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Citation for published version (APA):Mozaffari, M., Nash, R., & Tucker, A. (Accepted/In press). Anatomy and development of the mammalian externalauditory canal: Implications for understanding canal disease and deformity. Frontiers in Cell and DevelopmentalBiology.

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Download date: 21. Apr. 2022

Anatomy and development of the mammalian external auditory canal: 1

Implications for understanding canal disease and deformity 2

Mona Mozaffari1*, Robert Nash2, Abigail S. Tucker1 5

1Centre for Craniofacial and Regenerative Research, King’s College London, Floor 27, Guy’s 7

Hospital, London SE1 9RT 8

2Department of Paediatric Otolaryngology, Cochlear Implants, Great Ormond Street Hospital for 9

Children NHS Trust, London, UK 10

* Correspondence: 12

Corresponding Author 13

monireh.mozaffari@kcl.ac.uk 14

Key words: Hearing; Deafness; External Ear; Ear Canal; Congenital Aural Atresia 17

Anatomy and development of the external auditory canal

This is a provisional file, not the final typeset article

Abstract 25

The mammalian ear is made up of three parts (the outer, middle and inner ear), which work together 26

to transmit soundwaves into neuronal signals perceived by our auditory cortex as sound. This review 27

focuses on the often-neglected outer ear, specifically the external auditory meatus (EAM), or ear 28

canal. Within our complex hearing pathway, the ear canal is responsible for funnelling soundwaves 29

toward the tympanic membrane (ear drum) and into the middle ear, and as such is a physical link 30

between the tympanic membrane and the outside world. Unique anatomical adaptations, such as its 31

migrating epithelium and cerumen glands, equip the ear canal for its function as both a conduit and a 32

cul-de-sac. Defects in development, or later blockages in the canal, lead to congenital or acquired 33

conductive hearing loss. Recent studies have built on decades-old knowledge of ear canal 34

development and suggest a novel multi-stage, complex and integrated system of development, 35

helping to explain the mechanisms underlying congenital canal atresia and stenosis. Here we review 36

our current understanding of ear canal development; how this biological lumen is made; what 37

determines its location; and how its structure is maintained throughout life. Together this knowledge 38

allows clinical questions to be approached from a developmental biology perspective. 39

Running Title

Introduction 49

Hearing places us within our external environment, allowing us to experience a multi-dimensional 50

world, to listen and to communicate. Sound is essentially a series of pressure waves in our airborne 51

environment. That we can ‘hear’ these waveforms involves a complex array of neurophysiological 52

mechanisms which begin at the outer ear and end at the auditory cortex. The mammalian ear is a 53

crucial and fascinating sensory organ formed from the integration of three parts (Figure 1). The pinna 54

or auricle directs soundwaves into the external auditory Meatus (EAM), which then funnels sound 55

waves towards the ear drum or tympanic membrane (TM), causing it to displace and move the 56

ossicular chain of bones in the air-filled middle ear. The middle ear ossicles connect the TM to the 57

much smaller oval window of the inner ear. This intricate leverage mechanism corrects the 58

impedance mismatch between gas and liquid allowing airborne sound waves to move hair cells in the 59

fluid-filled cochlea, generating neural signals that are transmitted to the auditory cortex via the 60

cochlear nerve. 61

Hearing loss is most commonly caused due to pathology in the inner ear, referred to as sensorineural 63

hearing loss, which consequently receives the lion’s share of scientific research (1). However, there 64

are limited options for treating sensorineural hearing loss with many potential treatments in the 65

experimental phase (2). Currently, the clinician’s arsenal is best equipped to treat middle ear disease, 66

which consequently receives the lion’s share of clinical attention (1). This leaves the outer ear, which 67

is also indispensable for hearing, but is comparatively overlooked. Pathologies of the ear canal can 68

cause hearing loss, recurrent infection, and complications including cranial nerve palsy and 69

intracranial sepsis (3). Management of hearing loss in this part of the ear may be particularly 70

challenging, as the interaction between the anatomy and physiology of the canal, and the pathologies 71

that affect it, may complicate the two most commonly used techniques of hearing rehabilitation – 72

hearing aids and hearing restoration surgery. 73

In this review we focus in on the ear canal or EAM. We discuss the anatomy of the canal and the 75

epithelial dynamics involved in canal homeostasis. We then turn to the congenital and acquired 76

defects of the canal, and how they are currently treated, before investigating canal development. As 77

anatomical and molecular details are added to our understanding of ear development, compelling 78

questions are beginning to emerge. What factors determine the development of the ear canal’s 79

specialised epithelium? And how does the canal integrate with the middle ear to form a functioning 80

