Investigating the Effect of Ethanol on Wnt7a and Its Potential Implications in Fetal Alcohol Spectrum Disorder

University of Central Florida STARS

Honors Undergraduate Theses UCF Theses and Dissertations

2020

Investigating the Effect of Ethanol on Wnt7a and its Potential Implications in Fetal Alcohol Spectrum Disorder

Erika Lytle University of Central Florida

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Recommended Citation Lytle, Erika, "Investigating the Effect of Ethanol on Wnt7a and its Potential Implications in Fetal Alcohol Spectrum Disorder" (2020). Honors Undergraduate Theses. 730. https://stars.library.ucf.edu/honorstheses/730 INVESTIGATING THE EFFECT OF ETHANOL ON WNT7A AND ITS

POTENTIAL IMPLICATIONS IN FETAL ALCOHOL SPECTRUM DISORDER

ERIKA LYTLE

A thesis submitted in fulfillment of the requirements for the Honors in the Major Program in Biomedical Sciences in the Burnett School of Biomedical Sciences and in the Burnett Honors College at the University of Central Florida Orlando, Florida

Spring Term, 2020

Thesis Chair: Steven Ebert, PhD

i ABSTRACT

Fetal Alcohol Spectrum Disorders (FASDs), are caused by maternal alcohol consumption during pregnancy [3]. FASD encompasses a wide variety of cardiac and neural anomalies, while also associated with improper limb development, abnormal craniofacial features, problems within the central nervous system (CNS), and disabilities in learning and communication. Gene- regulating FASDs have not been well studied during the crucial phases of early embryonic development. Genes within the Wnt/Beta-catenin pathway control a vast amount of embryonic developmental processes. Among them is the Wnt7a gene, a significant downstream gene regulator which positively controls neural stem cell proliferation and cardiomyocyte differentiation on a large scale during early embryonic development. This project will serve to provide potential insight into the genes involved in FASD. We hypothesize that ethanol administration to early embryonic mice will suppress Wnt7a expression in the heart and brain, leading to FASD development. RNA-sequencing (RNA-Seq) and real-time quantitative PCR

(qPCR) were used to measure Wnt7a gene expression within the early embryonic mouse heart and brain. After evaluation of RNA-Seq data and a comparative analysis using the 2-ΔΔCT method, it is evident Wnt7a is present in embryonic mouse age E10.5 heart and brain samples, and Wnt7a is suppressed at age E10.5 in embryonic mouse heart, but not brain, when induced with ethanol.

TABLE OF CONTENTS

LIST OF FIGURES ...... v

LIST OF TABLES ...... vi

LIST OF ABBREVIATIONS ...... vii

BACKGROUND ...... 1

Fetal Alcohol Spectrum Disorder ...... 1

Clinical Diagnosis of FASD ...... 2

Effects of FASD on the Embryonic Heart ...... 3

Wnt7a ...... 3

Wnt7a in the Embryonic Brain ...... 5

Wnt7a in the Embryonic Heart ...... 6

Effects of Ethanol on Wnt7a ...... 7

Significance ...... 8

METHODS ...... 10

Ethanol Administration to Wild-Type C57/BL6J Mice ...... 10

Testing for Wnt7a Presence in Early Embryonic Heart and Brain ...... 10

Determining Wnt7a Suppression in Early Embryonic Mouse Heart and Brain Post- Ethanol Administration ...... 11

RESULTS ...... 14

A Review of Preliminary Studies ...... 14

Wnt7a Presence in Early Embryonic Heart and Brain ...... 16

Determining Wnt7a Suppression ...... 17

Wnt7a in the Embryonic Heart ...... 18

Wnt7a in the Embryonic Brain ...... 20

iii DISCUSSION ...... 23

APPENDIX A WNT7A CT VALUES AND EXPRESSION FOLD CHANGE IN HEART AND BRAIN E10.5 SAMPLES ...... 25

WORKS CITED ...... 28

LIST OF FIGURES

Figure 1: An overview of Wnt signaling pathway activation...... 5 Figure 2: Experimental protocol for control vs. ethanol administration in wild-type C57/BL6J mice [32]...... 10 Figure 3: Diagram of experimental method...... 13 Figure 4: RNA-Seq analysis of gene expression in E10.5 mouse hearts, control vs. ethanol treated at E9.5 [32]...... 15 Figure 5: 2% agarose gel highlighting Wnt7a gene in control E10.5 embryonic mouse heart samples at proper band size 307 bp [35]...... 17 Figure 6: CT Values for Beta-actin and GAPDH of embryonic heart samples age E10.5, where error bars represent standard deviation from the mean...... 19 Figure 7: Expression fold change (2-ΔΔCT) for control and ethanol-treated samples with Wnt7a and housekeeping gene, Beta-actin, in embryonic mouse heart age E10.5, where error bars represent standard deviation from the mean...... 20 Figure 8: CT Values for Beta-actin and GAPDH of embryonic brain samples age E10.5, where error bars represent standard deviation from the mean...... 21 Figure 9: Expression fold change (2-ΔΔCT) for control and ethanol-treated samples with Wnt7a and housekeeping gene, Beta-actin, in embryonic mouse brain age E10.5, where error bars represent standard deviation from the mean...... 22

