Protein translation rate determines neocortical neuron fate

The mammalian neocortex comprises an enormous diversity regarding cell types, morphology, and connectivity. In this work, we discover a post-transcriptional mechanism of gene expression regulation, protein translation, as a determinant of cortical neuron identity. We find specific upregulation of protein synthesis in the progenitors of later-born neurons and show that translation rates and concomitantly protein half-lives are inherent features of cortical neuron subtypes. In a small molecule screening, we identify Ire1α as a regulator of Satb2 expression and neuronal polarity. In the developing brain, Ire1α regulates global translation rates, coordinates ribosome traffic, and the expression of eIF4A1. Furthermore, we demonstrate that the Satb2 mRNA translation requires eIF4A1 helicase activity towards its 5’-untranslated region. Altogether, we show that cortical neuron diversity is generated by mechanisms operating beyond gene transcription, with Ire1α-safeguarded proteostasis serving as an essential regulator of brain development.


Statistics
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Sample size
All detailed information on statistic, the exact numerical values and the sample size determination can be found in Supplementary Table S1.All reported replicates We did not predetermine the sample size.The sample size for our experiments were derived from our previous published studies and the standards in the field of sequencing and mass spectrometry according to practicality and financial feasibility.
Data exclusions No data were excluded.

Replication
The number of replicates in our experiments are listed in Supplementary Table S1.
Randomization Given the nature of labeling (IUE, transfection etc.) and nature of collected material (embryonic cortex), all samples randomly fall into analyzed categories.Embryonic and postnatal mice used in our experiments were utilized without sex distinction.Animals were randomly pooled across litters for the experiments.When pooling was required, this was also performed independent of the sex, per a biological replicate.

Blinding
Blinding was used in experiments requiring classification (axon numbers) and fluorescence measurements.For computational data analyses, we were not blinded to a sample identity.

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We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.The axis labels state the marker and fluorochrome used (e.g.CD4-FITC).
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Methodology Sample preparation
Right after trituration, E13.5 embryonic cortical neurons prepared from Satb2Cre/+ mice, were nucleofected with pCAG-EGFP and pCAG-loxP-Stop-loxP-tdTomato plasmids and seeded at 120 000 cells per well of 96-well plate.Two hours post-plating, cultures were treated with compounds at two concentrations, in technical duplicates.Cells were then cultivated until DIV2, when the proportion of Satb2tdTom neurons normalized to EGFP positive cells was determined using FACS.Cells were washed with PBS, briefly incubated with 0.25% trypsin solution, followed by trituration in the presence of FBS before feeding into the instrument.Sample preparation description can also be found in PMID: 28782628.
The embryonic cerebral cortices were isolated and placed in ice-cold HBSS with MgCl2 and CaCl2 (HBSS+/+, Gibco).Before trypsinization, brains were washed by centrifuging at 600 rpm for 1 minute.Then, the supernatant was removed and replaced with 5 ml of fresh HBSS+/+.The tissue was digested by adding 500 μl of 2.5 % trypsin, 0.02 mg/ml DNase, and 1 μl of 25U/μl benzonase to 5 ml of HBSS +/+ and following incubation for 20 min at 37°C.The reaction was stopped by adding 2 ml of FBS.The samples were washed three times with FACS buffer (2% FCS, 0.02 mg/ml DNase, 1 μl of 25U/μl benzonase).Next, the cortices were carefully triturated in 300 μl of FACS buffer.Subsequently, samples were transported on ice in FACS buffer to the cell sorting facility.Cell sorting was performed by the BIH Cytometry Core Facility on a BD FACSAria Fusion (BD Biosciences, San Jose, CA, USA), configured with 5 lasers (UV, violet, blue, yellow-green, red).Samples were collected after sorting in 1.5 ml of FACS buffer.The cells were then centrifuged for 15 minutes at 1000 rpm.The samples were subsequently frozen and stored at -80°С until further use.

Instrument
BD FACSCanto™ II and BD FACSAria Fusion Software BD FACSDiva™ Software

Cell population abundance
The cells expressing EGFP and tdTomato were quantified in the post-sort fraction based on the measured fluorescence.
Gating strategy FSC/SSC gates, as well as the cutoff values for EGFP, tdTomato expression and FT label were set prior to the screening experiments with control, untreated neurons.Gating was then unchanged for all tested compunds.The procedure is described in Ambrozkiewicz et al., J Neurosci Meth, 2017.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.