Pathophysiology of aniridia-associated keratopathy: developmental aspects and unanswered questions

The Ocular Surface 22 (2021) 245–266 Available online 11 September 2021 1542-0124/© 2021 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Pathophysiology of aniridia-associated keratopathy: Developmental aspects and unanswered questions L. Latta a , b , * , F.C. Figueiredo c , R. Ashery-Padan d , J.M. Collinson e , J. Daniels f , S. Ferrari g , N. Szentm´ ary a , S. Sol´ a h , R. Shalom-Feuerstein i , M. Lako j , S. Xapelli k , l , D. Aberdam m , n , ** , N. Lagali o , p , *** a Dr. Rolf. M. Schwiete Center for Limbal Stem Cell and Aniridia Research, Saarland University, Homburg, Saar, Germany b Department of Ophthalmology, Saarland University Medical Center, Homburg, Saar, Germany c Department of Ophthalmology, Royal Victoria Infirmary, Newcastle Upon Tyne, United Kingdom d Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel e School of Medicine, Medical Sciences and Nutrition, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom f Cells for Sight, UCL Institute of Ophthalmology, University College London, London, EC1V 9EL, UK g The Veneto Eye Bank Foundation, Venice, Italy h Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal i Department of Genetics and Developmental Biology, The Rappaport Faculty of Medicine and Research Institute, Technion - Israel Institute of Technology, Haifa, Israel j Biosciences Institute, Faculty of Medical Sciences, Newcastle University, Newcastle Upon Tyne, UK k Instituto Farmacologia e Neurociˆ encias, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal l Instituto de Medicina Molecular Jo˜ ao Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal m Centre de Recherche des Cordeliers, INSERM U1138, Team 17, France n Universit´ e de Paris, 75006, Paris, France o Department of Biomedical and Clinical Sciences, Link¨ oping University, Link¨ oping, Sweden p Department of Ophthalmology, Sørlandet Hospital Arendal, Arendal, Norway ARTICLE INFO Keywords: Aniridia-associated keratopathy (AAK) PAX6 Anterior chamber Development Limbal stem cells (LSC) Aniridia Aniridia animal and cellular models Clinical research Research strategies Limbal niche ABSTRACT Aniridia, a rare congenital disease, is often characterized by a progressive, pronounced limbal insufficiency and ocular surface pathology termed aniridia-associated keratopathy (AAK). Due to the characteristics of AAK and its bilateral nature, clinical management is challenging and complicated by the multiple coexisting ocular and systemic morbidities in aniridia. Although it is primarily assumed that AAK originates from a congenital limbal stem cell deficiency, in recent years AAK and its pathogenesis has been questioned in the light of new evidence and a refined understanding of ocular development and the biology of limbal stem cells (LSCs) and their niche. Here, by consolidating and comparing the latest clinical and preclinical evidence, we discuss key unanswered questions regarding ocular developmental aspects crucial to AAK. We also highlight hypotheses on the potential role of LSCs and the ocular surface microenvironment in AAK. The insights thus gained lead to a greater appreciation for the role of developmental and cellular processes in the emergence of AAK. They also highlight areas for future research to enable a deeper understanding of aniridia, and thereby the potential to develop new treatments for this rare but blinding ocular surface disease. 1. Introduction Aniridia is a rare, pan-ocular, bilateral and congenital disease affecting the normal development and function of almost all eye structures. Aniridia is inherited in an autosomal dominant manner, with high penetrance and yet with variable phenotypic expressivity even within the same family. A variable degree of iris hypoplasia or total absence of iris is the hallmark of the disease. Over 90% of cases of aniridia involve haploinsufficiency of the PAX6 gene, commonly due to heterozygous non-sense mutations on one copy of the gene. Well over 500 PAX6 * Corresponding author. Dr. Rolf. M. Schwiete Center for Limbal Stem Cell and Aniridia Research, Saarland University, Homburg, Saar, Germany. ** Corresponding author. Centre de Recherche des Cordeliers, INSERM U1138, Team 17. *** Corresponding author. Department of Biomedical and Clinical Sciences, Link¨ oping University, Link¨ oping, Sweden. E-mail addresses: [email protected] (L. Latta), [email protected] (D. Aberdam), [email protected] (N. Lagali). Contents lists available at ScienceDirect The Ocular Surface journal homepage: www.elsevier.com/locate/jtos https://doi.org/10.1016/j.jtos.2021.09.001 Received 10 April 2021; Received in revised form 19 July 2021; Accepted 8 September 2021 The Ocular Surface 22 (2021) 245–266 246 unique mutations have been identified in patients with familial or sporadic aniridia (http://LOVD.nl/PAX6) occurring in all exons and in noncoding regions of the gene. They include point mutations leading to amino acid substitution, deletions, insertions, premature termination, splicing defects or loss of the entire gene. Aniridia can occur either as an isolated malformation or as part of a syndrome such as WAGR (also known as WAGR complex, Wilms tumour-aniridia syndrome, aniridiaWilms tumour syndrome) caused by large deletions that affect both PAX6 and the adjacent WT1 gene. WAGR is characterized by Wilms tumour, Aniridia, Genitourinary anomalies and developmental delay (formerly ‘mental Retardation’) [1,2]. Aniridia