Roles of the Human Specific Gene ARHGAP11B on Human Neocortex Expansion

Appearance and evolution of the neocortex in mammals, especially in primates, is perceived to be the reason for their advanced cognitive function [1]. Yet, neocortices of different species differ widely in many ways such as size, shape, and the number of neurons [2]. These differences altogether are presumably reflected through the organization of neural progenitor cells during embryonic development. Understanding neocortical evolution could be a huge step in revealing the cognitive development of non-human primates. But since the fossil records of the brain -which are soft tissues- are highly restricted, attempts on understanding neocortical development and differentiation at a cellular level have been limited to comparisons of living species [3].

However, because rodents’ neocortices are small and lissencephalic, their ability to reveal the development of the mechanisms of large and gyrocephalic neocortex, like the Homo sapiens, is highly limited.

Figure 1: Lissencephalic mouse neocortex and gyrocephalic chimpanzee neocortex [4].

Even though our knowledge of mouse cortical development goes beyond of other species, recent studies of the developing neocortex in humans, primates, marsupials, and carnivores begin to reveal how differences in neural progenitor cell (NPC) populations can cause differences in the shapes and sizes of neocortex [5,6,7].

Neocortex expansion is a hallmark of human evolution. This situation now makes it necessary to answer a question: What caused the expansion of the neocortex in humans?

Increased proliferation of neural progenitor cells leads to an abundant number of neurons being generated during human cortical development thus leading to the evolutionary expansion of the human neocortex [3,8,9].

A group of scientists from the Max Planck Institute of Molecular Cell Biology and Genetics searched through genes expressed in the progenitor cells that build the neocortex and detected one gene found in humans but not in mice: ARHGAP11B [10].

How was it Detected? 

Scientists started examining aborted human fetal tissue and embryonic mice to reveal which genes were active during neocortical development. The cortical expansion has been linked to increased generation of basal progenitors from apical radial glia and their greater and prolonged proliferation, resulting in enlargement of the subventricular zone [3,9].

In light of this, they decided to isolate the neural progenitor cell types from the fetal human neocortex and compare them with the ones from the embryonic mouse neocortex. To execute these cells, they either used transgenic Tubb3-Green Fluorescent Protein (GFP) mouse embryos or, for the fetal human neocortex, they performed vital DNA staining of the cell suspension with a fluorescent dye to distinguish neurons from NPCs based on their different DNA content [10,11].

With fluorescent markers, they used fluorescence-activated cell sorting for isolating certain cell populations from the neocortex of embryonic mice. After RNA sequencing of each cell fragment, differential gene expression analysis indicated that in mice, basal radial glia (bRG) are very similar to basal intermediate progenitors and neurons but are distinct from apical radial glia (aRG) [12–14].

As opposed to this, in humans, fewer genes were differently expressed between basal radial glia and apical radial glia. Therefore, they looked out for functional clues in the group of genes that have similar expression levels in human bRG and aRG but are down-regulated in mouse bRG compared to aRG [10].

After that, they searched through sets of differentially expressed human genes and then eliminated genes that had mouse orthologs that were expressed in mouse cortical neural progenitor cells. This series of processes left the group with only one gene that suited these criteria: ARHGAP11B.

The gene seems to differentiate humans from chimpanzees and also conducts the proliferation of the key progenitor cells. ARHGAP11B appeared in the evolutionary lineage of humans after the chimpanzee and the human lineage had been cleaved apart by partial duplication of the gene ARHGAP11A. Even though ARHGAP11B does not exist in non-human primates, it existed in Neandertals and Denisovans [10,11,15,17].

To explore the function of ARHGAP11B in neocortex development, ARHGAP11B was expressed in the mouse neocortex by in-utero electroporation [18]. Expression of ARHGAP11B increased basal mitoses yet it has not caused an abundance in apical mitoses and basal progenitors. It also increased the subventricular zone thickness [14].

In contrast, overexpression of the gene ARHGAP11A in mouse neocortex did not result in the abundance of basal progenitors.

