Author response: Species-specific maturation profiles of human, chimpanzee and bonobo neural cells (2018)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Comparative analyses of neuronal phenotypes in closely related species can shed light on neuronal changes occurring during evolution. The study of post-mortem brains of nonhuman primates (NHPs) has been limited and often does not recapitulate important species-specific developmental hallmarks. We utilize induced pluripotent stem cell (iPSC) technology to investigate the development of cortical pyramidal neurons following migration and maturation of cells grafted in the developing mouse cortex. Our results show differential migration patterns in human neural progenitor cells compared to those of chimpanzees and bonobos both in vitro and in vivo, suggesting heterochronic changes in human neurons. The strategy proposed here lays the groundwork for further comparative analyses between humans and NHPs and opens new avenues for understanding the differences in the neural underpinnings of cognition and neurological disease susceptibility between species. https://doi.org/10.7554/eLife.37527.001 Introduction Since the split from the last common ancestor of hominins (Homo) and African apes (Gorilla and Pan spp), human brain evolution has been characterized by several waves of increases in cranial capacity (Carlson et al., 2011; Falk et al., 2000) and selective expansion of regions implicated in complex cognition (Semendeferi et al., 2010; Semendeferi and Damasio, 2000). The increase in the cranial capacity in fossil hominins has been tied to behavioral changes, including the appearance of the first stone tools and their subsequent elaboration, increases in population size, and the spread of hominins into ecologically challenging habitats (Ambrose, 2001; Stout, 2011). Equally important were the subtle changes in brain organization at the microscopic level. While these changes cannot be inferred directly from the fossil specimens, comparative analyses of cortical organization between extant primate species suggest that the human brain indeed differs from the brain of other hominid species in several important microstructural aspects (Semendeferi et al., 2001; Barger et al., 2007; Semendeferi et al., 2011). One of these is the dendritic morphology of cortical pyramidal neurons, which differs between humans and the common chimpanzee (Pan troglodytes) (Bianchi et al., 2013a). Since cortical pyramidal neurons represent the most common type of neuron in the cortex and form basic units of cortical microcircuitry (DeFelipe and Fariñas, 1992), comparative analyses directed specifically at pyramidal neurons can yield insights into the organization of the microcircuitry that is typical of each species. Differences observed in the adult phenotype between humans and chimpanzees likely reflect differences in timing and/or rate of cortical development. Developmental differences between species represent an important component in evolutionary studies, as small changes in the timing of development translate into morphological differences in adulthood, often with important functional implications. In the case of human brain evolution, sequential hypermorphosis, a type of heterochrony characterized by prolongation of all stages of brain development compared to the ancestral state (McNamara, 2002; Vrba, 1998; McKinney, 2002), has been proposed as an evolutionary mechanism underlying cerebral expansion in humans. Humans and apes share a pattern of prolonged postnatal growth in brain size that sets them apart from Old World monkeys (Leigh, 2004). Similarly, the maturation of pyramidal neurons appears to be protracted in hominids compared to macaques (Cupp and Uemura, 1980; Petanjek et al., 2011; Sedmak et al., 2018) and in humans compared to chimpanzees (Bianchi et al., 2013a; Teffer et al., 2013), possibly accounting for longer and more branched dendrites with higher numbers of dendritic spines in humans (Bianchi et al., 2013b; Petanjek et al., 2008). Because there is little evidence that the simple addition or subtraction of genes is sufficient to explain the observed differences (Hill and Walsh, 2005), changes in the regulation (levels and patterns of expression) of genes shared between humans and chimpanzees have been proposed to play an important role (King and Wilson, 1975; Enard et al., 2002). In particular, the expression of genes relevant to developmental events such as migration and dendritic maturation in pyramidal neurons can provide important insights into the underlying mechanisms shaping the differences in the organization of neuronal networks observed in humans and other primates. Although studies using post-mortem brains from human and NHPs have provided important insights into developmental differences across species, the availability of specimens often limits the extent of the hypotheses that can be addressed. Recent advances in somatic reprogramming technology make comparative studies possible even in the absence of post-mortem specimens (Marchetto et al., 2013). The work presented here specifically addresses neural progenitor cell (NPC) migration and the development and functional maturation of cortical neurons in humans and chimpanzees. While changes in neural migration and cortical layering may pathologically affect early