Ectodysplasin A (EDA), encoded by the EDA gene positioned in the X chromosome, is a member of the tumor necrosis factor (TNF) superfamily that contribute to cell death, proliferation or differentiation [1]. However, EDA is a unique member of the TNF ligand because of its limited sequence homology to other TNF-like molecules except for the conserved TNF motif [2]. The EDA gene governs the morphogenesis of various ectodermal organs such as the teeth, hairs, and mammary glands during prenatal development [3]. A literature survey revealed that, among several signaling pathways, the EDA pathway was the first pathway to be utilized for the stimulation of tooth modifications. EDA gene mutations are widely studied in X-linked hypohidrotic ectodermal dysplasia (XLHED) and anhidrotic/hypohidrotic ectodermal dysplasia (HED), which is the most common genetic disorder of ectodermal development in humans resulting in hypotrychosis, hypodontia, heat intolerance, dry skin and dry eyes, the susceptibility to airway infections and crusting of various secretions.

2. Function of EDA in Physiology and Pathology

EDA is expressed in various organs and tissues, including the heart, kidney, pancreas, brain, lung, liver, skeletal muscle, teeth, as well as the skin during both embryonic development and adulthood ^[4][34]. Ever since its discovery in 1996 by D. Schlessinger, numerous studies have determined the role of EDA in the development of ectodermal structures such as the teeth, hair and several exocrine glands including the sweat, mammary and meibomian glands ^[5][6][8,35]. Recently, the expression of the EDA/EDAR receptor system has been extensively studied in the ocular surface as it is a regulator of the ectodermal organs. A clinical phenotype associated with EDA gene mutation is X-linked hypohidrotic ectodermal dysplasia (XLHED), also named as anhidrotic/hypohidrotic ectodermal dysplasia (HED), which is the most common genetic disorder of ectodermal development in humans resulting in hypotrychosis, hypodontia, heat intolerance, dry skin, susceptibility to airways infections and crusting of various secretions ^[7][36]. In the ocular surface, the abnormal expression of EDA mostly resulted in dry eye disease; that is, the pathology of the lacrimal functional unit (lacrimal gland, cornea, conjunctiva, meibomian glands and so on).

3. The Homeostasis of Ocular Surface

The ocular surface is a complicated system, constituting the cornea, conjunctiva, meibomian glands, lacrimal glands and the neural network, which complement each other in maintaining the ocular surface homeostasis ^[8][54]. The cornea is the transparent and avascular tissue that serves as a mechanical barrier and refractive surface of the eye. In addition to the tear film, the corneal epithelium is the outermost layer constantly exposed to the external environment. Conjunctiva plays an important role in protecting the eye by producing mucin and the presence of immune cells ^[9][55]. The conjunctival epithelium acts as a barrier similar to the corneal epithelium. The lacrimal gland renders lubrication and protects the ocular surface by the secretion of tears consisting of water, electrolytes, lipocalin, lactoferrin and mucus. The function of the lacrimal glands is also necessary for the homeostasis of normal vision ^[10][56]. The meibomian glands are the largest sebaceous glands that secrete various lipids including cholesterol, cholesterol esters, wax esters, triglycerides, phospholipids, free cholesterol and free fatty acids. The meibum and aqueous tears make up the stratified structure of the tear film. During the blink reflex, the meibum, aqueous and mucin mix to form the tear film on the ocular surface ^[11][57]. The homeostasis of the ocular surface plays an important role in maintaining the health of the eye. The destruction of the homeostasis of the ocular surface results in a variety of diseases, such as meibomian gland dysfunction, corneal epithelium abnormalities, dry eye disease, and etc.