LIST OF TABLES

Table 1: RNA-Seq data collection from Preliminary Studies...... 16 Table 2: CT Values for Beta-actin and GAPDH of embryonic heart samples age E10.5...... 26 Table 3: CT Values for Beta-actin and GAPDH of embryonic brain samples age E10.5...... 27

LIST OF ABBREVIATIONS

EtOH- Ethanol

FASD- Fetal Alcohol Spectrum Disorder

FAS- Fetal Alcohol Syndrome

ARND- Alcohol-Related Neurodevelopmental Disorder

ARBD- Alcohol-Related Birth Defects

CNS- Central Nervous System

CHDs- Congenital Heart Defects

TOF- Tetralogy of Fallot

PKC- Protein Kinase C

E3.5- Embryonic Day 3.5

E5- Embryonic Day 5

ADH- Alcohol Dehydrogenase

ALDH- Aldehyde Dehydrogenase

E9.5- Embryonic Day 9.5

E10.5- Embryonic Day 10.5

E11.5- Embryonic Day 11.5

E12.5- Embryonic Day 12.5

RT-PCR- Reverse-Transcription Polymerase Chain Reaction qPCR- Real-Time Quantitative Polymerase Chain Reaction cDNA- Complimentary DNA

RNA-Seq- Genomic RNA-Sequence

vii bp- Base Pair (size)

GAPDH- Glyceraldehyde 3-phosphate dehydrogenase

E15- Embryonic Day 15

IQR- Interquartile Range

viii

BACKGROUND

Fetal Alcohol Spectrum Disorder

Maternal consumption of ethanol (EtOH) during pregnancy has been found detrimental to proper fetus development on a systemic scale [1]. Typical teratogenic effects due to alcohol consumption have been implicated in the medical diagnosis Fetal Alcohol Spectrum Disorder

(FASD), a term adopted to describe a wide range of effects due to fetal alcohol-related incidences that occur due to maternal consumption of alcohol during pregnancy [2]. FASD presents itself with a variety of symptoms and systemic defects, including abnormal facial features, thin upper lip, microcephaly, learning disabilities, vision or hearing problems, and improper limb, kidney, and heart development [1, 2]. With further understanding of FASDs, scientists and medical professionals have agreed that the act of binge drinking (consuming 3-5 drinks or more in one occasion) has shown to be most detrimental to fetal development, where one drink is considered one 12 oz. beer, one 5 oz. glass of wine, or one 1.5 oz. shot of distilled spirits [3, 4]. In recent studies, it has been recognized that FASDs are the leading cause of preventable developmental disabilities in the world, where it has also been found that the prevalence of FASDs in the United

States is much higher than previously believed [3]. Although it is difficult to determine the exact number of children born with FASD, researchers have estimated that approximately 40,000 babies may be born with FASD in the United States each year [5]. A recent study by Philip et al. created current estimates of FASD based on large, diverse United States population samples which showed estimated prevalence of FASD ranged from 1.1% to 5.0%, or 1 to 5 per 100 school children [6].

1 Clinical Diagnosis of FASD

The adverse effects of alcohol on a fetus present a spectrum of abnormalities ranging from behavioral and neurocognitive disabilities, to physical abnormalities of the limbs, craniofacial region, heart, and kidneys, symptoms that when in conjunction, can be most accurately termed Fetal Alcohol Spectrum Disorder (FASD) [1, 2, 7]. To aid in diagnosis and treatment, medical professionals have further differentiated FASD into three separate diagnoses, each with a more specific evaluation of symptoms, allowing medical practitioners a better understanding and development of treatment plan for patients affected by FASD. The three distinct diagnoses of FASD include Fetal Alcohol Syndrome (FAS), Alcohol-Related

Neurodevelopmental Disorder (ARND), and Alcohol-Related Birth Defects (ARBD) [8]. It has been established that children at the severe end of the spectrum (meaning the child exhibits the complete phenotype) are defined to have Fetal Alcohol Syndrome (FAS) [7, 8]. FAS presents with abnormal craniofacial features, growth problems, central nervous system (CNS) problems, and potential inadequacy in learning, communication, vision, and hearing problems, with the most extreme outcome of FAS being fetal death. In the lesser extreme cases of FASD are

ARND, which presents most commonly with intellectual, behavioral, and learning disabilities, and ARBD, which mostly comprises of physical abnormalities or problems within the heart, kidneys, bones, and potentially hearing [8]. With medical professionals having a clearer understanding of the distinct forms of FASD, it is easier to diagnose and create a treatment plan, however, there is no cure for fetal alcohol-related abnormalities as they are present during the developmental stages of embryonic life, where these effects cannot be reversed and the fetus thus suffers lifetime consequences [2, 8].

Effects of FASD on the Embryonic Heart

FASD presents itself with a wide range of symptoms and can be further specified into three categories for diagnostic procedures. The subset FASD that most commonly effects the heart is Fetal Alcohol Syndrome (FAS), where it has been found prenatal exposure to alcohol has a direct impact on embryo cardiogenesis, seeing that alcohol engenders a higher risk of cardiac anomalies and most commonly leads to congenital heart defects (CHDs) [9, 10]. More specifically, prenatal alcohol exposure has been found to cause a wide variety of CHDs such as cardiac transposition of great vessels, coarctation of the aorta, Tetralogy of Fallot (TOF), and pulmonary valve stenosis. Quite commonly discovered in newborns affected by maternal consumption of alcohol is the incidence of ventricular and atrial septal defects, otherwise known as hole in the heart, where a hole develops and does not properly close between the left and right ventricles or atria [9, 11]. Furthermore, the assortment of cardiological developmental complications due to prenatal alcohol exposure is known to contribute to heart disease later in adulthood [9, 10]. These severe heart defects are detrimental to a developing embryonic heart.

