The Role of The Role of Islet1Islet1 in the in the Development of the Auditory Development of the Auditory

and Vestibular Pathwaysand Vestibular Pathways

Nicole DoddNicole Dodd

1515thth May 2014 May 2014Laboratory of Molecular PathogeneticsLaboratory of Molecular Pathogenetics

Institute of Biotechnology AS CRInstitute of Biotechnology AS CR

•Rationale

•Auditory and Vestibular systems

•Transcription factor Islet1

•Overexpression Model

•What we know so far..

•Results

•Molecular mechanisms?

•Conclusions thus far & future work

Overview

Rationale

• Unravel the role of Islet1 in neurosensory development

• Due to early KO lethality, role in neurosensory development is unclear

• Propose Islet1 molecular interactions

Inner Ear

Auditory apparatus - Cochlea

Both the bony labyrinth and the cochlear duct are coiled in a ‘snail-like’ shape

Vestibular system – vestibule & 3 semicircular canals

Structures dedicated to sense of balance

The Inner Ear: Auditory

•Hair cells transduce vibrations into receptor potentials•Inner hair cells communicate via the neurotransmitter glutamate

• A specific wave frequency causes fibres to rapidly vibrate

• Short waves (lower frequencies) at the basal part, long waves (higher frequencies) at the apical segments

The Inner Ear: Vestibular

3 major components:•Peripheral sensory apparatus•Central processor•Mechanism for motor output

Peripheral apparatus sends information to the CNS – specifically the vestibular nucleus and the cerebellum

The Vestibulocochlear Nerve VIII

The vestibulocochlear nerve enters the brainstem at the pontomedullary junction

• Each lobule receives different sensory input

• Lobules III, IV and V receive major afferent input through the ventral spinocerebellar tract

• Lobule X has extensive vestibular input

Cerebellum

Numbered I to X, rostral to caudal

• Represents 10% of brain volume, but contains over half the neurons

• One of the first structures to differentiate

• Coordination centre, fine tunes movement/balance

Transcription Factor Islet1

• Insulin Gene Enhancer Protein

• LIM domain for protein/protein interactions, homeodomain for binding to DNA control elements

• Expressed in multiple organs

• Expression studies suggest Islet1 plays a role in cell fate decision of neurosensory cells in the inner ear

• Islet1 KO is lethal at E10.5

• Exact role in neurosensory development

unknown

Pronuclear injection of Pax2-isl1 construct

Pax2 promoter

LIM

Homeodomain

DNA binding

Protein binding

Islet1

FVB

T2

T1 -/+

-/+

LIM

T1/52

T2/300

Pax2-Isl1 transgenic mouse: What we know so far…

• Reduced survival rate

• Circling phenotype consistent with vestibular dysfunction

• Reduced hearing, especially in mid-range

• Deaf by 6 monthsN

um

ber

of

pu

ps/

litte

r

Transgenic Islet1 expression at P3

Lobule X Islet1 Pax2

WT

Tg+/-

• Islet1 not endogenously expressed in the cerebellum

• White cells indicate transgenic Islet1 positive cells

• Pax2 endogenously expressed by a subset of cerebellar GABAergic interneurons and their precursors

• Pax2 expressed from E12 until the end of cerebellar development (postnatal day 15)

ABR data shows auditory defects

• Increase rate of hearing loss

• Hearing loss more profound in midrange frequencies

• More profound in the 6-9 month group

WT

Tg+/-

Nissl stain

A

B

Decrease in number of SG neurons

Calretinin staining of the Vestibular ganglion

WT Tg+/-

B

• No calretinin positive cells in Tg

• Apoptosis?

• Differentiation?

• Calcium regulation?

Decrease number of Unipolar Brush Cells

Lobule IX & X calretinin

WT

Tg+/-

• UBCs express the calcium binding protein calretinin

• Excitory glutamatergic interneuron found in the granule layer of cerebellar cortex

• Abundant in regions liked to vestibular input especially lobules X & a portion of IX

• Amplify inputs form vestibular ganglia and nuclei

Pax2 Calbindin

Decrease size of cerebellum

WT

Tg+/-

• Overall size reduced

• Disruption in Purkinje cell layer

• Purkinje cells control the output of the cerebellum

Lipophilic dye-tracing of neuronal fibres

• Each neuron is surrounded by a lipid bilayer that allows filling those profiles with lipophilic dyes

• Allows selective labelling of distinct fibre types• Multicolour dye approach to verify projections

otherwise indistinguishable• Antibody ‘stains’ can lead to false negatives in

mutant mice owing to limited presence of antigen

Professor Bernd Fritzsch, leading expert in developmental neuroscience & specialist in carbocyanine dyes

Lipophilic dye tracing

Ut

Ac Hc

CN

VN

Sa

Sg

FN

Vestibular > Cb & Bs

Ac

FN

Ut

Sa

AE

AE

Ig

Eff

Cerebella injection

WT

Tg+/-

VIIIth

Cb

Bs

FN

PC

Cb

VIIIth

S

S/I VG

PC

Bs

Inner ear injection Cerebellar injection

WT

Tg+/-

Lipophilic dye tracing

Molecular mechanisms?

• Unique combinations of LIM-HD TFs form a transcriptional ‘LIM Code’

• Contributes to cell type specification

• Calcium binding proteins in homeostasis- No calretinin expression in tg vestibular

ganglion- Decrease expression in UBCs- No expression difference in auditory system

Conclusions

•Peripheral - hearing defects

•Central - projection aberration

•Vestibular ganglion - calretinin negative

•Reduction in UBCs in the cerebellum

•Reduction in overall size

•Initial premature, more rapid differentiation in the presence of additional islet1

•Overexpression effects are context dependent

Future work

• Quantify calretinin expression and intensity profile

• Determine stage differences occur

• Neuronal fibre projection atP1 using dye-tracing vestibular to cerebellumE10.5 (tubulin ICH)

• qPCR quantify islet1 OE in inner ear and cerebellum

• RNDr. Gabriela Pavlínková, Ph.D. (Institute of

Biotechnology AS CR)

Ing. Romana Bohuslavová, Michaela Lišáková

• Prof. Bernd Fritzsch (University of Iowa)

• Prof. Josef Syka (Institute of Experimental Medicine AS

CR): Mgr. Tetyana Chumak, Dr. Daniela Buckiova,

Acknowledgements