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Spring 4-27-2017 NEURAL EXPRESSION AND FUNCTION OF AUTISM CANDIDATE IN ZEBRAFISH Elyse P. Kite University of Texas at Tyler

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Recommended Citation Kite, Elyse P., "NEURAL EXPRESSION AND FUNCTION OF AUTISM CANDIDATE GENES IN ZEBRAFISH" (2017). Biology Theses. Paper 41. http://hdl.handle.net/10950/560

This Thesis is brought to you for free and open access by the Biology at Scholar Works at UT Tyler. It has been accepted for inclusion in Biology Theses by an authorized administrator of Scholar Works at UT Tyler. For more information, please contact [email protected]. NEURAL EXPRESSION AND FUNCTION OF AUTISM CANDIDATE

GENES IN ZEBRAFISH

by

Elyse Ping Kite

A thesis submitted in partial fulfillment of the required for the degree of Master of Science Department of Biology

Brent Bill, Ph.D., Committee Chair

College of Arts and Sciences

University of Texas at Tyler May 2017

© Copyright 2017 by Elyse Ping Kite All rights reserved.

Acknowledgements

I would like to thank all my family and friends back home for all their love and support. I would also like to thank Brad Low and Alan Lizarraga for their help with ordering all the materials needed for my project. Thank you to the Essner Laboratory for giving me the pkT3TS-goldyTALEN vector. I would like to thank all the esteemed members of my committee Dr. Blake Bextine, Dr. Kate Hertweck, and Dr. Dustin Patterson for their guidance and assistance throughout my time at UT Tyler. I would also truly like to thank all the wonderful, supportive loving friends I have met in Tyler, Texas. My time at UT

Tyler would not have been as memorable and rewarding without them. Lastly, and most importantly, I would like to thank the Dr. and Mrs. Bill. Without Ashely and Brent none of my success would have been possible.

Table of Contents List of Figures ...... ii Abstract ...... iii Chapter 1: Introduction ...... 1 Autism ...... 1 Zebrafish: A model organism for developmental disorders ...... 2 Dipeptidyl-Peptidase 6 ...... 5 26S Subunit, Non-ATPase 12 ...... 9 Structure of 26S proteasome ...... 9 The Proteasome System (UPS) ...... 11 Functions of Ubiquitin modification ...... 11 Neurons and the UPS ...... 12 Chapter 2: Material and Methods ...... 15 DPP6 Results ...... 22 PSMD12 Results ...... 26 Chapter 4 Discussion ...... 39 DPP6 Discussion ...... 39 Human DPP6 and Zebrafish orthologs compared ...... 39 DPP6 expression in zebrafish ...... 40 Future Studies for DPP6 ...... 42 PSMD12 Discussion ...... 42 Zebrafish and Human PSMD12 comparison ...... 42 Specific PSMD12 Antibody for zebrafish ...... 42 Chromosomal editing of zebrafish PSMD12 ...... 43 Future studies for PSMD12 ...... 46 Appendix A: Antibodies ...... 56 Appendix B: Primers ...... 57 Appendix C: Solutions ...... 58

i List of Figures

Figure 1. Schematic representation of a TALEN pair binding to its target sequence and cleaving double stranded DNA...... 4

Figure 2. in situ hybridization dpp6b RNA expression demonstrates widespread neural expression and IHC with DPP6 antibody recognizes similar regions...... 8

Figure 3. Scaled synteny mapping of human DPP6 and D. rerio dpp6a, dpp6b...... 23

Figure 4. DPP6 antibodies epitope alignment of human and zebrafish orthologs using Clustal Omega...... 24

Figure 5. DPP6 (ab41811) shows non-specific binding in adult WT zebrafish neuronal tissue...... 25

Figure 6. A 50 -sliding window of human PSMD12 and zebrafish psmd12 show seven syntenic genes shared...... 27

Figure 7. PSMD12 antibody epitope alignments against human and zebrafish using Clustal Omega...... 28

Figure 8. PSMD12 ab156604 shows more specificity when proteins are extracted with RIPA buffer...... 30

Figure 9. Highly conserved regions targeted in zebrafish Psmd12...... 32

Figure 10. TALEN genotyping assay...... 34

Figure 11. Colony PCR of TALEN pFUS_A and pFUS_B constructs...... 35

Figure 12. Colony PCR of TALEN products successfully inserted into pKT3TS- goldyTALEN...... 36

Figure 13. Western blots of PSMD12 ATG MO injected embryos...... 37

Figure 14. Average Psmd12 level and viability of MO injected zebrafish embryos 3dpf...... 38

Figure 15. Golden Gate reactions. Using type IIS restriction endonucleases (BsaI and ESp3I) plasmids are digested and ligated together in a specific sequential manner...... 44

ii Abstract

NEURAL EXPRESSION AND FUNCTION OF AUTISM CANDIDATE GENES IN ZEBRAFISH

Elyse Ping Kite

Thesis Chair: Brent Bill, Ph.D.

The University of Texas at Tyler April 2017

The Autism Spectrum Disorders (ASD) refer to a large heterogeneous set of developmental disorders defined by two behavioral domains: 1) Impairment of social comprehension and abnormal development of social communication, and 2) the presence of stereotypical and repetitive behaviors. The etiology of ASD is complex, with both genetic and environmental components. De novo and inherited mutations within DPP6 and PSMD12 have been identified within individuals diagnosed with ASD suggesting an etiologic role. Understanding the functionality of these genes could lead novel treatments for ASD. Zebrafish were utilized as a model system for the assessment of functionality of the orthologous genes dpp6a, dpp6b, and psmd12. Zebrafish are an ideal model system for neurological disorders because they develop quickly, share many fundamental brain structures and capacities with humans, and are genetically malleable. This work demonstrates commercially available antibodies designed to DPP6 bind promiscuously; therefore, necessitating a continued search for specific antibodies. In addition, three TALE Nucleases (TALENs) were designed to specifically target the psmd12 gene. In order to elucidate psmd12 mutants, genotyping assays were designed and a specific antibody was ascertained. These tools will be critical in the development of an allelic series and functional assessment of psmd12.

iii

Chapter 1: Introduction

Autism ! Leo Kanner originally described autism in 19431; however, variability in presentation later led to the collation of several disorders such as Asperger’s Syndrome2, pervasive developmental disorder not otherwise specified3, Fragile X Syndrome4, and

Rett Syndrome (RTT)5 under a much broader definition of the Autism Spectrum

Disorders (ASD)6. The current diagnosis of ASD refers to a large heterogeneous set of developmental disorders defined by two behavioral domains: 1) Impairment of social comprehension and abnormal development of social communication, and 2) the presence of stereotypical and repetitive behaviors6.

The prevalence of ASD is about 1% in North America, Asia and Europe7; however, recent studies conducted in South Korea and the United States note prevalences of 2.6%8 and 1.5%, respectively, of children born9. Complicating prevalence are differences between male and female occurrence. ASD is on average three times more common in males than females10. In those individuals classified as high functioning, presence of speech and lack of co-occurring intellectual delay, the male to female ration may be as high as 8:1, while in those classified as low functioning individuals the male to female ratio is closer to 1:111. It is not currently known if this is due to neurologic or genetic protection afforded by sex, or if traditional diagnostic tools that were developed using male behaviors are under diagnosing females with ASD. Changing diagnostic

1 metrics have been previously implicated in increased prevalence of ASD within the population, and may continue to impact epidemiologic numbers for this population12.

The etiology of ASD is complex, with both genetic and environmental components. Twin studies demonstrated high concordance rates in monozygotic twins, with 50-90% trait heritability13-18, while it is only 0-30% heritable for dizygotic twins, comparable to the recurrence rate for non-twin siblings (3-26%)11,19. Recent studies have led to the definition of approximately 800 genetic loci linked to ASD based on the observation of recurrent chromosomal aberrations, common and rare copy number variants (CNVs), single nucleotide variations20-22, or differences in observed in the cortex of ASD post mortem brains23-29. While the identification of these genes has progressed rapidly, the characterization of their neurobiological function and role in ASD has lagged behind. This thesis will utilize the zebrafish (Danio rerio) to identify and accelerate the assignment of gene functionality of two new candidate genes,

DPP6 and PSMD12.

Zebrafish: A model organism for developmental disorders

To look at functionality of ASD genes, Danio rerio (D. rerio) were utilized. D. rerio is an ideal model system for neurological disorders as they are small, robust, develop quickly, and share many fundamental brain structures and capacities with humans30. They produce up to 700, externally fertilized, eggs that generate transparent embryos per mating, that are genetically malleable31. There exist several well-developed transgenic and targeted gene knockdown (KD) or knockout (KO) methods for D. rerio; therefore, multi or single-gain-or-loss-of-function mutations can be efficiently generated to define gene interaction networks and novel regulatory connections32.