Considering that the precursor cardiac progenitor cells develop and differentiate in the third week of gestation, giving rise to three embryonic germ layers: ectoderm, endoderm, and mesoderm, contribute to explaining why alcohol consumption during pregnancy prompts fetal damage [1, 9-11].

Wnt7a

The Wnt gene family consists of structurally related genes that encode secreted signaling proteins involved in oncogenesis and various developmental processes, especially during

3 embryogenesis [12]. One specific member of this gene family is Wnt7a, which in humans, is a gene found on chromosome 3, specifically at the cytogenic location 3p25.1 [13]. Wnt7a is a gene commonly known to play a large role in embryonic development, assisting in dorsal versus ventral patterning of limb, skeletal, and urogenital tract development of the embryo, while also showing as pertinent to CNS angiogenesis, blood-brain barrier regulation, and a required gene for normal neural stem cell proliferation [12, 14]. Additionally, the Wnt7a gene is a known ligand for members of the frizzled family of seven transmembrane receptors working within the

Wnt/Beta-catenin pathway, where Beta-catenin is a dual function protein, known to be involved in cell to cell adhesion and gene transcription [12]. The Wnt family of genes has been determined as a group of evolutionarily conserved, secreted Cys-rich proteins, where there are 19 total Wnt genes within humans that couple to a number of receptors, thereby activating various downstream pathways [12, 15]. Definitively, early studies have established said pathways as either canonical (Beta-catenin-dependent) or noncanonical (Beta-catenin-independent) [14, 15].

In the canonical pathway, Beta-catenin has been recognized as the key effector responsible for signal transduction to the nucleus while triggering transcription of Wnt-specific genes that work on a cellular level. In the noncanonical pathway, it is noted that Wnt signaling is independent of

Beta-catenin signaling function, contributing mostly protein kinase C (PKC) dependent pathways

[14]. Amongst the two methods of Wnt signaling, the canonical, Beta-catenin-dependent pathway has been distinguished as following a signal transduction cascade, where highly transforming members of this cascade include Wnt1, Wnt3, Wnt3a, and Wnt7a [15, 16]. Due to

Wnt genes primarily driving tissue stem cells and working closely within the canonical pathway, the Wnt signal transduction cascade has been established as a main regulator of development.

Nonetheless, mutations within the Wnt pathway are a primary reason behind growth-related pathologies and cancerous developments on a cellular level [16]. An overview of Wnt signaling pathway activation is shown in Figure 1 below.

Figure 1: An overview of Wnt signaling pathway activation. On the left, Wnt signaling is inactivated due to absence of the Wnt ligand. Beta-catenin is then phosphorylated, resulting in Dishevelled (DVL), Axin, APC and GSK3β complex, ensuing proteosomal degradation. On the right, canonical Wnt signaling activation occurs after Wnt ligand binding. Unphosphorylated Beta-catenin enters the nucleus to drive transcription of downstream genes, which pictured here include C-MYC, SOX9, CD44. Reprinted from Targeting Wnt Signaling for Multifaceted Glioblastoma Therapy, by M. McCord et al., 2017, https://doi.org/10.3389/fncel.2017.00318 [17].

Wnt7a in the Embryonic Brain

Wnt7a is expressed in both neural progenitor cells and neurons at notably high expression levels. Correspondingly, recent experimental data has shown Wnt7a regulates neural stem cell proliferation and neuronal differentiation by the activation of downstream target genes within the

5 canonical Beta-catenin pathway, indicating Wnt7a specifically stimulates neural stem cell proliferation by activation of the canonical cascade. Equally important, studies have identified that knockdown of Wnt7a, and thus Wnt7a expression, resulted in cell proliferation inhibition, where reduced neural stem cell renewal and inhibition of hippocampal dentate neuron production and maturation was observed [18].

Wnt7a in the Embryonic Heart

Soon after fertilization in utero, vertebrate embryos grow quite rapidly, with the heart forming as the first functional organ [19]. Developmental processes begin as the heart forms from mesodermal regions of the anterior region of the heart [9-11, 19]. Specifically, the Wnt7a and Wnt7b genes work canonically in the Wnt pathway in formation of cerebral vascular and pulmonary vascular smooth muscle development, both by use of the canonical pathway and both regulating cardiomyocyte differentiation depending on the stage of development [20-23].

