(B) Quasibinomial fit line of representative marker gene expression across cells ordered by correlation with PC1. Despite many similarities between human and chimp, the external appearance and behavioural differences are prominent. As can be seen in Figure 2A, the vast majority of these cells resided in the VZ (distinguishable as a germinal zone in the DAPI-stained images), consistent with them being APs. There appears to be a misunderstanding. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. Datasets are the same as in Figure 5B and C. Left side: APs in a slice of a D30 human cerebral organoid from iPSC line SC102A-1. PC1 and PC2 described cell cycle phases, and the top 50 correlating and anticorrelating genes were used to infer an intercellular correlation network for human and chimp APs, human iPSCs, and a human endothelial cell line. The difference between human and chimpanzee APs with regard to the progression of mitosis (Figure 5) is in line with the longer S-phase finding, as the longer metaphase plate stage may similarly impact the mode of AP division and thus the fate of the AP progeny. These genes are APOLD1, BICC1, EFNB1, GSTM1, IFI44L, ITGB8, SDK2, SEMA5A, SLC35F1, ZNF516. The 1.2% chimp-human distinction, for example, involves a measurement of only substitutions in the base building blocks of those genes that chimpanzees and humans share. Here, we have generated cerebral organoids from chimpanzee-derived induced pluripotent stem cells (iPSCs), and used single-cell transcriptomics, immunohistofluorescence and live imaging to compare relevant features of chimpanzee NSPCs to human NSPCs in cerebral organoids and fetal neocortex. This amounts to about 40 million differences in our DNA, half of which likely resulted from mutations in the human ancestral line and half in the chimp line since the two species diverged. Furthermore, the ~5 min longer prometaphase-metaphase in human than chimpanzee APs constituted only a fraction of the total duration of their mitosis. To study the earliest stages of brain development, researchers often use human brain cells grown in the laboratory. These small differences nevertheless provide a set of clues as to which NSPC features may underlie the differential extent of neocortex expansion in humans versus apes, and are consistent with a scenario in which the accumulation of such small differences during evolution may have resulted in the distinct chimpanzee and human neocortices. Discordant sites and indels including 6 bp upstream and downsteam of the indel position were masked (replacing the base with N). This is why we have pointy chins whereas chimps have receding chins – we attach our many lip muscles to the prominent lower chin, but chimpanzees lack many of these muscles and so do not need a protruding chin. Page points out that this is not all about gene decay or loss. Although the human diet is markedly different from the diets of closely related primate species, the influence of diet on phenotypic and genetic differences between humans and other primates is unknown. Together, these data allowed reconstruction of the chimpanzee organoid cerebral cortex from single-cell transcriptomes. 2015, they divided AP cells into S/G2/M and G1 clusters and not G1 and G2/M. For example, we have about half the same DNA as a banana, and yet people do not use this to emphasize how similar bananas are to us! When comparing these results to bulk RNA-seq data from mouse APs and neurons (Florio et al., 2015), we find that 75% of the genes with expression specific to APs or neurons in humans are also specific to each cell type in the mouse, suggesting that these gene expression programs were already established and likely present in the common ancestor of mouse, human and chimpanzee some 90 million years ago (Figure 3F). We found that nearly all genes upregulated in human APs in G2-M compared with human APs in G1 were also upregulated during G2-M in iPSCs and endothelial cells (Figure 8C). The resulting pellet was resuspended in 30–50 μl (for cortical slices) or 250–500 μl (for whole organoids) of Diff +VA medium. and that of the present study are very different from one another. The Max Planck Institute for Evolutionary Anthropology has an institutional permit for the transport of biological material derived from endangered species (DE216-08, see http://cites.org/common/reg/si/e-si-beg.shtml). It would be very interesting indeed to compare the various mitotic phases of human and chimpanzee basal progenitors by live high-resolution time-lapse imaging. In brief, the consensus genome was constructed based on the chained and netted pairwise alignment of human (hg38) and chimpanzee (panTro4) obtained from UCSC. In conclusion, the major proportion of the variation in these data is not between in vitro and in vivo tissues or between species, but among cell states during neurogenesis, confirming that the major