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A study of matr3 and its effects on the neural progenitor cells of Xenopus laevis tadpoles
Kendall Branham
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Recommended Citation Branham, Kendall, "A study of matr3 and its effects on the neural progenitor cells of Xenopus laevis tadpoles" (2020). Undergraduate Honors Theses. Paper 1564. https://scholarworks.wm.edu/honorstheses/1564
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A study of matr3 and its effects on the neural progenitor cells of Xenopus laevis tadpoles
A thesis submitted in partial fulfillment of the requirement for the degree of Bachelor of Science in Neuroscience from The College of William and Mary
by
Kendall Robyn Branham
Accepted for Honors
Robin Looft-Wilson Robin Looft-Wilson, Ph.D, Director
_____Jennifer Bestman______Jennifer Bestman, Ph.D
____Diane Shakes______Diane Shakes, Ph.D
Dana Willner Dana Willner, Ph.D
Williamsburg, VA May 5, 2020
Acknowledgements
The process of this thesis coming to a close has been very unexpected and unusual, but I am so grateful for the support I have received during this time.
I would first like to thank Dr. Jennifer Bestman for always believing in me and offering me continuous advice since I entered her lab in 2018. She has always encouraged my curiosity in neuroscience and pursuit of my interests as well as provided me the means to engage in meaningful research. The personal and academic growth I have experienced in my time spent in the Bestman lab means so much to me and I am so thankful for Dr. Bestman’s unwavering support during my time at William & Mary.
I would also like to thank the other members of my lab; Martin Feng, Alyssa Luz-Ricca,
Adithi Ramakrishnan, and Sophia Hernandez. I spent many hours alongside them and am very thankful for their input, assistance, UNO games, and laughter throughout this project.
Thank you to Dr. Diane Shakes and Dr. Dana Willner for being willing to review my thesis, even in these unforeseen circumstances. Some of my favorite classes have been taught by these two professors and I am grateful for their contributions to my academic success and scientific understanding.
Finally, I would like to thank my family for their continuous support and love throughout this entire process. Without their help, I would not have been able to complete this thesis and for their encouragement I am forever grateful.
Table of Contents
Acknowledgements………………………………………………………………………………………………………………….2
List of Figures……………………………………………………………………………………………………………………….....4
List of Tables…………………………………………………………………………………………………………………………….5
Abstract……………………………………………………………………………………………………………………………………6
Introduction…………………………………………………………………………………………………………………………….7
Materials and Methods…………………………………………………………………………………………………………..16
Results…………………………………………………………………………………………………………………………………….23
Discussion……………………………………………………………………………………………………………………………….32
Conclusions…………………………………………………………………………………………………………………………….35
References………………………………………………………………………………………………………………………………36
List of Figures
Figure 1. Proliferation of Radial Glial Cells…………………………………………………………………………..7
Figure 2. Position of a RGC in the X. laevis Optic Tectum……………………………………………………..9
Figure 3. Effects of Inability to Phosphorylate Matrin-3………………………………………………………12
Figure 4. Anatomy of a X. laevis tadpole ……………………………………………………………………………13
Figure 5. Electroporation Technique…………………………………………………………………………………..20
Figure 6. Multiple Sequence Alignment of Matrin-3 protein……………………………………………….23
Figure 7. Progenitor Cells in the Left Tectum………………………………………………………………………24
Figure 8. Neuron in the Right Tectum…………………………………………………………………………………25
Figure 9. Matr3 Isolation and BP Recombination……………………………………………………………….26
Figure 10. KB012 and KB014 Plasmids……………………………………………………………………………….27
Figure 11. Expected Result of Phospho-matrin-3 overexpression in Radial Glial Cells…………29
Figure 12. Matrin-3 Overexpression Induces Cell Cycle Alterations…………………………………….30
List of Tables
Table 1. PCR Specifications for Matr3 Isolation………………………………………………………………….18
Table 2. Gateway Reaction Reagents………………………………………………………………………………..18
Table 3. Primers………………………………………………………………………………………………………………..19
Abstract
Understanding the genes and mechanisms that regulate the highly important process of neurogenesis in the developing nervous system is crucial to uncovering why disease arises when this system is disrupted. Neural progenitor cells and their multipotent nature allow for the growth of a large pool of stem cells in the early stages of development, but the signal that leads these cells to the irreversible fate of becoming a neuron is unclear. The gene matr3 has been implicated in having a role in the maintenance of undifferentiated neural stem cells but its role needs further investigation to fully reveal how its nuclear protein matrin-3 may be affecting this process. Overexpression of this gene through the introduction of plasmid DNA in vivo is expected to maintain the pool of progenitor cells for an extended period of time in the brains of Xenopus laevis tadpoles. Whole brain electroporation and live imaging studies will hopefully reveal the effects of this gene’s overexpression on radial glial progenitor cells within the X. laevis optic tectum and would allow for an increase in the knowledge of how matr3 potentially plays a role in human brain development .