Explicitly, studies have shown that in the early stages of development, Wnt signals specifically promote cardiogenesis while inhibiting hematopoiesis, whereas in the late stages of development, Wnt signals inhibit cardiomyocyte differentiation [20-23]. Even so, it is quite evident that activation of the canonical Wnt pathway is required for the induction of cardiac transcription factors and cardiomyocyte differentiation during the crucial stages of embryo development [19-23]. Specifically, in the early stages of cardiological development in mice embryo, Wnt7a is first detectable in the myocardium of the outflow tract and ventricles by embryonic day 3.5 (E3.5) and makes a vast appearance throughout the entirety of the heart by embryonic day 5 (E5), whereby after E5, Wnt7a was primarily found only in the ventricular

6 endocardium and interventricular septum [24, 25]. Concerning the variety of Wnt family genes within the canonical pathway, Wnt11 shows transient and prominent expression in the cardiac conduction system, while Wnt7a expression was found chiefly in areas of peripheral conduction cells, with both genes up-regulated consistently with conduction cell development in vitro [25].

Equally important, studies have established that ectopic Wnt signals can repress cardiac formation from anterior mesoderm in vitro and in vivo, where inhibition of Wnt signaling would promote anterior lateral mesoderm formation, and Wnt signaling in the posterior lateral mesoderm will promote blood cell development and differentiation [26].

Effects of Ethanol on Wnt7a

As previously stated, Wnt7a is a pivotal gene within the Beta-catenin canonical pathway, important for neural stem cell proliferation and required for the induction of cardiac transcription factors and cardiomyocyte differentiation in the fetus [18, 21-23]. With much experimentation in neurodevelopmental studies, it has been found that ethanol decreases Wnt7a expression [27-30].

Looking closely at the breakdown of ethanol in bodily metabolic processes may help in distinguishing how ethanol is determined as being harmful in various aspects. The body processes and eliminates ethanol in a series of steps using enzymes and producing intermediate metabolites found to be harmful to the body. Most ethanol that is ingested is broken down by the liver enzyme alcohol dehydrogenase (ADH), which transforms ethanol to acetylaldehyde, a known carcinogen. Next, acetylaldehyde is further broken down by the enzyme aldehyde dehydrogenase (ALDH) to form acetate, a less toxic compound that is metabolized into water and carbon dioxide for easy elimination [31].

With better understanding of the breakdown of ethanol and how the metabolic reaction yields harmful intermediates, it will become more apparent how maternal binge alcohol consumption can induce a cascade effect on embryonic development. In particular, it has been found that expression of Wnt7a is decreased by acute alcohol exposure in the developing embryonic mouse, where Wnt7a was expressed at low levels during the first week of life and rose several-fold higher in the following week [27]. With this idea, researchers investigated ethanol exposure to Wnt7a in developmental processes and found that maternal consumption of ethanol had a direct effect on regulation of neural stem cell proliferation due to inhibition of the

Wnt signaling pathway [27-29]. It was found ethanol significantly altered neural differentiation in pathway-related gene expression and Wnt signaling proteins were discovered as suppressed after ethanol exposure, specifically the Wnt7a gene [27-30].

Significance

Wnt7a has shown to play a significant role within the Beta-catenin canonical pathway as a downstream gene regulator, while also proving to be an imperative gene in the regulation of neural stem cell proliferation and cardiomyocyte differentiation during early embryonic development. Previous studies have shown the Wnt7a gene is suppressed with ethanol exposure, causing a canonical knockdown of downstream genes during neural, and potentially cardiac, development. In testing the effects of ethanol on Wnt7a expression in the early embryonic heart and brain, one can further investigate the relationship between maternal alcohol consumption and the canonical genes present and affected by ethanol addition during early embryonic development in both the heart and brain. Specifically, testing the effects of ethanol on Wnt7a

8 within the early embryonic heart, then testing if these effects happen simultaneously within the embryonic brain, can possibly assist in better understanding cardiac and neural anomalies present in Fetal Alcohol Spectrum Disorder.

METHODS

Ethanol Administration to Wild-Type C57/BL6J Mice

The binge alcohol animal model established by Webster et al. exemplified congenital heart defects (CHDs), which commonly occur in FASD after maternal alcohol administration

[32, 33]. Thus, this model was used throughout experimentation in order to standardize ethanol administration. In this model, wild-type C57/BL6J mice (age 8-10 weeks) were mated at age

E9.5 and administered orally with a single dose of alcohol (5g/kg) or saline as control [32, 33].

The embryonic hearts and brains were then isolated and flash-frozen in liquid nitrogen, allowing for future RNA extraction. A summary of this experimental protocol is shown below in Figure 2.

Figure 2: Experimental protocol for control vs. ethanol administration in wild-type C57/BL6J mice [32].

Testing for Wnt7a Presence in Early Embryonic Heart and Brain

Wnt7a presence in early embryonic heart and brain was tested using wild-type C57/BL6J mice at embryonic age E10.5. This age was chosen to better conclude any changes in Wnt7a expression between ethanol and control samples at an early embryonic developmental stage,

10 which per the binge alcohol animal model, would be synonymous to a human embryo aged about

4 weeks [32]. In doing so, one can potentially better understand how Wnt7a expression tested with and without ethanol in the early embryonic heart and brain may contribute to cardiac and neural anomalies present in Fetal Alcohol Spectrum Disorder.