may also occur in Gillespie syndrome, caused by mutation in a different gene, ITPR1, consisting of aniridia, cerebellar ataxia and intellectual impairment [3]. Although nearly all PAX6-aniridia patients develop progressive opacification of the cornea termed aniridia-associated-keratopathy (AAK), glaucoma and cataract, the underlying molecular mechanisms and physiological causes of the many pathological features of the disease have not yet been elucidated. A main reason for this is that ocular development, maintenance and regeneration involve crosstalk between different tissues, and complex interactions with the immune, nervous, and metabolic systems. AAK, also sometimes termed aniridia-related keratopathy (ARK) or aniridic keratopathy, is a highly prevalent condition in PAX6heterozygotes that is potentially painful and severely limits functional vision [4]. As the cornea is readily accessible and can be partly or fully replaced, AAK is a prime target for therapies to improve vision in aniridia. Even a small reduction in the severity of AAK can translate into significant benefits in vision and ocular surface symptoms. Moreover, AAK is progressive, so it is worthwhile to concentrate efforts into understanding its pathogenesis and pathophysiology, because the potential may exist for slowing, altering, or even arresting its progression in younger subjects. Symptoms of AAK include breakdown of the corneal surface, with epithelial thinning or loss, inflammation with immune cell infiltration, vascularization and chronic progressive opactification. These symptoms overlap partly or wholly with those that arise when the stem cells at the periphery of the cornea – limbal stem cells (LSCs) – are deficient due to disease or injury. Traditionally, it has been widely believed that AAK is a consequence of a progressive limbal stem cell deficiency (LSCD) [5,6], although to date, there is no definitive proof of loss or degradation of limbal stem cells (LSCs) or their niche as the causal event triggering AAK. Given the complexity of the pathology present in AAK [7], it is more likely that PAX6 controls multiple physiological and biological factors that act together, and that their dysregulation in aniridia leads to AAK development. Identifying the possible underlying pathogenic mechanisms leading to AAK development and progression holds the potential for establishing new therapeutic options for the single greatest unmet need of patients severely affected by aniridia. In this review, we highlight key questions of importance – many still unresolved – concerning developmental aspects and the emergence of AAK. We show that to regard AAK as purely a limbal stem cell deficiency is to ignore the multiple developmental and pathogenic events in aniridic eyes, affecting multiple tissues, that may contribute to the onset or progression of the disease. We highlight some potential therapeutic strategies that may arise from a fuller understanding of the developmental basis of AAK. The topics discussed are also relevant more generally to diverse types of environmental or congenital corneal pathologies that involve LSCD, corneal neovascularization and opacification. Finally, we present recent findings on translation of results from animal models to humans as a critical step in developing future therapies to treat AAK and understanding their relevance to other corneal diseases. 2. Are developmental deficiencies involved in the emergence of AAK? PAX6 1 codes for a key transcription factor that is essential for eye development and maintenance. PAX6 is expressed at the earliest stages of eye development and in multiple eye tissues throughout life. Several Pax6-heterozygous mutant murine strains (collectively ‘small eye’ or ‘Sey’ mice) have been characterized and used as in vivo models of aniridia, to study the roles of the gene and the developmental and pathological aspects of the disorder [8]. The mouse models represent a spectrum of mutations. Some such as the Harwell allele Pax6 Sey−H and ‘Dickies small eye’ Pax6 Sey−Dey are large deletions affecting Pax6 and surrounding genes, and show severe phenotypes that are perhaps not ideal for studying human aniridia and AAK [9–12]. Others, including Pax6 Sey (=Pax6 SeyMH ), Pax6 Sey−Neu (=Pax6 Sey−Neu1 ), Pax6 ADD4802 , Pax6 Coop , Pax6 Aey11 , Pax6 AEY18 and the allelic series Pax6 2Neu to Pax6 10Neu and Pax6Leca1 – 4 include point mutations leading to premature termination, frameshifts or splice defects [9,13–16]. There are also engineered deletions (Pax6 LacZ ) and floxed alleles (Pax6 flox ) that yield large deletions upon action of Cre recombinase [17–19]. With the exception of some hypomorph alleles (Pax6 4Neu , Pax6 7Neu and Pax6 Coop ) and a gain of function (Pax6 ADD4802 ), most alleles listed above are thought or known on the basis of nonsense-mediated RNA decay, phenotypes, and/or allelic complementation studies, to be null for Pax6. Unless stated otherwise, all Pax6 mouse mutants discussed below represent null alleles. Pax6-knockout results in early failure in lens placode development and anophthalmia (eye absence) but heterozygotes display a phenotype that resembles human aniridia, including a progressive AAK [14,28]. Although the phenotype of Pax6 mouse mutants is affected by genetic background and there is individual variation in severity even within litters, overall they are remarkably consistent models of AAK. Concordance with human aniridia, however, is not complete. For example, Pax6 +/− mice exhibit microphthalmia with a 10% reduction in eye diameter and reduced lens size; while microphthalmia can also occur in human aniridia, most patients have a relatively normal eye and lens size [20]. The extent to which microphthalmia in mice may modulate the anterior segment dysgenesis associated with Pax6 is not known, but it should be noted that the transgenic ‘PAX77’ mouse that overexpresses 5–6 copies of human PAX6 exhibits microphthalmia (including microcornea) without an AAK phenotype [21,22]. Typically, the Pax6 heterozygous newborn mice exhibit morphological alterations in the corneal epithelium. Separation of the lens from the cornea during development is delayed in Pax6-mutant mice and iridocorneal and/or lens-cornea adhesions similar to Peters’ anomaly may be observed at birth, dependent on mouse strain and the Pax6 allele [23–26]. The gross abnormalities these mice can exhibit at birth are small eyes and/or an opacity of the central cornea due to delayed or failed detachment of the lens from the cornea. In contrast to the murine models, the ocular surface of the majority of aniridia patients does not exhibit any apparent gross abnormality at birth. Exceptionally, rare cases of Peters anomaly are caused by PAX6 mutations [27] but in nearly all cases of congenital aniridia, a proper separation of the cornea and lens is observed [28]. Despite a seemingly normal cornea observed early in life, however, closer inspection of the central cornea in a 4-year-old children with congenital aniridia indicated reduced sensitivity to mechanical touch, reduced sub-basal nerve density and elevated presence of antigen-presenting dendritic cells [29]. Moreover, the corneal thickness is known to be pathologically increased in aniridia, even in childhood [29]. As outlined below, PAX6 is expressed in many eye structures during development (section 2.1). Therefore, it is probable that AAK is 1 Human nomenclature is used whenever applicable. If findings are speciesrelated the gene/protein nomenclature of the species is used according to the literature source. L. Latta et al. The Ocular Surface 22 (2021) 245–266 247 at least partly influenced by developmental defects and not solely due to postnatal emergence of pathological corneal function. AAK penetrance is full, but its phenotypic expressivity is highly variable between individuals, even between siblings [30]. This suggests that strong environmental and stochastic components, and/or modifier genes that act in concert with PAX6 and vary between individuals can influence the expression of the disease. This could also be true for epigenetic differences between individuals that may modulate AAK severity. It would moreover be important to understand how the different eye structures influence each other during corneal development, and the role of PAX6 levels in influencing the onset and severity of AAK. PAX6/Pax6, the mammalian orthologue of the Eyeless gene (Ey) in Drosophila, is a paired and homeodomain transcription factor which is essential for eye development. The activity of PAX6 in eye development is evolutionarily conserved as the human or mouse Pax6 genes can induce ectopic formation of the compound eye from Drosophila imaginal discs as well as in Xenopus embryos [31,32]. PAX6, however is also important for brain, gut and pancreas development [33–35]; see review [36]. This multiple organ involvement partially explains why haploinsufficiency of PAX6 causes in adults, not only aniridia phenotype, but also several non-ocular conditions such as obesity, glucose intolerance and diabetes, and anosmia [37,38]. Sleep disorders are also reported in patients and PAX6 may impact brain structures such as the pineal gland [39]. The importance of PAX6 for different structures of the eye is highlighted by the fact that aniridia patients suffer from multiple eye abnormalities in both anterior and posterior segments of the eye. PAX6 is expressed in the multiple ocular cell types from the earliest stages of eye development and throughout life (Figs. 1-3). The exact role of PAX6 in lens and optic cup derivatives has been systematically investigated using conditional mutagenesis (reviewed in Ref. [40]). By contrast, the impact of Pax6 on corneal development continues to be further explored, requiring efficient genetic deletion in the various corneal cell types [41]. 2.1. How is the anterior segment of the eye formed? In vertebrate eye development, Pax6 expression is detected in the anterior neural plate in the eye field region [42] and subsequently in both the neuroectoderm and surface ectoderm progenitors of the eye [18,33,42,43]. The surface ectoderm gives rise to the lens and corneal epithelial layers (lens, limbus and corneal epithelium), while the neuroectoderm populates the optic vesicles that undergo morphogenesis to form the optic cups. The outer layer of the optic cups is populated by the retinal pigmented epithelium progenitors (RPE), while the inner layers of the optic cup differentiate to form all of the retinal neurons and the Müller glia cells. The anterior optic cup rim gives rise to the iris and ciliary body pigmented epithelia Fig. 1). The ocular mesenchyme surrounding the optic cup rim eventually contributes to the iris and corneal stroma. The high and continuous expression of Pax6 in cells that derive from surface ectoderm and optic cup (lens, corneal epithelium, iris and ciliary epithelium) is required for the expression of genes encoding transcription factors, structural