# Investigating how bHLH circuits integrate signals for cell fate decisions

> **NIH NIH K99** · HARVARD MEDICAL SCHOOL · 2024 · $124,740

## Abstract

Project Summary
Multipotent stem cells in animals integrate information from various extracellular signals to choose between fates.
Signal integration enables robust, context-specific decisions, while errors in this process underlie developmental
disorders and cancers. To be effective, signal integration must be tightly linked to coordination between fates,
i.e., the activation of a target fate program and inactivation of alternative fates. How is this achieved?
 My recent postdoctoral work suggested that, in cultured neural stem cells (NSCs), a gene regulatory
circuit of basic helix-loop-helix (bHLH) transcription factors enables the integration of two signals to
simultaneously activate astrocyte differentiation and suppress alternative fates. Transcriptional interactions
among bHLHs are an important component of this circuit, but they alone could not account for signal integration.
 I hypothesize that two other key features of bHLHs play an essential role: protein-level dimerization and
oscillatory dynamics of bHLHs. Here, I will investigate how these features contribute to signal integration by the
NSC circuit using quantitative measurements of dimerization and dynamics complemented by precise
perturbations. Moreover, I will analyze the role of a bHLH circuit in the developing zebrafish spinal cord to
understand how principles of circuit function obtained using in vitro systems extend to an in vivo context.
 In Aim 1, I will investigate the role of bHLH dimerization by designing novel dimerization mutants based
on computational sequence co-evolution analysis (in collaboration with Dr. Debora Marks), validating them using
a quantitative yeast-based measurement platform that I have developed, and analyzing their impact on signal
integration in NSCs. In Aim 2, I will use a combination of timelapse imaging and multiplexed RNA-FISH in NSCs
to analyze how oscillatory dynamics in the bHLH Hes1 is controlled by upstream signals and subsequently
impacts other bHLHs in the circuit as well as downstream fate outcomes. I will also assess how ectopically
modulating Hes1 dynamics affects circuit behavior. In Aim 3, I will determine whether and how a bHLH circuit in
stem cells of the zebrafish neural plate integrates two developmental signals to enable an early fate choice in
this tissue. Specifically, I will first characterize how signaling activity in individual cells impacts their fate using a
combination of in vivo timelapse microscopy and targeted signaling perturbations. I will then investigate how
interactions in the bHLH circuit mediate the effects of signals on fate choice.
 bHLH factors are expressed in most stem cells during development and in adult tissues. bHLH circuits
could therefore play a ubiquitous role in integrating signaling information to enable cell fate decisions. This work
seeks to broadly understand how they function, leveraging quantitative approaches both in vitro and in vivo with
guidance from my mentors Dr. Galit Lahav and Dr. S...

## Key facts

- **NIH application ID:** 10873289
- **Project number:** 5K99GM151398-02
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Nagarajan Nandagopal
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $124,740
- **Award type:** 5
- **Project period:** 2023-07-01 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10873289

## Citation

> US National Institutes of Health, RePORTER application 10873289, Investigating how bHLH circuits integrate signals for cell fate decisions (5K99GM151398-02). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10873289. Licensed CC0.

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