2 Morpholino oligonucleotides (MO) are used for temporary KD of protein function. MO were first introduced in 1996 as a method to inhibit the translation of RNA transcripts in vivo33, as they interfere with post-transcriptional RNA processing. They are injected into the yolks of developing one- to two-cell stage embryos. MO bind to a complementary sequence of their target mRNA resulting in inhibition of translation initiation. MO can also be designed to inhibit pre-mRNA splicing by preventing spliceosome formation. MO offer a quick transient KD of a protein, which may allow researchers to ascertain a phenotype allowing researchers to determine if a permanent KO is worth creating. There are, however, some disadvantages to MO. MO only provide a temporary inhibition of protein synthesis. As the embryo grows and develops the concentration of the injected MO will decrease and no longer effectively block translation of the protein of interest33,34. Injection of the MO also causes a set of characterized non- specific effects due to the chemistry itself35. New genome engineering techniques, for example, Transcription Activator-Like Effector Nucleases (TALENs), have rapidly accelerated the process of site directed mutagenesis, reducing the initial barriers to permanent genome editing. Some researchers have proposed other techniques may replace MO entirely.

Transcription Activator-Like (TAL) effectors were initially found in plant pathogen Xanthomonas spp36,37. TALs are proteins secreted by Xanthomonas spp will bind to specific nucleotides. The first TAL effector gene was discovered in 1989 during the cloning of a virulence protein Bs3 (avrBs3) by Bonas and colleagues38. TALENs are the fusion of a TAL effector DNA-binding domain and a FokI nuclease catalytic domain.

The fusion of TAL and FokI allows for targeted DNA double-strand breaks in vivo. TAL

3 effectors can enter the nucleus, and bind to extremely specific sequences of DNA39

(Figure 1). TALEN genome editing has been used successfully in zebrafish39 and livestock40.

Figure 1. Schematic representation of a TALEN pair binding to its target sequence and cleaving double stranded DNA. Each TALEN is made of multiple TALEs at the amino acid terminus and a FokI nuclease at the carboxyl terminus. Each TALE repeat is comprised of 33-35 amino acids and recognizes a single , called the repeat variable diresidue (RVD).

4 Just like with MO, TALENs are injected into one- to two-cell stage embryos; however, TALENs are targeted toward the nucleus of the cell instead of the middle of the yolk. Capped RNA encoding for the two TALENs is titrated and co-injected to optimize mutagenicity. Each TALEN complementarily binds its target sequence allowing for FokI nucleases to be brought within close enough proximity for cleavage. Subsequently, the nuclease causes a double-strand break at the target site. Imperfect repair by the error- prone non-homologous end-joining (NHEJ) mechanism results in small insertions or deletions called indels. Indels that result in mutagenic frameshift changes or the introduction of an early stop codon are then selected to establish KO alleles.

Dipeptidyl-Peptidase 6 ! Dipeptidyl-Peptidase 6 (DPP6), also known as DPPX, was highlighted as an autism candidate gene after, de novo41 and inherited CNVs were found within the DPP6 gene in individuals with ASD42-46. Mutations in DPP6 were observed in 5/2,588 autism cases and 1/2,690 neuro-typical controls. Four of the five cases were duplications in

DPP6, while there was one case of DPP6 deletion47. In addition, Bock and colleagues identified a missense mutation in DPP6 that segregated with individuals with autistic phenotype in a multiplex family48. DPP6 is co-expressed with other autism candidate genes such as Astn2 (Astrotactin 2), Ptchd1 (Patched Domain Containing 1) and Galnt13

(polypeptide N-acetylgalactosaminyltranserase 13)49.

In humans, DPP6 is expressed in muscle, heart, and neuronal tissue50,51. DPP6 is also expressed in mouse neuronal tissue, mouse facial tissue and monkey kidney (COS7 cells)52-54. Dpp6 expression has been investigated extensively in mice. Dpp6 mRNA is

5 expressed in several peripheral tissues in addition to the brain, such as ovary, prostate, testis and kidney. In the brain, DPP6 is expressed in the granule cell layers of the cerebellar cortex and in pyramidal neurons of the hippocampus55. Specific isoforms of

DPP6 display unique patterns of expression. DPP6-L is exclusively expressed in the brain. While DPP6-S is expressed in olfactory regions and in high levels in hippocampal complex, DPP6-L has low expression in these regions56. DPP6-E is expressed in hippocampal pyramidal cell, cerebellar granule neurons and in neurons in the super colliculus55. DPP10, another autism candidate gene, which is closely related to DPP6 is expressed in the brain, pancreas, adrenal glands, spinal cord, Purkinje cells and

GABAergic interneurons57,58.

DPP6 is an auxiliary subunit of the voltage gated (Kv)-mediated A-type K+ channels that blocks ion flow following activation of the channel52. DPP6 is a type II membrane glycoprotein that has a single membrane-spanning domain, a large extracellular C-terminal domain, and a short alternately spliced intracellular N-terminal59.

DPP6 plays an important role in Kv4.2 expression by promoting potassium channel trafficking to the dendrites50, and the loss of DPP6 can cause a decrease in the A-Type current, resulting in hyper-excitability of the dendritic tree by enhancing dendritic action potential back propagation, calcium electro-genesis and the induction of long-term synaptic potentiation60.

Mouse models have been informative for function of this potassium channel regulator; however, a zebrafish model could provide a vehicle for high-throughput pharmaceutical screens or gene-gene interaction studies32. Zebrafish underwent a partial

6 genome duplication 300 million years ago, which resulted in two DPP6 orthologs, dpp6a and dpp6b61.

qRT-PCR and in situ hybridization have shown dpp6b expression as early as 2 days post fertilization (dpf) and continuing through adult. In situ hybridization for dpp6b expression was observed within the zebrafish hypothalamus, optic tectum of the periventricular gray zone, thalamus, and torus longitudinalis. Immunohistochemistry

(IHC) analysis utilizing a rabbit anti-DPP6 (abcam: ab41811) antibody, showed expression in brain areas similar to that of the in situ hybridization data mentioned above; however, broader staining in the cerebellum, fasciculus retroflexus, and several sensory- associated neurons was also observed (unpublished data, Dr. Brent Bill) (Figure 2).

7

Figure 2. in situ hybridization dpp6b RNA expression demonstrates widespread neural expression and IHC with DPP6 antibody recognizes similar regions. (A) In, the torus longitudinalis, periventricular gray, and hypothalamus stains prominently, and confirms staining observed by IHC (B, C, and Figure E respectively). IHC was performed on 6- micron sections of adult brain. Staining was observed in the (D) fasciculus retroflexus, (E) hypothalamus, and (G) optic nerve.

8 Proteasome 26S Subunit, Non-ATPase 12

PSMD12 (Proteasome 26S Subunit, Non-ATPase 12), also known as Rpn5 or P55, is part of the non-ATPase subunits of the 26S proteasome. The proteasome was first discovered in 1977 by Etlinger and Goldberg62. The proteasome degrades misfolded, damaged, older or oxidized proteins63. It has multiple protease activities, and is capable of breaking down proteins into 7-8 amino acid long peptides. The targeting and degradation of proteins is characterized by two discrete steps: (1) conjugation of multiple ubiquitin moieties to a target protein in order to generate a polyubiquitin degradation signal, and (2) destruction of the targeted tagged protein by the 26S proteasome complex.

Structure of 26S proteasome ! The 26S proteasome is a 2,000 kDa protein complex64 composed of two sub- complexes, the 20S proteolytic core particle (20S-CP) and the 19S regulatory particle

(19S-RP)65. The barreled shaped 20S-CP consists of four stacks of seven membered rings; the subunits are highly homologous. The proteasome contains an interior chamber, and the inner surface of this chamber promotes unfolding of proteins66. The proteolytic active sites are inside the 20S-CP barrel and access to these sites is regulated by a gated axial channel67-69. The 19S-RP is located at the ends of the 20S-CP. The 19S-RP is made up of at 19 subunits: 6 AAA-ATPase subunits (Rpt1-6) and 13 non-ATPase subunits:

Rpn1-3, Rpn5-13, Rpn1570, and the DUB (deubiquitylating ) Rpn1171. It is the job of theses subunits to recognize, unfold, and translocate the target protein into the core of the proteasome72,73. The 19S-RP can be broken down further into two subcomplexes:

(1) the lid and (2) the base74,75. The base contains at least 10 subunits, six homologous

9 ATPases, two scaffold subunits Rpn1 and Rpn275 and two proteasomal Ub receptors

Rpn1075,76 and Rpn1377. Rpn10 and Rpn13 are located on the apical part of the 26S proteasome78, and they recognize poly-ubiquityled substrates77,79. The six distinct regulatory particles known as AAA-ATPases: Rpt1-6 form a hexameric ring and constitute the molecular motor of the proteasome. PSMD12 resides in the lid of the 19S-

RP. The asymmetric lid subcomplex contains 7 other non-ATPase subunits: Rpn3, Rpn6-

9, Rpn11 and Rpn1274. Rpn11, also called PSMD14, is a metalloprotease that deubiquinates substrates preceding their degradation. The activity of PSMD14 is very important for efficient protein degradation80,81. PSMD14 activity can be regulated by

PSMD1282.