structure? 81

Ear canal anatomy and function 83

With certain exceptions, the EAM is similar across all mammals; a deep-set structure on either side 84

of the skull ending in an ear drum cul-de-sac medially and opening to a pinna laterally (Figure 1). In 85

adult humans, the EAM is typically 25mm in length and 8 mm in diameter (4). Rather than a 86

horizontal tube its shape is that of a soft sigmoid, tilted to face downwards and inwards as it projects 87

medially (5). This curved path varies between individuals (5). Finite element modelling of the ear 88

has highlighted the importance of a patent EAM in hearing higher frequency sound (which is crucial 89

for speech recognition) (4). Lower frequencies with wavelengths larger than the canal itself are not 90

affected by variations in canal diameter, however, as soundwaves concertina at higher frequency, 91

variations in canal dimension have a discernible effect on sound transmission. In particular, the 92

segment of canal closest to the TM plays an important part in high frequency sound transmission (4). 93

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Variation in this pretympanic area, namely a deeper pretympanic recess, therefore can influence 94

acquisition of high frequency sound, and has also been linked to increased susceptibility to chronic 95

otitis externa (5). Such biomechanical modelling at a patient-level would provide valuable 96

information in understanding stenotic canal disease and measuring treatment outcomes. 97

In contrast to the deep canal observed in most mammals, in sauropsids (reptiles and birds) and 99

amphibia, the ear canal is typically shallow or non-existent (Figure 2A,B). Indeed, we can consider a 100

deep canal to be a specific mammalian trait. It has been proposed that a cavitated middle ear and 101

tympanic membrane evolved multiple times in land vertebrates (tetrapods), therefore the deep 102

mammalian ear is not homologous to the shallow ear canal of other land vertebrates (6). A deep 103

canal is observed in both therian mammals (placentals and marsupials) and monotremes (egg laying 104

platypus and echidna), however monotremes do not have pinna, suggesting that this was a later 105

therian adaptation as monotremes diverged from other mammals relatively early in evolutionary time 106

(Figure 2C,F) (7). The deep canal means that the mammalian tympanic membrane is protected within 107

the head and therefore potentially at less risk of damage. Despite this, damage to the mammalian 108

tympanic membrane is fairly common, with perforations being caused by loud noises, bangs to the 109

head and from middle ear infections (8). A deep canal also provides a means for funnelling and 110

thereby focusing sound waves, as discussed above. 111

Depending on the position of the middle ear with respect to the exit position of the canal, the relative 113

length of the canal can vary across mammals. For example, in the platypus the ear canal exits high on 114

the head, next to the eye, so that both the eye and ear are not submerged while the animal is 115

swimming (7). This results in the development of a very long canal (Figure 2C). How the developing 116

ear canal reaches the middle ear, finding its way to varying exit sites in different species, remains a 117

largely unanswered question. 118

A deep-set lumen requires structural support. In humans, the inner two-thirds of the external auditory 120

meatus is bony (typically 16mm in length in the human adult) and the outer third is cartilaginous 121

(around 8mm in length), encasing the canal with a semi-rigid wall (9). How the mammalian ear canal 122

evolved remains an intriguing question. Did pinna cartilage elongate inwards to meet the bony canal 123

or did the cartilaginous canal evolve separately from the pinna? The latter appears more likely as 124

monotremes have no pinna but still have a cartilaginous support that forms around the developing 125

EAM (Figure 2E) (10). Cartilage support for the EAM, therefore, likely predates pinna evolution. 126

The newborn human ear canal is soft and rudimentary along its length (11). The osseous canal is not 128

evident during embryonic development so appears to develop postnatally (12). The specifics of its 129

development, however, remain vague. It is not yet known whether the tympanic ring, which is a 130

membranous bone, extends posterolaterally to take part in creation of the osseous canal, or whether 131

the osseous canal forms by endochondral ossification of an initially fibrocartilaginous scaffold. Both 132

scenarios are described in textbooks and articles, however there are as yet no published studies 133

demonstrating the process (9,11). The cartilaginous canal appears prior to and apparently 134

independent of the osseous canal (12). From around 15 gestational weeks, a chain of bar like 135

cartilages form inferiorly between the tragus and helix. The anterior cartilage of the tragus and the 136

posterior cartilage of the helix develop early and are consistent in shape; ring and plate-like 137

respectively (12). Rather than fibrocartilage, glial fibrillary acid protein (GFAP) positive elastic 138

