The question of what makes modern humans unique has long been a driving force for researchers. Comparisons with our closest relatives, the Neandertals, therefore provide fascinating insights. The increase in brain size, and in neuron production during brain development, are considered to be major factors for the increased cognitive abilities that occurred during human evolution. However, while both Neandertals and modern humans develop brains of similar size, very little is known about whether modern human and Neandertal brains may have differed in terms of their neuron production during development. Researchers from the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden now show that the modern human variant of the protein TKTL1, which differs by only a single amino acid from the Neandertal variant, increases one type of brain progenitor cells, called basal radial glia, in the modern human brain. Basal radial glial cells generate the majority of the neurons in the developing neocortex, a part of the brain that is crucial for many cognitive abilities. As TKTL1 activity is particularly high in the frontal lobe of the fetal human brain, the researchers conclude that this single human-specific amino acid substitution in TKTL1 underlies a greater neuron production in the developing frontal lobe of the neocortex in modern humans than Neandertals.

Only a small number of proteins have differences in the sequence of their amino acids – the building blocks of proteins – between modern humans and our extinct relatives, the Neandertals and Denisovans. The biological significance of these differences for the development of the modern human brain is largely unknown. In fact, both, modern humans and Neandertals, feature a brain, and notably a neocortex, of similar size, but whether this similar neocortex size implies a similar number of neurons remains unclear. The latest study of the research group of Wieland Huttner, one of the founding directors of the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden, carried out in collaboration with Svante Pääbo, director at the Max Planck Institute for Evolutionary Anthropology in Leipzig, and Pauline Wimberger of the University Hospital Dresden and their colleagues, addresses just this question. The researchers focus on one of these proteins that presents a single amino acid change in essentially all modern humans compared to Neandertals, the protein transketolase-like 1 (TKTL1). Specifically, in modern humans TKTL1 contains an arginine at the sequence position in question, whereas in Neandertal TKTL1 it is the related amino acid lysine. In the fetal human neocortex, TKTL1 is found in neocortical progenitor cells, the cells from which all cortical neurons derive. Notably, the level of TKTL1 is highest in the progenitor cells of the frontal lobe.

Microscopy picture of a dividing basal radial glial cell, a progenitor cell type that generates neurons during brain development. Modern human TKTL1, but not Neandertal TKTL1, increases basal radial glia and neuron abundance. © Pinson et al., Science 2022 / MPI-CBG

Modern human TKTL1, but not Neandertal TKTL1, leads to more neurons in embryonic mouse neocortex

Anneline Pinson, the lead author of the study and researcher in the group of Wieland Huttner, set out to investigate the significance of this one amino acid change for neocortex development. Anneline and her colleagues introduced either the modern human or the Neandertal variant of TKTL1 into the neocortex of mouse embryos. They observed that basal radial glial cells, the type of neocortical progenitors thought to be the driving force for a bigger brain, increased with the modern human variant of TKTL1 but not with the Neandertal variant. As a consequence, the brains of mouse embryos with the modern human TKTL1 contained more neurons.

More neurons in the frontal lobe of modern humans

After this, the researchers explored the relevance of these effects for human brain development. To this end, they replaced the arginine in modern human TKTL1 with the lysine characteristic of Neandertal TKTL1, using human brain organoids – miniature organ-like structures that can be grown from human stem cells in cell culture dishes in the lab and that mimic aspects of early human brain development. “We found that with the Neandertal-type of amino acid in TKTL1, fewer basal radial glial cells were produced than with the modern human-type and, as a consequence, also fewer neurons,” says Anneline Pinson. “This shows us that even though we do not know how many neurons the Neandertal brain had, we can assume that modern humans have more neurons in the frontal lobe of the brain, where TKTL1 activity is highest, than Neandertals.” The researchers also found that modern human TKTL1 acts through changes in metabolism, specifically a stimulation of the pentose phosphate pathway followed by increased fatty acid synthesis. In this way, modern human TKTL1 is thought to increase the synthesis of certain membrane lipids needed to generate the long process of basal radial glial cells that stimulates their proliferation and, therefore, to increase neuron production.

“This study implies that the production of neurons in the neocortex during fetal development is greater in modern humans than it was in Neandertals, in particular in the frontal lobe,” summarizes Wieland Huttner, who supervised the study. “It is tempting to speculate that this promoted modern human cognitive abilities associated with the frontal lobe.”

Anneline Pinson, Lei Xing, Takashi Namba, Nereo Kalebic, Jula Peters, Christina Eugster Oegema, Sofia Traikov, Katrin Reppe, Stephan Riesenberg, Tomislav Maricic, Razvan Derihaci, Pauline Wimberger, Svante Pääbo, Wieland B Huttner: “Human TKTL1 implies greater neurogenesis in frontal neocortex of modern humans than Neandertals”, Science. 09. September 2022. Link

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For successful organ formation, it is very important that cells generate unique shapes and architecture from which these organs develop. Also during the development of the vertebrate eye, cells need to actively migrate to the correct locations to generate the right tissue shape. The lab of Caren Norden at the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) showed that such cell movements are crucial already at early developmental stages. If cells do not reach the right place at the right time, all further eye development is impaired.

