The ear is a highly sophisticated structure that compartmentalizes into three parts: the external, middle, and inner ear. The external ear functions to direct sound towards the tympanic membrane and consists of the auricle and the external auditory canal. The tympanic membrane forms the boundary between the external and middle ear. It is the point at which energy from sound waves becomes converted into mechanical energy that travels through the ossicular chain. The middle ear cavity is home to the three auditory ossicles, the malleus, incus, and stapes, all of which serve to transmit vibrations towards the inner ear. Together, the external and middle ear serves to amplify and transmit sound from the environment towards the inner ear. The inner ear houses the organs of hearing and balance and arises from a pair of transient ectodermal thickenings that form during the fourth week of development. The cochlea, semicircular canals, and otolith organs develop appropriately in time and space under the direction of essential morphogens, genes, and transcription factors that regionalize neurogenic activity and establish the axial identity of the ear.

External Ear

The external auditory meatus arises from the first pharyngeal cleft. It begins as an invagination of ectoderm between the first and second pharyngeal arches that extend inwards towards developing middle ear structures. At week 5 of embryonic development, this ectodermal diverticulum extends towards the pharynx and contains proliferating ectodermal cells that form a meatal plug that fills the entire lumen. At ten weeks, the bottom of the meatal plug expands circumferentially to create a disk-like structure. By the thirteenth week, this disk-like plug comes into contact with the primordial malleus medially, contributing to the future formation of the tympanic membrane. By the fifteenth week, the disk-like plug splits, leaving behind a thin ectodermal cell layer of the immature tympanic membrane. A continuation of the thin skin of the pinna lines the entire external auditory meatus and also the outer surface of the tympanic membrane. The external auditory meatus is completely patent and expands to its complete form by the eighteenth week.

By the end of the 4th week of development, the auricle develops from 6 mesenchymal proliferations/swellings known as hillocks derived from the first and second pharyngeal arches that surround the first pharyngeal cleft.  There are three auricular hillocks on each side of the external meatus that eventually fuse to form the auricle.  The first three auricular hillocks emerge from the first pharyngeal arch and give rise to the tragus, helix, and cymba concha. The last three auricular hillocks arise from the second pharyngeal arch and give rise to the concha, antihelix, and antitragus.  The external ears begin their embryological development in the lower neck region and gradually ascend posterolaterally to the level of the eyes as the mandible develops.[1]

Middle Ear

The tympanic cavity and Eustachian tube originate from an extension of the endoderm of the first pharyngeal pouch called the tubotympanic recess. During week 5 of development, the tubotympanic recess extends laterally until it reaches the floor of the first pharyngeal cleft. The endoderm of the tubotympanic recess and the ectoderm of the first pharyngeal cleft are adjacent to one another at this point, with a fibrous layer derived from mesenchyme called the lamina propria sandwiched in between. Resulting in the formation of a trilaminar tympanic membrane made up of three separate germ layers consisting of ectoderm, mesoderm, and endoderm. The dorsal portion of the tubotympanic recess expands to form the tympanic cavity and is filled with loose mesenchymal tissue, while the ventral portion develops into the Eustachian tube. Anatomically, the tympanic cavity divides into upper (attic) and lower (atrium) chambers that surround the ossicles and other structures of the middle ear. The tympanic cavity is considered an expansion of the pharynx and is lined by pharyngeal endoderm epithelium that expands to line the mastoid antrum as well. The tympanic cavity connects to the oral cavity via the Eustachian tube, whose function is to ventilate and drain the tympanic cavity. At birth, the Eustachian tube is more horizontal, shorter, and narrow than in adults and is a major reason infants have recurrent ear infections. The Eustachian tube demonstrates the most growth during weeks 16 to 28 of the fetal period. [2]

Middle Ear Ossicles and Muscles

At the 6th week of development, a condensation of mesenchyme at the dorsal end of the tubotympanic recess appears that gives rise to the middle ear ossicles. The cartilage origin of the three middle ear ossicles arises from neural crest-derived mesenchyme of the first and second pharyngeal arches. The malleus and incus develop from Meckel's cartilage of the first pharyngeal arch, while the stapes arises from Reichert's cartilage of the second pharyngeal arch. The early stages of ossicle development occur within the mesenchyme of the first two pharyngeal arches until the 8th month of development. As the tympanic cavity develops, the cartilages ossify via the process of endochondral ossification, which continues throughout the entire fetal period. During the 8th and 9th months of fetal life, the mesenchyme holding the ossicles in place undergoes resorption via programmed cell death resulting in an air-filled tympanic cavity at birth. 

