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An early cell shape transition drives evolutionary expansion of the human forebrain

Silvia Benito-Kwiecinski, Stefano L. Giandomenico, Magdalena Sutcliffe, Erlend S. Riis, Paula Freire-Pritchett, Iva Kelava, Stephanie Wunderlich, Ulrich Martin, Greg Wray, Madeline A. Lancaster

Preprint posted on July 04, 2020 https://www.biorxiv.org/content/10.1101/2020.07.04.188078v1

How Humans Got Big Brains: A delayed cell-state transition in neurogenesis leads to a larger human brain

Selected by Monica Tambalo, Teresa Rayon, Maiko Kitaoka

Background

How has the human brain evolved its unique features? Such a fascinating question remains yet widely unexplored. Among the human brain’s remarkable features, its size is perhaps one of the most evident, with roughly a 3-fold increase in human brain size compared to chimpanzee and gorilla. A conspicuous amount of studies, mainly using rodent model systems, have contributed to our current understanding of brain embryonic development. Such approaches are excellent to teach us about conserved mechanisms but are not suitable for informing on human specific brain features and primate evolution.

A revolutionary approach to study human brain development and its evolution has been the advent of human brain organoids derived from human pluripotent stem cells [1],[2], and the invaluable possibility of extending this approach to virtually any species.

A fundamental process in brain development is neurogenesis. Within the cerebral cortex a neurogenic precursor, also known as neuroepithelial (NE) cell, is characterized by a columnar morphology and divides symmetrically during its proliferative phase. This proliferation is essential for the enlargement of the neocortex in primates. NE cells are highly epithelial, with tight and adherens junctions at their apical surface. NE progenitors will then transition into neurogenic radial glia (RG) by losing epithelial features, thinning and elongating the bipolar processes, and switching to asymmetric cell divisions, where one daughter cell self-maintains itself as RG and the other starts the neurogenic differentiation. This mechanism has been mainly studied using murine models while little is known about how it works in apes. Correlative evidence points to early changes in NE behaviour as a key mechanism for human cortical expansion, however direct evidence is poor.

To address directly this issue, Benito-Kwiecinski, Giandomenico, and colleagues [3] derived brain organoids from human, gorilla and chimpanzee and directly compared their neurogenetic properties, prior to neuron formation. This approach has given the authors the advantage of manipulating candidate genes and the ability to address their involvement in human and ape brain evolution.

(Top panels) 5-week organoids stained for neural progenitor marker SOX2 (red), dorsal telencephalic/intermediate progenitor marker TBR2 (grey), neuronal markers TUJ1 (human) and HuCD (gorilla) in yellow, and DAPI (blue) showing human derived organoids become larger in overall size than gorilla and chimpanzee organoids. Scale bar: 1 mm. From Figure 1A.

(Lower panels) Schematic summarizing the morphological changes in neural progenitor cells observed in human and gorilla organoids. Progenitor cells of both species undergo a gradual transition from NE to tNE to RG-like shapes. Human cells maintain columnar NE-characteristics for a longer period while gorilla cells show tNE morphologies (blue background) earlier than human. From Figure 2H.

Key points

  • The authors developed comparable human, gorilla and chimpanzee brain organoids to study NE development prior to the onset of neurogenesis. Perhaps as expected, human brain organoids were consistently larger than the gorilla or chimp organoids.
  • Human organoids show an enlarged ventricular apical surface at early stages of NE expansion, before the onset of neurogenesis.
  • The transition from NE (non neurogenic) to RG (neurogenic) in human and ape occurs during several days, and ape NE cells make this transition more rapidly than human.
  • Cell shape changes occur before the change in cell identity and before the onset of neurogenesis.
  • Identification of a new intermediate cell morphology state, the tNE.
  • By comparing RNA-seq data from each species, they focused on the differential expression of the ZEB2 gene, particularly due to its well-known role as a key mediator of the EMT transition and found that the ZEB2 gene drives the NE to RG transition.
  • Delayed onset of ZEB2 expression extends the NE stage, compared to ape, and may be a major contributor to neocortical expansion in humans
  • They generated an inducible-ZEB2 human brain organoid, demonstrating the powerful advantages of organoids to allow unprecedented control to manipulate genes involved in neurogenesis. By overexpressing human ZEB2 prematurely to match the expression levels and timing of gorilla organoids, the authors could trigger an earlier NE transition that led to a nonhuman, ape-like organoid morphology! This suggests that ZEB2 is a key regulator of species-specific brain development.