In 1997, Saito and colleagues cloned the p55 subunit of the human 26S proteasome and found that the disruption of their counterpart gene in yeast resulted in lethality. Yen and colleagues concluded the function of Rpn5, the homolog of PSMD12, is a highly conserved by rescuing the phenotype of a yeast rpn5Δ using human RPN5.

Furthermore, they show Rpn5 potentially regulates assembly of the 26S proteasome in two different ways: 1) nuclear import and 2) assembly at the nuclear membrane83. In

Arabidopsis thaliana there are two forms of RPN5 (RPN5a and RPN5b)36, and they are essential for the proper gametogenesis, sporophyte development and complex assembly.

Homozygous rpn5a and rpn5b null mutants could not be generated. Single rpn5b mutants appeared wild-type, while single rpn5a mutants displayed many morphological defects.

PSMD12 has gained further interest as it has been identified as the causative gene found with in a multiplex (multiple children with ASD family)84,85. Recently Küry reported reductions in optic tectum in zebrafish CRISPR/Cas9 mosaics. This suggests it is a

10 potential ASD susceptibility gene, as are other previously identified components of the ubiquitin proteasome system.

The Ubiquitin Proteasome System (UPS)

Functions of Ubiquitin modification

Covalent modification of lysine residues with ubiquitin, ubiquitination, was discovered in the early 1980’s as a form post-translational modification84,86-89. It regulates many critical cellular functions including: modulation of the progression of the cell cycle90,91, DNA damage repair92, membrane protein maintenance93, apoptosis94,95, transcription regulation91, protein location96, protein function97, protein degradation (see below), and facilitation of protein-protein interactions98. The best studied is the process of polyubiquitination, which marks proteins for degradation.

In 2005, Avram Hershko, Aaron Ciechanover and Irwin A. Rose won the Nobel

Prize in Chemistry for their description of the UPS99. In eukaryotes, the UPS is the major process regulating intracellular protein degradation100. The role of is highly regulated in cells, it is crucial for cellular signaling, homeostasis and fate determination101-103. Proteins ligated with multiple are localized to the 26S proteasome for degradation.

Ubiquitin ‘labels’ target proteins in an energy-dependent, three-step pathway: (1) activation, (2) conjugation, and (3) ligation86. In the first step a high energy thioester linkage between Ub and E1 is formed when ubiquitin-activating enzyme (E1) binds to

ATP, and Ub is transferred to a cysteine residue104. In the second step, conjugation occurs with a ubiquitin-conjugated enzyme (E2) that catalyzes the transfer of the activated Ub

11 from E1 to a cysteine residue on E2 via a trans(thio)esterification105. The final step is ligation that is catalyzed via ubiquitin-protein ligase (E3). An isopeptide bond is formed between the C-terminal glycine of Ub and a lysine residue in the target protein106. Within the there are two E1 , at least 38 E2 enzymes, and thousands of

E3 enzymes predicted107. The E3 ligases confer specificity for the final ubiquitination of the target protein108. There are three classes of E3 ligases as characterized by a conserved structural domain and by the mechanism by which Ub is transferred from the E2 to the target protein109.

Neurons and the UPS

The plasticity of brain circuits plays a large role in our ability to memorize and learn. Pharmacological inhibition of protein synthesis has been shown to impair behavior and synaptic plasticity. In order for the nervous system to effectively carry out tasks and function plastically proteins must be transcribed, modified and degraded. Therefore, regulation of protein synthesis and degradation are crucial for normal neuronal function110.

The UPS has been shown to play a numerous roles in neuronal morphology and function111. Morphology is regulated by modifications of axonal growth and morphogenesis, dendritic outgrowth, and dendritic arborization. Neuronal function regulation is primarily mediated through regulation of neurotransmitter release through intracellular trafficking112-114, and protein homeostasis, availability and receptor function in synapses and mRNA translation110,115.

12 UPS enzymes have been observed in neurons, specifically near synapses and in the dendrites116-118, and associated or implicated in many diseases that involve the nervous system, such as: Alzheimer’s disease (AD), Parkinson’s disease (PD), , and Huntington’s disease (HD)119. For many of these disorders, the mechanism of disease appears to be the inability for the UPS to target misfolded proteins, thus forming aggresomes that are toxic to the neurons and lead to cell death120,121. Supporting this hypothesis, overexpression of ubiquitin is capable of reducing protein aggregates and extending the lifespan of HD transgenetic mice122.

Angelman syndrome (AS) is a neurodevelopmental disorder whose main features are microcephaly, intellectual disability, seizures and lack of speech123. RTT is an X- linked dominant disorder exclusive to females, which shows similar phenotypes as AS including microcephaly, cognitive impairment, seizures, and ataxia124. AS and RS

(syndromic members of the ASD spectrum), and several non-syndromic autism spectrum disorders have been associated with the UPS124-126. The strongest association has been with the gene, UBE3A (Ubiquitin Protein Ligase E3A) that encodes for the HECT domain E3 protein127. UBE3A is located at 15q11-13128; this region is flanked by three low copy repeats leading to common recombination of this loci, and association with three neurodevelopmental disorders: autism, AS, and Prader-Willi Syndrome (PWS). A duplication or triplication of the maternally inherited 15q11-13 is one of the most common cytogentic events associated with autism126,129. De novo deletions of maternal

15q11-q13 are responsible for about 70% of AS cases130,131; 5-10% of the cases are caused by mutations in the maternal 15q11-q13 region132; and 2-3% are caused by paternal uniparental disomy of 15133. Deletion of the paternal copy of the

13 15q11-q13 region leads to PWS129. Complete penetrance of autism is seen in individuals who have four copies of the gene and partial penetrance in those who have three copies129,134. In mice the loss of maternal UBE3A causes AS135,136 and the increase of

UBE3A is associated with autism traits137,138. Therefore, based on the converging lines of evidence, the UPS could be a critical pathway in the etiology of ASD.

14 Chapter 2: Material and Methods

Zebrafish maintenance

Farm raised wild-type (WT) zebrafish were maintained, and used for experiments in accordance with terms and conditions of the University of Texas at Tyler standards

(IACUC Protocol #112). The fish were kept on a 14-hour light/10-h dark cycle at 28°C in reverse osmosis (RO) purified water (pH 7.0) salted with Instant Ocean Sea Salt to a specific gravity of 1.004 (salinity 5.9). They were fed brine shrimp twice a day.

Extraction of Zebrafish Protein

Adult WT zebrafish or embryos were euthanized and tissue was harvested. The adult eyes and/or brains were stored on ice, and homogenized with 750ul of RIPA buffer

(ThermoFisher, Cat#89901) and protease inhibitors (ThermoFisher Scientific,

Cat#78441B). Lysates were then spun by centrifugation at 11,500 x g for 30 minutes at

4°C to collect S1 fractions. Total protein concentration was determined by comparing protein extracts to a bovine serum albumin standard curve using the BCA assay kit

(ThermoFisher Scientific, Cat#23225).

Western Blotting

Conventional Western blotting procedures were used to detect target proteins. Protein was loaded into pre-cast 7.5% SDS–PAGE gels (Biorad, Cat#456-1023). The gels were submerged in tris-glycine electrophoresis running buffer (Appendix C) and run for 60 minutes at 175V. The proteins were transferred to a PVDF (polyvinylidene difluoride)

15 membrane (Millpore, Cat#IPVH00010) using a liquid transfer 280mA, run for 2 hours.

Membranes were blocked in 5% milk block (Appendix C) for 2 hours or overnight.

Membranes were probed with antibodies (Appendix A) and incubated overnight, 4°C in

5% milk block. The membranes were washed three times in TBST, incubated with secondary antibodies (Appendix A) for 1 hour and washed three times in TBST

(Appendix C). To visualize membranes NBT (1%) and BCIP (1.5%) substrate in alkaline phosphate (AP) buffer (Appendix C) were used. Band intensities were quantified with

ImageJ software139.

