Boys Town National Research Hospital (BTNRH) Genetic & Molecular Biology Labs Auditory Neurobiology Vestibular Neurogenetics Neurochemistry Retinal Neurobiology Gene Expression Lymphatic Research Gene Marker & Identification Cell Signaling & Angiogenesis

Goal is to understand the basic mechanisms underlying auditory system development in normal and pathological systems.  Role of thyroid hormone in cochlear development  Role of Vlgr1 in stereociliary bundle development  Development of amino acid neurotransmission among neurons of the caudal brainstem Auditory Neurobiology Lab Edward Walsh, JoAnn McGee Hz dB SPL

Techniques  Single cell electrophysiology  Evoked potential  Sensory cell transduction  Inner ear and CNS histology and ultrastructure  Immunocytochemistry and histochemistry  Otoacoustic emissions  Microiontophoresis Auditory Neurobiology Lab Edward Walsh, JoAnn McGee

Goal is to identify the molecular and biochemical mechanisms by which endogenous angiogenesis inhibitors prevent tumor development and growth  Identify the mechanism through which the carboxy terminal of type IV collagen, specifically, the noncollagenous domain 1 of the  1 chain of type IV collagen (  1(IV)NC1), inhibits angiogenesis and tumor growth (type IV collagen is the major component of vascular basement membranes)  Determine mechanism whereby  1β1 integrin regulates hypoxia- associated factors associated with the antiangiogenic activity of  1(IV)NC1  Identify the cell signaling pathway involved in the non-integrin mediated antiangiogenic activity of a1(IV)NC1  Identify natural proteins that can be used as therapeutic agents for the treatment of cancer Cell Signaling & Angiogenesis Lab Sudhakar Akulapalli

Techniques  In vivo tumor burden studies  Cell culture systems (mouse lung endothelial cells, mouse lung fibroblast preparations)  Migration and proliferation assays  Immunoprecipitation and immunoblotting to study protein interactions  Immunohistochemistry Sudhakar et al. (2005) Cell Signaling & Angiogenesis Lab Sudhakar Akulapalli

Study of molecular mechanisms underlying progressive pathologies resulting from mutations in basement membrane proteins; the role(s) of cell adhesion and integrin signaling proteins, cytokines and chemokines  Alport Syndrome (Type IV collagen)  Renal pathology  Inner ear pathology  Usher Syndrome Type IIa (Usherin)  Retinal pathology  Inner ear pathology Gene Expression Lab Dominic Cosgrove, Velidi Rao

Techniques  Gene knockout mouse models for Alport syndrome and Usher syndrome type IIa  Immortalized cell lines derived from the retina, the stria vascularis, and the renal glomerulus of both normal and mutant mice  Biochemical and molecular techniques  Treatment strategies

Identification of genes associated with hearing loss and localization to the human gene map  Syndromic:  Usher Syndrome (Type I, Type II and Type III)  Branchio-Oto-Renal (BOR) Syndrome  Cleft Palate  Nonsyndromic:  Dominant Progressive Hearing Loss  Autosomal Recessive Hearing Loss (Auditory Neuropathy – otoferlin) Gene Marker & Identification Lab Bill Kimberling, Dana Orten, Phil Kelley Usher Syndrome USH1b – myosin VIIa USH1c – harmonin USH1d – cadherin 23 USH1f – protocadherin 15 USG1g - sans USH2a – usherin USH2c – vlgr1 USH3 – clarin1 Hereditary Hearing Loss Syndromic (20%) Usher Syndrome Nonsyndromic (80%) Dominant (15%) Recessive (80%)

Gene Marker & Identification Lab Bill Kimberling, Dana Orten, Phil Kelley Techniques  Construct physical maps of the critical region of the human genome associated with hearing loss, identify candidate genes in that region, and search for mutations in candidate genes  Linkage mapping and haplotype mapping to identify regions of human genome linked to hearing loss genes  Positional candidate cloning strategy to identify genes causing hearing loss  Treatment strategies

Study of the lymphatic system as it relates to human inflammatory and malignant disease. Study of the molecules and mechanisms that contribute to lymphatic growth assessed by lymphangiogenesis Lymphatic vessels drain extracellular fluid and improve the capacity for pathogen surveillance by directing fluid towards regional lymph nodes Lymphatic Research Laboratory Richard Tempero

Techniques  Murine model of respiratory inflammation (e.g., Mycoplasma pulmonis infection)  Study molecules and mechanisms important for lymphangiogenesis Confocal image of normal mouse trachea lymphatics (LYVE-1 red) blood vessels (CD31 green) Lymphatic Research Laboratory Richard Tempero

The role of neurotransmitters and receptors in the establishment of neural pathways and the formation of maintenance of synaptic connections.  The role of acetylcholine and acetylcholinesterase in the development and maturation of the primary neurotransmitters in the auditory brainstem.  Understanding how acetylcholine drives the auditory system before the onset of hearing.  Understanding how nicotinic acetylcholine receptors function in outer hair cells and spiral ganglion cells. Neurochemistry Lab Barbara Morley

Techniques  Pharmacological and immuno- cytochemical characterization of neurotransmitter receptor subunits and assembled receptors  Expression of receptor subunits using in situ hybridization and real time RT-PCR  Construction of mice and cell lines that are have null mutations for cochlea-specific genes Neurochemistry Lab Barbara Morley

Molecular mechanisms of hereditary retinal degeneration, particularly retinitis pigmentosa  Mechanism of retinal degeneration in Usher syndrome type IIa; the role of usherin in cell signaling  Mechanism by which rod-specific genetic disorders lead to the degeneration of genetically-normal cone photoreceptors  Role of plasticity in bipolar cell connections during retinal degeneration Retinal Neurobiology Lab You-Wei Peng

Techniques  Immunofluorescence  In situ hybridization  Real time RT-PCR  Retinal histology and ultrastructure Rod marker, rhodopsin (red), Cone marker, cone transducin γ (green)

Study of the molecular and cellular mechanisms of otoconia development in the vestibular system Otoconia are composite crystals that overlie and provide optimal stimulus input to the sensory epithelium of the balance system. The crystals are critical for spatial orientation and balance. Vestibular Neurogenetics Lab Yesha Lundberg Deletion of Oc90 gene leads to abnormal otoconia formation and balance deficits

Vestibular Neurogenetics Lab Yesha Lundberg Techniques  Gene targeting  Protein biochemistry  Histological and ultrastructural analyses  Cell culture  Physiological studies (collaborative)

 Lymphatic Research  R Tempero  Neurochemistry  B Morley  Retinal Neurobiology  Y Peng  Vestibular Neuro- genetics  Y Lundberg BTNRH: Genetic and Molecular Biology Laboratories  Auditory Neurobiology  E Walsh, J McGee  Cell Signaling and Angiogenesis  S Akulapalli  Gene Expression  D Cosgrove, V Rao  Gene Mapping and Identification  B Kimberling, D Orten, P Kelley