and signaling molecules, which are critical for the morphogenesis and differentiation of the neuronal, pigmented and the transparent cornea and lens eye lineages. The role of Fig. 1. Eye development and PAX6 expression in the mouse. The green box shows the developing murine eye at different stages. The color code marks the embryonic origin of these tissues. (Blue: surface ectodermal, Green: Neural ectodermal; Grey: Neural crest; Yellow: Mesodermal). The yellow Box below shows the relative PAX6 expression in these different structures. The neural ectoderm forms the optic cup as a double-layered structure and is important for lens placode development of the surface ectoderm. The distal tips of the optic cup will develop iris structures, and this coincides with very high PAX6 expression levels. The inner layer of the optic cup will form the neural retina the outer layer develops to retinal pigment epithelium. Upon separation of lens and surface ectoderm several waves of neural crest and mesodermal cells migrate into the anterior segment contributing to the corneal stroma, corneal endothelium, iris stroma and anterior chamber. Most of the data displayed derive from studies on murine development [19,33,43]. *Note that endothelium and stroma differentiation is displayed for mouse where endothelium and stroma differentiate from the same cell mass migrated in a first wave. However, it is unknown if endothelial cells are already specified prior migration [111]. A second migration wave later appears in the angle between future cornea and optic cup and differentiate into stroma of iris and ciliary body [47]. In human and birds three migration waves are observed. First endothelium is specified then mesenchyme migrates between epithelium and endothelium to differentiate to stroma and the third wave contributes to iris and ciliary body [46]. Graphics adapted from different sources [44,47,112]. L. Latta et al. The Ocular Surface 22 (2021) 245–266 248 Pax6 in each of these structures and the signaling cues that mediate their coordinated development during the formation of the anterior segment of the eye, as relevant to the onset of AAK, is briefly summarized in the next section. The anterior segment of the eye includes the cornea, conjunctiva anterior chamber, iris, lens and associated structures. In development of these structures, the neural and surface ectodermal cells interact with mesenchymal cells of neural crest and mesodermal origin. These various and complex interactions are briefly described below. For a detailed description of the associated processes, see reviews [44–47]. The putative influence of PAX6 dosage on these different processes in development of the corneal phenotype is discussed in separate sections. 2.1.1. Early development – surface ectoderm and optic vesicle Morphologically, the development of the eye is evident with the formation of the optic vesicles. The optic vesicles are PAX6-expressing bilateral evaginations from the diencephalon that give rise to the optic cup through morphogenesis (reviewed in Refs. [48,49]). The optic cup interacts with surface ectoderm, lens and migrating mesenchymal cells. Disruption of developmental processes has been described by manipulating signaling pathways. A saucer-shaped optic cup, ventral coloboma, or a deficiency of periocular mesenchyme were observed by manipulating either Wnt-, Lrp6-, or retinoic acid signaling [50]. The peripheral rim of the optic cup contain progenitors that will give rise, during post natal stages to the pigmented and non-pigmented layers of the iris and ciliary body (reviewed in Ref. [51]). Wnt ligands and BMP signaling from the surface ectoderm elicit Wnt/GSK3β-response in retinal pigment epithelium progenitors (RPE) and are crucial for production of the correct number of RPE cells and proper curvature of the optic cup [50,52]. 2.1.2. Lens As soon as the optic vesicle forms, in mice at E8.5, Pax6 is expressed in neuronal and surface ectoderm progenitors of the eye (See E8.5 Fig. 1). The lens derives from Pax6-expressing lens-competent facial ectoderm which is contacted by the optic vesicle [53]. After the lens placode has been induced, it invaginates to form the lens vesicle. The detailed mechanisms of lens induction and genetic and signaling networks are reviewed in Ref. [54]. In the lens and optic cup, Pax6 expression is specified independently, by cis-regulatory elements [40,54]. Pax6 autoregulates its own expression and the Pax6 surface ectodermal enhancer element driving PAX6 autoregulation interacts with another transcription factor, SOX2 [55]. Sox2 expression is induced in the surface ectoderm upon an inductive signal from the optic vesicle and determines lens placode formation (See E9.5 Fig. 1) [56]. The cells from the anterior pole of the lens vesicle give rise to the lens epithelial cells, whereas the posterior cells differentiate into lens fiber cells. PAX6 continues to be expressed during lens invagination and Fig. 2. Aligned developmental stages of critical events in anterior eye development in several species. * Embryonic days or weeks of gestation (WG). A1,2,3). Earliest expression of PAX6 in surface ectoderm and neuroectoderm is further observed and sustained in several emerging structures such as optic vesicle, optic cup developing lens and surface epithelium [33,43,113,114]. Note that given time points for each species showed the earliest observed expression in the cited studies. B) Neural crest migration is important for contributing