Immunohistochemistry (IHC)

Embryos were collected at 2 and 3dpf, euthanized, and fixed with 4% paraformaldehyde

(PFA) (Sigma, Cat#30525-89-4) overnight at 4ºC. Embryos were permeabilized four times for 5 minutes in phosphate-buffered saline (PBS) with 0.1% Triton-X (PBST)

(Appendix C). The embryos were blocked in IHC blocking solution (Appendix C) for 2 hours at room temperature. Subsequently, the embryos were incubated in rabbit anti-

DPP6 (ab41811) (1:500) or rabbit anti-PSMD12 (ab156604) (1:500) overnight at 4°C, washed in PBST three times, and incubated with secondary antibody goat anti-rabbit IgG

Alexa Fluor® 546 (ThermoFisher, Cat#A-11010) at 4°C overnight. Embryos were mounted on slides and imaged with Carl Zeiss Axioskop 2 mot plus with a LSN 5 Pascal confocal attachment.

16 Bioinformatics

DPP6: Sequences for human Dpp6 (Gene ID: 1804), zebrafish Dpp6a (Gene ID:

00007104) and Dpp6b (Gene ID: 566832) were obtained from Genbank140, and aligned in

Clustal Omega141 using default parameters. Synteny was established using a previously validated synteny database142 maps constructed with the source genome set as D. rerio with Homo sapiens (H. sapiens) as the outgroup.

PSMD12: Protein sequences for Homo sapiens, Mus musculus, Gallus gallus, Xenopus laevis, Danio rerio, Drosophila melanogaster, and Saccharomyces. cerevisiae were identified through Unigene, and downloaded into the main CLC Workbench (https:// www.qiagenbioinformatics.com). Primers to verify expression and for genotyping were designed utilizing IDT Primerquest based on areas of high amino acid identity across all seven species examined.

Bacterial strains, media and growth conditions

All bacterial strains used were chemically competent. The strains used were: JM109

(Promega, Cat#L2005), DH5⍺™ (ThermoFisher, Cat#18265017) and One Shot®

Mach1™ T1 phage-resistant (ThermoFisher, Cat# C862003) cells were grown up following standard protocols. Liquid cultures were grown overnight in Luria broth (LB)

(BD, Cat#244620) with appropriate antibiotics at 37°C, 225 rpm. Agar plates were made with LB+Agar (Appendix C) and appropriate antibiosis, if necessary.

17 Transformation procedures

JM109 (Promega, Cat#L2005), DH5⍺™ (ThermoFisher, Cat#18265017) and One Shot®

Mach1™ (ThermoFisher, Cat# C862003) bacterial cells were transformed according to provided standard protocols.

Cloning of Psmd12

RNA was harvested from WT zebrafish using Zymogen Direct-zol™ RNA MiniPrep Kit

(Cat#R2050). RNA was used for first strand cDNA synthesis using both the oligod(T) and Random hexamer primers with the Promega GoScript™ Reverse Transcription

System (Cat#A5000). The cDNA was amplified using gene specific primers (Appendix

B) and Promega Go Taq PCR kit (Cat#M7805).

The full-length zebrafish psmd12 sequence was ligated into Promega pGEM®-T vector

(Cat#A362A) and transformed into JM109 (Promega, Cat#L2004) following the Promega pGEM® Vector Systems instructions manual (Cat#A1360). Bacteria were grown on

Luria-Bertani + ampicillin (100ug/ml) agar plates, colonies were selected, purified using

Qiagen MiniPrep Kit (Cat# 27106) and sequenced by Eurofins SimpleSeq

(http://www.eurofinsgenomics.com/).

18 Morpholino oligonucleotides (MO)

Morpholino Design

Three different 24 base pair (bp) MO were designed. Two 24 base pair (bp) sequences were identified at the 1st exon and one at the 3rd exon. The splice acceptor (PSMD12

E3SA) was created against exon 3 and the two translational blocker MO were designed against exon 1: start of translation (PSMD12 ATG), or 14 bp upstream of the translational start (PSMD12 5p). All MO designed did not show the ability to form hairpins, and were unique to psmd12 (next nearest blast hit was 15/15 bp match). They also had a GC of ~50% without four guanines in a row. A negative control MO

(PSMD12 ATG MS) was designed; it differs from the translational start morpholino

(PSMD12 ATG) by 5 bp. The MO constructs will sterically hinder the production of

PSMD12 RNA in zebrafish in order for us to observe phenotype changes in the nervous system (See Appendix B for MO primer table).

Designed MO:

1.! PSMD12 ATG: Translational blocker. Starts 14 bp in front of the PSMD12 start

codon and covers 10 bp in to the start codon The ATG and PSMD12 5p MO will

block the binding of the small ribosomal subunit.

2.! PSMD12 5p: Translational blocker, further upstream 5’UTR.

3.! PSMD12 ATG Mismatch (MS): Negative control that is identical to the PSMD12

ATG MO, except for a 5 bp mismatch difference. Therefore, it will not bind to the

mRNA.

4.! PSMD12 E3SA: Splicesome blocker. 100% complementary to 17 bp in PSMD12

exon 3. This MO blocks the splicesome from cleaving exon 3.

19 Morpholino Injection

Injections were done following the methods described in Bill and colleagues35. We injected with and without the tP53 MO to account for background phenotypes due to the upregulation of the pathway. Efficiency of injection was determined using a positive control chordin:fluorescein MO.

TALEN Creation

GoldyTALENs Design

TALENs were designed using Mojo Hand software143. Highly conserved areas of the genome were targeted using CLC Workbench and the specificity of primers confirmed using NCBI Primer-BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Three sites were targeted: PSMD12E3 site 1, PSMD12E3 site 127 and PSMD12E6 site 11. Two primers were designed for each site surrounding the targeted loci by approximately 1000 bp

(Figure 9 and 10). RFLP’s within these amplicons are the basis for genotyping assays.

The TALEN targeting sequences are shown below. Targeting sequences were validated by reverse transcriptase PCR of WT zebrafish RNA, and we demonstrated a lack of polymorphisms or sequencing errors for each site.

Specific TALEN RVDs and Restriction Enzymes:

PSMD12E3 site 1: Exon 3 TAL1: NG HD NN NN NI NG NI NG NN NN NG NN NG HD NG TAL2: NI NI HD NG NN NI NI HD NG NI NG NG NN HD HD NI HD Restriction enzyme: AlwI (www.neb.com/R0513)

20 PSMD12E3 site 127: Exon 3 TAL-1: HD NG NN NI NI HD NN NI NI NI NI HD NI NG HD NI TAL-2: NN HD NG NG HD NI NN HD NG NN NN HD NG NG HD NG NG Restriction enzyme: BbvI (www.neb.com/R0173)

PSMD12E6 site 11: Exon 6 TAL-1: NN NN NI NN NI NI NN NI NI NI NN NI NN NI NI NN NN TAL-2: NI HD NI HD NI NN HD HD NG HD NI NG HD NG NN HD Restriction enzyme: BspCNI (www.neb.com/R0624)

Synthesis of GoldyTALENs

DNA fragments encoding engineered TALE repeat arrays were constructed using the

Golden Gate TALEN and TAL Effector Kit 2.0 (Figure 15).

(http://www.addgene.org/taleffector/goldengatev2)144. The destination vector used was pkT3TS-goldyTALEN with LacZ145. The higher active RVD NN was used to target guanine instead of the less active NK146 and NH147 RVDs. In the first Golden Gate reaction, TALE repeats 1-10 were synthesized into pFUS_A or pFUS_B vectors. Colony

PCR was run, to verify proper insertion of RVDs, primers: pCR8_F1 and pCR8_R1

(Appendix B). Correct plasmids were transformed into competent bacterial cells.

In the second Golden Gate reaction, the correct TALE repeats were synthesized into a pFUS_B4 vector. The two TALE pFUS vectors and the last nucleotide in either pLR-NI,

-HD, -NG, or -NN were combined in the final Golden Gate reaction into the expression vector pkT3TS-goldyTALEN145 (Figure 15). The circular GoldyTALEN constructs were linearized and turned into RNA using mMESSAGE mMachine® T3 Kit (Life

Technology, Cat#AM1340).

21 Chapter 3: Results

DPP6 Results

Examination of Zebrafish DPP6 orthologous proteins

Comparison of zebrafish gene sequences indicate there was a whole genome duplication, hundreds of years ago61, leading to two zebrafish orthologs of DPP6, dpp6a

(chromosome 24, 63.40% identity) and dpp6b (chromosome 2, 59.02% identity).

Chromosomal gene arrangement is an indicator of evolutionary history; therefore, synteny was investigated for both paralogs using the zebrafish section of Synteny

Database142,148. The dpp6a region maintains higher synteny (5 gene loci, Figure 3a) to human DPP6 chromosomal region than the dpp6b chromosomal region (3 gene loci,

Figure 3). Expansion of the sliding gene window from 100 to 200 genes further demonstrates a closer relationship between human chromosome 7 and D. rerio chromosome 24 (data not shown).

