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Honors College Theses

2017

The Influence of utismA Linked 3B (Top3B) on Neural Development in Zebrafish

Sydney Doolittle Georgia Southern University

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Recommended Citation Doolittle, Sydney, "The Influence of utismA Linked Gene Topoisomerase 3B (Top3B) on Neural Development in Zebrafish" (2017). Honors College Theses. 563. https://digitalcommons.georgiasouthern.edu/honors-theses/563

This thesis (open access) is brought to you for free and open access by Digital Commons@Georgia Southern. It has been accepted for inclusion in Honors College Theses by an authorized administrator of Digital Commons@Georgia Southern. For more information, please contact [email protected]. The Influence of Autism Linked Gene Topoisomerase 3β (Top3β) on Neural Development in Zebrafish

An Honors Thesis Submitted in partial fulfillment of the requirements of the Honors in the Department of Biology

By: Sydney Doolittle

Under the mentorship of Vinoth Sittaramane

Abstract

Autism Spectrum Disorder is a class of developmental disabilities characterized by a spectrum of social, communication, and behavioral impairments in affected individuals. Studies have shown these defects stem from abnormal brain development during critical periods during early development. The underlying genetic cause of these impairments is not well understood but is believed to be a combination of a complex pairing of genetic and environmental factors. One of the genetic factors that has been recognized to influence the phenotypic symptoms of ASD is the enzyme topoisomerase 3β (top3β.) are responsible for the prevention of supercoiling during DNA replication. Top3β is responsible for breaking single strands to be exchanged by adjacent and involved in DNA and RNA catalytic activities. Due to their unique RNA catalytic capabilities, further research into this specific topoisomerase has been conducted and found to promote a mediator complex (TDRD3) that has been previously associated with mental disorders. These hindered neural circuits could potentially contribute to the cognitive deficits in Autism. Our current research aims to better understand the specific roles of top3β in early neurological development through an in vivo study using zebrafish embryos as a vertebrate model system. To do this we created a top3β deficiency in zebrafish embryos and conducted a behavioral analysis comparing total average duration of movement, velocity, total distance travelled, and integrated visualization. Analysis of behavioral revealed a decrease in velocity and total distance travelled, as well as, a unique, repetitive spinning pattern expressed by only the top3β deficient embryos. Antibody staining of these embryos revealed defects in spinal motor nerves in the trunk with excessive branching patterns and misconnection of synapses to their target muscles. Analyzing the top3β deficient zebrafish model would help us better understand this spectrum disorder and its potential sources, as well as, potential drug screening target to discover therapies and treatments for Autism Spectrum Disorders.

Thesis Mentor: ______Dr. Vinoth Sittaramane Honors Program: ______Dr. Steven Engel April 2017 Department of Biology University Honors Program Georgia Southern University Table of Contents

Acknowledgments…………………………………………………………………4

1. Introduction……………………………………………………………....………..5

1.1. Brain Development in Autism……………………………………..6

1.2. Spinal Motor Neurons……………………………………………..8

1.3.Other Links to ASD………………………………………………..9

1.4. Hypothesis and Predictions ………………………………………12

2. Materials and Methods…………………………………………………………..12

2.1. Zebrafish……………………………………………..……………12

2.2. Zebrafish Husbandry…………………………………...………….13

2.3. Morpholinos……………………………………………...………..13

2.4. Live Tracking……………………………………………………...13

2.5.Analysis of Live Tracking………………………………………….14

2.6. Antibody Staining………………………………………………....15

2.7. Mounting…………………………………………………………..15

2.8. Imaging………………………………………………………….... 15

3. Results and Discussion……………………………………………………………16

3.1. Decreased Distance Travelled…………………………………….16

3.2. Decreased Velocity………………………………………………. 16

3.3. Limited Exploration of Arena…………………………………….17

3.4. Spinning Behaviors………………………………………………. 18

3.5. Successful Staining of Neural Circuitry…………………………. 19

3.6.Axonal Deficiency and Decreased Trunk Neurons………………. 20

2

3.7. Cranial Motor Neuron Deficiency………………………………..21

4. Conclusion………………………………………………………………………..21

4.1. Future Studies…………………………………………………….22

5. References………………………………………………………………………...23

3

Acknowledgments

The following project and results were funded by the Sittaramane Lab, Georgia

Southern Honors Program, Biology Department and Chandler Scholarship. The original

