Relative Growth of Ganglia in ​D.

melanogaster ​and the Effects of the Mutant SLC36

Transporter Pathetic

Brendon Davis, Alex Sova, Siobhan Duffy, Jade Lopez

BIOL 413 A
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Introduction ​As was determined in Lin, W. et al. (2015), the mutant form of the Pathetic gene,
which encodes an SLC36 amino acid transporter, affects development of c4da neurons, with
fewer dendritic arbors and smaller neurons seen in the absence of the wildtype gene. It was also
demonstrated that ​path​ affects nutrient signalling and protein homeostasis in these neurons, but it
does not have a significant effect on smaller sensory neurons. However, the effect of ​path​ on the
central nervous system of ​D. melanogaster​ has not been exhaustively documented. While no
significant phenotypic results of mutant ​path​ have been shown to exist in the central nervous
system, it remains to be seen if the large neurons associated with this system, specifically in the
ventral ganglion, may have growth dependent on the Pathetic gene.
It has been previously documented that genes which affect sensory neuron growth may
also affect the central nervous system. To take some examples, cell-polarity markers for
Drosophila​ photoreceptors are also used in a number of other mitotic processes, including in
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brain development, pathways regulating dendritic pruning have been shown to be markedly
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similar among the central and peripheral nervous system, and programmed cell death of certain
neurons during embryonic and larval development influences the shape and function of the
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central nervous system. It is thus reasonable that a gene necessary for large neuron growth in
sensory neurons may also be necessary for specific neurons in the brain.
The brain of ​D. melanogaster​ is composed of a huge number of interconnected neurons
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of varying sizes. Due to the connected nature of this web of cells, if growth of any is hindered, it
follows that the brain could condense to maintain synapses between neurons. Hence, measuring
brain size could indicate the effect of ​path​ on the neurons of the central nervous system.
Brain growth and development is well-studied across many organisms. In mammals, the
brain continues to grow throughout early post-birth development and then, especially in humans,
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continues to neurologically develop into adulthood. To a further extreme, fishes undergo
extensive brain growth throughout their early somatic development and then continue to display
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phases of neurogenesis throughout their adult lives. Most directly for this study, insects see
continued neuron proliferation throughout most of the larval stage, if not through their whole

1
​Lin, W., Williams, C., Yan, C., & Parrish, J. (2015). Functions of the SLC36 transporter Pathetic in
growth control. ​Fly,​ ​9​(3), 99-106.
2
​Laffafian, A., & Tepass, U. (2019). Identification of Genes Required for Apical Protein Trafficking in
Photoreceptor Cells. ​G3 (Bethesda, Md.),​ ​9​(12), 4007-4017.
3
​Lin T, Kao H-H, Chou C-H, Chou C-Y, Liao Y-C, Lee H-H (2020) Rab11 activation by Ik2 kinase is
required for dendrite pruning in ​Drosophila​ sensory neurons. PLoS Genet 16(2): e1008626.
4
Pinto-Teixeira, F., Konstantinides N., Desplan, C. (2016). Programmed cell death acts at different stages
of ​Drosophila n ​ eurodevelopment to shape the central nervous system. ​FEBS Lett, ​590(15):2435-2453.
5
​Mizutani, R., Saiga, R., Takeuchi, A., Uesugi, K., & Suzuki, Y. (2013). Three-dimensional network of
Drosophila brain hemisphere. ​Journal of Structural Biology,​ ​184​(2), 271-279.
6
​Workman, A., Charvet, C., Clancy, B., Darlington, R., & Finlay, B. (2013). Modeling transformations of
neurodevelopmental sequences across mammalian species. ​The Journal of Neuroscience : The Official
Journal of the Society for Neuroscience,​ ​33​(17), 7368-7383.
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Ganz, J., & Brand, M. (2016). Adult Neurogenesis in Fish. C​old Spring Harbor Perspectives in Biology,
8(​ 7), Cold Spring Harbor perspectives in biology, 01 July 2016, Vol.8(7).
 Brain Size 2

lifetime. In ​D. melanogaster​, neuroblasts continue to divide during the entire larval stage of the
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animal, indicating continuous brain growth and development over time until the pupal stage. All
of the previous information being considered, brain growth may be noticeably tracked over time,
and the effects of environmental and genetic factors on central neuronal development may be
monitored in this way.