Things we like

This work elegantly demonstrates how the evolution of the neocortex can be studied systematically and quantitatively comparing stem cell models/organoids across species, providing perturbations that wouldn’t be feasible in human embryos. Previous 2D and 3D comparisons using stem cell models of primate cortical development had primarily focused on the differences in composition and proliferation rates of radial glial cells (RG) of macaque, human and chimpanzees [4]–[7]. Instead, this work centres its attention on the role of NE cells in cortical expansion for the first time and adds gorilla stem cells to the primate cerebral organoid zoo. Finally, we really appreciate that this work applies a classical embryology approach to organoids, where the authors start from a morphological observation and have begun to pin down the molecular mechanism.

Questions for the authors 

Q1. How complex was it to establish reliable differentiation protocols for human, gorilla and chimpanzee brain organoids? And how consistent were the phenotypes observed across cell lines?

Q2. Is ZEB2 regulated differently in gorilla vs human vs chimp? How so? Ie, what triggers its expression, and how might this be regulated in a species-specific way?

Q3. Mutations in the ZEB2 gene cause Mowat-Wilson syndrome. Do the authors think that the early role that they uncovered for ZEB2 is related to the microcephaly and intellectual disability of the patients?

Q4. Mutations in the ZEB2 gene cause Mowat-Wilson syndrome. Do the authors think that the early role that they uncovered for ZEB2 is related to the microcephaly and intellectual disability of the patients?

Q5. Considering the exceptional current times, it has been challenging for everyone to work from home at a usual pace. How was the process of writing this preprint during lockdown?

References

  1. Lancaster, M. A.; Knoblich, J. A. Generation of Cerebral Organoids from Human Pluripotent Stem Cells. Nat. Protoc. 2014, 9 (10), 2329–2340. https://doi.org/10.1038/nprot.2014.158.
  2. Lancaster, M. A.; Renner, M.; Martin, C. A.; Wenzel, D.; Bicknell, L. S.; Hurles, M. E.; Homfray, T.; Penninger, J. M.; Jackson, A. P.; Knoblich, J. A. Cerebral Organoids Model Human Brain Development and Microcephaly. Nature 2013, 501 (7467), 373–379. https://doi.org/10.1038/nature12517.
  3. Benito-Kwiecinski, S.; Giandomenico, S. L.; Sutcliffe, M.; Riis, E. S.; Freire-Pritchett, P.; Kelava, I.; Wunderlich, S.; Martin, U.; Wray, G. A.; Lancaster, M. A. An Early Cell Shape Transition Drives Evolutionary Expansion of the Human Forebrain. bioRxiv 2020, 2020.07.04.188078. https://doi.org/10.1101/2020.07.04.188078.
  4. Neurogenesis, C.; Fiddes, I. T.; Lodewijk, G. A.; Mooring, M.; Salama, S. R.; Jacobs, F. M. J.; Haussler, D.; Fiddes, I. T.; Lodewijk, G. A.; Mooring, M.; Bosworth, C. M.; Ewing, A. D. Human-Specific NOTCH2NL Genes Affect Notch Article Human-Specific NOTCH2NL Genes Affect Notch Signaling and Cortical Neurogenesis. Cell 2018, 173 (6), 1356-1369.e22. https://doi.org/10.1016/j.cell.2018.03.051.
  5. Otani, T.; Marchetto, M. C.; Gage, F. H.; Simons, B. D.; Livesey, F. J.; Otani, T.; Marchetto, M. C.; Gage, F. H.; Simons, B. D.; Livesey, F. J. 2D and 3D Stem Cell Models of Primate Cortical Development Identify Species-Specific Differences in Progenitor Behavior Contributing to Brain Size Article 2D and 3D Stem Cell Models of Primate Cortical Development Identify Species-Specific Differences in Progenitor Behavior Contributing to Brain Size. Stem Cell 2016, 18 (4), 467–480. https://doi.org/10.1016/j.stem.2016.03.003.
  6. Suzuki, I. K.; Gacquer, D.; Heurck, R. Van; Polleux, F.; Detours, V.; Vanderhaeghen, P. Human-Specific NOTCH2NL Genes Expand Cortical Neurogenesis through Delta / Notch Regulation Article Human-Specific NOTCH2NL Genes Expand Cortical Neurogenesis through Delta / Notch Regulation. 2018, 1370–1384. https://doi.org/10.1016/j.cell.2018.03.067.
  7. Pollen, A. A.; Bhaduri, A.; Andrews, M. G.; Nowakowski, T. J.; Meyerson, O. S.; Mostajo-Radji, M. A.; Di Lullo, E.; Alvarado, B.; Bedolli, M.; Dougherty, M. L.; Fiddes, I. T.; Kronenberg, Z. N.; Shuga, J.; Leyrat, A. A.; West, J. A.; Bershteyn, M.; Lowe, C. B.; Pavlovic, B. J.; Salama, S. R.; Haussler, D.; Eichler, E. E.; Kriegstein, A. R. Establishing Cerebral Organoids as Models of Human-Specific Brain Evolution. Cell 2019, 176 (4), 743-756.e17. https://doi.org/10.1016/j.cell.2019.01.017.

