Post-translational Tuning of Human Cortical Progenitor Neuronal Output

Background

Proneural genes such as those that are part of the neurogenin (NGN) family encode basic helix-loop-helix transcription factors. These factors bind to DNA in heterodimeric complexes to activate gene transcription. In rodent cortices, Neurog2 is the main proneural gene and plays an important role in neuronal differentiation. It is involved in reorganizing the chromatin landscape at several levels and promotes the differentiation of radial glial cells (RGCs) into neurons. During development, its activity is regulated by post-translational modifications (PTMs). Such modifications, including the phosphorylation of serine residues, can lead to the activation of gene cascades important for proper neuronal development and fate commitment. Whether the gene encoding Neurogenin-2 in humans, NEUROG2, is required for human cortical neurogenesis or whether its function has diverged is still unclear. As such, the authors of the preprint highlighted here decided to investigate if this gene has evolved species-specific functional plasticity in human RGCs.

Key findings

Predominant expression of NEUROG2 in RGCs and impaired neurogenic outputs in NEUROG2 KO organoids

Following differentiation of human cortical organoids (hCOs), the authors developed and performed a high-throughput deep learning-based image analysis in these organoids, as well as in fetal neocortex, and found that the majority of NEUROG2+ cells corresponded to RGCs.
Then they examined the outcomes of the loss of NEUROG2 on neurogenesis by employing CRISPR/Cas9 to generate NEUROG2 KO hCOs. In these organoids, they found a significant increase in progenitors in comparison to control clones. Moreover, these KO organoids exhibited a shift toward producing neurons typically found in the deep layers of the cortex.

Increased neurogenesis in phospho-mutant NEUROG2 hCOs

Given the importance of the phosphorylation of the NEUROG2 T149 residue in mice, the authors investigated the consequences of a phospho-mutant T149 in hCOs. Following replacement of T149 with Alanine (TA/TA) by genome editing, they observed a reduction in progenitors in early (D70) and late (D140) neurogenesis, accompanied by a decrease in deep-layer identity at D140.
This increased neurogenic potential was further observed in TA/TA RGCs which showed a higher capacity to generate mature neurons than WT/WT cells.

Chromatin landscape remodeling driven by phospho-mutant NEUROG2

By combining RNAseq and ATAC-seq, the authors connected distal candidate cis-regulatory elements (cCREs) to their putative target genes and found an enrichment
of AP-1 (JUN/FOS) motifs in TA/TA RGCs in comparison to control, while these motifs were less enriched in TA/TA intermediate progenitors (IPs).
This was further demonstrated by footprinting analysis to detect the physical binding of TF to their motifs, which revealed that the JUN footprint is stronger in TA/TA RGCs.

Promotion of neurogenesis via the AP-1 complex

The enrichment of AP-1 motifs prompted the authors to better understand AP-1’s involvement in increased neurogenesis. Thus, they performed an in silico knock-down of JUN and FOS. This resulted in a reduced differentiation potential of TA/TA RGCs and IPs, which reverted to a WT/WT-like transcriptional state. Then, they treated 2D cultures of human RGCs with an AP-1 inhibitor, which led to a decrease in the number of immature neurons.

What I like about the preprint

I found this preprint very intriguing as it highlights the functional relevance of PTMs in neurogenesis and their conserved role during species-specific cortical evolution. I appreciated the methodological rigor throughout the study, in particular the 3D differentiation protocol as part of which the authors characterized the presence of RGCs prior to downstream analyses. I also appreciated the development of a custom image analysis pipeline, combined with the integration of deep learning and machine-learning. The followed workflow was well documented making the work highly reproducible and aligned with FAIR data principles.

Future directions and questions for the authors

• In a previous work from your lab (Mora N et al., 2018), you reported that self-amplifying neural stem cells (NCSs) in Drosophila arise by a spatiotemporal conversion of classical self-renewing NSCs. Given that the T149 residue is conserved down to Drosophila Tap as noted in the manuscript, do you think that similar phosphorylation dependent modulation could occur for Tap or other genes present in invertebrates ?
• The in silico knockdown of JUN/FOS showed a clear effect on differentiation. Are you considering conducting an in vitro knock down by siRNA or shRNA to further explore these findings ?
• The outcomes of the AP-1 inhibition 2D RGC cultures are very promising. Is it possible to inhibit AP-1 in the organoids as well ? If so, what would be the anticipated phenotypes in a 3D context ?

References

• Vasan, L., Chinchalongporn, V., Saleh, F., Zinyk, D., Ke, C., Suresh, H., Ghazale, H., Belfiore, L., Touahri, Y., Oproescu, A., Patel, S., Rozak, M., Amemiya, Y., Han, S., Moffat, A., Black, S. E., McLaurin, J., Near, J., Seth, A., . . . Schuurmans, C. (2024). Examining the NEUROG2 lineage and associated gene expression in human cortical organoids. Development, 152(2). https://doi.org/10.1242/dev.202703
• Miranda-Negrón, Y., & García-Arrarás, J. E. (2022). Radial glia and radial glia-like cells: Their role in neurogenesis and regeneration. Frontiers in Neuroscience, 16, 1006037. https://doi.org/10.3389/fnins.2022.1006037
• Hulme, A. J., Maksour, S., Glover, M. S., Miellet, S., & Dottori, M. (2021). Making neurons, made easy: The use of Neurogenin-2 in neuronal differentiation. Stem Cell Reports, 17(1), 14–34. https://doi.org/10.1016/j.stemcr.2021.11.015
• Quan, X., Yuan, L., Tiberi, L., Claeys, A., De Geest, N., Yan, J., Van Der Kant, R., Xie, W. R., Klisch, T. J., Shymkowitz, J., Rousseau, F., Bollen, M., Beullens, M., Zoghbi, H. Y., Vanderhaeghen, P., & Hassan, B. A. (2016). Post-translational control of the temporal dynamics of transcription factor activity regulates neurogenesis. Cell, 164(3), 460–475. https://doi.org/10.1016/j.cell.2015.12.048

Tags: epigenetics, neurodevelopment, phosphorylation

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