Introduction
Neurogenesis
Neurogenesis is the complex process that establishes the structure and function of the developing brain and requires strict regulation. The multipotent, proliferative neural stem cells
(NSCs) that initiate this process are key to the proper and complete development of the central nervous system (CNS)(Gotz & Huttner, 2005). NSCs are capable of producing the major cells of the nervous system, neurons and glia, but the mechanisms that control this process remain unclear. Neural stem cells undergo various types of divisions which allows for the growth of the
nervous system into well-defined layers.
These cells begin by undergoing
symmetric division to produce more stem
cells before shifting into asymmetric
divisions and symmetric neurogenic
divisions (Borrell & Calegari, 2014). These
initial divisions allow for the thickening of
cortical walls as well as the formation of
distinct layers from the most medial
ventricular zone (VZ) to the lateral
marginal zone (MZ) in mammals
(Penisson, Ladewig, Belvindrah, &
Francis, 2019). This then begins the production of neurons as well as intermediate progenitors cells (IPCs) that undergo symmetric division at the basal membrane to produce pairs of IPCs or neurons, and radial glial cells which divide at the apical or ventricular surface to self-renew as more radial glia or asymmetrically divide (Gotz & Huttner, 2005)(Figure 1). It was originally thought that radial glia were purely structural, but through imaging studies, it is now understood that these cells are true progenitors that play a large role in the developing nervous system (Noctor et al., 2001). Understanding the underlying cellular mechanisms that govern these cells’ divisions, is crucial to fully comprehending the process of nervous system development.
One of the main features of radial glial cells that is most notable, is the long slender process that connects its soma to the ventricular surface, and its endfoot to the pial surface of the brain (Penisson, Ladewig, Belvindrah, & Francis, 2019)(Figure 2). In mammals, throughout the cell cycle the soma of these cells is known to move throughout the ventricular zone before undergoing symmetric or asymmetric division (Pinto & Gotz, 2007). If a radial glia cell is to commit to the fate of a neuron, studies have shown a shorter time in S phase as a marker of this transition (Arai et al., 2011). While factors such as these have been shown to influence the neurogenic fate of radial glia, cellular pathways and regulatory genes involved are constantly being discovered. A potential gene involved in the maintenance of the multipotent cells is matr3, whose nuclear protein matrin-3 has been seen in the ventricular and subventricularzones of mouse embryonic tissue (Niimori-Kita, Tamamaki, Koizumi, & Niimori,
2018).
The goal of this research is to understand the role of the gene Matr3 during early nervous system development and how its actions may affect the radial glial cell progenitor pool.
The gene matr3
Matr3 is a gene well studied for its association with Amyotrophic Lateral Sclerosis (ALS) when its normal function is disrupted, but its role in the process of neurogenesis is not well understood (Malik et al., 2018). In humans, it is located on chromosome 5 and encodes for the nuclear matrix protein matrin3 which can be found across numerous organisms, including
Xenopus laevis. This gene is in the family small nucleolar RNA (snoRNA) protein coding host genes, a subgroup of snoRNA host genes. In particular, human Matr3 hosts the snoRNA U19. This family contains 145 known protein coding genes which serve as hosts for snoRNAs in their introns including other notable genes like Translocase of Inner Mitochondrial Membrane 23
(TIMM23) (HUGO Gene Nomenclature Committee, 2019). In X. laevis this gene is found on chromosome 3 and while it is of the same family as human matr3, the same U19 snoRNA is not being hosted.
