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Molecular determination of novel genes and pathways required for vestibular morphogenesis in zebrafish

Dissertation
Author: Jessica Ann Petko
Abstract:
The vestibular system of the vertebrate inner ear functions to detect gravity and motion in order to maintain balance. Functionally there are two divisions of the vestibule: the semicircular canals which detect angular acceleration and the otolith organs which perceive linear movements. Each division, although structurally distinct, detects movement and gravity though the deflection of mechanosensory hair cells. The semicircular canals are three fluid filled tubes arranged in different orthogonal planes. Angular rotation of the head creates uneven flow of fluid through the canals and over the sensory hair cells that lie at each terminal, which can be translated to the brain via the eighth cranial nerve. The otolith organs, including the utricle and the saccule, are each composed of a patch of sensory hair cells associated with an ear stone composed of calcium carbonate. The goal of my research is to use zebrafish as a tool for the identification and characterization of genes that participate in vestibular development. I have discovered several genes that are expressed in the zebrafish otic vesicle during embryogenesis, and I have shown that these genes are essential for the formation of vestibular structures. The first genes that I analyzed were the two zebrafish orthologs of Pten, a well studied tumor suppressor gene that is involved in many human cancers. This gene is known to control cell growth and proliferation through its negative regulation of the Akt pathway. The zebrafish orthologs, ptena and ptenb are expressed during embryonic development, and their gene products are able to regulate the levels of phorphorylated Akt in embryogenesis. The two genes show distinct expression patterns, and predictably, knockdown of the individual genes also produce distinct phenotypes. ptena, but not ptenb, is expressed in the otic vesicle. Knockdown of ptena produces otolith defects at 24 hpf (hours post fertilization) and semicircular canal defects at 48 and 72 hpf. Knockdown of ptenb does not affect inner ear development. These results suggest that the function of one ancestral Pten may have been divided between two orthologs after the genome duplication in teleost fish. I also conclude that control of cell growth and survival may play a role in the formation of these vestibular structures. I have identified and investigated a novel zebrafish ortholog of a mammalian gene involved in otolith development. The mammalian otolith organs contains thousands of minute biomineralized particles called otoconia, whereas the inner ear of teleost fish contains three large ear stones called otoliths that serve a similar function. Otoconia and otoliths are composed of calcium carbonate crystals condensed on a core protein lattice. Otoconin-90 (Oc90) is the major matrix protein of mammalian and avian otoconia, while Otolith Matrix Protein-1 (Omp-1) is the most abundant matrix protein found in the otoliths of teleost fish. This difference in major matrix protein composition has been hypothesized to account for the morphological differences observed between mammalian otoconia and zebrafish otoliths. Therefore, it was unexpected that orthologs for these matrix proteins would be found other species. I have identified a novel gene, otoc1, which encodes the zebrafish ortholog of Oc90. Expression of otoc1 is detected in the ear between 15 hpf and 72 hpf, and is restricted primarily to the macula. During embryogenesis, expression of otoc1 mRNA precedes the appearance of omp-1 transcripts. Knockdown of otoc1 mRNA translation with antisense morpholinos produces a variety of aberrant otolith phenotypes. My results suggest that Oc90 orthologs may serve to nucleate calcium carbonate mineralization of zebrafish otoliths, and that this protein is not strictly involved in determining mammalian otoconial morphology. Previous experiments have shown that a zebrafish ortholog of Neuronal Calcium Sensor-1 (Ncs-1) is required for the formation of semicircular canal hubs in zebrafish otogenesis (Blasiole et al. 2005). In order to gain further insight into the pathways involved in this Ncs-1 dependent process, I studied the role of Ncs-1 interacting proteins (NIPs) in vestibular development. A yeast-2-hybrid screen was performed to identify novel NIPs. Several of these newly identified and other previously known interactors were analyzed as candidates for otogenic genes. I determined that many zebrafish NIP orthologs are expressed in the developing semicircular canal structure. Morpholino knockdown of three of these genes, arf1, pi4kβ and dan has demonstrated that these genes are indeed important for vestibular morphogenesis. Combinatorial knockdowns have also been used to show that arf1, pi4kβ, and ncs-1a functionally interact. These functional interactions and direct physical associations suggest that these genes are involved in a unified pathway during inner ear development. (Abstract shortened by UMI.)