to corneal structures such as endothelium and stroma as well as iris stroma. In mice two migration waves are observed and in avian and human there are three reported waves, although there are inconsistencies in the literature [44–47,111]. B1) In mice neural crest cells increase at E13 [115]. In a second wave, mesenchymal cells migrate and contribute to stroma of the iris (E15.5) [47]. B2) In humans the first wave is in the 7th WG as no endothelial cells can be observed before [116]. In birds, the stroma is built up by mesenchymal cells migrating between epithelium and endothelium around the 8th WG [117]. Although there are descriptions of anterior chamber angle development, note that the migration path of these mesenchymal cells has not been addressed in those studies [118,119]. B3) In chicken there are three waves of NCC. The first wave leads to formation of the endothelium [120]; the second wave develops into corneal keratocytes [120] and the third wave of NCC contributes to iris and mesodermal cells become distinguishable from endothelium at the seventh day [121]. C) The timing of limbus formation, specification and maturation at the molecular level is still enigmatic C1) Before keratin surface markers become distinguishable (See D), expression changes could lead to lineage segregation of corneal and conjunctival cells. In the rat, CX43 expression is lost at E12 and could explain a spatial separation of limbus and cornea epithelium [74]. C2) In the human, single cell analysis identified clusters of corneal, conjunctival and limbal cell lineages [70]. KRT15 starts to become restricted to limbal epithelium five weeks later at WG17 in humans [71]. At a similar timepoint, single cell analysis also identifies different stem cell and progenitor markers [70]. C3) In the chicken, a diffusion barrier is established in the limbal region before cell lines can be distinguished by cytokeratin expression changes. Interestingly, the limbal barrier is established prior to expression changes in CX43 [73]. D1,2) As apparent in the timeline, the changes in surface expression of keratins or integrins become clearer with further stratification. These processes occur much later than the first evidence of lineage segregation of conjunctiva, limbal and corneal epithelium (See Section 2.1.3). (Time points depicted in the graphics are obtained from cited literature. Stages are aligned with Carnie Stage comparison). Gestation Week (WG), E (Embryonic), P (Postnatal) [308]. L. Latta et al. The Ocular Surface 22 (2021) 245–266 249 differentiation [19,57] as reviewed by Cvekl A et al. [58] and is maintained in the lens epithelium throughout life. 2.1.3. Corneal stroma and endothelium After the lens, optic cup and ocular surface ectoderm have been specified, neural crest cells and mesodermal cells migrate between these structures. The corneal stroma is formed by neural crest-derived cells from the periocular mesenchyme, a population of mesenchymal cells located near the optic cup and presumptive lens. Fate mapping of mesoderm-derived cells showed a contribution to the corneal endothelium and stroma [59,60]. In humans (and birds), three waves of neural crest cells (NCCs) migration are reported in contrast to two waves in mice (Note that Fig. 1 refers to mouse development). A first wave of NCCs migrate between the lens and the surface ectoderm to form the corneal endothelium. A second wave forms the stromal keratocytes. The third wave of NCCs contributes to the ciliary body and iris structures (see Section 2.1.4). In mice a single wave of neural crest migration gives rise to both endothelium and stroma. Studies isolating primary stromal cells from transgenic mouse corneas revealed similarities in mRNA expression profiles (Twist, snail, Slug and Sox9) between neural crest-derived precursors and the isolated corneal precursor cells. This finding was regarded as evidence for a neural crest origin of these cells, which are important for the turnover of stromal tissue [61]. The corneal endothelium provides an important pump function, actively maintaining a fluid and electrolyte balance between the anterior chamber and corneal stroma to prevent corneal swelling, thus maintaining corneal transparency. The cellular density of this single endothelial cell layer is critical for maintaining an adequate pump function. From a histologic analysis of human fetuses, it has been shown that the cellularity of the endothelium rapidly decreases in the prenatal period from 16 weeks of gestation to term, at the same time the cornea grows in size [62]. The reduction in endothelial cell density during this period is about 50%, while the density reduces further by a third during the first two years of life. A putative effect of PAX6 levels on stromal, limbal stromal and endothelial development and how this could influence AAK is discussed in Section 2.6. 