Introduction

Adult hearing embryonic origins.

Otic placodes (Stage 11 dorsal view)

Historic images - The membranous labyrinth

 The inner ear is derived from a pair of surface sensory placodes (otic placodes) that appear in
human development during week 4 (GA week 6) in the head region lying behind the second
pharyngeal arch.

These otic placodes fold inwards forming initially a depression, then pinch off entirely from
the surface forming an epithelium surrounding a fluid-filled sac or vesicle (otic
vesicle, otocyst, auditory vesicle). The vesicle sinks into the head mesenchyme some of
which closely surrounds the otocyst forming the otic capsule.
The otocyst finally lies close to the early developing hindbrain (rhombencephalon) and the
developing vestibulo-cochlear-facial ganglion complex.

The otocyst epithelium then undergoes a series of morphological changes, forming the
primitive membranous labyrinth. During the human fetal period this will differentiate into the
inner ear components for hearing (cochlea) and balance (semi-circular canals).
The adult cochlear has a "snail-shell" appearance, with the total number of turns differing
between species. The adult human cochlear is typically described as having 2.5 turns, but this
can vary up to 2.75 or even 3 turns.[1]
The "organ of corti" that develops within the cochlea was first identified by Alfonso Giacomo
Gaspare Corti (18221876), an Italian anatomist, in 1851.

Some Recent Findings

Role of BDNF and neurotrophic receptors in human inner ear development[2] "The
expression patterns of the neurotrophin, brain-derived neurotrophic factor, BDNF, and the
neurotrophic receptors-p75NTR and Trk receptors-in the developing human fetal inner ear
between the gestational weeks (GA) 9 to 12 are examined via in situ hybridization and
immunohistochemistry. BDNF mRNA expression was highest in the cochlea at GA 9 but
declined in the course of development. In contrast to embryonic murine specimens, a decline
in BDNF expression from the apical to the basal turn of the cochlea could not be observed.
...Our findings suggest that BDNF and neurotrophin receptors are important players during
early human inner ear development. In particular, they seem to be important for the survival
of the afferent sensory neurons."

Lineage tracing of Sox2-expressing progenitor cells in the mouse inner ear reveals a
broad contribution to non-sensory tissues and insights into the origin of the organ of
Corti[3] "The transcription factor Sox2 is both necessary and sufficient for the generation of
sensory regions of the inner ear. ...We find that Sox2-expressing cells in the early otocyst
give rise to large numbers of non-sensory structures throughout the inner ear, and that Sox2
only becomes a truly prosensory marker at embryonic day (E)11.5. Our fate map reveals the
organ of Corti derives from a central domain on the medial side of the otocyst and shows that
a significant amount of the organ of Corti derives from a Sox2-negative population in this
region." Developmental Signals - Sox | Mouse Development

Development of the stria vascularis and potassium regulation in the human fetal
cochlea[4] "We present an investigation on the development of the stria vascularis in the
human fetal cochlea between 9 and 18 weeks of gestation (W9-W18) and show the cochlear
expression dynamics of key potassium-regulating proteins. At W12, MITF+/SOX10+/KIT+
neural-crest-derived melanocytes migrated into the cochlea and penetrated the basement
 membrane of the lateral wall epithelium, developing into the intermediate cells of the stria
vascularis. These melanocytes tightly integrated with Na+ /K+ -ATPase-positive marginal
cells, which started to express KCNQ1 in their apical membrane at W16. At W18, KCNJ10
and gap junction proteins GJB2/CX26 and GJB6/CX30 were expressed in the cells in the
outer sulcus, but not in the spiral ligament."
Distribution and development of peripheral glial cells in the human fetal cochlea[5] "The
adult human cochlea contains various types of peripheral glial cells that envelop or myelinate
the three different domains of the spiral ganglion neurons: the central processes in the
cochlear nerve, the cell bodies in the spiral ganglia, and the peripheral processes in the
osseous spiral lamina. ...The developmental dynamics of the peripheral glial cells in the
human fetal cochlea is in support of a neural crest origin. Our study provides the first
overview of the distribution and maturation of peripheral glial cells in the human fetal
cochlea from W9 to W22." Neural Crest Development
Auditory ganglion source of Sonic hedgehog regulates timing of cell cycle exit and
differentiation of mammalian cochlear hair cells[6] "Neural precursor cells of the central
nervous system undergo successive temporal waves of terminal division, each of which is
soon followed by the onset of cell differentiation. The organ of Corti in the mammalian
cochlea develops differently, such that precursors at the apex are the first to exit from the cell
cycle but the last to begin differentiating as mechanosensory hair cells. ...The dynamic
relationship between the restriction of Shh expression in the developing spiral ganglion and
its proximity to regions of the growing cochlear duct dictates the timing of terminal mitosis
of hair cell precursors and their subsequent differentiation." Sonic hedgehog
Tbx1 and Brn4 regulate retinoic acid metabolic genes during cochlear
morphogenesis.[7]"These results indicate that Tbx1 expression in the POM regulates cochlear
outgrowth potentially via control of local retinoic acid activity."