vii TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................... x   LIST OF TABLES ....................................................................................................... xii   LIST OF COMMON ABBREVIATIONS .................................................................. xiii   ACKNOWLEDGEMENTS ......................................................................................... xv   Chapter 1 Literature Review ....................................................................................... 1   1.1 Structure and Function of the Vertebrate Vestibular System ......................... 2   1.1.1 Overview of the Inner Ear .................................................................... 2   1.1.2 Mechanosensory Hair Cells .................................................................. 2   1.1.3 Otolith Organs ...................................................................................... 3   1.1.4 Semicircular Canals .............................................................................. 5   1.2 Zebrafish as a Developmental Model ............................................................. 5   1.2.1 General Model System Information ..................................................... 6   1.2.2 Zebrafish as a Model for Inner Ear Development ................................ 11   1.2.2.1 Evolutionary Conservation of Inner Ear Anatomy .................... 11   1.2.2.2 Structural development of the Zebrafish Inner Ear. ................... 14   Otic Vesicle Formation ................................................................... 14   Statoacoustic Gangion Development .............................................. 15   Otolith and Macula Formation ....................................................... 16   Semicircular Canal and Cristae Development ............................... 18   1.2.2.3 Genetic determination the Vestibular System ............................ 19   Otic Induction ................................................................................. 19   Genes Required for otoconia or otolith development ..................... 25   Genes required for semicircular canal duct and crista development ............................................................................. 31   1.3 Phosphatase and Tensin Homolog .................................................................. 35   1.4 Otoconins ........................................................................................................ 36   1.5 Neuronal Calcium Sensor-1 ............................................................................ 40   1.6 Rationale and Hypothesis ............................................................................... 44   Chapter 2 ptena and ptenb Genes Play Distinct Roles in Zebrafish Embryogenesis ...................................................................................................... 48   2.1 Introduction ..................................................................................................... 49   2.2 Experimental Procedures ................................................................................ 51   2.2.1 Identification and Characterization of Zebrafish pten Genes ............... 51   2.2.2 mRNA Expression Analysis ................................................................. 51   2.2.3 Antisense Morpholino Knockdowns .................................................... 52   2.2.4 Phosphorylated Akt Assay ................................................................... 54  

viii 2.3 Results ............................................................................................................. 54   2.3.1 Identification and Characterization of Zebrafish pten Genes ............... 54   2.3.2 Expression of ptena and ptenb Genes in Zebrafish Embryos ............... 57   2.3.3 Antisense Morpholino Knockdown of ptena and ptenb Expression .... 61   2.3.4 Biological Activity of Ptena and Ptenb ................................................ 71   2.4 Discussion ....................................................................................................... 72   Chapter 3 Otoc1: a Novel Otoconin-90 Ortholog Required for Otolith Mineralization in Zebrafish .................................................................................. 77   3.1 Introduction ..................................................................................................... 78   3.2 Experimental Procedures ................................................................................ 81   3.2.1 Cloning of zebrafish otoc1 gene ........................................................... 81   3.2.2 Phylogenetic analysis ........................................................................... 82   3.2.3 otoc1 mRNA expression ....................................................................... 82   3.2.4 Antisense morpholinos ......................................................................... 83   3.2.5 Immunofluorescence analysis of tether cells ........................................ 84   3.3 Results ............................................................................................................. 84   3.3.1 Identification of otoc1 .......................................................................... 84   3.3.2 Phylogenetic analysis ........................................................................... 86   3.3.3 Expression of otoc1 mRNA .................................................................. 90   3.3.4 Morpholino knockdown of otoc1 mRNA translation ........................... 94   3.4 Discussion ....................................................................................................... 102   Chapter 4 A Screen for Ncs-1 Interacting Proteins and Their Role in Semicircular Canal Development .............................................................................................. 107   4.1 Introduction ..................................................................................................... 108   4.2 Experimental Procedures ................................................................................ 112   4.2.1 Yeast-two-hybrid screen ....................................................................... 112   4.2.2 Ortholog identification and cloning ...................................................... 113   4.2.3 Whole-mount in situ hybridization ....................................................... 114   4.2.4 Morpholino Knockdowns ..................................................................... 114   4.2.5 Protein interaction assays ..................................................................... 116   4.3 Results ............................................................................................................. 117   4.3.1 Identification of human NIPs ............................................................... 117   4.3.2 Identification and cloning of zebrafish NIP orthologs ......................... 118   4.3.3 Expression analysis of zebrafish NIP mRNAs ..................................... 123   4.3.4 Morpholino Knockdown of Individual NIPS ....................................... 124   4.3.4.1 Morpholino Knockdown of arf-1 .............................................. 128   4.3.4.2 Morpholino Knockdown of pi4kβ ............................................. 129   4.3.4.3 Morpholino Knockdown of dan ................................................ 130   4.3.5 Functional interaction between ncs-1a and multiple NIPs ................... 134   4.3.6 Direct interaction of Ncs-1 and NIP orthologs ..................................... 135   4.4 Discussion ....................................................................................................... 137  