 

Tags: brain organoids, evodevo, neurodevelopment

Posted on: 31st July 2020

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    Q1. How complex was it to establish reliable differentiation protocols for human, gorilla and chimpanzee brain organoids? And how consistent were the phenotypes observed across cell lines?

    The author team

    We have found that regardless of species, any given pluripotent stem cell (PSC) line can produce brain organoids, as long as it successfully undergoes neural differentiation. However, like many others in the field, we have observed that some PSC lines simply do not reliably generate neural identity, and are therefore not adequate for brain organoid differentiation. Indeed, several reports have indicated epigenetic and transcriptional signatures (Kim et al., 2011) and developmental biases (Strano et al., 2020) that cause some PSC lines to be restricted in their differentiation ability. Thus, we screened several cell lines from both human and non-human apes, and ultimately only used those that reliably formed neural tissues, while also paying attention to the sex of the cell lines (all lines used in our study were female) so as not to introduce confounding variables.

    With the different species PSC lines, we sought to use identical protocols, both in terms of culture conditions (media formulation, number of cells seeded, etc.) and timing. However, while culture conditions did not have to be adjusted uniquely for the different species, we found that the earliest stage of organoid differentiation, namely the embryoid body (EB) stage, had to be slightly sped up for chimpanzee PSCs.  This was evident in the morphology, and quantity of non-neural tissue produced. We found that if we followed the human timing with chimpanzee PSCs, non-ectodermal lineages developed during the EB stage into more differentiated cell fates prior to neural induction. To correct this, we simply moved chimpanzee EBs to neural induction two days earlier than the other apes. This is very interesting given the more rapid gestation of chimpanzees (34 weeks).

    Kim, K., Zhao, R., Doi, A., Ng, K., Unternaehrer, J., Cahan, P., Hongguang, H., Loh, Y.H., Aryee, M.J., Lensch, M.W., et al. (2011). Donor cell type can influence the epigenome and differentiation potential of human induced pluripotent stem cells. Nat. Biotechnol. 29, 1117–1119.

    Strano, A., Tuck, E., Stubbs, V.E., Livesey, F.J. (2020). Variable Outcomes in Neural Differentiation of Human PSCs Arise from Intrinsic Differences in Developmental Signaling Pathways. Cell Rep. 31, 107732.

    Q2. Is ZEB2 regulated differently in gorilla vs human vs chimp? How so? Ie, what triggers its expression, and how might this be regulated in a species-specific way?

    The author team

    At present we don’t know what signalling pathway is upstream of ZEB2 but appealing candidates would be FGF family members, as they play key roles early in neurodevelopment and have the potential to induce ZEB2 expression (JeongGoo, L. et al, 2018). ZEB2 is a very interesting gene in the context of neurodevelopment as it has been shown to be involved in a number of processes ranging from pluripotency exit to axon pathfinding and myelination. The ZEB2 locus contains and is surrounded by a number of human accelerated regions, sequences of the genome that show extremely high conservation across vertebrates but are strikingly different in humans. In addition, recent single-cell ATAC-seq experiments (Kanton, S. et al, 2019) have revealed a number of differentially accessible genomic regions between human and chimp organoids, suggesting that the gene is differentially regulated in these two species. Thus, changes in the enhancer and silencer regions associated with ZEB2 might orchestrate the different expression dynamics we observed in human and gorilla brain organoids. While genomic-level differences are the obvious suspect, it should not be ruled out that differences in ZEB2 expression could potentially also come down to changes in post-transcriptional regulation. For example, the 3’ UTR of ZEB2 is recognised by miRNAs of the miR-200 family both in the context of metastasis and neurogenesis (Hill, L. et al., 2013, Yang, S. et al., 2018).

    Yang, S., Toledo, E.M., Rosmaninho, P. et al. A Zeb2-miR-200c loop controls midbrain dopaminergic neuron neurogenesis and migration. Commun Biol 1, 75 (2018). https://doi.org/10.1038/s42003-018-0080-0

    Louise Hill , Gareth Browne, Eugene Tulchinsky. ZEB/miR-200 feedback loop: at the crossroads of signal transduction in cancer. Int J Cancer 132(4):745-54 (2013).

    JeongGoo Lee, Eric Jung and Martin Heur. Fibroblast growth factor 2 induces proliferation and fibrosis via SNAI1-mediated activation of CDK2 and ZEB1 in corneal endothelium. Journal of Biological Chemistry 293(10): 3758–3769 (2018).