Otic Placode

otic placode
 Stage 12 otic placode
The embryonic surface sensory placode associated with hearing and balance. This will be lost
from the embryo surface to form the otocyst or otic vesicle.

Stage 11 - single layer of ectodermal cells organized in a columnar epithelium, which differs in
cell shape from the surrounding cuboidal epithelia that will contribute the epithelia of the skin.
zebrafish model - a number of specific genes are involved in initial induction of the otic placode
including both growth factors (fgf3 and fgf8) and transcription factors (dlx3b, dlx4b, and foxi1).[8]
Proliferation of the otic placode cells leads to an inward folding, or invagination, giving the
external appearance of a depression on the lateral sides of the early developing neck region.
The epithelium is still a single layer of cells, which continues to invaginate until the edges of the
disc of cells come into apposition on the embryo surface.
mouse model - placodal invagination but not specification requires placodal expression of the
transcription factor Sox9.[9]
 Sensory Placodes

Week 4 a series of thickened surface ectodermal patches form in pairs rostro-caudally in the
head region.
Recent research suggests that all sensory placodes may arise from common panplacodal
primordium origin around the neural plate, and then differentiate to eventually have different
developmental fates.

Each pair of sensory placodes will later contribute key components of each of our special senses
(hearing, vision, smell and taste).
Otic Placode - one of the first to form and contributes inner ear structures.
Optic (Lens) Placode - lies on the surface, adjacent to the outpocketing of the nervous system
(which will for the retina) and will form the lens.
Nasal Placode - 2 components (medial and lateral) and will form the nose olefactory epithelium.
Other species have a number of additional placodes which form other sensory structures (fish,
lateral line receptor).
Note that their initial postion on the developing head is significantly different to their final position
in the future sensory system.

Links: Placodes | Week 4

Otocyst
The otocyst will differentiate to form all components of the membranous labyrinth. The organ
of Corti forms initially from the central domain on the medial side of the otocyst.[3]Sox2
regulates organ of Corti prosensory progenitor development, through Jag1 (Notch ligand)
signaling.

Otocyst historic drawings
Week 5
Stage 13 embryo (week 5) showing otocyst that will form the inner ear.
A. Ventrolateral view of the whole
embryo with 5-mm scale bar. At
this stage of development no
middle or external ear structures
are apparent and will be derived
later from pharyngeal arches one
and two (labeled).
Stage 13 otic vesicle now
lies beneath the embryo
surface.
Stage 13 - the otic placode
has sunk from the surface
ectoderm to form a hollow
epithelial ball, the otocyst
(otic vesicle), which now
lies beneath the surface
surrounded by
mesenchyme (mesoderm
and neural crest).
The epithelia of this ball
varies in thickness and has
begun to distort, it will
eventually form the inner
ear membranous labyrinth.
forms otocyst
branches form and
generate endolymphatic
duct and sac
forms vestibular (dorsal)
and cochlear (ventral)
regions
differentiation of otic vesicle
to membranous labyrinth
B. The gray bar through the head indicates the plane
of cross-section, which is a cross-section of the
head showing the size and position of the otic
vesicles. At this stage of development they lie
within the head mesenchyme behind pharyngeal
arch one and two and in close apposition to the
developing hindbrain. Note the close position of
 the otic vesicle to the rhombomeres, hindbrain
folds that represent the initial segmentation of the
hindbrain. Also shown are developing cranial
ganglia and blood vessel lying adjacent to the otic
vesicles. The wall of the otic vesicle at this stage
is a simple epithelium.

Week 7

Week 8
Stage 22 embryo (week 8) showing the embryo near the end of the embryonic period.

