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○ Females on average had more crystal cells present than males.○ The genes Ncc69 and Cbp53E were investigated because of the
varying amounts of crystal cell based on gender.○ The gene Unc-89 was also investigated to determine if it had a
role in the varying amounts of crystal cells throughout all thegenotypes
●
● Banerjee, Utpal, et al. “Drosophila as a Genetic Model for Hematopoiesis.” Genetics, vol. 211, no. 2, 1 Feb. 2019, pp. 367–417., doi:10.1534/genetics.118.300223.
● FlyBase. FlyBase Gene Report: Dmel\Unc-89, flybase.org/reports/FBgn0053519.● Katzemich, Anja, et al. “Binding Partners of the Kinase Domains in Drosophila Obscurin and Their Effect on the Structure of the
Flight Muscle.” Journal of Cell Science, The Company of Biologists Ltd, 15 Sept. 2015, jcs.biologists.org/content/128/18/3386.● “The Hippo Pathway Regulates Hematopoiesis in Drosophila Melanogaster.” Current Biology, Cell Press, 30 Oct. 2014,
www.sciencedirect.com/science/article/pii/S0960982214013402.● Cattenoz, Pierre B et al. “Functional Conservation of the Glide/Gcm Regulatory Network Controlling Glia, Hemocyte, and
Tendon Cell Differentiation in Drosophila.” Genetics vol. 202,1 (2016): 191-219. doi:10.1534/genetics.115.182154● Makhijani, Kalpana, and Katja Brückner. “Of blood cells and the nervous system: hematopoiesis in the Drosophila larva.” Fly vol.
6,4 (2012): 254-60. doi:10.4161/fly.22267● Hagel KR, Beriont J, Tessier CR (2015) Drosophila Cbp53E Regulates Axon Growth at the Neuromuscular Junction. PLoS ONE
10(7): e0132636. https://doi.org/10.1371/journal.pone.0132636● Michele Markstein, “Drosophila workers unite!”
Crystal Cell Formation○ The average number of crystal cells melanized were similar between each gender,
except for in line 28179.○ The sex of the larva had an effect on line 28179. The average number of
melanized crystal cells in female larva was more than males within the samegenome. This could be due to a large variation in one of the genders.
Genes● Unc-89
○ The gene Unc-89 encodes a protein located at the midline of the muscle thickfilaments that is needed for assembly of a symmetrical sarcomere. It is involvedin myogenesis and the Hippo signalling pathway.(Katzemich, et al. 2015)
○ The hippo pathway was found to regulate Hematopoiesis. The hippo pathwayturns on the protein lozenge and the protein lozenge causes blood stem cells todifferentiate into crystal cells. So, if there was more of the protein lozenge beingproduced then there could be an increase in the number of crystal cells, and viseversa.(Milton, et al. 2014)
Gene Differences Between Sexes● Cpb53E
○ The gene Cbp53E, which is involved in the negative regulation of response to wounding and neuronal growth, was more likely to have a single nucleotide polymorphism(SNP) in females than males.
○ This gene might affect crystal cell melanization because of its role in the negative regulation of response to wound healing since crystal cells facilitate wound healing by melanizing.
○ Cbp53Es’ role in neuronal growth may also influence blood cell development because hemocytes rely on the peripheral nervous system to localize and proliferate.(Brückner, et al. 2012; Hagel, et al. 2015)
○ Furthermore the SNP on this gene might have an affect on crystal cell melanization because it is a minor allele in the genotype 28137, which has the highest average number of crystal cells, with females having more crystal cells on average than males.
● Ncc69○ Ncc69 has alleles that manifest in reticular associated glial cells. Microglial, a
type of glial cell, acts as the primary defense to immune responses of the nervous system. When considering crystal cell developmental factors the alleles associated with glial cells in the Ncc69 genome may be the reason for line 28179 having a drastically high crystal cell difference and line 28247 having a slightly higher crystal cell difference in females. (Cattenoz, 2016)
○ In addition, GCM is likely to trigger the expression of target genes being that it is known to control the gene loco in late glial cell differentiation. Therefore, if GCM plays a large role in glial cell development then Ncc69 could be another one of its possible target genes. This may be the reason for higher crystal cell amounts in females due to the microglial correlation with immune responses. (Cattenoz, 2016)
○ Treat the larva with methoprene and compare thenumber of crystal cells that melanize.
○ Investigate further to find if the genes arespecifically associated with crystal cellmelanization.
○ Overexpress GMC in females and males thatboth have the Ncc69 gene.
A
○ Heat Shocking: Thethird instar larvae arebeing heated at 70degrees celsius for 10minutes in the DNAThermal Cycler. Thenthe crystal cellsmelonized.
○ Scoring: The number ofcrystal cells weremanually-counted on thedorsal, ventral, andlateral sides using aclicker.
○ Drosophila have almost asmany genes as humans do,14,000 compared 21,000genes in humans. They alsohave a homolog of over65% of genes associatedwith human diseases.(Markstein )
○ Drosophila have a 6-10 daylife cycle from an embryoto an adult.
○ There are three differenttypes of blood cell typesthat are found in Drosophilawhich are cystall cells,plasmatocytes, andlamellocytes.(Banerjee, et al. 2019)
○ Crystal cells facilitatewound healing, innateimmunity, and the hypoxicresponse. Under stressconditions crystal cellsmelanize.(Banerjee, et al. 2019)
○ Blood cells develop in thelarval hematopoietic organ(aka Lymph Gland)and theDorsal vessel.(Banerjee, et al. 2019)
○ A genome-wide associationstudy(GWAS) scansmarkers across completesets of genomes to findgenetic variationsassociated with a disease. AGWAS of drosophila can beused to find if certain genesinfluence the number ofcrystal cells in drosophila ofdifferent genotypes.
Natural Variations in Blood Composition of DrosophilaNatalie Santana, Suzaul Sobhan, Anwar Benmallouk, and Rebecca Spokony
Department of Natural Sciences Baruch College, CUNY, 17 Lexington Avenue, New York, NY 10010
ACKNOWLEDGMENTSWe would like to thank Areeba Choudhury, Jessica Sparacio, Marium Sarder for training and supervising us throughout our research.
FUTURE DIRECTIONS○ Further visual analysis of larvae reveals that the average number of crystal cells differ in different genotypes. With
genotype 28137 having the highest number of crystal cells on average and genotype 28223 having the lowest numberof crystal cells on average.
RESULTS
INTRODUCTION
METHODS REFERENCES
DISCUSSION
○ The average number of crystal cells differ between the males and females of each genotype. Females from genotype28179 on average had higher amounts of crystal cells compared to the males. Despite the variation between the malesand females, the genotype 28137 has the highest average number of crystal cells in both sexes.
Number Of Crystal Cells In A Variety Of Drosophila Genotypes
Genotypes
25174 28196 28137 28223 28179 28247 55707
Male
Female
OVERVIEW
Figure 3: Heat shocking Process
Figure 2: Hematopoietic Organ
Mean Number of Crystal Cells Varies Between Different Drosophila Genotypes
Mean Number of Crystal Cells in Males and Females in Different Drosophila Genotypes
Figure 1: Life Cycle of Drosophila Melanogaster