In addition to skeletal structures, the tympanic cavity is also home to 2 of the smallest muscles in the human body, the tensor tympani and stapedius muscles. The middle ear muscles serve to protect the inner ear by attenuating vibrations of the malleus and stapes in response to loud and damaging noises. The tensor tympani muscle attaches to the handle of the malleus and reflexively retracts the malleus away from the tympanic membrane to reduce vibrations of the eardrum. The stapedius muscle attaches to the neck of the stapes and also serves to dampen vibrations. The tensor tympani muscle originates from the mesoderm of the first pharyngeal arch and therefore receives innervation from the mandibular branch of the trigeminal nerve. The stapedius muscle originates from the mesoderm of the second pharyngeal arch and is innervated by the facial nerve.[3][4]

Inner Ear

The inner ear originates from the invagination of the otic placodes during the fourth week of development. The otic placodes are sensory placodes, which are a series of transiently thickened surface ectodermal patches that form pairs rostrocaudally in the head region during week 4 of development. Sensory placodes are involved in the development of special sensory systems like vision, olfaction, and hearing. The otic placodes are one of the first sensory placodes to form and contribute to the formation of the inner ear structures associated with hearing and balance. The otic placodes are located behind the second pharyngeal arch and give rise to the otic pits by invaginating into the mesenchyme adjacent to the rhombencephalon during the fourth week of development. Towards the end of the fourth week, the otic pits break off from the surface ectoderm to form a hollow piriform shaped structure lined with columnar epithelium called the otic vesicle. At this point, the otic vesicle lies beneath the surface ectoderm enveloped in the mesenchyme, forming the otic capsule. The statoacoustic ganglion also forms during the formation of the otic vesicle and splits into cochlear and vestibular portions. The otic vesicle differentiates to form all the components of the membranous labyrinth and ultimately gives rise to the inner ear structures associated with hearing and balance. As the otic vesicle develops into the membranous labyrinth, its epithelium undergoes variations in thickness and begins to distort. The otic vesicle divides into a dorsal utricular portion and ventral saccular portion, with the dorsal utricular portion giving rise to the vestibular system and the ventral saccular portion giving rise to inner ear structures involved in hearing. The ventral saccular portion develops into the cochlear duct and saccule. The dorsal utricular portion forms into the utricle, semicircular canals, and endolymphatic tube.[5][6]

Saccule, Cochlea, and Organ of Corti

In the 6th week of development, the ventral saccular component of the otic vesicle penetrates the surrounding mesenchyme in a spiraling fashion. It completes two and a half turns to form the cochlear duct by the end of the 8th week. At this point, the saccule connects to the utricle via the ductus reuniens and mesenchyme surrounds the entire cochlear duct. The mesenchyme surrounding the cochlear duct forms cartilage. During the tenth week of development, this cartilaginous shell undergoes vacuolization to create the two perilymphatic spaces of the cochlea, the scala vestibule, and the scala tympani. Two membranes separate the cochlear duct proper, which is also known as the scala media, from the scala tympani and scala vestibule. The basilar membrane demarcates the scala media from the scala tympani, while the vestibular membrane separates the scala media from the scala vestibule. Laterally, the cochlear duct is attached to the surrounding cartilage via a connective tissue structure called the spiral ligament. The Organ of Corti is located within the scala media of the cochlear duct and resides on the basilar membrane. The organ of Corti is composed of mechanosensory cells and supporting cells. The arrangement of the mechanosensory cells is as outer and inner hair cells along rows. The outer hair cells are separated by supporting cells and form three rows, while the inner hair cells form a single row. The tectorial membrane covers these mechanosensory hair cells and, in combination with each other, constitute the organ of Corti. Shifting of the tectorial membrane in response to endolymph fluid motion displaces the stereocilia of sensory hair cells. Stereocilia displacement results in the generation of impulses that transmit to the spiral ganglion and reach the central nervous system via the auditory fibers of the vestibulocochlear nerve. The capsular cartilage that surrounds the membranous labyrinth becomes ossified between 16 and 23 weeks gestation to form the true bony labyrinth.[7]

The Utricle and Semicircular Canals

The utricle and semicircular canals are the organs of balance and originate from the dorsal utricular portion of the otic vesicle. During the 6th week of development, three flattened outpouchings of epithelium extend from the dorsal utricular portion of the membranous labyrinth that eventually give rise to the semicircular canals. At the central portions of each pouch, the epithelium of the pouches two opposing walls approach each other to form a fusion plate.  These fusion plates ultimately degenerate via programmed cell death to shape three semicircular canals. One end of each of the semicircular canal dilates to form the crus ampullare, while the other end, the crus nonampullare, does not dilate. At this point, five crura enter the utricle, two without an ampulla and three with an ampulla. The dilated ampulla contains sensory hair cells that form a crest called the crista ampullaris. Similar sensory areas form in the walls of the saccule and utricle.  The crista ampullaris senses changes in angular acceleration and is the sensory organ of rotation. Impulses generated in the sensory cells of the crista ampullaris reach the brain via the vestibular fibers of the vestibulocochlear nerve.[8]

Endolymphatic Duct and Sac

On the 12th day of gestation, after otic vesicle formation, the otic vesicle elongates to form a tube-like structure called the endolymphatic appendage. Soon after that, an indenting groove demarcating a tubular diverticulum on the medial side of the endolymphatic appendage forms. This tubular diverticulum differentiates into the endolymphatic duct and sac and continues to grow until the age of four years old.[9]

