# Identification of new components of the Toll receptor signaling pathway in Drosophila

> **NIH NIH R03** · UNIVERSITY OF TEXAS AT AUSTIN · 2022 · $79,250

## Abstract

First discovered for its role in embryonic dorsal/ventral (DV) patterning, studies of Toll receptor signaling in
Drosophila have led to major advances in our understanding of innate immunity and inflammation in
humans, and of the regulation of eukaryotic gene expression. In the early single-cell fly embryo, Toll is
distributed throughout the plasma membrane, but only activated ventrally. This is mediated by three
sequentially-acting extracellular serine proteases, the last of which, Easter, cleaves a precursor form of the
Toll ligand on the ventral side of the embryo. Ventral activation of Easter is controlled by Pipe, a Golgi-
localized sulfotransferase that is expressed in ventral cells of the follicular epithelium that surrounds the
developing oocyte. Pipe generates a sulfated cue that becomes embedded ventrally in the eggshell. Many
aspects of this pathway remain to be elucidated, including the precise molecular nature of the direct
substrate of Pipe (protein, carbohydrate, or lipid?), and the mechanism through which the Pipe target directs
the ventral activation of Easter. In addition to the ventral restriction of pipe expression, uniform low levels of
pipe expression around the developing oocyte results in embryos with residual DV polarity, indicating that
there exists another, previously undetected mechanism that can define embryonic DV polarity. This
application requests support for a pilot project, the objective of which is to utilize a novel approach, proximity
labeling by in vivo biotinylation, to identify new components of the Toll receptor signaling pathway. The
rationale is that because most of the known components were identified in screens for strict maternal effect
mutants, genes encoding pathway components required for viability of females to fertile adulthood are likely
to have been overlooked. Proximity labeling identifies factors that interact or colocalize with proteins-of-
interest and is not limited to proteins that are dispensable for survival. The following specific aims will be
pursued: 1. To identify new regulators and effectors of the serine proteases operating in the
perivitelline space we will express them in the female germline as fusions to newly-developed, highly
active biotin ligases, then perform proximity labeling in progeny embryos. 2. To identify proteins that
interact with or colocalize in the Golgi with Pipe we will perform proximity labeling with Pipe-biotin ligase
fusions expressed in ovarian follicle cells. For both specific aims, a subset of the identified presumed
interactors will be tested for effects upon embryonic DV patterning. The identity of previously unappreciated
pathway components will provide further insight into the control of Toll activity and stimulate new avenues of
research. In addition, this project will also serve as a feasibility study of the extent to which proximity
labeling can effectively identify bona fide protein/protein interactions in developing embryos and the extent
to whi...

## Key facts

- **NIH application ID:** 10360133
- **Project number:** 1R03HD107458-01
- **Recipient organization:** UNIVERSITY OF TEXAS AT AUSTIN
- **Principal Investigator:** DAVID S. STEIN
- **Activity code:** R03 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $79,250
- **Award type:** 1
- **Project period:** 2022-02-15 → 2024-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10360133

## Citation

> US National Institutes of Health, RePORTER application 10360133, Identification of new components of the Toll receptor signaling pathway in Drosophila (1R03HD107458-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10360133. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*