The programming of cell fate by transcription factors requires precise regulation of their time and level of expression. The LIM-homeodomain transcription factor Islet1 (Isl1) is involved in cell-fate specification of motor neurons, and it may play a similar role in the inner ear. In order to study its role in the regulation of vestibulo-motor development, we investigated a transgenic mouse expressing Isl1 under the Pax2 promoter control (Tg +/- ). The transgenic mice show altered level, time, and place of expression of Isl1 but are viable. However, Tg +/- mice exhibit hyperactivity, including circling behavior, and progressive age-related decline in hearing, which has been reported previously. Here, we describe the molecular and morphological changes in the cerebellum and vestibular system that may cause the hyperactivity of Tg +/- mice. The transgene altered the formation of folia in the cerebellum, the distribution of calretinin labeled unipolar brush cells, and reduced the size of the cerebellum, inferior colliculus, and saccule. Age-related progressive reduction of calbindin expression was detected in Purkinje cells in the transgenic cerebella. The hyperactivity of Tg +/- mice is reduced upon the administration of picrotoxin, a non-competitive channel blocker for the γ-aminobutyric acid (GABA) receptor chloride channels. This suggests that the overexpression of Isl1 significantly affects the functions of GABAergic neurons. We demonstrate that the overexpression of Isl1 affects the development and function of the cerebello-vestibular system, resulting in hyperactivity.

Department of Biology University of Iowa Iowa City IA USA

Institute of Biotechnology CAS Prumyslova 595 Vestec Prague West District 25242 Czech Republic

Institute of Experimental Medicine CAS Prague Czech Republic

Institute of Physiology CAS Prague Czech Republic

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Forbes PA, Siegmund GP, Schouten AC, Blouin J-S. Task, muscle and frequency dependent vestibular control of posture. Front Integr Neurosci. 2014;8:94. PubMed PMC

Beisel KW, Wang-Lundberg Y, Maklad A, Fritzsch B. Development and evolution of the vestibular sensory apparatus of the mammalian ear. J Vestib Res Equilib Orient. 2005;15(5-6):225–241. PubMed PMC

Maklad A, Fritzsch B. Partial segregation of posterior crista and saccular fibers to the nodulus and uvula of the cerebellum in mice, and its development. Brain Res Dev Brain Res. 2003;140(2):223–236. doi: 10.1016/S0165-3806(02)00609-0. PubMed DOI

Barmack NH, Yakhnitsa V. Functions of interneurons in mouse cerebellum. J Neurosci. 2008;28(5):1140–1152. doi: 10.1523/JNEUROSCI.3942-07.2008. PubMed DOI PMC

Bermingham NA, Hassan BA, Wang VY, Fernandez M, Banfi S, Bellen HJ, Fritzsch B, Zoghbi HY. Proprioceptor pathway development is dependent on Math1. Neuron. 2001;30(2):411–422. doi: 10.1016/S0896-6273(01)00305-1. PubMed DOI

Llinas R, Negrello MN. Cerebellum. Scholarpedia. 2015;10(1):4606. doi: 10.4249/scholarpedia.4606. DOI

Straka H, Fritzsch B, Glover JC. Connecting ears to eye muscles: evolution of a ‘simple’ reflex arc. Brain Behav Evol. 2014;83(2):162–175. PubMed

Goodworth AD, Mellodge P, Peterka RJ. Stance width changes how sensory feedback is used for multi-segmental balance control. J Neurophysiol. 2014 PubMed PMC

Stiles L, Smith PF. The vestibular-basal ganglia connection: balancing motor control. Brain Res. 2015;1597:180–188. doi: 10.1016/j.brainres.2014.11.063. PubMed DOI

Antoine MW, Hubner CA, Arezzo JC, Hebert JM. A causative link between inner ear defects and long-term striatal dysfunction. Science. 2013;341(6150):1120–1123. doi: 10.1126/science.1240405. PubMed DOI PMC