2.1.4. Iris and anterior chamber angle The anterior chamber angle is the angle between the iris and the corneal endothelium in the limbal region. The iridocorneal angle contains important aqueous humor drainage structures such as the trabecular meshwork and Schlemm’s canal. The contiguous iris and ciliary body epithelia derive from the rim of the optic cup while the stromal layers derive from the ocular mesenchyme [26]. A Pax6 expression gradient is observed in the optic cup with the highest level from the distal (close to the lens) to proximal side (close to the optic nerve [33] (Fig. 1, E 15.5). For the iridocorneal angle, lineage tracing experiments in mice revealed the contribution of NCCs to the ciliary muscles, ciliary blood vessels, anterior iris, trabecular meshwork and Schlemm’s canal in the iridocorneal angle [59,63,64]. Mesodermal cells also contribute to structures such as the lining of Schlemm’s canal and the iris stroma, but not to the ciliary muscles [59]. Pax6 may participate in regulation of the factors required for the migration of NCC into the eye [65] (see also reviews on neural crest of the eye [44,66]). In mouse embryos (E 15.5), iris and ciliary body progenitor cells can be molecularly distinguished from cells which form the presumptive neural retina. Thus the proper development of the iris relies on the correct compartmentalization of the optic cup (See review in Ref. [26] for molecular details). Fig. 3. Postnatal PAX6 gene expression in humans. The green Box (A–D) shows the different eye structures in human tissue. The color code marks the embryonic origin of these tissues. (Blue: surface ectodermal; Green: Neural ectodermal; Grey: Neural crest; Yellow: Mesodermal). The yellow box below shows the same ocular tissues which exhibit sustained PAX6 expression during postnatal stages, thus likely also playing a role in adulthood. Note some Data from other model organisms are also transferred to the human structures shown here (E– H). There is evidence for Pax6 expression in lacrimal and meibomian glands based on expression arrays [122] (A, E). Pax6 is expressed in lens epithelium, ciliary body (B, F), limbal, corneal and conjunctival epithelium (C, G). In the adult retina, Pax6 is expressed in the ganglion cell layer and the amacrine cells of the inner region of inner nuclear layer (D, H). Graphics freely adopted from different Internet sources [306,307]. L. Latta et al. The Ocular Surface 22 (2021) 245–266 250 Although the molecular mechanism responsible for the normal development of the iridocorneal angle has not yet been elucidated, a series of developmental steps are described in the mouse where final maturation of Schlemm’s canal and the trabecular meshwork extends postnatally to P42 [67]. In humans, the developmental processes are similar, with all rudimentary structures developed at birth [68]. Further maturation and reorganization take place, likely due to mechanical stress and aqueous humor flow and this process could last 1–8 years postnatally [69]. The impact of changing PAX6 levels on anterior chamber development and possible impact on the ocular surface is discussed in Section 2.5. 2.1.5. Limbus and limbal stem cells After the lens has formed, the PAX6-positive cells of the surface ectoderm segregate and give rise to the lineages of the anterior ocular surface epithelia, including conjunctival and limbal/corneal lineages [19] (Fig. 1, E8.5-E15.5).). In humans (but not mice), specialized anatomical structures, the ‘palisades of Vogt’, develop at the limbus – the boundary ring around the periphery of the cornea where the stem cells reside in adult life. The developmental aspects of their formation is of utmost interest, as they are important for stem cell homeostasis and are affected in AAK. To make the overview easier to understand, a comparison of the key events of the anterior chamber development in different animal models is shown (Fig. 2). At 8.5 WG (week of gestation), the human fetal cornea is still continuous with the surface ectoderm. At 12–22 WG, in turn, individual components such as the conjunctiva, cornea and limbus can be distinguished by gene expression [70]. In 12 WG fetal corneas, the presumptive limbus is observed as ‘ridge like’ feature or a ‘dimpling’ in the epithelium [71,72]. In human fetal corneas, the palisades of Vogt are not yet detected and are probably formed postnatally. During 8–22 WG, the limbal epithelium starts to become thicker than in the surrounding cornea and conjunctiva. Single cell RNA-seq studies of the developing human cornea indicate the presence of a proliferating epithelial progenitor cluster with highly expressed corneal epithelial stem and progenitor markers TP63, CLDN1, CLDN4 and TXNIP at 18 WG, indicating the first presence of “a peripheral limbal-like region” harboring the limbal stem and/or progenitor cells [70]. Due to the lack of detailed molecular and mechanistic studies of cell fate determination in the mammalian limbus, corneal epithelium and conjunctiva, here we refer also to functional studies done in chickens, to elucidate the time points when the cornea and conjunctiva become spatially separated by a limbus. In the chickens, a diffusion barrier between the corneal epithelium and conjunctiva is established at embryonic day 8 (See Fig. 2 for species comparison) [73]. This observation is consistent with recent transcriptional data from the developing human cornea at 12 WG, where there are two separate clusters of ocular surface and conjunctival epithelial cells [70]. This separation in chicken may be achieved through the differential expression of a connexin, CX43, or in response to other events allowing differential responses to, as yet unknown, inductive signals [73–75]. CX43 is present in the conjunctiva and is also strongly expressed in the corneal epithelium, but is absent at the limbal border region. This feature is thought to isolate limbal cells from signals in the surrounding tissues and to play a role in their maintenance in an undifferentiated state [73]. The detailed signaling pathways underlying these processes, however, remain to be further elucidated. Interestingly, several markers of adult LSCs are initially expressed throughout the entire corneal epithelium and it is only after stratification that their labeling pattern becomes restricted to the limbal epithelium. In common with other adult stem cell systems, it thus appears that the adult stem cells are a spatially restricted subpopulation of a progenitor population that is specified during embryogenesis. Although the limbus is specified prenatally, there may be some overlap in stratification of corneal epithelium and further maturation of limbal structures. Thus, it could be hypothesized that signals from the stratifying corneal epithelium are also necessary for further maturation of the limbus (See Section 2.3), as could influence underlying corneal stromal cells (See Section 2.6). 