idea for the project stemmed from Kori Williams and Danielle Lott who were both

influential lab members that mentored me in continuation of these experiments. Dr.

Vinoth Sittaramane was a dedicated mentor who inspired and critiqued my research techniques and encouraged me through my undergraduate experience. I thank each one of them for their guidance through my undergraduate research career and encouraging me to

pursue my future career goal to attend medical school.

4

1. Introduction

The nervous system is a complex web of neurons and neural circuits that are intertwined and connected during early development to coordinate movement and initiate sensation of the outside world to the body. Early development of this system is a critical time frame where formation of new neurons and extension of axons is hypercritical to normal neural circuit formation and cognitive functioning. Disruptions of this process could lead to various neurological disorders that could result in mental retardation, loss of motor control and coordination, and cognitive learning disabilities such as autism spectrum disorders (Rice and Barone 2000.) Autism Spectrum Disorders (ASD) are a class of neurodevelopmental disorders characterized by reduced social reciprocity and communication skills as well as unusual, repetitive behaviors and includes autistic disorder, Asperger disorder, and pervasive developmental disorder (Autism Spectrum

Disorder Symptoms 2014).

The prevalence of these disorders has been steadily increasing over the last 20 years due to better diagnostic tests and guidelines aimed at identifying undiagnosed school-aged children who fell through the cracks. Certain developmental milestones are key indicators of developmental delays and can be analyzed through a series of tests that look at relatability, sensitivity, specificity, and predictive power of children at 18 to 24 months (Kleinman et al. 2001). As of 2012, the CDC released that 1 in 68 children are affected by autism spectrum disorders, indicating that prevalence has doubled over the last ten years. This drastic increased in prevalence makes research of this disease more important than ever (Christensen et al. 2016). The biological mechanisms responsible for this class of disorders is widely unknown. Many genetic variances have been identified in

5 association with the disorder and primarily affect the synaptic connections during early development; however, the rapid increase in prevalence is indicative of environmental factors also playing a role (Won et al. 2013).

1.1 Brain Development in Autism

The developmental delays characterized by ASD include reduced social, emotional, and communicative skills that have traditionally all been attributed to developmental disruptions in the brain. These communicative delays have been observed across the board from child to adulthood with these disorders. Children with autism have deficits in the perception of eye gaze, poor eye contact during conversation, and difficulties assessing the social cues associated with mental states of others (Zilbovicius et al. 2006). Verbal and social behaviors are some of the most difficult tasks to learn

when synaptic connections are

disrupted. Individuals with autism

have scattered abnormalities in

brain development and dysfunction

of neuronal pathways in the frontal

lobe and the amygdala (Critchley

2000).

Early neural development Figure 1. Neurotransmission from one nerve cell to begins around three weeks after another through synaptic cleft. Modified from Synaptic Cleft: Definition & Function fertilization and begins with gastrulation, the forming of the three germ layers. The notochord then develops to provide the primitive axis for the embryo and future site of the vertebral column. During

6 neurulation, the original ectoderm layer divides into the neural tube, epidermis of the skin, and neural crest cells. These cells migrate and differentiate into a multitude of different cell types and interact with extra cellular molecules, such as fibronectin, that guide these cells on their respective migratory paths Figure 2. Synaptic cleft releasing neurotransmitters through the presynaptic membrane to neighboring (Gilbert 2000). such as the Pax3, dendrites. Modified from John Wiley & Sons, Inc. openbrain, and sonic hedgehog are essential for proper neural tube development which will further divide the tube into the forebrain, midbrain, and hindbrain aided by cell adhesion molecules to properly position cells (Alberts 2008).

