Abstract
The goal of this project is to elucidate long non-coding RNA Cyrano’s role in the neural differentiation pathway of embryonic stem cells (ESCs). Currently, long non-coding RNAs (lncRNAs) are known to be prolifically expressed, with nonprotein-coding transcripts consisting of a large portion of the human transcriptome, but their functions are understudied and not well understood. The studies that have been executed show that lncRNAs are impactful on gene expression and developmental processes. Further, misregulation of lncRNAs has been implicated in many diseases, including neurodegenerative disorders. It is crucial to investigate Cyrano as a key player in neural differentiation to delineate the function of a paradigmal lncRNA to build the foundation of understanding of lncRNA functions as a whole. Additionally, the implication of Cyrano in neural differentiation will further understanding of the gene expression network
necessary for neural differentiation. Although further studies will be necessary, investigation of Cyrano may prove to be an informative step in understanding gene regulation of neural
differentiation. Our data points to a suppressor role for Cyrano in neural differentiation, with a loss of Cyrano function resulting in an increase in neural differentiation and may suggest an interplay with miR-7 in this context.
Introduction
The plasticity of embryonic stem cells (ESCs) conveys significant potential for their use in regenerative medicine therapies and developmental biology studies. ESCs have the ability to generate cells of each of the three germ layer lineages and hence can derive every cell type of an organism and demonstrate immortality in their ability to self-renew indefinitely in vitro
(Hoffman and Carpenter, 2005; Murry and Keller, 2008). In human development, pluripotent cells equivalent to ESCs exist from day four to day seven post-fertilization, after which they
differentiate to form the developing embryo (Chen et al., 2009). For the purposes of scientific investigation, both human and murine ESC cell lines have been derived from the inner cell mass of blastocysts (Reubinoff et al., 2000). Pluripotency can be induced in somatic cells in certain conditions, prominently through the overexpression of pluripotency factors such Myc, Klf4, and Sox2 (Takahashi et al., 2007). These induced pluripotent (iPS) cells display many of the
characteristics of ESCs and are preferable for regenerative medicine techniques because they can be patient-derived (Yu et al., 2007).
Pluripotent stem cells (PSCs) including ESCs and iPS cells are potentiated to be integral to future regenerative medicine techniques because diseases leading in lethality, such as diabetes and heart and neurodegenerative disorders result from organismal inability to regenerate certain cell types in vivo (Murry and Keller, 2008). It is therefore of interest to efficiently guide lineage commitment of PSCs to replace these absent cell types in patients (Murry and Keller, 2008).
While protocols exist to manipulate culture media to induce differentiation towards either
endoderm, mesoderm, or ectoderm, and their derivatives (Borowiak et al., 2009; Cai and Grabel, 2007; Mae et al., 2013), it is imperative to understand epigenetic changes in ESCs during
differentiation in vitro and how to translate the cellular environment needed for the generation of the targeted lineage-committed population of cells (Murry and Keller, 2008).
A precisely regulated epigenetic landscape is necessary to maintain the pluripotency and self-renewal capacity of ESCs, as well as to guide ESC differentiation (Shen et al., 2009). Well- characterized are the protein-coding transcripts that contribute to these ESC characteristics, including Oct4, Sox2, and Nanog, that code for transcription factors that modulate early cell fate decisions, as well as certain protein-coding genetic markers, such as Sox1 (transcription factor that signals for ectoderm), Brachyury (transcription factor in early mesoderm), and Sox17
(transcription factor in premature endoderm) that indicate early differentiation of ESCs into specific germ layers (Wang et al., 2013). However, due to development in recent transcriptome analysis techniques, it is clear that the majority of transcripts in the mammalian genome do not code for proteins, even though the majority of nucleotides in the genome is transcribed to some degree (Dinger et al., 2008). These other cellular players, noncoding RNAs such as microRNA and long noncoding RNA (lncRNA), have been implicated in the pluripotency and
differentiation of embryonic stem cells, but are less understood in both function and mechanism of action (Dinger et al., 2008).
