What Makes Us Human: Insights from the Evolution and Development of the Human Neocortex.

"What makes us human?" is a central question of many research fields, notably anthropology. In this review, we focus on the development of the human neocortex, the part of the brain with a key role in cognition, to gain neurobiological insight toward answering this question. We first discuss cortical stem and progenitor cells and human-specific genes that affect their behavior. We thus aim to understand the molecular foundation of the expansion of the neocortex that occurred in the course of human evolution, as this expansion is generally thought to provide a basis for our unique cognitive abilities. We then review the emerging evidence pointing to differences in the development of the neocortex between present-day humans and Neanderthals, our closest relatives. Finally, we discuss human-specific genes that have been implicated in neuronal circuitry and offer a perspective for future studies addressing the question of what makes us human.

Role of cell metabolism in the pathophysiology of brain size-associated neurodevelopmental disorders.

Cell metabolism is a key regulator of human neocortex development and evolution. Several lines of evidence indicate that alterations in neural stem/progenitor cell (NPC) metabolism lead to abnormal brain development, particularly brain size-associated neurodevelopmental disorders, such as microcephaly. Abnormal NPC metabolism causes impaired cell proliferation and thus insufficient expansion of NPCs for neurogenesis. Therefore, the production of neurons, which is a major determinant of brain size, is decreased and the size of the brain, especially the size of the neocortex, is significantly reduced. This review discusses recent progress understanding NPC metabolism, focusing in particular on glucose metabolism, fatty acid metabolism and amino acid metabolism (e.g., glutaminolysis and serine metabolism). We provide an overview of the contributions of these metabolic pathways to brain development and evolution, as well as to the etiology of neurodevelopmental disorders. Furthermore, we discuss the advantages and disadvantages of various experimental models to study cell metabolism in the developing brain.

Functional synergy of a human-specific and an ape-specific metabolic regulator in human neocortex development.

Metabolism has recently emerged as a major target of genes implicated in the evolutionary expansion of human neocortex. One such gene is the human-specific gene ARHGAP11B. During human neocortex development, ARHGAP11B increases the abundance of basal radial glia, key progenitors for neocortex expansion, by stimulating glutaminolysis (glutamine-to-glutamate-to-alpha-ketoglutarate) in mitochondria. Here we show that the ape-specific protein GLUD2 (glutamate dehydrogenase 2), which also operates in mitochondria and converts glutamate-to-αKG, enhances ARHGAP11B's ability to increase basal radial glia abundance. ARHGAP11B + GLUD2 double-transgenic bRG show increased production of aspartate, a metabolite essential for cell proliferation, from glutamate via alpha-ketoglutarate and the TCA cycle. Hence, during human evolution, a human-specific gene exploited the existence of another gene that emerged during ape evolution, to increase, via concerted changes in metabolism, progenitor abundance and neocortex size.

High-resolution 3D ultrastructural analysis of developing mouse neocortex reveals long slender processes of endothelial cells that enter neural cells.

The development of the neocortex involves an interplay between neural cells and the vasculature. However, little is known about this interplay at the ultrastructural level. To gain a 3D insight into the ultrastructure of the developing neocortex, we have analyzed the embryonic mouse neocortex by serial block-face scanning electron microscopy (SBF-SEM). In this study, we report a first set of findings that focus on the interaction of blood vessels, notably endothelial tip cells (ETCs), and the neural cells in this tissue. A key observation was that the processes of ETCs, located either in the ventricular zone (VZ) or subventricular zone (SVZ)/intermediate zone (IZ), can enter, traverse the cytoplasm, and even exit via deep plasma membrane invaginations of the host cells, including apical progenitors (APs), basal progenitors (BPs), and newborn neurons. More than half of the ETC processes were found to enter the neural cells. Striking examples of this ETC process "invasion" were (i) protrusions of apical progenitors or newborn basal progenitors into the ventricular lumen that contained an ETC process inside and (ii) ETC process-containing protrusions of neurons that penetrated other neurons. Our observations reveal a - so far unknown - complexity of the ETC-neural cell interaction.

Development and evolution of the primate neocortex from a progenitor cell perspective.

