Expression Heterogeneity in Nuclei

Ashleigh Van Deusen

Molecular, Cellular and Developmental Biology University of Colorado, Boulder

Defense: April 3, 2019

Thesis Advisor: Dr. Bradley B. Olwin, Department of Molecular, Cellular and Developmental Biology

Defense Committee: Dr. Jennifer Martin, Honors Council, Department of Molecular, Cellular, and Developmental Biology Dr. Bradley B. Olwin Department of Molecular, Cellular and Developmental Biology Dr. Alison Vigers, Department of Molecular, Cellular and Developmental Biology and Neuroscience Dr. Heidi Day, Department of Neuroscience

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Table of Contents

Abstract

Introduction

Chapter 1: Single Muscle Nuclear Isolation and Characterization

I. Introduction

II. Methods

III. Results

IV. Discussion

Chapter 2: Single Nucleus RNA Sequencing in Skeletal Muscle

I. Introduction

II. Methods

III. Results

IV. Discussion

General Discussion 3

Abstract

Skeletal muscle is required for breathing and voluntary movement and loss of muscle mass is a significant contributor to humans’ inability to stay active throughout their entire lifespan. Skeletal muscle consists of multinucleated cells and there is no comprehensive data on the of the hundreds of nuclei located in a single myofiber required for contraction, attachment to tendons and force generation. Typical methodologies to examine gene expression of single cells cannot be performed on skeletal muscle because individual cells are large and multinucleated. Single nucleus sequencing can be exploited to examine differential gene expression that likely occurs among the hundreds of myonuclei organized within the myofiber. Isolation of nuclei from skeletal muscle previously done masked variability that exists in different muscles and types. I devised a protocol to isolate nuclei from a single muscle. I validated the method and performed it in multiple muscle groups, allowing the study of different expression profiles from a single mouse. The established protocol was employed in conjunction with single nucleus RNA Sequencing to investigate differential gene expression in the nuclei of adult, aged, and injured muscle. Preliminary analysis of adult muscle indicates clusters of myonuclei and mononucleated cells. There was widespread misregulation in aged muscle, myonuclei were expressed in all clusters and some of the mononucleated populations were undetected. Satellite nuclei present in uninjured aged muscle have become activated, impairing the longevity of muscle maintenance. This work could provide new therapeutic targets to prevent the age induced loss of muscle regeneration. 4

Introduction

Aging is an inevitable part of being human. The goal of a large body of research is attempting to better understand aging so ultimately it can be delayed. For most, a significant portion of the 79-year average lifetime will not be lived actively or disease free. The average health span is nearly 20% shorter than overall lifespan at about 63.3 years, meaning individuals are unable to perform desired activities (World Health Organization, 2016). Interventions that slow the types of processes that limit health span would be a major step towards helping people live longer, healthier lives. One approach to delay aging targets muscle wasting, a major contributor that shortens health span. Sarcopenia is the age-associated loss of function and muscle mass unrelated to disease, but leads to loss of strength, immobility and frailty. Both men and women experience sarcopenia, which results in 20-40% loss in strength for adults in their seventies and eighties (Doherty, 2003). All organ systems are affected by aging and understanding the distinct changes that occur in skeletal muscle as humans age could lead to new therapeutic targets to combat the impacts of aging.

Nearly 40% of human body mass is skeletal muscle, which is required for voluntary movement. Skeletal muscle consists of two distinct muscle cell populations, satellite cells, and the terminally differentiated multinucleated myofibers (Dumont et al., 2015; Zeng et al., 2016).

Proper functioning of these cells is dependent on gene expression regulation, but until recently cell to cell variation has been difficult to study. Recent advances in high-throughput sequencing technologies makes studying gene expression regulation more accessible. In particular, RNA sequencing (RNA-Seq) has allowed the investigation of differential gene expression in tissues.

The method is advantageous for studying RNA because it is more sensitive to subtle differences by capturing both known and novel features and having a specific target is not required 5

(Bengston et al., 2018). A comprehensive view of gene expression is provided, all of the transcripts are averaged from the entire sample so cell to cell variation is lost. Single cell RNA- seq (scRNA-Seq) and single nucleus RNA-seq (snRNA-Seq) have unique advantages over RNA- seq by allowing examination of cell to cell variation and eliminating the possible masked heterogeneity of bulk samples (Illumina, 2018). Heterogeneity in gene expression is the variation of expression between cells and is masked in bulk samples because averages are taken from all of the cells, so individual cell-to-cell variation is not exposed. Single cell and single nucleus sequencing provide information into any variation that occurs in a sample and allow the study of complex tissues in environmental or time-dependent processes.

