Introduction

Pharyngeal pouches are endodermal out-pockets occurring between the pharyngeal arches in embryological development. Various transcription factors regulate the mechanical bending of the endodermal tube, resulting in pharyngeal pouch formation.[1] The pouches give rise to tissues responsible for the formation of the middle ear cavity and eustachian tube, palatine tonsils, thymus, parathyroid glands, and parafollicular cells of the thyroid. Malformations in the development of the pharyngeal pouches can cause DiGeorge syndrome, branchio-oto-renal (BOR) syndrome, cyst formation in the neck, and concerns related to the respective structures of individual pouches. Proper pharyngeal pouch formation is essential to individual pharyngeal arch separation and proper organismal development during embryogenesis.[2]

Development

Pharyngeal pouches emerge during the segmental phase of pharyngeal arch development, forming partitions between each pharyngeal arch.[2] The pharyngeal arches develop in the fourth week of embryological development in vertebrates and are composed of ectoderm externally, endoderm internally, and a mesoderm core containing both mesoderm and neural crest cells.[3] Segmentation of pharyngeal arches is a controlled process of outward folding of endoderm towards the ectoderm, regulated by various transcription factors. As embryogenesis continues, the lateral side of the pharynx evaginates, creating an out pocket between each arch, which forms the pharyngeal pouches. The medial side invaginates, forming the pharyngeal clefts. The formation of pharyngeal pouches starts anteriorly, in the most posterior region of the pharynx, and proceeds sequentially in a posterior direction. There are six pharyngeal pouches in mammals and birds. Humans have four pharyngeal pouches, as the fifth and sixth pharyngeal pouches are comprised within the fourth pharyngeal pouch. 

Pharyngeal pouch development is hypothetically independent of neural crest migration towards the endoderm.[4] Neural crest ablation experiments performed demonstrated endodermal pharyngeal pouch formation not being affected by neural crest ablation.[5][2] Expression of transcription factors within the endoderm drives out pocketing and pouch formation in between each pharyngeal arch.

The first pharyngeal pouch lies between arches one and two. The second pharyngeal pouch sits between the second and third pharyngeal arches. In the same fashion, the third pouch is in between the third and fourth arches. Finally, the fourth pharyngeal pouch lies between the fourth arch and the terminal, sixth, arch. The fifth pharyngeal arch disappears during embryonal development.[6] The endodermal out-pockets migrate towards midline as a final position in the adult following the completion of pouch formation. The resulting pouches give rise to the middle ear cavity, Eustachian tube, mastoid air cells, palatine tonsils, thymus, parathyroid, and parafollicular cells of the thyroid.[7] Each of these tissues develops from their respective pharyngeal pouch, or pouches, in the case of the parathyroid gland.

Cellular

Pharyngeal pouches form in distinct positions along the anteroposterior axis of the developing embryo. Assorted extracellular matrix proteins are involved in the formation and stabilization of pharyngeal pouches. Pouch formation is governed by regions of endoderm containing high amounts of actin fibers, directing pouch expansion towards ectoderm. 

Adaptor proteins stimulate signal transduction pathways within the endoderm, driving protein complex formation. A subtype of adaptor proteins from the Ripply family of genes, Ripply3, is critical for pouch formation.[8] The expression of Ripply3 activates extracellular matrix protein, Integrin Beta 1, to bind fibronectin. Fibronectin is an extracellular matrix protein that forms connections between the endoderm and ectoderm. Ripply3 accumulates in regions of focal adhesions facilitated by integrin to establish contact points between the endoderm and ectoderm.[9] Regions with high levels of expression of Ripply3 correlate to areas in which endodermal bending occurred, resulting in pouch formation.[8] In addition to fibronectin, actin bundles in the apical side of the epithelium induce bending of the endoderm, whereas the basal side relaxes and weakens, assisting in the formation of the pharyngeal pouch. After endodermal evagination occurs, the actin fibers in the pouches will form cables, regulating pouch expansion and morphogenesis.

Mutations in the Ripply3 gene result in loss of flexural strength of the epithelial sheet, producing defects in adhesion contacts between the two extracellular matrices.[8] As a result, embryos deficient in Ripply3 will not form pouches and instead form a single continuous layer of endodermal epithelium. 

Molecular Level

Many signaling molecules expressed in the endoderm contribute to pharyngeal pouch formation. An assortment of transcription factors discussed below contributes to the development of pharyngeal pouches and respective tissue derivatives.

