Canonical Analysis of Fluorescent Timer-Anchored Transcriptomes Resolves Joint Temporal and Developmental Progression

Cell-state transitions during development are shaped by multiple concurrent processes, yet single-cell transcriptomic analyses often rely on static molecular profiles or infer time without an experimental anchor. We developed mCanonicalTockySeq, a systems-level framework that reconstructs temporally resolved developmental state spaces by combining signalling history with single-cell RNA sequencing. Using the Nr4a3-Tocky Fluorescent Timer system in developing thymic T cells, we combined scRNA-seq with a molecular clock of strong T-cell receptor signalling to establish an experimentally anchored temporal reference. Using Timer-defined landmark populations, mCanonicalTockySeq constructs a shared state space in which temporal progression and developmental maturation are jointly represented, identifying a Tocky-defined temporal manifold while resolving progression toward CD4 and CD8 single-positive states. This framework recovered biologically coherent dynamics of immediate TCR-response genes, lineage-associated regulators, and agonist-selection-associated programmes. We then extended the framework across species by translating human thymic single-cell transcriptomes into one-to-one mouse ortholog space and projecting them into the mouse Nr4a3-Tocky reference. The projected human cells occupied an interpretable temporal-developmental geometry, and the inferred Tocky-equivalent temporal coordinate showed a significant donor-level association with chronological age. Together, these results establish mCanonicalTockySeq as a general framework for modelling how signalling history and developmental progression are jointly organised in single-cell state space, and illustrate how experimentally anchored reference systems can support comparative analyses across species.