Altered temporal sequence of transcriptional regulators in the generation of human cerebellar granule cells

Abstract

Brain development is regulated by conserved transcriptional programs across species, but little is known about divergent mechanisms that create species-specific characteristics. Among brain regions, human cerebellar histogenesis differs in complexity compared with non-human primates and rodents, making it important to develop methods to generate human cerebellar neurons that closely resemble those in the developing human cerebellum. We report a rapid protocol for the derivation of the human ATOH1 lineage, the precursor of excitatory cerebellar neurons, from human pluripotent stem cells (hPSC). Upon transplantation into juvenile mice, hPSC-derived cerebellar granule cells migrated along glial fibers and integrated into the cerebellar cortex. By Translational Ribosome Affinity Purification-seq, we identified an unexpected temporal shift in the expression of RBFOX3 (NeuN) and NEUROD1, which are classically associated with differentiated neurons, in the human outer external granule layer. This molecular divergence may enable the protracted development of the human cerebellum compared to mice.

Data availability

Sequencing data have been deposited in GEO under accession code: GSE163710. For reviewers only, a temporary password has been generated: ejkpqqeupdkplcx.Upon publication, the data will be released publicly.

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Hourinaz Behesti

    Laboratory of Developmental Neurobiology, Rockefeller University, New York, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9383-9929
  2. Arif Kocabas

    Laboratory of Developmental Neurobiology, Rockefeller University, New York, United States
    Competing interests
    No competing interests declared.
  3. David E Buchholz

    Laboratory of Developmental Neurobiology, Rockefeller University, New York, United States
    Competing interests
    No competing interests declared.
  4. Thomas S Carroll

    Bioinformatics Resouce Center, Rockefeller University, New York, United States
    Competing interests
    No competing interests declared.
  5. Mary E Hatten

    Laboratory of Developmental Neurobiology, Rockefeller University, New York, United States
    For correspondence
    hatten@rockefeller.edu
    Competing interests
    Mary E Hatten, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9059-660X

Funding

National Institute of Neurological Disorders and Stroke (1R21NS093540-01)

  • Mary E Hatten

The Rockefeller University Center for Clinical and Translational Science (Pilot award)

  • Hourinaz Behesti
  • Mary E Hatten

Starr Foundation (Tri-Institutional Stem Cell Initiative Grant)

  • Mary E Hatten

Department of Defense US Army Medical Research Acquisition Activity Grants (W81XWH1510189)

  • Mary E Hatten

The Robertson Therapeutics Development Fund

  • Mary E Hatten

Renate, Hans, and Maria Hofmann Trust

  • Mary E Hatten

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocol (#14746-H) of the Rockefeller University. All surgery was performed under hypothermia, and every effort was made to minimize suffering.

Human subjects: Fixed de-identified human tissue were acquired from the Human Developmental Biology Resource (http://www.hdbr.org/) following institutional policies.

Copyright

© 2021, Behesti 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

  • 2,002
    views
  • 292
    downloads
  • 18
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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. Hourinaz Behesti
  2. Arif Kocabas
  3. David E Buchholz
  4. Thomas S Carroll
  5. Mary E Hatten
(2021)
Altered temporal sequence of transcriptional regulators in the generation of human cerebellar granule cells
eLife 10:e67074.
https://doi.org/10.7554/eLife.67074

Share this article

https://doi.org/10.7554/eLife.67074

Further reading

    1. Developmental Biology
    2. Neuroscience
    Taro Ichimura, Taishi Kakizuka ... Takeharu Nagai
    Tools and Resources

    We established a volumetric trans-scale imaging system with an ultra-large field-of-view (FOV) that enables simultaneous observation of millions of cellular dynamics in centimeter-wide three-dimensional (3D) tissues and embryos. Using a custom-made giant lens system with a magnification of ×2 and a numerical aperture (NA) of 0.25, and a CMOS camera with more than 100 megapixels, we built a trans-scale scope AMATERAS-2, and realized fluorescence imaging with a transverse spatial resolution of approximately 1.1 µm across an FOV of approximately 1.5×1.0 cm2. The 3D resolving capability was realized through a combination of optical and computational sectioning techniques tailored for our low-power imaging system. We applied the imaging technique to 1.2 cm-wide section of mouse brain, and successfully observed various regions of the brain with sub-cellular resolution in a single FOV. We also performed time-lapse imaging of a 1-cm-wide vascular network during quail embryo development for over 24 hr, visualizing the movement of over 4.0×105 vascular endothelial cells and quantitatively analyzing their dynamics. Our results demonstrate the potential of this technique in accelerating production of comprehensive reference maps of all cells in organisms and tissues, which contributes to understanding developmental processes, brain functions, and pathogenesis of disease, as well as high-throughput quality check of tissues used for transplantation medicine.

    1. Developmental Biology
    2. Genetics and Genomics
    Mehul Vora, Jonathan Dietz ... Cathy Savage-Dunn
    Research Article

    Smads and their transcription factor partners mediate the transcriptional responses of target cells to secreted ligands of the transforming growth factor-β (TGF-β) family, including those of the conserved bone morphogenetic protein (BMP) family, yet only a small number of direct target genes have been well characterized. In C. elegans, the BMP2/4 ortholog DBL-1 regulates multiple biological functions, including body size, via a canonical receptor-Smad signaling cascade. Here, we identify functional binding sites for SMA-3/Smad and its transcriptional partner SMA-9/Schnurri based on ChIP-seq peaks (identified by modEncode) and expression differences of nearby genes identified from RNA-seq analysis of corresponding mutants. We found that SMA-3 and SMA-9 have both overlapping and unique target genes. At a genome-wide scale, SMA-3/Smad acts as a transcriptional activator, whereas SMA-9/Schnurri direct targets include both activated and repressed genes. Mutations in sma-9 partially suppress the small body size phenotype of sma-3, suggesting some level of antagonism between these factors and challenging the prevailing model for Schnurri function. Functional analysis of target genes revealed a novel role in body size for genes involved in one-carbon metabolism and in the endoplasmic reticulum (ER) secretory pathway, including the disulfide reductase dpy-11. Our findings indicate that Smads and SMA-9/Schnurri have previously unappreciated complex genetic and genomic regulatory interactions that in turn regulate the secretion of extracellular components like collagen into the cuticle to mediate body size regulation.