A. Lateral view of the whole embryo B. Cross-section of the head at the plane of the skull
with 5 mm scale bar. Note the base and oral cavity to the top. The otic capsule is
well developed external ear with well formed by this stage containing all the
simplified adult structure and membranous labyrinth structures. It is still a
 narrower meatal opening. The cartilaginous structure ventral to the brainstem and
grey bar through the head lying behind the oral cavity. The tongue occupies
indicates the plane of cross- the floor of the oral cavity with the unfused palatal
section for (B) and (C). shelves lying lateral and the auditory tubes clearly
shown on the posterior wall. The external ear is
visible on the right hand side of the head with a
band of cartilage (dark stain) within the auricle.
C. The gray box indicates this region: detail of inner
and middle ear development. The middle ear cavity
has not yet formed and the ossicles (malleus shown)
are embedded in mesenchyme that is being lost. The
tensor tympani muscle is differentiating in the adjacent
mesenchyme. The inner ear membranous labyrinth has
formed its adult external structure. The section through
the turns of the cochlear duct shows the internal
cochlea structure is still underdeveloped; in contrast,
the balance region is more developed.
The Membranous Labyrinth

Fetal
Week 8.4

Human fetal cochlea basal turn week 8.4 Gestational Week GA 10.4 (Stain - Haematoxylin
Eosin)[10]

Week 10

Human fetal cochlea basal turn week 10 Gestational Week GA 12 (Stain - Haematoxylin
Eosin)[10]

Week 18 - 22
Developmental data from an EM study of the human organ of corti.[11] Original GA data has
been recalculated as fertilisation ages.
WeekEvent
18 stria vascularis, tectorial membrane, afferent nerve ending
20 Nuel space, stereocilia maturation, efferent nerve ending
22 tunnel of corti, kinocilium, space between hair and supporting cell
Week 18 Week 20 Week 22
tunnel of corti - GA 24
Nuel space - 20 weeks (GA 22
stria vascularis - 18-20 weeks week
(GA 20 week 3 days - 22 week 0 days - 24 week) Space between hair and
week) Efferent nerve ending - 20 weeks
supporting cell - 24 week
tectorial membrane - 18 (GA 22 week 0 days) Presence of kinocilium -
weeks (GA 20 week 3 days) Maturation of stereocilia - 20 weeks
GA 24 week 0 days
Afferent nerve ending - 18 (GA 22 week 0 days - 24 week 0
 weeks (GA 20 week 3 days) days)

Historic
A series of historic wax-plate reconstructions of the membranous labyrinth and the
surrounding periotic tissue-spaces showing development stages (median and lateral views)
of these spaces at the same scale (ages are only approximations calculated on CRL).
Human Fetal Membranous Labyrinth
Development
Fetus 50 mm CRL 10 weeks (GA 12)

lateral view median view

Fetus 85 mm CRL 14 weeks (GA 16)
lateral view median view
Fetus 130 mm CRL 15 weeks (GA 17)

lateral view median view

Links: Growth of the Otic Capsule (1918) | Fetal CRL | Fetal Development

Fetal Cochlea Molecular

Human fetal cochlea basal turn by Gestational Week GA[10]

Abbreviations
SGN - spiral ganglion neuron
IHC - inner hair cell
O1 - first row of outer hair cells
O2 - second row of outer hair cells
Gestational Week GA
Week 10 (W10) - SOX2
identifies the
prosensory domain
within the
SOX9/SOX10+
cochlear duct
epithelium. Neurites
from the adjoining
TUBB3+/PRPH+SGNs
do not yet penetrate
into the epithelium.
Week 11 (W11) -
Penetration starts prior
to hair cell
differentiation.
Week 12 (W12) - The
first MYO7A+/SOX9-
/SOX10-/SOX2+ (inner)
hair cell can be seen,
and is contacted by
multiple TUBB3+ and
PRPH+neurites.
Penetrating neurites
are also found at the
location of the future
OHCs.
Week 14 (W14) - Both
the IHCs and OHCs
have differentiated, and
neurites underneath the
OHCs start to run in a
 O3 - third row of outer hair cells spiral direction. At this
OHC - outer hair cell. stage, hair cells still
express SOX2.