Organogenesis depends on the orchestrated interplay of molecular and cellular changes as well as overall tissue rearrangements. To understand how this is regulated at these different scales and how cellular and tissue behaviour feed back onto each other is a main interest of Caren Norden’s lab at the MPI-CBG. To reach this goal the group uses the small zebrafish as it has the advantage of fast development and unmatched imaging possibility due to the fact that the larvae are translucent. This means that developmental processes can be observed while they happen where they happen in the live embryo.

The lab is particularly interested to understand how the vertebrate eye forms during development. A recent project focused on the emergence of the optic cup. From this early developmental structure the later retina develops. This cup, like the future retina, has a hemispheric architecture. So far, it was assumed that the hemispheric shape is reached by cells constricting and pulling additional cells into the cup. The Norden group however showed that mere passive pulling is not enough to get a sufficient amount of cells into the cup but that instead a significant amount of cells undergoes active migration. They further identified the molecular mechanisms that drive this cell migration. When this cell migration is impaired, the optic cup does not acquire its hemispheric shape, but instead of a c-shaped cup an s-shaped cup is formed. This can lead to a massive coloboma and in extreme cases even secondary lens formation.

This study is a prime example of what Caren Norden’s group is very excited about. To understand how the right cells reach the right place in development is one of the core questions in diverse projects in the group. Cell movements play a crucial role in many developmental contexts in diverse examples of organ formation so that understanding how cells get where they later function is one of the important questions to ask when one wants to understand development of organisms including humans.

Original Publication:
Jaydeep Sidhaye, Caren Norden:
Concerted action of neuroepithelial basal shrinkage and active epithelial migration ensures efficient optic cup morphogenesis
eLife, 4 April 2017
https://elifesciences.org/content/6/e22689

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What distinguishes humans from monkeys and apes? The gene ARHGAP11B is probably among the things that make humans special: This gene is only present in humans and contributes to the amplification of brain stem cells. Researchers at the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) have now made a spectacular finding: It is a single base pair substitution in the ARHGAP11B gene that ultimately is responsible for the ability of the ARHGAP11B protein to amplify brain stem cells, a process thought to underlie the expansion of the neocortex in modern humans.

During evolution, the human genome has undergone subtle changes that underlie the expansion of a particular region of the brain called the neocortex. This part of the brain is responsible for cognitive functions like speaking and thinking. Wieland Huttner, MPI-CBG Research Group Leader and Director, and his team have recently shown that the gene ARHGAP11B is key to the regulation of brain size. This gene is only found in humans and in our closest relatives, the Neanderthals and Denisova-Humans, but not in chimpanzees, as collaborator Svante Pääbo from the Max Planck Institute of Evolutionary Anthropology in Leipzig has shown. Moreover, ARHGAP11B induces an increase in a specific subpopulation of brain stem cells called basal progenitors, which have been implicated in neocortex expansion, and can trigger folding of the neocortex in mouse.

C becomes G – a tiny change that makes a huge difference

The human-specific gene ARHGAP11B arose through a partial duplication of the ubiquitous gene ARHGAP11A approximately five million years ago along the evolutionary lineage leading to Neanderthals, Denisovans, and present-day humans, and after this lineage had segregated from that leading to the chimpanzee. However, the big leap in neocortex expansion began later, about two million years ago. How, then, could ARHGAP11B possibly be a key player in increasing neocortex size, researchers wondered.

There is more to ARHGAP11B that makes it unique: Not only is the gene as such human-specific, but the protein encoded by the gene contains a sequence of 47 amino acids that is only found in humans and that is due to a shift in the reading frame caused by the absence of 55 nucleotides in the ARHGAP11B messenger RNA.

The startling discovery

Wieland Huttner and Marta Florio, PhD student in Huttner’s group, first thought that the absence of these 55 nucleotides would go back to when ARHGAP11B arose by partial gene duplication five million years ago. This is when they got lost, the researchers assumed. But then they realized, to their astonishment, that the stretch of 55 nucleotides is indeed present in the ARHGAP11B DNA and only disappears when the messenger RNA of ARHGAP11B is produced – they are spliced out. This is triggered by a single C-to-G base substitution in the ARHGAP11B gene. A really tiny change at the molecular level, given that the human genome consists of more than three billion base pairs. The consequences, however, are not tiny at all: The single mutation leads to eliminating the 55 nucleotides from the ARHGAP11B messenger RNA, which in turn leads to the 47 amino acid sequence in the ARHGAP11B protein that is human-specific – leading to an increase in the abundance of basal progenitors, thought to be key to the evolutionary expansion of the human neocortex.