Using Qiagen RNeasy kits, RNA extraction and cleanup were performed on unaffected, control embryonic mouse heart and brain tissue samples age E10.5. A reverse-transcription polymerase chain reaction (RT-PCR) was then performed to amplify the mouse RNA samples, where the RNA was thereby reverse transcribed to complimentary DNA, or cDNA, in a reaction also termed RT-reaction [34]. The embryonic mouse heart and brain samples were purified by use of Qiagen PCR purification kits and the samples were nanodropped to ensure a baseline

DNA concentration of 20-nanograms per microliter. This procedure was followed by a real-time quantitative polymerase chain reaction (qPCR) with the newly transcribed mouse cDNA, coupled with the Wnt7a gene primer. This process allowed for the detection of Wnt7a presence in embryonic mouse heart and brain samples at age E10.5. A 2% agarose gel was then prepared and completed to confirm possible findings of the Wnt7a gene, which appeared as bands of 307 bp [35].

Determining Wnt7a Suppression in Early Embryonic Mouse Heart and Brain Post-Ethanol Administration

Once it was established that Wnt7a is normally present in embryonic mouse heart and brain samples age E10.5, ethanol and control samples, both coupled with Wnt7a, were to be tested. Similar to finding Wnt7a presence in early embryonic heart and brain, RNA was extracted and cleaned by use of Qiagen RNeasy kits, however, from experimental (ethanol) and control

(saline) samples age E10.5. A RT-PCR was then performed to reverse transcribe the mouse samples from RNA to cDNA, then concurrently, Qiagen PCR purification kits were used to purify the embryonic mouse heart and brain samples. Once the samples were purified, a nanodrop was performed to establish a baseline DNA concentration of 20-nanograms per microliter. qPCR was performed using the mouse cDNA, coupled with the Wnt7a gene, which provided CT values. Comparative analysis using the 2-ΔΔCT method normalized to same-sample housekeeping genes, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Beta-actin, facilitated in dissecting potential differences between the experimental and control samples of embryonic mouse heart and brain samples at age E10.5. A 2% agarose gel was then prepared and completed to confirm possible findings.

By completing qPCR and a comparative analysis using the 2-ΔΔCT method, it was possible to determine if Wnt7a expression was suppressed in embryonic mouse heart and brain samples age E10.5. This was possible because qPCR assays work to detect the accumulation of a fluorescent signal, where the given CT value is defined as the number of cycles required for the fluorescent signal to cross threshold and exceeds background level of expression. Moreover, CT values are inversely proportional to the amount of target nucleic acid (Wnt7a) in the sample, where the lower the CT value, the greater the amount of target nucleic acid in the sample [36]. A summary of this experimental protocol is shown in Figure 3.

Figure 3: Diagram of experimental method.

RESULTS

A Review of Preliminary Studies

For the development of this study, preliminary data from previous undergraduate and graduate researchers within Dr. Steven Ebert’s lab at the Lake Nona (UCF) campus was acquired and further analyzed. Prior research within Dr. Steven Ebert’s lab developed genomic RNA-

Sequence (RNA-Seq) data analysis that showed Wnt7a, as well as other genes within the Beta- catenin pathway, to downregulate with the addition of ethanol at embryonic day 10.5 (E10.5). To acquire this data, RNA was isolated from E10.5, E11.5, and E12.5 embryonic hearts and RNA-

Seq data was analyzed to identify at least 14 genes that were substantially (2-fold or greater) and significantly (P<0.05) decreased in the embryonic heart within 24 hours of maternal alcohol administration. These results are displayed in Figure 4, where use of the log2 scale allowed for the representation of the relative expression for each gene tested, shown as a heat map. In this figure, each square represents one heart sample, while each row represents the relative expression for each gene. These results suggest that gene expression changes in the heart occur primarily within the first 24 hours after a single binge administration of ethanol at E9.5 [32].

When analyzing this set of data, one can conclude that Wnt7a expression drastically decreased

24 hours after ethanol addition, suggesting it as an important gene to consider for further analysis.

Control E10.5 EtOH

Figure 4: RNA-Seq analysis of gene expression in E10.5 mouse hearts, control vs. ethanol treated at E9.5 [32].

Isolating the Wnt7a gene from the RNA-Seq data allowed for a better interpretation of how the addition of ethanol to embryonic mouse heart tissues age E10.5, E11.5, and E12.5 may affect expression values of Wnt7a. As shown in Table 1, the RNA-Seq data proved Wnt7a suppression due to ethanol administration in embryonic heart tissue samples of wild-type

C57/BL6J mice, where Wnt7a expression values are suppressed most at the E10.5 level, then

E12.5, and E11.5.

Age Expression Expression Average difference Value Value (control-ethanol) (control) (ethanol) 84 6 E10.5 42 3 9 5 29 3 AVG: 10.25 AVG: 4.25 6 6 8 E11.5 20 14

10 7 11 11 AVG: 11.75 AVG: 10 1.75 11 10 E12.5 5 10

20 10 10 5 AVG: 11.5 AVG: 8.75 2.75

Table 1: RNA-Seq data collection from Preliminary Studies. Data shows the expression values of embryonic mouse heart tissue samples ages E10.5, E11.5, and E12.5, control vs. ethanol, with average difference to elucidate the differences in control vs. ethanol at specific ages with Wnt7a.