The generation of neurons in the developing neocortex is a major determinant of neocortex size. Crucially, the increase in cortical neuron numbers in the primate lineage, notably in the upper-layer neurons, contributes to increased cognitive abilities. Here, we review major evolutionary changes affecting the apical progenitors in the ventricular zone and focus on the key germinal zone constituting the foundation of neocortical neurogenesis in primates, the outer subventricular zone (OSVZ). We summarize characteristic features of the OSVZ and its key stem cell type, the basal (or outer) radial glia. Next, we concentrate on primate-specific and human-specific genes, expressed in OSVZ-progenitors, the ability of which to amplify these progenitors by targeting the regulation of the cell cycle ultimately underlies the evolutionary increase in upper-layer neurons. Finally, we address likely differences in neocortical development between present-day humans and Neanderthals that are based on human-specific amino acid substitutions in proteins operating in cortical progenitors.

Neocortical neurogenesis in development and evolution-Human-specific features.

In this review, we focus on human-specific features of neocortical neurogenesis in development and evolution. Two distinct topics will be addressed. In the first section, we discuss the expansion of the neocortex during human evolution and concentrate on the human-specific gene ARHGAP11B. We review the ability of ARHGAP11B to amplify basal progenitors and to expand a primate neocortex. We discuss the contribution of ARHGAP11B to neocortex expansion during human evolution and its potential implications for neurodevelopmental disorders and brain tumors. We then review the action of ARHGAP11B in mitochondria as a regulator of basal progenitor metabolism, and how it promotes glutaminolysis and basal progenitor proliferation. Finally, we discuss the increase in cognitive performance due to the ARHGAP11B-induced neocortical expansion. In the second section, we focus on neocortical development in modern humans versus Neanderthals. Specifically, we discuss two recent findings pointing to differences in neocortical neurogenesis between these two hominins that are due to a small number of amino acid substitutions in certain key proteins. One set of such proteins are the kinetochore-associated proteins KIF18a and KNL1, where three modern human-specific amino acid substitutions underlie the prolongation of metaphase during apical progenitor mitosis. This prolongation in turn is associated with an increased fidelity of chromosome segregation to the apical progenitor progeny during modern human neocortical development, with implications for the proper formation of radial units. Another such key protein is transketolase-like 1 (TKTL1), where a single modern human-specific amino acid substitution endows TKTL1 with the ability to amplify basal radial glia, resulting in an increase in upper-layer neuron generation. TKTL1's ability is based on its action in the pentose phosphate pathway, resulting in increased fatty acid synthesis. The data imply greater neurogenesis during neocortical development in modern humans than Neanderthals due to TKTL1, in particular in the developing frontal lobe.

A role for the serotonin 2A receptor in the expansion and functioning of human transmodal cortex.

Integrating independent but converging lines of research on brain function and neurodevelopment across scales, this article proposes that serotonin 2A receptor (5-HT2AR) signalling is an evolutionary and developmental driver and potent modulator of the macroscale functional organization of the human cerebral cortex. A wealth of evidence indicates that the anatomical and functional organization of the cortex follows a unimodal-to-transmodal gradient. Situated at the apex of this processing hierarchy-where it plays a central role in the integrative processes underpinning complex, human-defining cognition-the transmodal cortex has disproportionately expanded across human development and evolution. Notably, the adult human transmodal cortex is especially rich in 5-HT2AR expression and recent evidence suggests that, during early brain development, 5-HT2AR signalling on neural progenitor cells stimulates their proliferation-a critical process for evolutionarily-relevant cortical expansion. Drawing on multimodal neuroimaging and cross-species investigations, we argue that, by contributing to the expansion of the human cortex and being prevalent at the apex of its hierarchy in the adult brain, 5-HT2AR signalling plays a major role in both human cortical expansion and functioning. Owing to its unique excitatory and downstream cellular effects, neuronal 5-HT2AR agonism promotes neuroplasticity, learning and cognitive and psychological flexibility in a context-(hyper)sensitive manner with therapeutic potential. Overall, we delineate a dual role of 5-HT2ARs in enabling both the expansion and modulation of the human transmodal cortex.

Targeted Microinjection and Electroporation of Primate Cerebral Organoids for Genetic Modification.