LncRNA display specific expression profiles that are distinct amongst cell types suggesting context-dependent roles (Saunders et al., 2013). LncRNAs are an RNA class that consists of transcripts 200 nt or more in length and have little to no coding potential (Dinger et al., 2008). LncRNAs vary in conservation amongst different transcripts, but the biological investment in their stabilization, particularly in polyadenylation, 5’ capping, and splicing from precursors, indicates they may hold functional roles (Smith et al., 2017). Previously identified as transcriptional noise, lncRNAs have been tied to the maintenance of pluripotency, differentiation into each germ layer, as well as disease states such tumorigenesis and metastasis (Ponting et al., 2009). Several lncRNAs, such as Malat1 and Xist, have been thoroughly investigated, while the vast majority remain under-studied (Batista and Chang, 2013). There are different classes of lncRNAs based on their transcription direction and location relative to other genes (Yan et al., 2017). These classes can be indicative of potential mechanisms by which the lncRNA functions (Yan et al., 2017). One possible mechanism includes interaction with microRNAs; microRNAs are approximately 22 nt transcripts that function with different proteins such as Argonaute to repress gene expression post-transcriptionally (Heinrich and Dimmeler, 2012). MicroRNAs and
associated proteins assemble to form the RNA induced silencing complex, or RISC complex; the microRNA serves as a guide to find the correct mRNA transcripts, which are degraded in
proportion to the degree of complementarity with the microRNA (Gregory et al., 2005).
Cyrano (OIP5-AS1 in humans) is a lncRNA first discovered in zebrafish (Ulitsky et al.,
2011). It was originally investigated because 67 nt of the lncRNA was shown to be highly
conserved in humans and mice, indicating it may have vital functionality in mammals (Ulitsky et al., 2011). The mouse ortholog of Cyrano is expressed in ESCs and in our previous work, we determined it is integral to the maintenance of ESC pluripotency through inhibition of the differentiation-associated microRNA, mir-7, which targets Nanog, a known pluripotency marker (Smith et al., 2017). In zebrafish, humans, and mice, Cyrano is highly expressed in the nervous system, suggesting a role in nervous tissue ((Ulitsky et al., 2011) Gtex) (Figure 1). Specifically in zebrafish, Cyrano was determined to be crucial for neurogenesis; a knockdown resulted in phenotypes of small heads, decreased abundance of NeuroD-positive neurons in the retina and tectum, and defective neural tube opening, among other deficiencies that led to premature death (Ulitsky et al., 2011). A reintroduction of Cyrano rescued normal neural development, further capitulating Cyrano as the responsible variable for normal neural development (Ulitsky et al., 2011). Based on these data that assign functionality of Cyrano in ESCs and neural development, we investigated the role of Cyrano in the neural differentiation of embryonic stem cells.
Results
Preliminary investigation into Cyrano expression in the developing mammalian brain utilizing publically available RNA-seq data revealed that in neural cell types, Cyrano’s
expression is highest in neurons and neural progenitors cells (Figure 2A, Smith, unpublished).
Expectedly, Cyrano displays high expression in the areas of the brain that contain these cell
types, particularly in the cerebellar hemisphere and cortex, which are neuronal dense (Figure 1B, Gtex, Dr. Keriayn Smith, unpublished), and the subventricular and ventricular zones, which contain neural progenitors. Notably, Cyrano has positive relative expression in these brain segments similar to Hes1, an established neural progenitor marker, while a known non- proliferative marker, FIRRE, has negative relative expression in the subventricular and
ventricular zones (Yang et al., 2015). This expression profile suggested that Cyrano may play a role in the progression to neural cell types of ESCs, in which Cyrano is also expressed and plays a role in maintaining ESC pluripotency (Figure 2A, Smith et al., 2017).