features of the genetic programs regulating the NSPC-to-neuron lineage are conserved between human and chimpanzees, and are recapitulated in cerebral organoids. The genes similarly expressed within such a cell group generally correlate well with previously described cell type marker genes and we therefore think we have the resolution to discretely classify APs, BPs, and neurons. This assignment was consistent with an unbiased assignment using the method published by (Scialdone et al. Cells with high AP specificity scores are in yellow in the main scatter plot. This kind of experiment would support the data presented in Figure 2B. How these species differences arise is not clear, but it likely occurs in the earliest phases of development when brain stem and progenitor cells divide and give rise to cerebral cortex cells in the growing brain. To identify genes differentially expressed between chimpanzee and human cortex-like cells, we remapped all single-cell transcriptome reads to a consensus human-chimpanzee genome and used human annotations to identify 1-to-1 orthologous genes. Compared to chimps, humans are about 38% taller, are 80% heavier, live 50% longer, and have brains that are about 400% larger (1330 ccs compared to 330 ccs). Protocols to generate structured cerebral tissue (cerebral organoids) from pluripotent stem cells in vitro constitute a major advance for studying neocortex development, in particular with regard to humans and non-human primates where fetal brain tissue is hard or impossible to obtain and manipulate (Kadoshima et al., 2013; Lancaster and Knoblich, 2014; Lancaster et al., 2013; Mariani et al., 2015; Qian et al., 2016). Or it may simply be a genetic mutation with no purpose – white around the iris is seen in some chimpanzees also. As to the issue how reliably that clock operates from culture to culture: We have analyzed human cerebral organoids from two independent iPSC lines and chimp cerebral organoids from two independent iPSC lines, and find that cortical development proceeds reproducibly from culture to culture. Instead, they differ remarkably in their structure and gene content. Demultiplexed reads were mapped using TopHat v2.0.14, and FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values per gene were quantified using Cufflinks v.2.2.1 (Trapnell et al., 2012). We then generated a weighted adjacency network graph using the graph.adjacency() command and visualized cells as vertices connected to other cells via edges if the pairwise correlation between two cells was higher than 0.4. Common chimpanzees do not engage in recreational sex, and mating only takes ten or fifteen seconds, often whilst eating or doing something else. (A) Cryosections of cortical regions from human and chimpanzee organoids at day 28 and day 52 immunostained for PAX6 (magenta) and TBR2 (green) combined with DAPI staining. How many cells are counted per sample? In contrast to APs, human and chimpanzee iPSCs had similar prometaphase-metaphase lengths (Figure 6A,B,E; Figure 5—source data 1). The portmanteau word humanzee for a human–chimpanzee hybrid appears to have entered usage in the 1980s. The humans and chimpanzees were not 50% similar genetically, or 60%, or even 80%, they were 98 to 99% similar, nearly identical. Cell lines were regularly tested for mycoplasma using a PCR-based test (Minerva Biolabs) and found to be negative. How could the authors account for this age difference in vitro in organoids? Key Terms: Chimpanzee Genome, Human Genome, Number of Chromosomes, Size What is Human Genome The human genome is the collection of human DNA. These cortical-like regions are often patterned as dorsal telencephalon (FOXG1 and OTX2-), however we have observed cortical regions that express ventral telencephalon or hindbrain markers (Camp, Badsha et al. Scale bars, 200 μm. This shows that nearly all genes enriched in G2-M phase of the AP cell cycle are not specific to APs, but also enriched in G2-M of mitotic iPSCs and endothelial cells. The total duration of mitosis was the sum of these phases. Observation in 1960 of chimpanzees using sharpened twigs to fish for termites has since changed this. This figure may still sound impressive, but most DNA is used for basic cellular functions which all living things share. (E–G) Time between the start of chromosome congression and anaphase onset (referred to as 'prometaphase + metaphase') (E), between the start of chromosome congression and the formation of a metaphase plate (referred to as 'prometaphase') (F), and between the formation of a metaphase plate and anaphase onset (referred to as 'metaphase') (G). We have included this information into the Methods section under the heading “Differential gene expression analysis” and added an additional supplemental figure (Figure 8—figure supplement 1). The reviewers, however, identified a number of technical and conceptual issues that