Genetic variations in the gene matr3 have been shown to cause mutations to its protein leading to the neurodegenerative disorder ALS. Researchers have identified two primary locations within the protein where these mutations are most likely to occur in humans. Amino acids 66-154 are known to contain six possible mutation sites while amino acids 610-787 contain five others. These alterations in matrin-3 have been shown to interfere with its protein- protein interactions and role in mRNA export (Boehringer et al., 2017). Mutations within other
RNA binding proteins associated with ALS have been shown to affect mRNA translation in X. laevis, but how matr3 mutations affect this organism is not well studied (Bray, 2015).
In the cell, matrin-3 is solely localized to the nucleus and nuclear matrix and serves as both a structural and functional role in cellular activities (Coelho, Attig, Ule, & Smith, 2016). As a nuclear matrix protein, matrin-3 plays a role in DNA organization and replication as well as gene transcription and expression. It has shown to enhance transcription, it binds to specific miRNAs to prevent their nuclear export and affect aspects mRNA splicing (Weiss, Treiber,
Meister, & Schratt, 2019; Malik et al., 2018; Coelho et al., 2015). It contains two tandem RNA recognition motif (RRM) RNA binding domains, two zinc finger (Znf) domains, and a nuclear localization signal (NLS) as well as many interspersed intrinsically disordered regions. The protein also has a nuclear export signal (NES) despite no evidence of having a function in the cytoplasm that has been discovered (Coelho, Attig,Ule, & Smith, 2016). These domains are conserved across humans and Xenopus laevis.
The most common post-translational modifications matrin3 undergoes are ubiquitination and phosphorylation (Iradi et al., 2018). This phosphorylation is potentially one of the key mechanisms underlying this protein’s role in maintaining undifferentiated progenitor cells. In a study by Niimori-Kita, Tamamaki, Koizumi, & Niimori in 2018, researchers discovered phosphorylation by ATM kinase at the Ser208 residue of matrin-3 results in radial glia cells maintaining an undifferentiated state for longer in mice. Typically, ATM kinase is associated with DNA damage but it plays a different role in the phosphorylation of matrin-3. Part of the mechanism that is required for this phosphorylation to occur is the presence of fibroblast growth factor 2 (FGF2). In mammals, phospho-Matrin-3 is found in abundance in SVZ and VZ layers where progenitor cells originate. It is theorized that the phosphorylation by ATM kinase is crucial to the localization of matrin-3 to these particular areas during development. In the absence of FGF2, progenitor cells are seen undergoing neuronal differentiation, and matrin-3 is exported from the nucleus of the newly formed neuron (Niimori-Kita, Tamamaki, Koizumi, &
Niimori, 2018). In summary, for matrin-3 to maintain radial glial cells in an undifferentiated state in mammals, it requires FGF2 as well as ATM kinase activity. The serine residue in this protein is conserved from humans to X. laevis in some isoforms of the protein, and in Xenopus
FGF is known to bind to FGF receptors to signal various cellular events including kinase activity
(Brunsdon & Isaacs, 2020) (Figure 3) . These similarities may allow for an identical mechanism of phosphorylation mediated activity in both mammals and Xenopus.
The Xenopus laevis Animal Model
Xenopus laevis or the African Clawed frog are widely recognized for their use as a model organism in research due to their similarities to humans. This amphibian has been used in developmental biology studies for over 50 years, allowing for techniques using these animals to be fine-tuned over time. In addition, 90% of the organism's genes have been shown to be homologs to those in humans with a high amount of sequence conservation (Blum & Ott, 2018)
(Vergara & Del Rio-Tsonis, 2009). Another important aspect besides being genetically similar is that X. laevis have similar developmental patterns to humans. Understanding this process of
neural progenitor differentiation and the
regulatory mechanisms of this process in X.
