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DOT1L activity affects cell lineage progression in the developing brain by controlling metabolic programs

Bismark Appiah, Camilla L. Fullio, Christiane Haffner, Patrice Zeis, Martin Treppner, Patrick Bovio, Arquimedes Cheffer, Ilaria Bertani, Harald Binder, Dominic Grün, Nereo Kalebic, Elena Taverna, Tanja Vogel

Preprint posted on 12 April 2022 https://www.biorxiv.org/content/10.1101/2022.04.08.487591v2.article-info

Enough neurons at the right moment: how cell division, epigenetics and metabolism interact to ensure that the brain is big enough.

Selected by Laura Celotto

Why I chose this preprint

I used to think of cortical development as a sequence of steps, each governed by a network of transcription factors that eventually promote progenitor differentiation to neurons. However, the identity of a cell is more than a group of molecules operating at the DNA level, and I think that this preprint nicely develops this point. I enjoyed the linearity of the story, which explores and tries to unify several aspects of a cell identity, including metabolism. I enjoyed the elegance of the experiments and the authors’ effort to bring a new, more coherent, perspective on neuronal development investigation.

Background

The balance between asymmetric, self-renewing divisions of apical progenitors (APs) and symmetric, neurogenic divisions of basal progenitors (BPs) ensures the correct development of the mammalian brain cortex. When such balance is impaired, brain diseases like microcephaly can occur and lead to reduced brain size and cognitive impairment. In this study, Appiah and colleagues investigate how the histone methyltransferase Disruptor of telomeric silencing like 1 (DOT1L) affects AP cell divisions, differentiation and neuron production during mouse mid-neurogenesis (E14.5). They found that DOT1L preserves AP identity by preventing their premature consumptive, symmetric neurogenic divisions and by inhibiting the premature occurrence of the metabolic state of differentiated neurons.

Key findings

DOT1L inhibition results in increased AP delamination, neurogenic commitment and symmetric division

The authors studied the role of DOT1L during mouse mid-neurogenesis (E14.5) by applying the specific DOT1L pharmacological inhibitor EPZ to mouse brain hemispheres and/or cortical sections put in culture for 24 hours.

  1. Because of DOT1L inhibition, APs delaminated more basally in the cortical wall. Accordingly, TUBG1-positive centrioles and ARL13B-positive primary cilia were positioned more basally in the cortical wall of EPZ-treated cultures than in untreated controls. In mouse neurogenesis, basal delamination is concomitant with the acquisition of different cell fates that include BPs or neurons. The researchers investigated the possibility that inhibition of DOT1L leads to increased neurogenesis, at the expense of AP identity.
  2. Accordingly, basal delamination of APs upon DOT1L inhibition was paralleled by increased proportion of neurogenic TIS21-GFP-positive cells. Increased neurogenic commitment of delaminating APs was confirmed at a single cell level via microinjection of Dextran-Alexa555 (Dx-A555) in cultured cortical slices. Counterstaining of Dx-A555-injected sections with EOMES (a marker of BPs) and TUBB3 (a marker of neurons) showed that TUBB3-positive cells were more numerous in EPZ-treated than in untreated sections. Instead, the number of EOMES-positive cells did not change between treated and untreated samples. Hence, DOT1L inhibition results in increased AP delamination and increased production of neurons in the developing mouse cortex.
  3. The authors wondered whether increased neurogenesis was due to increased symmetric divisions at the expense of asymmetric, self-renewing divisions of APs upon DOT1L inhibition. Hence, they microinjected Tis21(Btg2)-Gfp mice with Dx-A555 and followed cell doublets in cultured cortical sections with and without EPZ treatment. Here, GFP- expression in both cells of a doublet was considered a proxy of symmetric division of their progenitor. Accordingly, doublets in which both Dx-A555 daughter cells were also GFP-positive were more numerous in EPZ-treated than in untreated samples, meaning that DOT1L inhibition results in increased symmetric and neurogenic divisions of APs.

To summarize, during normal cortical development DOT1L activity preserves the pool of APs by promoting AP asymmetrical, self-renewing division and impeding premature neurogenesis.

How does DOT1L actually work to preserve AP identity?

DOT1L inhibition results in epigenetic and metabolism alterations of neural progenitors

  1. To gain mechanistic insights on the role of DOT1L, the authors performed single cell RNA sequencing (scRNA-seq) of FAC-sorted APs and AP-derived cells from EPZ-treated as well as untreated cortical samples. scRNA-seq revealed clusters of APs, BPs, and cells with a rare, transient transcriptional state (TTS) in the developing mouse cortex of both EPZ-treated and untreated samples. Gene ontology analysis showed that cells in the TTS express epithelial-like markers, which relate them to APs, but have reduced proliferative capacity. More intriguingly, EPZ-treated samples showed a larger TTS cluster and a reduced fraction of APs as compared to untreated controls.
  2. Interestingly, the TTS of EPZ-treated samples highly expressed genes that correlate with oxidative phosphorylation, like Asns. This gene encodes for asparagine synthetase, an enzyme that metabolizes glutamine to glutamate and eventually feeds the Krebs cycle, which, in turn, feeds oxidative phosphorylation in mitochondria. Since increased oxidative phosphorylation is a hallmark of the metabolic state of neurons as compared to progenitors, the researchers propose that DOT1L inhibition results in premature neurogenesis by shifting the metabolism of APs to the more neuronal-like metabolic state of TTS cells. Indeed, ASNS overexpression in cultured cortical slices was sufficient per se to increase the number of TUBB3-positive AP-derived cells as compared to untreated controls. Conversely, Asns inhibition in cultures treated with EPZ rescued the premature AP neuronal differentiation as compared to EPZ-treated only controls.
  3. The researchers propose an epigenetic link between DOT1L function and ASNS-mediated metabolic regulation. Indeed, they found that inhibition of DOT1L results in decreased methylation of the Asns gene locus in neuronal progenitors. The authors propose that DOT1L inhibition reduces the access of EZH2 at the level of the Asns locus. EZH2, which is a component of the polycomb repressor complex 2, normally promotes the methylation of H3K27 at a gene locus, repressing its expression. Because of EZH2 reduced access, transcription of Asns increases, and this might lead to increased ASNS activity. Subsequently, the Krebs cycle and oxidative phosphorylation are enhanced in neuronal progenitors, which thus acquire a more neuronal-like metabolic state.

Questions for the authors

  1. Did you think of live imaging APs to track the increased symmetric mode of cell divisions upon DOT1L inhibition?
  2. The number of EOMES-positive BPs does not decrease significantly, despite a strong increase in the number of TUBB3-positive cells upon DOT1L inhibition. You argue that DOT1L inhibition favours the acquisition of a neuronal state over the BP ones, but how can we relate this finding to an almost unchanged amount of cells in the BP pool?
  3. Did you think of performing AP lineage tracing using a specific CreLoxP mouse?

 

Posted on: 26 August 2022

doi: https://doi.org/10.1242/prelights.32573

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