Abstract Lumbar intervertebral disc degeneration is a cascade of cellular, structural and mechanical changes that is strongly implicated as a cause of low back pain. Current therapies for painful disc degeneration, conservative or surgical, focus only on alleviating symptoms. There is a critical need for new therapies that restore disc structure and mechanical function by directly addressing the underlying biological causes. A key challenge to developing effective treatments for disc degeneration is the need to recapitulate the structural complexity and specialized extracellular matrix (ECM) of the component tissues, which comprise cells of multiple developmental lineages. The central nucleus pulposus (NP) is implicated in the initiation of disc degeneration. The developmental origin of NP cells was until recently a point of contention, but was resolved though fate mapping studies in mice that demonstrated conclusively that these cells in their entirety are descendants of embryonic notochordal cells. During growth and aging, NP cells progressively lose their notochordal characteristics to be replaced by smaller, chondrocyte-like “mature” NP cells. The disappearance of notochordal cells has long been considered to contribute to progressive disc degeneration later in life, however the underlying molecular mechanisms are not understood. With degeneration, the NP undergoes an inflammation-mediated, fibrous transformation that compromises disc biomechanical function; however the underlying cellular events that initiate and drive this process are poorly understood. Single cell RNA-Seq (scRNA-Seq) is an emerging tool that permits high resolution transcriptome analysis of individual cells, facilitating identification of rare subpopulations. In combination with cutting-edge computational tools, scRNA- Seq can elucidate transitional states and relationships between cell subpopulations to predict cell differentiation trajectories. The overall objective of this proposal is to use scRNA-Seq to investigate the emergence of distinct NP cell subpopulations during intervertebral disc development and growth (Aim 1), and establish the fate and function of these subpopulations during injury and degeneration (Aim 2). For both Aims, using the top cluster-specific genes, the presence and localization of distinct NP cell subpopulations will be confirmed using immunofluorescence and qPCR. The results of this study will have important implications for the development of novel cell-based therapies for disc regeneration. For example, establishing NP progenitor- specific markers through cluster-specific gene analysis may allow subsequent identification and enrichment of therapeutic cell populations in human disc tissue, and defining differentiation trajectories may allow reprogramming of such cells for therapeutic application.