dendritic organization and microcircuitry formation in humans (Pramparo et al., 2015; Reiner et al., 2016; Muraki and Tanigaki, 2015; Brennand et al., 2015), previous research has not examined migration and early development of pyramidal neurons from an evolutionary perspective. We therefore hypothesized that the neurodevelopmental differences between humans and chimpanzees would become visible during distinct processes of NPC migration and during the initial establishment of the organization of dendritic trees and functional neuronal maturation in the neocortex. To test this hypothesis, we utilized induced pluripotent stem cell (iPSC) technology to model NPC migration in Homo and Pan spp. (chimpanzee and bonobo) and in the early development of cortical neurons. We found differential migration patterns in human NPCs compared to those of chimpanzee and bonobo based on RNA expression profile analysis and live-cell imaging. Next, we observed morphological and functional developmental differences between human and chimpanzee neurons, suggesting differences in the timing of neuronal maturation between the two species. We report here in vitro and in vivo comparative analyses of the neural development of two closely related primate species. The strategy applied in this work can be utilized for further studies addressing human brain evolution and the mechanisms underlying the cellular and molecular aspects that are unique to the human brain. Results Analysis of the expression profiles of human, chimpanzee and bonobo NPCs shows differentially regulated genes related to cell migration Fibroblasts from chimpanzees (Pan troglodytes), bonobos (Pan paniscus) and humans (Homo sapiens) were reprogrammed to their pluripotent state as previously described (Brennand et al., 2015) and then differentiated into NPCs. Briefly, we initiated neural differentiation by plating neuralized embryoid bodies (EBs) for a week until neural rosettes became apparent in the dish. Dissociated rosettes formed a homogeneous population of NPCs that continued to proliferate in the presence of the mitogen FGF2 and were positive for the pan-neural progenitor marker Nestin (Figure 1A and B) and cortical neural progenitor markers Pax6 and Foxg1 (Figure 1—figure supplement 1A–C). To determine whether there was a difference in cortical layer identity between species at the population level, we performed single cell RNA sequencing on NPCs derived from a human, bonobo, or chimpanzee sample. The results revealed that each species had similar proportions of cortical markers, indicating that within the cortical-fated cells there was no enrichment for any given layer between the species (Figure 1—figure supplement 2A–C, see Materials and methods for details). Figure 1 with 2 supplements see all Download asset Open asset Characterization of neural progenitor cells (NPCs) derived from human (Homo), chimpanzee and bonobo (Pan) iPSCs. (A) Scheme showing the main steps and timeline involved in the generation of NPCs from iPSCs. (B) Morphological aspects of neural rosettes derived from embryoid bodies (EBs) (BF: Bright Field). Neural rosettes are positive for the early neural marker Nestin (red). In the presence of FGF2, dissociated rosettes form a homogeneous monolayer population of proliferating Nestin-positive NPCs. Upon FGF2 withdrawal, NPCs differentiate readily in Tuj-1-positive neurons (green). Scale bars: 50 µm. (C) Heat map representation of mapped reads corresponding to protein coding genes. Chimpanzee and bonobo NPCs clustered closer to each other than to human NPC lines and to human embryonic stem cell-derived NPCs (HUES6 and H9 ES lines). (D) Venn diagram showing pairwise comparison of protein-coding genes. Red or green: significantly differentially expressed genes (FDR < 0.05 and fold change >2). Red and green: total of expressed genes with no significant differences in mRNA levels between compared species. (E) Principal component analysis (PCA) of human, chimpanzee and bonobo NPCs showing human samples (green circles) clustering separately from NHPs (red and pink circles) on the first principal component (33% of the variance). (F) Heat map showing comparison between Homo and Pan NPC expression profiles of genes related to cell migration. https://doi.org/10.7554/eLife.37527.002 We also investigated whether human and Pan NPCs were similarly specified to cortical and hindbrain fates. For this analysis, we determined the proportion of NPCs expressing well-known cortical and hindbrain markers (Figure 1—figure supplement 2C) SOX2 and NESTIN expression were used to identify NPCs; PAX6, NEUROD6 and TBR1 to identify cortical NPCs; and EN1, EN2, ISL1, and LHX3 to identify hindbrain NPCs. We identified similar proportions of all NPCs, cortical NPCs, and hindbrain NPCs in all species. This analysis further indicated that there was no detectable species-dependent bias in regional patterning. To gain insights into differences in gene expression between human, chimpanzee and bonobo NPCs, we performed high-throughput RNA sequencing (RNA-seq) analyses on eight human and five nonhuman primate (chimpanzee and bonobo) NPC lines. We assessed the RNA-sequencing data from NPCs for developing brain regional signatures. Using BrainImageR tool (Linker et al., 2019), we calculated the enrichment of each differentially expressed gene (described on Supplementary file 1) within tissues of the developing brain. Genes upregulated in Homo NPCs exhibited similar regional enrichment to genes upregulated in Pan (chimpanzee and bonobo) NPCs, indicating that the differences observed in RNA sequencing were not influencing broad fate specification between NPCs from Homo versus Pan (Figure 1—figure supplement 1D). The expression profiles of NPCs from chimpanzee and bonobo clustered closer to each other than to human NPC lines derived from iPSC or human embryonic stem cell (ES) (Figure 1C–E). The Venn diagram in Figure 1D represents protein-coding expressed genes with non-significant differences between species, differentially regulated genes with estimated false discovery rates (FDR) of less than 5% and a fold change greater than 2-fold (434 genes for human and 762 genes for chimpanzees and bonobos). Importantly, a principal component analysis (PCA) plot confirmed that human samples clustered separately from nonhuman primates (Pan) unbiasedly. The first principal component (33% of the variance) separated human from chimpanzee and bonobo samples (Figure 1E). We next focused on genes differentially expressed between Homo and Pan NPCs and found that, among the 1196 differentially expressed genes (Supplementary file 1), 52 genes were involved in pathways related to cellular migration (Fisher’s exact test, p<1.07e-24) (Figure 1F, Figure 1—figure supplement 1E and Supplementary file 2 and 3). Differential migration properties in human compared to chimpanzee and bonobo NPCs in vitro We performed a scratch assay, that is wound healing assay, (see Materials and methods) to further evaluate the migration properties of NPCs (Liang et al., 2007). In Figure 2A, representative images taken at the time of the scratch (0) and after 12 and 24 hr for the different species are shown. While both chimpanzee and bonobo NPCs were confluent by 24 hr, a gap remained at 24 hr in the human NPCs (Figure 2A, bottom panels). To better explore the migration differences between species, we quantified two features of NPC migration: 1) the path taken by NPCs in response to the scratch and 2) the dynamics of the cells as they moved along that path. Individual NPCs were randomly selected from the population in each experiment and tracked manually (Figure 2—figure supplement 1A) for the three species. A total of 340 human, 165 chimpanzees, and 186 bonobo NPCs were analyzed from 57 human, 36 chimpanzee, and 34 bonobo movies (see Materials and methods). Representative cell tracks for each species are shown (Figure 2B–D). The starting point for each example cell in the track was marked with a colored circle (Figure 2B, human = black, chimpanzee = red and bonobo = blue), the path taken was marked with a colored line and the final location was marked with a gray circle. To test if the cells migrated toward the scratch, the distance difference (DD) for each NPC of each species was calculated and compared to the DD for simulated random migration. For all three species, migration movement was directed toward the scratch when compared to random migration (Figure 2—figure supplement 1B–D). Therefore, we determined that the scratch assay can be used to compare directional migration of NPCs from humans, chimpanzees and bonobos. Figure 2 with 1 supplement see all Download asset Open asset NPC differential migration patterns in humans, chimpanzees and bonobos. (A) DIC (differential interference contrast) (left) and fluorescent (GFP) (right) images of time-lapse NPC migration from humans, chimpanzees, and bonobos at 0 hr (top row), 12 hr (middle row), and 24 hr (bottom row) following a scratch made with a glass pipette in the center of the field of view (at 0 hr). (B–D) Individual tracks with NPC origin marked with a black circle for human NPCs (B), red circle for chimpanzee NPCs (C), and blue circle for bonobo NPCs (D). Migration paths for each NPC are marked with a line and the end of the migration is marked with a gray circle. The scratch location is marked with a gray line. Inserts contain vectors for each cell showing net migration (from beginning of the track to the end – colored arrows for each NPC) relative to the scratch (gray line). (E) Cumulative distance from each NPC over time from all human (black lines), chimpanzee (red lines) and bonobo (blue lines). Also shown is the mean cumulative distance for each species (green line). (F) Cumulative histogram of motion coefficients of NPCs from three species and mean motion coefficient for NPCs from three species (inset) (**p<0.01, Mann-Whitney U test), showing that chimpanzee and bonobo NPCs migrate longer distances at higher speeds compared to human NPCs. (G–H) Migration analysis using neurosphere outgrowth assay. (G) Representative images of iPSC forebrain neurospheres from human and chimpanzee showing differential outgrowth migration of NPCs. The average distance between the radius of the inner neurosphere and outer circumference of cells was calculated using (DAPI)-stained nuclei (blue). Four measurements were taken and averaged for each neurosphere (examples of distances shown in red). (H) Graph showing quantification of distances migrated by human, chimpanzee and bonobo NPCs. The data confirmed that NPCs from chimpanzees and bonobos migrate longer distances than human NPCs. Comparative statistics as