Quantitative Identification of Ligand and Receptor Pairs that Drive Astrocyte Development Open Access
Voss, Anna (Spring 2022)
Abstract
Astrocytes, the most abundant cell-type in the central nervous system (CNS), are star-shaped cells that have numerous roles in CNS physiology including neurotransmitter uptake at the synapse, the formation of the blood brain barrier and regulation of neural circuit formation. Many of these functions are developmentally regulated and are tied to the process of astrocyte maturation, but much remains unknown about the mechanisms that trigger this transformation. An important open question about astrocyte development asks what extrinsic signals cause immature astrocyte progenitors to transition into mature, quiescent cells. We hypothesize that ligands, signaling molecules secreted by other cell types in the brain, regulate astrocyte maturation by binding to receptors on astrocyte progenitors and activating downstream mature astrocyte genes. However, a difficult obstacle is identifying (1) which ligands are present in the developing brain that (2) have cognate receptors expressed in astrocyte progenitors and (3) would likely trigger the expression of mature astrocyte genes. In this study, we use quantitative, genome-wide computational tools to decipher these candidate ligand/receptor pairs whose interactions may initiate astrocyte maturation. Using RNA-Seq data of purified CNS cell populations and NicheNet, an algorithm that computationally matches the regulatory potential of ligands and receptors, we identify ten ligand/receptor pairs with corroborating expression data and whose activation is predicted to mediate expression of mature astrocyte genes. To further refine and confirm the ligands and receptors of interest, we use single-cell human data to add cell type specificity and to more confidently validate the candidacy of putative ligand-receptor pairs. Finally, we use this data to systematically test drivers of astrocyte maturation by exposing cortical organoids and purified human fetal astrocytes to candidate ligands and use bulk RNA-Seq to observe the ligands’ effects on maturation. Understanding how astrocyte maturation occurs may help elucidate their contribution to neurodevelopmental disorders, and the tools we develop in this work can be applied more broadly to understand how cell-cell communication drives cellular processes throughout the human body.
Table of Contents
INTRODUCTION 1
METHODS 4
Candidate Ligand Identification 4
Generation of Cortical Organoids 5
Organoid Ligand Exposures 5
RNA-Sequencing Library Preparation 6
RNA-Sequencing Processing and analysis 6
Immunopanning organoid-derived and fetal human astrocytes 7
Immunocytochemistry 8
Astrocyte morphology quantification 8
Immunohistochemistry 8
qPCR 9
RESULTS 9
NicheNet predicts ligand and receptor pairs that influence
astrocyte development 9
Targeted RNA-Sequencing reveals increased astrocyte
signature following ligand exposures 11
Bulk RNA-Sequencing confirms upregulation of
astrocyte genes and downregulation of neuron genes 12
The gliogenic switch occurs between organoid day 70-110 12
Ligand exposures affects astrocyte development
before and after the gliogenic switch 13
Ligands work synergistically, but not individually,
to influence astrocyte development 14
Purified fetal astrocytes validate astrocyte
development in ligand-exposed organoids 15
Ligands drive mature astrocyte morphology
in purified fetal astrocytes 16
DISCUSSION 17
Ongoing Experiments 19
Future Directions 20
FIGURES
1. Determining candidate neuronal ligands and measuring
their effect on astrocyte development 21
2. Organoid ligand exposures at crucial developmental intervals 23
3. Synergistic and individual contributions of candidate ligands 25
4. Impact of ligand 6-10 exposures on fetal tissue gene 27
signatures and morphology 29
REFERENCES 39
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