    Kanton, S., Boyle, M.J., He, Z. et al. Organoid single-cell genomic atlas uncovers human-specific features of brain development. Nature 574, 418–422 (2019). https://doi.org/10.1038/s41586-019-1654-9

    Q3. Mutations in the ZEB2 gene cause Mowat-Wilson syndrome. Do the authors think that the early role that they uncovered for ZEB2 is related to the microcephaly and intellectual disability of the patients?

    The author team

    In our study we tested the effect of ZEB2 heterozygous loss-of function on brain organoid development. In this background early in brain organoid development we observed an upregulation in epithelial cell-cell junction markers including E-cadherin and Occludin. Concomitantly, we saw a decrease in the number of TBR2+ intermediate progenitor cells produced. We did not explore the long-term effect of these changes but they are likely to have an impact on the overall organoid neuronal output.

    Mowat-Wilson syndrome (MWS) is a complex disorder that manifests itself as an array of brain developmental defects with variable penetrance. This complex pathology is caused by de novo heterozygous loss-of-function mutations including nonsense mutations, frameshift mutations, and deletions in ZEB2. Although we were primarily interested in the evolutionary role of ZEB2, the mutation we introduced to test for a role in early neuroepithelial differentiation mimicked that seen in patients with MWS. Thus, it may well be that our model captures certain aspects of the pathology, but more work would be needed to conclusively say. Having said this, the early role that we uncovered for ZEB2 is likely to have important implications for brain size determination and neuronal output. Therefore, it is not unreasonable to speculate that ZEB2 mutations that lead to impairment of this neurodevelopmental process in humans could contribute to MWS pathogenesis.

    Q4. Mutations in the ZEB2 gene cause Mowat-Wilson syndrome. Do the authors think that the early role that they uncovered for ZEB2 is related to the microcephaly and intellectual disability of the patients?

    The author team

    It would be difficult both technically and ethically to study this process in vivo in human or ape embryos. First, the behaviour of neural progenitor cells we have characterized occurs at early pre-neurogenic stages in fetal development, corresponding to less than a month post-conception – a stage where many mothers are not even aware of their pregnancy. Although it is possible to culture primary brain tissue as an organotypic slice culture (Subramanian et al., 2017), samples have not been collected early enough to observe pre-neurogenesis stages where neural progenitor cells would still be purely expanding in behaviour and neuroepithelial in morphology. Second, it is unethical to experiment on apes, given their protected status, so a study of ape embryos would be impossible at any stage, unless through a spontaneous abortion, which is rare and difficult to acquire intact. Thus, although it would be highly beneficial to confirm our findings with primary specimens, evidence from later stage organoids suggests a high degree of overlap with in vivo development (Camp et al., 2015; Luo et al., 2016) suggesting that it is a safe assumption that the rest of the self-organization process of brain organoid formation follows human brain development with a high fidelity. Therefore, brain organoids offer a window into an otherwise black box in human and ape brain development.

    Camp, J.G., Badsha, F., Florio, M., Kanton, S., Gerber, T., Wilsch-Bräuninger, M., Lewitus, E., Sykes, A., Hevers, W., Lancaster, M., et al. (2015). Human cerebral organoids recapitulate gene expression programs of fetal neocortex development. Proc. Natl. Acad. Sci. U. S. A. 112, 15672–15677.

    Luo, C., Lancaster, M.A., Castanon, R., Nery, J.R., Knoblich, J.A., Ecker, J.R. (2016). Cerebral Organoids Recapitulate Epigenomic Signatures of the Human Fetal Brain. Cell Rep. 17, 3369-3384.

    Subramanian, L., Bershteyn, M., Paredes, M.F., and Kriegstein, A.R. (2017). Dynamic behaviour of human neuroepithelial cells in the developing forebrain. Nat. Commun. 8.

    Q5. Considering the exceptional current times, it has been challenging for everyone to work from home at a usual pace. How was the process of writing this preprint during lockdown?

    S.B-K.: It was a little challenging to convert the home into a workplace and get used to writing from a kitchen chair versus a comfortable library seat. But the writing-up process fortunately did not require physical access to the lab and we had great communication throughout via regular Zoom meetings and e-mail correspondence.

    S.L.G.: They were certainly trying times but I feel that having the manuscript to focus on was an extremely fortunate coincidence. Working in a team with Madeline and Silvia and the other authors, having regular meetings and deadlines, really helped me to deal with the daily struggles and keep spirits high.

    M.A.L.: Like many with small children, I found it quite challenging to juggle childcare and work. And finding a solid chunk of time for writing was often very difficult. But thankfully, I have an amazing team, and Silvia and Stefano (the co-first authors) worked together beautifully, making the writing process for this manuscript seamless.

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