22

Figure 3. Scaled synteny mapping of human DPP6 and D. rerio dpp6a, dpp6b. Zebrafish Synteny Database outputs demonstrate that Z) Human chromosome 7 (Hsa7, Green) and D. rerio chromosome 24 (Dre24, blue) have a closer evolutionary relationship, as they share five syntenic genes, while Zb) Hsa7 (green) and D. rerio chromosome 2 (Dre2, blue) share three syntenic genes. Orange lines connect the orthologs, and curved edges indicate directionality of the gene.

23 DPP6 ab41811 Antibody in zebrafish

In order to ascertain concentration and localization of Dpp6 in D. rerio, two antibodies were tested: rabbit anti-DPP6 (ab41811) and rabbit anti-DPP6 (ab198506).

Figure 4. DPP6 antibodies epitope alignment of human and zebrafish orthologs using Clustal Omega. Human DPP6 (NP_570629.2), Dpp6a (XP_009295542.1), Dpp6b (XP_005163346.1) and (A) abcam 41811 (human 765-865 (amino acids) (aa)). (B) abcam 198506 (human 450-550aa). Asterisk indicates perfectly conserved, colon indicates amino acids with different properties, but still slightly conserved, and a no annotation means amino acids in that positon are not conserved.

Dpp6a is 810 amino acids (aa) long, and is expected to create a band on Western blots at 92.8 kDa. Dpp6b is 857aa long, and a band should be observed around 97.4 kDa.

In mouse, DPP6 (abcam: ab41811) antibody forms a band around 100kDa149. Protein was extracted using two, different, buffers: RIPA buffer (ThermoFisher, Cat#89901) with protease inhibitor (PI) (ThermoFisher Scientific, Cat#78441B) (Figure 5A) extraction buffer with PI lane 1: 4 WT zebrafish eyes, lane 2: 3 WT zebrafish brains. In Figure 5A, protein bands were observed at 89.5 kDa, 55.1 kDa, 47.8 kDa and 39 kDa (lane 1), while in lane 2, bands are observed at 109.5 kDa, 84.2 kDa, 55.1 kDa, 46.8 kDa, 38.5 kDa and

33.9 kDa.

24 In Figure 5B lane 1 (brain tissue) there are bands at: 90.6 kDa, 73.1 kDa, 58.9 kDa, 55.6 kDa and 39.6 kDa. Lane 3 (eye tissue) bands are visible at: 120.3 kDa, 57.8 kDa, 46.9 kDa, 40.8 kDa and 34.2 kDa. Lanes 2 and 4 are the acetylated-tubulin control, bands are observed at 49 kDa and 46.9 kDa respectively.

Figure 5. DPP6 (ab41811) shows non-specific binding in adult WT zebrafish neuronal tissue. S1 fraction separated on a 7.5% SDS-PAGE gel. DPP6 (ab41811) 1:5000, rabbit (rb) anti-goat AP-conjugated (secondary) 1:5000. Protein extracted (A) with RIPA buffer and PI. Lane 1: 2 brains, lane b: 4 eyes, (B) with extraction buffer (Appendix C). Lane 1 and 3: rb anti-DPP6 (ab41811) 1:500, Lanes 2 and 4: mouse (ms) anti-acetylated !- tubulin 1:2000.

25 Specific binding was not observed with ab41811, as Western blots in mice had shown higher specificity of the antibody52,149. A second antibody was experimented with:

DPP6 RabMAb® (abcam:198506). The same batch of protein was utilized for Figure 6B and the DPP6 RabMAb® Western blot. No bands were observed for either zebrafish brain or eye neuronal tissue (data not shown).

PSMD12 Results

Zebrafish and Human PSMD12 comparison

Mutations in PSMD12 cause specific ASD behavioral phenotypes84; therefore, we propose that PSMD12 is not a critical component of all , but is a specific regulatory component of neuronal proteasomes. Previous work by Thisse and coworkers demonstrated that psmd12 in zebrafish is expressed within the head region of embryonic zebrafish150; however, as this was a high throughput screen, detailed analysis of expression has not been performed. Therefore, our first goal was to describe Psmd12 expression both temporally and spatially.

BLAST alignments of human PSMD12 to the zebrafish genome identified a PSMD12 ortholog. The protein exhibits 86.84% sequence identity (NCBI: AIC49518.1,

AAI65732.1). PSMD12 amino acid sequences compared between yeast and human suggest high conservation throughout evolution; therefore, we propose a common function within the 26S proteasome.

Synteny mapping using the zebrafish Synteny Database142,148 shows 30 paired genes between zebrafish psmd12 and human PSMD12. BLAST of zebrafish genomic sequences confirmed only one copy of psmd12 present (data not shown).

26

Figure 6. A 50 gene-sliding window of human PSMD12 and zebrafish psmd12 show seven syntenic genes shared. Zebrafish Synteny Database outputs demonstrate that A) Human (Hsa17, green) and D. rerio chromosome 6 (Dre6, blue) share seven syntenic genes, orange lines connect the orthologs, and curved edges indicate directionality of the gene.

27 Specific PSMD12 Antibody for zebrafish

In order to ascertain concentration and localization of psmd12 in D. rerio two antibodies, designed against human PSMD12, were tested: rabbit anti-PSMD12 (ab156604) and rabbit anti-PSMD12 (ab137932).

Figure 7. PSMD12 antibody epitope alignments against human and zebrafish using Clustal Omega. Red amino acids show completely mismatched amino acids, and green show mismatched amino acids with similar properties. (A) NP_002807.1 (human PSMD12), NP_963872.1 (zebrafish Psmd12) and abcam 137932 (human 137-186aa). B) NP_002807.1, NP_963872.1 and abcam 156604 (human 276-304aa).

PSMD12 ab156604 was designed against human PSMD12 (274-304aa). A Clustal

Omega alignment of human, D. rerio and the ab156604 epitope shows, 89.66% match, D. rerio has a three amino acid differences: amino acid 277 tyrosine (Y) instead if a phenylalanine (F), 292 threonine (T) instead of a glycine (G) and 303 arginine (R) instead of a lysine (K).

28 PSMD12 ab137932 was designed against human PSMD12 (137-186aa). A Clustal

Omega alignment of human, D. rerio and the ab137932 epitope shows, 90% match, D. rerio has five amino acid differences: aa 151 histidine (H) instead if a threonine (T), 156 serine (S) instead of an asparagine (N), 164 alanine (A) instead of a serine (S), 184 lysine

(K) instead of an arginine (R), and 183 alanine (A) instead of a valine (V).

Western blots were run using neuronal protein extracted from either one, two, or three zebrafish with either extraction buffer or RIPA buffer and blotted with antibody ab155604 (Figure 8). Rabbit anti-goat conjugated AP was used as the secondary antibody at a concentration of 1:5000 (Appendix A). Zebrafish Psmd12 is 456aa long; therefore, we expected to observe a band in Western blots at 52.82 kDa. In figure 7A, bands are observed at 52 kDa, 50 kDa, 51 kDa and 50 kDa in lanes 1 through 4 respectively.

However, unexpected bands were observed in all lanes at 115 kDa, 72 kDa (doublet band) and 38 kDa. In lane 2, there were four unexpected bands at 58.4 kDa, 41.2 kDa,

33.8 kDa and 30 kDa. In lanes 3 and 4 there was one unexpected band each at 37.7 kDa and 59.3 kDa respectively. A more stringent buffer was used in order to break the bonds associating with our protein of interest. Decreasing the non-specific binding seen in

Figure 8A.

When the protein was extracted using RIPA buffer (Figure 8B) only one unexpected band was observed at 32 kDa in eye tissue and no unexpected bands were observed in brain tissue. The PSMD12 ab156604 antibody showed a specific band at 55 kDa in eye tissue, and 54 kDa in brain tissue.

The PSMD12 ab137932 antibody did not show remarkable bands. There was a faint band observed around 50 kDa. Despite larger amounts of protein isolated from one,

29 two or three adult WT zebrafish neuronal tissue and an increased primary antibody concentration of 1:250, no notable bands were observed (data not shown).

Figure 8. PSMD12 ab156604 antibody shows further specificity when proteins are extracted with RIPA buffer. WT zebrafish neuronal tissue S1 fractions run in 7.5% SDS- PAGE. PSMD12 (ab156604) 1:500, AP-conjugated goat anti-rabbit (Jackson Labs 111- 055-003) 1:2000. Bands observed at expected size 52 kDa. (A) Protein extracted with standard extraction buffer (Appendix C) and PI. Lane 1: 6 eyes, lane 2: 3 brains, lane 3: 4 eyes, and Lane 4: 2 brains. (B) RIPA buffer with PI. Lane 1: 3 brains, lane 2: 2 brains, lane 3: 1 brain, lane 4: 6 eyes, lane 5: 4 eyes and lane 6: 2 eyes.