Synapse assembly begins when axons approach their targets and establish contact with dendritic anchors or soma of their target neurons and create a synaptic cleft ready for signals and message to pass through (Cohen-Cory 2002) (Figure 1.). Proper synaptic assembly is critical at this stage for proper signaling through action potentials down axons to neighboring neurons. Neurotransmitters are passed from one nerve to the next through the synaptic cleft and are taken in by receptors on neighboring dendrites which pick up in impulses and send the messages through the axon to the next neuron (Figure

2.). Between the time of birth and age two or three, the brain creates twice as many synapses as it will keep in adulthood that are gradually eliminated through a process called pruning that occurs during childhood and adolescence. In this process the brain fine-tunes itself according to the environmental input it receives with synapses

7 strengthened with repeated use and weakened when rarely used. The strength of these synapses contributes to the connectivity and efficiency of the neural networks that support learning, memory, and other cognitive abilities (Li 2003). Excessive synapses have been observed in ASD brains due to a slowdown of this pruning process. Figure 3. Confocal image of all three primary spinal motor neurons (left) with tracing of each A closer look at the brain cells showed neuron using Neurolucida. Arrows indicate axons and arrowheads indicating neural cell bodies. damaged parts as well as disturbances in the Image modified from Issa et al. 2012. autophagy of unneeded synapses (Tang 2014).

1.2 Spinal Motor Neurons

During vertebrate embryogenesis, the critical step of the formation of motor circuits begins by the migration of motor axons from the spinal cord along predetermined paths into the periphery somites (Zeller et al. 2002). Primary motor axons are larger and exit the spinal cord during the first day of development and guide secondary motor neurons down the correct points throughout the rest of axogenesis. The three main primary motor neurons can be easily identified and serve a specific function for movement. The caudal primary motor neurons (CaP MNs), the blue arrow/ tracing in figure 3, are responsible for the innervation of the ventral trunk. The middle primary motor neurons (MiP MNs), identified by the green arrow/tracing in figure 3, are responsible for innervation of the dorsal trunk. And finally, the rostral primary motor neurons (RoP MNs), identified by the red arrow/tracing in figure 3, are responsible for

8 innervation of the muscle fibers in between the caudal and middle motor neurons (Babin et al. 2014). Together these primary motor neurons are responsible for movement in all directions and guiding secondary motor neurons to their predetermined destinations accurately.

1.2 Other Links to ASD

Autism spectrum disorders are etiologically heterogeneous in nature with numerous genes contributing to the wide spectrum of symptoms. Differential development between individuals with ASD due to the genetic diversity is one of the most difficult barriers for research of these disorders. One of the aims for research is to identify “core” mechanisms that, when combined, contribute to a substantial number of

ASD symptoms. One such mechanism, excitatory synaptic transmission, involves development of these excitatory synapses is regulated by synaptic adhesion molecules, synaptic scaffolding , and actin-regulatory proteins and can be regulated by synaptic trafficking and stabilization of AMPA receptors (Won 2013). Identification of adhesion molecules involved in synaptic outgrowth on Schwann cells may point research in the direction for developing pharmaceuticals for regeneration of underdeveloped synapses (Seilheimer 1988).

