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Written by | November
The generation of complex brain organoids from human stem cells under in vitro culture conditions provides new tools and hope
for understanding the basic molecular mechanisms of human brain development.
Different neurons in the brain can develop from embryonic stem cells or induced pluripotent stem cells, and the fate and state of each different cell are the end result
of complex regulation of different transcription factors.
Transcription factors confluence to regulatory elements and interact with chromatin to achieve precise control of gene expression [1,2].
The advent of single-cell sequencing methods provides a way to analyze gene expression and chromatin accessibility in individual cells, and a comprehensive cell atlas of mouse and human brain cells provides a reference
for understanding the composition and development of organoids.
Brain organoids can now be used to mimic microcephaly, periventricular ectopia, autism, and other neurodevelopmental disorders
that can affect different regions of the human brain.
However, the gene regulatory networks (GRNs) that coordinate early brain development in humans under normal and abnormal conditions are not fully understood
.
On October 5, 2022, the Barbara Treutlein Research Group, Zhisong He Research Group and the Basel Institute of Molecular and Clinical Ophthalmology at the Federal Institute of Technology in Basel, Switzerland.
The Gray Camp research group co-published a paper entitled Inferring and perturbing cell fate regulomes in human brain organoids, by establishing a Pando analysis framework that integrates multi-omics data and transcription factor binding site prediction.
This enables the description
of the comprehensive gene regulatory network of brain organoids.
There is some understanding of the signaling pathways that coordinate the formation of core signaling factors and gene regulatory programs in vertebrate brain regions, including SHH, WNTs, BMP, FGF, NOTCH, Neuregulins, and R-spondins
。 Much of these signaling pathways that regulate brain morphogenesis have been validated in non-human model biological systems, but it is unclear how different nerve cell lineages in the human brain diverge
.
To study the mechanisms of human brain development, the authors generated a database of single-cell transcriptome and single-cell chromatin accessibility assays during brain organoid development (Figure 1).
The database contains gene expression and chromatin accessibility data
from 11 different cell lineages at different time points from 3 human induced pluripotent stem cells and 1 from embryonic stem cells.
These time points cover embryonic formation, neuroectoderm induction, neuroepithelialization, neural progenitor cell patterning, and neurogenesis
.
At each time point, the authors isolated organoid tissues of four cell lines, ran single-cell RNA-seq sequencing and single-cell ATAC-seq on the same cell suspension, further multiplexed the sequencing data using single nucleotide variants specific to each individual, and integrated the two modes
for each row and time point using typical correlation analysis.
In addition, the authors used clustering methods to integrate and visualize
metacells.
From this, the authors revealed a relatively continuous distribution of cellular states throughout the time course in human brain organoids (Figure 1).
The analytical models developed by the authors can be used to identify marker genes and gene regulatory regions
in different cellular states.
Figure 1 Multiomics Atlas of Human Brain Organoids reveals key stages
of cell fate determination In order to infer the gene regulatory network of human brain organoid development, the authors developed an algorithm called Pando, which utilizes multimodal single-cell genomic measurements and models gene expression
through transcription factor interactions 。 The authors found that the accessibility of gene expression regulatory regions and transcription factor expression vary with the stage of organoid development and are separated
during brain regionalization.
In order to better understand chromatin accessibility and gene regulatory network activity in different brain regions, the authors divided the global gene regulatory network into region-specific regulatory networks
.
These analyses provide a rich resource
for further study of gene regulatory programs that control human brain regionalization and transcription factor-mediated cellular programming.
To better understand the mechanisms that regulate cell fate and state during human brain development, the authors designed a library of lentiviral gRNAs targeting 20 transcription factors, each with three corresponding gRNAs, which are key factors
expressed in human brain organoids and early human cerebral cortex at different stages.
By restoring gRNAs, the authors found that recycled gRNAs have region-specific distribution characteristics, indicating that transcription factor perturbations are region-specific
.
On the basis of these indicators, the authors focused on the genes HES1 and GLI3
.
The authors found that GLI3-activated genes were positively correlated with cortical metastasis probabilities, while HES1 had an inhibitory effect
on such genes.
GLI3 is a well-known medium of SHH signaling, and its loss-of-function mutations can cause the mouse cortex to fail to form [3].
In humans, mutations in GLI3 are associated with Greig Syndactyly syndrome and Pallister Hall syndrome, in which patients exhibit different brain malformations depending on the specific mutation [4].
。 In order to confirm GLI3's involvement in cell fate in the human brain and unravel the underlying developmental mechanisms, the authors used CRISPR-Cas9 gene editing technology to construct a lineage of GLI3-knocked human induced pluripotent stem cells, and generated wild-type and GLI3-knockout human brain organoids
, respectively.
The authors found that GLI3 knockout affected ventral brain cell status
.
The results confirm that GLI3 is necessary in the establishment of neuronal fate in the human cortex, and its absence affects the development
of the ventral telbrain.
In general, this work obtains gene expression and chromatin accessibility data from human brain organoids generated from pluripotent stem cells and embryonic stem cells through single-cell genomic technology, providing an important database and algorithm platform
for studying the gene regulatory network of human brain development, that is, the regulatory group (Regulomes).
In addition, in the case of the transcription factor GLI3 study, the authors found that the key role of GLI3 in the establishment of the fate of the human cortex is consistent
with the research conducted in mammalian model systems.
Therefore, the study provides a working example
of how human developmental biology can be reconstructed using human model systems and single-cell techniques.
Original link:
https://doi.
org/10.
1038/s41586-022-05279-8
Pattern maker: Eleven
References
1.
Eiraku, M.
et al.
Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals.
Cell Stem Cell 3, 519–532 (2008).
2.
Lancaster, M.
A.
et al.
Cerebral organoids model human brain development and microcephaly.
Nature 501, 373–379 (2013).
3.
Ruiz i Altaba, A.
, Palma, V.
& Dahmane, N.
Hedgehog-Gli signalling and the growth of the brain.
Nat.
Rev.
Neurosci.
3, 24–33 (2002).
4.
Biesecker, L.
G.
The Greig cephalopolysyndactyly syndrome.
Orphanet J.
Rare Dis.
3, 10(2008).
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