Examining what is actively being transcribed in the nucleus, versus transcripts in the nucleus and already existing transcripts in the , can help identify changes that may occur during aging. Single nucleus sequencing could be useful in identifying factors that contribute to muscle wasting, but isolation needs to be performed in a single muscle. Skeletal muscle fibers exist in a multinucleated syncytium surrounded by tough connective tissue making isolation of nuclei difficult and yields low(Kuehl, 1975). Previous isolation protocols in skeletal muscle have only used pooled samples, either from multiple muscle groups in a single mouse or multiple mice but this limits the variability that would be exposed in a single muscle. I sought to devise a protocol to isolate nuclei from a single muscle. The method was validated and performed in multiple muscle groups, allowing the possibility to study many different states and expression profiles from a single mouse.

The established protocol was employed in conjunction with snRNA-Seq to investigate the differential gene expression that occurs during age and injury in the hindlimb muscles of individual mice. The goal of my study was to examine the heterogeneity of nuclei in a single 6 muscle, predicting that the sequencing would uncover a few nuclei that may be large contributors to the aging phenotype. The nuclei are clustered into populations based on their gene expression and differences in the aged muscle were determined by examining the populations in the adult muscle. I expected to see many nuclear populations in the muscle including myogenic cells, satellite cells, neuromuscular junction (NMJ), myotendinous junction, and non-muscle cell types endothelial, immune, fibroblasts. I also expected mis-regulations in nuclei of the aged muscle present in one or all of the given populations. The atlas that was created of the adult, aged and injured muscles allowed me to determine the extent of heterogeneity in muscle and non-muscle nuclei and whether aged muscle differs from young adult muscle.

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Chapter 1: Single Muscle Nuclear Isolation and Characterization

Introduction

Skeletal muscle is required for breathing and voluntary movement. Investigation of gene expression in a skeletal muscle lends insight into cellular needs and how they can be mis- regulated. Previous analysis methods have not been able to identify distinct cell populations coming from a single mouse or muscle, they have been in bulk providing only averages. There is need for a unique tool that could show nuclear specific transcripts and what is being actively transcribed for the cell. A nuclear isolation method from a single muscle would provide the ability to detect mis-regulations in gene expression that occur in aging and provide targets to combat dysfunction.

The isolation of nuclei is advantageous for studying active transcription, and has been performed in the brain (Matevossian and Akbarian, 2008). Nuclei have been isolated in bulk skeletal muscle samples (Cutler et al., 2017) but has not been performed on a single muscle.

Nuclear isolation from skeletal muscle is particularly difficult because there is a lower concentration of nuclei than in other cell types, all of the nuclei share a cytoplasm in each myofiber, and individual myofibers are held together with connective tissue making the muscle tough to break apart and release nuclei without damaging them (Fig. 1) (Kuehl, 1975). An investigation of the different nuclear populations in a single skeletal muscle could inform us about heterogenous transcription among myonuclei and what occurs in diseased and aged states. 8

Skeletal muscle consists of two distinct muscle cell populations, satellite cells, and the

terminally differentiated multinucleated myofibers. Myofibers contain the contractile unit of

muscle and form an ordered structure with connective tissue (Fig. 1A). Myofibers are created by

hundreds of mononucleated myocytes, differentiated myoblasts created from satellite cells, that

fuse together and exist in a syncytium. Once in a syncytium the myofibers are no longer able to

divide, they depend on satellite cells to maintain the muscle. Satellite cells are mononucleated

Figure 1: Skeletal Muscle Structure and Maintenance A. Skeletal muscle consists of multinucleated muscle fibers held together by connective tissue (Cummings, 2001) B. Following activation satellite cells can self-renew their population or progress to myoblasts which can differentiate into myocytes and fuse into the multinucleated myofiber. 9 cells located under the basal lamina that repair muscle through myogenesis (Yin et al., 2013).

Long-term skeletal muscle maintenance is monitored by satellite cells that remain inactive until stimulated by injury or growth factors. Once activated, satellite cells either divide to replenish the myonuclei in terminally differentiated myofibers or self-renew to maintain their population

(Fig. 1B) (Dumont et al., 2015; Pawlikowski et al., 2015). Muscle mass and maintenance are dependent on satellite cells’ regenerative ability, which declines with age. This loss of efficiency contributes to the loss of muscle mass associated with aging, but our knowledge of the mechanism remains incomplete.

In the present study, I isolated nuclei from a single muscle, allowing use of sequencing technologies to make comparisons between individual muscles and conditions. The isolation method was performed in whole muscle in adult, injured, and aged mice to characterize gene expression to further elucidate the mechanisms of muscle loss and repair. Differential gene expression among nuclei from mononucleated cells and myofiber nuclei were compared through snRNA-Seq in a whole muscle.

Methods

Mice

Mice were housed and bred at the University of Colorado Boulder in a pathogen-free facility. All protocols involving animal subjects were approved by the Institutional Animal Care and Use

Committee (IACUC). A variety of genotypes, ages and genders of mice were used in optimizing nuclear isolation. Isoflurane followed by cervical dislocation was used to euthanize mice.