2.1.6. Specification of conjunctival-, corneal and limbal epithelia Despite their proximity within a contiguous ocular surface epithelium and their common and persistent expression of Pax6 during development and throughout life, the corneal epithelial cells and the conjunctival epithelial cells belong to two distinct lineages [76,77] arising from different populations [78]. These two lineages arise simultaneously from Pax6 positive ectodermal cells that remain on the embryonic ectodermal surface of the developing eye once the lens vesicle has formed [79,80]. In vivo studies with ocular epithelial cells isolated from rabbits and transplanted into mice have shown that limbal and corneal epithelial cell-derived cysts contained only stratified squamous-type epithelial cells. In contrast, conjunctival epithelial cell-derived cysts contained stratified columnar-type epithelial cells interspersed with Periodic Acid Schiff (PAS) staining-positive cells with goblet-like structure [78]. Despite the fact that such isolated cells might not contain stem cells or may be influenced by differentiated co-transplanted cells, such findings would support the hypothesis that corneal and limbal epithelial cells originate from a different embryonic lineage than conjunctival epithelial cells, and that goblet cells originate from the conjunctival compartment and not following external modulation, as originally proposed [78]. Nevertheless, lineage tracing of these hypothesized progenitor populations will be needed to unequivocally address this issue in wild type mice and furthermore, explore the ocular lineage specification in PAX6-mutated mice. In spite of their importance, relatively little is known about the factors regulating conjunctival goblet cell development. Conjunctival epithelial cells and goblet cells derive from a common bipotent progenitor [81,82]. SAM Pointed Domain-Containing ETS Transcription Factor (SPDEF) has been described as a crucial transcription factor for goblet cell differentiation [83]. Goblet cell differentiation and mucin secretion appear to be directly related to the eyelid opening. In humans, the eyelids are fused until the 5th-6th month of intrauterine life, and goblet cells appear in the fornix extending toward the palpebral and bulbar regions from the 8th to 9th week of gestational age [84,85]. Studies are currently underway to evaluate when the neural regulation of goblet cell secretion becomes functional [86] and the hierarchical network of transcription factors regulating goblet cell development in healthy and pathophysiological conditions (reviewed in Ref. [87]). Some signals which prevent the conjunctival phenotype in the central cornea epithelium are known [88,89]. The extent of PAX6 involvement in these processes will be discussed further in Section 2.2. In contrast to the poorly known mechanisms determining cell fates, the tissue specific markers (mostly cytokeratins) for corneal limbal and conjunctival epithelia are well described, although species-specific differences in some of these markers exist. The conjunctival, limbal and corneal epithelium are composed of one to two cell layers before eyelid opening, after which the thickness of these cell layers increases to 4–5 cell layers (see Fig. 2 for animal model comparison). Cells become stratified and distinguishable by their morphology, with basal cells having a cuboidal shape and being attached to the epithelial basement membrane. The intermediate wing cells (anterior to the basal cells) are present, and are in turn covered by flattened superficial squamous epithelial cells. In the fetus, only minor keratin expression is visible in the superficial shedding cells [72,90]. These processes are similar in different model organisms and have been previously summarized [91]. Early murine corneal epithelial differentiation takes place at embryonic day 15 (E15), as expression of the Pax6-target gene Krt12 becomes apparent and is a specific marker of the corneal epithelium throughout life [92,93]. This is likely also true in humans, as the neutral counterpart of KRT12, KRT3, becomes visible in human fetal corneas L. Latta et al. The Ocular Surface 22 (2021) 245–266 251 from 12 WG to 17 WG in superficial cells. By 20 WG, KRT3 can be detected in the superficial cells of limbus and cornea, similar to the adult cornea [71]. Notably, the sequential appearance of the KRT3 and KRT12 pair is different in chicken and rabbit animal models [94]. In mice the developing limbus matures upon corneal epithelial stratification that is associated with eyelid opening around postnatal day 10–14 (P10-14). KRT12 is expressed in the superficial epithelial cells and extends to all suprabasal