Single-Cell Analysis of Normal and Perturbed Early T-Cell Developmental Processes

Author: Zhou, Wen

Year: 2021

Degree: Dissertation (Ph.D.)

Advisor: Rothenberg, Ellen V.

Committee Members: Wold, Barbara J.; Bronner, Marianne E.; Cai, Long; Rothenberg, Ellen V.

Option: Biochemistry and Molecular Biophysics

DOI: 10.7907/733t-mg82

Abstract

Early T-cell development converts multipotent precursors to committed pro-T cells, silencing progenitor genes while inducing T-cell genes. However, both the underlying steps of developmental progression and the regulations involved have remained obscure. Although some of the expressions of important regulators in early T-cell development have been studied in bulk populations, the nature of heterogeneity in this constantly refreshed developmental continuum makes it difficult to understand the developmental trajectories that the cells have undergone using bulk analysis, both in natural conditions and under gene perturbations.

Combining droplet-based single cell RNA sequencing (scRNA-seq), deep-sequenced whole-transcript scRNA-seq, and seqFISH for key regulatory genes, we established regulatory phenotypes of sequential ETP subsets; confirmed initial co-expression of progenitor- with T-cell specification genes; defined stage-specific relationships between cell-cycle and differentiation; and generated a pseudotime model from ETP to T-lineage commitment, supported by RNA velocity and transcription factor perturbations. This model was validated by developmental kinetics of ETP subsets at population and clonal levels. The results imply that multilineage priming is integral to T-cell specification in natural developing pro-T cells in the thymus.

Moreover, we examined the functional implications of some of the transcription factors (TFs) through bone marrow (BM) derived ex-vivo differentiation systems. Using scRNA-seq, Cell Hashing, and a pool-based CRISPR/Cas9 perturbation system, we established the normal and perturbed developmental trajectories before and after the T-lineage commitment stages. Our analysis revealed that, without the essential lineage commitment TF, Bcl11b, the developing early T cells immediately realized the lack of the essential regulator around the proliferating late DN2a stage. But instead of pushing the developmental path backwards to resemble the earlier stage of uncommitted cells, cells lacking Bcl11b underwent a diverging route of accumulation of 'non-T' genes that are not naturally expressed in earlier stages, potentially leading to the eventual loss of Notch responses. Our results also revealed the complex regulations by TFs that set up the earliest T-lineage progression and commitment conditions. The SCENIC analysis suggested that Gata3 and Tcf7, despite both being important regulatory factors for T-lineage progression, have very different regulatory roles in controlling proliferation and suppressing myeloid lineages. Furthermore, pseudotime analysis also showed that some of the stem and progenitor genes and 'multilineage' associated genes expressed by early pro-T cells potentially hold back the T-lineage differentiation speed. In summary, our study leveraged both in vivo thymic pro-T cells' developmental trajectory obtained through single-cell analysis and ex-vivo derived T cells for internal-controlled perturbations, and revealed some profound roles of TFs in regulating early T-cell differentiation processes.

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