Tsuchida T, Ensini M, Morton SB, Baldassare M, Edlund T, Jessell TM, Pfaff SL. Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes. Cell. 1994;79(6):957–970. doi: 10.1016/0092-8674(94)90027-2. PubMed DOI

Nichols DH, Pauley S, Jahan I, Beisel KW, Millen KJ, Fritzsch B. Lmx1a is required for segregation of sensory epithelia and normal ear histogenesis and morphogenesis. Cell Tissue Res. 2008;334(3):339–358. doi: 10.1007/s00441-008-0709-2. PubMed DOI PMC

Shirasaki R, Pfaff SL. Transcriptional codes and the control of neuronal identity. Annu Rev Neurosci. 2002;25:251–281. doi: 10.1146/annurev.neuro.25.112701.142916. PubMed DOI

Cai CL, Liang X, Shi Y, Chu PH, Pfaff SL, Chen J, Evans S. Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart. Dev Cell. 2003;5(6):877–889. doi: 10.1016/S1534-5807(03)00363-0. PubMed DOI PMC

Pfaff SL, Mendelsohn M, Stewart CL, Edlund T, Jessell TM. Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell. 1996;84(2):309–320. doi: 10.1016/S0092-8674(00)80985-X. PubMed DOI

Liang X, Song MR, Xu Z, Lanuza GM, Liu Y, Zhuang T, Chen Y, Pfaff SL, et al. Isl1 is required for multiple aspects of motor neuron development. Mol Cell Neurosci. 2011;47(3):215–222. doi: 10.1016/j.mcn.2011.04.007. PubMed DOI PMC

Lu KM, Evans SM, Hirano S, Liu FC. Dual role for Islet-1 in promoting striatonigral and repressing striatopallidal genetic programs to specify striatonigral cell identity. Proc Natl Acad Sci U S A. 2014;111(1):E168–E177. doi: 10.1073/pnas.1319138111. PubMed DOI PMC

Ehrman LA, Mu X, Waclaw RR, Yoshida Y, Vorhees CV, Klein WH, Campbell K. The LIM homeobox gene Isl1 is required for the correct development of the striatonigral pathway in the mouse. Proc Natl Acad Sci U S A. 2013;110(42):E4026–E4035. doi: 10.1073/pnas.1308275110. PubMed DOI PMC

Li H, Liu H, Sage C, Huang M, Chen ZY, Heller S. Islet-1 expression in the developing chicken inner ear. J Comp Neurol. 2004;477(1):1–10. doi: 10.1002/cne.20190. PubMed DOI

Huang M, Sage C, Li H, Xiang M, Heller S, Chen ZY. Diverse expression patterns of LIM-homeodomain transcription factors (LIM-HDs) in mammalian inner ear development. Dev Dyn. 2008;237(11):3305–3312. doi: 10.1002/dvdy.21735. PubMed DOI PMC

Hans S, Liu D, Westerfield M. Pax8 and Pax2a function synergistically in otic specification, downstream of the Foxi1 and Dlx3b transcription factors. Development. 2004;131(20):5091–5102. doi: 10.1242/dev.01346. PubMed DOI

Pfeffer PL, Payer B, Reim G, di Magliano MP, Busslinger M. The activation and maintenance of Pax2 expression at the mid-hindbrain boundary is controlled by separate enhancers. Development. 2002;129(2):307–318. PubMed

Maricich SM, Herrup K. Pax-2 expression defines a subset of GABAergic interneurons and their precursors in the developing murine cerebellum. J Neurobiol. 1999;41(2):281–294. doi: 10.1002/(SICI)1097-4695(19991105)41:2<281::AID-NEU10>3.0.CO;2-5. PubMed DOI

Christophorou NA, Mende M, Lleras-Forero L, Grocott T, Streit A. Pax2 coordinates epithelial morphogenesis and cell fate in the inner ear. Dev Biol. 2010;345(2):180–190. doi: 10.1016/j.ydbio.2010.07.007. PubMed DOI PMC