laevis could allow for advancements in
knowledge of how this process occurs in
humans. One of the key similarities between
these two species is the presence of radial glial
cells and their role as the major progenitors
that establish the developing brain (Howard et
al., 2008). In the X. laevis tadpole, the optic
tectum, the region where visual neurons called
retinal ganglion cells begin synapsing to
establish a visual circuit, is a site of cell
proliferation throughout tadpole development
(Figure 4). This being a region so crucial to the organism’s survival, many new cells must be produced in the optic tectum to allow for proper visual system development (Kaslin, Ganz, & Brand, 2008). Due to the distinct stages involved in
X. laevis development, it is very accessible to visual these rapidly dividing cells using techniques such as fluorescent tagging. Sox2 and Transfection
One mechanism to transfect these progenitor cells is through electroporation techniques. Whole brain electroporation allows various cells of the brain to take up specific genetic material to be expressed without the need of creating an entire transgenic animal (Haas et al., 2002). To ensure neural progenitor cells in the tectum are targeted through this technique, the expression vector takes advantage of the Sex-determining Region Y Box 2 (Sox2) transcription factor binding domain that is expressed in radial glia (Pevny & Nicolis, 2010). Sox2 belongs to the SoxB family of the 20 Sox genes, all of which have a HMG box domain in common. It is known to regulate and maintain the multipotent nature of neural progenitor cells. In the mammalian brain when Sox2 is not present, progenitor cells exit the cell cycle prematurely and migrate away from the VZ (Gong et al., 2020). Cells that contain endogenous
Sox2 will be targeted by the introduced plasmid to express a specific gene, matr3, as well as green fluorescent protein to serve as a reporter. To further enhance GFP expression the Sox2 promoter is repeated six times and then activates a GAL4 binding protein. This GAL4 unit then binds to the following UAS sequence which repeats fourteen times. This further increases the expression of the Sox2 promoter. In addition, FGF promoter regions are interspersed through this Sox2 construct. These tools are extremely useful for modeling overexpression of matr3 in vivo to visualize its effects on radial glial cell differentiation.
Research Question Summary
The gene matr3 and its nuclear matrix protein have been implicated for a role in numerous cellular processes. When this protein is mutated in humans, it causes the neurodegenerative disease ALS. While extensive research has been done to understand the protein’s actions in neurodegeneration, its lesser known role in neurodevelopment is yet to be investigated. This widely conserved gene will be studied in Xenopus laevis to uncover more knowledge into how its protein may be altering the differentiation of radial glial cells.
Understanding the mechanisms of this protein in the early stages of development will hopefully lead to more information as to how neurogenesis is regulated in many organisms.
Materials and Methods
Xenopus laevis Rearing
Albino X. laevis were mated in the vivarium of the Integrated Science Center at William
& Mary. Two pairs of females and males were injected with human chorionic gonadotropin hormones to encourage mating. These animals were left in a chamber above a dish in which any eggs dropped by the females, were to be collected. 12 hours after initial injection, eggs were collected and sorted to assess if fertilization had occurred. Eggs that had been fertilized would exhibit divisions and these would be kept to develop into tadpoles. Tadpoles were stored in 12-hour light/12-hour dark cycle incubators at temperatures of 23°C in Steinberg’s media.
Tadpoles were staged according to “The Normal Table of Xenopus laevis” as developed by
Nieuwkoop and Faber. All procedures were compliant with the Institutional Animal Care and
Use Committee of William and Mary.
Xenopus laevis Genome Alignments
The tetraploid nature of the X. laevis genome results in numerous variations in the sequence of the gene matr3. To ensure the most abundant and conserved sequence, alignments of these variants were performed using the NCBI Basic Local Alignment Search Tool
(BLAST). The variant matr3.S was selected for its abundance in the central nervous system and availability in a commercial product for isolation. This variant produces a protein called matrin-
3 which was fully encoded in the cloned product purchased from Horizon Discovery.
Plasmid Construction
To express matr3 DNA in the cells of the X. laevis tadpole, a cloning mechanism that uses multiple fragments recombined into one transfectable vector was used. This method combines a 5’ promoter element, middle entry (ME) clone which contains my gene of interest, and a 3’ fluorescence element for visualization within the cell. Once introduced to the cells of the host organism, transcription and translation of this entire product will occur. To ensure that both the gene of interest’s protein and the fluorescent protein are translated into two discrete molecules, a self-cleaving peptide called P2A separates the two elements after translation. The use of a P2A element was found to be a more suitable method in this experiment rather than a matrin-3:GFP fusion protein as the two proteins would be translated separately.