Variations in protein concentrations have been studied to establish their correlation to ASD symptoms and have been found to contribute greatly to early neural development. Two of the major observations in ASD children is that the amygdala and frontal lobes are larger than expected at their age, but that this overgrowth stops earlier during childhood. The frontal lobe and amygdala are crucial for proper higher order social and motor skills, so this arrest in growth could point to some of the psychological

9 symptoms seen in patients with ASD. Structural organization of dendric spines during development is regulated by SHANK3 gene, located on 22, that has been found to be mutated in those diagnosed with autism and Asperger’s syndrome (Durand

2007). A closer look at reveals production of a class of proteins essential for DNA replication, topoisomerases. Topoisomerases are enzymes involved in the prevention of supercoiling as helicase separates DNA during replication. Topoisomerase

I relaxes DNA by simultaneously cleaving and rejoining one strand of DNA to release the torsion introduced during replication (European Bioinformatics Institute Protein

Information Resource SIB Swiss Institute of Bioinformatics 2017). Topoisomerase II acts on the double-strand helix as a whole, breaking it to allow other portions of the duplex to move through and reseal the double helix (Lodish 1970). Topoisomerase III are involved in the breaking of single strands to be exchanged by adjacent chromosomes. This type of topoisomerase also plays a role in DNA and RNA catalytic activities.

The regulation of RNA catalytic activities is unique to this type of topoisomerase and has caused more research to be done on this type of topoisomerase. Top3α forms a complex with BLM and functions in the regulation of recombination in somatic cells

(Top3A 2017). Top3β RNA metabolism activities has been shown to be promoted by a mediator protein complex known as TDRD3 (Tudor domain-containing protein 3). This protein complex has been previously correlated to other mental disorders such as Fragile

X Syndrome, Schizophrenia, and Autism (Siaw et al. 2016). Top3β binds multiple mRNA’s encoded with genes involved in neuronal functions which suggests that Top3β may contribute to the pathogenesis of mental disorders. Expression of some of these genes has resulted in defective synapse formation in Top3β mutant flies indicating that it

10 acts as an RNA topoisomerase to promote expression of mRNAs crucial for neurodevelopment and mental health (Xu et al. 2013).

Association of neurodevelopmental defects have been observed through an isolated population in Northern Finland due to repeated bottlenecking of the Finish population leading to an extreme genetic drift (Peltonen et al. 1999). Due to the isolation of this population, they have been used to identify how different genetic variances affect phenotypic abnormalities. The interaction of Top3β and TDRD3 complex also combine with the Fragile X mental retardation protein (FMRP) to form a complete protein complex that, if disrupted or mutated, can cause downregulation and disruption of protein complex functions (Stoll et al. 2013). Further exploration of the neurological impacts of

Top3β abnormalities in zebrafish 24 to 48 hours post fertilization revealed disorganization of notochord cell integrity, morphological abnormalities of the trunk, and axonal abnormalities resulting in faulty synapse connections (Williams 2015).

Behavioral observations of embryos 1 to 2 days post fertilization revealed Top3β deficient embryos moved at a slower velocity, travelled shorter distances, and did not explore their arenas as much as the control embryos. Another unique behavioral abnormality found in Top3β deficient embryos is the pattern of movement that these morphants display. Top3B has previously been associated with the phenotypic behavioral effects associated with neurodevelopmental disorders (Stoll et al. 2013). Live tracking during alternating light stimulus showed that while the light stimulus was turned on, the control zebrafish traveled along the edge of the plate and explored their environment while the morphants swam in tight circles. These observations set the stage for further research in to the later effects of Top3β deficiency in zebrafish embryos which is the aim

11 of the present study. Behavioral observations of zebrafish five days post fertilization as well as antibody staining of the axonal development as well as glimpsing at possible rescue of defective synapse connections will be conducted during this study.

1.3 Hypothesis and Predictions

Based on the previous evidence, we are hypothesizing that the genetic defects of the topoisomerase 3β gene will result in altered behavioral observations and circuit defects. Our prediction is that if the genetic defects in the motor neurons of the embryo are present then the mutant fish will present with altered behaviors and staining of the neural circuitry will result in circuit defects in the head and trunk.