follows: Human versus Chimpanzee, p=0.0058; Human versus Bonobo, p=0.0108; Chimpanzee versus Bonobo, p=0.6831. *p<0.05, **p<0.01, Welch’s t test). https://doi.org/10.7554/eLife.37527.005 Next, we characterized features of the migration process itself and focused on the speed of NPC migration (Figure 2). For single NPCs, we first characterized the speed at each frame with respect to both space (Figure 2—figure supplement 1E–G) and over time (Figure 2—figure supplement 1H–J). Although cells from all species showed periods of slow migration (between 0 μm /min and 1 μm /min), only chimpanzee and bonobo NPCs had periods of rapid migration (1.25 μm /min to 2.5 μm /min). As a result, the distribution of speed for chimpanzee and bonobo NPCs had a heavy tail corresponding to these periods of rapid migration. To compare the speed profiles across the three species, we first calculated the cumulative distance covered by each cell and the mean cumulative distance covered for each species and calculated the migration speed per cell (Figure 2E,F). The distribution of migration speed of cells from the three species differed, with human NPCs moving significantly more slowly than either chimpanzee or bonobo NPCs (mean migration speed: human = 0.46 ± 0.19 μm /min, chimpanzee = 0.70 ± 0.31 μm /min, bonobo = 0.72 ± 0.35 μm /min, Figure 2F). By contrast, we did not find significant differences in the migration speeds between chimpanzee and bonobo NPCs. We next confirmed the cell migration differences between species by performing an independent assay, the neurosphere outgrowth assay. We measured the distance that NPCs migrated radially from a plated neurosphere. We compared outgrowth from human-, chimpanzee- and bonobo-derived spheres generated by culturing NPCs in non-adherent conditions for 48 hr and then plating them on coated plates, as previously described (Brennand et al., 2015; Delaloy et al., 2010). We observed significantly reduced outgrowth from human neurospheres (331.7 ± 14.24 μm, total of 123 neurospheres analyzed) relative to chimpanzee and bonobo (392.7 ± 18.86 μm for chimpanzees and 404.3 ± 20.98 μm for bonobos; total of 135 neurospheres analyzed) (Figure 2G and H). To evaluate if differential migratory properties were influenced by the presence of cells from other brain regions due to loss of cell purity during migration, we post-stained human and NHP cells after neurosphere migration experiment with cortical progenitor marker (PAX6) and neuroblast migration marker (doublecortin, DCX). We did not observe significant differences between species, even though the migration distances covered by human NPCs were significantly shorter than the distances covered by NHP cells (Figure 2—figure supplement 1K–M). In conclusion, individual NHP NPCs reached higher instantaneous speeds, had higher migration speeds and migrated further than human NPCs. Taken together, differences in migration speed and neurosphere outgrowth demonstrated that human NPCs migrated more slowly in vitro than chimpanzee and bonobo NPCs. Differential migration properties in human compared to chimpanzee NPCs in vivo We then asked if the difference seen in the migration speeds was influenced by the in vitro culture conditions or represented a bona fide difference in underlying developmental programs that have changed during evolution. To address this question, we grafted NPCs from two human and two chimpanzee NPC lines into the developing postnatal mouse cortex (Figure 3A). Chimpanzee and human NPCs expressed different fluorescent markers (GFP and TdTomato) and were mixed together at a 1:1 ratio prior to transplantation. Mice were sacrificed at two weeks post-transplantation (PST) and two different migration parameters were analyzed: (1) the area covered by migrating NPCs and (2) the distance migrated by NPCs from the injection site (Figure 3B,C and D and Figure 3—figure supplement 1A,B). Once again, chimpanzee NPCs showed an increase in area coverage and distance traveled from injection site. In summary, our data show that chimpanzee and bonobo NPCs migrated faster and covered longer distances than human NPCs, independent of culture conditions. We conclude that faster migration represents a cell autonomous feature of NHP NPCs that could influence the differences in brain size and brain organization between species. Figure 3 with 1 supplement see all Download asset Open asset In vivo migration comparative analysis. (A) Scheme of the transplantation experimental procedure to detect in vivo cell migration. Fibroblasts from human and chimpanzee were reprogrammed to iPSCs and differentiated to NPCs and then transduced with lentiviral vectors expressing either green fluorescent protein (GFP) or TdTomato. NPC lines from human and chimpanzee were mixed and grafted into the developing postnatal mouse cortex. Mice were sacrificed at two weeks post-transplantation. (B) Representative image of grafted human (green) and chimpanzee (red) NPCs migrating away from the injection site (indicated by the white dashed line). (C) Representation of the area covered by human and chimpanzee migrating NPCs (D) Distance migrated by single NPCs from the injection site. (****p<0.0001, Mann-Whitney U test). https://doi.org/10.7554/eLife.37527.007 Differential neuronal maturation of human and chimpanzee cells after transplantation Neuronal