# Wnt pathway dynamics and their role in generating self-organized ectodermal patterns

> **NIH NIH R01** · RICE UNIVERSITY · 2020 · $322,947

## Abstract

Project summary
The Wnt pathway plays a crucial role in embryonic development and disease. During early
development, Wnt signaling is required for gastrulation and mesendoderm differentiation, and,
slightly later, for the differentiation of neural crest cells within the ectodermal layer. While the
detailed biochemistry of signal transduction has been studied extensively, much less is known
about the dynamics of pathway activity. We have used CRISPR-Cas9 genome engineering to
create a novel reporter for Wnt pathway dynamics in both human embryonic stem cells (hESCs)
and human cancer cells with temporal resolution on the time scale of minutes. Using this tool,
we have found that stimulation by Wnt ligands yields transient localization of the signaling
effector β-catenin to the cell nucleus. This was surprising as previous work has shown, and we
have confirmed with our reporter, that total β-catenin levels are stably elevated by Wnt
signaling. Here, we propose to use a combination of automated fluidic control for manipulating
ligand dynamics, quantitative measurements with Wnt pathway and cell fate reporters, and
mathematical modeling to dissect the relationship between ligand inputs, signal transduction by
β-catenin, and the resulting cell fate decisions. We will examine the role of endogenous Wnt
signaling in human ectodermal patterning using a novel hESC culture platform. In this system,
cells are grown in controlled geometries using micropatterning technology, differentiated to
ectodermal fates, and then treated with the ligand BMP4. We have shown that BMP4 response
leads to epidermal fates near the colony border, and that it induces secondary, endogenous
Wnt signals that lead to neural crest differentiation at a particular radius within the colony. Cells
at the center of colony do not receive these signals, and adopt a neural fate. We will combine
this assay with live cell measurements of Wnt and BMP pathway activities and cell fate markers
to understand how the interplay between the supplied BMP signal and the induced Wnt signal
creates self-organized patterns of ectodermal cell fates. This project will provide essential
information on Wnt pathway dynamics, which is crucial for understanding its role in embryonic
development, harnessing it in directed differentiation protocols, and circumventing it in cases
where Wnt signaling drives the growth of cancers. Further, understanding the role of Wnt in the
in vitro ectodermal patterning assay will reveal principles of generating self-organized tissues in
vitro, a central goal in regenerative medicine.

## Key facts

- **NIH application ID:** 9840478
- **Project number:** 5R01GM126122-03
- **Recipient organization:** RICE UNIVERSITY
- **Principal Investigator:** Aryeh Warmflash
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $322,947
- **Award type:** 5
- **Project period:** 2018-01-01 → 2022-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9840478

## Citation

> US National Institutes of Health, RePORTER application 9840478, Wnt pathway dynamics and their role in generating self-organized ectodermal patterns (5R01GM126122-03). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/9840478. Licensed CC0.

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