Running Title

cartilage makes up the outer cartilaginous canal (12). The cartilages transform morphology via 139

intermediate circular and triangle shapes to become H-shaped cartilage wrapping their arms upwards 140

around the developing EAM epithelium (12). Interestingly, these bars of cartilage appear to develop 141

along a fascial condensation linking Reichert’s cartilage and the tragus (12). No evidence of a 142

corresponding fascial plane extending out from Meckel’s cartilage has been described that would 143

potentially precede a superior wall (Figure 1 insert). Unlike the anterior canal, the posterosuperior 144

canal is usually deficient of cartilage in the adult (9). There is significant individual variation in canal 145

shape, which is perhaps not surprising given the complex of irregular shaped cartilages that interlink 146

to form the cartilaginous ear canal (13). Clinically, this variability appears to be of little consequence. 147

Inevitable gaps between the jigsaw of connecting cartilages does however provide a likely 148

explanation for the aetiology of congenital auricular pseudocysts that can occur in multiples between 149

the perichondrium and the skin of the external ear, requiring drainage and pinna reconstruction (14). 150

The lining of the ear canal is continuous with skin covering the pinna laterally and the outer layer of 152

the tympanic membrane medially (15). Being an anatomical cul-de-sac ending at the TM, the ear 153

canal is lined with specialised skin capable of migrating, such that it can self-clean with the 154

epidermal layer sloughing off and moving outwards – as opposed to upwards in skin elsewhere 155

(16,17). A failure of this self-cleaning mechanism leads to the build-up of sloughed off keratinocytes 156

in the canal, causing hearing impairment from blockage and localised tissue damage from chronic 157

inflammation (18). Indeed, failure to remove ear canal skin is also observed in keratosis obturans and 158

ear canal cholesteatoma, which are uncommon but well recognised diseases of the external ear 159

discussed further below (18,19). 160

Experiments tracing the movement of ink-tattooed ear canal skin cells in mammals (including 162

humans) show these labelled cells moving laterally, out of the canal over time (16,20). This 163

movement occurs roughly at the rate of fingernail growth with anterior canal skin demonstrating the 164

fastest migration (16). Does this migratory pattern have an epicentre at the tympanic membrane? 165

Dated dye experiments suggest a radial pattern of epidermal cell migration spoking away from the 166

umbo, at the centre of the TM (21). More recent experiments using BrdU to chase and label 167

proliferating cells suggest a more involved and dynamic source of stem and progenitor cells that 168

migrate in different directions in different parts of the TM (22,23). Frumm et al., add further detail to 169

the migratory nature of the keratinocytes in the tympanic membrane’s outer layer. Using a 170

combination of lineage tracing, live imaging and single cell sequencing, they demonstrate that the 171

TM epidermis has distinct stem cell and committed progenitor regions, located close to supporting 172

mesenchyme (the manubrium and annulus respectively). The progeny of these cells migrate out 173

across the TM, maintaining a thin vibratory surface (24). 174

There are distinctions in the morphology of the skin that covers the bony canal versus the skin that 176

covers the cartilaginous canal in humans. The former is thin and flush against the canal wall bone, 177

whilst the latter is thickened with a spongey subcutaneous layer possessing modified sebaceous 178

glands called cerumen glands. The bony canal skin, does not possess ceruminous glands or hair 179

follicles (9). Our observations of human cranial skin verses ear canal skin development suggest the 180

latter develops precociously in relation to skin (development paper). This has interesting implications 181

for experimental comparisons drawn between ear canal skin and skin elsewhere, as well as clinical 182

implications, where skin from the limbs is used to reconstruct the canal lumen. If a different 183

Running Title

developmental timeline leads to different skin biology, transplanting skin into a reconstructed ear 184

canal may not be the ideal graft tissue. 185

The cerumen glands are responsible for the production of ear wax. Serous secretions and sebum from 187

the cerumen glands mix with sloughed keratinised squames. A natural lubricant, ear wax aids the 188

self-cleaning function of the ear canal, and is thought to have antimicrobial properties (25). 189

Interestingly, a little like tongue rolling ability, ear wax type is a dimorphic trait. Whether your wax 190

is wet or dry is determined by one single-nucleotide polymorphism (SNP) of a single gene 191

(ABCC11) (26). Patients with dry wax, which lacks oily components, are at higher risk of suffering 192

from cerumen impaction. Impacted ear wax is the single most common ear canal ailment to affect 193

patients and may require regular removal, especially in those using hearing aids (27). The human 194

ABCC11 gene has no orthologous gene in mammals, except for primates. In mice, cerumen 195

production is from a single large gland, the glandula ceruminosa, which opens into a single duct near 196

the TM (28). Layout of glands and secretion type is likely to be species specific and relate to size and 197

position of the canal. In mice, this gland is present from E15 (28). Human ceruminous glands are 198

noted at around 19 gestational weeks in published literature (29), although we see them even earlier 199

at 13 weeks (unpublished data from Tucker lab). It is likely that ceruminous glands evolved from 200

modified sebaceous glands, as like sebaceous glands these are holocrine glands, were the cells burst 201

to release their content (30). Sebaceous glands fail to develop normally in patients with hypohidrotic 202

ectodermal dysplasia (XLHED), suggesting involvement of the ectodysplasin (EDA) receptor 203

signalling pathway in the development of sebaceous glands (31). Similarly, Eda mutant mice have 204

defects in sebaceous glands, with addition of Eda stimulants able to rescue the defect (32). 205