ix Chapter 5 Closing Discussion ..................................................................................... 143   5.1 ptena is involved in ear development ............................................................. 144   5.2 Otoc1 is a potential otolith matrix protein in zebrafish .................................. 147   5.3 Ncs-1a mediated pathway in semicircular canal development ....................... 151   5.4 Human Vestibular Dysfunction ...................................................................... 159   REFERENCES ............................................................................................................ 161  

x LIST OF FIGURES Figure 1.1: Hair cell structure and function. ............................................................... 5   Figure 1.2: Anatomy of the Vestibular System ........................................................... 8   Figure 1.3: Mechanisms of morpholino functionality. .............................................. 10   Figure 1.4: Chemical structure of nucleic acids vs morpholinos. .............................. 10   Figure 1.5: Anatomical structure of the inner ear. ...................................................... 13   Figure 1.6: Development of the larval zebrafish ear. .................................................. 13   Figure 1.7: Two-phase model summarizing genetic interactions during otic placode induction. ................................................................................................. 22   Figure 1.8: The compartment boundary model. .......................................................... 24   Figure 1.9: Expression of ncs-1a mRNA during zebrafish embryogenesis. ............... 43   Figure 1.10: Expression of ncs-1a gene is essential for semicircular canal formation. .............................................................................................................. 45   Figure 1.11: Aberrant semicircular canal formation in 5 dpf ncs-1a morphants. ....... 46   Figure 2.1: Comparison of human Pten and zebrafish Pten polypeptides. ................ 56   Figure 2.2: Expression of zebrafish ptena mRNA during embryogenesis. ............... 58   Figure 2.3: Expression of zebrafish ptenb mRNA during embryogenesis. ............... 60   Figure 2.4: Specificity of pten antisense morpholinos. .............................................. 62   Figure 2.5: Effect of ptena-MO1 morpholino on zebrafish development. ................. 66   Figure 2.6: Effect of ptena-MO1 morpholino on zebrafish inner ear development. ......................................................................................................... 67   Figure 2.7: Effect of ptenb-MO1 morpholino on zebrafish development. ................ 69   Figure 2.8: Lipid phosphatase activity of zebrafish pten genes. ................................ 70   Figure 3.1: Nucleotide and deduced amino acid sequence of zebrafish otoc1. ......... 87   Figure 3.2: Comparison of Otoconin orthologs. ........................................................ 90  

xi Figure 3.3: Phylogenetic analysis of PLA2L Otoconins. ........................................... 91   Figure 3.4: Expression of zebrafish otoc1 mRNA during embryogenesis. ............... 93   Figure 3.5: Specificity of otoc1 antisense morpholino. ............................................. 95   Figure 3.6: Expression of otoc1 is necessary for normal otolith development. ......... 97   Figure 3.7: Phenotypes of otoc1 morphants. .............................................................. 99   Figure 3.8: Tether cells are present in otoc1 morphant ears. ..................................... 100   Figure 3.9: Phenotypes of embryos co-injected with sub-effective doses of otoc1-ATG and omp-1 morpholinos. .................................................................... 101   Figure 4.1: Comparison of human Ncs-1 interacting protein sequences and their zebrafish orthologs. ............................................................................................... 123   Figure 4.3: Expression of arf1 is necessary for normal semicircular canal development. ......................................................................................................... 131   Figure 4.4: Expression of pi4kβ is necessary for normal semicircular canal development. ......................................................................................................... 132   Figure 4.5: Expression of dan is necessary for normal semicircular canal development. ......................................................................................................... 133   Figure 4.6: Phenotypes of embryos co-injected with sub-effective doses of ncs- 1a, arf1,and pi4kβ, morpholinos. .......................................................................... 138   Figure 5.1: NIP model of intracellular trafficking. .................................................... 158  

