight in the high protein diet,
suggesting a unique role for Tra in the nutrient-body size axis. Currently, we are investigating how Tra
activity is nutrient-dependently modified.
4. Investigating sex differences in the Drosophila fat body. Celena Cherian, Rideout Lab, University of British Columbia.
Sexual dimorphism has fascinated biologists for centuries and differences in external appearances,
behaviour and reproduction have been well documented across many species. However, there is a lack of
knowledge surrounding sex differences in developmental and physiological processes. One such
physiological aspect is systemic growth. Males and females of many species across the animal kingdom
differ in body size and studies using Drosophila have begun to unravel the mechanisms behind this sexual
size dimorphism. In 2015, our lab published a paper showing that the sex of the larval fat body, an
endocrine organ equivalent to mammalian adipose tissue and liver, contributes to the male-female
difference in body size. Yet, we lack detailed knowledge of male-female differences in the fat body that
would explain why the sexual identity of this cell type regulates body size. Clues into how the fat body might
differ between the sexes to influence body size emerge from more than 25 years of studies on larval
growth. These studies show that the correct regulation of mRNA translation and mitochondrial function are
essential for flies to achieve a normal body size. Unfortunately, none of these studies included both sexes.
This study aims to understand whether sex differences exist in mRNA translation and mitochondrial
function, and whether these differences contribute to male-female differences in body size.
CELL BIOLOGY AND DEVELOPMENT
5. Understanding the role of translational control in oocyte function. Kaicheng Ma, Greenblatt Lab, University of British Columbia.
The control of protein translation in both time and space is essential for animal development and for the
continual function of cells such as neurons and germ cells. Mutations in the (fragile X messenger
ribonucleoprotein 1 (FMR1) gene, encoding the RNA binding protein FMRP, lead to fragile X syndrome
(FXS) and fragile X primary ovarian insufficiency (FXPOI). Our lab found that in the Drosophila female
germline, FMRP acts at the level of translation initiation to boost protein production from mRNAs encoding
unusually large proteins, many of which are encoded by the fly orthologs of human autism-associated
genes. The goal of my research project is to gain fundamental insights into translational control
mechanisms by understanding the mechanism by which FMRP promotes translation of its targets. To gain
mechanistic insights into FMRP's function, I took advantage of a novel assay which allows us to visualize
FMRP activity in vivo. FMRP-dependent translation drives the formation of microscopically visible particles
of one of its targets, a ~590 kDa ubiquitin ligase Poe in developing Drosophila nurse cells. By screening for
the loss of Poe particles using single-molecule fluorescence in situ hybridization (smFISH), I discovered a
potential requirement for two RNA binding proteins, lingerer (lig) and ovarian tumour (otu), which also
contain domains that either bind to or remove ubiquitin groups. Based on my preliminary data, we
hypothesize that FMRP functions as part of a larger complex of RNA-binding proteins that bind to and/or
modify the ubiquitination status of core translation regulatory factors.
6. Characterizing Robinow Syndrome DVL1 mutations in Drosophila melanogaster. Gamze Akarsu, Verheyen Lab, Simon Fraser University.
Insights into how development is regulated by sophisticated and interconnected signaling networks can be
gained from studies on developmental diseases. Deciphering the effects of mutations that result in
abnormal development through these networks can be difficult due to their complexity. Since flies have little
genetic redundancy and are substantially easier, cheaper, and faster to grow than other vertebrate models,
using Drosophila melanogaster can help solve the challenge of investigating such human disorders. In this
study we are focusing on characterizing the altering effects on signaling of 3 Dishevelled 1 (DVL1) variants
obtained from Robinow Syndrome (RS) patients. RS is a rare developmental disorder characterized by
short-limb dwarfism and fetal facies like broad nasal tips and widely spaced eyes phenotypes. RS has been
linked to certain mutations in different genes which are components of the non-canonical/Planar Cell
Polarity (PCP) pathway of Wnt signaling. Wnt signaling plays a role in both homeostasis and embryonic
development. DVL is a relay molecule for both canonical and non-canonical/PCP Wnt signaling cascades.