Meibomian glands, another example of a modified sebaceous gland, also fail to develop in rodent 206

models carrying hypomorphic mutations in the Edar signalling pathway consistent with an XLHED 207

phenotype, with increased Edar signalling leading to larger meibomian glands (33). Recently Edar 208

has been shown to be expressed in developing rat cerumen glands, highlighting a conserved role for 209

this pathway in these holocrine glands (31). In addition to the Eda pathway, Wnt and hedgehog 210

signalling have been shown to play a role in skin sebaceous gland development and therefore may 211

have a role also in cerumen gland development(34,35). Further study into the developmental 212

mechanisms of these unique glands would certainly be revealing. 213

Development of the ear canal and how it reaches its target 215

The ear canal develops in two parts, the outer part or primary canal, and the inner part or meatal plug 216

(36) (Figure 3). At around Carnegie stage 17 (6 weeks post conception and equivalent to E12.0 in 217

mice), the primary canal begins to form either by the first cleft deepening or as a new invagination 218

within the 1st arch. Supporting the latter scenario is a recent study unexpectedly suggesting the 219

murine ear canal forms entirely within the first arch (37). This is unexpected because it had 220

previously been assumed that the ear canal forms within the first cleft (38). The meatal plug then 221

forms as a plug of cells adjacent to this cleft. (20). This plug of cells will continue to extend inwards 222

into the surrounding mesenchyme becoming a finger-like projection pointing to the developing 223

middle ear cavity (MEC) (Figure 3B) (39). Temporal and situational differences in cell proliferation 224

appear to influence the extension and directional growth of the meatal plug as it begins to develop 225

(36). Both human and mouse display a distinctive duality in EAM development: the outer, primary 226

canal (which will become the cartilaginous canal) develops differently to the inner, meatal plug 227

(which will become the osseous canal) (36). Initially open, the primary canal closes for some time 228

before opening again along with the meatal plug, to form one long lumen (36) [figure 3C,E]. This 229

closure has been linked to the removal of periderm (36). In mouse embryos, a superficial layer of 230

Running Title

periderm, denoted by keratin 8 staining, acts as a non-stick Teflon layer to keep the primary canal 231

open in early development (36). 232

Programmed cell death, or apoptosis, is a ubiquitous event in development. Its role in ear canal 234

development has been previously alluded to in experiments noting the emergence of TUNEL-positive 235

cells at E15.5 in mice with the surprising lack of them around the time of canal opening, up to 12 236

days postnatally(40). Inhibiting apoptosis in culture and staining K8 positive peridermal cells 237

demonstrated that it is the apoptotic death of the periderm that allows the primary canal to close at 238

around E15.5 in mice (36). Indeed, in Grhl3 null mice with deficient periderm, the primary canal 239

closed prematurely, providing a hopeful avenue for further research into the aetiopathology of CAA 240

(36). 241

The canal finally opens by the creation of a central lumen (Figure 3E). This occurs at around 16 243

weeks in human fetal samples, and is preceded by a precocious programme of differentiation, ahead 244

of developing skin elsewhere, and involves the apoptotic cell death of the cornified loricrin-positive 245

layer of squamous epithelium (Figure 3D). The complex and multistage development of the ear canal 246

may account for the variability, both in form and occurrence, of congenital aural atresia. 247

As with much of craniofacial development, the EAC is in essence an epithelial structure taking shape 249

in relation to, and influenced by, its surrounding structures. How does the developing ear canal find 250

its path to the middle ear cavity? Current knowledge tells us some detail about these relative relations 251

and signalling pathways, putting the tympanic ring in a lead role. When tympanic ring formation is 252

disrupted using retinoic acid, the EAC also fails to form (41). And when the tympanic ring is 253

duplicated in Hoxa2 null mutant mice, a duplication of the EAC is also noted, as the second arch 254

transforms to a first arch fate (42). In keeping with this phenotype, overexpression of Hoxa2 in the 255

first arch neural crest leads to a duplication of the pinna and loss of the EAM (37). Additionally, 256