30 Constructs to elucidate the function of PSMD12

PSMD12 and PSMD14 interact in the regulatory lid of the proteasome inhibiting de-ubiquitination of proteins151 by obstructing the PSMD14 active site82. Unfortunately,

PSMD12’s function in vertebrates has not been examined, and specific roles for PSMD12 within the nervous system were only recently suggested due to its association with ASD.

Proteasome dysfunction in other neurologic diseases leads to protein aggregations, and cell death; therefore, we hypothesize that PSMD12 will alter cell viability of specific neuron classes that have previously been observed to be involved in ASD. Our long-term goal will be to quantify the viability of developing GABAergic and serotonergic neurons in the developing zebrafish.

In order to determine the function of PSMD12, PSMD12 was cloned into an expression vector in order to overexpress or rescue a KO, while MO and TALENs were created to knockdown or KO the protein function.

Full length PSMD12 cloned in an expression vector

The full length, zebrafish, PSMD12 gene was cloned in to a Promega pGEM®-T vector (Cat#A362A). The construct was transformed into JM109 bacterial cells, and

DNA harvested using Qiagen minipreps (Cat#27106). The clones were verified using

Sanger sequencing (Eurofins SimpleSeq) using the T7 and SP6 primers, and sequences verified using NCBI gene bank (ID: BC165732.1).

31 TALENs

TALEN Design and Screening

The goal of the TALEN is to disrupt the protein sequence enough to impair its translation. A frameshift mutation from the TALEN may cause a noticeable phenotype.

Specific sequences in zebrafish Psmd12 were targeted due to their high conservation from zebrafish to human (Figure 9).

Figure 9. Highly conserved regions targeted in zebrafish Psmd12. Top image is of 11 exons in zebrafish psmd12 with TALEN targeted regions in purple. Summation of conservation regions of PSMD12 protein from Saccharomyces cerevisae (S. cerevisae). to H. sapiens. PSMD12 homologs compared against seven species and the amount of conservation of each amino acid was analyzed, depicted in a bar graph from CLC Workbench. Purple arrows indicate where the TALEN will bind to DNA.

32 There was a high amount of variability in the inclusion of exon 1 and exon 2. We wanted to KO regions of the chromosome that were conserved from human to yeast.

Therefore, we targeted exon 3, to ensure we hit all isoforms.

Gene models show variably within exon 1 and 2; therefore, exon 3 was targeted to remove all isoforms of psmd12. Two TALENs were created for exon 3, and one for exon

6. TALENs in exon 3 should disrupt the protein early; therefore, we are hoping for a complete null phenotype. TALEN 3 is targeted to exon 6. This is to KO a conserved region in exon 6 (I-L-E) that is conserved from humans all the way to S. cerevisae

(Figure 9).

Genomic screening for psmd12 mutation

In order to genotype mutations caused by TALEN 1 (TAL1), TALEN 2 (TAL2) and TALEN 3 (TAL3), restriction enzymes sites (AlwI, BbvI and BspCNI respectively) were designed within the TALEN spacer region, so that when deleted these sites would be lost. TAL 1 and 2 are designed within exon 3; therefore, primers were designed against genomic DNA to yield a band at 1,000 bp, while primers flanking will produce a band at 1,189 bp (Figure 10A). In wild-type animals, AlwI will cut genomic DNA and 2 bands will be observed at 668 bp and 399 bp, BbvI will cut producing two bands at 668 bp and 500 bp; and BspCNI will cut producing three bands at 800 bp, 400 bp and 168 bp.

This allows for rapid and cost effective identification of mutation through loss of restriction sites and a long-term solution for genotyping.

33

Figure 10. TALEN genotyping assay. 1% agarose gel with 5% EtBr. The top image shows the 11 exons in zebrafish psmd12 in green are the PCR primer targeting sites. (A) PCR of genomic DNA for either exon 3 (target sites for TAL1 and TAL2), or exon 6 (Target site for TAL3) (B) Restriction enzyme digests of the PCR products in (B) AlwI is utilized for TAL1, BbvI for TAL2, and BspCNI for TAL3. Black arrows indicate the 3 bands when PCR product is digested with BspCNI.

Creation of TALENs targeting Exon 3 and Exon 6 of psmd12

Colony PCR confirmed successful insertion of RVDs into either pFUS_A or pFUS_B vectors (Figure 11). PCR primers: pCR8_F1and pCR8_R1 (Appendix B).

Successful pFUS_A inserts will form a band around 1200 bp and pFUS_B will form a band around 750 bp. We expected to see the “laddering” effect because primers used

34 should bind to each individual RVD. Incorrect insertion of RVDs will show a single, solid band around 250 bp.

Figure 11. Colony PCR of TALEN pFUS_A and pFUS_B array constructs. 1% agarose gel with 5% ethidium bromide (EtBr). pFUS_A constructs form a band around 1200 bp, pFUS_B constructs band around 750 bp and both produce a ‘laddering’ effect below band. (A) Lane 1: TAL 1-1, 10 RVDs in pFUS_A plasmid. Lane 2: TAL1-1, 5 RVDs in pFUS_B plasmid. Lane 3: TAL1-2, 10 RVDs in pFUS_A plasmid. Lane 4: TAL 1-2, 7 RVDs in pFUS_B plasmid. (B) Lane 1: TAL 2-1, 10 RVDs in pFUS_A plasmid. Lane 2: TAL2-1, 6 RVDs in pFUS_B plasmid. Lane 3: TAL2-2, 10 RVDs in pFUS_A plasmid. Lane 4: TAL 2-2, 6 RVDs in pFUS_B plasmid. (C) Lane 1: TAL 3-1, 10 RVDs in pFUS_A plasmid. Lane 2: TAL3-1, 7 RVDs in pFUSB plasmid. Lane 3: TAL3-2, 10 RVDs in pFUS_A plasmid. Lane 4: TAL 3-2, 6 RVDs in pFUS_B plasmid.

35 Creation of full TALEN constructs in pKT3Ts-goldyTALEN

In order to identify correct assembly of pFUS_A, pFUS_B, pLR-X and pKT3Ts- goldyTALEN (Golden Gate reaction two) (Figure 15) colony PCR was run using primers: TAL_F1 and TAL_R2 (Appendix B).

Successful assembly of individual TALENs will form a single band just below

2,000 bp with “laddering” effect below. The “laddering” effect is observed because primers are expected to bind to each RVD. Failed assembly will show two bands: one between 3,000-2,000 bp and the other around 650 bp, with no “laddering”. All TALENs in Figure 12 were sequence verified via Sanger sequencing (Eurofins SimpleSeq), primers used for sequencing: TAL_Seq_5-1 and TAL_R3 (Appendix B).

Figure 12. Colony PCR of TALEN products successfully inserted into pKT3TS- goldyTALEN. 1% agarose gel with 5% EtBr. Primers: TAL_F1 and TAL_R2. (A) TALEN 1: Lanes 1-3: TAL1-1 (upstream), Lanes 4+5: TAL1-2 (downstream). (B) TALEN 2: Lanes 1+2: TAL2-1 (upstream), lanes3-6: TAL2-2 (downstream). (C) TALEN 3: Lane 1: TAL3- 1 (upstream), Lane 2+3: TAL3-2 (downstream).

36 MO Results

Knockdown of PSMD12 ATG

Bands were observed in all injected and uninjected conditions. The amount of

Psmd12 observed differed in each condition. In Figure 13D, a second band was observed around 62 kDa in all conditions except in 10.5 ng. In Figure 13E, protein was extracted from 2 dpf embryos and a doublet band was observed, around 62 kDa, in both PSMD12

ATG MO and the mismatch injection. GAPDH was used as a loading control in all blots.

Figure 13. Western blots of PSMD12 ATG MO injected embryos. S1 fraction run in a 7.5% SDS-PAGE. Primary antibody: PSMD12 (ab156604) used at concentration 1:500, and GAPDH (loading control) used at a concentration of 1:3000. Secondary antibody: rabbit anti-goat AP conjugated used at a concentration of 1:5000. Bands visualized via AP buffer (Appendix C), 1% BCIP and 1.5% NBT. (A-D) 3dpf embryos (E) 2dpf embryos. UC: uninjected control (wild-type), C1: collection one, C2: collection two.

37

Figure 14. Average Psmd12 level and viability of MO injected zebrafish embryos. Quantified protein was normalized to GAPDH control and normalized a second time to the mismatch (MS) protein levels of PSMD12. Viability was determined by calculating the difference of the total number of embryo injected and total number of surviving embryos one day after injection. One-way ANOVA: F(2,9)=32.8, P=7.35x10-5. Tukey HSD shows MS to 3 ng, (p=0.001), mismatch (MS) to 6 ng, (p=0.001) and 3 ng to 6 ng (p=.475). ** indicates a p<0.001.