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Immunofluorescence

An antibody to a nuclear transmembrane protein specific to skeletal muscle, TMEM38a, was used to visualize muscle nuclei from other nuclei. Before the staining protocol samples were mounted in agar on the coverslip. Nuclei were incubated in blocking buffer (5% Goat serum,

0.5% BSA, 94.5% 1X Phosphate Buffered Saline) overnight no rocking. The following day nuclei were incubated for 1.5 hours at room temperature in a Rabbit anti-TRIC-A antibody (06-

1005 Lot 3013335) (1:100 in blocking buffer) (Bleunven et al., 2008). Nuclei were washed six times with PBS and incubated for 45 minutes in conjugated secondary antibody Donkey-anti- rabbit IgG 555 (Ref A3 572 Lot 1454443) (1:500 in blocking buffer). Three more washes in PBS were performed and then nuclei were stained for 4’,6-diamidino-2-phenylindole (DAPI), which binds to AT rich DNA. Nuclei were mounted onto glass slides with Moiwol to be imaged.

Fluorescent microscopy was performed on an Olympus IX81 Inverted Widefield

Microscope and scoring was performed using ImageJ software (1.52h).

Results

The efficacy of the nuclear isolation protocol was validated prior to performing any sequencing experiments; the nuclei were characterized visually by immunofluorescence, yields were calculated with a BioRad TC20 automated cell counter, which also confirmed the purity of the nuclei prep, and RNA isolation with Trizol reagent was performed to ensure the nuclei had RNA associated with them.

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Nuclear Isolation

I first performed the nuclear isolation protocol in cultured chicken fibroblast cells to validate the efficacy of the sucrose gradient for isolation. Optimization for isolation from a single tibialis anterior (TA) muscle began utilizing a protocol in pooled muscle from multiple mice

(Cutler et al., 2017). The method employed was homogenization, using a tissue grinder to release nuclei from the muscle. The tibialis anterior (TA) muscle was dissected, weighed, and placed in a microcentrifuge tube. The muscle was chopped with scissors (FST Item No.15024-10. 8mm cutting edge, 0.2mm tip diameter) for 30 seconds. 0.5mL of homogenization buffer (10mM

HEPES, 60mM KCl, 2mM EDTA, 0.5mM EGTA, 300mM sucrose, qH2O) was added to the tube and the muscle was chopped for another 30 seconds.

The mixture was placed into a 2mL tissue homogenizer and ground with the loose pestle

(clearance 0.114mm). Throughout homogenization a 96 well plate was used to observe myofiber size and destruction by placing 100µl of sample every 5 strokes and observing under a brightfield microscope. Samples used for optimization were processed at many numbers of strokes 25-60 (intervals of 5) (Fig. 3).

A B C

Figure 2: Nuclear isolation was optimized using a 96 well plate for visualization during and after homogenization(A-C) 96 well images of TA muscle, 25 strokes, 35 strokes, 45 strokes respectively. 12

Following homogenization, the mixture was put through a 100µm cell strainer and centrifuged at 1000xg for 10 minutes. The pellets were resuspended in 3.5mL of homogenization buffer. To separate nuclei from other cellular debris, the sample was placed onto a discontinuous sucrose gradient (2.8M and 2.1M sucrose: 50mM HEPES, 25mM KCl, 5mM MgCl2, qH2O) and centrifuged for 3 hours at 186,232xg at 4ºC.

The nuclei were no longer visible following sucrose separation. After centrifugation the nuclei were located at the 2.0/2.8M sucrose interface, and the sample was not visible (Fig. 3).

The interface was removed with a pipette and resuspended in resuspension buffer (20mM

HEPES, 10mM KCl, 1.5mM MgCl2, 0.2mM

EDTA, qH2O) at a 1:100 dilution. The sample was then centrifuged for 15 minutes at 3000g and aspirated to 1.5mL. The pellet was placed in a microcentrifuge tube. The samples were centrifuged for 5 minutes at 2100xg, aspirated Figure 3: Nuclear Isolation Protocol-The muscle of down to 100µl, resuspended and allowed to sit on interest is dissected and homogenized. Following centrifugation nuclei are isolated from the 2.0M/2.8M ice for 5-10minutes. The nuclei were fixed in 2.5% interphase of the sucrose gradient and can then be utilized (Cutler 2017). paraformaldehyde for 10 minutes, spun a final time and resuspended in 12µl PBS for staining and mounting.

A mounting protocol for staining was developed to prevent clumping and loss of nuclei.

Small circular collars were placed on square coverslips using multiple coats of nail polish. The nuclei were placed in the center of the circle and incubated for 30 minutes to allow them to 13 sediment to the coverslip by gravity. After the first 15 minutes 2% Agarose was heated and placed in a 37ºC bead bath along with pipette tips. After 30 minutes, 50µl of the agarose was placed over the nuclei and allowed to solidify. Prior to staining 75µl of 1XPBS with 0.02% sodium azide was placed over the samples and stored at 4ºC.

Visualization of Nuclei

Immunofluorescence was used to confirm the nuclear isolation protocol was effective. The samples were stained with DAPI (1:1000 in PBS) and imaged on a fluorescent microscope. It was determined via DAPI-stained images that 35 strokes yielded the most nuclei from the tibialis anterior (TA) muscle (~0.06g) (Fig. 4).