Prx1, Gas1, Tshirt (Tsh) and Goosecoid (Gsc) have all been shown to cause EAC defects in the 257

presence of hypoplastic or absent tympanic rings (43–46). Whilst EAC defects are observed in mouse 258

mutants of Prx1 and Gas1, TSH and GSC have been corroborated in human genome wide sequencing 259

studies also (47). Interestingly, mutations in Gsc and Prx1 also cause hypomorphism of the malleal 260

manubrium - an essential link in sound transduction from outside world to cochlea. Experiments in 261

knock out mice as well as associative data corroborating human CT scans and intra-operative 262

findings, indicate an essential role for the EAC in the induction and proper positioning of the 263

manubrium within the eardrum ((15,48)). Not unlike an anatomical love triangle, these findings 264

indicate that formation of the EAM depends on formation of the tympanic ring. In turn formation of 265

the manubrium depends on formation of the EAM. 266

Congenital disease of the external auditory meatus 268

Congenital aural atresia (CAA) is a spectrum of defects affecting the EAM at birth. The EAM may 269

fail to form partially, completely or be narrowed. Occurring in around 1 in 10,000 to 20,000 live 270

births, CAA affects males more than females and is typically unilateral, involving the right side more 271

commonly than the left (49). Over the years, a number of systems for classifying CAA have been 272

proposed (50–52). More typically, clinicians group CAA patients into complete atresia (complete 273

obliteration of the canal usually in combination with a bony atretic plate), stenosis (in which there is 274

a continuous lumen to an at least partial tympanic membrane) or partial atresia (variable presence of 275

Running Title

a canal lumen lateral to an atretic plate) (Figure 4)(53). Complete atresia and stenosis are much more 276

common than partial atresia, which is rare (54). Severity of canal atresia has been linked with an 277

early arrest in EAM development, although this does not account for the striking difference in the 278

incidence of complete verses partial atresia (11,54). Recent studies described above, examining EAM 279

development at a range of timepoints and using molecular cues suggest a more involved aetiology at 280

play, which may, for example, begin to explain the surprising rarity of partial in comparison to 281

complete atresia (36). 282

Genetic basis of CAA 284

Congenital atresia is often associated with pinna and middle ear defects (55). Less frequently inner 285

ear deformities and deformities of the stapes footplate are also present (56). Associations with other 286

craniofacial abnormalities such as facial asymmetry and cleft lip/palate are also common (11). In 287

some patients, these craniofacial defects are part of syndromic conditions such as Treacher-Collins, 288

de Grouchy (chromosome 18q deletion) and Branchio-Oto-Renal syndrome (Figure 4D) (11). 289

Genetic analysis of syndromic patients can provide valuable genetic background to the aetiology of 290

both monogenic and syndromic CAA. For example, Genotype-phenotype mapping of chromosome 291

18q deletions (responsible for de Grouchy syndrome) has consistently shown the critical region for 292

CAA in these patients to be 18q22.3 (47). Further delineating critical regions have highlighted 293

candidate genes to investigate, with a loss of function mutation in the gene TSHZ1, known to be 294

essential for murine middle ear development, being responsible for CAA both in isolated cases and in 295

18q deletion syndrome (45) . A similar approach in a cohort of patients with the syndrome SAMS 296

(short stature, auditory atresia, mandibular hypoplasia, and skeletal abnormalities) has identified 297

mutations in the Goosecoid(GSC) gene as a causative factor (57). GSC is an important regulator of 298

neural crest/mesoderm cell fate and has been shown to modulate tympanic ring development (58). A 299

canal defect in these patients may therefore be secondary to a tympanic ring defect, assigning a key 300

signalling role to the tympanic ring (41). 301

Mutations in HOXA2 have been characterised in a number of families with microtia (59). Only one of 303

these families, however, were noted to have additional EAM defects, with shortened and stenotic 304

canals (59). Mouse studies analysing hypomorphic Hoxa2 mutants with 45% the normal level of 305

Hoxa2, and conditional mice, where loss was activated relatively late at E12.5, did not show any 306

defects other than stapes and pinna deformity respectively (37,41). This is consistent with 307

observations that Hoxa2 requirement in craniofacial development is temporal (60). It may be that low 308

Hoxa2 levels only effect EAM development in the early embryonic period by affecting arch fate, 309

converting second arch to a first arch fate. That Alasti et al., report stenotic and shorted canals rather 310

than completely atretic canals is interesting because it contradicts previous thinking that earlier 311

mutations in EAM development lead to more severe anatomical defects (11,54). It is likely that a 312

more complex molecular basis is at play. 313

Genes discussed thus far play an indirect role in ear canal development, presumably due to their 315

effect on the tympanic ring. Foxi3, in contrast, is likely involved in the development of the ear canal 316

epithelium itself, making it a particularly interesting gene in the study of outer ear embryology (61). 317