However, there are still many things that should be investigated in PCP signaling, which regulate
cytoskeletal processes and directs cell polarity within the epithelial plane. In vertebrates there are 3 DVL
proteins while Drosophila has a common ortholog, Dsh. The 3 DVL1 variants we study have distinct
frameshift mutations that cause the C-terminus to be replaced by the same new peptide sequence with no
known homology. The GAL4-UAS system is used to express human wildtype DVL1, and the variants found
in patients. Our studies have demonstrated that these DVL1 patient variations ectopically stimulate
PCP/JNK signaling, trigger apoptosis, and interfere with the stability of Armadillo/b-catenin, thus inhibiting
canonical Wnt signaling. Additionally, the mutations cause a number of novel phenotypes in wing tissue,
including anomalies in the anterior cross vein, ectopic bristles, and vein thickening. These suggest new
roles of mutant DVL1 in other conserved signaling pathways. We gain insight into the underlying
mechanisms of non-canonical Wnt signaling and, more broadly, how development is affected in people with
RS by knowing how conserved signaling pathways are disrupted by these DVL1 variations. Future
therapies for RS patients may be influenced by the data provided by our studies.
7. The Influence of Basigin on Focal Adhesion Complexes at The Perineurial Glial Membrane. Sophie Roth, Auld Lab, University of British Columbia.
In Drosophila melanogaster, glial cells play an important role in ensheathing and protecting nerves from
damage. The perineurial glia are a conserved class of glia that ensure proper ensheathment of nerves
through interactions with the extracellular matrix (ECM). Glia-ECM interactions occur through focal
adhesion complexes (FACs) comprised of Integrins and intracellular adapter proteins including Talin. Loss
of FACs leads to the loss of the glial sheath and disruption of nervous system function. Integrins also
associate with the transmembrane protein Basigin (aka Neuroplastin) in the glia. Loss of Basigin leads to
deformation of the glial membrane, cytoskeleton and the overlying ECM. Since the loss of Basigin
phenotype can be rescued by loss of Integrin, we hypothesize that Basigin negatively regulates Integrin
activation in order to modulate focal adhesion strength across the glial membrane. This leads to the
questions: 1. Does the increase in Integrin activation with Basigin knockdown lead to changes in tension
across the membrane? 2. What is the role of Basigin in glial FACs? To investigate these questions, a FRET
(fluorescence resonance energy transfer) sensor in the protein Talin will be used to visualize and quantify
changes in Integrin activation when Basigin is knocked down. The sensor includes donor and acceptor
fluorophores connected by a flexible linker peptide. Increased Integrin activation will lead to a decrease in
FRET as the two fluorophores become separated. FRET acceptor photobleaching will be performed in the
nerves of live larvae to compare controls with Basigin knockdown. This study will help elucidate the role of
Basigin in glial FACs and provide insight into the nature of the interaction between Basigin and Integrin.
Overall, investigation into focal adhesion complex maintenance in glia is an important concept in all animals
because of their role in establishing and maintaining glial sheath formation and protecting nervous system
function.
8. Exploring human CDK19 variants and the effects of N-acetylcystine amide using Drosophila as a model organism. Katie Sew, Verheyen Lab, Simon Fraser University.
Cyclin-dependent kinase 8 (Cdk8) is a serine/threonine kinase that has been well characterized as a
mediator in the regulation of gene transcription. We aim to investigate Cdk8’s potential mediator-
independent functions, along with the functions of it’s human ortholog, CDK19 in collaboration with the
Bellen Lab. We found that expression of Cdk8 can modulate mitochondrial morphology under physiological
conditions. In muscles, expression of Cdk8 leads to a fragmented mitochondrial phenotype, whereas
depletion of Cdk8 leads to an elongated phenotype. A kinase dead version of Cdk8 further enhances the
elongated phenotype, which suggests the change in mitochondrial morphology is likely a kinase-dependent
event. The Bellen Lab identified in their previous publication two de novo mutations in CDK19 that are
responsible for patients’ epilepsy as well as neurodevelopmental delays. Their preliminary data showed that
the wild-type CDK19 (CDK19WT) was able to rescue the neuronal defects caused by the depletion of Cdk8,
whereas two identified de novo mutations further enhanced the defective phenotypes. To further validate
their findings, we examined the mitochondrial morphology using the same lines, and found that CDK19WT
resulted in fragmented mitochondria whereas we found elongated mitochondria with expression of the
mutant CDK19 variants. Interestingly, the elongated mitochondrial phenotype is also seen in fibroblast
samples taken from a patient with a de novo CDK19 mutation, which is consistent with what is modeled in
Drosophila. In addition to the mitochondrial phenotypes, we observed that knocking down Cdk8 caused
defects in climbing ability. Using the drug, N-acetylcysteine amide (NACA), that has been previously proven
to be protective against mitochondrial and neuronal defects, we wanted to see if NACA could revert the
defective phenotypes caused by depletion of Cdk8. Our preliminary data has shown that Cdk8 knock-down
flies reared on food supplemented with NACA have improved climbing ability compared to the ones reared
on normal food. By exploring the roles of Cdk8, CDK19, and the effects of NACA, we hope to provide
therapeutic insights to patients with CDK19 mutations.