TA 35 Strokes DAPI 10X

Homogenization

Figure 4: Nuclear isolation was optimized using DAPI staining for visualization during and after homogenization 35 strokes were determined to be optimal for the TA muscle by visualization of nuclei with DAPI following isolation with a sucrose gradient

The TA sample was used to determine what the muscle sample should look like during homogenization. The 96 well images from the TA were used for looking at myofiber size and destruction when isolating nuclei from other muscle groups, including the diaphragm, tongue, soleus. Using the TA as a guide for subsequent experiments in other muscle groups, it was 14

Tongue 35 strokes ~0.06g

TA 35 strokes ~0.06g

Diaphragm 35 strokes Diaphragm 60 strokes ~0.1g ~0.1g

Figure 5: Nuclear isolation was optimized using a 96 well plate and DAPI staining for visualization during and after homogenization. 96 well images of myofiber fragments and sizes from the TA were used to optimize isolation from other muscle groups-the tongue and diaphragm.

determined 35 strokes was sufficient for the tongue (0.06g) and 60 strokes for the diaphragm

(~0.1g). (Fig. 5).

Nuclei Yield and RNA Isolation

To obtain sufficient nuclei for use in sequencing experiments, nuclei concentrations had to be

determined. Following the isolation protocol, 10µl of nuclei was used to calculate the yield on an

automated cell counter. The yields (30,000-85,000 nuclei) for all samples were sufficient for

sequencing experiments and the pictures produced from the cell counter show the nuclear 15

1050 nuclei/µl Total Yield: 84,000nuclei RNA Yield: 126.9ng/µl

Figure 6: Nuclei yield and diameter: Nuclei yield for all samples were calculated using the BioRad TC20 automated cell counter, along with nuclei diameter and purity of the samples. The amount of RNA isolated from the nuclei was calculated using a nanodrop.

preparations are free of debris and unlikely to clog downstream fluidics in the 10X Genomics

workflow. Prior to RNA sequencing, Trizol RNA isolation was performed and quantified with a

nanodrop spectrophotometer after isolation to confirm that the nuclei had RNA associated (Fig.

6).

Myogenic nuclei in adult and old mice

The cell counter confirmed nuclei were intact following isolation, but what nuclei were

isolated was unknown. To examine differential expression among nuclei in myofibers, nuclei

from fibers must be present. Myonuclei were visually characterized using an antibody specific to

the nuclear membrane of contractile cells. A comparison of the myonuclei in adult and old mice

was achieved by using a muscle specific antibody, TMEM38a, that binds the nuclear envelope

calcium channels in contractile cells. Staining procedures provided information about the shape 16

of the nuclei, myofiber nuclei are elongated, others are round. The nuclear membrane was still

intact because the antibody binds the nuclear envelope. Images were processed with ImageJ and

DAPI TMEM38a Merge

Young Uninjured ld Uninjured O

Figure 7: Adult and Old Myogenic Nuclei: Nuclei were scored for TMEM + and TMEM – nuclei. Arrowheads represent TMEM- nuclei. Nuclei not stained for TMEM would be from mononucleated cells, satellite cells and non- muscle cells. fields scored for DAPI (+) and TMEM38a (+) and TMEM38a (-) nuclei to determine the

percentage of myogenic nuclei (Fig. 7). Samples were blinded for scoring to avoid bias toward

young or aged counts. Nuclei not in the plane of the image were not counted.

The number of DAPI (+) nuclei was used as the total when calculating percentages of

myogenic nuclei. While there were only two mice used for the study, in order to establish

reliability, 17 images were scored for each mouse. The variance was much broader in the aged

sample. The average percentage of myonuclei in the adult muscle was 84.96% (±6.48%) and 17

aged 81.14% (±10.75%). These numbers are comparable to calculations of the expected amount

of myonuclei in a single TA muscle, 60 ± 20% previously (Brewer, 2015). An unpaired t-test

was performed (Prism V8) and there was no significant difference between the proportion of

myonuclei in adult and old mice (t=1.681, p=0.102) (Fig. 8).

Young versus Old %myogenic cells

110

100

90

80 %TMEM + 70

60 Young Old

Figure 8: Percentage of Myonuclei in Young and Old TA Mouse Muscle-Overall Percentage of Myonuclei in all blinded Images 82.42%. Percentages between young and old myogenic nuclei are not significant: Unpaired T- test performed in Prism, P=0.102

Discussion

I have established a method to isolate nuclei from a single skeletal muscle. Isolation of

nuclei from skeletal muscle is difficult because the cells are not mononucleated and myofibers

are highly intertwined with connective tissue. There are limitations when isolating nuclei from

skeletal muscle because as it is easy to destroy nuclei during homogenizing. Isolated nuclei are

highly enriched and free of cellular debris because of the sucrose gradient, but some nuclei are

also likely lost in this step. If a certain sub-population of nuclei are lost due to their frailty, we 18 are unaware of it, but the yield of nuclei was sufficient for performing sequencing experiments, determined using an automated cell counter. Nuclei were characterized visually prior to sequencing to ensure the isolation procedure did not destroy them.