Bouchard M, de Caprona D, Busslinger M, Xu P, Fritzsch B. Pax2 and Pax8 cooperate in mouse inner ear morphogenesis and innervation. BMC Dev Biol. 2010;10:89. doi: 10.1186/1471-213X-10-89. PubMed DOI PMC

Fritzsch B, Jahan I, Pan N, Elliott KL. Evolving gene regulatory networks into cellular networks guiding adaptive behavior: an outline how single cells could have evolved into a centralized neurosensory system. Cell Tissue Res. 2015;359(1):295–313. doi: 10.1007/s00441-014-2043-1. PubMed DOI PMC

Wang VY, Zoghbi HY. Genetic regulation of cerebellar development. Nat Rev Neurosci. 2001;2(7):484–491. doi: 10.1038/35081558. PubMed DOI

Zordan P, Croci L, Hawkes R, Consalez GG. Comparative analysis of proneural gene expression in the embryonic cerebellum. Dev Dyn. 2008;237(6):1726–1735. doi: 10.1002/dvdy.21571. PubMed DOI

Chumak T, Bohuslavova R, Macova I, Dodd N, Buckiova D, Fritzsch B, Syka J, Pavlinkova G (2015) Deterioration of the medial olivocochlear efferent system accelerates age-related hearing loss in Pax2-Isl1 transgenic mice. Mol Neurobiol PubMed

Simmons D, Duncan J, de Caprona DC, Fritzsch B. Development of the inner ear efferent system. In: Ryugo DK, Fay RR, Popper AN, editors. Auditory and vestibular efferents. New York: Springer; 2011. pp. 187–216.

Duncan J, Kersigo J, Gray B, Fritzsch B: Combining lipophilic dye, in situ hybridization, immunohistochemistry, and histology. Journal of visualized experiments 2011(49). PubMed PMC

Tonniges J, Hansen M, Duncan J, Bassett M, Fritzsch B, Gray B, Easwaran A, Nichols MG. Photo- and bio-physical characterization of novel violet and near-infrared lipophilic fluorophores for neuronal tracing. J Microsc. 2010;239(2):117–134. PubMed

Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001;29(9):e45. doi: 10.1093/nar/29.9.e45. PubMed DOI PMC

Bohuslavova R, Kolar F, Kuthanova L, Neckar J, Tichopad A, Pavlinkova G. Gene expression profiling of sex differences in HIF1-dependent adaptive cardiac responses to chronic hypoxia. J Appl Physiol. 2010;109(4):1195–1202. doi: 10.1152/japplphysiol.00366.2010. PubMed DOI

Khan Z, Carey J, Park HJ, Lehar M, Lasker D, Jinnah HA. Abnormal motor behavior and vestibular dysfunction in the stargazer mouse mutant. Neuroscience. 2004;127(3):785–796. doi: 10.1016/j.neuroscience.2004.05.052. PubMed DOI

Brooks SP, Dunnett SB. Tests to assess motor phenotype in mice: a user’s guide. Nat Rev Neurosci. 2009;10(7):519–529. doi: 10.1038/nrn2652. PubMed DOI

Carter RJ, Lione LA, Humby T, Mangiarini L, Mahal A, Bates GP, Dunnett SB, Morton AJ. Characterization of progressive motor deficits in mice transgenic for the human Huntington’s disease mutation. J Neurosci. 1999;19(8):3248–3257. PubMed PMC

Cristina C, Diaz-Torga G, Baldi A, Gongora A, Rubinstein M, Low MJ, Becu-Villalobos D. Increased pituitary vascular endothelial growth factor-a in dopaminergic D2 receptor knockout female mice. Endocrinology. 2005;146(7):2952–2962. doi: 10.1210/en.2004-1445. PubMed DOI