9. Investigating the Role of Cdk8 in a Drosophila Model of Parkinson Disease. Claire Shih, Verheyen Lab, Simon Fraser University.
Cyclin-dependent kinases 8 (Cdk8) are serine-threonine kinases that function as transcriptional regulators,
acting as part of a conserved kinase module in association with the Mediator complex. While its mediator
function as gene regulator has been well-characterized, potential mediator-independent functions of Cdk8
have not been fully investigated. When Cdk8 is ubiquitously knocked down in flies, progeny with desired
genotype have phenotypic effects including held-up and droopy wing postures, reduced life span, and
defects in both flight and climbing abilities. Interestingly, these phenotypic effects are characteristic of flies
with mutations in either PTEN-induced putative kinase 1 (Pink1) or Parkin, two well-established players
associated with Parkinson’s disease (PD). Pink1 and Parkin normally regulate the homeostasis of
mitochondria in a process known as mitophagy. Impaired or dysfunctional mitochondria will be recognized
by Pink1 and Parkin and targeted for degradation by autophagy. Since tissue specific knock down of Cdk8
also resulted in impaired climbing ability, we hypothesized that Cdk8 functions in a common pathway with
Pink1 and Parkin in mitophagy. Muscle-specific expression of Cdk8 was able to suppress defects in
climbing ability and thorax indentation caused by the loss of function Pink1 allele, pink1B9; relative to
pink1B9 mutants. Furthermore, mitochondrial and muscle fiber morphologies were restored when Cdk8 was
overexpressed in the pink1B9 mutant background. Having determined that Cdk8 in muscle can suppress
Pink1B9 mutant phenotypes, we next asked how the modulation of Cdk8 expression might influence the
CNS. Neuron-specific knockdown of Cdk8 in flies caused a significant decrease in the climbing ability and
longevity compared to a white RNAi control; with a particularly strong effect on males, suggesting potential
sex-specific effects for future studies. Altogether, we show that Cdk8 has mediator-independent roles in
muscle and neuronal tissue and is able to revert the defective phenotypes caused by pink1B9 mutant allele.
NEUROSCIENCE
10. How to wrap a nerve: glia-ECM communication. Katie Clayworth, Auld Lab, University of British Columbia.
Peripheral nervous system (PNS) health is largely dependent on proper glial cell functioning during
development. Myelinating and non-myelinating Schwann cells (MSCs and NMSCs, respectively) are glial
cells in the PNS that ensheathe and protect axons. Communication between Schwann cells and the
extracellular matrix (ECM) is essential for PNS development. The ECM protein laminin is important for MSC
development, however very little is known about the mechanisms underlying the role of laminin in NMSC
development. We use developing Drosophila wrapping glia (WG), which ensheathe axons similarly to
NMSCs, as a model to study the role of laminin in NMSC development. We found that laminin appears to
be the only ECM protein expressed around WG, as perlecan, viking (collagen), and nidogen are not
expressed in this region of the nerve. We found strong expression of LanA (one of two laminin alpha
subunits in Drosophila), around WG. Wing blister, the other laminin alpha subunit, is not strongly expressed
the peripheral nerve. Knockdown of LanA in WG eliminated LanA expression around WG and caused WG
swellings, suggesting that LanA is expressed by WG. Preliminary data suggests that LanA is most often
found at the adaxonal WG membrane (between WG and axons), rather than the abaxonal WG membrane
(between WG and its adjacent glial layer, subperineurial glia). These results potentially indicate a form of
WG polarization, a feature that has not been well understood in WG thus far. Finally, further preliminary
data suggests LanA is localized preferentially around motor axons rather than sensory axons, potentially
reflecting a role of WG-derived laminin in animal movement. Due to the highly conserved nature of laminins,
our results have implications for NMSC development—thus improving our understanding of the factors
underlying glia-ECM communication in all animals.
11. Targeted RNA sequencing to investigate the neurobiological and molecular basis of Aedes aegypti smell and taste. Leisl Brewster, Matthews Lab, University of British Columbia.