DAPI staining provided information about the shape of the nuclei and myogenic cells were visualized with a nuclear envelope antibody specific to contractile skeletal muscle cells.

Adult and aged muscle were compared, and there was not a significant difference in the percentage of myogenic nuclei in the two populations, although the aged sample had greater variance in the amount of TMEM38a in the images. One adult and one aged mouse were used,

17 images were taken for each mouse, but to identify biologically relevant differences more mice need to be used for scoring. The staining procedures allowed me to observe that nuclei were still intact and how many myogenic nuclei are in the samples. RNA isolation from the samples provided the final validation that the nuclei were not compromised and contained RNA.

The nuclear isolation method that I created can be utilized to answer questions about the heterogeneity of the nuclei of a single muscle. Nuclei were isolated from adult, aged, and injured mice to perform snRNA-Seq and create a transcriptional atlas of whole skeletal muscle. The transcriptional atlas from an adult muscle allows aging and other diseased states to be examined to explore which, and how many nuclei are mis-regulated and if they can be targeted for manipulation. My protocol can be used to isolate nuclei from multiple muscle groups, including the tibialis anterior, the diaphragm, soleus, and tongue. In the future experiments examining the differential expression of nuclei in fast and slow twitch muscles, developmentally different muscles or diseased muscles can be explored.

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Chapter 2: Single Nucleus RNA Sequencing in Skeletal Muscle

Introduction

Single cell RNA-Seq has been used to study the transcriptomes of isolated satellite cells

(Cho and Doles, 2017). Satellite cells are a heterogenous population. Once activated they exit quiescence to proliferate into myoblasts for fusion into new and existing fibers, or undergo self- renewal to maintain the satellite cell population (Kuang et al., 2007)(Fig. 1). There is a decrease in the proportion of satellite cells that maintain muscle mass during aging (Day et al., 2010).

Studies done in satellite cells from adult uninjured mice showed that there is a great deal of heterogeneity in individual satellite cells at different states indicating they may exist along a continuum rather than one discrete state or another (Cho and Doles, 2017). Heterogeneity exists in satellite cells, but the expression that occurs in myofiber nuclei or fusing nuclei or an aged muscle have not been examined. The goal of the present study is to identify the changes that occur in gene expression in the whole muscle in these different states.

Single nucleus RNA-Seq (snRNA-Seq) was originally established as a method to examine the transcriptomes of highly connected neurons in the central nervous system where whole cells are difficult to isolate and keep intact. This method is useful to further our understanding of the cellular basis of behavior, allows in depth analysis of transcriptional states in vivo, and eliminates possible transcriptome changes that occur as a result of enzymatic isolation (Aevermann et al., 2016; Bakken et al., 2018; Sathyamurthy et al., 2018). This technology proves useful in skeletal muscle to examine global gene expression because myofibers are too large to be processed by scRNA-Seq, they will clod microfluidics channels.

SnRNA-Seq has been performed on an immortalized human myoblast cell line to characterize myoblast differentiation. These results could be used to determine cell fate and 20 potential differences in disease conditions (Zeng et al., 2016). Previous studies in skeletal muscle were limited to use in either mononucleated muscle cells or snRNA-seq on cell lines. These studies are not able to capture the whole muscle, since myonuclei from fibers were not included and cells came from multiple samples. snRNA-seq has previously not been possible on whole muscle to give in vivo information about all the nuclei contained in a muscle, including satellite cells, myonuclei and non-muscle nuclei. I established a protocol to isolate nuclei from a single muscle and can use it to analyze global gene expression of all the nuclear populations in whole muscle.

Using the isolation protocol I optimized, as described in Chapter 1, to perform snRNA-

Seq on nuclei of adult, aged, and injured muscle to examine the heterogeneity of gene expression. The atlas that was created for each condition displays what is being actively transcribed and whether muscle and non-muscle nuclei have homogenous or heterogenous gene expression and how they differ in an aged muscle. The injury timepoints can provide information about satellite cell efficiency and whether nuclei that have recently fused into myofibers will become homogenous or heterogenous with existing nuclei in fibers or remain mononucleated cells that failed to fuse.

A major hallmark of aging involves the accumulation of senescent cells that inhibit proliferation of damaged cells, preventing tumor growth but damaging the tissue structure and function where they are located (Baker, et al., 2011). It is possible that in a syncytial cell a few nuclei become senescent and have dysregulation of certain transcripts that injure the entire myofiber. I predicted aged muscle would have greater heterogeneity of transcripts compared to the adult and senescent nuclei that were injuring their respective fibers and subsequently the 21 entire muscle. This uncontrolled regulation of transcripts could be contributing to age induced muscle loss.

Methods

Mice Injuries

For sequencing experiments two injury timepoints and an uninjured timepoint were analyzed.

