# Cellular and molecular mechanisms of vertebrate testis homeostasis

> **NIH NIH R35** · UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH · 2021 · $381,250

## Abstract

PROJECT SUMMARY
Primordial germ cells seed the somatic gonad early in vertebrate development, and develop together to yield a
functional testis replete with spermatogonial stem cells (SSCs). This population of SSCs maintains itself while
generating astonishing numbers of differentiated sperm across the lifetime of the animal. We lack an
understanding of the cellular and molecular mechanisms in SSCs that maintain an active population of stem
cells. Studies of SSCs in vertebrates have been limited to population or single-cell snapshot studies, which fail
to capture the contributions of individual SSCs to this process.
Here, I propose to apply recently developed CRISPR lineage tracing, CRISPR mutagenesis, and single-cell
RNA sequencing methods to uncover the mechanisms that maintain the vertebrate testis. First, we will
investigate the dynamics of individual germline stem cells in testis homeostasis. We will use CRISPR lineage
tracing in zebrafish to determine the contributions of individual SSCs to self-renewal and differentiation, and
serial sampling from individual animals to track long-term dynamics. Second, we will characterize new
regulators of SSC homeostasis. Using single-cell atlases of SSC gene expression, we will use our rapid
CRISPR methods to mutagenize candidate regulator genes and phenotype using single-cell phenotyping and
high-resolution imaging. Third, we will examine the evolutionary mechanisms that have generated testis
phenotypic diversity across the vertebrate subphylum. We will generate single-cell, spatially resolved atlases of
the juvenile, adult and aged testis from species across a phylogenetic tree of teleosts, and use comparative
gene expression analysis to determine cellular and molecular differences in SSCs and niche cells.
These projects will define the molecular and cellular mechanisms that maintain stem cell populations within the
testis, with implications for human infertility. They will also uncover general principles of stem cell homeostasis
in tissues and organs.

## Key facts

- **NIH application ID:** 10276262
- **Project number:** 1R35GM142950-01
- **Recipient organization:** UTAH STATE HIGHER EDUCATION SYSTEM--UNIVERSITY OF UTAH
- **Principal Investigator:** James Alan Gagnon
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $381,250
- **Award type:** 1
- **Project period:** 2021-08-01 → 2026-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10276262

## Citation

> US National Institutes of Health, RePORTER application 10276262, Cellular and molecular mechanisms of vertebrate testis homeostasis (1R35GM142950-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10276262. Licensed CC0.

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