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Cellular and Molecular Explorations of Anthropogeny
Friday, September 29, 2017Abstracts
Abstracts
Understanding the evolutionary mechanisms underlying expansion and reorganization of the human brain is essential to comprehend the emergence of the cognitive abilities typical of our species. Comparative analyses of neuronal phenotypes in closely related species (Homo sapiens; human, Pan troglodytes; chimpanzees and Pan paniscus; bonobos) can shed light onto neuronal changes occurring during evolution, the timing of their appearance and the role of evolutionary mechanisms favoring a particular type of cortical organization in humans. The availability of post-mortem brains of endangered primates is limited and often does not represent important species-specific developmental hallmarks. We used induced pluripotent stem cell (iPSC) technology to model neural progenitor cell and neurons both functionally and genomically. This presentation provides a cellular and molecular analysis of comparative neural development in closely related hominids. The strategy proposed here lays the groundwork for further comparative analysis between human and non-human primates and opens new avenues for understanding the differences in the neural underpinnings of cognition and neurological disease susceptibility between species.
Variations in cerebral cortex size and complexity are thought to contribute to differences in cognitive ability between humans and other animals. We are using primate stem cell systems to understand the cellular and molecular mechanisms underlying species differences in cerebral cortex development. Directed differentiation to cerebral cortex of human and non-human primates pluripotent stem cells (PSCs) in adherent two-dimensional (2D) and organoid three-dimensional (3D) culture systems enables comparative studies of neurogenesis in different species, including chimpanzee and macaque. Using these approaches, we have identified mechanisms for controlling cerebral cortex size that are regulated cell-autonomously, suggesting that primate cerebral cortex size is regulated at least in part at the level of individual cortical progenitor cell clonal output. We are using a range of methods, including single cell RNA sequencing and comparative genomics, to identify candidate genes and pathways that regulate the output of each cortical progenitor cell, and thus contribute to species differences in brain size.
The expansion of the human neocortex, which constitutes a basis for our cognitive abilities, is due to an increased abundance and proliferative capacity of neural stem and progenitor cells (NPCs) during fetal cortical development. Specifically, NPCs in a secondary germinal zone called the subventricular zone, referred to as basal progenitors (BPs), are thought to be key for neocortex expansion. Here, a particularly important role has been attributed to one specific BP type called basal (or outer) radial glia (bRG).
In search for human-specific genome changes underlying neocortex expansion, we have found that the human-specific gene ARHGAP11B is specifically expressed in bRG and the NPCs from which bRG are derived. When expressed in embryonic mouse neocortex, ARHGAP11B causes amplification of BPs and is able to induce folding of the embryonic mouse neocortex, which is normally smooth. The ability of ARHGAP11B to amplify BPs is based on a single C-to-G base substitution that, due to a reading-frame shift resulting from altered mRNA splicing, is ultimately responsible for the appearance of a novel, human-specific 47-amino acid sequence in ARHGAP11B that is thought to be essential for BP amplification.
The developing human brain contains a huge number of cells whose identities have not yet been fully explored but whose specific molecular and functional features lead to the development of human specific abilities. We are using single cell approaches to establish an integrative definition of cell types in the developing human neocortex. Our single cell genomics analysis has revealed the molecular identity of a key human progenitor cell type, termed an outer radial glia cell (oRG). The developing human cortex contains a massively expanded progenitor region that is enriched in oRG cells that are thought to contribute to the developmental and evolutionary increase in cortical size and complexity of the human brain. We sequenced mRNA from single human progenitor cells and found that oRG cells preferentially express genes involved in growth factor signaling and self-renewal pathways, suggesting that oRG cells establish a self-sustaining proliferative niche. Using single cell clonal lineage analysis, we found that oRG cells can generate hundreds of daughter neurons, establishing the extensive proliferative and neurogenic capacity of this cell type. Surprisingly, we also discovered that the mTOR signaling pathway, known to promote cell growth and proliferation in a wide variety of cell types, is selectively active in oRG cells. This finding highlights a previously unappreciated cellular pattern of selective vulnerability and may have implications for our understanding of human diseases associated with MTOR pathway mutations, such as autism.