Mice were anesthetized with 3% isoflurane to be unconscious during the injury procedure. 50µl of 1.2% BaCl2 in a 27 ½ G tuberculin syringe was injected in the left tibialis anterior muscle in parallel with the muscle for injuries. Wild type C57BL/6 were used for the adult samples and male Pax7iCreERT2;LSL:TVA mice (2-2.5yr) were used for aged samples. Four male mice, two adult (6mo) and two aged (2-2.5yr), were injured: an adult and aged 7 days prior to isolation and an adult and aged 4 days prior to isolation. Six samples were used for nuclei comparisons: adult uninjured, adult 4 days post injury (dpi), adult 7dpi, aged uninjured, aged 4dpi, and aged 7dpi.

Nuclear Isolation

Nuclear isolation was performed as previously established in Chapter 1with minor changes. Cell strainers were pre-wet with Homogenization Buffer before samples were placed on them. 7.5µl

RNase OUT was added to prevent RNA degradation. 0.1µl of 10% BSA was added to the 100µl sample in the final step for a final concentration of 0.1%. The nuclei were pelleted at 2100g for 5 minutes and aspirated to 50µl and counted with a BioRad TC20 Automated Cell Counter

(Catalog #145-0101).

22 snRNA sequencing snRNA sequencing was carried out using the 10X Genomics Chromium Single Cell 3’ Protocol,

Accessories and Kits (CG000183 Rev A). Eight samples were used for processed: Adult uninjured (AUI), Adult 4dpi (A4), Adult 7dpi (A7), Old uninjured (OUI), Old 4dpi (O4), Old

7dpi (O7). AUI and OUI were run in duplicate. In Step 1 GEMS were created, 1600 nuclei were loaded per reaction in the chromium controller for a targeted recovery of 1000 nuclei (Table 1).

Table 1: Concentration of nuclei used for GEM generation Sample AUI A4 A7 OUI O4 O7 AUIb OUIb Nuclei 427 362 506 1050 366 903 427 1050 Concentration (nuclei/µl) Volume used 3.74µl 4.41µl 3.16µl 1.52µl 4.37µl 1.77µl 3.74µl 1.52µl to get 1600 nuclei

Nuclei not used in the reaction were fixed and mounted for TMEM38a staining. Samples were stored in 4ºC overnight following Step1. In Step 2 14 cycles, instead of 12, were done for cDNA amplification because nuclei have lower RNA content compared to cells. Prior to Step 3 samples were analyzed on a Bioanalyzer 2100 at the BioFrontiers Sequencing Core to determine cDNA concentration. Only 25% of each sample was used for generating gene expression libraries

(Table 2).

Table 2: Concentration of cDNA for Library Preparation Sample AUI A4 A7 OUI O4 O7 AUIb OUIb cDNA 171.2 92.8 39.3 200.8 115.2 16.9 167 170.4 concentration (ng) Concentration 42.8 23.2 9.83 50.2 28.8 4.23 41.8 42.6 for library generation (ng)

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Eight unique sample indices from PN-220101 Chromium i7 Sample Index 153024 were added to each sample (Table 2) for identifying each sample after sequencing and 14 cycles were performed for sample index PCR in Step 3.

Table 3-Chromium i7 Sample Indices AUI SI-GA-A2 TTTCATGA ACGTCCCT CGCATGTG GAAGGAAC

A4 SI-GA-A3 CAGTACTG AGTAGTCT GCAGTAGA TTCCCGAC

A7 SI-GA-A4 TATGATTC CCCACAGT ATGCTGAA GGATGCCG

OUI SI-GA-A5 CTAGGTGA TCGTTCAG AGCCAATT GATACCGCC

O4 SI-GA-A6 CGCTATGT GCTGTCCA TTGAGATC AAACCGAG

O7 SI-GA-A7 ACAGAGGT TATAGTTG CGGTCCCA GTCCTAAC

AUIb SI-GA-A8 GCATCTCC TGTAAGGT CTGCGATG AACGTCAA

OUIb SI-GA-A9 TCTTAAAG CGAGGCTC GTCCTTCT AAGACGGA

Library quality and quantification was performed prior to sequencing at the BioFrontiers

Sequencing Facility on the Illumina Nextseq500. The goal read depth per nucleus was 50,000 reads for a targeted recovery of 1000 nuclei. Sequencing was 1x28bp for the 10X barcode and unique molecular identifier, 1x91bp for the cDNA fragment, and an 8bp index for identifying samples. 1203 nuclei were captured from AUI and 1351 nuclei for OUI.

Bioinformatics

Cellranger (3.0.1) mkfastq was used for demultiplexing the DNA sequences for the samples and the count module was used for alignment, filtering, barcode counting and UMI counting. The reference genome used was mm10. Cell Loupe Browser (3.0.1) was utilized to clustering the nuclear populations. A list of known muscle and non-muscle genes were compiled to investigate gene expression within and across clusters. 24

Results

Myogenic Nuclei in Adult Aged and Injured Muscle

Nuclei not used for library preparation were mounted and stained for TMEM38a. Due to the high amount of background fluorescence in the DAPI channel only four section images were taken for each of the six samples. The number of DAPI (+) nuclei was used as the total when calculating percentages of myogenic nuclei. The percentage of TMEM (+) nuclei ranged from

62% (±5.65%) to 86% (±9.71%) and is comparable to previous data in Chapter 1 (Table 3, Fig.

