Exploring the Driving Factors of Human Astrocyte Development Restricted; Files Only
Vera Pimentel, Natali (Spring 2024)
Published
Abstract
Human brain development is a complex and highly orchestrated process involving multiple components. Early brain development involves neural stem cells, also known as radial glia, generating neurons and eventually switching to generating astrocytes, a glial cell, in a time-dependent manner. This cell fate transition is termed the “gliogenic switch”. Disruption to this switch may contribute to neurodevelopmental disorders, such as autism spectrum disorder (ASD) and Down syndrome. Various intrinsic and extrinsic signals, such as transcription factor (TF) activity and cytokine signaling, are essential for initiating the gliogenic switch. We are interested in manipulating TFs to better understand the driving factors behind human astrocyte development. We first focused on knocking down neurogenic genes by using short-hairpin RNA (shRNA) lentiviruses. Knocking down master regulators of neurogenesis at different time points before the gliogenic switch can provide insight into the specific roles that neurogenesis has on astrogenesis. We chose to knock down NEUROGENIN2 (NGN2) by creating NGN2 shRNA lentiviruses and infecting human cortical organoids (hCOs) and human induced pluripotent stem cell (hiPSC)-derived NPCs. Due to complications with our positive control, we were unable to properly assess the impact of NGN2 knockdown. We identified gliogenic TFs through paired RNA-seq and ATAC-seq analyses and were interested in overexpressing these TFs to observe the effect on astrocyte production. We started with RFX4 and SOX21 overexpression lentiviruses and, unfortunately, ran into fluorescence issues that prevented further investigation of these two TFs. We next focused on LHX2 and began infecting hiPSC-derived NPCs and human fetal tissue at different time points. The hiPSC-NPCs were infected with LHX2 at different stages of differentiation and showed a suppression of neurogenic genes and a promotion of astrocytic genes at early time points. The human fetal tissue was infected with LHX2 at different time points before and after the gliogenic switch and presented significant morphological changes after the gliogenic switch. The combined trends from the LHX2 infections suggest this TF assumes different neuronal and glial responsibilities based on timing. This project provides insights into the role of these specific TFs on the gliogenic switch, further developing our understanding of human astrocyte development.
Table of Contents
Chapter 1: General Introduction 1
Chapter 2: Approaches for Knocking Down Neurogenic Genes 5
Chapter 3: Overexpression of Transcription Factors 7
Chapter 4: Methods 9
Chapter 5: Results 17
Chapter 6: Discussion and Future Directions 32
References 36
About this Honors Thesis
| School | |
|---|---|
| Department | |
| Degree | |
| Submission | |
| Language |
|
| Research Field | |
| Parola chiave | |
| Committee Chair / Thesis Advisor | |
| Committee Members |
Primary PDF
| Thumbnail | Title | Date Uploaded | Actions |
|---|---|---|---|
|
|
File download under embargo until 24 May 2025 | 2024-04-09 18:20:03 -0400 | File download under embargo until 24 May 2025 |
Supplemental Files
| Thumbnail | Title | Date Uploaded | Actions |
|---|