Wnt7a Presence in Early Embryonic Heart and Brain

After analyzing the preliminary studies data and findings, it became evident that the

Wnt7a gene is present in the embryonic mouse heart at age E10.5. To ensure the gene primer properly worked, a test-run was completed. The experimental procedure was the same as previously described, where RNA was isolated from E10.5 embryonic mouse heart tissues which had not been administered with ethanol, followed by RT-PCR and qPCR, to test if the expression of Wnt7a was found to be present within the mouse embryonic heart at age E10.5. After analysis of a 2% agarose gel that was loaded with the completed qPCR samples, Wnt7a showed on the gel at proper band size (307 bp) when amplified with E10.5 cDNA at 20-nanograms per microliter concentration, meaning Wnt7a was present in embryonic mouse hearts age E10.5 [35].

Figure 5 shows the 2% agarose gel, highlighting bands of E10.5 embryonic mouse heart samples coupled with the Wnt7a gene at 307 bp.

Figure 5: 2% agarose gel highlighting Wnt7a gene in control E10.5 embryonic mouse heart samples at proper band size 307 bp [35].

The Wnt7a gene was then tested for presence and expression within E10.5 embryonic mouse brain samples following the same experimental procedure. After RT-PCR and qPCR, a

2% agarose gel was performed and showed bands once again for the Wnt7a gene at proper band size, exemplifying Wnt7a was present within the embryonic mouse brain tissue at age E10.5.

Determining Wnt7a Suppression

By completing qPCR and a comparative analysis using the 2-ΔΔCT method, it is possible to determine if Wnt7a expression is suppressed in embryonic mouse heart and brain samples age

E10.5. Concurrently, this age was chosen to better conclude any changes in Wnt7a expression between ethanol and control samples at early embryonic developmental stages [32]. In doing so, one can potentially better understand how Wnt7a expression tested with and without ethanol in

17 the early embryonic heart and brain may contribute to cardiac and neural anomalies present in

Fetal Alcohol Spectrum Disorder.

Wnt7a in the Embryonic Heart

Following the same experimental method, embryonic heart samples of both control and ethanol-administered wild-type C57/BL6J mice were isolated at age E10.5, where RT-PCR and qPCR were performed. The housekeeping genes, Beta-actin and GAPDH, were analyzed for consistency, where fluctuations between control and ethanol samples in the individual housekeeping genes were evaluated, as the individual genes should show a similar CT value throughout the test samples. If fluctuations between the control and ethanol samples occurred, the data was viewed as inconsistent as fluctuations signify potential interactions between the housekeeping genes and the ethanol, providing conflicting data for comparison of the embryonic samples. The CT values for control and ethanol in Beta-actin and GAPDH of embryonic heart samples age E10.5 are shown in Figure 6.

Beta-actin Heart E10.5 CT Values GAPDH Heart E10.5 CT Values

Control Control 30 Ethanol 30 Ethanol

25 25 CT Value CT Value

20 20 Control Ethanol Control Ethanol (p=0.08) (p=0.71) A B

Figure 6: CT Values for Beta-actin and GAPDH of embryonic heart samples age E10.5, where error bars represent standard deviation from the mean. (A) CT Values for Beta-actin control and ethanol-treated samples embryonic heart age E10.5. (B) CT Values for GAPDH control and ethanol-treated samples embryonic heart age E10.5.

From the data above, it was evident the more consistent housekeeping gene for comparison of the embryonic heart samples age E10.5 was Beta-actin, as the CT values between the control and ethanol samples were more consistent than those of the GAPDH, having less standard deviation between samples. Beta-actin was then used to compare embryonic heart samples age E10.5. Comparative analysis using the 2-ΔΔCT method normalized to same-sample

Beta-actin control facilitated in examining any differences between the experimental and control samples to determine if Wnt7a was suppressed in embryonic heart samples age E10.5. The expression fold change (2-ΔΔCT) of the samples is displayed in Figure 7.

Expression Fold Change of Heart E10.5

2.0 Control Ethanol 1.5

1.0

0.5

Expression Fold Change 0.0 Control Ethanol (p=0.002)

Figure 7: Expression fold change (2-ΔΔCT) for control and ethanol-treated samples with Wnt7a and housekeeping gene, Beta-actin, in embryonic mouse heart age E10.5, where error bars represent standard deviation from the mean.

As displayed in Figure 7, Wnt7a expression was downregulated when interacted with ethanol in embryonic mouse heart age E10.5, where the average expression fold change between control and ethanol samples decreased 2-fold, a substantial decrease in expression [32]. A two- tailed test was then completed to further statistical analysis of the samples with p-values. Wnt7a normalized to same-sample Beta-actin control in E10.5 heart samples proved significant

(P=0.002).

Wnt7a in the Embryonic Brain

The same experimental method was applied to embryonic brain tissue samples of both control and ethanol-administered wild-type C57/BL6J mice isolated at age E10.5, where RT-

PCR and qPCR were performed. The housekeeping genes, Beta-actin and GAPDH, were analyzed once more for consistency, where fluctuations between control and ethanol samples in the individual housekeeping genes were evaluated, as the individual genes should show a similar

CT value throughout the test samples. The CT values for control and ethanol in Beta-actin and

GAPDH of embryonic brain samples age E10.5 are shown in Figure 8 below.