9). The aged samples trend towards more myogenic nuclei than the adult, the percentage of myonuclei in the 7dpi samples was much less than the uninjured but statistics were not performed between populations because there were not enough images taken per sample. The percentage of myonuclei was compared to the number of myonuclei that resulted from sequencing. Within the TMEM38a population, I predicted to see multiple sub-populations; myotendinous junction, neuromuscular junction, that could be identified with Cell Loupe 25

Percent Myogenic Nuclei in Adult Aged and Injured Muscle 100% 90% Sample TMEM+ TMEM- 80% Adult Uninjured 0.7997 0.2003 Old uninjured 0.8628 0.1372 70% A4dpi 0.8169 0.1831 60% O7dpi 0.7777 0.2223 50% A7dpi 0.6205 0.3795 40% O7dpi 0.7016 0.2984 30% Table 3: Percentage of Myogenic Nuclei in 20% Samples Used for snRNA-Seq 10% 0% AUI OUI A4dpi O4dpi A7dpi O7dpi % TMEM + % TMEM -

Figure 9: Percentage of Myogenic Nuclei in Adult Aged and Injured Muscle is comparable to previous results (Ch. 1). Blue bars represent myogenic nuclei, statistics were not performed to compare populations.

Adult Nuclei Gene Expression

A t-distributed stochastic neighbor embedding (T-SNE) plot was created through the

bioinformatics pipeline that graphically clustered nuclei based on similar gene expression

patterns. The T-SNE plot takes a high dimensional plot and reduces it to a low dimensional 2D

plot by clustering nuclei based on their similarities. Four nuclei clusters were produced from the

pipeline and populations of myonuclei and mononucleated cells were determined by inputting

skeletal muscle gene markers. Heterogeneity was expected to exist in the different populations of

mononucleated cells (blood, endothelial, satellite cells, etc.) and in the myonuclei population. 26

Nuclei near the neuromuscular junction (NMJ) transcribe acetylcholinesterase (AChE) for breaking down acetylcholine and acetylcholine receptors (CHRNA1) that are required for , and these genes are undetectable in denervated areas of muscle fibers (Newlands et al., 1998). The expression of AChE and CHRNA1 in the adult muscle clustered to a very small population of nuclei, as would be expected. TMEM38a expression was present in all of the myonuclei populations and consisted of 79% of the total nuclei, comparable to previous calculations.

The myonuclei had homogenous expression of some genes (actin, myosin light chain, titin, TMEM38a, etc.) as expected, but there were three distinct populations of myonuclei that each expressed different myosin heavy chains. Myh7 is expressed in slow twitch myofibers and is associated with oxidative metabolism, while Myh1, Myh2, and Myh4 are present in fast twitch myofibers and perform glycolytic and oxidative metabolism (Quiat et al., 2011). The TA is predominantly fast twitch and expresses Myh1, Myh2, Myh4 in three different populations.

Myh7 acted as a negative control. It would not be expected to be expressed in a fast twitch muscle, and it is not (Fig. 10).

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A Myonuclei Single cell nuclei MyoD, Myf6, Myf5, Myosin light chain 1 Actin1, Mef2c, Titin, Tmem38a, Nebulin Glycolytic Myh4 Pfkfb3 Glycolytic Myh1 Myoglobin

NMJ Oxidative AChE, Chrna1 Myh2 Lpl Satellite Cells Pax7 Endothelial Myf5 Pecam, Gbp4

Fibroblasts Blood tenascin-X Kcnj8 CD34 Pdgfrb Pdgfra Vitronectin

B- AChE/CHRNA1

28

C-TMEM38a

Figure 10: Nuclear atlas of adult uninjured TA- A) Four clusters of nuclei populations were produced from the TA muscle. Myonuclei and single cell nuclei are displayed as 2D T-SNE plot. The myonuclei form three distinct populations while all express genes myonuclei are expected to produce including TMEM38a, myosin, actin and titin. B) AChE and CHRNA1 cluster in a small population at the NMJ C) TMEM38a expression in nuclei is abundant in all three populations in the myonuclei.

Aged Nuclei Gene Expression

Nuclear transcripts in aged muscle differed extensively when compared to the adult. The

Pax7 population is lost, but instead nuclei were identified expressing MyoD, meaning the nuclei

have differentiated and are unable to go back to quiescence. There are still distinct populations of

fibroblasts and blood, but other mononucleated populations have disappeared, dispersed, or are

expressed at undetectable levels. The myonuclei specific genes present in the adult sample are

observed in all clusters, including TMEM38a. In the aged sample one of the glycolytic

populations is no longer clustered (Fig. 11). Regulation of gene expression is not as tightly 29

controlled as it is in the adult muscle, causing genes to be dispersed in all of the clusters. This

may contribute to the lost efficiency in muscle regeneration that accompanies aging.

