Periodic Reporting for period 4 - LinPro (Principles of Neural Stem Cell Lineage Progression in Cerebral Cortex Development)
Reporting period: 2022-06-01 to 2022-11-30
How to build a brain of correct size is an outstanding and fundamental question in Neuroscience. The cerebral cortex is responsible for higher cognitive functions and 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 major unsolved question. In a pursuit to obtain definitive insights into this question we assessed 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 used multidisciplinary experimental approaches to pursue a research program with the following major objectives: We 1) Functionally dissected the intrinsic genetic requirements and effects from the environment in NSC lineage progression; 2) Defined the principles of lineage progression in human NSCs in situ using MADM technology in cerebral organoid system; 3) Deciphered the logic and mechanisms of glia lineage progression in the neocortex. Altogether we achieved our ultimate goal, to establish a definitive quantitative framework and mechanistic model of lineage progression in cortical NSCs. In a broader context, our results also 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 project we have established and validated a number of genetic MADM tools, and resources that were essential for the entire project. These MADM resources have been made available to the global mouse genetics community as a direct output of the project. During the entire project we have also been engaged in many outreach activities and promoted open science initiatives such as the SCOPES project (Sparking Curiosity Through Open-Source Platforms in Education and Science). These resources shall contribute to the education of the next generation of STEM students already in primary school. We published technical protocols to ease the use of the MADM technology. We have successfully established RNA sequencing protocols to analyze transcriptomes at single cell level in MADM paradigm. Further, we established cell culture protocols and successfully used MADM paradigm in the context of cerebral organoids for the study of NSC lineage progression in self-organizing systems. These efforts also provide now a basis for future follow-up studies 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 signaling pathways controlling RGP-mediated cortical neuron and glia genesis. A key aspect of the project was the functional analysis of candidate genes to define their role in NSC lineage progression. Along this line we have found many novel functions for a list of critical candidate genes, that also included epigenetic regulators, in NSC lineage progression. In collaboration we also investigated lineage progression of adult stem cells, established mathematical models by using MADM data sets and probed the principles generating neuronal diversity in distinct stem cell niches. Based on these foundations and the obtained key results during the project we basically fulfilled the ultimate goal of the entire project to establish a definitive quantitative framework and mechanistic model of neural stem cell lineage progression during cortical development.
The most innovative aspect of this proposal lied 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 offered an unparalleled solution and permitted quantitative clonal analysis, concurrent with genetic manipulation, of precise division patterns and lineage progression at unprecedented individual progenitor cell resolution. The completed research along the LinPro project led to significant new insights, and provided an inaugural but definitive quantitative mechanistic understanding of neural stem cell lineage progression and cortical development at single cell resolution. Our findings are currently being translated to other brain regions and new research projects will determine the generality of our findings. In a broader context, our findings along LinPro also provide deeper understanding of brain function and why human brain development is so sensitive to disruption of particular signaling pathways in pathological neurodevelopmental disorders. Our obtained results also contributed to our knowledge of cortical neuron and/or glia specification and thus provide a basic foundation for prospective embryonic stem cell-based approaches in the context of directed brain repair.