Hypothesis ​Based on previous studies, we hypothesize there is an established relationship

between body size and brain size. Specifically, there is an approximately linear positive
correlation between sizes​—​larvae with longer bodies on average have larger brains, while larvae
with shorter bodies on average have smaller brains. To be clear, we are not making a claim that
somatic and neural development are both controlled in the same manner, a fact that has been
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previously reported. Rather, we predict that by using body size as a proxy for the age of the
animal, we can then find brain size and plot this over body size to track brain growth over the
temporal progression of larval development.
We further hypothesize that Pathetic, in addition to its effects on sensory dendrite growth,
also affects brain size in larvae on the basis of previous findings as well as the experiments that
were performed in class earlier in the quarter. Since ​path m​ utants have less dendrite growth and
branching, we expect to see that mutant ​path ​larvae will specifically have overall smaller brain
sizes when compared to wildtype larvae that are of the same body size. In graphical form, this
would correlate to a positive trend similar to that of wildtype but that is shifted down. In other
words, the slope will remain unchanged when comparing wildtype with ​path​ mutants, but the
y-intercept for the ​path​ mutant model will be lower than that for wildtype flies.

Experimental Approach ​Since our main goal for the experiment is to test if there is a
correlation between body and brain size, our design is based around obtaining measurements of
both and then being able to compare them. To do this, we set up a system that involves imaging
the larvae, before we promptly separated the head from the body in order to isolate the brain and
image that as well. We implemented a processing software that will allow us to get readings on
both body and brain that are accurate and considerate of the larvae’s miniscule forms. Later, we
plotted that data onto a line graph, giving the clearest indication of whether there is a correlation
or not.

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Urbach, R., & Technau, G. (2003). Early steps in building the insect brain: Neuroblast formation and
segmental patterning in the developing brain of different insect species. ​Arthropod Structure and
Development, 32​(1), 103-123.
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Montgomery, S., Mundy, N., & Barton, R. (2016). Brain evolution and development: Adaptation,
allometry and constraint. ​Proceedings. Biological Sciences, 283​(1838), Proceedings. Biological sciences,
14 September 2016, Vol.283(1838).
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Materials/Methods

- Larvae: ​In all experiments, ​D. melanogaster ​larvae of various instar stages were
examined and sampled. Four main lines were prepared: wildtype (wt), homozygous
​ ​ mutants, a wt/​pathΔ​ heterozygote, and a ​path​dg50​/pathΔ ​heterozygote. The wild
pathdg50
type larvae will serve as our main control and to also establish any relationship there may
be between body size and brain size. The first heterozygote will be used as a secondary
control to determine if brain size can be changed due to one mutant LOF allele, while the
second heterozygote serves as a functional knockout so that there are no confounding
alleles in other genes that may be causing any size differences we observe. 20-30 samples
were to be taken from each line.
Due to time constraints, the heterozygote lines were not able to be imaged and
scored. A complete study would look at these strains in addition to the homozygote
backgrounds to completely characterize the effect of ​path​ on brain growth.

- Body Imaging: ​To get an accurate reading of length, all larvae were placed onto glass
slides and covered with glycerol in order to immobilize their movements and allow us to
straighten them. A smaller, thinner glass slide was then placed on top in order to keep the
larvae in place and reduce risk of them bending or performing any other movement that
skews the image taken and complicates getting an accurate reading on the size.
All images were obtained using the software Amscope, with the microscope’s dial
turned to its lowest setting of 0.7 magnification (Figure 1C). After images were obtained,
they were labeled with the dissector’s name, the strain it was from, and a number to keep
track of how many samples the dissector had done.