A Myonuclei Single cell nuclei

Glycolytic Myh4 Observed all Clusters Myh1, Mb, Mef2c, Pfkfb3 Myf6, Myosin light chain 1 Titin, Tmem38a, Nebulin

Undetected in Green cluster

Satellite Cells Pax7 Myf5

Endothelial Blood Pecam Kcnj8

Gbp4 Fibroblasts Pdgfrb tenascin-X Vitronectin CD34 Pdgfra

30

B-TMEM38a

Figure 11: Nuclear atlas of aged uninjured TA-The aged muscle has substantial differential expression, it is missing a satellite cell and endothelial population and has expression of genes in every cluster that were only present in myonuclei in the adult muscle. B) TMEM expression is present in all clusters and the percentage is higher than in the adult muscle.

Discussion

A gene expression atlas was created for adult, aged, and injured muscle using snRNA-

Seq. The samples were compared to see changes that may be occurring in injury and aging.

Populations of myonuclei and mononucleated cells were separated via gene expression patterns and populations were clustered, including blood, fibroblasts, endothelial and satellite cells. As was expected mRNA expression was not homogenous for every nucleus that was present in the myofibers. There were nuclei clustered tightly and expressing AChE and CHRNA1 mRNA, this 31 was expected to be the NMJ. Staining of myogenic nuclei allowed a comparison to the populations that came out of the sequencing run, to see if the number of myogenic cells are similar between the two experiments. The amount of myonuclei expressing TMEM38a mRNA appears comparable to the staining experiments, validating the populations of myonuclei. The myonuclei had three distinct populations all consistently expressing genes needed for proper functioning of myofibers (actin, myosin light chain, TMEM38a) that were not expressed in the mononucleated cluster. Each population of myonuclei expressed a different myosin heavy chain for fast twitch muscle, and slow twitch myosin, Myh7, was absent in all three populations.

The clusters present in the old muscle also had two distinct populations of myonuclei but the Myh1 population that was clustered in the adult was present everywhere in the aged muscle.

There were many genes that were expressed in all clusters or not present in any clusters that were present in the adult muscle. The aged muscle has substantial differences in expression compared to the adult, but the significance of these changes has not yet been determined. The genotypes of all the mice were not the same for the sequencing experiment. All mice were the same C57BL/6 strain, but the adult samples were wild-type and the aged mice were transgenic. There may be inherent differences that are showing up as differences in gene expression as a result of age but are really an artifact of being transgenic mice. A follow up study using all wild-type mice would confirm the different populations are due to age. The analysis performed here is preliminary, determining true differences in expression within a single muscle and between aged and injured states and their significance will be explored through further analysis of the samples.

32

General Discussion

A protocol to isolate nuclei from a single whole muscle was created and exploited to examine the active transcriptional profiles of adult, aged and injured muscles. SnRNA-Seq utilizing Illumina sequencing was used to carry out transcriptional profiling of the TA muscle, which previously had not been performed in skeletal muscle due to difficulty of nuclear isolation. Discovering heterogeneity of gene expression in these different conditions will provide information about dysregulation in an aged muscle. The atlas that was created will also allow more specific questions to be asked about the differential gene expression that is potentially harmful to proper muscle functioning.

The largest limitation of this study was the sample size. In both staining experiments the number of biological replicates should be increased to make more valid conclusions about the percentage of myogenic cells in different populations, however the amount of myonuclei expressing TMEM38a was comparable in the sequencing experiment. This study provides preliminary data on differences in expression of adult and aged muscle. The aged muscle appears to have substantial differences in gene expression compared to the adult muscle, but more detailed analysis and statistics of adult and aged muscle nuclei along with analysis of the injury timepoints needs to be performed to discover true differences in the populations. In the future more injury timepoints will be carried out with the snRNA-Seq to get a more complete picture of what is being transcribed during the injury and repair process.

The isolation protocol is versatile. It can be used in any number of muscle groups and sequencing can be performed on different muscle groups to make comparisons of fast versus slow twitch muscles, large muscles, or very small muscles because of the low input and high sensitivity of the sequencing. Directed studies can be carried out and questions about what is 33 actively transcribed in different populations can be answered. This system allows investigation of diseased states, development, or studies of specific subpopulations to be manipulated and possibly targeted for therapeutics. Muscle biopsies of patients with skeletal muscle diseases can be explored to create more targeted therapeutic manipulation.

34

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Acknowledgements

The mentorship provided by Dr. Hall made this project possible, along with contributions and insights from other members of the Olwin lab. I would like to thank Amber Scott at the

BioFrontiers Institute Next-Gen Sequencing Core Facility, which performed the Illumina sequencing and library construction. Additionally, I would like to thank Dongmei in the Yi lab for 10X equipment and training, and James Orth in the light microscopy core. This project was in part funded by the Undergraduate Research Opportunity Program and the Biological Sciences

Initiative.