The matr3 gene was isolated using primers designed using the NCBI primer blast tool and a polymerase chain reaction to amplify the desired transcript. These primers contained
Gateway system (Thermo-Fisher) specific sequences that allow for recombination in the future, while isolating the cDNA sequence of matr3. The gene was isolated from a donor pCMV-
SPORT6 vector sold by Horizon Discovery (Dharmacon) and renamed KB005. This product was sequenced to verify its validity before moving forward. And it was noticed to have a stop codon at the end of its transcript. This would result in the formation of the gene of interest in the final vector, but early exit from translation of the final fluorescent protein. The primers were edited to perform a site-directed mutagenesis to effectively eliminate the stop codon.
A polymerase chain reaction (PCR) using Phusion Green HotStart polymerase II was conducted and resulted in isolated matr3 cDNA (Table 1). This was verified to be matr3 by sequencing reactions and the stop codon was shown to be eliminated. Restriction enzyme digests using FastDigest enzyme HindIII (Thermo Fisher) were also utilized. This diagnostic revealed a product of the intended sizes 5573 and 662 base pairs when run on a 1% agarose gel.
This isolated cDNA product was combined with tol2kit 218 as a donor backbone for recombination using the Invitrogen Gateway System. The primers used to isolate the MATR3 sequence contained gateway specific attB1 and attB2 sites critical for recombination. This BP reaction is required to establish recombination sites for the final plasmid construction (Table 2).
The matr3-t2k218 construct, named KB012, was plated on kanamycin plates and again restriction enzyme digested for diagnostic purposes. The enzymes EcoR1 and Eco32I were used and revealed the expected sizes of 3256 and 876 base pairs when run on a gel.
After this plasmid was confirmed, it was then combined in ratio with premade stocks of a 5’ pSox2 promoter element, 3’ p2A-eGFP fluorescent protein, and a final donor destination vector tol2kit 394 (Table 2). The p2A self-cleaving peptide was utilized as a means to produce both the matrin-3 protein, and eGFP protein during translation of the plasmid within the X. laevis. This LR reaction resulted in the final recombination of the four elements to create a plasmid resistant to Ampicillin which was named KB014. This construct was again sequenced using primers 3, 4, 5, and 6 for verification (Table 3).
In addition, multiple restriction enzyme digests were employed using HindIII enzyme for expected sizes of 3893, 1884, 1879 and 1252 base pairs and EcoR1 enzyme for expected sizes of
4798, 1957, 1662, and 491 base pairs. All diagnostics revealed the successful construction of a plasmid containing psox2-matr3-GFP elements. To confirm this construct would be viable, the translation of this plasmid was tested using the programs SnapGene and A Plasmid Editor (APE) using the sequencing information generated. These programs ensured the elements were all in frame with no stop codons to cease translation. It was confirmed that the matr3 protein would be produced as well as the
GFP to allow for sox2 expressing cells to possess both overexpression of matr3 and green fluorescence.
Electroporation and Transfection
Stage 46 animals were anesthetized in a solution of MS-222 prior to transfection. Glass pipette tips were loaded with the pSox:matrin3-GFP KB014 plasmid mixed with Fast Green for visualization. To transfect tectal cells of X. laevis the pipette tip was inserted into the midbrain ventricle using a micro-manipulator set up. Pulses of plasmid were delivered until the ventricle was filled. A platinum electrode was then placed on either side of the ventricle and three pulses of -35V were delivered at each polarity (Figure 5).
This same technique was employed using the RFP control plasmid for a baseline of radial glial cell activity during the same developmental stages. The tadpole was then placed in recovery
Steinberg’s media for at least 24 hours for the cells to begin expressing the construct. This mechanism allows for visualization of individual radial glial cells as well as their progeny within the optic tectum of X. laevis tadpoles where proliferation at this stage should be occurring at a very high rate.