will require the authors' attention. Cerebral organoids were fixed with 1% PFA in 120 mM phosphate buffer pH 7.4 for 20 min at room temperature and subjected to cryosectioning (14 µm) and immunofluorescence as described (Camp et al., 2015). [xii] Look at … This simple piece of information may help understand if these organoids are indeed helpful to unravel differences between chimp and human cortex development, and if so where to look. The Seurat package (Macosko et al., 2015) implemented in R was used to identify cell populations present in chimpanzee organoids (Figure 1—figure supplement 2). Nucleolar Dbf2-Mob1 then phosphorylates Cfi1/Net1 and Cdc14, activating Cdc14. Human subjects: Human fetal brain tissue (11-13 weeks post conception (wpc)) was obtained with informed written maternal consent followed by elective pregnancy termination. Related to Figure 5B and C Live tissue imaging of mitotic phases, as reported by chromosomes, in organotypic slice culture of cerebral organoids. We added the following statement to make this point more clear: “Though this classification is convenient to describe the cell types present in the chimpanzee organoid, we note that many of the cells can be described as intermediates between APs, BPs, and different stages of neuron maturation.”. Spindle orientation analysis was performed as described (Mora-Bermudez et al., 2014). There are only two species of chimps described under the genus Pan, P. troglodytes (Common chimp) and P. paniscus (Bonobo). For Figure 4G, the maximal range of orientations per every mitotic AP was calculated from the formation of a metaphase plate to anaphase onset. (D) Heatmap shows the differential expression score between human and chimpanzee APs (z-score) and AP specificity score (Log2 normalized) of the same genes that are specific to APs relative to endothelial cells and iPSCs. Humans and chimpanzees evolved in Africa from a common ancestor millions of years ago. Humans and both chimpanzee species evolved from a common ancestor, possibly sahelanthropus tchadensis, between five and seven million years ago. The dynamics of prometaphase-metaphase is very different in mouse as compared to human or chimpanzee, therefore, it is difficult to relate that the lengthening of prometaphase-metaphase characterizes proliferating NPSCs in humans. In Figure 1E we calculated for each chimpanzee organoid cortex cell the Spearman correlation of its transcriptome (all genes) with bulk transcriptome data from each of 4 microdissected human cortical zones (VZ, iSVZ, oSVZ, CP, mean expression value of each gene across 4 replicates from 13 weeks post conception, data published in ([Fietz et al., 2012] GSE38805). However, our data revealed no clear differences in spindle orientation, either during metaphase (Figure 4C–G) or shortly after anaphase onset (Figure 4C–F, I–J). from the ventricular to the pial surface, using 100-µm wide fields, as stated in the Methods section. Like humans, chimpanzees’ game types and playmates change as they age; just as we grow out of “kiddie” games, chimpanzees’ preferences mature as well. By comparison, prometaphase-metaphase of APs in slice culture of mouse neocortex, a well-characterized model system for neurogenesis, lasted for only approximately half the amount of time than human APs (Figure 5D,E; Figure 5—source data 1). For sample numbers, please see response above. B) More details are needed to understand the variation within each specie. Further support for this notion was obtained by analysis of the interphase of the cell cycle, specifically S-phase. In sum, two independent lines of evidence, the detailed analysis of AP mitosis phase lengths and the determination of the proportions of the various NSPC types, support the concept that a longer neurogenic period (Lewitus et al., 2014), which in turn implies a longer phase of NSPC proliferation (Otani et al., 2016), contributes to the greater expansion of the neocortex in humans than the great apes. 2011), these data again are consistent with the concept that human APs exhibit a greater tendency for proliferative than differentiative divisions than chimpanzee APs. Finally, we have previously shown by single-cell RNA sequencing that the gene expression programs controlling neocortex development in human cerebral organoids are remarkably similar to those in the developing fetal tissue (Camp et al., 2015). 