- Dissections: ​After body images were obtained, dissections were then performed in order
to isolate the brain so a clear image of it could be taken. Early trials used a pinning
technique in PBS to dissect and filet larvae so that the body (Figure 1A) and brain (Figure
1B) could be imaged concurrently. This proved time consuming and introduced
variability into data, as assayers might stretch the worms to different tensions when
pinning and the optics were not high quality enough to get accurate measurements of the
relatively small brains.
Given the shortcomings of the first technique, a new form of dissection was
adopted. All dissections were done by grasping the larvae’s head and bottom half
between two pairs of tweezers, before the head was then promptly ripped from the body.
This method provides a simpler and less time consuming-way of getting to the brain than
filet dissection, and also creates less risk of damaging the brain that could come with
cutting the body open and separating all other internal organs. Dissections occurred in
either agar plates filled with PBS solution or another glass slide containing glycerol.
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- Brain Imaging: ​Given previous trials, brain images were ultimately obtained either in
PBS solution or in glycerol with the head ripping method. With PBS, after an image of
the body was obtained, the larvae were then moved to an agar plate filled with PBS and
then dissected and the brain, once isolated and positioned to give a clear indication of the
size, was then imaged.
A secondary method for imaging was to keep the larvae in glycerol and then
perform the dissection there (Figure 1D)​—​this could either be done on the same glass
slide that the body image was obtained from or the larvae could be moved to another
slide. After the brain was isolated, two thin square slides were placed at either end of the
glass slide, before the same thin slide that kept the larvae in place during body imaging
was then placed on top. This was done in order to keep the brain still and not float in the
glycerol without the risk of the top slide’s weight squishing the brain and damaging it.
Both methods proved to have advantages and disadvantages. PBS allowed for us
to be able to pin the larvae’s head down after pulling and keep it in place, which made it
easier to isolate the brain and had less risk of putting any weight on it. However, the
optics were quite noticeably lower in quality and it could be difficult getting the image
into focus. The glycerol slides gave much higher quality optics, particularly because this
allowed for magnification of the central ganglion of the brain and could give clearer
indication of the length of the brain. At the same time, though, it proved challenging at
times to be able to grasp the head and maneuver it so to get the brain alone.
All images were once again obtained through Amscope, this time with the
microscope dial being turned to its highest setting of 4.5 magnification (Figure 1D).
Upon imaging, photos were then labeled with the dissector’s initials, the strain of the
organism, and a number to keep track of how many samples the dissector had done.
Unlike our initial dissections, we also kept the brain entirely intact and did not
remove the optic lobes. This was mainly for simplicity’s sake, as it previously proved to
be very time consuming and difficult to try to cut the lobes without damaging the brain.

- Measuring Size: ​As a means to reduce bias in results, the researcher assigned to
measuring and recording brain and body lengths was given blind images. The labels of
which researcher had taken the picture and the strain of the larvae were removed. Brain
and body images were then separated into two separate files, and each image was
designated a 5-7 number as a means to keep track of the images.
Image measurements were done using the ImageJ processing program and
distance was recorded in mm. To set a scale, a ruler with mm tick marks was imaged
under both the 0.7 and 4.5 microscope magnifications, to be used for the body and brain
images, respectively. Body length was recorded as the distance from the extreme tips of
the anterior and posterior of each larva. Brain lengths were taken as the distance between
 Brain Size 5

the extreme points of high nerve density in the ganglion (the lobes were not included),
and brain widths were recorded as the distance across the widest section of the ganglion.

Results ​After graphing the wildtype results, we found that there seemed to be a very small
correlation between brain and body size. Our graphs had much less “noise” than our initial
dissections, but were still not as clean as we had expected. While the relationship between brain
and body size (in regards to both brain length and width) appears to be loose, there is still a mild
positive correlation, as predicted. It is possible that with more data points, the relationship
between brain and body size may become more apparent.
When wt and mutant ​path g​ raphs were compared for a body-brain relationship (Figure
2A, 2B), we found that ​path c​ lustering was very similar to wt, especially in the largest body size
(correlating to 3rd instar larvae), which was the bin for which we produced the most data. Due to
unforeseen current events, we were unfortunately unable to carry out experiments on either of
our heterozygote lines. Though the lines of best fit are somewhat different when comparing the
path ​homozygote to the control, these regressions do not correspond to any significant
differences between the groups, especially without more data taken for smaller mutant ​path
larvae.
For one final statistical comparison, only the larvae found to have bodies of between 4
and 5 mm were considered, since this bin had the most data and because it can be assumed that
these animals were in roughly the same developmental stage. There was some difference (~7%)
between corresponding averages for both brain length and width (Figure 2C), but they did not
show statistical significance in an unpaired t-test (p=.37 for brain length and p=.10 for brain
width).