2. Materials and Methods

2.1 Zebrafish

Zebrafish, Danio rerio, are rapidly becoming popular model organisms in the field of neuroscience helping to gain insight on the development of the brain and nervous system. Larval and adult zebrafish have been used to increase understanding of brain function, dysfunction, and their genetic contributors (Kalueff 2014). Highly motile dendritic projections of developing neurons in zebrafish embryos resemble those of mammalian developing central neurons undergoing synaptogenesis both in culture and in vivo (Cohen-Cory 2002). Because of this, zebrafish offer an alternative modelling system for primary motor neuron diseases and are suitable for exploring the functional effects of human mutations. They are also becoming newly popular in biological genetics studies because they are naturally social animals that show preference for the presence of conspecifics (Norton 2010). Their ability to show social preferences makes them prime

12 models for the genetics of social behavior for diseases such as autism spectrum disorders.

Manipulation through the knockdown of genes and fluorescent dyes allows us to identify locations and functions of different proteins and their contribution to different diseases.

2.2 Zebrafish Husbandry

Danio rerio are raised in a freshwater filtration system from AQUAneering

Incorporated, in a controlled environment maintained at 28°C. Transgenic lines were utilized. Embryos were collected from a cross-tank and placed in an incubator at 28.5°C.

Embryos were observed daily for development and fixed at appropriate stages.

2.3 Morpholinos

Heterozygous top3β gene knockdown embryos were created using CRISPR editing for observing behavioral and morphological neural development.

2.4 Live Tracking

Integrated visualization of zebrafish motor and behavioral response to the lack of top3β protein were

performed using DanioVision and the corresponding analysis Figure 4. Image of Daniovision Observation Chamber software, Ethovision 11.5 (Figure 4). A 6-well plate protocol was performed, either a control or morphant embryo were

transferred into each well, and the plate was placed inside A the instrument (Figure 5). Behavior recording occurred using a White Light Routine that included a 30-minute B acclimatization period followed by four alternating light Figure 5. 6-well plate used for live and dark periods lasting an hour each (Figure 6). tracking of embryos for control (row A) and treated (row B).

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Ethovision 11.5 analysis software was used to measure locomotion, total distance travelled, mean velocity, overall activity, and total time spent moving. After the routine was completed, the plate was removed and embryos were fixed for antibody staining.

Trial and group statistics and charts were produced from Ethovision for each trial to compare control and morphant and complete further analysis.

2.5 Analysis of Live Tracking

ffsd

Figure 6. Example of White Light Routine Setup for Daniovision live tracking experiment Once live tracking movement data had been acquired, the Ethovision 11.5 software allowed us to “nest” the acquired tracks to select the specific time frame desired.

For our purposes, we selected the entire three-hour duration of the experiment and a ten- minute interval for selective visualization of specific swimming patterns. The six acquired tracks were then split into three control and three treated groups according to their position in the 6 well plates previously recorded at the beginning of the experiment.

The software then evaluates each group individually determining the average duration of movement, velocity, total distance travelled, and integrated visualization for the control and treated groups.

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2.6 Antibody staining

Zebrafish embryos 5dpf were fixed in a 4% paraformaldehyde solution for

Immunostaining. Axons were labelled using a tubulin antibody to locate disturbances in neural development. A four-day staining protocol was applied. Day one, the 4% pfa solution was removed and embryos were washed with Incubation Buffer (IB) solution containing TritionX100 to prepare the membrane for the primary antibody [77451

SIGMA Monoclonal Anti-Acetylated Tubulin antibody produced in mouse; 1:500]. Day two, IB washes were repeated to eliminate unbound primary antibody and secondary fluorescent antibody appropriate for conjugation was added [A31621 Alexa Fluor 555 goat anti-mouse IgG; 1:500]. Day three, the embryos were washed with a PBS buffer solution and fixed in 4% pfa to set the staining. Day four, 4% pfa was removed and embryos were mounted in 70% glycerol until needed for imaging.

2.7 Mounting

All remaining yolk was removed from the 5 dpf embryos were mounted dorsally and laterally to visualize all neural circuits in the head and trunk. The stained embryos in

70% glycerol were mounted using Zeiss AX10 light microscope and supplies from Globe

Scientific Incorporated (tweezers, pins, slides, coverslips, glass pipettes, and bulbs).

