# Single-Cell Profiling and Lineage Tracing of Zebrafish Hematopoiesis

> **NIH NIH F30** · HARVARD MEDICAL SCHOOL · 2022 · $51,752

## Abstract

Project Summary:
Hematopoietic stem cells (HSCs) give rise to a diverse population of blood cells, including myeloid and
lymphoid cells, platelets, and erythrocytes. These cell types play important roles in defending against disease,
responding to tissue injury, and transporting vital cellular nutrients. Hematopoiesis, the generation of blood
cells from HSCs, is a carefully orchestrated process that is highly conserved among vertebrates. In recent
years, it has become apparent that HSCs are heterogenous and may be biased with respect to their production
of different blood cells. Further, somatic mutations acquired over the course of an organism’s life may alter
HSC output. If the mutations confer a survival or proliferative advantage, this may lead to the expansion of
cells derived from a specific HSC (clonal expansion) and clonal hematopoiesis. In 2014, a clinical entity called
clonal hematopoiesis of indeterminant potential (CHIP) was described in which specific genetic mutations
appeared to be promoting HSC clonal expansion without any obvious hematologic abnormalities. Individuals
with CHIP are at an increased risk for hematologic malignancy but also and more surprisingly at an increased
risk for developing coronary heart disease (CHD) secondary to increased inflammation. At present, scientists
do not fully understand how mutations in HSCs lead to increased inflammation nor know how to predict which
individuals with CHIP will progress to malignancy. The overarching goal of the proposed work is to gain
insight into HSC biology and immune cell development in unperturbed hematopoiesis and genetically-
induced clonal hematopoiesis through single-cell analyses and lineage tracing. In Aim 1, HSC output,
immune cell development, and hematopoietic lineage relationships will be studied in unperturbed zebrafish.
This will be accomplished with scGESTALT, a new method for in vivo cellular barcoding and fate mapping.
Adult kidney marrows and thymi from scGESTALT zebrafish will be dissected and single-cell RNA sequencing
will be performed to obtain transcriptional and ancestral information. Aim 2 will assess the impact of mutations
found in clonal hematopoiesis on HSC clonal output and immune cell development and differentiation.
CRISPR-Cas9 will be used to mutate zebrafish orthologs of human ASXL1, NRAS, EZH2, and RUNX1 at
genomic locations frequently mutated in patients. Transcriptional profiling and lineage tracing will be performed
on kidney marrows and thymi from scGESTALT zebrafish with and without expanded clones. The ability of
macrophages and lymphocytes to promote clonal expansion will be explored via targeted knockout/knockdown
approaches. Identifying altered cells, developmental and transcriptional programs, and regulatory networks in
clonal hematopoiesis will shed light on the contributing cells and mechanisms to both clonal expansion and
increased inflammation. Such knowledge has the potential to better inform clinical management of CHIP and
...

## Key facts

- **NIH application ID:** 10397698
- **Project number:** 5F30HL152628-03
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Sara Ann Rubin
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $51,752
- **Award type:** 5
- **Project period:** 2020-05-01 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10397698

## Citation

> US National Institutes of Health, RePORTER application 10397698, Single-Cell Profiling and Lineage Tracing of Zebrafish Hematopoiesis (5F30HL152628-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10397698. Licensed CC0.

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