Comparison of induced neurons reveals slower structural and functional maturation in humans than in apes

  1. Maria Schörnig
  2. Xiangchun Ju
  3. Luise Fast
  4. Sebastian Ebert
  5. Anne Weigert
  6. Sabina Kanton
  7. Theresa Schaffer
  8. Nael Nadif Kasri
  9. Barbara Treutlein
  10. Benjamin Marco Peter
  11. Wulf Hevers
  12. Elena Taverna  Is a corresponding author
  1. Max Planck Institute for Evolutionary Anthropology, Germany
  2. Radboud University Medical Center, Netherlands

Abstract

We generated induced excitatory neurons (iNeurons, iNs) from chimpanzee, bonobo and human stem cells by expressing the transcription factor neurogenin‑2 (NGN2). Single cell RNA sequencing (scRNAseq) showed that genes involved in dendrite and synapse development are expressed earlier during iNs maturation in the chimpanzee and bonobo than the human cells. In accordance, during the first two weeks of differentiation, chimpanzee and bonobo iNs showed repetitive action potentials and more spontaneous excitatory activity than human iNs, and extended neurites of higher total length. However, the axons of human iNs were slightly longer at 5 weeks of differentiation. The timing of the establishment of neuronal polarity did not differ between the species. Chimpanzee, bonobo and human neurites eventually reached the same level of structural complexity. Thus, human iNs develop slower than chimpanzee and bonobo iNs and this difference in timing likely depends on functions downstream of NGN2.

Data availability

Sequencing data for single cells have been deposited in ArrayExpress under the accession code E-MTAB-9233 and under Mendeley Data with doi: 10.17632/y3s4hnyvg6. To make our scRNAseq data accessible to the neuroscience community, we provide a ShinyApp-based web browser for data exploration, called iNeuronExplorer. https://bioinf.eva.mpg.de/shiny/iNeuronExplorer/ Morphological data for neurons and a custom made script for analysis have been deposited in GitHub under the URL: https://github.com/BenjaminPeter/schornig_ineuron.

The following data sets were generated
    1. Kanton S
    (2020) iNeuronExplorer
    MPI EVA webbrowser, shiny/iNeuronExplorer/.
The following previously published data sets were used

Article and author information

Author details

  1. Maria Schörnig

    Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Xiangchun Ju

    Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Luise Fast

    Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Sebastian Ebert

    Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Anne Weigert

    Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Sabina Kanton

    Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Theresa Schaffer

    Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  8. Nael Nadif Kasri

    Human Genetics, Radboud University Medical Center, Nijmegen, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  9. Barbara Treutlein

    Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
    Competing interests
    The authors declare that no competing interests exist.
  10. Benjamin Marco Peter

    Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2526-8081
  11. Wulf Hevers

    Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1881-5913
  12. Elena Taverna

    Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
    For correspondence
    elena_taverna@eva.mpg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2430-4725

Funding

This work was supported by the Max Planck Society.The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Human subjects: For this study we used three human (hiPS409-B2, SC102A1, HmRNA), three chimpanzee (SandraA, JoC, ciPS01) and one bonobo (BmRNA) iPS cell lines and one additional human ES cell line (H9). The human iPSC lines hiPS409-B2 and SC102A1 were purchased from the Riken BRC Cellbank and System Biosciences, respectively. The human iPSCs line HmRNA (generated in this study) was reprogrammed from human dermal fibroblasts using the StemMACS mRNA transfection kit. The cell line was validated for pluripotency markers by immunohistochemical staining using the Human Pluripotent Stem Cell 3-Colour Immunohistochemistry Kit and were differentiated into the three different germ layers using the Human Pluripotent Stem Cell Functional Identification kit and StemMACS Trilineage Differentiation Kit. Karyotyping was carried out using Giemsa banding at the Stem Cell Engineering facility, a core facility of CMCB at Technische Universität Dresden. Karyotypes were found to be normal. The human ES cell line H9 was purchased from WiCell. The chimpanzee iPSC lines SandraA and JoC as well as the bonobo iPSCs line BmRNA were generated in a previous study (Kanton et al., Nature, 2019). The chimpanzee iPSCs ciPS01 line was provided by the Max-Delbrück-Centrum für Molekulare Medizin, Berlin.The rtT A/Ngn2-positive iPSCs/ESCs hiPS409-B2_Ngn2, SandraA_Ngn2, BmRNA_Ngn2, H9_Ngn2, SC102A1_Ngn2, HmRNA_Ngn2, ciPS01_Ngn2 and JoC_Ngn2 were generated using lentiviral vectors to stably integrate the transgenes into the genome of the stem cells and differentiate the stem cells into neurons as previously described by Frega et al., Jove, 2017.Our cultures were regularly controlled for mycoplasma.Permission to work with human and non-human primate iPSC lines and Ngn2-inducible cell lines was obtained through the Sächsisches Staatsministerium für Umwelt und Landwirtschaft (Az.: 55-8811.72/26, Az.: 55-8811.72/26/350). The use of human ESCs was approved by the ethics committee of the Robert Koch Institut (https://www.rki.de/DE/Content/Gesund/Stammzellen/Register/reg-20161027-Paeaebo.html).