The cerebral cortex is the outermost brain structure and is responsible for the processing of sensory input and motor output; it is seen as the seat of higher-order cognitive abilities in mammals, in particular, primates. Studying gene functions in primate brains is challenging due to technical and ethical reasons, but the establishment of the brain organoid technology has enabled the study of brain development in traditional primate models (e.g., rhesus macaque and common marmoset), as well as in previously experimentally inaccessible primate species (e.g., great apes), in an ethically justifiable and less technically demanding system. Moreover, human brain organoids allow the advanced investigation of neurodevelopmental and neurological disorders. As brain organoids recapitulate many processes of brain development, they also represent a powerful tool to identify differences in, and to functionally compare, the genetic determinants underlying the brain development of various species in an evolutionary context. A great advantage of using organoids is the possibility to introduce genetic modifications, which permits the testing of gene functions. However, the introduction of such modifications is laborious and expensive. This paper describes a fast and cost-efficient approach to genetically modify cell populations within the ventricle-like structures of primate cerebral organoids, a subtype of brain organoids. This method combines a modified protocol for the reliable generation of cerebral organoids from human-, chimpanzee-, rhesus macaque-, and common marmoset-derived induced pluripotent stem cells (iPSCs) with a microinjection and electroporation approach. This provides an effective tool for the study of neurodevelopmental and evolutionary processes that can also be applied for disease modeling.

Response to Comment on "Human TKTL1 implies greater neurogenesis in frontal neocortex of modern humans than Neanderthals".

Herai et al. discuss the known fact that a low percentage of modern humans who lack any overt phenotypes carry the ancestral TKTL1 allele. Our paper demonstrates that the amino acid substitution in TKTL1 increases neural progenitor cells and neurogenesis in the developing brain. It is another question if, and to what extent, this has consequences for the adult brain.

Primary cilia are WNT-transducing organelles whose biogenesis is controlled by a WNT-PP1 axis.

WNT signaling is important in development, stem cell maintenance, and disease. WNT ligands typically signal via receptor activation across the plasma membrane to induce ß-catenin-dependent gene activation. Here, we show that in mammalian primary cilia, WNT receptors relay a WNT/GSK3 signal that ß-catenin-independently promotes ciliogenesis. Characterization of a LRP6 ciliary targeting sequence and monitoring of acute WNT co-receptor activation (phospho-LRP6) support this conclusion. Ciliary WNT signaling inhibits protein phosphatase 1 (PP1) activity, a negative regulator of ciliogenesis, by preventing GSK3-mediated phosphorylation of the PP1 regulatory inhibitor subunit PPP1R2. Concordantly, deficiency of WNT/GSK3 signaling by depletion of cyclin Y and cyclin-Y-like protein 1 induces primary cilia defects in mouse embryonic neuronal precursors, kidney proximal tubules, and adult mice preadipocytes.

Preventive herd management practices and their effect on lamb mortality in Ethiopia.

According to previous studies, lamb mortality is high in the Ethiopian highlands. The present study aims to evaluate the execution of preventive sheep herd health management practices with respect to if, and how, such practices are linked to occurrence of lamb mortality. Interviews were performed with 74 sheep-owning households participating in a capacity development program on livestock and 69 households not participating in such program. To evaluate the impact of combinations of performed practices, a scoring system was developed-the households retrieved a higher score the more desired routines were accomplished. To identify which practices had the highest impact on lamb mortality, a similar score was calculated for each phase of the sheep reproductive year, creating sub-scores for each phase. The results showed a significant (p < 0.05) negative correlation between the total number of performed practices and occurrence of lamb mortality, indicating a lower occurrence of lamb mortality the more desired practices implemented. Further analysis of sub-scores showed significant (p < 0.05) negative correlations between a higher number of performed desired practices during gestation period and during lambing. Conclusively, the study indicates that preventive herd management routines are beneficial for lamb survival, foremost when enforced during the gestation period and around lambing-hence, this is where to focus future interventions.

Human TKTL1 implies greater neurogenesis in frontal neocortex of modern humans than Neanderthals.