Autry AE, Adachi M, Nosyreva E, Na ES, Los MF, Cheng PF, Kavalali ET, Monteggia LM. NMDA receptor blockade at rest triggers rapid behavioural antidepressant responses. Nature. 2011;475(7354):91–U109. doi: 10.1038/nature10130. PubMed DOI PMC

Naydenov AV, Horne EA, Cheah CS, Swinney K, Hsu KL, Cao JK, Marrs WR, Blankman JL, et al. ABHD6 blockade exerts antiepileptic activity in PTZ-induced seizures and in spontaneous seizures in R6/2 mice. Neuron. 2014;83(2):361–371. doi: 10.1016/j.neuron.2014.06.030. PubMed DOI PMC

Smith AM, Wellmann KA, Lundblad TM, Carter ML, Barron S, Dwoskin LP. Lobeline attenuates neonatal ethanol-mediated changes in hyperactivity and dopamine transporter function in the prefrontal cortex in rats. Neuroscience. 2012;206:245–254. doi: 10.1016/j.neuroscience.2011.11.018. PubMed DOI PMC

Maklad A, Kamel S, Wong E, Fritzsch B. Development and organization of polarity-specific segregation of primary vestibular afferent fibers in mice. Cell Tissue Res. 2010;340(2):303–321. doi: 10.1007/s00441-010-0944-1. PubMed DOI PMC

Leonard RB, Kevetter GA. Molecular probes of the vestibular nerve. I. Peripheral termination patterns of calretinin, calbindin and peripherin containing fibers. Brain Res. 2002;928(1-2):8–17. doi: 10.1016/S0006-8993(01)03268-1. PubMed DOI

Desai SS, Ali H, Lysakowski A. Comparative morphology of rodent vestibular periphery. II. Cristae ampullares. J Neurophysiol. 2005;93(1):267–280. doi: 10.1152/jn.00747.2003. PubMed DOI

Desai SS, Zeh C, Lysakowski A. Comparative morphology of rodent vestibular periphery. I. Saccular and utricular maculae. J Neurophysiol. 2005;93(1):251–266. doi: 10.1152/jn.00746.2003. PubMed DOI

Park JJ, Tang Y, Lopez I, Ishiyama A. Age-related change in the number of neurons in the human vestibular ganglion. J Comp Neurol. 2001;431(4):437–443. doi: 10.1002/1096-9861(20010319)431:4<437::AID-CNE1081>3.0.CO;2-P. PubMed DOI

Melcher JR, Kiang NY. Generators of the brainstem auditory evoked potential in cat. III: identified cell populations. Hear Res. 1996;93(1-2):52–71. doi: 10.1016/0378-5955(95)00200-6. PubMed DOI

Resibois A, Rogers JH. Calretinin in rat brain: an immunohistochemical study. Neuroscience. 1992;46(1):101–134. doi: 10.1016/0306-4522(92)90012-Q. PubMed DOI

Lugli A, Forster Y, Haas P, Nocito A, Bucher C, Bissig H, Mirlacher M, Storz M, et al. Calretinin expression in human normal and neoplastic tissues: a tissue microarray analysis on 5233 tissue samples. Hum Pathol. 2003;34(10):994–1000. doi: 10.1053/S0046-8177(03)00339-3. PubMed DOI

Nunzi MG, Birnstiel S, Bhattacharyya BJ, Slater NT, Mugnaini E. Unipolar brush cells form a glutamatergic projection system within the mouse cerebellar cortex. J Comp Neurol. 2001;434(3):329–341. doi: 10.1002/cne.1180. PubMed DOI

Radde-Gallwitz K, Pan L, Gan L, Lin X, Segil N, Chen P. Expression of Islet1 marks the sensory and neuronal lineages in the mammalian inner ear. J Comp Neurol. 2004;477(4):412–421. doi: 10.1002/cne.20257. PubMed DOI PMC

