During development, regulatory factors appear in a precise order to determine cell fates over time. Consequently, to investigate complex tissue development, it is necessary to visualize and manipulate cell lineages with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here, we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labeling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation and inactivation of reporters and/or effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.

Cells regulate function by synthesizing and degrading proteins. This turnover ranges from minutes to weeks, as it varies across proteins, cellular compartments, cell types, and tissues. Current methods for tracking protein turnover lack the spatial and temporal resolution needed to investigate these processes, especially in the intact brain, which presents unique challenges. We describe a pulse-chase method (DELTA) for measuring protein turnover with high spatial and temporal resolution throughout the body, including the brain. DELTA relies on rapid covalent capture by HaloTag of fluorophores that were optimized for bioavailability in vivo. The nuclear protein MeCP2 showed brain region- and cell type-specific turnover. The synaptic protein PSD95 was destabilized in specific brain regions by behavioral enrichment. A novel variant of expansion microscopy further facilitated turnover measurements at individual synapses. DELTA enables studies of adaptive and maladaptive plasticity in brain-wide neural circuits.

Chemical synapses between axons and dendrites mediate much of the brain’s intercellular communication. Here we describe a new kind of synapse – the axo-ciliary synapse - between axons and primary cilia. By employing enhanced focused ion beam – scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between the serotonergic axons arising from the brainstem, and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, 5-hydroxytryptamine receptor 6 (HTR6), whose mutation is associated with learning and memory defects. Using a newly developed cilia-targeted serotonin sensor, we show that optogenetic stimulation of serotonergic axons results in serotonin release onto cilia. Ciliary HTR6 stimulation activates a non-canonical Gαq/11-RhoA pathway. Ablation of this pathway results in nuclear actin and chromatin accessibility changes in CA1 pyramidal neurons. Axo-ciliary synapses serve as a distinct mechanism for neuromodulators to program neuron transcription through privileged access to the nuclear compartment.