Imaging
After 24 hours, tadpoles were screened and imaged for GFP expressing cells in the left and right tectum. Tadpoles were screened using a fluorescence microscope to assess the efficacy of the electroporation prior to full-scale imaging. Locations of the fluorescence were noted and tadpoles were sorted into individual wells of a six well plate containing Steinberg’s media. Imaging was performed on a 3i spinning disk confocal microscope. Tadpoles were anesthetized in MS-222 solution and placed in a mold shaped to fit the tadpole so that its body would be stabilized, and the brain would be able to be viewed subdermally. The completed slide was then placed under the objective for imaging. The green 488 confocal laser was used to take 3D Stacks at 1μM thickness descending through the tissue into the deep brain. These stacks were then projected in 2D to form one cohesive image of the whole cell through the space of the optic tectum. Bright field images of the cells’ positions within each lobe of the tectum were also recorded to use as a reference. This was repeated at the same time each day for three consecutive days to establish a time lapse of these cells. Images were analyzed by quantifying the number of cells seen in each tectal lobe and classifying the cells as progenitors or neurons. Images were edited for brightness and contrast and figures were made using
ImageJ, Adobe Photoshop and Adobe Illustrator.
Results
Genome Analysis
An in-depth analysis of the sequence of Matr3 in both Xenopus laevis and Homo sapiens was done to find similarities in this homologous gene. X. laevis is a tetraploid organism while humans are diploid so there is an added level of genetic complexity when comparing variants in these genomes. Within the matrin-3 protein, the RRM and Znf domains are conserved across species making them functionally similar. The matr3.S variant in X. laevis was selected in this study to ensure the most homologous sequence would be expressed as it had the highest conservation of base pair identities. The X2 isoform analyzed did not have the same level of conservation as Matr3.S (Figure 6).
One of the key findings in the role of Matrin-3 during neurogenesis is the protein’s phosphorylation at its Ser208 residue (Niimori-Kita, Tamamaki, Koizumi, & Niimori, 2018).
Multiple alignments were performed using BLAST to find the variant of X. laevis Matrin-3 that had conservation at this amino acid. Many isoforms had asparagine residues at this site to which BLAST gives a score of (+), as this amino acid is deemed functionally similar to serine to act in similar ways and may serve as a phosphomimetic amino acid in X. laevis but requires more research to confirm its role (Chen & Cole, 2015). While phosphorylation has been shown to occur on this residue in some cases, selecting the protein with full conservation was the best approach in this experiment further encouraging that matr3.S would be the best option.
RFP Control Plasmids
In this experiment, red fluorescent protein (RFP) expressing radial glial cells were used as a control to compare normal radial glial cell morphology, movement of new progeny, and differentiation to those expressing matr3. This plasmid has the same regulatory control as the matr3 plasmid (KB014) that was designed. Typically, radial glia exhibits a position in the optic tectum with their soma along the midbrain ventricle and an endfoot reaching towards the pial surface. These two cellular features are connected by a long slender radial process. The RFP plasmid utilized expresses in the cytoplasm, revealing the full cell morphology(Figure 7).
The noticeable difference that occurs as a radial glial cell differentiates into a neuron is the loss of the endfoot, and generation of neuronal features such as a dendritic arbor or axon growth
(Figure 8).
The goal of using this plasmid was to establish a general timeline of differentiation in progenitor cells over three days. Progenitor cells were to be counted on the first day of imaging, 24 hours after initial transfection, in both the left and right tectum. On the following days, the cells recorded on day one would be identified, and assessed for any changes in morphology, divisions, and differentiation into another cell type. In addition, any new cells would be counted and assessed which progenitor cell they were the progeny of. This experiment after a number of trials would provide for a baseline of when progenitor cells undergo divisions or differentiation in the average X. laevis tadpole.
Matr3 Overexpression
The multisite gateway cloning system allows for the recombination of three elements, a
5’ promoter element, middle entry element which contains a gene of interest, and 3’ fluorescence element into one cohesive plasmid vector. The matr3 cDNA became the middle entry clone responsible for introducing overexpression of the gene and its associated protein and was named KB012 (Figure 9).
KB012 was then combined with other plasmids that had previously been constructed and contained the proper recombination sites. The 5’ element promoter Sox2 allows for expression in progenitor cells with the transcription element while the 3’ element contains GFP that will illuminate the entire cell. All three elements were combined into the donor backbone t2k394
(Figure 10).
