# The role of chromatin dynamics in stochastic cell fate specification

> **NIH NIH F31** · JOHNS HOPKINS UNIVERSITY · 2022 · $46,752

## Abstract

Project Summary: Stochastic cell fate specification, in which cells randomly choose between cell fates, is critical
during development. Stochastic mechanisms diversify visual and olfactory receptors, motor neurons, immune
cells, and stem cells. Breakdowns in these mechanisms lead to vision disorders, anosmia, immunodeficiency,
autism, and lymphomas. I study the developing Drosophila visual system as a paradigm to understand the
mechanisms controlling stochastic cell fate specification. Within the fly eye, R7 photoreceptors make a stochastic
binary fate choice between two subtypes, characterized by expression of light-detecting rhodopsins (Rh3 or
Rh4). This decision is controlled by the stochastic ON/OFF expression of the transcription factor Spineless (Ss).
SsON R7s express Rh4 and SsOFF R7s express Rh3. Each R7 independently makes this random decision with a
67% chance of taking the SsON R7 fate and 33% chance of taking the SsOFF R7 fate. Thus, retinas have consistent
subtype ratios, but unique, random patterns.
 We have identified a two-step mechanism controlling ss expression during development. In step one, an
early pulse of expression opens the ss gene locus in precursor R7s. In step two, the locus compacts to varying
degrees, determining the ability to reinitiate expression upon specification of terminal R7 subtypes. This final
expression decision is then maintained for the lifetime of the animal. I am investigating how dynamic chromatin
compaction at the ss locus influences stochastic R7 subtype specification. I hypothesize that R7 precursors
rapidly reorganize chromatin at the ss locus following the early pulse of expression to determine the ON/OFF
state of ss expression in terminal R7s.
 To test this hypothesis, I will use fixed and live imaging techniques to track ss compaction during R7
subtype specification. To visualize compaction in fixed images, I developed a three-color DNA-FISH strategy
that labels the ss locus, upstream region, and downstream region. Measuring the 3D distance between these
regions provides cell-type-specific quantification of compaction in developing R7s. Complementing this method,
I developed a live imaging approach utilizing the LacO/LacI reporter system. Inserting LacO repeats into the
regions upstream and downstream of ss, I will measure the 3D distance between the two LacI reporter punctae
in differentiating R7s, focusing on transition points that cannot be identified with fixed imaging. To facilitate
quantification, I developed an automated image analysis pipeline to evaluate chromatin compaction in fixed and
live tissue (Aim 1). I will complement these approaches by conducting ATAC-seq to identify chromatin
accessibility at the ss locus in normal and mutant conditions. I will focus specifically on the accessibility of the
late enhancer whose activity ultimately determines the ssON or ssOFF R7 fate (Aim 2). The results of this project
will elucidate the regulatory relationship between chromatin dynamics an...

## Key facts

- **NIH application ID:** 10326372
- **Project number:** 5F31EY032430-02
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Lukas Voortman
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $46,752
- **Award type:** 5
- **Project period:** 2021-01-16 → 2024-01-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10326372

## Citation

> US National Institutes of Health, RePORTER application 10326372, The role of chromatin dynamics in stochastic cell fate specification (5F31EY032430-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10326372. Licensed CC0.

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