To investigate Cyrano in neural differentiation, several protocols for the conversion of embryonic stem cells into neuroectodermal precursors were optimized for the R1 ESC line (Fluri et al., 2012). Several medium conditions were evaluated based on the resulting cell morphology for the efficiency of neural differentiation. ESCs cultured (1) leukemia-inhibitory factor (LIF) withdrawal with retinoic acid supplementation, (2) N2B27 (see materials and methods), and (3) N2B27 with retinoic acid supplementation were observed for morphological indications of neural differentiation, as all are methods commonly used (Figure 3A) (Ying et al., 2003). After day three of culture, N2B27 was eliminated; the cells in this condition displayed archetypal undifferentiated ESC morphology, including limited intercellular partitions and rounded colonies. At day five of culture, LIF withdrawal with retinoic acid supplementation was
eliminated; the ESCs cultured under these conditions demonstrated morphologies similar to non- neural cell types, including fibroblasts, rather than cells of the neural lineage. N2B27 and
retinoic acid was selected as protocol for neural differentiation; the cells cultured in this condition were elongated and appeared to have projections characteristic of neural cell types.
The differentiation protocol resulted in increased cell death and decreased cellular adherence that
necessitated further optimization for passaging. The experiment was then manipulated in regards to the starting density of cells and cell number after passaging. Cells cultured in N2B27 and retinoic acid were plated at 7,500, 15,000, and 30,000 cells per well and allowed to differentiate for three days. Each starting density was then passaged at varying dilutions, 1:5, 1:10, and 1:20 (Figure 4A). Wells were then selected for gene expression analysis based on elongated cell morphology, prevalence of single cells and low cellular adherence, and branching. Cells were collected at day 10, day 12, and day 14 post plating in N2B27 and retinoic acid. From these, RNA was extracted and cDNA was synthesized. Neural differentiation was confirmed via quantitative real-time polymerase chain reaction analysis (qRT-PCR); neural differentiation markers, from early ectoderm to mature neuron indicators, were upregulated relative to the ESC control in cells cultured in N2B27 and retinoic acid (Figure 4B). From the density and dilution options, 15,000 starting cell count and 1:10 dilution, and 30,000 starting cell count and 1:5 passage dilutions were selected going further based on the consistent upregulation of the majority of neural differentiation markers. Notably, while qRT-PCR analysis indicated that neural differentiation was successful, it also showed that this experimental system resulted in endoderm and mesoderm formation as well, as indicated by the upregulation of markers for these germ layers (Sox17 and Meox, Figure 4B). Because neural differentiation was confirmed,
Cyrano expression was then evaluated relative to an ESC control in samples cultured in N2B27 with retinoic acid supplementation and was found to be upregulated in conditions of neural differentiation (Figure 5B).
A more defined time course of neural differentiation was then pursued. Utilizing a 15,000 cell starting density and 1:10 passage dilution, cells were collected at days one through six in neural differentiation media conditions. Cyrano expression was then analyzed throughout the
time course and was shown to steadily increase with the progression of neural differentiation, apart from day four, which was perturbed by passaging (Figure 4B). Neural differentiation in this time course was ascertained through the upregulation of neural markers. Interestingly, Cyrano expression trends similarly to the expression of intermediate progenitor marker, Eomes, suggesting their roles in the process may intertwined (Figure 4C).
Considering this trend, the role of Cyrano in neural differentiation warranted further investigation. Cyrano was knocked down via short hairpin RNA transfection in ESCs, which were then transferred to N2B27 and retinoic acid. The knockdown had efficiencies of 80-90%
(Figure 6A). Interestingly, the cells under conditions of Cyrano suppression demonstrated morphology that indicated further progression in neural differentiation relative to the non- targeting control. Cells were more spread out, more likely to be individual, more rounded, and had more extensive branching (Figure 6B). This phenotype was apparent at day two (not shown) and continued to day four, when the cells were collected. Gene expression analysis via qRT-PCR revealed an upregulation in neural differentiation markers for the neuronal lineage (Nestin, Figure 6C, Pax6, Figure 6D, and Eomes, Figure 6E). Glial markers did not demonstrate a clear upregulation or downregulation (PDGFRA, Figure 6F). Taken together, these data indicate that Cyrano loss may speed progression of ESCs to a neuronal fate, further suggesting that Cyrano may inhibit the progression of neuronal differentiation.