By using novel markers that reveal the morphology of radial glia cell subtypes, we discovered that the radial glial scaffold, which has classically been viewed as a continuous structural framework upon which the cortex develops, becomes a discontinuous pathway entirely constructed by oRG cell fibers. This transformation occurs partway through human brain development and results in a different lineage for upper cortical layer neurons compared to deeper layer neurons. This developmental event may underlie primate-specific features of upper cortical layer neurons that have been related to higher cognitive functions in humans.
Together, our results highlight cellular features of human brain development that are not represented in animal models and may reflect human or primate-specific evolutionary adaptations. These findings also provide a roadmap for interpreting laboratory models of human brain development and evolution.
From Galapagos finches to anteaters, the remarkable diversity of craniofacial structures within the vertebrate species is a testament to the plasticity of development and resourcefulness of evolution. While craniofacial development requires interactions between multiple embryonic cell types, Cranial Neural Crest Cells (CNCCs) play a major role in establishing the central plan of facial morphology as well as determining its species-specific variation. I will discuss an approach we termed ‘cellular anthropology’ in which in vitro differentiation models using hominid iPSCs can be effectively applied to studies of both basic physiology as well as evolutionary questions. Specifically, I will focus on using cellular anthropology to understand how sequence variation in human CNCC regulatory elements produce quantitative and cell type-restricted transcriptional changes that can mediate morphological evolution and individual variation of the craniofacial form.
Multiple Genomic Events Altering Hominin Sialic Acid Biology Predated the Common Ancestor of Humans and Neanderthals
Naazneen Khan1, Stevan Springer1, Marc de Manuel Montero2, Stephane Peyrégne3, Kay Prüfer3, Tomas Marques-Bonet2, Pascal Gagneux1 and Ajit Varki1
1Center for Academic Research and Training in Anthropogeny (CARTA), Glycobiology Research and Training Center (GRTC), Departments of Medicine, Pathology, Cellular & Molecular Medicine and Anthropology, UC San Diego, La Jolla CA, USA.
2Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Baldiri i Reixac 4, Barcelona, Spain.
3Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany.
Using immunochemical approaches (Sarich & Wilson), and then protein sequencing (Goodman, Doolittle and others) it was conclusively shown by the 1970s that chimpanzees were our closest living evolutionary cousins. For the next two decades, the popular assumption (King & Wilson, 1975) was that humans and chimpanzees differed primarily in terms of expression of otherwise nearly identical genes and proteins. It was then suggested by Olson that gene loss might also be an engine of evolutionary change (“Less is More”). Contemporaneous with this suggestion came the first report of a human-specific and human-universal gene loss resulting in a clear structural and biochemical difference between humans and chimpanzees: pseudogenization of the CMAH gene, which resulted in loss of the common mammalian sialic acid (Sia) called Neu5Gc, and an excess of the precursor Sia called Neu5Ac. As sialic acids can have highly diverse presentations and are displayed at >100mM concentrations on most cell surfaces, this event constituted a major change in many aspects of biology, including “self-associated molecular patterns”, which are known to modulate innate immune cells via engagement of CD33-related Siglec receptors. This Alu-mediated mutation appears to have been fixed ~2-3 million years ago and may have contributed to the origin of the genus Homo. We now know that gene expression changes and gene loss are not the only mechanisms involved in human evolution, and (perhaps not surprisingly, in retrospect), all possible genomic mechanisms are operative.
