Date

1-2015

Document Type

Dissertation

Degree Name

Ph.D.

Institution

Oregon Health & Science University

Abstract

During central nervous system (CNS) development, neural progenitor cells undergo dramatic changes in gene expression to differentiate into diverse types of neurons. One of the fundamental challenges in neurobiology is to understand the molecular mechanisms that drive this drastic and thorough transformation of the gene expression profile. Previous research suggests that transcription factors are the primary regulators of gene expression changes during neurogenesis. However, recent studies have shown that microRNAs (miRNAs) are also important components of the gene regulatory networks that direct neuronal cell fate. The extent to which miRNAs collaborate with transcription factors in the gene network that determines neuronal identity remains unclear. Previous work in the Lee laboratory has shown that two LIM-homeodomain factors, LIM homeobox 3 (Lhx3) and Islet-1 (Isl1), form a transcription complex that is a potent driver of spinal motor neuron fate specification. Work in our laboratory and others have shown that the Isl1-Lhx3 complex directly upregulates genes that promote motor neuron characteristics. To determine whether miRNAs are also upregulated by Isl1-Lhx3, I performed a miRNA array in Isl1-Lhx3-induced mouse embryonic stem cells (Isl1- Lhx3 ESCs). This experiment showed that miR-218 is uniquely and highly upregulated during Isl1-Lhx3 ESC motor neuron differentiation. The fact that miR-218 is highly induced during Isl1-Lhx3-directed motor neurogenesis in Isl1-Lhx3 ESCs led us to investigate whether miR-218 expression is directly controlled by the Isl1-Lhx3 complex. The analysis of our chromatin immunoprecipitation deep sequencing (ChIP-seq) data from Isl1-Lhx3 ESCs uncovered potential Isl1-Lhx3-bound ChIP-seq peaks near both miR-218-1 and miR-218-2 genes. To validate these ChIP-seq peaks, we performed ChIP experiments in Isl1-Lhx3 ESCs and embryonic mouse spinal cord. We found that the ChIP-seq peak regions near both miR-218 genomic loci are occupied by the Isl1-Lhx3 complex in Isl1-Lhx3 ESCs and the developing spinal cord. Altogether, these results strongly suggest that miR-218 is directly upregulated by Isl1-Lhx3 during spinal motor neuron differentiation. The robust expression of miR-218 in Isl1-Lhx3 ESC-derived motor neurons led me to test whether miR-218 is upregulated in motor neurons in vivo. I performed in situ hybridization analyses in developing mouse and chick spinal cords, which showed that miR-218 is exclusively expressed in motor neurons throughout embryonic spinal cord development. Additionally, miR-218 expression began at the onset of motor neuron differentiation and endogenous miR-218 activity was sufficient to repress the expression of synthetic miR-218 target mRNAs specifically in spinal motor neurons. To examine whether miR-218 is important for motor neuron development, I designed a miR-218 sponge and a 2’O methyl RNA antisense inhibitor and performed loss-of-function studies. Using in ovo electroporation, I found that inhibition of miR-218 resulted in a small, but significant (10%) reduction of motor neurons in embryonic spinal cord. Additionally, we generated mouse ESC lines, which express either miR-218 sponge or scramble sponge in a doxycycline-dependent manner, and found that miR-218 was essential for the generation of motor neurons from ESCs. In order to understand the function of a miRNA, it is important to identify authentic target mRNAs. To determine direct miR-218 targets, I collaborated with the Goodman laboratory to perform RISC-trap screens in HEK293T cells. The RISC-trap experiments identified numerous novel miR-218 target mRNAs as well as previously known miR-218 targets. Remarkably, some of the miR-218 targets have well established roles in progenitor cell maintenance or interneuron differentiation in the developing spinal cord. Using target 3’UTR reporter assays both in vitro and in vivo, I further validated five miR-218 target mRNAs: TEA Domain Family Member 1 (Tead1), Solute Carrier Family 6 Member 1 (SLC6A1), B-Cell CLL/Lymphoma 11A (BCL11A), LIM homeodomain 1 (Lhx1), and Forkhead box protein 2 (Foxp2). Next, to test whether misexpression of miR-218 inhibits the expression of the newly identified miR-218 target mRNAs, as well as interneuron or progenitor fates, I designed a miR-218 overexpression construct and performed in ovo electroporation. Overexpression of miR-218 in the developing chick neural tube significantly decreased interneurons, but did not have a significant effect on the number of neural progenitors or motor neurons. Additionally, we generated mouse ESC lines that constitutively express miR-218 or a control miRNA. When we directed these miRNA-expressing ESCs to differentiate into interneurons, miR-218 repressed the expression of interneuron markers, while it did not affect the expression of a broad neuronal marker. Together, these gain-offunction experiments validated RISC-trap target the interneuron genes, and provided strong evidence that miR-218 downregulates the expression of genes that promote interneuron programs. Our data show that miR-218 is upregulated by the Isl1-Lhx3 complex and is important for the generation of motor neurons in vitro and in vivo. Our data also demonstrate that miR-218 downregulates target mRNAs that are important for interneuron differentiation. However, these experiments did not directly assess whether miR-218 activity is important for motor neuron fate specification downstream of the Isl1- Lhx3 complex. Previous work in the Lee laboratory has shown that electroporation of Isl1-Lhx3 generates ectopic motor neurons in the dorsal spinal cord. To assess whether miR-218, which is induced by Isl1-Lhx3, is important in the gene regulatory network that determines motor neuron fate, I performed co-electroporation experiments with Isl1 and Lhx3 and either miR-218 sponge or scramble sponge inhibitor. These experiments revealed that inhibition of miR-218 activity significantly reduces the ability of the Isl1- Lhx3 complex to generate ectopic motor neurons at the expense of interneurons. These data support our major finding that miR-218 is essential for motor neuron differentiation downstream of Isl1-Lhx3, and provide further evidence that miR-218 functions to establish motor neuron identity by repressing the expression of genes that promote interneuron characteristics. In addition to investigating the role of miR-218 in motor neuron development, I also identified other miRNAs that are upregulated during motor neuron differentiation. Further analysis of the activity of multiple motor neuron miRNA candidates revealed that numerous miRNAs may have dynamic spatiotemporal expression pattern in the developing spinal cord. In particular, miR-153 was identified as a promising motor neuron miRNA candidate, and further investigation of miR-153 expression suggests that miR-153 may play a role in spinal cord neurogenesis. Additionally, previous work in the Lee laboratory show that miR-218 and miR-153 co-regulate an axon guidance factor roundabout 2 (Robo2). I performed a Robo2 3’UTR sensor analysis and found that endogenous miR-218 and miR-153 combinatorially regulate Robo2 expression in motor neurons. This result suggests that combinatorial function of miRNAs is important to effectively repress target mRNAs.

Identifier

doi:10.6083/M4697299

Division

Neuroscience Graduate Program

School

School of Medicine

Available for download on Monday, January 15, 2018

Included in

Neurosciences Commons

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