Periodic Reporting for period 4 - CerebralHominoids (Evolutionary biology of human and great ape brain development in cerebral organoids)
Periodo di rendicontazione: 2023-01-01 al 2023-06-30
The human brain is greatly enlarged and it is thought that this expansion is at least in part responsible for its complex functionality and our higher cognitive capabilities. However, how this expansion arises during development, and what features of brain development are unique to humans remains to be explained. This question is of fundamental importance for our understanding of both the healthy and diseased brain, for it is often the very same human-specific features that are affected in patients with neurological and mental health conditions.
In order to gain a better understanding of developmental events unique to the human brain, we have used cerebral organoids, or brain organoids, as an in vitro model of the human developing brain. We have compared human organoids to those of other apes, to compare early brain development across these species. Our comparisons have revealed striking differences very early in brain development in terms of tissue morphogenesis and cell shape pointing to several key regulators of cell shape, most notably ZEB2, which we have determined can drive changes in cell shape of the early founder pool, leading to an increase in the number of neural stem cells and thus increased neuron production and size. Furthermore, perturbing timing of this gene can lead to an ape-like phenotype in the human context, and vice versa.
We have also discovered several key extrinsic regulators of human neurogenesis. In particular, we have discovered that testosterone can influence progenitors that give rise to excitatory neurons, but not those that give rise to inhibitory neurons, leading to a change in the balance of these populations, an effect that does not seem to be shared with rodents. We have also explored the role of the cerebrospinal fluid, by first producing a new organoid model of the tissue that generates cerebrospinal fluid, the choroid plexus. This has led to identification of several growth factors and secreted proteins that seem to be unique to human CSF during development. Finally, we have identified an important role for cell position and tissue architecture in neural stem cell fate determination. More specifically, we have performed various morphological perturbations of brain organoids resulting in disrupted cell position while maintaining the cell types. However, this results in a progressive disruption in the temporal fate of progenitors and a dysmaturity of neurons. Thus, our findings have revealed that cells use their spatial position to determine what time it is in development.
Overall, this work has shed light on why the human brain is larger than other apes, and uncovered key cellular and molecular mechanisms that govern this enlargement. The work has opened up a new line of enquiry and our future studies will be focused on the genetic and molecular mechanisms governing the difference in timing that we have uncovered.
In order to gain a better understanding of developmental events unique to the human brain, we have used cerebral organoids, or brain organoids, as an in vitro model of the human developing brain. We have compared human organoids to those of other apes, to compare early brain development across these species. Our comparisons have revealed striking differences very early in brain development in terms of tissue morphogenesis and cell shape pointing to several key regulators of cell shape, most notably ZEB2, which we have determined can drive changes in cell shape of the early founder pool, leading to an increase in the number of neural stem cells and thus increased neuron production and size. Furthermore, perturbing timing of this gene can lead to an ape-like phenotype in the human context, and vice versa.
We have also discovered several key extrinsic regulators of human neurogenesis. In particular, we have discovered that testosterone can influence progenitors that give rise to excitatory neurons, but not those that give rise to inhibitory neurons, leading to a change in the balance of these populations, an effect that does not seem to be shared with rodents. We have also explored the role of the cerebrospinal fluid, by first producing a new organoid model of the tissue that generates cerebrospinal fluid, the choroid plexus. This has led to identification of several growth factors and secreted proteins that seem to be unique to human CSF during development. Finally, we have identified an important role for cell position and tissue architecture in neural stem cell fate determination. More specifically, we have performed various morphological perturbations of brain organoids resulting in disrupted cell position while maintaining the cell types. However, this results in a progressive disruption in the temporal fate of progenitors and a dysmaturity of neurons. Thus, our findings have revealed that cells use their spatial position to determine what time it is in development.
Overall, this work has shed light on why the human brain is larger than other apes, and uncovered key cellular and molecular mechanisms that govern this enlargement. The work has opened up a new line of enquiry and our future studies will be focused on the genetic and molecular mechanisms governing the difference in timing that we have uncovered.
The project has involved several areas of research with important contributions to the scientific community and dissemination more broadly. Specifically, we have developed several new tools, as outlined in section 1 below, and used those tools to investigate evolutionary mechanisms of human brain expansion, as outlined in section 2 below.
1) Next generation brain organoids methods and their characterization
Over the course of this project, we have developed several new organoid methods, and carefully characterised the key aspects of their generation and use. First, we have developed a new method to mature brain organoids and achieve functional neural networks. This method was published in Nature Neuroscience in 2019, and disseminated at international meetings including the FENS summer school in 2018. The method is now being used within the lab, but also by at least a dozen labs in Europe and beyond to investigate human neuronal function.
Second, using these more mature new organoids, we developed a method to perform correlative light and electron microscopy using cryo tomography on organoids, specifically on the axons projecting from human neurons within. This allowed us to investigate the ultrastructure with very high resolutions, revealing unique endoplasmic reticulum morphologies and a paucity of ribosomes. The method resulted in a large number of datasets that can be used for further investigation of human axon biology, and was published in eLife in 2021, along with all the data which was made publicly available.