Reviewing Editor

  1. Anita Bhattacharyya, University of Wisconsin, Madison, United States

Publication history

  1. Received: May 26, 2020
  2. Accepted: January 19, 2021
  3. Accepted Manuscript published: January 20, 2021 (version 1)
  4. Version of Record published: February 8, 2021 (version 2)

Copyright

© 2021, Schörnig et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 3,816
    Page views
  • 406
    Downloads
  • 12
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Maria Schörnig
  2. Xiangchun Ju
  3. Luise Fast
  4. Sebastian Ebert
  5. Anne Weigert
  6. Sabina Kanton
  7. Theresa Schaffer
  8. Nael Nadif Kasri
  9. Barbara Treutlein
  10. Benjamin Marco Peter
  11. Wulf Hevers
  12. Elena Taverna
(2021)
Comparison of induced neurons reveals slower structural and functional maturation in humans than in apes
eLife 10:e59323.
https://doi.org/10.7554/eLife.59323

Further reading

    1. Developmental Biology
    2. Neuroscience
    Eleni Chrysostomou et al.
    Research Article

    Neurogenesis is the generation of neurons from stem cells, a process that is regulated by SoxB transcription factors (TFs) in many animals. Although the roles of these TFs are well understood in bilaterians, how their neural function evolved is unclear. Here, we use Hydractinia symbiolongicarpus, a member of the early-branching phylum Cnidaria, to provide insight into this question. Using a combination of mRNA in situ hybridization, transgenesis, gene knockdown, transcriptomics, and in-vivo imaging, we provide a comprehensive molecular and cellular analysis of neurogenesis during embryogenesis, homeostasis, and regeneration in this animal. We show that SoxB genes act sequentially at least in some cases. Stem cells expressing Piwi1 and Soxb1, which have a broad developmental potential, become neural progenitors that express Soxb2 before differentiating into mature neural cells. Knockdown of SoxB genes resulted in complex defects in embryonic neurogenesis. Hydractinia neural cells differentiate while migrating from the aboral to the oral end of the animal, but it is unclear whether migration per se or exposure to different microenvironments is the main driver of their fate determination. Our data constitute a rich resource for studies aiming at addressing this question, which is at the heart of understanding the origin and development of animal nervous systems.

    1. Developmental Biology
    Noah P Mitchell et al.
    Research Article

    Organ architecture is often composed of multiple laminar tissues arranged in concentric layers. During morphogenesis, the initial geometry of visceral organs undergoes a sequence of folding, adopting a complex shape that is vital for function. Genetic signals are known to impact form, yet the dynamic and mechanical interplay of tissue layers giving rise to organs' complex shapes remains elusive. Here, we trace the dynamics and mechanical interactions of a developing visceral organ across tissue layers, from sub-cellular to organ scale in vivo. Combining deep tissue light-sheet microscopy for in toto live visualization with a novel computational framework for multilayer analysis of evolving complex shapes, we find a dynamic mechanism for organ folding using the embryonic midgut of Drosophila as a model visceral organ. Hox genes, known regulators of organ shape, control the emergence of high-frequency calcium pulses. Spatiotemporally patterned calciumpulses triggermuscle contractions via myosin light chain kinase. Muscle contractions, in turn, induce cell shape change in the adjacent tissue layer. This cell shape change collectively drives a convergent extension pattern. Through tissue incompressibility and initial organ geometry, this in-plane shape change is linked to out-of-plane organ folding. Our analysis follows tissue dynamics during organ shape change in vivo, tracing organ-scale folding to a high-frequency molecular mechanism. These findings offer a mechanical route for gene expression to induce organ shape change: genetic patterning in one layer triggers a physical process in the adjacent layer - revealing post-translational mechanisms that govern shape change.