46. The Role of EDA in the Development of Ocular Surface

Most of the ocular surface tissues such as the meibomian gland, lacrimal gland and corneal epithelium, originate from the ectoderm ^[12][58]. As previously discussed, EDA is involved in the development of several ectodermal organs, including the teeth, hair and mammary glands ^[13][59]. Patients with defective EDA are also reported to have photophobia and a reduction in the lacrimal function ^{[14][15][16][17]}[60,61,62,63]. Kaercher et al. also observed that these patients presented with alterations in the meibomian glands irrespective of age, and corneal changes in some older patients. Other abnormalities, such as conjunctivitis, lacrimation and dry eye progressed gradually with age in these patients ^[18][64]. Similarly, in the naturally occurring animal model of XLHED, the integrity of the cornea, function of lacrimation and formation of the meibomian gland was destroyed ^[19][20][65,66]. The Tabby mice, a mice model generated by debilitating the EDA gene, also showed similar clinical characteristics seen in XLHED ^[21][22][67,68]. The mutation of EDA was elucidated to be the major cause of alternation of the meibomian gland in XLHED ^[21][22][67,68]. A further study found that the EDA-DKK4-Lrp6 axis plays a crucial role in the formation of the meibomian gland, and that EDA directly activates the major Wnt pathway modulator Dickkopf-4 (Dkk4) and its receptor Lrp6 during the meibomian gland’s induction ^[23][22]. During embryonic development of the lacrimal gland (LG) in mice, the EDA pathway is found to be active in both basal and supra-basal cell layers of the epithelial compartment ^[24][52]. However, Eda activity gradually decreased as development proceeded, and there were only a few positive cells in the lacrimal gland acinar domain of the 13-week-old mice ^[24][52]. The LG ductal and acinar compartment formation is not affected by the EDA pathway, while EDA is necessary for the terminal differentiation of LG cells and the secretory function of LG during development ^[24][52]. Compared to the wild-type mice, the terminal differentiation of cells was found to be altered in all of the LG compartments of EDA^−/− mice. Interestingly, the blinking rate remained consistently higher even in one-year-old EDA^−/− mice, indicating a long-term physiological defect of the ocular surface in EDA^−/− mutants. In addition to the Tabby mice, Takashi Kuramoto et al. generated an swh/swh rat model by inducing mutation of the Edar-associated death domain (Edaradd) gene, which showed a similar phenotype of meibomian gland and other ocular surface abnormalities in Tabby mice ^[25][69]. Collectively, the deficiency of EDA contributed to the deformation of the meibomian gland and the immaturity of the lacrimal gland.

54. The Role of EDA in Ocular Surface Homeostasis

54.1. Meibomian Gland

Meibomian gland dysfunction (MGD), a chronic abnormality, could induce dry eye, which affects the health and well-being of millions of people, with terminal obstruction and/or glandular secretion changes ^[26][72]. Mutation of the EDA gene induces the abnormal development of the meibomian gland in XLHED patients ^[27][73] and animal models of dog ^[20][66], mice ^[28][53] and rat ^[25][69]. Most of the EDA was contributed by the meibomian gland in the ocular surface ^[29][33]. The meibomian gland secretes EDA protein to the ocular surface, which in turn contributed to the health of the corneal and conjunctiva ^[29][33].

54.2. Lacrimal Gland

The LG secretes the aqueous layer of the tear film ^[30][74]. Although the EDA activity was observed to progressively decrease during development ^[24][52], the quantity and quality of tear production by the LG was dramatically alternated in progressive XLHED patients and animal models. The LG weight was increased in EDA^−/− mice compared with wild-type mice ^[24][52]. Moreover, the terminal differentiation of cells was found to be altered to an unmatured state in all the LG compartments including the epithelium of ducts and acinar, and myoepithelial cells in EDA^−/− mice ^[24][52]. Indeed, a proper terminal differentiation is crucial for physiological LG secretion ^[24][52]. The EDA pathway not only maintains appropriate cell differentiation but also mediates the expression of the protective secretory factors found in the tear film. Additionally, the growth factors and inflammatory cytokines such as growth differentiation factor 5 (Gdf5), C-X-C motif chemokine ligand 10 (CXCL10) known to be secreted in basal tears were downregulated in the EDA^−/− LG ^[24][31][52,71]. It is a remarkable fact that Gdf5 is involved in the inhibition of corneal epithelial cells’ proliferation ^[32][75], while CXCL10 is associated with dry eye ^[33][76]. Moreover, it is shown that EDA^−/− animals presented with delayed corneal wound healing ^[34][70], which could possibly be due to LG maturation defects, and the TGF-β1, FGF7 and HGF of the lacrimal gland showed abnormal expression during this process. Surprisingly, inhibiting EDA signaling in the LG epithelium seems to be part of a feedback loop between the cornea and LG, which allows the secretion of reflex tears supporting corneal wound healing ^[24][52]. Similar to the meibomian gland, the EDA could maintain the homeostasis of the LG and promote tear production to support the cornea and conjunctiva.

54.3. Cornea

The corneal epithelium weakly expresses EDA protein, whereas it significantly expresses EDAR ^[29][33]. Few researchers have reported that EDA signaling is inactive in the cornea during physiological and pathological conditions ^[24][52]. However, the corneal changes, such as corneal defect and keratitis was age dependent in patients and animal models with EDA mutation ^[17][18][24][52,63,64]. The corneal epithelial integrity was defective and the thickness was reduced in the early postnatal stage of EDA mutant Tabby mice, with the decrease in corneal epithelial proliferation and delayed corneal wound healing ^[29][33]. EDA-mutated Tabby mice also displayed significant inflammation of the ocular surface and corneal pannus during their adult stage ^[34][70]. Primarily, these defects were assumed to be induced by the alteration of the tear film lipid layer in MGD and the reduction in tear production by LG dysfunction ^[18][35][64,77]. More recently, researchers have concluded that these syndromes are a primary sign of XLHLED, i.e., EDA deficiency ^[36][78]. EDA contributes to the maintenance of the epithelial barrier function ^[34][70], with the upregulating of ZO-1 and claudin-1 expression through the activation of the sonic hedgehog signaling pathway ^[34][70].