To dissect a possible mechanism to explain this phenotype, the relationship between Cyrano and microRNA mir-7, which has previously been shown to regulate ESC pluripotency, was investigated (Smith et al., 2017). Mir-7 is differentiation-associated, and has perfect sequence complementarity to a highly conserved binding site in Cyrano (Smith et al., 2017).
Additionally, mir-7 has been functionally implicated in neural cell types, particularly in the
mammalian brain (Piwecka et al. 2017). We investigated mir-7 expression upon Cyrano loss, and found it to be upregulated relative to non-targeting controls in LIF-containing ESC maintenance media (Figure 7A, Smith, unpublished). Interestingly, in a neural differentiation time course, mir-7 expression increases upon early departure from the pluripotent state, but decreases as neural differentiation progresses (Figure 7B). This expression profile may indicate an antagonistic relationship between Cyrano and mir-7 in the context of neural differentiation.
Discussion
LncRNAs are vast in number and expression, but the functions of, and mechanisms by which most act are unclear, especially in the context of embryonic stem cell differentiation (Saunders et al., 2013). Here we describe a potential role of Cyrano, a canonical long noncoding RNA prevalent in ESCs and neural cell types, especially neural progenitors, previously shown to contribute to the maintenance of ESC self-renewal, in neural differentiation (Smith et al., 2017).
The loss of Cyrano in conditions that were demonstrated to induce neural differentiation of ESCs resulted in an upregulation of neuronal lineage markers, suggesting that Cyrano may repress the progression of neural differentiation in vitro. Our results are particularly compelling considering that, while displaying basal high expression, Cyrano is most highly expressed in neurons.
Additionally, Cyrano loss in zebrafish has resulted in less neurons in vivo throughout development. For these reasons, Cyrano was expected to support the conversion of ESCs to neural cell types, while our results indicate the opposite. Further analysis into the mechanism behind this function of Cyrano is needed to clearly understand Cyrano’s role in and the overarching precise epigenetic regulatory controlling neural differentiation. Particularly of promise, microRNAs have proven to be prominent cellular players in regulation of biological
processes, including differentiation (Smith et al., 2017). They have also been shown in numerous contexts to interact with lncRNAs in their mechanisms of action (Gregory et al., 2005). The opposing expression trends of Cyrano and mir-7 may indicated a potential axis by which neural differentiation is regulated. These data indicate that further investigation is necessary regarding Cyrano in neural differentiation, and the epigenetic regulation of stem cell differentiation as a whole.
Materials and Methods ESC Culture
Mouse R1 ESCs were sustained in LIF-containing 10% FBS, 10% KSR, 1mM sodium pyruvate, 2mM L-glutamate, 0.1 mM β-mecaptoethanol, 100U/mL penicillin-streptomycin in Dulbecco’s Modified Eagle Medium. They were grown on 0.1% gelatin-coated six well tissue culture plates and cultured at 37°C at 5% CO2. Media was changed every first or second day. The cells were passaged on every third day.
ESC Differentiation
Mouse R1 ESCs were cultured in the above media for 24 hr, after which they were sustained in 1:1 DMEM/F12 and neurobasal medium (Thermo) with N2 supplement, B27 supplement, 2mM L-glutamate, 0.1 mM β-mecaptoethanol, 100U/mL penicillin-streptomycin. When indicated, media also contained 10-8 M retinoic acid. They were grown on 0.1% gelatin-coated six well tissue culture plates and cultured at 37°C at 5% CO2. Media was changed every first or second day. The cells were passaged on day three.
Transfection
Cyrano was knocked down using three independent short hairpin RNAs. Plasmids were isolated from bacteria in glycerol stocks (OpenBio) using Zyppy Plasmid Miniprep Kit (Zymo). Plasmids were transfected using LTX Lipofectamine kits (Thermo) and GFP-positive cells were selected via puromycin.