xii LIST OF TABLES Table 1.1: Known otolith matrix proteins ................................................................... 38   Table 2.1: Occurrence of ptena and ptenb morphant phenotypes at 48 hpf. ............... 62   Table 3.1: Comparison of PLA2L Otoconins. ........................................................... 88   Table 4.1: Morpholino names and sequences. ........................................................... 114   Table 4.2: NIP ortholog indentification and cloning. ................................................. 115   Table 4.3: Human Ncs-1 interacting proteins. ............................................................ 119  

xiii LIST OF COMMON ABBREVIATIONS Ca 2+ Calcium Ions CO 3 2- Carbonate Ions cDNA Complementary Deoxyribonucleic Scid DM Distance Matrix DNA Deoxyribonucleic Acid dpf Days Post Fertilization EST Expressed Sequence Tag hpf Hours Post Fertilization kDa Kilodalton µg Microgram µl Microliter µM Micromolar mM Millimolar MO Morpholino Oligonucleotide MP Maximum Parsimony mRNA Messanger Ribonucleic Acid N Number ng Nanogram ORF Open Reading Frame PAGE Polyacrylamide Gel Elecrophoresis

xiv PBS Phosphate Buffered Saline PLA2 Secretory Phospholipase A2 PLA2L Secretory Phospholipase A2 Like PCR Polymerase Chain Reaction RACE Rapid Amplification of cDNA Ends RT Reverse Transcriptace SDS Sodium Dodecylsulphate Y2H Yeast-Two-Hybrid

xv ACKNOWLEDGEMENTS I would like to offer my sincerest gratitude to all of those who have contributed to the completion of my graduate work. First, I would like to thank Dr. Robert Levenson, my thesis advisor and mentor, for his unending support and encouragement and for providing an environment in which I could work and think independently. I am grateful to the members of my thesis committee, Drs. Victor Canfield, Keith Cheng, and Laura Carrel, for their advice and support throughout my graduate school career. I especially thank Dr. Victor Canfield for his technical advice and support.

Secondly, I would like to thank the past and present members of the Levenson Lab who have made the lab an enjoyable place to work. Each and every member of the lab has contributed to my education through their support, knowledge, and friendship.

I extend gratitude to my collaborators, including Drs. Isolde and Reudiger Thalmann, Drs. Bernard and Christine Thisse, Dr. Inna Hughes, Dr. Bruce Riley, and Bonny Millimaki, for their assistance and advice. I am also indebted to the faculty and staff of the Pharmacology Department and the Genetics Program. In particular, I thank Marie Duvall and Kathy Shuey for their guidance and friendship. I also appreciate Dr. Sarah Bronson for giving me the opportunity to help out with the genetics symposium and meet my idol, Nancy Hopkins.

xvi Finally, I want to acknowledge my friends and family members. I thank my parents and my husband for their continued love and support throughout my schooling. I am truly lucky to have a mother who has encouraged me to receive the favor that I deserve and to do all that is in my heart.

Chapter 1

Literature Review

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1.1 Structure and Function of the Vertebrate Vestibular System 1.1.1 Overview of the Inner Ear Although hearing and balance are functionally different senses, information about sound, motion, and gravity are processed by similar mechanisms within the vertebrate inner ear. These stimuli are all detected by the same type of specialized mechanosensory cells with the assistance of various extracellular structures. The functional diversity between auditory and vestibular systems is explained by the structural variation within the individual compartments of the inner ear. While the auditory system has evolved in structural complexity, the anatomy and function of the vertebrate vestibular apparatus has remained conserved. All vertebrates sense angular movement of the head through the semicircular canal system, and they sense linear movements and gravity through the otolith organs. 1.1.2 Mechanosensory Hair Cells The sensory epithelia of the vestibular and auditory organs are composed of the mechanosensory hair cells and the accessory supporting cells (Fig. 1.1, A). Sound, motion, and gravity are perceived by the mechanical stimulation of the sensory hair cells. Hair cells are named for the array of stereocilia extending from the apical end of the cell into the endolymphatic interior of the membranous labyrinth (Purves et al. 2008). These microvilli vary in size and are arranged in a graduated manner from shortest to tallest