Week 20 (W20) - SOX2

is downregulated in all
hair cells, as opposed
to the other cells in the
organ of Corti. PRPH
expression
distinguishes between
type I (PRPH-) and
type II (PRPH+)
neurites.

Organ of Corti
Within the cochlea, the specialised structure required for converting mechanical vibration
into an electrical signal occurs at the organ of corti.
The images (mouse) below show the detail of the specialised structure, the organ of
Corti, that develops through the fetal period.
 Endolymphatic Sac
the adult endolymphatic
sac is filled with
endolymphatic fluid
with a unique
composition of high
potassium and low
sodium ions
it is not known in
humans at what stage
of development this
ionic status is achieved
In the rat, adult sodium
levels are seen in the
first week after birth,
while both potassium
and chloride levels
were below the normal
adult levels.

Vestibular Sac
generates 3 expansions - form semicircular ducts
remainder forms utricle
epithelia lining generates - hair cells, ampullary cristae, utricular macula
Vestibular - Otoconia, otoconin- inner ear biominerals

Cochlear Sac
Cochlear Sac[4]

generates coiled cochlear duct (humans 2 1/2 turns)

remainder forms saccule
epithelia lining generates
hair cells
structures of organ of corti
saccular macula

Scala Media

three spiral passages of cochlea

(Latin, medius = middle) spiral of middle cochlear duct lying between scala vestibuli and
scala tympani, containing endolymph.

Scala Tympani
(Latin, tympanon = drum) the spiralling cochlear duct below spiral lamina,
containing perilymph and ending at round window near tympanic membrane.
Scala Vestibuli
(Latin, vestibulum = cavity at beginning of canal) the spiralling cochlear duct above spiral
lamina, containing perilymph, beginning near the vestbule and ending where it
communicates with the scala tympani at the helicotrema.

Endolymph

extracellular fluid secreted by the stria vascularis.

potassium is the main cation required for depolarizing electrical current in the hair cells.

Perilymph

extracellular fluid similar in composition to either plasma or cerebrospinal fluid.

sodium is the main cation.

Inner ear hair cells

Stria Vascularis
Anatomical (upper half) and compartmental
In the adult the stria vascularis functions to
synthesise and secretes endolymph.
The three main cell types within the stria
that have different embryological origins
and are connected by different forms of
cell junctions.
stria vascular histology
(lower half) model of the adult stria
vascularis showing the three cellular
layers and depicting the location of
potassium regulating channels. The stria
vascularis is electrochemically isolated
from neighboring structures by tight
junctions (black bars).[4]
 stria vascular 1

stria vascular 2

stria vascular 3

vascularis development
Marginal cells Intermediate cells Basal cells
line the lumen of the melanocyte-like cells lie between the mesenchymal spiral
cochlear duct marginal and basal cell layers ligament fibrocytes
derived from derived from otic
derived from the neural crest.
epithelia. mesenchyme.

Mouse SEM
The gallery below shows scanning electron micrographs of the developing mouse (E18.5)
cochlea.[13]


Cochlea overview E18.5

Base region

Mid-base and Apex region

Mid-base region

Adult Cochlea
Adult inner ear (Max Brdel 1934)
Magnetic Resonance Images of the Adult
Cochlea[14]
The investigators used a method to obtain magnetic resonance imaging (MRI) to measure
cochlear length from the temporal bones of 6 cadavers. By overlapping digitalized rulers on these
images it was possible to measure cochlear length. Adult cochlear length varied between 17 and
26.5 millimeters.[14]

Mouse cochlea (SEM) showing organization of the hair cells.[15]

Mouse Cochlea Links: Cochlea overview SEM | Base region SEM | Mid-base and Apex
region SEM | Mid-base region SEM | Mid-base hair cells SEM | Mouse Development

Bony Labyrinth
formed from chrondified mesoderm
Periotic Capsule
mesenchyme within capsule degenerates to form space filled with perilymph

Auditory Neural Pathway

Hearing sound localization circuits brainstem

Vestibulocochlear Nerve

Adult cochlea nerve glia cartoon[5]

Afferent (sensory) cranial nerve brainstem primary terminal nuclei

forms beside otocyst

from wall of otocyst and neural crest cells
bipolar neurons

Vestibular Neurons

outer end of internal acoustic meatus

innervate hair cells in membranous labyrinth
axons project to brain stem and synapse in vestibular nucleus

