Date of Award

Summer 7-16-2025

Document Type

Campus Access Thesis

Degree Name

Master of Science (MS)

Department

Biotechnology and Biomedical Science

First Advisor

Juliet Girard

Second Advisor

Alexey Veraksa

Third Advisor

Labib Rouhana

Abstract

Precise regulation of hematopoietic (blood) stem and progenitor cells (HSPCs) is essential for sustaining healthy lifelong blood production in metazoans. The Drosophila larval lymph gland is a well-established model for dissecting the mechanisms that govern HSPC maintenance, proliferation, and differentiation. Normal Wnt signaling supports homeostatic blood cell development by balancing HSPC self-renewal with differentiation, whereas aberrant Wnt signaling contributes to diseases such as leukemias. In the lymph gland, several Wnt ligands are expressed in HSPCs, including Wnt6 and Wnt5. Wnt6 prevents HSPC differentiation by promoting HSPC cell growth and pausing HSPCs in the G2 phase of the cell cycle through a β-catenin-independent pathway. In contrast, β-Catenin-dependent (canonical) signaling allows late HSPCs to escape the G2 pause and progress into G1 where HSPCs commit to differentiation. Thus, both canonical and non-canonical branches operate in HSPCs to balance HSPC maintenance and differentiation. In contrast to Wnt6, the role of Wnt5 in lymph gland hematopoiesis remains unclear, including whether Wnt5 acts redundantly with or distinctly from Wnt6. Here we investigated how modulating Wnt5 expression affects HSPC differentiation, cell size, and cell-cycle status, and we examined which downstream pathways mediate Wnt5 signaling.

We combined genetics, flow cytometry, quantitative reverse transcription PCR, and immunofluorescence analysis to investigate the effect of Wnt5 loss- and gain- of function. Wnt5 expression was manipulated using the GAL4/UAS system, which was either used to drive RNA interference (RNAi) to knockdown Wnt5 or drive Wnt5 overexpression. Progenitor and differentiated cells were quantified using the fluorescence of established reporters for HSPCs (domeMESO-EGFP) and differentiated cells (HmlD-DsRed). Knockdown of Wnt5 increased the proportion of differentiated blood cells and reduced HSPCs compared with the wild-type control. Conversely, Wnt5 overexpression had no effect on differentiation. Cell size analysis using forward-scatter measurements showed no change in HSPC cell size upon Wnt5 knockdown compared to wild-type, unlike Wnt6 knockdown, which significantly decreases cell size. Wnt5 overexpression, in contrast, aberrantly increased cell size compared to wild-type. Analysis of the DNA content of HSPCs using Hoechst fluorescence revealed that unlike Wnt6, Wnt5 depletion or overexpression has no effect on HSPC cell cycle. Given that Wnt5 seems to mediate a distinct mechanism of HSPC maintenance from Wnt6, we took a candidate approach to investigate which pathways act downstream of Wnt5. For this we examined 3 pathways that have been shown in other systems to act downstream of Wnt5, specifically: inhibition of canonical Wnt signaling (monitored by nkd and stg/Cdc25 transcription), activation of JNK signaling (assessed by MMP1 expression), and Wnt/Ca²⁺ signaling (tracked with a GCaMP6f reporter).

qPCR revealed that stg/Cdc25 transcript levels were significantly increased in both UAS-Wnt5 overexpression and Wnt5RNAi (KK) lymph glands, whereas nkd transcripts remained unchanged. No significant differences were detected in the JNK-responsive gene MMP1. Moreover, MMP1 immunostaining also showed no significant protein level difference. Preliminary data shows that the mean GCaMP6f fluorescence, a proxy for intracellular Ca²⁺ signaling, was significantly higher in UAS-Wnt5 overexpression and Wnt5RNAi (KK) compared to the controls, indicating that Wnt5 perturbation affects Ca²⁺ signaling. While Wnt5RNAi (BL) showed no significant difference in GCaMP6f fluorescence, it showed the same trend of increased GCaMP6f seen in UAS-Wnt5 and Wnt5RNAi (KK), which supports these findings. Together, these results suggest that Wnt5 restrains HSPC differentiation in a mechanism distinct from Wnt6. Our initial findings suggest that Ca²⁺-dependent non-canonical Wnt signaling mechanism and canonical Wnt modulation may act downstream of Wnt5.

Comments

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