Nakano Y, Jahan I, Bonde G, Sun X, Hildebrand MS, Engelhardt JF, Smith RJ, Cornell RA et al (2012) A mutation in the Srrm4 gene causes alternative splicing defects and deafness in the Bronx waltzer mouse. PLoS Genet 8(10):e1002966 PubMed PMC

Gerlai R, Pisacane P, Erickson S. Heregulin, but not ErbB2 or ErbB3, heterozygous mutant mice exhibit hyperactivity in multiple behavioral tasks. Behav Brain Res. 2000;109(2):219–227. doi: 10.1016/S0166-4328(99)00175-8. PubMed DOI

Vitali R, Clarke S. Improved rotorod performance and hyperactivity in mice deficient in a protein repair methyltransferase. Behav Brain Res. 2004;153(1):129–141. doi: 10.1016/j.bbr.2003.11.007. PubMed DOI

Graham DR, Sidhu A. Mice expressing the A53T mutant form of human alpha-synuclein exhibit hyperactivity and reduced anxiety-like behavior. J Neurosci Res. 2010;88(8):1777–1783. PubMed PMC

Altman J. Morphological and behavioral markers of environmentally induced retardation of brain development: an animal model. Environ Health Perspect. 1987;74:153–168. doi: 10.1289/ehp.8774153. PubMed DOI PMC

Mao Y, Reiprich S, Wegner M, Fritzsch B. Targeted deletion of Sox10 by Wnt1-cre defects neuronal migration and projection in the mouse inner ear. PLoS One. 2014;9(4):e94580. doi: 10.1371/journal.pone.0094580. PubMed DOI PMC

Fritzsch B, Pan N, Jahan I, Duncan JS, Kopecky BJ, Elliott KL, Kersigo J, Yang T. Evolution and development of the tetrapod auditory system: an organ of Corti-centric perspective. Evol Devel. 2013;15(1):63–79. doi: 10.1111/ede.12015. PubMed DOI PMC

Zuniga MG, Dinkes RE, Davalos-Bichara M, Carey JP, Schubert MC, King WM, Walston J, Agrawal Y. Association between hearing loss and saccular dysfunction in older individuals. Otol Neurotol. 2012;33(9):1586–1592. doi: 10.1097/MAO.0b013e31826bedbc. PubMed DOI PMC

Morita N, Kariya S, Farajzadeh Deroee A, Cureoglu S, Nomiya S, Nomiya R, Harada T, Paparella MM. Membranous labyrinth volumes in normal ears and Meniere disease: a three-dimensional reconstruction study. Laryngoscope. 2009;119(11):2216–2220. doi: 10.1002/lary.20723. PubMed DOI PMC

Cushing SL, Papsin BC, Rutka JA, James AL, Gordon KA. Evidence of vestibular and balance dysfunction in children with profound sensorineural hearing loss using cochlear implants. Laryngoscope. 2008;118(10):1814–1823. doi: 10.1097/MLG.0b013e31817fadfa. PubMed DOI

Hatten ME, Heintz N. Mechanisms of neural patterning and specification in the developing cerebellum. Annu Rev Neurosci. 1995;18:385–408. doi: 10.1146/annurev.ne.18.030195.002125. PubMed DOI

Sgaier SK, Lao Z, Villanueva MP, Berenshteyn F, Stephen D, Turnbull RK, Joyner AL. Genetic subdivision of the tectum and cerebellum into functionally related regions based on differential sensitivity to engrailed proteins. Development. 2007;134(12):2325–2335. doi: 10.1242/dev.000620. PubMed DOI PMC

Wurst W, Auerbach AB, Joyner AL. Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum. Development. 1994;120(7):2065–2075. PubMed

Timmer JR, Wang C, Niswander L. BMP signaling patterns the dorsal and intermediate neural tube via regulation of homeobox and helix-loop-helix transcription factors. Development. 2002;129(10):2459–2472. PubMed

