Identifying Drivers of Human Astrocyte Development Restricted; Files Only

Abstract

The development of the central nervous system (CNS) is a complex process characterized by the orchestration of highly specialized cells into intricate structures that form the architecture of the brain. Within this dynamic landscape, neural stem cells, also called radial glia, play a pivotal role, giving rise to the diverse array of cells that shape the CNS. With precise temporal resolution, radial glia produce neurons, followed by astrocytes and oligodendrocytes. Understanding this intricate process necessitates advanced human model systems that faithfully recapitulate the complexities of neurodevelopment. We first explore human stem cell models of glial development, including 2D cell cultures, 3D organoids, and bioengineered systems. We next delve into the extrinsic and intrinsic factors governing astrocyte fate commitment. We identified key ligand–receptor interactions and canonically neurogenic transcription factors involved in driving the gliogenic switch. Using published single cell sequencing data of the developing human brain, we identified 5 neuronally-secreted ligands that bind to radial glial receptors and drive changes in astrocyte gene expression: TGFβ2, NLGN1, TSLP, DKK1, and BMP4. These ligands synergistically cooperate to steer human astrogenesis with their combinatorial effects converging on the mTORC1 signaling pathway. Simultaneously, we employed paired expression and chromatin accessibility of human cortical organoids across development to identify key regulators of the gliogenic switch. We identified transcription factors canonically involved in neurogenesis that also drive astrogenesis, including FOXG1 and LHX2. These findings challenge the conventional roles of neurogenic factors by revealing their pivotal influence on astrocyte development. Overexpression experiments confirm the impact of these intrinsic regulators, unveiling gliogenic pathway activation and potential interactions with NFIA and SOX9. Altogether, leveraging human model systems to identify both extrinsic and intrinsic regulators of the gliogenic switch provide profound insights into the molecular mechanisms dictating human neurodevelopmental processes.

Table of Contents

Chapter 1: Introduction

1.1 Glial Development

1.1.1 Astrogenesis and Oligodendrogenesis

1.1.2 Microglial Ontogeny

1.1.3 Human-Specific Features of Glia

1.1.4 Limitations to Studying Human Glia

1.2 Human Stem Cell Models of Gliogenesis

1.2.1 Modeling Neurological Disorders

1.2.2 Validating Glial Identity

1.2.3 2D Models

1.2.4 3D Models

1.2.5 Bioengineered Models

1.3 Summary and Gaps

Chapter 2: Identification of ligand–receptor pairs that drive human astrocyte development

2.1 Abstract

2.2 Introduction

2.3 Results

2.3.1 NicheNet predicts ligand–receptor drivers of astrogenesis

2.3.2 The gliogenic switch occurs reproducibly around day 90 in hCOs.

2.3.3 Timing of ligand exposure affects astrocyte development

2.3.4 Cognate receptors of ligands are developmentally regulated

2.3.5 Ligands synergistically influence hCO astrocyte development

2.3.6 Candidate ligands impact human fetal astrocyte development

2.3.7 Ligands drive mature morphology in purified fetal astrocytes

2.3.8 Ligands act independently of LIF/CNTF activation paradigms

2.3.9 Ligand exposure biases toward astrocyte differentiation

2.3.10 Ligand cocktail converges on mTORC1 signaling to promote astrocyte development

2.4 Discussion

2.5 Methods

Chapter 3: Gliogenic capacity of neurogenic transcription factors LHX2 and FOXG1 during human astrogenesis

3.1 Abstract

3.2 Introduction

3.3 Results

3.3.1 Temporal Dynamics and Transcriptional Regulation in Human Astrocyte Development

3.3.2 FOXG1 and LHX2 Are Present in Fetal Human Astrocytes

3.3.3 FOXG1 and LHX2 Overexpression Biases Towards Astrogenesis

3.3.4 LHX2 Overexpression Does Not Activate MAPK or STAT3 Signaling Pathways.

3.3.5 LHX2 Induces Morphological Changes Around the Gliogenic Switch

3.3.6 Time Course of LHX2 Overexpression

3.3.7 LHX2 May Interact with NFIA and SOX9

3.4 Discussion

3.5 Methods

Chapter 4: Summary, Limitations, and Future Directions

4.1 Summary

4.2 Integrating Extrinsic Signaling and Intrinsic Transcriptional Regulation

4.3 Identifying Key Regulatory Pathways and Dynamic Changes

4.4 Overcoming Challenges with Astrocyte Markers

4.5 Elucidating LHX2 Interactions

4.6 Conclusion

Appendix A: Neuronally Secreted Signaling

References

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Genetics and Molecular Biology
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Biology, Genetics Biology, Neuroscience
Mot-clé	Gliogenic Switch Astrocytes Neurodevelopment
Committee Chair / Thesis Advisor	Steven Sloan, Emory University
Committee Members	Yue Feng, Emory University Jennifer Spangle, Emory University Kenneth Moberg, Emory University Bing Yao, Emory University

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