Beta-actin Brain E10.5 CT Values GAPDH Brain E10.5 CT Values

30 30 Control Control Ethanol Ethanol

25 25 CT Value CT Value

20 20

Control Ethanol Control Ethanol (p=0.10) (p=0.06) A B

Figure 8: CT Values for Beta-actin and GAPDH of embryonic brain samples age E10.5, where error bars represent standard deviation from the mean. (A) CT Values for Beta-actin control and ethanol-treated samples embryonic brain age E10.5. (B) CT Values for GAPDH control and ethanol-treated samples embryonic brain age E10.5.

From the data above, it was evident the more consistent housekeeping gene for comparison of the embryonic brain samples age E10.5 was Beta-actin, as the CT values between the control and ethanol samples were more consistent than those of the GAPDH, having less standard deviation between samples, similar to the embryonic heart samples examined prior.

Beta-actin was then used to compare embryonic brain samples age E10.5. Comparative analysis using the 2-ΔΔCT method normalized to same-sample Beta-actin control facilitated in examining any differences between the experimental and control samples to determine if Wnt7a was suppressed in embryonic brain samples age E10.5. The expression fold change (2-ΔΔCT) of the samples is displayed in Figure 9 below.

Expression Fold Change of Brain E10.5

2.0 Control Ethanol 1.5

1.0

0.5

Expression Fold Change 0.0 Control Ethanol (p=0.23)

Figure 9: Expression fold change (2-ΔΔCT) for control and ethanol-treated samples with Wnt7a and housekeeping gene, Beta-actin, in embryonic mouse brain age E10.5, where error bars represent standard deviation from the mean.

As displayed in Figure 9, Wnt7a expression was slightly upregulated when interacted with ethanol, where the average expression fold change between control and ethanol samples increased 0.9-fold, which is not considered a substantial increase in expression, seeing as it must be 2-fold or greater [32]. A two-tailed test was then completed to further statistical analysis of the samples with p-values. Wnt7a normalized to same-sample Beta-actin control in E10.5 brain samples proved insignificant (P=0.23).

DISCUSSION

After evaluation of RNA-Seq data and a comparative analysis using the 2-ΔΔCT method, it is evident Wnt7a is present in embryonic mouse age E10.5 heart and brain samples, and Wnt7a is suppressed at age E10.5 in embryonic mouse heart, but not brain, when induced with ethanol.

The RNA-Seq data in Figure 4 provided evidence that Wnt7a expression was substantially and significantly suppressed 24 hours after ethanol addition, where the gene was downregulated significantly and displayed an average expression fold change greater than 6-fold at age E10.5, as shown in Table 1. Thereafter, Wnt7a was found to be present in the embryonic mouse heart age E10.5, as expected, but also found in embryonic brain, both at the correct band size when run on 2% agarose gels. Wnt7a expression with ethanol was then tested to determine if gene suppression occurred simultaneously in embryonic heart and brain at embryonic age E10.5.

Overall gene expression was calculated by comparing average expression fold change, using the

2-ΔΔCT method, normalized to same-sample housekeeping gene Beta-actin. Beta-actin was used throughout the study as it was found to have more consistent CT values with less standard deviation between control and ethanol samples than GAPDH in both embryonic heart and brain.

Wnt7a was normalized to same-sample housekeeping gene Beta-actin, allowing for the comparison of CT values and calculation of expression fold change of embryonic mouse heart at age E10.5. As shown in Figure 7, Wnt7a expression in embryonic heart at age E10.5 was downregulated when interacted with ethanol, where the average expression fold change between control and ethanol samples decreased 2-fold, a substantial (2-fold or greater) and significant

(P=0.002) decrease in expression. The same protocol was followed for testing Wnt7a expression in embryonic brain at age E10.5. As shown in Figure 9, Wnt7a expression increased 0.9-fold (not

23 substantial) and the results were proven insignificant (P=0.23), so the null hypothesis was accepted, meaning Wnt7a expression showed no change when induced with ethanol in embryonic brain at age E10.5.

Limitations to this study include a lack of prior research studies, where previous studies focused on Wnt7a suppression due to ethanol within neurodevelopmental stages at embryonic age E15 [27, 28]. Additionally, having a larger sample size with more mice samples across different litters could better justify results and allow for more effective statistical analysis of data. One can also further this research in a multitude of ways. First, it is important to note the

RNA-Seq data from the preliminary studies showed a decrease in the expression of Wnt7a in embryonic heart samples at ages E11.5 and E12.5 as well. These later ages can be studied to further the experimentation of the effects of ethanol on Wnt7a expression to determine if similar results occur. Moreover, Wnt7a is involved in the limb, skeletal, and urogenital tract development of the embryo [12, 14]. With this in mind, one can further study Wnt7a expression within mouse trunk samples at embryonic ages E10.5, E11.5, and E12.5 to further the knowledge of Wnt7a presence in different tissues throughout various stages of embryonic development.

Wnt7a expression can also be tested with alterations made in the Webster et al. binge alcohol animal model, where differing ethanol or saline volumes can be administered to the maternal mice at various times, thereby expanding the study to include smaller or larger ethanol doses administered at various times, rather than only embryonic age E9.5. Lastly, experimentation can be extended to include cell cultures, where cardiomyocyte cell lines can be used and tested directly.

