Development of a novel lineage tracing tool using DNA damage as a cell marker

Abstract

Lineage tracing technologies are powerful tools that offer insight into embryonic development by tracking the inheritance of a cell ‘marker’ across cell divisions. Existing lineage tracing tools have successfully constructed broad phylogenies of cellular lineages providing insight into important developmental events. However, these phylogenies often feature large gaps due to information loss, caused by their marker not being inherited, failure to separately resolve divisions of the same lineage, or through the occurrence of cell death.

This thesis describes the development of a novel lineage tracing technique that overcomes this limitation by using inherited DNA damage as a cell ‘marker’. Lesion segregation describes the process through which DNA damage can be inherited across multiple cell divisions (Aitken et al. 2020). When cells experience a discrete mutagenic event, lesions form on the DNA strands. These lesions can evade repair, instead being replicated across during cell division, introducing base pair mismatches to the newly synthesised DNA strand. Since replication necessarily occurs with every cell division, the marker must be inherited by subsequent daughter cells.

Additionally, both DNA strands are independently damaged, generating complementary patterns of mutations. This complementarity creates an expectation that can be used to infer the loss of a lineage as for every pattern of mutations we observe, we expect to find another, complementary pattern. In contrast to the broad, inexact phylogenies generated by current tools, the method described in this thesis provides potentially complete resolution over a defined window of development with all subsequent lineages separately resolved.

To develop this method, I have introduced DNA damage to sperm or cleavage phase zebrafish embryos, a phase of development during which DNA repair is minimal. I have trialled both UV irradiation and ENU as sources of DNA damage, titrating exposures to identify optimal dosage for introducing mutations without perturbing developmental progress. I have then generated both single cell RNA sequencing and whole genome sequencing data from individual zebrafish and combined this data to identify resulting mutations in individual cells and assign cell identities. Finally, I have used shared patterns of inherited mutations to infer relationships between cells generating a simple phylogeny, and used the expectations from lesion segregation as a barometer against which phylogenies can be tested. In doing so, I provide an initial proof of concept for the use of lesion segregation to resolve cell lineages in the developing embryo.