# Mechanisms controlling stochastic gene expression during eye development

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2020 · $402,318

## Abstract

Project Summary
 Cell fate specification is driven by lineage, signaling, and stochastic regulatory inputs. The
mechanisms controlling stochastic fate specification, in which a cell randomly chooses between
two or more fates, are poorly understood. Stochastic fate specification is critical for diversifying
retinal neurons, olfactory sensory neurons, motor neurons, immune cells, and stem cells.
Breakdowns in these mechanisms cause debilitating human disorders, including vision
impairments, anosmia, autism, immunodeficiencies, and lymphoma. The main goal of this project
is to determine how chromatin state and transcriptional variability control stochastic fate
specification, using the random patterning of photoreceptor subtypes in the fly retina as a
paradigm.
 The fly eye contains a random mosaic of two color-detecting R7 photoreceptor subtypes,
defined by expression of Rhodopsin 4 (Rh4) or Rhodopsin 3 (Rh3). This fate decision is controlled
by the transcription factor Spineless (Ss), which is expressed in a random subset of mature R7s.
SsON R7s express Rh4, while SsOFF R7s express Rh3. Our data support a two-step mechanism
regulating ssON/OFF expression in mature R7s. In step 1, the early enhancer drives an early pulse
of ss transcription in R7 precursors that opens the chromatin at the ss locus. In step 2, the
transcriptional pulse ceases and chromatin variably closes, defining the accessibility of the late
enhancer. Depending on the degree of chromatin compaction, the late enhancer either turns on
(open chromatin) or remains off (closed chromatin) for the lifetime of the mature R7. How
regulation of transcription and chromatin compaction is integrated to turn genes randomly on or
off during development is poorly understood. We will use DNA FISH, genomics, CRISPR, and
lacO/LacI-based live imaging approaches to assess the role of chromatin dynamics and the
temporality of regulatory inputs in the two-step mechanism (Aim 1).
 Identification of the source of variability driving stochastic fate specification in metazoans
has not been achieved. Our data suggest that variability in transcription (initiation, elongation,
frequency, and duration) in individual cells influences terminal R7 fate specification. To test this
hypothesis, we will use nascent multi-color RNA FISH and MS2/MCP-based live imaging to
assess transcriptional parameters and relate them to R7 subtype fates (Aim 2). Successful
completion of these experiments will identify a source of variability that drives a cell fate decision
and inform how molecular noise is utilized to diversify cell types during metazoan development.

## Key facts

- **NIH application ID:** 9973446
- **Project number:** 2R01EY025598-06
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Robert John Johnston
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $402,318
- **Award type:** 2
- **Project period:** 2015-08-01 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9973446

## Citation

> US National Institutes of Health, RePORTER application 9973446, Mechanisms controlling stochastic gene expression during eye development (2R01EY025598-06). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/9973446. Licensed CC0.

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