Periodic Reporting for period 3 - LinPro (Principles of Neural Stem Cell Lineage Progression in Cerebral Cortex Development)
Reporting period: 2020-12-01 to 2022-05-31
The cerebral cortex consists of an extraordinary number and great diversity of neurons. Yet, how the cortical entity, with all its functional neuronal circuits, arises from the neural stem cells (NSCs) during development is a fundamental unsolved question in Neuroscience. In a pursuit to obtain definitive insights into this question we assess NSC lineage progression at unprecedented single cell resolution, using the unique genetic MADM (Mosaic Analysis with Double Markers) technology. MADM offers an unparalleled approach to visualize and concomitantly manipulate sparse clones and small subsets of genetically defined neurons. Within the scope of this project we use multidisciplinary experimental approaches to pursue a research program with the following major objectives: We will 1) Functionally dissect the intrinsic genetic requirements and effects from the environment in NSC lineage progression; 2) Define the principles of lineage progression in human NSCs in situ using MADM technology in cerebral organoid system; 3) Decipher the logic and mechanisms of glia lineage progression in the neocortex. The primary goal of the proposed research is to establish a definitive quantitative framework and mechanistic model of lineage progression in cortical NSCs. Ultimately, our results shall translate into a deeper understanding of brain function and why human brain development is so sensitive to disruption of particular signaling pathways in pathological neurodevelopmental and psychiatric disorders.
In this first period of the project we have established and validated a number of genetic MADM tools and resources that are essential for the entire project. We have successfully established RNA sequencing protocols to analyse transcriptomes at single cell level in MADM paradigm, and inaugurated cell culture protocols and started with the generation of cerebral organoids for the prospective study of NSC lineage progression in human context. We have also established novel quantitative MADM-based experimental paradigms at single RGP resolution to define the cell-autonomous functions of candidate genes and signalling pathways controlling RGP-mediated cortical neuron and glia genesis. A key aspect of the project is the functional analysis of candidate genes to define their role in NSC lineage progression. Along this line we have found novel functions for genes encoding epigenetic regulators in NSC lineage progression. In collaboration we also investigated lineage progression of embryonic and adult stem cells that are essential for cortical development, and established mathematical models by using MADM data sets. Altogether our efforts and results provide new insights into the logic of neural stem cell lineage progression.
The most innovative aspect of this proposal lies in its interdisciplinary approach to address a fundamental question in neuroscience: What are the cellular and molecular mechanisms in cortical stem cell progenitors regulating the balance between proliferation and differentiation into neurons and/or glia cells, to specify the cerebral cortex of the correct size and cellular composition? While previous efforts greatly contributed to our current framework of neocortical genesis, experimental paradigms were mostly based upon whole population approaches (e.g. full and/or conditional knockout studies). However, the lack of true single cell resolution of progeny fate vital for dissecting progenitor division patterns has previously precluded a definitive understanding. MADM offers an unparalleled solution and permits quantitative clonal analysis, concurrent with genetic manipulation, of precise division patterns and lineage progression at unprecedented individual progenitor cell resolution. The future research along the LinPro project promises significant new insights, and shall provide a definitive quantitative mechanistic understanding of neural stem cell lineage progression and cortical development at single cell resolution. Our findings may potentially also be translated to other brain regions. Ultimately, such advances can result in a deeper understanding of brain function and why human brain development is so sensitive to disruption of particular signalling pathways in pathological neurodevelopmental or psychiatric disorders. In a broader context, the anticipated results likely also contribute to our knowledge of cortical neuron and/or glia specification, and may reveal a logic that can generate cellular diversity; thus providing a possible foundation for prospective future embryonic stem cell-based approaches in the context of directed brain repair.