2.8 Imaging

Mounted embryos were imaged using Zeiss LSM710 confocal microscope with the compatible Zen Black 2011 software.

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3. Results and Discussion

3.1 Total Distance Travelled

Throughout the duration of the White Light Routine, center point detection of morphant embryos expressed a decrease in total distance travelled by a mean of 1.7mm as compared to control fish which had a mean of 2.7mm around the arena Figure 7. Cumulative time (seconds) spent moving around the (Figure 8). Although there was an arena for the control and Morphant (treated) group. Both groups engaged in the same amount of total cumulative overall decrease in distance travelled movement during the duration of the experiment. in the arena, the total cumulative movement for the entire duration of the experiment was the same for both the control and treated groups moving around 17000 seconds throughout the 18000 second total duration (Figure 7).

3.2 Decreased Velocity

During light on portion of White Light Routine, morphant embryos expressed a decrease in overall velocity when swimming around in the arena. When compared to the total time spent exploring the arena, both the control and the morphant groups were active in the arena for the same amount of time during the trials; however, the velocity and distance moved was significantly decreased for the morphant group which travelled at a mean of 50 mm/s compared to the control which travelled 80 mm/s (Figure 8). This decrease in overall velocity mimics the delayed behaviors expressed in Autism Spectrum

Disorders and is indicative of top3β contributing to synaptic development and

16

transmission during early

development. These results also

correlate with previous studies of

behavior in top3β deficient

embryos. Behavioral similarities

in this study between top3β

deficient embryos and

with ASD support the hypothesis

that top3β contributes to synaptic

development in early

neurological development and

contribute to the phenotypic Figure 8. Mean velocity and total distance moved defects in people with ASD. for both the control and morphant (treated) groups.

3.3 Limited Exploration of Arena

The total distance A B travelled was lower for the treated embryos than the control (Figure 8) and this can be explained through the mapped live tracking in

Figure 9 which tracked Figure 9. Mapped live tracking of entire duration of White Light Routine for control (A) and morphant (B) embryos. movements for the entire

17 duration of the experiment. The morphant embryos stayed mainly in one quadrant of the arena and stayed along the wall in that quadrant for the duration of the experiment. The control, however, explored the entire arena and tested the parameters of the arena all the way around. This lack of exploration suggests that top3β deficient embryos are less communicative and willing to explore and socialize which coincides with the behaviors expressed in humans with ASD that commonly express lack of ability to express themselves to others and have impaired social interactions.

3.4 Spinning Behavior

Live tracking analysis of 5 dpf embryos revealed a unique swimming pattern exhibited by the morphant embryos. When the White

Light Routine switched the light on, the control group A B explored the arena swimming all the way to the edges and circling around to test the boundaries of their surrounding area. The Figure 10. Graph comparison of Clockwise Rotation frequency morphants, however, exhibited for the entire duration of the experiment (top). Mapped live tracking of control (A) and morphant (B) embryos during 10 tight circling behaviors in minutes of light on stage of White Light Routine (bottom). Line tracking illustrates unique spinning pattern from treated group limited areas of the arena with compared to control.

18 an increased clockwise rotation frequency as compared to the control (Figure 10). These patterns were expressed numerous times throughout the behavior trials. This behavior mimics one of the major characteristics of Autism Spectrum Disorders, repetitive behaviors. These repetitive routines and behaviors allow people affected with ASD to reduce uncertainty and increase predictability of their environment. Spinning is a common presentation of these behaviors, so having the zebrafish exhibit similar behaviors presents a strong correlation of Top3β absence to ASD symptoms. Although the morphant embryos remained localized to small regions of the arena, the total amount of locomotion throughout the duration of the experiment was the same for both the control and the morphant groups (Figure 7). This shows that the activity level of both groups remained unchanged by the deletion of the top3β gene which is indicative that top3β does not have metabolic anabolism functions.