Endodermal out pocketing is widely regulated by the expression of fibroblast growth factor (FGF), T-box transcription factor (TBX1), retinoic acid, and Wingless related integration site (Wnt) protein.[10][2] FGF expression is important in the segmentation of the endoderm into pouches as it stimulates clusters of endodermal cells to migrate laterally, forming the pharyngeal pouches.[10] Tbx1 encodes for a T-box protein, which is present in all four pouches, stimulating tissue and organ formation during embryogenesis. A zebrafish study with Tbx1 mutants illustrated defects in pouch formation and subsequent caudal arch agenesis.[11] The various clinical presentations in DiGeorge's syndrome are a result of a mutation in the Tbx1 transcription factor.[12] Over 90% of DiGeorge syndrome cases are a result of a monosomic deletion of chromosome 22q11.2. This deletion produces a mutation in the Tbox1 transcription factor, altering the metabolism of retinoic acid. This active form of vitamin A is necessary for caudal endodermal pouch segmentation.[2] Variations in retinoic acid metabolism result in the aortic arch and conotruncal anomalies in also observed in DiGeorge syndrome. This topic receives further discussion in the "Pathophysiology" section. The absence of vitamin A results in decreased Tbx1 expression, producing errors in pouch formation.[2] More recently, studies have identified the role of Wnt signaling as another driver of pharyngeal pouch formation and arch segmentation.[2] Wnt 11r destabilizes endodermal epithelium to promote lateralization of the pouch, forming the endoderm. Subsequently, wnt4a induces the rearrangement of pouch cells into bilayers, stabilizing adherens junctions. Specific transcription factors correlate with the development of each pouch and are detailed below. 

The first pharyngeal pouch is highly dominated by the expression of the TBX1 and eyes absent 1 (EYA1) and 4 (EYA4) genes. The TBX1 gene induces the development of the first pharyngeal pouch, forming the tympanic membrane. Mutations in the EYA4 and EYA1 genes cause partial conductive hearing loss. Mutations in EYA4 lead to an undersized eustachian tube. branchio-oto-renal syndrome, characterized by branchiogenic malformation resulting in cyst formation, hearing loss, and various renal anomalies, is a result of a mutation in the EYA1 gene.[2][13] Mutations in the eyes absent (EYA1) and SOX1 genes cause the varying combinations of branchial cysts, fistulas, and sinuses as well as inner, middle, and outer ear and renal anomalies [14]. BOR syndrome is discussed in greater detail in the "pathophysiology" section below. 

The function of the third and fourth pharyngeal pouches is modulated by the roles of Homeobox A3 (HOXA3), Glial cell missing 2 (GCM2), Forkhead box protein N1 (FOXN1), EYA1, TBX1, and Paired box 9 (PAX9) genes. HOXA3 genes organize the spatial identity of the developing embryo along the anterior-posterior axis. Mutations in this gene result in partial parathyroid and thymus agenesis. GCM 2 is the earliest marker of the parathyroid and a derivative of the third and fourth pharyngeal pouches. The FOXN1 gene is necessary for thymic epithelial cell differentiation into the cortical and medullary epithelium. EYA1 also influences thymus and parathyroid development. TBX1 and PAX9 are involved in the successful development of the thymus and parathyroid from the third and fourth pharyngeal pouches.[2] Collectively, these genes work together, forming the derivatives of the third and fourth pharyngeal pouches, respectively. 

Function

Pharyngeal pouches derivatives produce tissues necessary for hearing, calcium homeostasis, and adequate immune response. The first pharyngeal pouch develops into the middle ear cavity and the eustachian tube, which joins the tympanic cavity to the nasopharynx. The inner surface of the eustachian tube is covered by a mucosal layer of ciliated cells, supporting cells, secretory cells, and connective tissue. The ciliated cells in the eustachian tube allow for secretions from the middle ear cavity to enter and drain into the nasopharynx. The primary function of the eustachian tube is to equilibrate pressures between ambient air pressure and the middle ear by permitting entry of air into the middle ear cavity. Failure of ciliated cells leads to pathologies such as otitis media with effusions, causing conductive hearing loss. 

The second pharyngeal pouch develops into the palatine tonsils, a secondary lymphoid organ playing a role in protecting the body from pathogens passing through the pharynx.

The third pharyngeal pouch develops into the thymus and inferior portion of the parathyroid. The thymus is a primary lymphoid organ that supports the development and selection of T cells. Host T-cell immunity is attributable to the development of the third pharyngeal pouch. Positive selection of T-cells takes place in the cortex of the thymus. The medulla of the thymus is responsible for self-tolerance education in T cells. Failure in the development of this pouch results in severe immunodeficiency against viral and fungal pathogens.   

The fourth pharyngeal pouch is responsible for the development of the superior region of the parathyroid and the ultimobranchial bodies. Together, the third and fourth pharyngeal pouches play a crucial role in the homeostasis of calcium and phosphate via the function of the parathyroid gland. The ultimobranchial cells develop into the C cells of the thyroid gland, which produce calcitonin in response to increased serum calcium levels. The fifth and sixth pharyngeal pouches combine with the fourth pharyngeal pouch.  

Cardiac neural crest cells arising from the dorsal neural tube migrate to the third and fourth pharyngeal pouches.  The cardiac cells proliferate and integrate into the parenchyma that eventually forms the aortic arches and great vessels.

Testing

DiGeorge syndrome is diagnosed using chromosomal microarray analyses. This type of analysis can identify a heterozygous deletion in chromosome 22q11.2.[15]

Pathophysiology

The pathophysiology of pharyngeal pouch malformations covers a broad range of diagnoses due to various derivatives of each pouch. Most recognizable anomalies in pharyngeal pouch development causing DiGeorge syndrome, branchio-oto-renal syndrome, and congenital cysts are discussed below.