Stoodley CJ, Schmahmann JD. Functional topography in the human cerebellum: a meta-analysis of neuroimaging studies. NeuroImage. 2009;44(2):489–501. doi: 10.1016/j.neuroimage.2008.08.039. PubMed DOI

Wang VY, Rose MF, Zoghbi HY. Math1 expression redefines the rhombic lip derivatives and reveals novel lineages within the brainstem and cerebellum. Neuron. 2005;48(1):31–43. doi: 10.1016/j.neuron.2005.08.024. PubMed DOI

Fink AJ, Englund C, Daza RA, Pham D, Lau C, Nivison M, Kowalczyk T, Hevner RF. Development of the deep cerebellar nuclei: transcription factors and cell migration from the rhombic lip. J Neurosci. 2006;26(11):3066–3076. doi: 10.1523/JNEUROSCI.5203-05.2006. PubMed DOI PMC

Schwarting RK, Huston JP. The unilateral 6-hydroxydopamine lesion model in behavioral brain research. Analysis of functional deficits, recovery and treatments. Prog Neurobiol. 1996;50(2-3):275–331. doi: 10.1016/S0301-0082(96)00040-8. PubMed DOI

Schirmer M, Kaiser A, Lessenich A, Lindemann S, Fedrowitz M, Gernert M, Loscher W. Auditory and vestibular defects and behavioral alterations after neonatal administration of streptomycin to Lewis rats: similarities and differences to the circling (ci2/ci2) Lewis rat mutant. Brain Res. 2007;1155:179–195. doi: 10.1016/j.brainres.2007.04.012. PubMed DOI

Smith AD, Llinás R, Kostyuk PG. Commentaries in the neurosciences. Oxford: Pergamon Press Ltd; 1980.

Mizuhara E, Minaki Y, Nakatani T, Kumai M, Inoue T, Muguruma K, Sasai Y, Ono Y. Purkinje cells originate from cerebellar ventricular zone progenitors positive for Neph3 and E-cadherin. Dev Biol. 2010;338(2):202–214. doi: 10.1016/j.ydbio.2009.11.032. PubMed DOI

Kalinichenko SG, Okhotin VE. Unipolar brush cells—a new type of excitatory interneuron in the cerebellar cortex and cochlear nuclei of the brainstem. Neurosci Behav Physiol. 2005;35(1):21–36. doi: 10.1023/B:NEAB.0000049648.20702.ad. PubMed DOI

Hulst T, van der Geest JN, Thürling M, Goericke S, Frens MA, Timmann D, Donchin O. Ageing shows a pattern of cerebellar degeneration analogous, but not equal, to that in patients suffering from cerebellar degenerative disease. NeuroImage. 2015;116:196–206. doi: 10.1016/j.neuroimage.2015.03.084. PubMed DOI

Berquin P, Giedd J, Jacobsen L, Hamburger S, Krain A, Rapoport J, Castellanos F. Cerebellum in attention-deficit hyperactivity disorder: a morphometric MRI study. Neurology. 1998;50(4):1087–1093. doi: 10.1212/WNL.50.4.1087. PubMed DOI

Courvoisie H, Hooper SR, Fine C, Kwock L, Castillo M. Neurometabolic functioning and neuropsychological correlates in children with ADHD-H: preliminary findings. J Neuropsychiatry Clin Neurosci. 2004;16(1):63–69. doi: 10.1176/jnp.16.1.63. PubMed DOI

Edden RA, Crocetti D, Zhu H, Gilbert DL, Mostofsky SH. Reduced GABA concentration in attention-deficit/hyperactivity disorder. Arch Gen Psychiatry. 2012;69(7):750–753. doi: 10.1001/archgenpsychiatry.2011.2280. PubMed DOI PMC

Breggin P. Talking back to Ritalin: what doctors aren’t telling you about stimulants and ADHD. Boston: Da Capo Press; 2007.

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