APPENDIX A WNT7A CT VALUES AND EXPRESSION FOLD CHANGE IN HEART AND BRAIN E10.5 SAMPLES

Samples CT (B) CT (W) ΔCT (W-B) ΔΔCT (ΔCT -ΔCT Control Avg) Expression Fold Change Avg Expression Fold Change Control 11.1 23.87 34.05 10.18 0.9 1 11.8 22.41 33.86 11.44 1.01 18.7 23.87 34.25 10.38 0.92 5.1 21.94 34.31 12.37 1.09 5.2 22.11 34.12 12 1.06 Ethanol 8.2 21.59 34.26 12.66 1.38 0.38 0.51 8.4 21.33 34.19 12.85 1.58 0.33 8.5 22.65 34.09 11.44 0.16 0.89 8.6 21.19 34.07 12.87 1.59 0.33 A 8.7 22.33 34.31 11.97 0.69 0.61

Samples CT (G) CT (W) ΔCT (W-G) ΔΔCT (ΔCT -ΔCT Control Avg) Expression Fold Change Avg Expression Fold Change Control 11.1 28.95 34.05 5.09 0.66 1 11.8 26.52 33.86 7.33 0.96 18.7 28.41 34.25 5.84 0.76 5.1 24.44 34.31 9.87 1.29 5.2 24.19 34.12 9.92 1.3 Ethanol 8.2 26.36 34.26 7.89 0.28 0.82 3.65 8.4 24.68 34.19 9.51 1.89 0.26 8.5 27.3 34.09 6.79 -0.82 1.76 0.92 (without outlier) 8.6 30.32 34.07 3.74 -3.86 14.57 B 8.7 26.44 34.31 7.86 0.25 0.83

Table 2: CT Values for Beta-actin and GAPDH of embryonic heart samples age E10.5. (A) CT Values for Beta- actin and Wnt7a in embryonic heart age E10.5. (B) CT Values for GAPDH and Wnt7a in embryonic heart age E10.5. Here, W (Wnt7a) CT values were subtracted from B (Beta-actin) CT values or G (GAPDH) CT values to find individual expression fold change (2-ΔΔCT), providing an average expression fold change between control (red) and ethanol-treated (blue) samples.

*indicates an outlier. Outliers found using interquartile range (IQR) calculations, where all values must fall within the 25th and 75th quartiles of data, otherwise they are indicated as outliers and excluded from the total average expression fold change (2-ΔΔCT).

Samples CT (B) CT (W) ΔCT (W-B) ΔΔCT (ΔCT -ΔCT Control Avg) Expression Fold Change Avg Expression Fold Change Control 18.2 19.97 26.73 6.76 1.01 1 18.3 18.96 26.52 7.56 1.13 18.4 19.36 26.56 7.2 1.08 18.5 19.36 26.14 6.77 1.01 18.9 19.76 26.16 6.39 0.96 11.7 20.54 26.04 5.5 0.82 11.6 20.17 26.22 6.04 0.9 11.5 19.24 26.12 6.87 1.03 5.8 19.07 25.86 6.79 1.02 Ethanol 27.6 19.52 26.11 6.59 -0.06 1.04 2.68 27.7 19.94 26.42 6.47 -0.18 1.13 27.8 24.03 27.18 3.14 -3.5 11.37 1.11 (without outlier) 12.1 20.11 26.76 6.64 -0.01 1 12.2 20.01 26.44 6.42 -0.23 1.17 12.3 20.46 26.35 5.89 -0.76 1.69 12.4 23.08 27.03 3.95 -2.7 6.51 12.5 19.67 26.22 6.55 -0.1 1.07 12.6 19.75 26.43 6.68 0.02 0.98 A 12.7 19.1 26.08 6.98 0.32 0.79

Samples CT (G) CT (W) ΔCT (W-G) ΔΔCT (ΔCT -ΔCT Control Avg) Expression Fold Change Avg Expression Fold Change Control 18.2 24.03 26.73 2.7 0.65 1 18.3 22.98 26.52 3.53 0.85 18.4 21.66 26.56 4.89 1.19 18.5 21.23 26.14 4.9 1.19 18.9 21.63 26.16 4.52 1.09 11.7 21.72 26.04 4.32 1.05 11.6 22.95 26.22 3.26 0.79 11.5 21.11 26.12 5 1.21 5.8 21.98 25.86 3.88 0.94 Ethanol 27.6 22.42 26.11 3.68 -0.43 1.34 3.27 27.7 23.43 26.42 2.99 2.99 0.12 27.8 27.92 27.18 -0.74 -4.85 29.02 0.41 (without outlier) 12.1 23.95 26.76 2.8 -0.04 1.03 12.2 22.24 26.44 4.19 4.19 0.05 12.3 23.01 26.35 3.34 3.34 0.09 12.4 26.83 27.03 0.2 0.2 0.86 12.5 22.25 26.22 3.97 3.97 0.06 12.6 22.31 26.43 4.11 4.11 0.05 B 12.7 22.15 26.08 3.93 3.93 0.06

Table 3: CT Values for Beta-actin and GAPDH of embryonic brain samples age E10.5. (A) CT Values for Beta- actin and Wnt7a in embryonic brain age E10.5. (B) CT Values for GAPDH and Wnt7a in embryonic brain age E10.5. Here, W (Wnt7a) CT values were subtracted from B (Beta-actin) CT values or G (GAPDH) CT values to find individual expression fold change (2-ΔΔCT), providing an average expression fold change between control (red) and ethanol-treated (blue) samples.

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