The yellow fever mosquito, Aedes aegypti, is a vector for viruses that have devastating impacts on global
health and economies. Mosquitoes rely on their senses of smell and taste to execute behaviours such as
host seeking or finding oviposition sites. Dozens to hundreds of chemoreceptors expressed in sensory
tissues such as the antennae, legs and proboscis bind to ligands in the environment, thereby activating
these sensory neurons; sending a signal to the brain and driving behaviour. However, it is not clear how
these chemoreceptors are expressed within and organized across sensory neurons in order to encode
complex chemical stimuli and drive behaviour. To address this, we are developing a targeted RNA
sequencing protocol in Ae. aegypti to profile gene expression in genetically identified populations of sensory
neurons. This will be done using the Q-binary expression system to express a nuclear-targeted green
fluorescent protein (GFP) that can be used to isolate labeled neurons from whole tissues using dounce
homogenization. This will be followed by fluorescence-activated cell sorting (FACS). We will generate
sequencing libraries from pooled nuclei to obtain “cell-type specific transcriptomes” from neuronal
populations of interest, as well as carry out single nucleus RNA sequencing on these populations.
Characterizing chemoreceptor expression within and across sensory neurons will help to elucidate the
mechanisms by which a limited receptor repertoire can encode the inordinate diversity of the chemical
world; shedding light on the evolution of sensory systems across insects and informing future mosquito
control strategies.
12. Taste coding and processing in Drosophila taste projection neurons. Jinfang Li, Gordon Lab, University of British Columbia.
Taste is an important survival cue which informs animals about the safety and nutritional qualities of food.
Therefore, the coding of taste in the nervous system needs to be robust and unambiguous. There is
currently a controversy on whether taste coding in the brain follows a labelled line or combinatorial model.
Here we investigate the Drosophila melanogaster taste system, which shares many commonalities with
mammalian taste, to gain more insights into its coding strategies. We focus on the taste projection neurons
(TPNs) that connect gustatory receptor neurons (GRNs) with higher order processing centres such as the
mushroom bodies (MB). We identified three distinct types of TPNs, each of which encodes a single taste
modality: sweet, high salt, or bitter. We found that each type of TPNs is more narrowly tuned compared to
GRNs. Moreover, behavioural and EM circuit tracing results showed that each TPN type is not only
modality specific, but also functionally specific by selectively synapsing with subpopulations of higher order
neurons that are known to play important roles in reinforcement learning and other complex behaviours. We
concluded that while the coding of some taste modalities in GRNs is combinatorial, the coding in the
identified TPNs mostly adheres to a labelled line model. This strategy allows parallel processing, which
facilitates decoding of complex taste information in the brain.
13. Investigating the function of the autophagy-related protein ATG4D using a Drosophila model. Emily McMann, Gorski Lab, Simon Fraser University.
Macroautophagy (hereafter “autophagy”) is a highly conserved intracellular recycling process that occurs at
basal levels in all eukaryotic cells. Autophagy serves to maintain cellular homeostasis by engulfing excess,
old, and damaged organelles and proteins in double-membrane structures (termed autophagosomes) and
delivers them to the lysosome for degradation. Among the 30+ proteins that regulate the process of
autophagy are the ATG4 cysteine proteases. In mammals, there are four ATG4 homologues (ATG4A-D)
that function to prime Atg8-family members for conjugation to phosphatidylethanolamine in the
autophagosomal membranes, as well as remove them from the outer membrane after autophagosome
formation. The ATG4 homologue ATG4D has been the least well-studied of the ATG4 family members as
initial investigations found its priming capabilities to be weak and limited to a single Atg8-family protein,
leading to the belief that ATG4D was redundant to the other ATG4 homologues. However, apparent loss of
function mutations in ATG4D have since been implicated in a canine neurodegenerative disease, termed
neurodegenerative vacuolar storage disease (NVSD), which is characterized by motor coordination defects,
progressive cerebellar ataxia and Purkinje cell loss, and vacuolization of neuronal and fibroblast cells. More
recently, NVSD-like phenotypes have been identified in human patients with mutations in ATG4D. It has yet
to be determined how exactly ATG4D loss of function contributes to the phenotypes observed in NVSD, or
even if the phenotypes are the result of a defect in autophagy or some other process. Drosophila provide an
excellent model to study human disease and protein function, and we aim to gain insights into the function
of ATG4D via the knockdown and knockout of the Drosophila homologue Atg4b. Additionally, we will utilize
the Gal4-UAS system to express human pathogenic variants of ATG4D in an Atg4b knockout background
to create a disease model for the in vivo study of NVSD.