Less is more: Single Cell Analysis of Zebrafish Blood Development

Project Information

ZF_Blood

Grant agreement ID: 677501

DOI

10.3030/677501

Project closed

EC signature date 16 February 2016

Start date 1 May 2016

End date 30 April 2022

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 500 000,00

EU contribution

€ 1 500 000,00

Coordinated by

THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE
United Kingdom

Periodic Reporting for period 4 - ZF_Blood (Less is more: Single Cell Analysis of Zebrafish Blood Development)

Reporting period: 2020-11-01 to 2022-04-30

Summary of the context and overall objectives of the project

Blood stem cells need to both perpetuate themselves (self-renew) and differentiate into all mature blood cells to maintain blood formation throughout life. However, it is unclear how the underlying gene regulatory network maintains this population of self-renewing and differentiating stem cells, and how it accommodates the transition from a stem cell to a mature blood cell. Our current knowledge of transcriptomes of various blood cell types has mainly been advanced by population-level analysis. However, the population of seemingly homogenous blood cells may include many distinct cell types with substantially different transcriptomes and abilities to make diverse fate decisions. To overcome these limitations, we used single-cell transcriptome sequencing of blood cells to reconstruct the regulatory networks that maintain the dynamic balance between different blood cell types. This was achieved by pursuing two main objectives:
1) To create a comprehensive atlas of single cell gene expression in blood cells and computationally reconstruct the blood lineage tree. We ordered cells according to their most likely developmental chronology and identified genes and gene regulatory networks that define distinct cell types.
2) The in-depth functional characterisation of a subset of novel key regulators of blood formation and identified cell types in vivo.
The results from these studies revealed complex relationships between the continuous spectra of blood cells, and provided unprecedented insight into the regulation of blood cell formation.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

My group applies an integrative strategy, combining genetic perturbation with novel computational sequencing and network analysis methods to reconstruct and validate gene regulatory networks in blood development. The most important results include:

1. Resolving the cellular hierarchy of lineage development and progression in haematopoiesis in zebrafish (Cell Rep. 2016; Nat Commun. 2017). The results from these two studies have advanced our understanding of how haematopoietic cell fate decisions are instigated and provided significant new understanding of the architecture and functioning of lower vertebrate haematopoiesis. In addition, I have generated a user-friendly cloud repository, BASiCz (Blood Atlas of Single Cells in zebrafish) for interactive exploration and visualisation of single-cell sequencing data from zebrafishblood cells (http://www.sanger.ac.uk/science/tools/basicz).
2. Development of a novel zebrafish model for the study of the rare haematological disorder Fanconi anaemia. The generated model was used to understand the underlying disease biology (PNAS 2017).
3. Characterisation of T and NK cells at the single-cell transcriptome level for the first time in a non- mammalian species. We proposed a model of high gene turnover and faster evolution of immune transmembrane genes, but at the same time conservation of core cytoplasmic immune genes from zebrafish to mammalian species (Genome Res. 2017). In addition, we were the first group to identify and characterise innate-lymphoid cells (ILCs) in zebrafish (Sci Immunol. 2018).
4. Characterised transcriptional, epigenetic and mutational changes that occur during differentiation of blood stem cells (Cell Stem Cell 2021, Nature 2021).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Together with collaborators, my group has pioneered the use of somatic mutations to
i) estimate the rate and time of mutation acquisition during normal development,
ii) reconstruct blood phylogenetic tree during development and,
iii) estimate the number of blood antecedents at different stages during development. In addition, we characterised transcriptional and epigenetic changes that occur during differentiation of blood stem cells. All these achievements significantly advanced a research field and went beyond the state of the art.

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