To analyze the effects of ARHGAP11B, they microinjected ARHGAP11B mRNA into a single aRG in an organotypic slice culture of the mouse neocortex [14].

After 24 hours, the same segment of aRG progeny was recognizable as identical cells upon control versus ARHGAP11B microinjection, indicating that ARHGAP11B did not affect aRG division as such. However, a greater percentage of aRG progeny showed immunoreactivity upon ARHGAP11B microinjection compared with control, impliying that ARHGAP11B promoted basal progenitor generation from aRG [14].

ARHGAP11B mRNA microinjection resulted in increased clone size of the aRG progeny. Compatible with this finding, ARHGAP11B electroporation (the use of high-voltage electric shocks to introduce DNA into cells) increased the proportion of cycling cells in the subventricular zone. With all of the results considered, this shows that ARHGAP11B promotes basal progenitor self-amplification [14]. 

At last, in half of the cases analyzed, ARHGAP11B expression in the lissencephalic mouse neocortex resulted in neocortex folding, evocative of gyrification and, quality of the human neocortex. Thus, they identified ARHGAP11B as a human-specific gene that boosts basal progenitors and is capable of causing neocortex folding in mice.

Figure 2: Neocortex of an embryonic mouse. ARHGAP11B was selectively expressed in the right half of the brain, which is visible by the folding of the neocortical surface [19].

This probably reflects a role for ARHGAP11B in the development and evolutionary expansion of the human neocortex [11], [12], [14].

With the same methodology used, other teams of scientists repeated the experiment on other animals for further knowledge about the human-specific gene and its effects.

Neocortical Expansion in Developing Ferret Neocortex

Three years forward, another group of scientists from the Max Planck Institute of Molecular Cell Biology and Genetics decided to repeat the experiment to observe the effect of ARHGAP11B on an animal -a carnivore- with an already gyrified neocortex: a ferret.

Using in utero electroporation [20], they expressed ARHGAP11B in the ferret neocortex starting at embryonic day 33, when the generation of upper layer neurons and the formation of the outer subventricular zone start [21,22].

The analyses of electroporated embryos were performed at four different developmental stages:

They detected ARHGAP11B transcript at all the stages analyzed and only in ferret embryos subjected to ARHGAP11B in-utero electroporation [21].

In addition to that, immunofluorescence staining at embryonic day 37 showed the specific presence of the ARHGAP11B protein in neural progenitors of the ferret embryos. They also observed an abundance of cells in the outer subventricular zone of the ARHGAP11B-expressing embryos compared with the control. The abundance of cells was increased in both the inner subventricular zone and outer subventricular zone, but this increase was especially strong in the outer subventricular zone [21].

These data altogether indicate that ARHGAP11B remarkably increases the abundance of basal progenitor cells in the embryonic ferret neocortex [21].

Increased Size and Folding of Primate Neocortex in the Fetal Marmoset

Over the years, overexpression studies of ARHGAP11B in developing mouse and ferret neocortex have implicated that the human-specific gene caused neocortical expansion, yet the relevance for primate evolution has not been studied. In this study, scientists have worked through ways to enlighten the human-specific ARHGAP11B gene’s role in human evolution [23].

As a result, ARHGAP11B expressed in the fetal neocortex of the marmoset under control of the gene’s own promoter increased the amount of basal radial glia progenitors in the marmoset outer subventricular zone, increased the numbers of upper-layer neurons, expanded the neocortex, and stimulated its folding.

Based on the results of the study, they conclude that the human-specific ARHGAP11B gene may have caused neocortical expansion in the course of human evolution [23].

References:

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Figure References:

  1. “Comparison of the relative volumes of neocortex and brain stem in mouse… | Download Scientific Diagram.” https://www.researchgate.net/figure/Comparison-of-the-relative-volumes-of-neocortex-and-brain-stem-in-mouse-Mus-musculus_fig1_266495421 (accessed Mar. 22, 2023).
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