Reconstruction of an ancestral version of the gene

The C-to-G base substitution in ARHGAP11B probably happened much later than when this gene arose about 5 million years ago, anytime between 1.5 million and 500,000 years ago. The Dresden researchers wanted to cross-check the significance of this base substitution for the function of the ARHGAP11B protein, and therefore reconstructed an ancestral version of the ARHGAP11B gene as it likely arose approximately 5 million years ago – without the single base pair substitution. Remarkably, when expressed in mice, the ancestral ARHGAP11B protein did not lead to an increase in the abundance of basal progenitors.

Implications of the results

The finding that a mere single point mutation may have causally contributed to the expansion of the neocortex is remarkable. Wieland Huttner says: “This change is tiny on a genomic scale but substantial in its functional and evolutionary consequences – it’s a single base substitution that likely drove brain size evolution and that may have set the stage for what makes humans special.” Point mutations are not rare, but in the case of ARHGAP11B its advantages seem to have immediately influenced human evolution.

Original Publication:
Marta Florio, Takashi Namba, Svante Pääbo, Michael Hiller, Wieland B. Huttner:
A single splice site mutation in human-specific ARHGAP11B causes basal progenitor amplification
Science Advances, 7 December 2016
http://advances.sciencemag.org/content/2/12/e1601941

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The retina consists of different layers of neurons. Emerging neurons have to migrate to find the right position in the appropriate layer. However, how these migration events are executed and orchestrated is poorly understood. Researchers at the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden now studied how the first layer of neurons, which later forms the optic nerve, is established. They revealed that retinal ganglion cells migrate across the retina and then set the stage for the formation of other retinal layers. The research team could also show that if retinal ganglion cells cannot migrate correctly, all following neuronal layer formation is perturbed.

To understand how neuronal migration works in the retina, the group of Caren Norden used the zebrafish as a model system of choice. This is due ti its small size and the fact that the embryos are transparent. Both facts make this system suitable for observing tissue in live samples under the microscope. The structure of the retina is the same in all vertebrates – fish and humans share the same principles of retina development. The team performed the experiments using Light Sheet Fluorescence Microscopy, which made the imaging of neurons in live fish much easier and reduces light-induced stress.

The team of researchers followed emerging retinal ganglion cells and revealed that the migration of these cells can be divided into two phases: First, cells move in a fast and persistent manner for almost 2 hours. In the second phase, they switch to a fine positioning with slower and more random movements for another 3 hours. The migration of retinal ganglion cells and the formation of the first layer of neurons is a crucial first step – if the cells fail to migrate, the entire development of the retina is disrupted.

Original Publication:
Jaroslav Icha, Christiane Kunath, Mauricio Rocha‐Martins, and Caren Norden:
Independent modes of ganglion cell translocation ensure correct lamination of the zebrafish retina
Journal of Cell Biology, 24 October 2016
doi: 10.1083/jcb.201604095
http://jcb.rupress.org/content/215/2/259

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When the right gene is expressed in the right manner in the right population of stem cells, the developing mouse brain can exhibit primate-like features. Researchers at the Max Planck Institute of Molecular Cell Biology and Genetics succeeded in mimicking the sustained expression of the transcription factor Pax6 as seen in the developing human brain, in mouse cortical progenitor cells. This altered the behavior of these cells to one that is akin to that of progenitors in the developing primate neocortex. Consequently, the mouse progenitors generated more neurons – a prerequisite for a bigger brain.

The developing neocortex contains different types of neural stem and progenitor cells, but one particular class, the basal progenitors, behave differently in small-brained animals such as mice than in large-brained animals such as humans. In humans, basal progenitors can undergo multiple rounds of cell division, thereby substantially increasing neuron number and ultimately the size of the neocortex. In mice, these progenitors typically undergo only one round of cell division, thus limiting the number of neurons produced. A potential cause underlying this difference in the proliferative capacity of basal progenitors could be the differential expression of Pax6 between species. Mouse basal progenitors, in contrast to human, do not express Pax6. “We were very curious to see what would happen if we were to change the expression pattern of Pax6 in developing mouse brain to mimic that observed in large-brained animals”, says Fong Kuan Wong, a PhD student in the lab of Wieland Huttner and first author of the study.

To this end, another PhD student in the lab, Ji-Feng Fei, generated a novel transgenic mouse line. This line provided the basis for altering the expression of Pax6 in the cortical stem cell lineage such that it would be sustained in basal progenitors. The researchers then introduced the Pax6 gene into the stem cells of these mouse embryos. Strikingly, sustaining Pax6 expression in mouse basal progenitors increased their capacity to undergo multiple rounds of cell division, as typically observed in primates. This not only expanded the size of the basal progenitor population in a way somewhat reminiscent to what is seen in large-brained animals. It also resulted in an increase in cortical neurons, notably those in the top layer, another characteristic feature of an expanded neocortex.