In this regard, human genomic events altering >10 other genes involved in Sia biology have been discovered, which also appeared to be human-specific, i.e., not found in then available limited chimpanzee genomic data––suggesting that Sia biology might represent a "hotspot" in hominin evolution. However, the possibility of ascertainment bias due to the number and quality of human genomes compared with those of other hominids could not be ruled out. Availability of many more hominid genomes including ancient DNA of extinct hominins now allows fresh analysis, and better evolutionary timing of these events. All known human genomic changes in CD33-related SIGLEC gene cluster (affecting 8 of 13 members of this class of genes) are found in African populations, indicating that they predated the common ancestor of modern humans. Comparisons with 147 “great ape” genomes indicate that all these changes are indeed unique to hominins. There was no evidence for strong selection after the Human-Neanderthal/Denisovan common ancestor, ~500 kya. Consistent with these observations, Neanderthal and Denisovan genomes include almost all the major changes found in modern humans. Genome-level analyses can miss domain-specific rapid evolution within functional regions of specific proteins. Indeed, Sia-binding domains of CD33-related Siglecs prominent on innate immune cells harbor higher rates of evolution relative to those of the other hominids and of the adjacent/underlying/more proximal structural C2-set domains. The CMAH mutation likely induced changes in self-associated molecular patterns, setting in motion multiple events, including emergence of human-specific pathogens that coat themselves with Neu5Ac-containing molecular mimics of human glycans, to suppress immune responses via Siglec engagement. The resulting “hot spot” in hominin Sia biology apparently occurred in the Homo lineage prior to the common ancestor of Humans and Neanderthals. Multiple genomic changes in the CD33rSIGLEC gene cluster may also be relevant to unusual human-specific expression change of CD33rSiglecs in locations such as placental trophoblast (SIGLEC6); pancreatic islets (SIGLEC7); ovarian fibroblasts (SIGLEC11/16); amniotic epithelium (SIGLEC5/14); microglia (SIGLEC11/16 and CD33); mucosal epithelial surfaces (SIGLEC12 and 13); NK cells (SIGLEC17); and, T cells (SIGLECs 5/14,7,9).
Two prominent hallmarks of the human brain are the prolonged maturation time of neuronal circuits and a significant increase in cortical neuron connectivity. These features have been hypothesized to underlie the emergence of higher cognitive functions in modern humans. However, little is known about the molecular changes that have led to the emergence of human-specific traits of cortical development and function. Our attention has recently focused on gene duplications that are unique to the human genome. One such gene is SRGAP2: the ancestral copy, SRGAP2A, promotes excitatory (E) and inhibitory (I) synapse maturation and limits the density of both E and I synapses made onto cortical pyramidal neurons (Charrier et al. Cell 2012; Fossati et al. Neuron 2016). Partial duplication of SRGAP2A resulted in a human-specific paralog, SRGAP2C, which binds to and inhibits SRGAP2A function. Deletion of SRGAP2A or expression of SRGAP2C in mouse cortical pyramidal neurons leads to the emergence of phenotypic traits characterizing human cortical neurons, including increased E and I synaptic density and a protracted period of synaptic maturation. However, how this increased density of synapses and prolonged maturation affects the structure and function of cortical circuits remains unknown and constitutes the main goal of our current project. I will present some new results probing changes in circuit structure and function upon SRGAP2A loss-of-function or ‘humanization’ of SRGAP2C expression in mouse cortical circuits. Our results provide new insights into the significance of the emergence of human-specific SRGAP2C gene on brain evolution by defining its impact on synaptic organization and circuit function.
Huxley and Darwin were among the first to appreciate the close evolutionary relationship of humans and other African great apes but also to ponder what genetic changes might make us human. Initial comparisons of human and chimpanzee genes, however, showed little difference (>99% identical) despite the numerous adaptations that must have occurred on the human lineage. Recent studies of more complex regions of our genome have revealed hotspots of rapid and dramatic evolutionary change. Embedded within these regions are hundreds of new duplicate genes, several of which appear to be important in unique human-specific neuroanatomical adaptations, including the expansion of the neocortex and increase in synaptic connectivity. Paradoxically, this genetic complexity has led to a high background rate of copy number mutations causing childhood diseases (e.g., autism, intellectual disability, and epilepsy) suggesting that human-specific genes and increased disease burden are tightly linked.