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(Fig. 1.1, A). Kinocilia, the tallest cilia, utilizes microtubules to maintain its rigid structure, while the remainder of the stereocilia is packed with actin (Flock and Cheung 1977; Sobkowicz et al. 1995). The apical end of the hair cell converts mechanical energy to receptor currents (Wever 1971; Gillespie and Walker 2001). Depolarization of the hair cell membrane allows for the release of neurotransmitters from the basal end of the cell onto the peripheral end of cranial nerve VIII (Gillespie and Walker 2001). The molecular basis of the hair bundles response to stimuli resides in the tip links, elastic filaments that connect the tip of each stereocilia to its neighboring cilia (Gillespie and Walker 2001). When the stereocilia are deflected towards the kinocilia, tension in the tip links allows for the opening of mechanically sensitive nonselective cation channels (Fig. 1.1, C), depolarization of the cell (Fig. 1.1, D), and increased transmitter release (Hudspeth and Corey 1977; Gillespie and Walker 2001). Deflection of the stereocilia away from the kinocilium (Fig. 1.1, B and D) decreases tip link tension which prevents the opening of transduction channels and generates a hyperpolarized state (decreases transmitter release). Hair cells are normally coupled to extracellular structures such as the gelatinous cupula in the semicircular canals, and the otolithic structures of the saccule and utricle (Purves et al. 2008). These structures assist in the deflection of hair cells during movement of the head. 1.1.3 Otolith Organs Gravity and linear acceleration are perceived through the otolith organs located in the saccular and utricular compartments of the vestibular system (Fig. 1.2, A). The saccule

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and the utricle each contain a single cluster of sensory hair cells and supporting cells called a macula. These sensory regions are associated with an extracellular mass of calcium carbonate. Mammalian, avian, reptilian and amphibian otolithic masses are composed of thousands of tiny polyhedral particles called otoconia that are embedded in a gelatinous membrane (Fig. 1.2, B). In contrast, teleost fish have large, singular otoliths that are smooth in appearance. Both otoliths and otoconia are composed of calcium carbonate (CaCO 3 ) crystals condensed on an extracellular protein matrix; however, the CaCO 3 crystal polymorph utilized and the protein composition of the organic matrix vary between species (Pote and Ross 1991). The inertial properties of this mass load during periods of linear motion allow deflection and activation of the hair cells of the maculae. 1.1.4 Semicircular Canals The semicircular canals consist of three fluid-filled tubes which are oriented in perpendicular planes (Fig. 1.2, A) (Purves et al. 2008). At the terminal end of each canal lies a cluster of sensory hair cells termed cristae. The cupula, an extracellular gelatinous membrane, covers the hair cells and deflects the stereocilia in response to fluid flow (Fig 1.2, C and D). Angular movement of the head causes a disproportional flow of Figure 1.1: Hair cell structure and function. (A) Cross section diagram of vesibular sensory region. (B) Deflection of cilia away from kinocilium leads to slackening of tip links. (C) Deflection of cilia toward kinocilium leads to increased tension of tip links and opening of mechanically gated ion channels. (D) Ionic basis for depolarization of hair cell in response to ciliary deflection. Adapted from Neuroscience (Purves et al. 2008).

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endolymphatic fluid through each canal and a differential activation of each cristae. These inequalities are translated from the hair cells to the brain via the eighth cranial nerve. 1.2 Zebrafish as a Developmental Model 1.2.1 General Model System Information Zebrafish, Danio rerio, are fresh water fish that originate from streams in the Ganges River basin of East India and Burma. In the late 1970’s, Dr. George Streisinger introduced the scientific community to the power of the zebrafish as a developmental model organism. Zebrafish are attractive for the study of vertebrate development as they are small enough to maintain in large numbers as adults, and yet their embryos are large enough for classical embryonic manipulations. Unlike mammals and some other fish species, in zebrafish fertilization occurs externally allowing simple physical, molecular, and/or genetic manipulations and developmental observation from the very beginning of embryogenesis. The zebrafish embryo remains transparent from zygote formation through embryonic stages. Chemical inhibition of pigmentation after 24 hpf (hours post fertilization) by phenythiourea (PTU) can extend the transparency of the fish through early larval stage (Karlsson et al. 2001). The transparent nature of the embryo and larva allow for observation and comparison of organogenesis between normal and genetically altered states. Embryo transparency has also contributed to the success of using whole

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mount in situ hybridization for determining gene expression during early development (Thisse and Thisse 1998).

Online resources for zebrafish have expanded significantly since the turn of the century. Sequencing of the zebrafish genome began in 2001 at the Sanger Institute in Cambridge, UK. The project incorporated two strategies: the traditional mapping and sequencing of BAC libraries and whole genome shotgun sequencing. As of January 12, 2008 the project is estimated to be 76% complete (Sanger 2008). The genome sequences are annotated and released semi-annually to Ensembl (www.ensembl.org) for use by the scientific community. There is also an extensive collection of Expressed Sequence Tags (ESTs) that have been generated, sequenced, and annotated by the Washington University Zebrafish Genome Resource (WUZGR) group. There are many sets of ESTs that were generated from different periods of development and/or different tissue types. These sequences are searchable on both GenBank (www.ncbi.nlm.nih.gov/) and Ensembl and are available for purchase.