“The evolutionary expansion of the neocortex is a hallmark of species with higher cognitive functions. Our findings contribute to our understanding of the molecular mechanisms underlying this expansion”, explains Wieland Huttner, the research group leader and director at the MPI-CBG. While the findings demonstrate how altering the expression of a single key gene can make a big difference to brain development, a future challenge will be to obtain a comprehensive, integrated view of all the molecular changes that made our brains big.

Original Publication:
Fong Kuan Wong, Ji-Feng Fei, Felipe Mora-Bermudez, Elena Taverna, Christiane Haffner, Jun Fu, Konstantinos Anastassiadis, A. Francis Stewart & Wieland B. Huttner
Sustained Pax6 expression generates primate-like basal radial glia in developing mouse neocortex
PLOS Biology (2015), August 6, 2015

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About 99 percent of human genes are shared with chimpanzees. Only the small remainder sets us apart. However, we have one important difference: The brain of humans is three times as big as the chimpanzee brain. During evolution our genome must have changed in order to trigger such brain growth. Wieland Huttner, Director and Research Group Leader a the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), and his team identified for the first time a gene that is only present in humans and contributes to the reproduction of basal brain stem cells, triggering a folding of the neocortex. The researchers isolated different subpopulations of human brain stem cells and precisely identified, which genes are active in which cell type. In doing so, they noticed the gene ARHGAP11B: it is only found in humans and in our closest relatives, the Neanderthals and Denisova-Humans, but not in chimpanzees. This gene manages to trigger brain stem cells to form a bigger pool of stem cells. In that way, during brain development more neurons can arise and the cerebrum can expand. The cerebrum is responsible for cognitive functions like speaking and thinking.

Wieland Huttner’s researchers developed a method that isolates and identifies special subpopulations of brain stem cells from the developing human cerebrum. No one has managed to do this so far. The scientists first isolated different stem and progenitor cell types from fetal mice and human cerebrum tissue. In contrast to the big and folded human brain, the brain of mice is small and smooth. After the isolation, the researchers compared the genes that are active in the various cell types and were able to identify 56 genes that are only present in humans and which play a role in brain development. “We noticed that the gene ARHGAP11B is especially active in basal brain stem cells. These cells are really important for the expansion of the neocortex during evolution,” says Marta Florio, PhD student in Wieland Huttner’s lab, who carried out the main part of the study.

The human-specific gene also works in mice

In the further course of the study, the researchers focused on the function of this special gene. The researchers suspected that if it was responsible for a bigger pool of brain stem cells in humans and thereby for an expanded cerebrum, then this human-specific gene should trigger a similar development in the smaller brain of a mouse. They introduced the gene into mice embryos and indeed: Under the influence of the human-specific gene, the mice produced significantly more brain stem cells and in half of all cases even a folding of the neocortex, which is typical for human brains. All these results suggest that the gene ARHGAP11B plays a key role in the evolutionary expansion of the human neocortex.

Data from researchers working with Svante Pääbo from the Max Planck Institute for Evolutionary Anthropology in Leipzig confirm that ARHGAP11B not only occurs in the human genome, but also existed in the Neanderthals and Denisova-Humans. Neanderthals had a similar big brain to humans. “ARHGAP11B is the first human-specific gene where we could show that it contributes to the pool of basal brain stem cells and can trigger a folding of the neocortex. In that way, we managed to take the next step in tracing evolution”, summarizes Wieland Huttner.

His research group has been interested in the secrets of human brain evolution for a long time. In the last years, his researchers made several discoveries that contributed to the understanding of how a big brain could develop during evolution. In the year 2010 for example, the researchers identified a new type of stem cell in the outer growth zones of the brain. The current project of Wieland Huttner and his team was performed together with Andreas Dahl from the DFG Research Center for Regenerative Therapies Dresden and Robert Lachmann from the University Hospital Carl Gustav Carus of the Dresden University of Technology.

Original Publication:
Marta Florio, Mareike Albert, Elena Taverna, Takashi Namba, Holger Brandl, Eric Lewitus, Christiane Haffner, Alex Sykes, Fong Kuan Wong, Jula Peters, Elaine Guhr, Sylvia Klemroth, Kay Prüfer, Janet Kelso, Ronald Naumann, Ina Nüsslein, Andreas Dahl, Robert Lachmann, Svante Pääbo, Wieland B. Huttner
Human-specific gene ARHGAP11B promotes basal progenitor amplification and neocortex expansion
Science (2015), February 26, 2015

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