Protein was normalized to GAPDH and normalized a second time to the mismatch levels of Psmd12. Data was scored blindly. We see a 52% KD of Psmd12 in 6 ng, MS to

3ng=0.001, MS to 6ng, p=0.001, and 3ng to 6ng, p=.475. A minimum of three replicates were run per condition. We see a significant reduction in protein (F(2,9)=32.8,

P=7.35x10-5) as determined by a one way ANOVA, a Tukey post-hoc test demonstrates significant KD at both 3 ng (p=0.01) and 6 ng (p=0.01). No significant difference was seen between 3 and 6 ng KD (p=0.475).

38 Chapter 4 Discussion

DPP6 Discussion

Human DPP6 and Zebrafish orthologs compared

There are many lines of evidence that suggest DPP6 may play a role in the etiology of ASD: 1) Mutations in DPP6 have been observed in autistic patients, 2) DPP6 has an altered expression pattern in post mortem brains of autistic individuals versus neuro-typical controls29,42,152 , 3) DPP6 and A2BP1 (another ASD candidate gene) are co- expressed in a dataset that contains over 14,000 different microarrays (unpublished data

Dr. Brent Bill), and 4) the work of Bowers and Konopka demonstrates A2BP1 and DPP6 are both co-regulated by FOXP2, a with known roles in speech and

ASD153. Given the defined expression pattern for DPP6 in mammals, we hypothesize expression of dpp6 is critical for its function, and it will be important to understand its functional role in neuro-typical brain development, and how that differs in ASD.

In adult zebrafish, dpp6b is expressed in brain regions similar to the expression of murine Dpp6, specifically within the habenula, cerebellum and olfactory bulb154,155.

Additionally, IHC of adult zebrafish identified expression of Dpp6 in the fasciculus retroflexus (axons of habenula) and areas associated with the optic and auditory sense modalities (unpublished data, Dr. Bill) (Figure 2). This data supports the use of zebrafish as a tool to explore Dpp6, and may highlight new neuronal systems. One common symptom of ASD is altered sensitivities to environmental stimuli, such as fluorescent versus incandescent lights, hypersensitivity to sound and touch. This suggests a need to look at these modalities behaviorally in zebrafish, as pharmaceuticals that could modulate sensitivities would be quite a powerful tool for ASD treatment.

39 In order to determine the suitability of the zebrafish homologs for these studies.

Human DPP6 resides on chromosome 7, zebrafish dpp6a resides on chromosome 2 and dpp6b resides on chromosome 24. Using Clustal Omega Dpp6a, Dpp6b and human DPP6 protein sequences were compared via a percent identity matrix. The results support our synteny mapping data by showing Dpp6a is 4.38% more closely related to DPP6.

Moreover, at the nucleotide level, Dpp6a is more allied to DPP6 than Dpp6b.

Dpp6a is located on chromosome 24 and has five genes within 0.94 mega bases

(Mb) of DNA that is syngeneic with DPP6. Dpp6b is located on chromosome 2 with three number of genes within 0.8 Mb of DNA that is syntenic with DPP6. Important cis regulatory transcription factors usually fall within 10 kb of the beginning of the gene, although regional regulators can be much further away. The conservation of these regulatory factors could provide valuable information regarding the expression of DPP6.

DPP6 expression in zebrafish We hypothesized the Western blot utilizing the DPP6 ab41811 antibody would bind specifically to Dpp6 protein in zebrafish. IHC showed staining of olfactory regions and the habenula in mouse and zebrafish, and in situ hybridization in the fish conformed localization of the mRNA in these locations. Dpp6 expression labeled by ab41811 showed staining of the axons in zebrafish (Figure 5). Expression in axons was widespread throughout the brain, so to determine the specificity of the antibody for Dpp6 Western blotting was performed. A single band or a double band between 90 kDa and 97 kDa was expected; however, the presence of additional, unanticipated, bands in Figure 5 suggests possible cross-reactions, protein degradation or protein complexes due to insufficient denaturation. Other commercially available antibodies and different extraction buffers

40 were assessed while working with zebrafish Dpp6 to determine if the additional bands were due to technique or due to non-specific binding. Based in our current data, this binding is non-specific.

The epitope alignment of ab198506 showed a 75.33% and 70.95% identity with

Dpp6a and Dpp6b respectively. However, the epitope was in the middle of the protein, human amino acids 450-550, which may be a reason why no bands were observed. In order to verify a sufficient amount of protein transferred to the PVDF membrane (data not shown) total protein was observed with Ponceau S (Appendix C). The fact that there were no visible bands was not due to a lack of protein in the membrane. Since the same protein worked in previous Western blots, we can also conclude it was not due to an extraction issue. In titrating the antibody, a concentration up to 1:250 was used, much higher than most Western blotting protocols. Taken together this antibody is not capable of recognizing zebrafish Dpp6.

Multiple bands were observed in Western blots with both extraction and RIPA buffer extracted protein blotting with antibody ab41811. No bands were observed in

Western blots with RIPA buffer extracted protein blotted with antibody ab198506. We can conclude that Dpp6 is capable of being extracted using mild conditions, even though it is a transmembrane protein156. Dpp6 is expressed in zebrafish neuronal proteins and is not capable of being recognized, in a specific manner, with DPP6 antibody ab41811 or ab198506.

41 Future Studies for DPP6 Based on conservation, expression, and the ability of the human antibody to cross react with the zebrafish dpp6, we proposed the zebrafish homologs will be functionally similar; therefore, a good tool for future studies.

Sequencing of bands from Figure 5 to determine their identity. Creation or discovery of a more specific antibody would helpful. Places where we have seen overlap in the in situ and IHC are places we would like to look (Figure 2).

PSMD12 Discussion

Zebrafish and Human PSMD12 comparison

Alignment of human and zebrafish Psmd12 protein showed an 86.84% identity match. psmd12 is located on chromosome 6 with seven numbers of genes within 1.74 Mb of DNA syngeneic with human PSMD12. Just like in DPP6, the conservation of these regulatory factors could provide valuable information as to the expression of Psmd12 due to the location of regulatory transcription factors.

Specific PSMD12 Antibody for zebrafish

From this data it is clear PSMD12 ab156604 is the better antibody for zebrafish neuronal Psmd12. The alignment of the antibody epitope against zebrafish Psmd12 showed only a 3aa difference (Figure 7). The antibody showed specific binding when proteins were extracted using a more stringent extraction buffer, RIPA (Figure 8).

Therefore, harsher extraction conditions are recommended when utilizing this antibody in zebrafish.

42 Chromosomal editing of zebrafish PSMD12

Three different chromosomal editing tools were created to help elucidate Psmd12 function and location in zebrafish. An overexpression vector, three TALENs and three

MO.

Zebrafish psmd12 was cloned into an expression vector and it can be used to overexpress Psmd12 in zebrafish. Utilizing this may allow us to understand what happens when Psmd12 is functionally redundant, and it can help rescue a phenotype if a KO mutation transpires to be detrimental157.

The three TALENs created can be used individually or in combination with the others. Therefore, major mutations can be introduced into Psmd12. Multiple TALENs have been successfully used in combination to removed multiple exons in zebrafish158.

43

Figure 15. Golden Gate reactions. Using type IIS restriction endonucleases (BsaI and Esp3I) plasmids are digested and ligated together in a specific sequential manner. Step 1: Ten or more module plasmids containing RVDs ligated into array plasmids (p_FUS_A or p_FUS_B). Step 2: pFUS_A, pFUS_B, last repeat plasmid and pkT3Ts-goldy TALEN are digested and ligated in the destination vector (pkT3Ts-goldyTALEN). RVD: Random repeat variable diresidue, Tet: tetracycline, Spec50: spectinomycin50, Kan: kanamycin.

44 Correct colony PCR samples were transformed into One Shot® Mach1™ and

DH5⍺ chemically competent bacterial cells. DH5⍺ bacterial cells were used to transform pFUS_B, however, we found when transforming the pFUS_A constructs the One Shot®

Mach1™ yielded a higher success rate. One Shot® Mach1™ cells have a transformation efficiency > 1x109. DH5⍺ cells have a transformation efficiency > 1x106. The pFUS_A constructs were all 10 RVDs in length, and the pFUS_B were anywhere between 5 to 7.

Therefore, we think due to the larger size of the pFUS_A constructs the higher efficiency bacterial cells were necessary.

The DH5⍺ cells worked extremely well with the pFUS_B constructs. We had a success rate of about 70-80%. The success rate of pFUS_A constructs with DH5⍺ cells was 0%, and it was about 25% with the One Shot® Mach1™ cells. However, DH5⍺ cells yielded the only successful transformations during Golden Gate reaction two (Figure 15).