While no data was able to be collected using the completed KB014 plasmid it is still possible to hypothesize the expected results. Based on previous studies, it has shown that the post-translational phosphorylation of matrin-3 at its Ser208 residue is crucial in maintaining progenitor cells in their undifferentiated state within mice, but why this is the case is unknown.
Knock out of Matr3 has shown to induce progenitor cells to begin extending their neurites and reduce levels of cell proliferation as they shift toward becoming neurons (Niimori-Kita,
Tamamaki, Koizumi, & Niimori, 2018). While the effects of decreased matrin-3 have been studied, overexpression and the interactions of this protein requires more research.
In the X. laevis tadpole, overexpression of matr3 in the radial glial cells would lead to an increased amount of matrin-3 protein. As long as phosphorylation of this abundance of protein is able to occur by ATM kinase, it is reasonable to think that progenitors would remain undifferentiated for an extended period of time. This could be measured using cell imaging experiments where progenitor cells are transfected with KB014 and then imaged for three consecutive days. Typically, within three days there is a 25% increase of new cells in the optic tectum, with a steady increase in neurons as progenitor cells begin to asymmetrically divide or differentiate into neurons (Bestman, Lee-Osbourne, & Cline, 2012). The number of radial glial cells and the progeny they produce, whether it is more progenitors or neurons, would be recorded each day and analyzed to assess the amount of change in the cellular composition of the optic tectum when matrin-3 is overexpressed. If cells are able to proliferate freely in the overexpressed matrin-3 brain, then a larger than normal pool of progenitor cells with far less neurons would be expected at the end of three days. If they cannot proliferate, the number of cells would remain constant and possibly result in quiescent progenitor cells, cell death, and under-developed brains in X. laevis tadpoles. This would be compared to a normal cell expressing RFP without any manipulation to view the natural time course of a progenitor cell until it differentiates into a neuron.
ATM kinase is known for its role in resolving double stranded breaks during the cell cycle, but it is also capable of phosphorylating downstream targets that play a role in cell cycle progression (Shiloh & Kastan, 2001). Matrin-3 phosphorylation could be pausing the cell cycle of neural progenitor cells, preventing their progression towards neurogenic fate. The pool of these progenitor cells would stay expanded for a longer period of time, which could alter the timeline of development compared to the control organism, and ultimately cause cell death or improper migration of new cells to developing areas of the brain (Figure 11).
Because transfection of neural progenitor cells with electroporation does not transfect all cells in the X. laevis optic tectum, the effects of matr3 overexpression would occur in some cells but not others. Seeing how overexpression of matrin-3 alters the course of development in radial glial cells expressing excess matrin-3 versus those that only express RFP could reveal how important strict regulation of these proteins can be during neurogenesis.
Studies have previously shown that after symmetric division, radial glial cells maintain their progenitor-like morphology and later differentiate into neurons in the X. laevis optic tectum. In addition, the progeny of these cells are unlikely to migrate away from each other, despite being fully capable of doing so (Bestman, Lee-Osbourne & Cline, 2012). Excess Phospho-
Matrin-3 could possibly extend the period that newborn radial glial cells remain undifferentiated after division. It would be useful to understand how this protein may alter localization of the new cells and if they would change the pattern of remaining together or if migration would occur. The distances between somata in the matrin-3 overexpressing cells could be measured throughout the time lapse process and compared to the RFP expressing cells of a control organism. The overexpression of matrin-3 may completely disrupt the temporal aspect of the progenitor cell life cycle, sending these cells into a state of quiescence or dormancy. In addition, fate decisions made during the cell cycle may be altered. Rather than exiting the cell cycle in G1 and differentiating to a neuron in G0, radial glial cells would most likely continue to undergo proliferative divisions (Figure 12).
Again, analyzing these cells through imaging experiments is the best strategy to understanding how matrin-3 affects brain development in vivo.
