During corticogenesis, distinct subtypes of neurons are sequentially born from ventricular zone progenitors. How these cells are molecularly temporally patterned is poorly understood. We used single-cell RNA sequencing at high temporal resolution to trace the lineage of the molecular identities of successive generations of apical progenitors (APs) and their daughter neurons in mouse embryos. We identified a core set of evolutionarily conserved, temporally patterned genes that drive APs from internally driven to more exteroceptive states. We found that the Polycomb repressor complex 2 (PRC2) epigenetically regulates AP temporal progression. Embryonic age–dependent AP molecular states are transmitted to their progeny as successive ground states, onto which essentially conserved early postmitotic differentiation programs are applied, and are complemented by later-occurring environment-dependent signals. Thus, epigenetically regulated temporal molecular birthmarks present in progenitors act in their postmitotic progeny to seed adult neuronal diversity.
During corticogenesis, ventricular zone progenitors sequentially generate distinct subtypes of neurons, accounting for the diversity of neocortical cells and the circuits they form. While activity-dependent processes are critical for the differentiation and circuit assembly of postmitotic neurons, how bioelectrical processes affect nonexcitable cells, such as progenitors, remains largely unknown. Here, we reveal that, in the developing mouse neocortex, ventricular zone progenitors become more hyperpolarized as they generate successive subtypes of neurons. Experimental in vivo hyperpolarization shifted the transcriptional programs and division modes of these progenitors to a later developmental status, with precocious generation of intermediate progenitors and a forward shift in the laminar, molecular, morphological, and circuit features of their neuronal progeny. These effects occurred through inhibition of the Wnt-beta-catenin signaling pathway by hyperpolarization. Thus, during corticogenesis, bioelectric membrane properties are permissive for specific molecular pathways to coordinate the temporal progression of progenitor developmental programs and thus neocortical neuron diversity.
This protocol describes a fluorescence birthdating technique to label, track and isolate isochronic cohorts of newborn cells in the central nervous system in vivo. Injection of carboxyfluorescein esters into the cerebral ventricle allows pulse-labeling of M-phase progenitors in touch with the ventricle and their progeny across the central nervous system, a procedure we termed FlashTag. Labeled cells can be imaged ex vivo or in fixed tissue, targeted for electrophysiological experiments, or isolated using Fluorescence-Activated Cell Sorting (FACS) for cell culture or (single-cell) RNA-sequencing. The dye is retained for several weeks, allowing labeled cells to be identified postnatally. This protocol describes the labeling procedure using in utero injection, the isolation of live cells using FACS, as well as the processing of labeled tissue using immunohistochemistry.intraventricular injection of a carboxyfluorescein ester (a procedure we termed "FlashTag", FT). This procedure allows fluorescent tagging of VZ progenitors and tracking of time-locked cohorts of their postmitotic progeny, including neurons, throughout corticogenesis and early postnatal development.
Birthdating techniques: an overviewThe first-developed and probably still most widely used technique to permanently label neurons based on their embryonic time of birth is 5-bromo-3′-deoxy-uridine (BrdU) birthdating 2,3 . BrdU is a thymidine analogue that is incorporated into cells during DNA synthesis, i.e. during S phase. Administration of BrdU (usually through intraperitoneal (i.p.) injection in pregnant dams or via drinking water) results in systemic labeling of all S-phase
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