This study investigates the role of the gene Arrowhead (Awh) in regulating intestinal stem cell proliferation and homeostasis during aging in Drosophila. The researchers hypothesize that Awh activation increases expression of regulatory peptides involved in regeneration and proliferation. Knocking down or mutating Awh leads to increased mitotic figures and larger stem cell clones, suggesting Awh pushes stem cells towards differentiation. Awh mutant flies generate larger clones but fewer clones per gut, and have increased maximum clone size with age. Awh is necessary for responding to proliferative stress and may inhibit a proliferation promoter complex, potentially through interactions in the Wnt pathway.

1. Introduction
The importance of properly maintained endogenous ISC regulation is the maintenance of epithelium.
Without ISCs there would be no cell responsible for maintaining gut barrier function through
regeneration. The intestinal epithelium of Drosophila has been used to study the regenerative
capacity of intestinal stem cells (ISCs). The ability to maintain barrier function is lost with age
influenced loss in genomic stability, and reducing ISC regenerative capacity.
A recent proliferation study demonstrates an increase in LHX4 mRNA as a function of age in
mammals (Z. Wang). Similarly, the HOX family gene, Awh, in Drosophila is orthologous to the
LHX family genes found in mammals (Sun 2006). LHX4 expression in malignant colorectal tissues
suggest a LHX mediated relationship between cancer development and age. A recent study in mice
demonstrated a novel approach to genetic silencing via inhibiting expression of LHX tumor cells
Lim-1 acts as an oncogene. (Dormoy).
We have shown in an aging genome wide association study that the gene Arrowhead (Awh) may
influence proliferative homeostasis as a function age. The fly homolog of the mammalian homeobox
gene family is a transcription factor for stress response, regeneration, and suppresses Wnt upstream
of known cancer promoting pathways (Sato and Shibuya, 2013). Here, we validate our GWAS
finding by observing the differences in proliferation through validated genotype with the Awh KD in
the ISC and EBs.
Hypotheses
I hypothesize that in ageing tissue, Awh is activated to increase gene expression of regulatory
peptides that lead to an increased regenerative capacity, stress resistance, and proliferative
homeostasis.
I predict that both Awh knockdown & Awh mutant will exhibit a gain of function in ISCs resulting
with increased mitotic figures and larger clones, respectively.
Method Results Continued
7-day post heat shock clone size frequency, we binned the data to compare the R squared values (Fig. 6G). We
bin the data for 15 days as well (Fig. 6H). Awh KD in EB and ISCs increases endogenous mitotic figures (Fig. 6
I-J). The Drosophila gut cells respond to the Ecc15 microbial Infection (Fig. 6I’-J’) (Fig. 6K).
Key Findings
•Awh Mutant Increases GFP+/Clone Ratio
•Awh Mutant Reduces Clone/Whole Gut Ratio
•Awh Mutant Shows an Age Related Increase in Maximum Clone Size
•Awh RNAi in Stem Cell & Progenitor Cells Increases Mitotic Figures During Proliferative Stress
•Awh RNAi in ISCs & EBs Increases Endogenous Mitotic Figures
Arrowhead (Awh) is Required for Intestinal Stem Cell Lineage &
Management of Proliferative Homeostasis with Age
Adam Ashton Kinion, LinLin Guo, Heinrich Jasper
The Buck Institute for Research on Aging
Dominican University of California
Acknowledgements:
Rachel B. Brem, Walter Shine, Graciela Maria Carranza, Kazutaka Akagi, Kenny Wilson, Mauricio Ortega, Subhash D. Katewa,
Jesse Simons, Sven Lang, Pankaj Kapahi, Joanne Van Kampen, Evelyn Crivello, Hai Lu, Imilce Rodriguez-Fernandez,
Hansong Deng, Hongjie Li, Suzy Jackson, Yanyan Qi, Rebecca Riley, Samantha Haller, Daniel Hu, Mia Konjikusic, Pedro
Victor-Souza, Nancy Barnett, Alexis Valdovinos, Ethan Annis, Regina Thomas
References:
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Results
Immunohistochemistry Staining for delta, prospero, b-catenin/armadillo and GFP at 7 days’ post heat shock (Fig. 6A-A’). Awh
mutant lines generating an average clone size that is much larger than the FRT2A (Fig. 6B). Awh mutant clones also show on
average that there are less clones per gut relative to the control (Fig. 6C). When we leave the genotype the same, but assay the
flies were assayed at 15 days’ post heat shock, and exhibited similar to the 7 day qualitative readouts (Fig 6D-D’). The 15-day
post heat shock Awh mutant produced larger clone size (Fig. 6E). Awh mutants continue to generate less clones for gut (Fig.
6F).
Discussion & Future Direction
An increase in the size of the clones suggests that Awh is required in proliferative homeostasis by possibly pushing
ISCs towards terminal differentiation rather than symmetric division. Ecc15 exposure suggesting that Awh is necessary
for endogenous proliferation stress responses. Awh may be an inhibitory component of a proliferation promoter
complex and may be mediated by the function of Awh in the Wnt pathway. Binning the data allows for a standard
curve of the of the experimental to be compared group relative to the control group. The area under the curves can be
statistically analyzed. In the future, , we will collaborate with other labs, i.e. Juila Cordero Lab, in order to place Awh in
the Wnt Pathway; hypothetically, we will use these curves to determine possible interactions with Awh and Wnt
pathway peptides, and if we see a shift in the experimental curve that mimics the control curve this will indicate a
functional interaction.
Supplemental Table Supplemental Figure
Experimental Approach
Induction of Proliferation & Recombination
MARCM Genetics
Tzumin Lee 1999
Buchon et. al