Dissecting the transcription factor networks controlling fetal T-lineage differentiation

Project Summary/Abstract Multiple sequential waves of T-cell development in the fetal and post-natal thymus give rise to discrete T cell subsets, each with its own functional properties. Compared with all subsequent waves, the first wave of T-cell differentiation proceeds very rapidly and gives rise to unique lineages of innate lymphocytes and innate-like γδT cells. These cell-intrinsic characteristics of first-wave fetal thymocytes suggests that they have their own separate developmental program. T-cell development is coordinated by a dynamic set of transcription factors (TFs) with stage-specific activity, through a complex network of regulatory interactions. Significant progress has been made in recent years in understanding the T-cell differentiation program; however, whether first-wave fetal thymocytes use the same T-lineage differentiation program is unknown. Through direct comparison of the transcriptional program of murine adult and first-wave fetal thymocytes by single-cell RNA-seq, as well as reanalysis of a public scRNA-seq atlas of thymic organogenesis (GSE107910; Kernfeld et al, 2018), I observed that the first wave of fetal T-cell development occurs simultaneously along two separate trajectories. One trajectory follows the well-established T-cell development pathway, while the other is used exclusively by first- wave fetal thymocytes and gives rise to innate lymphocytes and innate-like γδT cells. Cells on this trajectory appear to use a distinct differentiation program consisting of many TFs typically associated with innate lymphocyte development—including PLZF and Lmo4—alongside shared T/innate TFs, such as TCF1, GATA3, and Bcl11b; as well as GATA1 and Meis1, two TFs with no known role in differentiation of either lineage, and the pan-fetal hematopoiesis regulator Lin28b. I hypothesize that this computationally-inferred population consists of immediate precursors of innate lymphoid and innate-like γδT-lineage cells, and that their differentiation is guided by this unique TF network. In Aim 1, I will fate map the progeny of this hypothesized innate lymphocyte/innate-like γδT cell precursor. Within thymocytes, it is the only population that expresses Gata1; therefore I will use Gata1CreERT2 x Rosa26lsl-tdT mice to fate map the progeny of Gata1- expressing cells at embryonic gestational day E13.5. Additionally, I will use clonal barcoding and scRNA-seq at multiple timepoints to construct a full lineage hierarchy of fetal thymocyte differentiation, and I will perform scATAC-seq to determine whether lineage priming is occurring in this population or in an upstream precursor at the level of chromatin accessibility. In Aim 2, I will use CRISPR/Cas9 to delete constituent factors in the innate lymphocyte/innate-like γδT-lineage program in fetal progenitors and assess the impact of each on developmental speed and lineage choice. I will also perform scRNA-seq on TF-deleted cells in order to identify the effect each TF has on the overall program, including how it regulates other members of the program. In this way, I will use perturbations to construct a network of the regulatory interactions dictating the characteristic developmental speed and lineage choice of first-wave thymocytes.