Discussion ​In the future, increasing consistency with our assays (dissections and imaging) would
be our first goal. In tandem with this, we would like to additionally assay 2-D brain area (lobes
and ganglion together) using more high quality dissections/images. We were unable to do so in
this study because we had very few images that clearly showed the edges of the full brain, in part
due to the fact that neuron bundles began to fall apart when suspended in glycerol. Another
interesting assay to attempt would be dissecting adult flies. We could perform this assay by
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immobilizing the adult flies on ice and removing the brain in an ice-cold saline buffer. In
regards to any future assay regarding the brain’s response to genetic changes, animals should be
synchronized so that brain sizes can be taken without needing to rely on body size as a proxy for
developmental stage. Though this was not available to us due to time constraints, doing so would
eliminate the need to take a regression of data and rather just compare the averages in brain sizes,
since each data point would be from the same time in development.

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​Diesner, M., Predel, R., & Neupert, S. (2018). Neuropeptide Mapping of Dimmed Cells of Adult
Drosophila Brain. ​Journal of The American Society for Mass Spectrometry,​ ​29(​ 5), 890-902.
 Brain Size 6

Also, it would be interesting to run a GWAS analysis as we originally intended instead of

simply comparing the brain size of the wildtype and pathetic mutant. This, of course, would
require more time. We would need to analyze more samples from each strain and from more
strains in general. Additionally, we could do a staining of the ventral ganglion so that it was clear
where the fly brain began and where it ended. This would hopefully reduce the subjectivity of
our measurements and enhance our assay results. If we were able to accomplish this, we could
collect data on all strains available from the drosophila genetic reference panel11 (approximately
200) with a consistent assay. Next, we would analyze the data and identify strains that either had
a significantly larger brain-body ratio or a significantly smaller brain-body ratio compared to the
other isogenic lines. This would allow us to identify variants, or single nucleotide
polymorphisms (SNPs), that were present specifically in those strains showing unusual
brain-body ratios than the ones with average brain-body ratios. While these SNPs may not
necessarily be ​causal, ​they would give us an idea of where a potential critical variant may lie
within the ​Drosophila ​genome. Thus, we could determine a possible candidate gene and/or
mutation responsible for modified proportional brain growth in Drosophila.
To conclude, of the parts of our study that we were able to complete, we saw that there is
a loose (R​2​=0.103 for brain length) positive correlation between body size and brain size,
supporting our hypothesis to a mild degree. However, LOF ​path​ mutation likely does not
significantly affect brain growth since the homozygote knockout did not show a noticeable
difference from the wildtype strain. This does not necessarily mean that ​path​ does not play a role
in the development of any brain neurons, but rather that it does not affect them to an extent that
brain size is noticeably affected. As mentioned above, there is room to extend and perfect this
study in the future to further understand the nuances of brain growth and development in ​D.
melanogaster.​

Author Contributions ​All authors dissected larvae and took images for scoring, including doing
so outside of regular class hours. Images were “blinded” and data was graphed by Brendon
Davis. Scoring was performed by Alex Sova. All authors contributed to writing this manuscript.

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Trudy F. C. Mackay, Stephen Richards, Eric A. Stone, Antonio Barbadilla, Julien F. Ayroles, Dianhui
Zhu, . . . Richard A. Gibbs. (2012). The Drosophila melanogaster Genetic Reference Panel. ​Nature,
482​(7384), 173-178.
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Figures

A B

C D​

Figure 1: Images of different dissection techniques. Images A (full body) and B (zoomed) show
the original filet technique, which produced highly variable results. Images C (live larvae) and D
(dissected brain) display the result of the “head-ripping” technique that was ultimately adopted.
 Brain Size 8

A​

Figure 2: Graphs of body length versus brain length (Graph A) and body length versus brain
width (Graph B), as well as the corresponding averages for only animals between 4 and 5 mm
 Brain Size 9

long (Graph C). Blue corresponds to wildtype and red corresponds to mutant ​path​. Equations for
the lines of best fit as well as the correlation coefficients are shown above plots A and B. Both
comparisons of length in width in plot C were insignificant (p-values of 0.37 and .10,
respectively).