progenitor migration and neuronal maturation are distinct processes that could be interconnected; therefore, we chose to compare human and chimpanzee neuronal differentiation in vivo by looking at morphological characteristics that are indicative of neuronal developmental steps. To provide an enriched niche and environmental cues for proper neuronal development, we grafted NPCs from two human and two chimpanzee lines into the developing postnatal mouse brain (see Materials and methods for details). Mice were sacrificed at different time points post transplantation (PST), and the morphological characteristics of transplanted neurons were quantified (Figure 4, Figure 4—figure supplements). The selected duration of in vivo neuronal growth was 19 weeks, which, based on human post-mortem studies, spans most of the period of prenatal development of pyramidal neurons in the human cortex, that is from the emergence of the first cells with pyramidal morphology at 17 weeks until et al., with pyramidal morphology were identified and at each time point of the were by their from the cell and visible dendritic spines (Figure We identified pyramidal neurons across all cortical in and clustered given that the of this study is the development of cortical neurons, only the neurons with cell bodies in the cortex and were in the analysis (Figure Figure with 2 supplements see all Download asset Open asset Comparative analyses on transplanted neurons over (A) Scheme of the transplantation experimental NPCs were from both species and transduced with lentiviral vectors expressing either green fluorescent protein (GFP) or The NPCs from both species were dissociated together and mixed injection into brains of at postnatal The brains of the were after 4, and 19 weeks post transplantation brain was and the of and pyramidal neurons in the cortex was used for the analysis are (B) Representative images from human and chimpanzee pyramidal neurons eight weeks Scale = µm. (C) of pyramidal neurons used for the analysis at different time points following transplantation. changes in in human and chimpanzee pyramidal neurons over time after transplantation. dendritic mean dendritic and dendritic point in the represents an individual is shown between time points and between the and the time point after transplantation. The show lines (red) of with weeks of and are indicated for each analysis test). Comparative analyses on transplanted neurons showed significant difference between species on time dendritic 2 weeks, weeks, 19 weeks, mean weeks, weeks, 19 weeks, and dendritic weeks, **p<0.01, t test). neurons show changes in dendritic features over time We analyzed changes in dendritic parameters over time to investigate dendritic development. we measured total dendritic mean dendritic dendritic dendritic of dendrites and cell area (Figure The dendritic and and in neurons significantly across the time points examined for both species (Figure The between these and time was calculated analysis to the coefficient of and was determined for each analysis. The analysis to and study between two measurements and on Figure and Figure 4—figure supplement The that showed significant positive for both human and chimpanzee were and indicating that the of dendritic spines from transplanted neurons over neuronal maturation for both species. comparison revealed significant changes in timing of dendritic maturation The and the timing of developmental events between the species for most of the the extent of the differences (Figure and Figure 4—figure supplement between human and chimpanzee neurons revealed a significant of species for the and at different time The growth of quantified as increase in over the 19 week different in each species (Figure weeks from human transplanted neurons and continued until the last time point examined In contrast, the of dendrites in chimpanzee neurons at two weeks with little change The difference between the species was significant at and 19 Similarly, was significantly higher in human neurons compared to chimpanzee neurons at and 19 weeks (Figure The little difference between the species for the first weeks at and 19 weeks, the in the human neurons, significantly higher numbers of spines compared to the chimpanzee neurons at eight weeks (Figure Our results suggest that aspects of dendritic morphology at in chimpanzee neurons compared to human neurons and the pattern is not seen in all of the dendritic (Figure and Figure 4—figure supplement 2 and indicating that of dendrites with different may at different rates in each species. Differential functional maturation of human, chimpanzee and bonobo neurons We then asked if there were functional changes related to the differences observed in morphological neuronal To neuronal over we differentiated NPC lines from human, two chimpanzee and two bonobo of eight and two lines Figure The cells and neural processes became apparent in the dish. this neurons were positive for markers (Figure and and cortical markers (Figure supplement We did not observe significant differences in the of cells positive for these markers between human, chimpanzee and bonobo lines at or weeks of differentiation (Figure and Figure supplement To address neuronal development over we used the Figure The for of changes with events on the cell for the of functional changes during the