Discussion
Clear evidence for what mechanisms regulates the gene Matr3 have not yet been discovered. Since it belongs to the family of snoRNA protein-coding host genes, it is hypothesized it is regulated by similar factors to other genes in its family. Most often for intronic snoRNAs to be transcribed, transcription of its host gene is required since introns do not possess promoter regions. In some cases, these snoRNAs are capable of enlisting a feedback mechanism to its host gene to alter its transcript or splicing activity (Boivin,
Deschamps-Francoeur, & Scott, 2018). Transcription factors such as GA-binding protein (GABP) regulate the gene TIMM23. This gene is in the same family as Matr3 and could have parallels in regulation or similar transcription factors (Prieto-Ruiz et al., 2018). Discovering more about the
U19 snoRNA in humans and analyzing how other snoRNA protein-coding host genes are regulated may uncover more detail into Matr3 regulation.
Matin-3 is heavily influenced by post-translational modifications like SUMOylation, acetylation, ubiquitination, and phosphorylation, but the effects of these modifications on the protein have not been well researched (Iradi et al., 2018). However, the importance of the presence of Ser208 phosphomatrin-3 in the maintenance of neural progenitor cells has been shown in the study by Niimori-Kita et al. in 2018. Many X. laevis isoforms of matrin-3 do not have conserved serine residues shown to be important to phosphorylation. It would be interesting to see the effects of overexpression of matrin-3 when the protein does not contain the typical serine208 residue but rather an asparagine residue. Site directed mutagenesis to substitute phosphomimetic or non-phosphorylatable amino acids in matrin-3 at regions where crucial phosphorylation is said to occur may allow more information into the function of phospho-matrin-3 and its importance in neurogenesis. It would be useful to know if phosphomimetic matrin-3 is sufficient to act as endogenous phospho-matrin-3. Another consideration is that when matrin-3 is unable to be phosphorylated, it undergoes nuclear export to the cytoplasm (Niimori-Kita, Tamamaki, Koizumi, & Niimori, 2018). If the excess matrin-3 introduced by the expression vector I created is unable to experience ATM phosphorylation, it would be exported into the cytoplasm where its roles are completely unknown. Mutation of this crucial serine amino acid to a non-phosphorylatable residue could assist in understanding how cytoplasmic matrin-3 affects a radial glial cells survival and morphology.
Matrin-3 has been extensively studied for its role in neurodegeneration, but it is equally as important to research its actions during development. The initial periods of expansion within the brain are crucial for proper function throughout an organism’s life and understanding how matrin-3 may affect this process would allow more insight into the regulation of neurogenesis.
Studies in vivo allow for full visualization of how manipulations in protein levels can affect an entire system, as the cells in the optic tectum are responsible for broad signaling after differentiation and the organism can be subjected to varying external conditions to assess how the environment may alter its development. Matr3 regulation requires more research but may be mediated by calcium signaling. The calcium binding motif is partially overlapped in one of the RRM domains of the protein (Alexander Valencia, Ju, & Liu, 2007). Mutating these RRM or calcium binding domains to prevent calcium binding activity, could elucidate the importance of the calcium’s in matrin-3 regulation. These mutations would also be useful to study if this alters its nuclear localization, as its NLS is also within this area of the RRM and calcium binding domains. Calcium wave transients have been shown to alter radial glial cell proliferation during development (Weissman et al., 2004). Calcium modulation has been repeatedly shown to regulate neurogenesis and by using calcium reporters in addition to the matr3 overexpression vector more information about their interactions could be recorded. X. laevis model provides many opportunities for continuing to study this gene through protein overexpression, knockout studies, mutation of functional domains, and activity dependent mechanisms.
Conclusions
Without strict regulation of neurogenesis in early development, all organisms are at risk for developing neurodevelopmental diseases or having errors in the size of their brains at birth.
Studies have shown that neurogenesis is tightly regulated by both intrinsic and extrinsic factors.
Well known elements such as cyclin-dependent kinases that regulate the cell cycle influence when progenitor cells differentiate but understanding other genes that have a role in this process and the possible effects when these genes are altered is important (Calegari & Huttner,
2003). Matr3 is only a small factor in the number of genes and proteins that are collaborating to ensure proper neurogenesis during development but its role is still an important one. Hopefully if more is understood about genes like this one and what mechanisms are responsible for regulation of neural progenitor cells, advancements can be made in how neurodevelopmental diseases are assessed and treated.
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