How did the human brain get so big?

Over 3 million years of evolution, the human brain underwent a considerable increase in size and complexity, resulting in the exceptional cognitive abilities we have today. But what were the genetic changes that enabled this evolution to take place?

The brain’s enlargement was mainly due to an increase in the number of neurons in the cerebral cortex, the outer part of the brain. As noted by David Haussler, from the University of California, Santa Cruz and the Howard Hughes Medical Institute, “That has to be a fundamental substrate for us becoming human.” But since we share about 99% of our genome with that of our closest living relative, the chimpanzee, it has remained a daunting task for scientists to identify which human-specific gene changes were responsible for human brain evolution.

Haussler and his team were one of two groups who claim to have uncovered this mystery, both of whom have come to the same conclusion. The other was led by Professor Pierre Vanderhaeghen, from ULB and VIB-KU Leuven. Both groups have published their results in the journal Cell.

One of the drivers of evolution is the emergence of novel genes through duplication: an ancestral gene is duplicated and the copy evolves into a related, so-called ‘paralog’ gene. Vanderhaeghen and his team were particularly interested in duplicated genes that arose specifically in the lineage common to humans and great apes.

“Developmental biologists usually look at changes in the regulation of genes to explain evolutionary differences, and not so much at genes themselves, since we share so many of our genes even with simple organisms such as worms,” Vanderhaeghen said. “But gene duplication can lead to novel genes in a species, which could contribute to the rapid emergence of human-specific traits — like the increased size of the brain’s cortex.”

Several dozens of ‘human-specific’ genes have been found in the human genome, but their role has often remained unknown. Many of these genes are thought to be non-functional or redundant, and are not even appropriately annotated in genome databases. Searching for human-specific genes involved in brain development has thus proved challenging.

“One of the main difficulties is distinguishing the expression of ancestral genes (present in all species) and human-specific paralogs (present only in human DNA), as obviously they are extremely similar,” said Ikuo Suzuki, a postdoctoral researcher at Vanderhaeghen’s lab. “That is why we had to use a tailored RNAseq analysis for specific and sensitive detection of the human-specific genes of interest. In that way, we could identify a whole repertoire of duplicated genes that are involved in the development of the cerebral cortex in humans.”

This allowed the researchers to identify a repertoire of 35 genes unique to humans that are active during the development of the cerebral cortex in humans. Among these, the team focused on one particular family: NOTCH2NL, a cluster of human-specific paralogs of the NOTCH2 receptor. The Notch pathway is well known as a key player in organ development, including that of the brain, while the ancestral gene NOTCH2 is important in signalling processes that control whether cortical stem cells produce neurons or regenerate more stem cells.

To better understand what the genes do in humans, the team turned to an in vitro model of cortical development from human pluripotent stem cells to explore NOTCH2NL function. They found that NOTCH2NL genes stood out for their ability to promote the expansion of cortical stem cells, which in turn generated more neurons — a feature expected to distinguish between human and non-human cortical neurogenesis. This led them to hypothesise that NOTCH2NL genes could act as species-specific regulators of brain size.

“From one stem cell, you can either regenerate two progenitor cells, generate two neurons or generate one progenitor stem cell and one neuron. And what NOTCH2NL does is bias that decision in a slight way towards regenerating progenitors, which can later go on to make more neurons,” Vanderhaeghen explained. “It’s a small early effect with large late consequences, as often happens with evolution.”

Meanwhile, Haussler and his colleagues were comparing genes expressed during brain development in humans and macaque monkeys in stem cell-derived models when they realised that they could detect NOTCH2NL in human cells but not in those of the macaques. Looking at the DNA, they also didn’t see it in orangutans and found only truncated, inactive versions in our closest relatives: gorillas and chimpanzees.

Reconstructing the evolutionary history of NOTCH2NL genes revealed that a process called gene conversion was likely responsible for repairing a non-functional version of NOTCH2NL, which originally emerged as a partial duplication of NOTCH2. This repair happened only in humans, and they estimate it happened 3–4 million years ago — around the same time that the fossil record suggests human brains began to expand. After it was repaired, but before we diverged from our common ancestor with Neanderthals, NOTCH2NL was duplicated two more times.

The team also looked at what happened when NOTCH2NL wasn’t expressed: they deleted it from human stem cells and used them to grow patches of cortex called organoids. In the organoids derived from NOTCH2NL-depleted stem cells, differentiation occurred faster, but the organoids ended up being smaller.

“If you lose NOTCH2NL, it leads to premature differentiation of cortical stem cells into neurons, but at the same time the very important stem cell pool gets depleted,” said co-senior author Frank Jacobs from the University of Amsterdam.

“Taken together, our study and that of our colleagues in the US point to a selective repertoire of human-specific gene duplications that may act as key controllers of human brain size and function: fewer copies of NOTCH2NL would lead to reduced brain size, while more copies would lead to an increase in brain size,” Vanderhaeghen said.

NOTCH2NL’s location on the genome, incorrectly mapped until recently, is further support for its role in human brain size, and was of particular interest to both groups. Duplications or deletions at a genome region known as 1q21.1 are known to cause macrocephaly or microcephaly, respectively, and are associated with a range of neurodevelopmental disorders, including ADHD, autism spectrum disorder and intellectual disability. Haussler’s team looked at 11 patients with errors at this locus and found that NOTCH2NL was indeed being duplicated and deleted in the rearrangement events associated with larger and smaller brain size that resulted.

“We really wanted the gene to be in the 1q21.1 disease interval, because it made logical sense, but in the incorrect reference genome, it wasn’t,” said Haussler. “And then we found new data, and we realised that it was a mistake in the reference genome! It seldom happens that when you want something that appears to be false to be true, it turns out to actually be true.”

This part of the genome is challenging to sequence and read, with Haussler saying, “We’ve been looking under the lamppost in human genetics, as they say, by studying just the regions that were easy to sequence. There’s a lot of information in these other regions, and there’s a reasonable argument that they are the real cauldron for rapid change over the last few million years.”

The researchers acknowledge that NOTCH2NL is something of an evolutionary trade-off between larger brains and 1q21.1 disease susceptibility, but Sofie Salama of UC Santa Cruz looked at this in a positive light.

“What we found when we developed the technology to really sequence it in individuals is that there are multiple different alleles of this gene,” she said. “And it’s possible that that variation creates the subtlety and plasticity that is important in enabling humans to be human.”

There are still a lot of unknowns when it comes to NOTCH2NL. Haussler’s team points out that they were only able to look at the genomes of a small sample of patients and that their organoid models didn’t address the later stages of cortical development, at a time when NOTCH2NL might be even more important. Meanwhile, Vanderhaeghen’s team wants to address what other human-specific genes identified here (in particular, those also found in the 1q21.1 region or other genome areas associated with brain diseases) do during brain development. And although both teams were able to show that NOTCH2NL is involved in the Notch signalling pathway, Vanderhaeghen acknowledges that there is still uncertainty around the exact mechanism by which NOTCH2NL tips the balance between differentiation and regeneration.

“What’s amazing is that there are many signalling pathways that control the development of the embryo and are completely conserved between species. The Notch signalling pathway is the oldest one,” he said. “You can find it in every animal you look at. It has been used by developing embryos for as long as animals have existed. And yet, there is a very recent innovation in this pathway specifically in the human lineage, through NOTCH2NL.

“Intriguingly, the same region in the genome holds several other human-specific genes with unknown function,” he added. “It will be interesting to see if they control other aspects of human brain development.”

Image caption: Human cortical cells in vitro. Image credit: Suzuki et al.