DiGeorge syndrome is a result of a 22q11.2 chromosomal microdeletion causing subsequent malformations in derivatives arising from the third and fourth pharyngeal pouches. DiGeorge syndrome is the most common microdeletion syndrome in humans, with a prevalence of one to every 2000 to 4000 newborns.[16] A myriad of clinical presentations characterizes this syndrome as a result of microdeletion heterogeneity on chromosome 22. The more standard disease presentations include cardiac defects, thymic hypoplasia, abnormal faces, cleft palate, and hypocalcemia.[12] The cardiac defects are a result of neural crest cell migration to the third and fourth pharyngeal pouches during embryonal development, allowing for the creation of tetralogy of Fallot, ventricular septal defect, truncus arteriosus, or an interrupted aortic arch.[15] About 25% of patients with DiGeorge syndrome present with aortic arch abnormalities. Characteristic facial features in individuals with DiGeorge's syndrome are micrognathia, low seat ears, small upper lip, and a smooth philtrum. Less common facial features include cleft palate and bifid uvula. Malformation of the third and fourth pharyngeal arches results in thymic and parathyroid hypoplasia. Hypoplasia of the parathyroid gland results in no parathyroid hormone production to regulate calcium homeostasis. In response to low serum calcium, parathyroid hormone is excreted from the gland acting on bone, intestines, and kidneys to increase serum calcium levels. The lack of parathyroid hormone function on the kidneys results in increased calcium resorption from the urine and phosphate retention. Thymic agenesis results in absent functioning T-cells, causing recurrent viral and fungal infections in infancy and early childhood.[17][18]  

Branchio-oto-renal syndrome is an autosomal dominant disease, producing cyst formation in branchial arch remnants, auricular malformations, and renal anomalies.[19] The EYA1 gene on chromosome band 8q13.3 is expressed in pharyngeal pouches as well as brachial clefts, explaining the anomalies occurring outside of the pharyngeal pouch derivatives. Branchial cleft malformations are responsible for the presentation of cysts, fistulas, or pits. Expression of EYA1 in pharyngeal pouch formation is responsible for middle ear anomalies contributing to cochlear anomalies and hearing loss in BOR syndrome.[20] Additionally, Eya1 is expressed in kidney mesenchyme resulting in renal abnormalities presenting from renal hypoplasia to renal agenesis. Less severe renal malformations can present as end-stage renal disease later on in life.

Congenital cysts arising from the development of the third and fourth pharyngeal arches can present in newborns. The cysts tend to be large and filled with fluid and air, displacing organs such as the trachea and esophagus anteriorly since their location is in the anterior neck. In some cases, cysts become adherent to organs such as the thyroid gland and, in other cases, abscesses form, compressing the airway creating respiratory distress.[21]

Clinical Significance

Proper pharyngeal pouch development ensures appropriate tissue formation in vital processes such as hearing, appropriate immunity, and regulation of calcium homeostasis. Pharyngeal pouch development also plays a critical role in the correct formation of pharyngeal arches. Failure of development from any of the pharyngeal pouches has clinical consequences contributing to syndromes such as DiGeorge syndrome and branchio-oto-renal syndrome, as well as additional pathologies.

Hearing loss is a common consequence of failure in the first pharyngeal pouch formation. Failure to form the middle ear cavity lining inhibits the formation of ciliated epithelial cells. Consequently, middle ear debris cannot be removed into the nasopharynx, resulting in recurrent otitis media infections with effusions. Malformation of the middle ear lining surrounding the ossicles results in decreased vibration transmission from the stapes to the oval window of the inner ear. The cochlear nerve will receive less stimulation resulting in decreased quality of hearing. Failure of eustachian tube development causes damage to fragile inner ear structures with changing atmospheric pressure causing progressive hearing loss. 

Errors in the second pharyngeal pouch formation affect palatine tonsil formation. Failure of palatine tonsil formation results in a lack of lymphoid tissue use to stimulate an immune response at the junction in the respiratory and gastrointestinal tracts. 

The thymus is an essential lymphoid organ in which T cell maturation and selection occurs, orchestrating the adaptive immune response in humans. The thymus enhances cell-mediated immunity with the production of T-cells that can generate an adequate immune response to various viral and fungal pathogens.

The parathyroid gland secretes parathyroid hormone in response to lower plasma calcium levels. Failure of gland development results in an inappropriate response by the body to low calcium levels. Parathyroid hormone acts on the intestines, kidneys, and bones to increase serum calcium. Abnormally low plasma calcium levels can present as numbness or tingling of the feet, hands, or lips, muscle cramps, decreased heart rate, facial twitching, weak nails, and individuals that are more prone to fracturing bones. 

Failure of the fourth pouch results in the impaired formation of the ultimobranchial bodies of the thyroid. These cells are responsible for calcitonin secretion in response to high serum calcium levels, downregulating osteoclast function, and calcium reabsorption in the kidneys. Failure of parafollicular cell development could result in high serum calcium levels. Hypercalcemia affects brain function resulting in lethargy, fatigue, and confusion causes gastrointestinal upset, and can induce cardiac arrhythmias. On EKG, hypercalcemia is observable by a shortened QT interval (<300ms).