# Combining dynamics of ligand presentation with dynamics of hESC response in colonies with defined architecture

> **NIH NIH R01** · ROCKEFELLER UNIVERSITY · 2020 · $339,000

## Abstract

Project Summary
During early embryogenesis, pluripotent cells specify their fates by integrating multiple signals delivered at
different times, places, and with different dynamics. In the mouse embryo, interplay between 3 signaling
pathways - BMP4, Wnt, and Nodal - initiate the induction and patterning of embryonic germ layers. However,
the relevance of these pathways to human development is not understood. Furthermore, how multiple dynamic
signaling pathways are integrated to define cellular fate is unclear. Here we propose to use human embryonic
stem cells (hESCs) to understand how individual cells process these dynamic signals to generate discrete
fates. To address this, we developed 2 innovative technologies. First, microfluidics to precisely control the
timing of ligand application and follow the behavior of the signal transducer SMAD4, with which we
demonstrated that TGFβ signaling was adaptive. Second, micropatterns to control hESC colony size and
geometry, to show that in response to BMP4, hESCs cultured in circular colonies self-organize into radially
symmetric patterns of discrete embryonic germ layers. This remarkably recapitulates the proximal-distal axis of
the gastrulating mouse embryo. In this competitive renewal, we combine the strengths of both technologies to
deliver distinct dynamics of ligand presentation by microfluidics to CRISPR-edited hESC lines cultured in
micropatterned colonies. Four independent signaling-reporter hESC lines that fluorescently tag SMAD1,
SMAD2, SMAD4, and β-CATENIN will be used to visualize signaling, and one triple-tagged, fate-reporter
CRISPR-edited line that fluorescently tags SOX2, BRACHYURY, and SOX17, will be used to monitor fate
acquisition. Our CRISPR-reporter lines will be used to measure signaling dynamics and fate acquisition, with
single cell resolution and in real-time by video-microscopy, when cells are presented with BMP4, Wnt3A, and
Activin/Nodal either as a persistent step of defined concentration, or as one of defined duration. We propose
three specific aims. In aim1, the three ligands will be presented to our signaling-reporter CRISPR lines, to
follow the behavior of the four tagged signal transducers and to determine the dynamic behavior of each
pathway. In aim2, using the same approach, we will evaluate pathway output by measuring the activity of
transcriptional reporters for the three ligands, and use our triple fate-reporter CRISPR line to follow fate
acquisition. These two approaches will establish a quantitative link between signaling dynamics, transcriptional
output, and fate determination. In aim3, large datasets obtained from aims1 and 2, will be used to model the
kinetics of signal transduction, and provide a mathematical paradigm to explain hESC self-organization. The
resolution of our three aims will have a strong impact on our understanding of the dynamic integration of
signaling pathways underlying human cell fate specification with direct relevance to both basic underst...

## Key facts

- **NIH application ID:** 9857027
- **Project number:** 5R01GM101653-08
- **Recipient organization:** ROCKEFELLER UNIVERSITY
- **Principal Investigator:** ALI H BRIVANLOU
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $339,000
- **Award type:** 5
- **Project period:** 2012-07-19 → 2021-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9857027

## Citation

> US National Institutes of Health, RePORTER application 9857027, Combining dynamics of ligand presentation with dynamics of hESC response in colonies with defined architecture (5R01GM101653-08). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9857027. Licensed CC0.

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