Sequential Transcriptional Waves direct the Differentiation of Newborn Neurons

 

Science  25 Mar 2016: Vol. 351, Issue 6280, pp. 1443-1446

Sequential transcriptional waves direct the differentiation of newborn neurons in the mouse neocortex

Ludovic Telley, et.al.

Department of Basic Neurosciences, University of Geneva, Switzerland.

Department of Genetic Medicine and Development, University of Geneva, Switzerland.

Department of Psychiatry, Geneva University Hospital, Switzerland.

Clinic of Neurology, Geneva University Hospital, Switzerland.

Biomedical Research Foundation Academy of Athens, Greece.

Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Saudi Arabia.

Institute for Genetics and Genomics in Geneva (iGE3), University of Geneva, Switzerland.

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During corticogenesis, excitatory neurons are born from progenitors located in the ventricular zone (VZ), from where they migrate to assemble into circuits. How neuronal identity is dynamically specified upon progenitor division is unknown. Here, we study this process using a high-temporal-resolution technology allowing fluorescent tagging of isochronic cohorts of newborn VZ cells. By combining this in vivo approach with single-cell transcriptomics in mice, we identify and functionally characterize neuron-specific primordial transcriptional programs as they dynamically unfold. Our results reveal early transcriptional waves that instruct the sequence and pace of neuronal differentiation events, guiding newborn neurons toward their final fate, and contribute to a road map for the reverse engineering of specific classes of cortical neurons from undifferentiated cells.

During neocortical development, distinct classes of neurons assemble to form local and long-range circuits. Although class-specific genes and features identify cortical neuron types relatively late in differentiation,    early postmitotic fate specification programs have been inaccessible. Here, we describe the dynamic transcriptional activity controlling layer 4 (L4) excitatory neuron birth and differentiation in the mouse neocortex.

Mammalian cortical progenitor cells in the ventricular zone (VZ) undergo DNA synthesis [S-phase, susceptible to bromodeoxyuridine (BrdU) labeling] at the basal border of the VZ and mitosis (M-phase, lasting about an hour at midcorticogenesis in mice) when their soma is apically located, adjacent to the ventricular space. At this location, mitotic cells are susceptible to labeling by intraventricular injection of carboxyfluorescein esters [“FlashTag” (FT)], which bind to and fluorescently label intracellular proteins. The short extracellular half-life of FT in the mouse ventricular space ensures effective pulse-labeling of juxtaventricular dividing cells. Intracellularly, FT is linearly diluted at each mitosis, such that fluorescence reflects the number of cell divisions that have occurred since the time of labeling. FT⁺ newborn cells synchronously moved away from the ventricular wall within 3 hours of labeling, reached the subventricular zone (SVZ) within 12 hours, and entered the cortical plate (CP) 24 to 48 hours after mitosis. Isochronic cohorts of VZ cells born at the time of injection can thus be specifically identified and tracked during their initial differentiation.

Our data provide a comprehensive transcriptional blueprint outlining the dynamic acquisition of neuronal identity in vivo. We show that early neuronal differentiation is directed by a series of transcriptional waves whose proper sequence is critical for normal progression through development. These waves provide discrete time windows during which specific transcriptional complexes are present simultaneously and can interact. These transient combinatorial transcriptional niches could act as sequential checkpoints during the course of differentiation, combinatorially coding for specific cell fates. These results build a road map for reverse engineering of cortical neuron subtypes from undifferentiated cells and provide a set of genetic targets for identification and directed differentiation of progenitors and nascent neurons.

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