Several procedures and technologies have been developed to alter gene expression or protein function in zebrafish. The most popular technique for reverse genetics in Figure 1.2: Anatomy of the Vestibular System (A) An anatomical representation of the inner ear. (B) Cross section of the utriculae macula showing hair bundles projecting into the extracellular otoconial membrane. (C) The position of the cupula without (left) and during (right) angular acceleration. Adapted from Neuroscience (Purves et al. 2008).

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zebrafish is knockdown of gene expression using morpholinos. Morpholinos (MOs) are antisense oligonucleotides that bind complementary sequences in mRNA (Stirchak et al. 1989). This binding results in a steric blockage of the translation initiation complex or pre-mRNA splicing machinery, which, in turn, leads to decreased translation products or altered splice products, respectively (Fig. 1.3). In addition, morpholinos can bind and block the activity of miRNAs and ribozymes. Morpholinos are favorable over other RNAi techniques because their mode of action does not require RNAse H or RISC mediated mRNA degradation (Summerton 2007).

Morpholinos are different from natural nucleic acids in that the ribose or deoxyribose sugar moieties are replaced with a morpholine ring (Fig. 1.4) and the anionic phosphates are substituted with non-ionic phosphodiamidate linkages. These modifications allow suitable positioning of the DNA bases to bind its complementary site on mRNA via Watson Crick base pairing (Kang et al. 1992). Also, the morpholino oligo is not recognized by cellular enzymes or nucleases making them stable and unable to trigger an innate immune response (Hudziak et al. 1996). Morpholinos are delivered via microinjection into the yolk of a newly fertilized zebrafish zygote (Nasevicius and Ekker 2000). Gene knockdown is effective for 48-72 hours post fertilization (hpf) at which time the morpholino becomes too dilute due to cellular division. Therefore, morpholinos are most useful for determining gene function during early development from embryogenesis to the early phases of larval development. Electroporation techniques have been utilized by some researchers to introduce morpholinos to particular tissues later in development or adulthood (Thummel et al. 2006).

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Figure 1.3: Mechanisms of morpholino functionality. (A) Normal translation. The ribosome (purple) bound to the mRNA (blue) is translating the sequence into a peptide (orange). (B) Translation blockage by a morpholino. A morpholino (green) that binds the mRNA at the translation initiation codon or the 5’ untranslated region will block the ribosome from translating a peptide. (C) Splice-blocking morpholino. A morpholino targeting a splice donor or acceptor site in pre-mRNA will block the activity of the spicing machinery at that site. Splice site morpholinos may result in mature mRNA that is missing an exon, including an unspliced intron, or another abnormal spicing event due to a cryptic splice site.

Figure 1.4: Chemical structure of nucleic acids vs morpholinos. (A) Structure of a ribose ring. (B) Structure of a deoxyribose ring. (C) Structure of a morpholine ring. (Summerton 2007)

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1.2.2 Zebrafish as a Model for Inner Ear Development 1.2.2.1 Evolutionary Conservation of Inner Ear Anatomy The inner ear of all vertebrates is composed of two functional divisions, the vestibular and auditory systems. However, structurally and evolutionarily the inner ear can be divided into the pars superior and the pars inferior. The term pars superior refers to the more anterior and dorsal structures, namely the semicircular canals and the utricle. The pars inferior is more ventral and posterior and is comprised of the saccule, lagena, and macula neglecta in fish; the sacule, lagena, basilar papilla and amphibian papilla in amphibians; the saccule, cochlea, and lagena in birds; and the saccule and cochlea in mammals. Schematics of the inner ear for each of these species are shown in Fig. 1.5 (Riley and Phillips 2003). The pars superior has changed very little in structure and function throughout evolution; however, the pars inferior has evolved a more complex structure and become adapted for sound detection in a variety of habitats.

In bony fish the structures of the inner ear are primarily cartilaginous and incorporate bone to a small degree (Carey and Amin 2006). In teleost fish, such as zebrafish, auditory sensitivity has been associated with all of the sensory regions in the pars inferior: the saccule, the lagena, and the macula neglecta. The zebrafish is a member of the Ostaryophysans, a group of fish that couple the saccule to the swim bladder through a set of bones called the webberian ossicles (Bang et al. 2001). This system transfers sound induced vibration of the swim bladder to the inner ear to enhance hearing sensitivity.