Morpholinos

Exon 3 was targeted because when looking at the protein alignment map, (Figure

9) you start to see conservation of sequence through the species around exon 3. IHC, of

!-acetylated tubulin, on 2 dpf MO injected embryos showed no noticeable differences in sensory neurons (data not shown). However, Küry and colleagues (2017) showed axon guidance defects and decreased optic tecta size with the mosaic CRISPR85. Therefore, we expected to see phenotypic differences if we look at deep neurons vs. superficial sensory neurons. If neuroanatomical and behavioral phenotypes were observed the animals would have been assessed and it would provide information for the transgenic lines used for

TALEN outcrosses.

45 Future studies for PSMD12

Quantification will be performed within transgenic zebrafish lines that express fluorescent molecules within specific neurons: Gad1-1:mCherry (hindbrain and spinal

GABAergic neurons), DLX5a-6a:GFP (forebrain and midbrain GABAergic neurons), and Th2:GFP (brain and gut serotonergic neurons). As with other disorders, we expect protein aggregations will precede cell death in the neurons that lack Psmd12.

Alternatively, if cell death is not observed, further analyses of neuron morphology and function will be performed.

In order to elucidate a phenotype of our MO and TALENs, IHC using similar procedures Küry and colleagues (2017) should be done. Therefore, in the future deep neurons should be targeted and embryos 3 dpf should be used. Studies like these will add in unraveling the role PSMD12 plays when it is KD. From this we can begin to understand how proteasome remolding caused by PSMD12 KD can lead to a neurodevelopmental syndrome.

46 Literature Cited

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55 Appendix A: Antibodies

Primary Antibodies: abcam: Anti-DPP6 antibody - Catalytic domain rabbit anti-DPP6 (ab41811) WB: 1:500 abcam: Anti-DPP6 antibody RabMAb® rabbit anti-DPP6 (ab198506): monoclonal WB: 1:1000 abcam: Anti-26S proteasome non-ATPase regulatory subunit 12 antibody rabbit anti-PSMD12 (ab156604): polyclonal WB: 1:500 abcam: Anti-26S proteasome non-ATPase regulatory subunit 12 antibody rabbit anti-PSMD12 (ab137932): polyclonal WB: 1:250-1:500

Sigma Aldrich: Product #: T6793 mouse anti-acetylated α-tubulin: monoclonal anti-tubulin, acetylated antibody produced in mouse WB: 1:2000 abcam: Anti-GAPDH antibody rabbit anti-GAPDH (ab209856): polyclonal to GAPDH Predicted 37 kDa WB: 1:500-1:3000 IHC: 1:100-1:1000

Secondary Antibodies: Alkaline Phosphatase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson, Cat#111-055-003) WB: 1:5000

Alkaline Phosphatase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson, Cat#112-055-175) WB: 1:5000

56 Appendix B: Primers

PSMD12 Primers.

Primer Name Sequence 5’-3’ OD nmol ug Len MW Tm gPSMD12-373F CGTGCTCATATTTCGTAGTTTACAT 15.1 65.6 499.7 25 7613.1 59.7 gPSMD12-1392R TCAAGAGCACAAGAGAGATTCC 14.7 63.1 426.4 22 6761.5 60.8 gPSMD12-3295F CTTGGGCTCTCCTACTCAATTT 14.3 75.3 499.1 22 6627.4 60.8 gPSMD12-Exon7R CTTCCAGAATACAAGGAGTGTCATA 13.8 53.9 413.9 25 7674.1 61.3

Morpholino Primers.

Name Sequence MW Molar Abs. Weight OD Holdback PSMD12ATG CATCAGACATTGTTGCAGCTAGTA 8128 252230 2.44 75.67 300 nmol PSMD12E3SA GACACCATATCCGAAGCCTGGAAA 8116 255990 2.43 76.80 300 nmol PSMD125p GCAGCGAATGTGACAACTACGATC 8147 254360 2.44 76.31 300 nmol PSMD12ATGms CATCCGACGAAGTTGCACCTCGTA 8074 246550 2.42 73.97 300 nmol

TALEN primers.

pCR8_F1: TTGATGCCTGGCAGTTCCCT pCR8_R1: CGAACCGAACAGGCTTATGT TAL_F1: TTGGCGTCGGCAAACAGTGG TAL_R2: GGCGACGAGGTGGTCGTTGG TAL_Seq_5-1: CATCGCGCAATGCACTGAC (use this for sequencing in place of TAL_F1) TAL_R3: GGCTCAGCTGGGCCACAATG

57 Appendix C: Solutions

Protein Extraction Buffer Recipe for 500 mL: 50 mM Tris HCl (1M) pH8 25mL Tris HCl 0.1% NP-40 500ul NP-40 0.05% Triton X-100 250ul Triton-X 150 mM NaCl (5M) 15mL NaCl (5M) Nanopure water to a final volume of 500 mL

6X SDS Loading Buffer Recipe for 15 mL: 10% SDS (sodium dodecyl sulfate) 1g of 10% SDS 30% glycerol 3mL of 100% glycerol 375 mM Tris-HCl (pH 6.8) 7mL of .5 M Tris-HCl, pH 6.8 0.2% Bromphenol Blue 1.2mg of Bromophenol Blue Nanopure water to a final volume of 15 mL 10X Tris-Glycine Running Buffer Recipe for 1L: 25 mM Tris-base 30.3g Tris-base 250 mM glycine 144g glycine 0.1% SDS 10.08g SDS pH 8.3 Distilled water to a final volume of 1L

10X Tris-buffered saline (TBS) Recipe for 1L: 20mM Tris-base pH 7.5 24g Tris-base 150mM NaCl 88g NaCl Dissolve in 900mL nanopure water pH to 7.6 Nanopure water to a final volume of 1L

Tris-buffered saline, 0.1% Tween-20 (TBST) Recipe for 1L: 20mM Tris-base pH 7.5 100mL of TBS 10X 150mM NaCl 900mL distilled water 0.1% Tween 20 1mL Tween 20 Nanopure water to a final volume of 1L

Ponceau S Stain Recipe for 50 mL: 0.2% Ponceau S .5g Ponceau S (FisherScientific, Cat#6226-79-5) 5% glacial acetic acid Dissolve in 2.5 mL glacial acetic acid 47.5mL nanopure water

58 Appendix C (Continued)

5% Milk Block 2.5g powder milk 50mL TBST

Bjerrum Schafer Nielsen (BSN) Transfer Buffer 25 mM Tris-base Recipe for 1L: 190 mM glycine 5.82g Tris-base 20% methanol 2.93g glycine Dissolve in 500mL of nanopure water 200mL methanol Nanopure water to a final volume of 1L Tris-acetate-EDTA (TAE) Buffer Working solution 1X: Recipe for 1L: 40 mM Tris (pH 7.6) 4.82g Tris-base 20 mM acetic acid 11.42mL glacial acetic acid 1 mM EDTA 2mL 0.5 M EDTA (pH 8.0) Nanopure water to a final volume of 1L

Stock solution 50X Recipe for 1L 2 M Tris (pH 7.6) 242g Tris-base .100 M acetic acid 571.1mL glacial acetic acid 50 mM EDTA 100mL 0.5M EDTA (pH 8.0) Nanopure water to a final volume of 1L Nitro blue tetrazolium chloride (NBT) 1ml 70% DMF (Dimethylformamide) 30mg NBT

5-Bromo-4-chloro-3-indolyl phosphate (BCIP) 15mg BCIP 1mL of DMF

Alkaline Phosphate (AP) Buffer

3.5 mM MgCl2 Recipe for 500mL: .1 M Tris-base 165mg MgCl2 6.05g Tris-base pH 9.5 Nanopure water to a final volume of 500mL

59 Appendix C (Continued)

Phosphate Buffered Saline (PBS) Stock solution (20X) Recipe for 1L: 2.7 M NaCl Stock solution (20X) .05 M KCl 160g NaCl .2 M Na2HPO4 4g KCl .036 M KH2PO4 2.88g Na2HPO4 4g KH2PO4 pH to 7.4 Distilled water to a final volume of 1L

Working solution (1X) Recipe for 1L: 137 mM NaCl Working solution (1X) 2.7 mM KCl 8g NaCl 10 mM Na2HPO4 0.2g KCl 1.8 mM KH2PO4 1.44g Na2HPO4 .24g KH2PO4 pH to 7.4 Distilled water to a final volume of 1L

Phosphate Buffered Saline 0.1% Triton X-100 (PBST) 1X PBS 0.1% Triton X-100

Immunohistochemistry Blocking Solution 1X PBST 1% DMSO 1% Normal goat serum 1% BSA

LB+Agar Plates (25 plates) 500mL Di-water 12.5g LB (BD, Cat#244620) 9.38g Agar Autoclave

Antibiotic working concentrations: Tetracycline (10 ug/mL) Kanamycin (50 ug/mL) Ampicillin (100 ug/ml) Spectinomycin 50 (50 ug/mL)

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