Project 1 - Molecular mechanisms controlling TEC dynamics and lineage hierarchies in the perinatal thymus

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Project Summary/Abstract Thymic epithelial cells (TECs) provide growth and differentiation signals that are indispensable for the development of T cells with a diverse, yet self-tolerant TCR repertoire. The number of TECs increases exponentially in newborns as the thymus grows to support increased production of naïve T cells that are exported to colonize peripheral lymphoid organs. The perinatal period is a time of dynamic changes in TEC subset composition, differentiation, organization and function. Although the conversion of perinatal thymus growth to juvenile thymus homeostasis is well established, the specific changes that occur in the composition of transcriptionally distinct TEC subsets and identification of signaling pathways that initially promote and subsequently limit expansion and remodeling of the TEC compartment are poorly understood. Furthermore, the TEC progenitors in which such molecular re-programming events occur have not been defined. Our preliminary data suggest that the Cyclin D1-retinoblastoma (RB)-E2F pathway is a key molecular switch that modulates TEC proliferation during the perinatal to adult transition. We have shown that expansion of the perinatal TEC compartment can be maintained into adulthood by deleting Rb family members or by expressing a keratin 5 driven Cyclin D1 (K5.D1) transgene to inactivate RB function. Our overall hypothesis is that perinatal and juvenile TECs express distinct transcriptional profiles that coordinate TEC proliferation and differentiation, and that the Cyclin D1-RB-E2F pathway regulates both processes. We propose a series of experiments that will test this hypothesis and provide new insights into currently unresolved questions regarding the perinatal TEC compartment. These questions include: 1) what changes occur in the composition of TEC subsets during the perinatal to juvenile transition; 2) how do the transcriptional signatures of TEC subsets change across the transition; 3) what molecular mechanisms and pathways regulate these changes; 4) what are the identities and lineage hierarchies of TEC progenitors during the transition; and 5) do comparable cellular and molecular changes occur in human TECs. The overall goal of RP1 is to resolve these knowledge gaps. In Aim 1, we will use scRNA-seq to determine changes in the transcriptional profiles and advanced imaging to map localization of transcriptionally distinct TEC subsets during the perinatal to juvenile transition. Parallel scRNA-seq and imaging analyses will be performed on human TECs and thymus tissue. In Aim 2 we will perform in vitro and in vivo assays to directly test the differentiation potential of candidate TEC progenitors identified in Aim 1. In Aim 3 we will determine whether molecular pathways that regulate TEC proliferation and differentiation are coordinately linked by performing proteomic screens coupled with ChIP- seq analyses.