Famously identified in hairless dogs, the Foxi family of genes are expressed in the ectoderm and play 318

important roles in early arch patterning and thus early craniofacial development (62). To date, 319

mutations in Foxi3 have been implicated in ear canal defects in mice, dog and human, suggesting it 320

Running Title

plays a role in cases of canal atresia (61). What is currently known about the role of Foxi3 in EAC 321

development is limited and associational. Unfortunately, Foxi3 homozygous null mutants do not 322

survive postnatally, and heterozygous mutants do not show a phenotype. A conditional mouse model 323

would provide an intriguing avenue for further research, in particular into the mechanisms underlying 324

the initiation of a meatal plug in early EAM development. 325

Given that CAA is a phenotype observed in multiple syndromes, it is not surprising that multiple 327

genes have been associated with its aetiopathology. The exciting next step will be to harness this 328

information, using conditional knock outs or culture setups, to answer questions such as, what are the 329

signalling pathways allowing the tympanic ring, ear canal and manubrium to communicate? 330

Treatment of congenital ear canal disease 332

Dividing canal atresia into complete, partial and stenotic is important to the clinician as it maps 333

patients to key treatment considerations: hearing outcome, likelihood of cholesteatoma (erosive 334

keratin-filled cysts that form when EAM skin cannot migrate out as normal) and the feasibility of 335

achieving a self-cleaning ear should atresiaplasty be considered (63). 336

Aesthetics aside, hearing loss is the main problem in complete atresia, as the absence of a lumen 338

precludes the possibility of cholesteatoma forming. Despite significant advances in surgical 339

technique and patient selection, atresiaplasty remains a difficult operation with typically poor 340

outcomes and risk to the facial nerve, which is characteristically laterally placed, and therefore 341

unexpectedly closer to the surgeon’s drill (63). Surgical atresiaplasty predates the use of bone 342

anchored hearing aids (BAHAs), and it is still performed in healthcare settings with limited access to 343

audiological services (64). Complications include restenosis, recurrent keratinous debris build up, 344

poor hearing outcomes (in one study as many as 93% of patients still required a hearing aid post-op) 345

and facial palsy resulting from altered and variable anatomy of the facial nerve in CAA (65). Once 346

the neo-lumen is created, it is covered with skin grafts taken from elsewhere or pedicled from 347

postauricular skin. Restenosis is prevented by performing a wide canalplasty, although this may have 348

cosmetic implications. As there is no native canal skin to use, the neo-lumen has no capacity to clear 349

desquamated debris, requiring regular aural microsuction (66). Hearing outcomes are typically poor 350

despite the opening of the canal and patients may still need a hearing augmentation device as well. 351

Meatoplasty, where the stenotic canal is widened, is more frequently considered in cases of 352

congenital aural stenosis. These patients carry a much greater risk of cholesteatoma to the presence of 353

a narrow canal, which may inhibit epithelial migration(69). Furthermore, native canal skin is often 354

available for canal reconstruction reducing chances of post-operative complications, making surgery 355

a more straightforward decision(69) 356

Given the difficulties realting to surgical correction of ear canal atresia, bone conduction devices and 358

auditory implants are often considered instead of atresiaplasty (67). Bone conduction devices bypass 359

the outer and middle ear by transmitting sound vibrations to the cochlea via the skull. Their 360

connection to the skull may be temporary, using methods such as a headband or adhesive; or it may 361

involve the placement of an auditory implant (Figure 4E). Auditory implants can be classified into 362

percutaneous devices, active and passive transcutaneous devices, and middle ear implants. Patient 363

factors, middle ear anatomy and clinician preference determines which devices can be offered (68). 364

Running Title

The option of implantable hearing aids is attractive, however, they require near to normal middle ear 365

anatomy and thus are not suitable for many CAA patients.. 366

In reality, in Europe and the United States, the majority of patients receive bone anchored hearing 368

aids (64). Whilst these provide good hearing rehab, they are visible, can cause skin irritation and 369

infection and of course do not create a continuous hearing response (they are taken on and off), 370

which is particularly important for spontaneous learning in young patients (64). Therefore, a surgical 371

approach which reconstructs the ear canal with few complications and without the need for further 372

device-assisted hearing, is attractive. Recent research investigating epithelial dynamics in the 373

developing ear canal may provide new avenues to explore the biological basis of atresiaplasty 374

complications (36). For example, surgeons note the greater frequency of restenosis in the lateral 375

reconstructed canal, which is cartilage-lined (70). As the bony and cartilaginous parts of the canal 376

develop in different ways (primary canal verses an extending meatal plate), the development may 377

explain these differences in response after surgery. It would therefore be interesting to study whether 378

there is an inbuilt signalling difference in the outer and inner parts of the EAM that influences their 379

response to injury. A better understanding of EAM development would also further clarify the 380

variable anatomy in CAA, e.g. the route of the facial nerve, easing technical challenges faced by 381

surgeons. 382

Acquired disease of the external auditory meatus 384

Acquired disease of the EAM includes of course the breadth of traumatic, infective and rarely 385

neoplastic disease and its comprehensive review is beyond the scope of this review. These can all 386

lead to a post-inflammatory canal atresia, sometimes referred to as stenosing otitis externa (71). 387

