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Less is more: Single Cell Analysis of Zebrafish Blood Development

Periodic Reporting for period 2 - ZF_Blood (Less is more: Single Cell Analysis of Zebrafish Blood Development)

Reporting period: 2017-11-01 to 2019-04-30

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, I will use single-cell transcriptome sequencing of zebrafish blood cells. I will apply an integrative strategy, combining genetic perturbation with computational sequence and network analysis methods, to reconstruct the regulatory networks that maintain the dynamic balance between different blood cell types. This will be achieved by pursuing two main aims:
1) I will create a comprehensive atlas of single cell gene expression in adult zebrafish blood cells and computationally reconstruct the blood lineage tree. I will order cells according to their most likely developmental chronology and identify genes and gene regulatory networks that define distinct cell types. The completion of the first aim will be followed by a more ambitious long-term one that is based on:
2) The in-depth functional characterisation of a subset of novel key regulators of blood formation and identified cell types in vivo. To achieve this I will generate a number of loss-of-function and transgenic zebrafish lines.
By sequencing thousands of single cells, this study is poised to go beyond traditional approaches in examining the complex relationships between the continuous spectra of blood cells, and will provide unprecedented insight into the regulation of blood cell formation.
Significant progress has been made on all areas of the project. The key early objective for the project is the single cell sequencing of large numbers of zebrafish blood cells. Sorting and processing of cells has proceeded very rapidly (1400 cells processed in the first three months of the project). Sequencing has produced the required data. The second major focus of the project has therefore been the analysis of this data. This has proceeded successfully and we are moving in the direction of our ultimate aim of producing an atlas of single cell gene expression in adult zebrafish blood cells. Two manuscripts have been written based on the data generated. Productive work has also been done on reconstructing the blood lineage tree. Focus has moved most recently to the lymphoid branch. The third strand of the project is transplantation experiments with zebrafish to ultimately identify novel key regulators of blood formation. Following on from the initial sequencing work, it has also made good progress and the we have established experimental workflow.
Although our knowledge of transcriptomes of blood cell types has been advanced by population-level analysis, populations of blood cells may include many distinct cell types with substantially different transcriptomes and the ability to make diverse cell fate decisions. This project is going beyond the state of the art by employing wide scale single-cell transcriptome sequencing of zebrafish blood cells. By sequencing thousands of single cells, it is on track to produce the following important results:
1) A comprehensive atlas of single cell gene expression in adult zebrafish blood cells.
2) A computational reconstruction of the blood lineage tree.
3) The in-depth functional characterisation of a subset of novel key regulators of blood formation and identified cell types in vivo.
These results will provide new knowledge which has important wider societal implications. The process of blood cells differentiation is subverted in the generation of blood pathologies. It is therefore of great importance that it be understood better. The construction of an atlas of single cell gene expression in adult zebrafish blood cells and a blood lineage tree is a very important step towards a better understanding. Furthermore, the characterisation of novel regulators of this process will illuminate novel disease-causing genes and inform approaches aimed at manipulating the expansion, directed differentiation or reprogramming of stem cell fate for therapeutic advantage.