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Quantitative analysis of variability and robustness in spatial pattern formation

Periodic Reporting for period 3 - SPACEVAR (Quantitative analysis of variability and robustness in spatial pattern formation)

Reporting period: 2020-01-01 to 2021-06-30

During embryonic development a single cell turns into a complex organism. This process is characterized by an antagonism between variation and stability. On the one hand, development is a tightly controlled process; tissues need to be specified at the right time, at the correct spatial position, and with a defined size. On the other hand, regulation should not be too rigid, since embryos need to adjust to environmental perturbations and correct errors caused by noisy gene expression. Furthermore, it is remains unclear to what extent vertebrate embryos can tolerate perturbations of lineage relationships. We study variation and stability during pattern formation in the zebrafish heart. We seek to understand the origin of embryo-to-embryo variability as well as robustness against perturbation.

The zebrafish heart is a powerful model system for studying variability, since heart positioning is inverted along the left/right axis in 5-10% of wildtype embryos. We aim to identify the mechanism underlying variability in heart positioning and understand its function. To this end, we combine light microscopy and gene expression analysis. To expand our study of embryo-to-embryo variability, we have developed a method for high-throughput single-cell lineage tracing based on CRISPR-Cas9, which we continue to improve. This novel approach will allow us to study embryo-to-embryo variability in developmental lineage specification systematically and on a massively parallel level. We will use this strategy to explore the corrective capacity of the zebrafish heart upon perturbation of progenitor cell pools, and to determine which mechanisms for error correction are activated in the embryo.
These quantitative experiments will provide unprecedented insight into variability and robustness during development. The concepts developed here will also be relevant for improving our understanding of variable outcomes in human disease.
The project is proceeding successfully, with minor deviations from the proposed strategy. In WP1, we set out to determine the source of stochastic heart laterality fluctuations in the zebrafish. We discovered that this phenomenon is linked to global left/right signaling in the zebrafish embryo, and we have strong evidence suggesting that fluctuations in the number of dorsal forerunner cells, a small cell population specified at gastrulation stages, is responsible for stochastic heart inversion. Specifically, we could show by live microscopy that those embryos in which the number of dorsal forerunner cells is smallest develop laterality defects with much higher probability. We found that these fluctuations are largely stochastic, with only a minor genetic component. This work was published during the reporting period (Moreno-Ayala et al., Cell Reports, 2021).

In WP2, we proposed to develop a method for simultaneous cell type identification and lineage tracing in thousands of single cells. This project has progressed exceedingly well and already led to a high-level publication (Spanjaard et al., Nature Biotech, 2018). Specifically, we developed experimental approaches for generating lineage barcodes in zebrafish embryos by CRISPR/Cas9 and for detecting these barcodes by single cell transcriptomics. Importantly, we characterized the diversity of the barcodes and the dynamics of cell labeling. Furthermore, we developed computational methods for lineage tree reconstruction based on this data. In summary, we now have a method that allows simultaneous cell type identification and lineage tracing in thousands of single cells.

However, it also turned out that the resolution of the current method is not yet sufficient to fully address the scientific questions of WP3 (principles of buffering mechanisms that are activated upon developmental perturbations). We therefore continue to improve the method on the experimental and computational level in order to increase the resolution. While the effects of subtle perturbation on lineage trees cannot be resolved yet, we are already successfully using our approach for studying the effects of larger perturbations. Specifically, we use high-throughput lineage tracing to systematically identify the origin of transient cell types in the adult zebrafish heart, which arise during regeneration after injury. We specifically focus on non-myocytes like fibroblasts and macrophages, both of which show an unexpectedly high degree of cell type diversity. This work is almost finished and the manuscript is currently in revision.
WP1 led to the identification of a remarkable phenomenon: Fluctuations in cell numbers at an early developmental stage are not buffered or corrected, but instead manifest themselves by a macroscopic phenotype – a laterality defect. This is an important conceptual novelty, which may have profound consequences also for human genetics, since similar phenomena might underlie incomplete penetrance of mutations in the human population. In the remaining funding period, we aim to further understand the origin of the stochastic fluctuations of dorsal forerunner cell numbers. We have strong indications that the origin of the cell number fluctuations is related to the timing of the maternal-to-zygotic transition. We are currently performing experiments to corroborate this hypothesis, and we expect that this part of the project will lead to a publication in the next 6 months.

In WP2, we developed a method for high-throughput lineage tracing (Spanjaard et al., Nature Biotech, 2018), which allows to identify the lineage origin of thousands of single cells from the same animal. Together with several other papers, this manuscript was selected by Science as the "Breakthrough of the Year 2018". We continue to improve this method on the experimental and computational level by e.g. introducing additional barcodes, extending the period of lineage recording, optimizing barcode recovery during single-cell RNA-seq, and by improving algorithms for lineage tree reconstruction. We expect that this method development will lead to one or two additional publications during the remaining funding period.

In WP3, we study how the zebrafish heart reacts to perturbation. We currently focus on analyzing the origin of transient cell types that arise during regeneration after injury in the adult heart. We expect that this work will lead to a publication within the next 6-12 months. This work is so far based on the published version of high-throughput lineage tracing. However, we also plan to use improved versions of the method to study changes in lineage trees upon perturbation in greater detail, in either the embryo or in the adult heart.