3.5 Successful Staining of Neural Circuitry

Antibody staining of axonal circuitry was successfully achieved in both head a trunk of the embryos. Figure 11 illustrates the staining of the neural circuitry and shows

A1 B1 C1

A2 B2 C2

Figure 11. Bright light tissue imaging of head and trunk (A1 and A2). Overlay of neural circuitry staining on bright light tissue image of head and trunk (B1 and B2). Neural circuitry staining of head and trunk (C1 and C2). Comparison of A and C images indicate successful staining of axons in both head and trunk.

19 complete staining of neural axons without staining of the exterior tissues shown in the bright light imaging. This provides sufficient evidence that further staining of mutant and control fish will accurately depict neural circuitry defects in the embryos.

3.6 Axonal deficiency and Decreased Trunk Neurons

A B C

Figure 12. Antibody staining of axons in trunk of control (A) and morphant (B and C) embryos 5 dpf. Staining reveals a decrease in overall neurons, decreased integrity of neurons, abnormal branching patterns of caudal primary spinal motor neurons, and misconnections of axons in morphant embryos in the middle and rostral motor neurons.

Antibody staining of axons revealed loss of integrity within the trunk of embryos

5dpf in the absence of top3β (Figure 12). These axons are important in movement and require extensive neural circuitry. During early development, proper connections and extension of axons is vital for proper signal transmission during action potential. The decrease in integrity indicates that, in absence of top3β, the neural circuitry developments incorrectly in early development of the embryos resulting in further dissociation as the fish age. These misconnections indicate that the action potentials through these axons may be dulled or even directed to wrong targets through false synaptic formations. As seen in Figure 12, the large caudal primary neurons seen in the control embryo have one main branch that extends down the trunk that is repeated down the entire trunk. The top3β deficient embryos, however, have multiple, abnormal branching patterns that

20 extend in all directions. These misaligned caudal primary motor neurons could potentially be influencing the targeting of the middle and rostral motor neurons affecting a multitude of other synaptic connections. The primary motor neuron defects could be responsible for the behavioral patterns seen in the live tracking with the top3β deficient embryos expressing repetitive, circling movements. This loss of axonal integrity and resulting behavioral defects are indicative that top3β is a key component in early axonal development.

3.7 Cranial Motor Neuron Deficiency

Upon further investigation of neuronal circuitry in top3β deficient embryos, cranial motor neurons were found in fewer frequency than those of control embryos. This is indicative of failed axonal and synaptic connections during the specialization of cranial nerves in the forebrain, midbrain, and hindbrain of the embryo. These are congruent to previous studies indicating loss of axonal strength at 48hpf causing delayed development that continues throughout maturity.

4. Conclusion

Overall, top3β plays an important role in early neurological development. It is a key component in the formation and strengthening of axons critical for behaviors such as head and neck movements, speech, and facial expressions. Manifestation of ASD symptoms typically present themselves by the age of three and new diagnostic procedures are allowing diagnosis to be made earlier by comparing key developmental milestones.

This is indicative of alteration of early neurological circuitry of which top3β is a key component. The abnormal development of primary motor axons as well as the behavioral

21 patterns both provide evidence that top3β plays a vital role in early neurological development. Lack of axonal integrity, decreased speed, and repetitive behaviors each support the vital role that top3β plays in early synaptic formation and connection and the connection it has to the symptoms presented in Autism Spectrum Disorders.

4.1 Future Studies

Further studies should focus on rescue of these defective neural axons in zebrafish

5dpf and compare behavioral studies between top3β deficient and injected human mRNA of top3β to previously knocked down individuals. If axonal connections improve and behaviors normalize with rescue, then the similarities between human and zebrafish top3β function could result in approval for research into treatment for ASD through zebrafish embryo studies. Quantification of total neuron reduction in the trunk could also be done to further clarify the influence top3β has on early neurological development.

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