RNA Extraction and Quantitative RT-PCR
Total RNA was isolated using the RNeasy Mini Kit (Qiagen) or the Quick-RNA MiniPrep Kit (Zymo), followed by DNase treatment (Ambion) and reverse transcription using the iScript reagent (Bio-Rad). qRT-PCR was completed with SsoFast Evagreen Supermix (Bio-Rad).
miRNAs were extracted with the Quick-RNA MiniPrep Kit (Zymo) and reverse transcription carried out with the TaqMan MicroRNA Reverse Transcription Kit.
Figure Captions
Figure 1. Cyrano expression in mammalian tissue. A) Cyrano expression in whole mouse analyzed by qRT-PCR, displays highest expression in the brain. B) RNA-Seq data from human tissue taken from the Gtex portal shows Cyrano expression is highest in the brain.
Figure 2. Cyrano is highly expressed in proliferative neural cell types and neurons. A)
Expression reads derived from Brainspan indicate that Cyrano is highest of neural cell types in neural stem cells and neurons (Dr. Keriaryn Smith, unpublished). B) Cyrano shows moderate relative expression in the ventricular and subventricular zones of the developing brain, which contain neural stem cells. Data extracted from Brainspan and visualized by the author using RStudio.
Figure 3. Optimization of neural differentiation of ESCs. A) 10X images of cellular morphology in different media conditions aimed to induce neural differentiation. Day in media is indicated
per image. B) Sample morphology and colony organization for cells passaged with a 1:10 dilution.
Figure 4. Neural markers upregulated in N2B27 and retinoic acid supplementation. d=day in N2B27 and retinoic acid upon collection, 1-3 indicates different starting densities and passage dilutions.
Figure 5. Cyrano expression increases during neural differentiation. A) Cyrano is upregulated in late neural differentiation relative to ESC control. B) Cyrano steadily increase in time course of neural differentiation, d=day in N2B27 and retinoic acid. C) Cyrano trends similarly to Eomes, an intermediate progenitor marker.
Figure 6. Cyrano loss causes aberrant neural differentiation. A) Cyrano knockdown efficiency was 80-90%. B) Differences in morphology upon Cyrano loss in neural differentiation. Images taken at d4 in N2B27 and retinoic acid. C) Nestin upregulated relative to non-targeting control in N2B27 and retinoic acid. 200 indicates 200,000 cell starting density. LIF samples indicate Cyrano knockdown in ESC maintenance media for comparison. D) Pax6 upregulated relative to non-targeting control in N2B27 and retinoic acid. 200 indicates 200,000 cell starting density. LIF samples indicate Cyrano knockdown in ESC maintenance media for comparison. E) Eomes upregulated relative to non-targeting control in N2B27 and retinoic acid. 200 indicates 200,000 cell starting density. LIF samples indicate Cyrano knockdown in ESC maintenance media for comparison. F) PDGFRA with no clear trend relative to non-targeting control in N2B27 and retinoic acid. 200 indicates 200,000 cell starting density. LIF samples indicate Cyrano knockdown in ESC maintenance media for comparison.
Figure 7. Mir-7 expression dynamics in Cyrano KD and neural differentiation. A) Mir-7 expression increases in ESC maintenance media relative to non-targeting control. B) Mir-7 increases then decreases in neural differentiation induced by N2B27 and retinoic acid.
Acknowledgements
I would like to thank my mentor, Dr. Keriayn Smith, for dedicating the time to help me become a scientific researcher, Dr. Terry Magnuson, for his continued support in my education and career as a researcher, and the rest of the lab for helpful commentary, instruction, and support.
Additionally, I would like to thank Dr. Jesse Raab for his continued help developing my computational and genetics skills. The funding for the project was provided in part by the Summer Undergraduate Research Fellowship and the Sigma Xi Grant-in-Aid of Research.
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