The external ear is composed of the external ear canal and the auricle, both of which are lined with keratinized squamous epithelium. Tiny hairs and cerumen producing apocrine glands also line the external ear canal. The tympanic membrane has a trilaminar structure with contributions from all three germ layers and separates the external ear from the tympanic cavity. The outer epithelial layer of the tympanic membrane originates from the ectoderm of the first pharyngeal cleft. It is composed of keratinized stratified squamous epithelium that runs continuously with the surrounding skin. A fibrous connective tissue layer derived from mesoderm composed of collagen and elastic fibers called the lamina propria forms the middle layer of the tympanic membrane. The inner mucosal layer of the tympanic membrane is derived from the endoderm of the first pharyngeal pouch and covered with simple cuboidal epithelium that is continuous with the lining of the tympanic cavity. The utricle and saccule are otolith organs located in the vestibule that detect movement in different planes. The utricle and saccule consist of sensory areas called maculae comprised of supporting cells and hair cells covered in a gelatinous acellular matrix called the otolithic membrane. The otolithic membrane is embedded with calcium carbonate crystals called otoliths. The crista ampullaris of the semicircular ducts have a sensory epithelium similar to that of the macula, also consisting of hair cells and supporting cells. The hair cells of the cristae project into a gelatinous material called the cupula, which does not contain otoliths, and serve to detect rotational acceleration. The organ of Corti is located on the basilar membrane and consists of a variety of supporting cells and two groups of hair cells: inner hair cells and outer hair cells.  The inner hair cells account for approximately 95% of the sensory input into the auditory system and arrange in one line along the entire basilar membrane. The outer hair cells account for about 5% of sensory input and serve primarily as acoustical pre-amplifiers. The outer hair cells receive efferent input and contract when stimulated, resulting in amplified sound waves. The supporting cells include Hensen cells, Corti pillars, Deiters cells, and Claudius cells. The supporting cells play essential roles in the function and maintenance of the inner ear and primarily serve structural and homeostatic functions.[10]

Proper formation and axial positioning of the components of the ear occurs through complex reciprocal interactions between the epithelium and mesenchyme of the pharyngeal arches and hindbrain. These complex interactions involve a wide variety of essential genes, morphogens, and transcription factors that ultimately determine the fate of cells in the inner ear. Members of the Wnt, Sonic Hedgehog (SHH), and Fibroblast-Growth-Factor (FGF) families, in combination with retinoic acid signals, regulate transcription factor genes within the primordial inner ear to regionalize neurogenic activity and establish the axial identity of the ear.

Otic placode induction is dependent on Wnts and FGFs provided by the hindbrain and surrounding head mesenchyme. After induction, the otic placode continues to be influenced by signaling information from surrounding tissues that determine its positional identity along the dorsal-ventral, anterior-posterior, and medial-lateral axes. The anterior-posterior axis is the first axis to be specified. It requires retinoic acid, a key morphogen, to confer proper anterior and posterior identities of the inner ear. Somites express high levels of Raldh2, a retinoic acid synthesizing enzyme that serves as the primary source of retinoic acid for patterning the inner ear. Retinoic acid signaling results in proper anterior-posterior patterning of the inner ear and establishes the neural-sensory-competent domain (NSD) in the anterior otic cup. The neural-sensory-competent domain gives rise to neurons of the cochleovestibular ganglion, as well as prosensory cells of the inner ear that differentiate into supporting cells or sensory hair cells. Neurogenin1 is a proneural gene that encodes a basic helix-loop-helix region (bHCH) transcription factor and is one of the earliest molecular markers that determines the neurogenic fate of cells in the inner ear. The anterior portion of the NSD contains Ngn1-positive cells that ultimately leave the otic epithelium and coalesce to become neurons of the cochleovestibular ganglion. The remaining sensory epithelium of the NSD develops into supporting cells, sensory hair cells, and some nonsensory cells.[11][12][13]

Proper patterning of the inner ear dorsal-ventral axis involves the secretion of Wnts transcription factors from the dorsal hindbrain and the release of Sonic hedgehog from the notochord and ventral floor plate. The patterning of the medial-lateral axis of the inner ear has not been well studied and is thought to involve hindbrain signaling mediated by the transcription factor Gbx2 from the otic epithelium.

Sonic Hedgehog is not only imperative in determining the dorsal-ventral axis of the inner ear, but it is also responsible for regulating and determining auditory cell fates within the inner ear. Sonic Hedgehog is released from the notochord and ventral hindbrain and allows for proper cochlear duct and semicircular canal development. The mesenchyme encasing the developing inner ear is also essential for shaping the semicircular canals and cochlear duct into their final form and does so through both structural and molecular means. Although the mechanisms and molecules involved in the process of semicircular canal formation are largely unexplored, studies have implicated a variety of mesenchymal genes in canal formation, such as Prx and Pou3f4. Proper extension and outgrowth of the cochlear duct is dependent on sonic hedgehog secretion from the notochord, and the release of transcription factors called Tbx1 and Pou3f4 from the otic mesenchyme. Studies have shown that an absence of Pou3f4 or Tbx1 in the otic mesenchyme results in abnormal shortening or coiling of the cochlear duct.[14][15][16]