Current treatment involves regular aural hygiene or meatoplasty to expand the canal opening. There 388

is experimental evidence in Guinea pigs for the use of Mitomycin C (an inhibitor of fibroblastic 389

activity) in preventing post-inflammatory canal stenosis (72). Human studies, however, have had 390

contradictory results and further controlled trials are needed to evaluate its potential role in 391

preventing restenosis (73,74). Here, we focus on ear canal diseases that may be further understood 392

through the lens of developmental biology. 393

Specific to the specialised biology of ear canal skin and its ability to migrate are two distinct 395

diseases: keratosis obturans (KO) and ear canal cholesteatoma. KO is a rare disease in which 396

squamous debris builds up in the ear canal, often leading to an expansion of canal diameter before 397

presenting with acute pain. Current treatment is regular microsuction to maintain a patent ear 398

canal(19). As with healthy patients, the TM of individuals diagnosed with KO can be labelled with 399

dye to observe the migration of the epidermal layer. One such study suggests that either delayed or 400

disrupted epidermal migration may be the cause of KO (75). External ear canal cholesteatoma 401

(EECC) is also rare and was for a long time grouped together with keratosis obturans as the same 402

disease (76). Whilst ear canal skin in KO is intact with layers of squames building up above it, in 403

EECC there are focal ulcerations in canal skin and the organisation of keratinous squames is random. 404

Importantly there is bony necrosis with focal areas of deep sequestered bone (76). As such, drawing a 405

diagnostic distinction is important, as whilst KO can be managed conservatively, EECC typically 406

requires surgical removal of all the cholesteatoma and reconstruction of the ear canal (76). The 407

aetiopathology of EECC remains uncertain with disordered epithelial migration and/or cerumen 408

gland dysfunction thought to play a role (77). Localised differences in epithelial cell migration in the 409

Running Title

tympanic membrane have recently been proposed to underly propensity to middle ear cholesteatoma 410

formation and may provide clues to the aetiology of canal cholesteatoma also (24). The underlying 411

pathology of another category of cholesteatoma, congenital cholesteatoma of the middle ear, also 412

remains controversial. Whilst current consensus rests with an epidermoid formation origin within the 413

middle ear, another proposed theory suggests that an errant developing EAC extending beyond the 414

tympanic ring and into the mastoid cavity may be at fault (78). What determines epithelial behaviour 415

in the ear canal, driving it to focal erosion in EECC rather than circumferential upward layering into 416

the lumen as in KO? Further understanding the epithelial dynamics that bestow EAM skin its 417

migratory potential and its regenerative capacity will certainly begin to answer this question. 418

Future Questions 420

Of the three-part mammalian ear, the external auditory canal receives arguably the least attention. 421

Although this has not always been the case. Until the mid-twentieth century and the advent of 422

hearing aids, an exaggerated external ear in the form of an ear trumpet was the most commonly used 423

hearing device, emphasising the valuable role of the external ear in hearing. Congenital disease of 424

the EAM is thankfully rare but crucially important. Infancy is an irretrievable period in which to 425

develop speech and language, which can determine the rest of a child’s life. Clarity and spontaneity 426

of hearing, as well as decibels gained, at this age is critical. Thus, current treatment options, BAHAs 427

or complication-prone canalplasties, leave room for improvement. Better understanding the 428

mechanisms underlying EAM development will provide valuable knowledge towards improving 429

treatment options both for congenital canal atresia and also treatment of acquired canal disease that 430

involves epithelial dysfunction, such as keratosis obturans or canal cholesteatoma. 431

Recent research has pushed forward valuable but dated, largely histology-based, knowledge of EAC 433

development and created new questions. How exactly does the ear canal open along its length, 434

creating innermost a single-cell layer thin tympanic membrane, a thinly lined osseous canal and a 435

spongier outer cartilaginous canal? What determines the different behaviour of epithelium along the 436

canal postnatally? During development, how do the different part of the complex mammalian ear 437

communicate and eventually integrate? Answering these questions will pave the way to answering 438

the clinical quandaries that affect patients’ lives. How to create a self-cleaning ear? How to recreate 439

the external ear epithelium? Which pathways can be modified in the face of congenital ear disease? 440

