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

Hourinaz Behesti,Arif Kocabas,David E Buchholz,Mary E Hatten,Thomas S Carroll

doi:10.7554/elife.67074

Abstract

Brain development is regulated by conserved transcriptional programs across species, but little is known about the divergent mechanisms that create species-specific characteristics. Among brain regions, human cerebellar histogenesis differs in complexity compared with nonhuman 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 (hPSCs). 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.

Highlights

Understanding the development of the human brain is an emerging area of neuroscience
We report a rapid protocol for the derivation of the human ATOH1 lineage, the precursor of excitatory cerebellar neurons, from human pluripotent stem cells
We report a method for the scalable derivation of the human ATOH1 neuronal lineage from human pluripotent stem cells (hPSCs) that yields cerebellar progenitors by day 16 and granule cells (GCs) within 48 days in chemically defined medium

Summary

Introduction

Understanding the development of the human brain is an emerging area of neuroscience. (Wingate and Hatten, 1999) with a primary germinal zone that produces the Purkinje neurons and interneurons, and a secondary germinal zone, marked by the ATOH1 transcription factor, that emerges from the rhombic lip and generates cerebellar granule cells (GCs) (Hatten and Heintz, 1995). The importance of understanding the human ATOH1 lineage is underscored by the fact that GCPs are a known cell of origin for medulloblastoma; the most common metastatic childhood brain tumor (Behesti and Marino, 2009; Marino et al, 2000), and GCs are implicated in neurodevelopmental disorders including autism (Bauman, 1991; Kloth et al, 2015; Menashe et al, 2013)

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