Cochlear Neurons

cell bodies lie in modiolus

central pillar of cochlear
innervate hair cells of spiral organ
axons project to cochlear nucleus

Links: Hearing - Neural Pathway

Cochlea Glial

Cochlea glial lineage [5] Adult cochlea nerve glia cartoon[5]

Links: Hearing - Neural Pathway

Inner Ear Genes

hindbrain segmentation occurs at same time placode arises
otocyst adjacent to rhombomere 5
may influence development
Hoxa1, kreisler, Fgf3
genes regulating neural crest cells (neural genes)
Pax2 Ko affects cochlear and spiral ganglion, but not vestibular apparatus
nerogenin 1 affects both ganglia
LIN28B - RNA-binding protein times auditory prosensory cell cycle withdrawal and
differentiation through both let-7dependent and independent mechanisms.[16]

Semicircular canal
Otx1- cochlear and vestibular normal

Hmx3, Prx1, Prx2

Sensory Organs

thyroid hormone receptor beta

Zebrafish-mindbomb mutant has excess hair cells but not supporting cells, Notch-
Delta signaling

Gene Expression-inner ear

 Brn-3c and Hair cell development
Supporting Cells- p27kip
Thyroid Hormone
Ganglion neurons require growth factors
vestibular neurons- BDNF, NT3
survival not development

Sox9 20346939 Sox2 20071536

Other Species Overview

Comparison of zebrafish and late stage chick and mouse

embryos[17]
Zebrafish Development Chicken Development | Mouse
Development

(a) Formation of
the pre-
placodal
 region (PPR),
otic placode
and otocyst
(otic vesicle)
from cranial
ectoderm.
The otocyst is
the source of
nearly all cell
types of the
mature ear.
(b) Otic
neurogenesis:
neuroblasts are
specified from
otic vesicle
epithelium, but
delaminate from
it and accumulate
beneath the ear
in a transit
amplifying
population (light
blue). Neurons
(dark blue)
differentiate from
this population,
and innervate
sensory hair cells
in the overlying
otic epithelium.
The ganglion
develops in close
association with
neural crest cells
(green), which
give rise to glia.
(c) Early otolith
formation in the
zebrafish otic
vesicle. At least
three distinct
populations of
cilia can be
distinguished:
immotile hair cell
kinocilia (red),
which tether the
otolith at early
stages; motile
cilia (blue) in the
vicinity of the
sensory hair
cells, which do
not bind otolithic
material, and
shorter immotile
cilia (green).
(d) Schematic
comparison of
semicircular
canal formation in
the zebrafish ear
(top row) and a
generalised
amniote ear
(bottom row). A
single canal is
illustrated for
clarity. Epithelia
adhere at a
fusion plate, from
which cells are
cleared to make
the duct. The end
result of both
events is the
same (right hand
image), but the
fusion plate is
much smaller in
the zebrafish.
(e) Comparative
sketches of inner
ears from adult
zebrafish and late
stage chick and
mouse embryos.
Sensory (red),
neuronal (blue)
and endolymph-
regulating
(yellow) cells are
shown for the
mouse ear.

Abbreviations: A
, ampulla; BP,
basilar papilla;
HC, hair cell; L,
lagena; LM,
lagenar macula;
MN, maturing
neurons; NB,
neuroblasts;
NCC, neural crest
 cells; NP, neural
plate; Nt,
notochord; ooC,
organ of Corti;
Ot, otolith; OV,
otic vesicle; PPR,
preplacodal
region; S,
saccule; SVG,
spiral and
vestibular
ganglion; TA,
transit amplifying
population of
neuroblasts; U,
utricle.
(text from original
figure legend)

Mouse

Dual embryonic origin of the mammalian otic vesicle forming the inner
ear[18] "The inner ear and cochleovestibular ganglion (CVG) derive from a specialized
region of head ectoderm termed the otic placode. During embryogenesis, the otic
placode invaginates into the head to form the otic vesicle (OV), the primordium of
the inner ear and CVG. Non-autonomous cell signaling from the hindbrain to the OV
is required for inner ear morphogenesis and neurogenesis. In this study, we show
that neuroepithelial cells (NECs), including neural crest cells (NCCs), can contribute
directly to the OV from the neural tube. ...This study defines a dual cellular origin of
the inner ear from sensory placode ectoderm and NECs, and changes the current
paradigm of inner ear neurosensory development."