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The amphibian pars inferior contains the sacule, lagena, basilar papilla and amphibian papilla (Bang et al. 2001). The lagenar pouch is reduced as compared to those of bony fish, and the saccule takes on a more anterior position. Although the saccule plays roles in motion and gravity sensation, it has also demonstrated the ability to detect sound stimuli (Lewis and Narins 1998). The papillae are the principle acoustic detectors in amphibians (Wever 1985; Elephandt 1996). Interestingly, the basilar papilla is often tuned to a component of the animals mating call, whereas the amphibian papilla is a “general purpose acoustic sensor” (Lewis and Narins 1998).

The primary auditory endorgan in birds and mammals is the cochlea. The cochea is believed to have arisen from extension of the basilar papilla observed in amphibians (Carey and Amin 2006). The saccule plays a strictly vestibular role in birds and mammals. The lagena is still present at the end of the cochlea in birds and is of unknown function (Riley and Phillips 2003). In contrast, mammals do not have a lagena and the cochlea is much longer and more coiled than in birds.

The studies I report in this thesis concentrate on the development of the semicircular canals and the otoliths of the saccule and utricle. The semicircular canals and the utricule strictly play vestibular roles, but the saccule is involved in sound detection in zebrafish. This is important, because the identification of genes involved in saccular function can be attributed to auditory and vestibular function. Studies of this organ may lead to further

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Figure 1.5: Anatomical structure of the inner ear. Representation of adult inner ear structures of zebrafish, Xenopus, chick, and mouse. Each diagram is a lateral view with the anterior to the left. Auditory regions are shaded in blue. Abbreviations: ap, amphibian papilla; bp, basilar papilla; c, cochlea; l, lagena; s, saccule; u, utricle. This figure was adapted from Riley and Phillips (2003).

Figure 1.6: Development of the larval zebrafish ear. The development of the zebrafish otic vesicle from placode to semicircular canal duct formation. All diagrams are lateral views with anterior to the left.

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functional characterization of genes known to be involved in deafness and vestibular dysfunction in humans. 1.2.2.2 Structural development of the Zebrafish Inner Ear. An overview of zebrafish inner ear development from placode formation to the completion of semicircular canal formation at 72 hpf is shown in Fig. 1.6. Otic Vesicle Formation Development of the inner ear begins in the mid-somite stage of zebrafish embryogenesis with the formation of the otic placode, an ectodermal thickening visible on either side of the hindbrain by 15 hpf (Haddon and Lewis 1996). The zebrafish otic vesicle is formed by cavitation of the otic placode into a hollow ball of epithelium. To begin this process, the placode thickens into a solid ovoid ball just below the surface of the embryo. The initial cells in this complex have already become polarized as indicated by a concentration of actin in the center of the mass (Haddon and Lewis 1996). Formation of the lumen is accomplished by the movement of the cell nuclei to the periphery of the thickened placode, and a loss of cell junctions at the placode center (Haddon and Lewis 1996). The lumen first appears as a slit at about 18 hpf, and expands maintaining its simple ovoid shape until 24 hpf. This process differs in amniotes such as birds and mammals in which the otic vesicle develops from a folding of ectodermal tissues and the pinching off a vesicle (Haddon and Lewis 1996). Interestingly the formation of the otic

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vesicle resembles the formation of the neural tube in these species. While fish develop the otic vesicle and the neural keel through a hollowing process, amniotes form the otic vesicle and the neural tube through a folding and pinching process (Haddon and Lewis 1996). The otic vesicle will derive all the structures of the vestibular labyrinth and the neurons of the statoacoustic ganglion (the VIIIth cranial nerve). Statoacoustic Gangion Development Neurons of the statoacoustic gangion are derived from neuoblasts which delaminate from the ventral side of the otic vesicle and migrate to an area just ventral of the vesicle. Delamination begins at about 22 hpf and over the next few hours, the number of cells that delaminate and migrate increases with the peak period being between 22 and 30 hpf (Haddon and Lewis 1996). This delamination process, which generates a few hundred neuronal precursors, slows and can no longer be observed after 42 hpf. As the cells delaminate, they collect in the developing ganglion. Between 36 and 48 hpf the ganglion becomes a separate compact mass oriented ventromedially to the otic vesicle that extends beneath the anterior and the posteriomedial sensory regions. Differentiation of ganglion neuroblasts to mature neurons begins between 24 and 30 hpf. By 3 dpf many neurons have developed and their dendrites contact the hair cells within the sensory regions.

