RESUMO
Neuroanatomists have long speculated that expanded primate brains contain an increased morphological diversity of inhibitory neurons (INs)1, and recent studies have identified primate-specific neuronal populations at the molecular level2. However, we know little about the developmental mechanisms that specify evolutionarily novel cell types in the brain. Here, we reconstruct gene expression trajectories specifying INs generated throughout the neurogenic period in macaques and mice by analysing the transcriptomes of 250,181 cells. We find that the initial classes of INs generated prenatally are largely conserved among mammals. Nonetheless, we identify two contrasting developmental mechanisms for specifying evolutionarily novel cell types during prenatal development. First, we show that recently identified primate-specific TAC3 striatal INs are specified by a unique transcriptional programme in progenitors followed by induction of a distinct suite of neuropeptides and neurotransmitter receptors in new-born neurons. Second, we find that multiple classes of transcriptionally conserved olfactory bulb (OB)-bound precursors are redirected to expanded primate white matter and striatum. These classes include a novel peristriatal class of striatum laureatum neurons that resemble dopaminergic periglomerular cells of the OB. We propose an evolutionary model in which conserved initial classes of neurons supplying the smaller primate OB are reused in the enlarged striatum and cortex. Together, our results provide a unified developmental taxonomy of initial classes of mammalian INs and reveal multiple developmental mechanisms for neural cell type evolution.
Assuntos
Evolução Biológica , Corpo Estriado , Desenvolvimento Embrionário , Macaca , Neurogênese , Neurônios , Bulbo Olfatório , Animais , Corpo Estriado/crescimento & desenvolvimento , Neurônios Dopaminérgicos , Feminino , Macaca/crescimento & desenvolvimento , Mamíferos , Camundongos , Neurogênese/fisiologia , Bulbo Olfatório/fisiologia , Gravidez , PrimatasRESUMO
Human pluripotent stem cells hold significant promise for regenerative medicine. However, long differentiation protocols and immature characteristics of stem cell-derived cell types remain challenges to the development of many therapeutic applications. In contrast to the slow differentiation of human stem cells in vitro that mirrors a nine-month gestation period, mouse stem cells develop according to a much faster three-week gestation timeline. Here, we tested if co-differentiation with mouse pluripotent stem cells could accelerate the differentiation speed of human embryonic stem cells. Following a six-week RNA-sequencing time course of neural differentiation, we identified 929 human genes that were upregulated earlier and 535 genes that exhibited earlier peaked expression profiles in chimeric cell cultures than in human cell cultures alone. Genes with accelerated upregulation were significantly enriched in Gene Ontology terms associated with neurogenesis, neuron differentiation and maturation, and synapse signaling. Moreover, chimeric mixed samples correlated with in utero human embryonic samples earlier than human cells alone, and acceleration was dose-dependent on human-mouse co-culture ratios. The altered gene expression patterns and developmental rates described in this report have implications for accelerating human stem cell differentiation and the use of interspecies chimeric embryos in developing human organs for transplantation.
Assuntos
Quimerismo , Células-Tronco Embrionárias Humanas , Neurogênese , Células-Tronco Pluripotentes , Animais , Células Cultivadas , Biologia Computacional , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/fisiologia , Humanos , Camundongos , Neurogênese/genética , Neurogênese/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Especificidade da Espécie , Transcriptoma/genéticaRESUMO
Pluripotent stem cells retain the developmental timing of their species of origin in vitro, an observation that suggests the existence of a cell-intrinsic developmental clock, yet the nature and machinery of the clock remain a mystery. We hypothesize that one possible component may lie in species-specific differences in the kinetics of transcriptional responses to differentiation signals. Using a liquid-handling robot, mouse and human pluripotent stem cells were exposed to identical neural differentiation conditions and sampled for RNA-sequencing at high frequency, every 4 or 10 minutes, for the first 10 hours of differentiation to test for differences in transcriptomic response rates. The majority of initial transcriptional responses occurred within a rapid window in the first minutes of differentiation for both human and mouse stem cells. Despite similarly early onsets of gene expression changes, we observed shortened and condensed gene expression patterns in mouse pluripotent stem cells compared to protracted trends in human pluripotent stem cells. Moreover, the speed at which individual genes were upregulated, as measured by the slopes of gene expression changes over time, was significantly faster in mouse compared to human cells. These results suggest that downstream transcriptomic response kinetics to signaling cues are faster in mouse versus human cells, and may offer a partial account for the vast differences in developmental rates across species.