2014 and unpublished observations), we do not think that this clustering pattern of chimpanzee cerebral organoid cells is related to the age of the respective organoid. Genetic features distinguishing us from chimpanzees and making us humans are still of a great interest. Even two completely unrelated humans are usually genetically more similar than two sibling chimpanzees. The cells that we see as intermediate or transiting cells were distributed between G2/M, S, and G1-phase, whereas the clusters we used in our analysis showed a clear assignment as G2/M or G1. The prometaphase-metaphase lengthening that we observed is a natural difference among three hominids and one rodent species, whereas Pilaz et al. We have now clarified this issue in the revised text (Introduction, second paragraph and subsection “Spindle orientation dynamics are similar in human and chimpanzee NSPCs”). Humans walk upright since infancy and have evolved bowl-shaped pelvises to support their internal organs while doing so. We thank the Services and Facilities of the Max Planck Institute of Molecular Cell Biology and Genetics for outstanding support, notably Jussi Helppi and his team of the Animal Facility, and Jan Peychl and his team of the Light Microscopy Facility. Chimpanzees develop different cultural practices depending on their environment, and transmit their culture as learned behaviour. Brain size alone, however, is not an absolute indicator of intelligence. These cell populations were therefore only analyzed by tissue immunofluorescence and RNA-seq (Figures 1–2). Chimps or chimpanzees are a type of apes and the closest extant relative to the humans. Compared to iPSCs, the length of prometaphase-metaphase is extended for both human and chimpanzee cerebral organoid APs (Figure 6). Cerebral organoid APs include apical radial glia-like NSPCs that contact a ventricle-like lumen, express radial glia marker genes, undergo interkinetic nuclear migration, and divide at the apical surface, similar to their in vivo counterparts, and cerebral organoid BPs comprise both basal radial glia-like and basal intermediate progenitor-like NSPCs (Lancaster et al., 2013). All of the differences between us and them, must relate to the 2%. their body) facilitates the execution of these decisions. Reads from human and chimpanzee were mapped to a consensus genome, and human gene annotations were used for expression counting. To investigate potential functions of prometaphase-metaphase lengthening, we asked whether mitotic phases were different between proliferating and neurogenic APs. But then, will they find similar differences between human and chimp when comparing organoids from other tissues (i.e. The authors nicely compare metaphase in blood B cells from chimp and human and don't find differences. We hierarchically clustered (Pearson’s correlation distance metric) cells based on their correlation coefficient with germinal zones and visualized the clustering in a heatmap showing correlation coefficients scaled across zones (mean-centering and dividing by standard deviation). Each fetal, human organoid, and chimpanzee organoid cortex cell was scored for the NSPC or neuron signature by summing the number of genes from each signature that have an expression greater than log2 FPKM of 5, and normalizing by the number of all genes expressed above log2 FPKM of 5 for each cell. Cdc15, the protein kinase that activates Dbf2-Mob1 at SPBs, also regulates its nuclear access. Mora-Bermúdez and colleagues address the fundamental question of what distinguishes human and chimpanzee brain stem progenitor cells leading to the dramatic difference in brain size between these species. In line with this, we have preliminary data suggesting that cortical development in chimpanzee cerebral organoids proceeds slightly faster than in human cerebral organoids (unpublished data). Primate blood samples used to generate iPSCs were obtained by certified veterinarians during annual medical examinations or other necessary medical interventions, meaning that no invasive procedures were performed on primates for the sole purpose of our research project. In the past, many scientists tried to argue that there were several species of human, and would often hasten to add that they themselves belonged to the ‘superior’ species. We thank David Andrijevic and Anne Weigert for help with maintenance and characterization of iPSC lines. (C) Cryosections of cortical regions from human and chimpanzee organoids at D53 immunostained for KI67 (yellow) combined with DAPI staining (blue). Furthermore, the chimpanzee brain is more symmetrical while the human brain has a more asymmetric shape. The determination of the chimpanzee genome sequence provides a means to study both structural and functional aspects of the evolution of the human genome. The brain of a chimpanzee has a volume of 370mL on average. Live tissue imaging of mitotic phases, as reported by chromosomes, in organotypic slice culture of E14.5 mouse neocortex. Once in the nucleus, priming phosphorylation of Cfi1/Net1, the nucleolar anchor of Cdc14, by the Polo-like kinase Cdc5 targets Dbf2-Mob1 to the nucleolus. If this is the main goal of this manuscript, it may be more informative to put more emphasis on the human and chimpanzee PAX6 TBR2 NSPCs / PAX6 TBR2- NSPCs, which were not analyzed due to their low numbers. 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