Full document contains 192 pages
Abstract: The vestibular system of the vertebrate inner ear functions to detect gravity and motion in order to maintain balance. Functionally there are two divisions of the vestibule: the semicircular canals which detect angular acceleration and the otolith organs which perceive linear movements. Each division, although structurally distinct, detects movement and gravity though the deflection of mechanosensory hair cells. The semicircular canals are three fluid filled tubes arranged in different orthogonal planes. Angular rotation of the head creates uneven flow of fluid through the canals and over the sensory hair cells that lie at each terminal, which can be translated to the brain via the eighth cranial nerve. The otolith organs, including the utricle and the saccule, are each composed of a patch of sensory hair cells associated with an ear stone composed of calcium carbonate. The goal of my research is to use zebrafish as a tool for the identification and characterization of genes that participate in vestibular development. I have discovered several genes that are expressed in the zebrafish otic vesicle during embryogenesis, and I have shown that these genes are essential for the formation of vestibular structures. The first genes that I analyzed were the two zebrafish orthologs of Pten, a well studied tumor suppressor gene that is involved in many human cancers. This gene is known to control cell growth and proliferation through its negative regulation of the Akt pathway. The zebrafish orthologs, ptena and ptenb are expressed during embryonic development, and their gene products are able to regulate the levels of phorphorylated Akt in embryogenesis. The two genes show distinct expression patterns, and predictably, knockdown of the individual genes also produce distinct phenotypes. ptena, but not ptenb, is expressed in the otic vesicle. Knockdown of ptena produces otolith defects at 24 hpf (hours post fertilization) and semicircular canal defects at 48 and 72 hpf. Knockdown of ptenb does not affect inner ear development. These results suggest that the function of one ancestral Pten may have been divided between two orthologs after the genome duplication in teleost fish. I also conclude that control of cell growth and survival may play a role in the formation of these vestibular structures. I have identified and investigated a novel zebrafish ortholog of a mammalian gene involved in otolith development. The mammalian otolith organs contains thousands of minute biomineralized particles called otoconia, whereas the inner ear of teleost fish contains three large ear stones called otoliths that serve a similar function. Otoconia and otoliths are composed of calcium carbonate crystals condensed on a core protein lattice. Otoconin-90 (Oc90) is the major matrix protein of mammalian and avian otoconia, while Otolith Matrix Protein-1 (Omp-1) is the most abundant matrix protein found in the otoliths of teleost fish. This difference in major matrix protein composition has been hypothesized to account for the morphological differences observed between mammalian otoconia and zebrafish otoliths. Therefore, it was unexpected that orthologs for these matrix proteins would be found other species. I have identified a novel gene, otoc1, which encodes the zebrafish ortholog of Oc90. Expression of otoc1 is detected in the ear between 15 hpf and 72 hpf, and is restricted primarily to the macula. During embryogenesis, expression of otoc1 mRNA precedes the appearance of omp-1 transcripts. Knockdown of otoc1 mRNA translation with antisense morpholinos produces a variety of aberrant otolith phenotypes. My results suggest that Oc90 orthologs may serve to nucleate calcium carbonate mineralization of zebrafish otoliths, and that this protein is not strictly involved in determining mammalian otoconial morphology. Previous experiments have shown that a zebrafish ortholog of Neuronal Calcium Sensor-1 (Ncs-1) is required for the formation of semicircular canal hubs in zebrafish otogenesis (Blasiole et al. 2005). In order to gain further insight into the pathways involved in this Ncs-1 dependent process, I studied the role of Ncs-1 interacting proteins (NIPs) in vestibular development. A yeast-2-hybrid screen was performed to identify novel NIPs. Several of these newly identified and other previously known interactors were analyzed as candidates for otogenic genes. I determined that many zebrafish NIP orthologs are expressed in the developing semicircular canal structure. Morpholino knockdown of three of these genes, arf1, pi4kβ and dan has demonstrated that these genes are indeed important for vestibular morphogenesis. Combinatorial knockdowns have also been used to show that arf1, pi4kβ, and ncs-1a functionally interact. These functional interactions and direct physical associations suggest that these genes are involved in a unified pathway during inner ear development. (Abstract shortened by UMI.)