Looking to developmental biology for the answers will be key. 441

Acknowledgements 443

Monotreme images were provided by the zoological museum in Cambridge University and the Hill 444

Collection at the Berlin Museum fur Naturkunde. Thanks to Neal Anthwal for discussions on the 445

monotreme ear. Thanks to Francis Smith for insightful conversations on syndromic ear canal 446

deformity. 447

Conflict of Interest 449

The authors declare that the research was conducted in the absence of any commercial or financial 450

relationships that could be construed as a potential conflict of interest. 451

Funding 453

Running Title

MM is supported through a clinical training fellowship award from the Medical Research Council 454

(MR/P019730/1). AST is funded through a Wellcome Trust Investigator award (102889/Z/13/Z). 455

Author Contributions 457

All authors read and contributed to the manuscript and discussion. MM was responsible for 458

conception and writing of the manuscript. AST contributed to the manuscript conception and 459

reviewed the manuscript throughout the writing process. RN provided clinical images and reviewed 460

clinical sections of the manuscript. 461

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Figure legends 654

Figure 1 656

Schematic diagram of the three-part ear; the external ear, which includes the pinna and ear canal; the 657

middle ear which includes the tympanic membrane (TM), middle ear cavity (MEC) and the ossicles. 658

The malleus inserts into the TM at the umbo. The middle ear connects to the nasopharynx via the 659

eustachian tube (ET). The inner ear (IE) includes the cochlea and semi-circular canals. The ear canal 660

Running Title

is enclosed in its inner two-thirds by a bony wall and its outer third by a cartilaginous wall, which is 661

lined by hair-bearing skin. The ear canal lumen varies in shape but typically has a pretympanic recess 662

and meatal opening. Inset shows anterior, posterior, superior and inferior axes as referred to in the 663

text. 664

Figure 2 666

Variations in the non-mammalian ear canal 667

(A) Bearded dragon (Pogona vivceps) with superficial tympanic membrane outlined by arrow. (B-E) 668

Histology sections (B) Sagittal trichrome section of gecko ear showing a shallow ear canal (star) and 669

superficial tympanic membrane (arrow). (C and D) Developing platypus (Ornithorhynchus anatinus) 670

EAM (C) Frontal section of platypus ear at 2 days post hatching (2 p.h.). The developing ear canal 671

(arrows) is long extending from the malleus to the top of the head. There is no pinna at the canal 672

opening (marked by *). (D) Frontal section of developing ear canal in 10 p.h. day platypus. The 673

EAM develops in a sheath of condensed mesenchyme (arrows). (E and F) Developing echidna 674

(Tachyglossus aculeatus) EAM (E) Horizontal section through the EAM of x p.h. echidna 675

demonstrating cartilage support around the developing EAM (arrows). (F) Horizontal trichrome 676

section through EAM of an echidna at 18 p.h. days showing a tortuous path to the opening (star). P, 677

Pinna; MEC, middle ear cavity; IE, inner ear, IE; EAM, external auditory meatus. 678

Figure 3 680

Schematic of mammalian ear canal development 681

(A) Invagination of the primary canal (marked by *) with a meatal plug at its tip. (B) Extension of the 682

meatus towards the forming middle ear, driven by a potential signal from the tympanic ring. At the 683

same time we see selective loss of periderm in the primary canal caused by apoptosis. (C) Closure of 684

the primary canal (following loss of periderm layer) and further extension of the 685

meatal plug to reach the tympanic ring. (D) Loricrin expression (red dashed line) marks the site of 686

opening of the canal. (E) Following keratinocyte differentiation, the whole external ear canal opens, 687

with the upper/rostral wall of the meatal plate forming the outer surface of the tympanic membrane. 688

Vertical black dashed lines between brown and blue regions represent the two distinct parts of the ear 689

canal. 690

Figure 4 692

Patient photograph and scans of congenital aural atresia 693

(A) Congenital aural stenosis. Coronal CT scan shows a normal, patent right ear canal and a stenotic 694

left ear canal marked by *. (B) Complete congenital aural atresia. Coronal CT scan shows a normal 695

ear canal on the right and an atretic plate on the left with no ear canal. (C) Congenital aural stenosis 696

and cholesteatoma. Axial CT scan of a patient with congenital aural stenosis and left-sided 697

cholesteatoma. The bony ear canal has been widened and remodelled by the collecting keratin 698

(marked by *). (D) Photograph of a patient with Treacher-Collins syndrome and complete aural 699

atresia of the left ear canal. The patient has been fitted with an osseointegrated hearing device visible 700

behind the pinna. (E) Example of an osseointegrated device. Computer-generated image of a BAHA 701

auditory meatus; IE, inner ear; MAC, mastoid air cells. 703

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