Assuntos
Diferenciação Celular/genética , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , RNA-Seq/estatística & dados numéricos , Animais , Linhagem Celular , Biologia Computacional , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Cinética , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Medicina Regenerativa , Especificidade da EspécieRESUMO
How species-specific developmental timing is controlled is largely unknown. By following human embryonic stem (ES) cell and mouse epiblast stem (EpiS) cell differentiation through detailed RNA-sequencing time courses, here we show that pluripotent stem cells closely retain in vivo species-specific developmental timing in vitro. In identical neural differentiation conditions in vitro, gene expression profiles are accelerated in mouse EpiS cells compared to human ES cells with relative rates of differentiation closely reflecting the rates of progression through the Carnegie stages in utero. Dynamic Time Warping analysis identified 3389 genes that were regulated more quickly in mouse EpiS cells and identified none that were regulated more quickly in human ES cells. Interestingly, we also find that human ES cells differentiated in teratomas maintain the same rate of differentiation observed in vitro in spite of being grown in a mouse host. These results suggest the existence of a cell autonomous, species-specific developmental clock that pluripotent stem cells maintain even out of context of an intact embryo.
Assuntos
Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Células-Tronco Embrionárias/citologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos SCID , Neurônios/citologia , Especificidade da Espécie , Teratoma/patologia , Fatores de TempoRESUMO
Mammalian brains vary in size, structure, and function, but the extent to which evolutionarily novel cell types contribute to this variation remains unresolved1-4. Recent studies suggest there is a primate-specific population of striatal inhibitory interneurons, the TAC3 interneurons5. However, there has not yet been a detailed analysis of the spatial and phylogenetic distribution of this population. Here, we profile single cell gene expression in the developing pig (an ungulate) and ferret (a carnivore), representing 94 million years divergence from primates, and assign newborn inhibitory neurons to initial classes first specified during development6. We find that the initial class of TAC3 interneurons represents an ancestral striatal population that is also deployed towards the cortex in pig and ferret. In adult mouse, we uncover a rare population expressing Tac2, the ortholog of TAC3, in ventromedial striatum, prompting a reexamination of developing mouse striatal interneuron initial classes by targeted enrichment of their precursors. We conclude that the TAC3 interneuron initial class is conserved across Boreoeutherian mammals, with the mouse population representing Th striatal interneurons, a subset of which expresses Tac2. This study suggests that initial classes of telencephalic inhibitory neurons are largely conserved and that during evolution, neuronal types in the mammalian brain change through redistribution and fate refinement, rather than by derivation of novel precursors early in development.
RESUMO
Primate brains vary dramatically in size and organization, but the genetic and developmental basis for these differences has been difficult to study due to lack of experimental models. Pluripotent stem cells and brain organoids provide a potential opportunity for comparative and functional studies of evolutionary differences, particularly during the early stages of neurogenesis. However, many challenges remain, including isolating stem cell lines from additional great ape individuals and species to capture the breadth of ape genetic diversity, improving the reproducibility of organoid models to study evolved differences in cell composition and combining multiple brain regions to capture connectivity relationships. Here, we describe strategies for identifying evolved developmental differences between humans and non-human primates and for isolating the underlying cellular and genetic mechanisms using comparative analyses, chimeric organoid culture, and genome engineering. In particular, we focus on how organoid models could ultimately be applied beyond studies of progenitor cell evolution to decode the origin of recent changes in cellular organization, connectivity patterns, myelination, synaptic development, and physiology that have been implicated in human cognition.
Assuntos
Evolução Biológica , Encéfalo , Organoides , Animais , HumanosRESUMO
The aim of the present study was to test sample reproducibility for model neural tissues formed on synthetic hydrogels. Human embryonic stem (ES) cell-derived precursor cells were cultured on synthetic poly(ethylene glycol) (PEG) hydrogels to promote differentiation and self-organization into model neural tissue constructs. Neural progenitor, vascular, and microglial precursor cells were combined on PEG hydrogels to mimic developmental timing, which produced multicomponent neural constructs with 3D neuronal and glial organization, organized vascular networks, and microglia with ramified morphologies. Spearman's rank correlation analysis of global gene expression profiles and a comparison of coefficient of variation for expressed genes demonstrated that replicate neural constructs were highly uniform to at least day 21 for samples from independent experiments. We also demonstrate that model neural tissues formed on PEG hydrogels using a simplified neural differentiation protocol correlated more strongly to in vivo brain development than samples cultured on tissue culture polystyrene surfaces alone. These results provide a proof-of-concept demonstration that 3D cellular models that mimic aspects of human brain development can be produced from human pluripotent stem cells with high sample uniformity between experiments by using standard culture techniques, cryopreserved cell stocks, and a synthetic extracellular matrix. Impact statement Pluripotent stem (PS) cells have been characterized by an inherent ability to self-organize into 3D "organoids" resembling stomach, intestine, liver, kidney, and brain tissues, offering a potentially powerful tool for modeling human development and disease. However, organoid formation must be quantitatively reproducible for applications such as drug and toxicity screening. Here, we report a strategy to produce uniform neural tissue constructs with reproducible global gene expression profiles for replicate samples from multiple experiments.