Neanderthal brains were similar in size to those of modern humans. We sought to investigate potential differences in neurogenesis during neocortex development. Modern human transketolase-like 1 (TKTL1) differs from Neanderthal TKTL1 by a lysine-to-arginine amino acid substitution. Using overexpression in developing mouse and ferret neocortex, knockout in fetal human neocortical tissue, and genome-edited cerebral organoids, we found that the modern human variant, hTKTL1, but not the Neanderthal variant, increases the abundance of basal radial glia (bRG) but not that of intermediate progenitors (bIPs). bRG generate more neocortical neurons than bIPs. The hTKTL1 effect requires the pentose phosphate pathway and fatty acid synthesis. Inhibition of these metabolic pathways reduces bRG abundance in fetal human neocortical tissue. Our data suggest that neocortical neurogenesis in modern humans differs from that in Neanderthals.

Human-specific ARHGAP11B ensures human-like basal progenitor levels in hominid cerebral organoids.

The human-specific gene ARHGAP11B has been implicated in human neocortex expansion. However, the extent of ARHGAP11B's contribution to this expansion during hominid evolution is unknown. Here we address this issue by genetic manipulation of ARHGAP11B levels and function in chimpanzee and human cerebral organoids. ARHGAP11B expression in chimpanzee cerebral organoids doubles basal progenitor levels, the class of cortical progenitors with a key role in neocortex expansion. Conversely, interference with ARHGAP11B's function in human cerebral organoids decreases basal progenitors down to the chimpanzee level. Moreover, ARHGAP11A or ARHGAP11B rescue experiments in ARHGAP11A plus ARHGAP11B double-knockout human forebrain organoids indicate that lack of ARHGAP11B, but not of ARHGAP11A, decreases the abundance of basal radial glia-the basal progenitor type thought to be of particular relevance for neocortex expansion. Taken together, our findings demonstrate that ARHGAP11B is necessary and sufficient to ensure the elevated basal progenitor levels that characterize the fetal human neocortex, suggesting that this human-specific gene was a major contributor to neocortex expansion during human evolution.

The relationship between external and internal load parameters in 3 × 3 basketball tournaments.

PURPOSE: 3 × 3 basketball games are characterized by high-intensity accelerations and decelerations, and a high number of changes of direction and jumps. It is played in tournament form with multiple games per day. Therefore, optimal regeneration is crucial for maintaining a high performance level over the course of the tournament. To elucidate how load of a match affects the athletes' bodies (i.e., internal load), muscular responses to the load of 3 × 3 games were analyzed. We aimed to investigate changes in contractility of the m. rectus femoris (RF) and m. gastrocnemius medialis (GC) in response to the load of single 3 × 3 games and a 3 × 3 tournament. METHODS: Inertial movement analysis was conducted to capture game load in 3 × 3. Changes in contractility were measured using tensiomyography (TMG). During a two-day tournament, TMG measurements were conducted in the morning and after each game. Additionally, off-game performance analysis consisting of jump and change-of-direction (COD) tests was conducted the day before the tournament. RESULTS: Significant changes of the muscle contractility were found for GC with TMG values being higher in the baseline than in the post-game measurements. In contrast to athletes of the GC group, athletes of the RF group responded with either decreased or increased muscle contractility after a single 3 × 3 game. A significant correlation between external and internal load parameters could not be shown. Concerning off-game performance, significant correlations can be reported for COD test duration, CMJ height and ∆Vc as well as COD test duration and ∆Dm. No systematic changes in muscle contractility were found over the course of the tournament in RF and GC. CONCLUSION: The athletes' external 3 × 3 game load and their performance level did not seem to affect muscular contractility after a single 3 × 3 game or a complete 3 × 3 tournament within this investigation. This might indicate that elite athletes can resist external load without relevant local muscular fatigue. With respect to the course of the tournament, it can therefore be concluded that the breaks between games seem to be sufficient to return to the initial level of muscle contractility.

Longer metaphase and fewer chromosome segregation errors in modern human than Neanderthal brain development.

Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals.

What Are the Human-Specific Aspects of Neocortex Development?

When considering what makes us human, the development of the neocortex, the seat of our higher cognitive abilities, is of central importance. Throughout this complex developmental process, neocortical stem and progenitor cells (NSPCs) exert a priming role in determining neocortical tissue fate, through a series of cellular and molecular events. In this Perspective article, we address five questions of relevance for potentially human-specific aspects of NSPCs, (i) Are there human-specific NSPC subtypes? (ii) What is the functional significance of the known temporal differences in NSPC dynamics between human and other great apes? (iii) Are there functional interactions between the human-specific genes preferentially expressed in NSPCs? (iv) Do humans amplify certain metabolic pathways for NSPC proliferation? and finally (v) Have differences evolved during human evolution, notably between modern humans and Neandertals, that affect the performance of key genes operating in NSPCs? We discuss potential implications inherent to these questions, and suggest experimental approaches on how to answer them, hoping to provide incentives to further understand key issues of human cortical development.

