Nature: Human brain development map

The human brain, probably the most complex organ in the entire biological world, has long been the subject of a fascination for researchers
.
However, studying the brain, especially the genes and molecular switches that regulate and guide brain development, is not an easy task
.

So far, scientists have used animal models, mostly mice, but their findings cannot be transferred directly to humans
.
Mice have different brain structures and do not have the groove surfaces
typical of human brains.
Until now, cell culture has had limited value in this field, as cells tend to spread to large areas as they grow in a dish; This does not fit the natural three-dimensional structure
of the brain.

A team of researchers led by Professor Barbara Treutlein of Basel's Department of Biosystems Science and Engineering has now taken a new approach to studying the development of the human brain: They are cultivating and using organoids — a millimeter-sized three-dimensional tissue that can be cultured
from so-called pluripotent stem cells.

As long as these stem cells are properly stimulated, researchers can program them to turn them into cells of any kind in the body, including neurons
.
When stem cells aggregate into a small tissue ball and then exposed to the appropriate stimulation, they can even self-organize, forming a three-dimensional brain organ
with complex tissue structures.

Treutlein and her colleagues have now studied thousands of individual cells
within a brain organ at different points in time and in great detail.
Their goal is to describe cells in terms of molecular genetics: in other words, the total number of all gene transcripts (transcriptomes) is used as a measure of gene expression, while also using the accessibility of the genome as a measure
of regulatory activity.
They successfully represented the data as a kind of map showing the molecular fingerprints
of every cell within the organoid.

However, this process yields a huge data set: each cell in an organoid has 20,000 genes, and each organoid is made up
of thousands of cells.
"This creates a huge matrix, and the only way we can solve it is with the help of the right programs and machine learning,"
explains Jonas Fleck, a PhD student on Treutlein's team and co-lead author of the study.
To analyze all of this data and predict the mechanisms of gene regulation, the researchers developed their own program
.
Fleck said: "We can use it to generate a complete interaction network for each gene and predict what
will happen in real cells when the gene fails.
"

The goal of this study was to systematically identify gene switches that have a significant impact on neuronal development in different areas of brain organoids
.

With the help of the CRISPR-Cas9 system, ETH researchers selectively turned off one gene in each cell, for a total of about 24 genes in the entire organoid to turn off
at the same time.
This allows them to find out the role of their respective genes in the development of brain organoids
.

"This technique could be used to screen for genes
associated with disease.
In addition, we can observe the effects of these genes on the development of different cells within the organoids, explains Sophie Jansen, who is also a doctoral student in Treutlein's team and the second co-first author
of the study.

To test their theory, the researchers chose the GLI3 gene as an example
.
This gene is the blueprint for the eponymous transcription factor, a protein that docks at a specific site in DNA to regulate another gene
.
When GLI3 is turned off, cellular mechanisms cannot read the gene and transcribe it into an RNA molecule
.

In mice, mutations in the GLI3 gene cause malformations
of the central nervous system.
Its role in human neurodevelopment has not been explored before, but it is known that mutations in this gene can cause diseases
such as Greig cephalodermia and finger malformation and Pallister Hall syndrome.

Silencing the GLI3 gene allowed the researchers to both validate their theoretical predictions and directly determine in cell culture how the loss of this gene affects further development of brain organoids
.
"We demonstrated for the first time that the GLI3 gene is involved in the formation
of human forebrain patterns.
This has only been shown in mice before," Treutlein said
.

"What's exciting about this study is that it allows you to use whole genome data from so many individual cells to hypothesize what a single gene does
," she explains.
Equally exciting in my opinion is that these model systems made in Petri dishes do reflect the developmental biology
we know from mice.
”

Treutlein also found an interesting phenomenon that the medium can produce self-organizing tissue that is structurally similar to the human brain — not only at the morphological level, but also (as the researchers demonstrated in their latest study) at the level of gene regulation and pattern formation
.
"Organoids like this are really an excellent way to study the biology of human development," she noted
.

One advantage of studying organoids made up of human cellular material is that the results can be transferred to humans
.
They can be used not only to study basic developmental biology, but also to study the role
of genes in diseases or developmental brain disorders.
For example, Treutlein and her colleagues are studying this type of organoid to study the genetic causes of autism and ectopic malformations; In the latter case, neurons appear outside
the usual anatomical locations of the cerebral cortex.

Organoids can also be used to test drugs and may be used to culture transplantable organs or parts of
organs.
Treutlein confirmed that the pharmaceutical industry is very interested in these cell cultures
.
However, growing organoids takes both time and effort
.
In addition, each cell cluster develops independently, rather than in a
standardized way.
That's why Treutlein and her team are working to improve organoids and automate
their manufacturing process.

Inferring and perturbing cell fate regulomes in human brain organoids