Third, we have developed a new organoid of the choroid plexus, the brain region responsible for generating cerebrospinal fluid. These organoids form a selective barrier, much like the actual barrier protecting the brain and spinal cord from blood-borne factors and pathogens, as well as secreting cerebrospinal fluid that can be used to investigate normal function as well as biomarkers. This new method was published in Science in 2020, presented at various meetings including the International Society of Stem Cell Research. It has been used in the lab to investigate how the virus causing COVID-19, SARS-CoV-2, enters and infects the brain, which we published in Cell Stem Cell in 2020 and was widely disseminated also in the popular press.
Finally, this project investigated how well different organoids model the human brain and which features of their morphology are key to their proper development. This work led to identification of several key methodological factors that lead to better quality organoids, and reveal an important role for tissue architecture in temporal progression of cell fate decisions. This study was published in Cell Stem Cell in 2023 and disseminated at several international meetings including an EMBO conference in 2022.
2) Using organoids to uncover human-specific features of brain development
Using these state-of-the-art organoid methods, we have uncovered new biological insight into human-specific neurodevelopment. First, by generating brain organoids from human cells and those of closely related apes, we uncovered a difference in neural stem cell behaviour, whereby human neural stem cells go through the early transition to neurogenesis more slowly that apes, leading to a larger founder progenitor pool that is able to generate more neurons. This explains how humans end up with a greater number of neurons than other apes, and thus answers an age-old question. This work was published in Cell in 2021, and was covered in various news outlets and presenting at several international meetings. Second, we investigated the potential role for sex hormones in brain organoids, revealing a role for androgens in the production of excitatory neurons, a role that seems to diverge in humans compared with mice. Thus, again, organoids have revealed evolutionary unique biology that may shed light on important differences in brain development in the presence of androgens like testosterone. This work was published in Nature in 2022 and presented at several international meetings.
1) Next generation brain organoids methods and their characterization
Over the course of this project, we have developed several new organoid methods, and carefully characterised the key aspects of their generation and use. First, we have developed a new method to mature brain organoids and achieve functional neural networks. This method was published in Nature Neuroscience in 2019, and disseminated at international meetings including the FENS summer school in 2018. The method is now being used within the lab, but also by at least a dozen labs in Europe and beyond to investigate human neuronal function.
Second, using these more mature new organoids, we developed a method to perform correlative light and electron microscopy using cryo tomography on organoids, specifically on the axons projecting from human neurons within. This allowed us to investigate the ultrastructure with very high resolutions, revealing unique endoplasmic reticulum morphologies and a paucity of ribosomes. The method resulted in a large number of datasets that can be used for further investigation of human axon biology, and was published in eLife in 2021, along with all the data which was made publicly available.
Third, we have developed a new organoid of the choroid plexus, the brain region responsible for generating cerebrospinal fluid. These organoids form a selective barrier, much like the actual barrier protecting the brain and spinal cord from blood-borne factors and pathogens, as well as secreting cerebrospinal fluid that can be used to investigate normal function as well as biomarkers. This new method was published in Science in 2020, presented at various meetings including the International Society of Stem Cell Research. It has been used in the lab to investigate how the virus causing COVID-19, SARS-CoV-2, enters and infects the brain, which we published in Cell Stem Cell in 2020 and was widely disseminated also in the popular press.
Finally, this project investigated how well different organoids model the human brain and which features of their morphology are key to their proper development. This work led to identification of several key methodological factors that lead to better quality organoids, and reveal an important role for tissue architecture in temporal progression of cell fate decisions. This study was published in Cell Stem Cell in 2023 and disseminated at several international meetings including an EMBO conference in 2022.
2) Using organoids to uncover human-specific features of brain development
Using these state-of-the-art organoid methods, we have uncovered new biological insight into human-specific neurodevelopment. First, by generating brain organoids from human cells and those of closely related apes, we uncovered a difference in neural stem cell behaviour, whereby human neural stem cells go through the early transition to neurogenesis more slowly that apes, leading to a larger founder progenitor pool that is able to generate more neurons. This explains how humans end up with a greater number of neurons than other apes, and thus answers an age-old question. This work was published in Cell in 2021, and was covered in various news outlets and presenting at several international meetings. Second, we investigated the potential role for sex hormones in brain organoids, revealing a role for androgens in the production of excitatory neurons, a role that seems to diverge in humans compared with mice. Thus, again, organoids have revealed evolutionary unique biology that may shed light on important differences in brain development in the presence of androgens like testosterone. This work was published in Nature in 2022 and presented at several international meetings.
This project not only developed new state-of-the-art methods now being used by dozens of labs, but it also revealed new insight into one of the most fundamental questions in biology: what makes us human. Our findings have opened up a new direction of research, and although the project is now finished, the research will continue with future efforts being focused on the genetic and molecular mechanisms underlying the cell biological differences we have uncovered.