Progenitor-Based Cell Biological Aspects of Neocortex Development and Evolution.

During development, the decision of stem and progenitor cells to switch from proliferation to differentiation is of critical importance for the overall size of an organ. Too early a switch will deplete the stem/progenitor cell pool, and too late a switch will not generate the required differentiated cell types. With a focus on the developing neocortex, a six-layered structure constituting the major part of the cerebral cortex in mammals, we discuss here the cell biological features that are crucial to ensure the appropriate proliferation vs. differentiation decision in the neural progenitor cells. In the last two decades, the neural progenitor cells giving rise to the diverse types of neurons that function in the neocortex have been intensely investigated for their role in cortical expansion and gyrification. In this review, we will first describe these different progenitor types and their diversity. We will then review the various cell biological features associated with the cell fate decisions of these progenitor cells, with emphasis on the role of the radial processes emanating from these progenitor cells. We will also discuss the species-specific differences in these cell biological features that have allowed for the evolutionary expansion of the neocortex in humans. Finally, we will discuss the emerging role of cell cycle parameters in neocortical expansion.

From stem and progenitor cells to neurons in the developing neocortex: key differences among hominids.

Comparing the biology of humans to that of other primates, and notably other hominids, is a useful path to learn more about what makes us human. Some of the most interesting differences among hominids are closely related to brain development and function, for example behaviour and cognition. This makes it particularly interesting to compare the hominid neural cells of the neocortex, a part of the brain that plays central roles in those processes. However, well-preserved tissue from great apes is usually extremely difficult to obtain. A variety of new alternative tools, for example brain organoids, are now beginning to make it possible to search for such differences and analyse their potential biological and biomedical meaning. Here, we present an overview of recent findings from comparisons of the neural stem and progenitor cells (NSPCs) and neurons of hominids. In addition to differences in proliferation and differentiation of NSPCs, and maturation of neurons, we highlight that the regulation of the timing of these processes is emerging as a general foundational difference in the development of the neocortex of hominids.

Primary Cilia and Centrosomes in Neocortex Development.

During mammalian brain development, neural stem and progenitor cells generate the neurons for the six-layered neocortex. The proliferative capacity of the different types of progenitor cells within the germinal zones of the developing neocortex is a major determinant for the number of neurons generated. Furthermore, the various modes of progenitor cell divisions, for which the orientation of the mitotic spindle of progenitor cells has a pivotal role, are a key parameter to ensure the appropriate size and proper cytoarchitecture of the neocortex. Here, we review the roles of primary cilia and centrosomes of progenitor cells in these processes during neocortical development. We specifically focus on the apical progenitor cells in the ventricular zone. In particular, we address the alternating, dual role of the mother centriole (i) as a component of one of the spindle poles during mitosis, and (ii) as the basal body of the primary cilium in interphase, which is pivotal for the fate of apical progenitor cells and their proliferative capacity. We also discuss the interactions of these organelles with the microtubule and actin cytoskeleton, and with junctional complexes. Centriolar appendages have a specific role in this interaction with the cell cortex and the plasma membrane. Another topic of this review is the specific molecular composition of the ciliary membrane and the membrane vesicle traffic to the primary cilium of apical progenitors, which underlie the ciliary signaling during neocortical development; this signaling itself, however, is not covered in depth here. We also discuss the recently emerging evidence regarding the composition and roles of primary cilia and centrosomes in basal progenitors, a class of progenitors thought to be of particular importance for neocortex expansion in development and evolution. While the tight interplay between primary cilia and centrosomes makes it difficult to allocate independent roles to either organelle, mutations in genes encoding ciliary and/or centrosome proteins indicate that both are necessary for the formation of a properly sized and functioning neocortex during development. Human neocortical malformations, like microcephaly, underpin the importance of primary cilia/centrosome-related processes in neocortical development and provide fundamental insight into the underlying mechanisms involved.