cells and in later embryonic stages to all layers of the epithelium upon maturation [95,96]. The corneal epithelium continues to mature up to 3–6 months postnatally [92]. Transcriptional changes in the corneal epithelium upon eye opening in mice, which are similar to humans, have been summarized [91]. One week after birth, α 9 integrin, which marks the limbal region in humans and in mice [97], is equally distributed across the entire corneal epithelium and becomes restricted to the limbus in mice and rats at 8 weeks postnatally [90,98]. Similarly, keratins can be used to observe limbal maturation. KRT19 is found to be expressed throughout the entire murine corneal epithelium prior to eyelid opening, however after stratification, the expression of this keratin becomes restricted to the mouse limbal epithelium [99]. KRT19 is also reported to be concentrated in human limbal epithelium but species differences during maturation of corneal epithelium are likely [100]. In early human development (8–14 WG), KRT15 is expressed across the entire ocular surface including conjunctiva, limbal and corneal epithelia, but becomes restricted to the limbal epithelium from 17 WG, prior to eye opening (See Fig. 2 C 2 ) [71]. These data seem to suggest that, before stratification of the ocular surface epithelium, stem cells are not restricted to the limbus but rather distributed throughout the entire limbal/corneal epithelium. (This would also point to the important role of stromal stem cell niche in maintaining stem cell capacity in adults as described in 3.1 Controversy on limbal stem cells). Additional support for this hypothesis came from the use of a X-chromosomal LacZ transgene, which allows random and irreversible labeling of embryonic blastocyst cells in female mice based on X-chromosome inactivation mosaicism at early embryonic blastocyst stage. Interestingly, in the first 3–4 weeks after birth, a disorganized mosaic pattern of LacZ + -labelled patches of cells appeared dispersed throughout the entire limbal/corneal epithelia. However, from P30 onward, a typical radial stripe pattern of cell migration into the cornea became evident as the stem cells became restricted to the limbus [101]. These elegant studies [101,102] together with other studies mentioned above, indicate that the corneal epithelium is self-sustained by its own pool of stem cells, these stem cells probably differentiate before P30 and from that stage, the limbus becomes the unique stem cell location. Notably, the original ‘XLacZ’ mouse model has limitations due to the fact that tracing is of blastocyst-stage (E3-4) cells and because it does not allow cell type-specific tracing in a clonal, temporal manner by vital microscopy. However, advanced quantitative lineage tracing studies has further shown that this concept is correct, revealing additional aspects of limbal stem cell biology (discussed in section 3.1 below [103–108]). 2.1.7. Corneal nerves In mice, branching nerve bundles cover the entire corneal stroma by E16.5. The corneal epithelium is first innervated at E16.5 and nerves subsequently form a swirl pattern in the subbasal nerve plexus at about three weeks postnatally [109]. The ophthalmic nerves arise from the trigeminal ganglion which is both derived from neural crest and ectodermal cells (see the review in Ref. [45]. At least in chickens, the cornea is innervated solely by the neural crest derived neurons of the trigeminal ganglion [110]. 2.2. Could disturbed segregation and separation of corneal and conjunctival cell lineages contribute to AAK? Lineage segregation of corneal epithelium from the conjunctiva in humans appears after sustained PAX6 induction at the time of surface ectoderm specification. In mice, PAX6 is found in the ocular surface ectoderm at E8, and in humans at embryonic day 42 (Fig. 2). Altered PAX6 levels in the corneal and conjunctival epithelium during development could impact signals required to define borders and selfmaintenance of corneal and conjunctival tissue identity. Deeper understanding of conjunctival and corneal differentiation is therefore essential to distinguish between the developmental and postnatal role of PAX6. In the mouse, the regulatory network of Pitx2 and downstream Dkk2 expressed in the mesenchyme inhibits Wnt/β-catenin signaling resulting in the inhibition of the conjunctival fate in the central cornea [88,89,123]. PITX2 is also described to integrate retinoic acid (RA) signaling from the surface ectoderm, optic cup and lens. Since RA signaling is altered in Pax6 Sey/Sey (Pax6 −/− ) mouse eyes [124], this could affect the crosstalk between the periocular mesenchyme and the surface ectoderm. In addition, transcriptional analysis in human subjects with aniridia indicates that RA metabolism could be altered in the conjunctival cells due to PAX6 mutation [125], but the mechanism requires further confirmation. Whether PAX6 protein levels could impact proper segregation of corneal and conjunctival tissue during development, and therefore potentially influence the development of AAK, remains unknown; however, recent studies are beginning to address this question. A recent single-cell RNA-seq analysis of human corneal development [70] encompassing 12–23 weeks of gestation indicated low overall PAX6 expression throughout the developing cornea; however, in all cases the highest expression was observed in the epithelial layer. The same was also true for the adult cornea with highest PAX6 expression observed in the corneal and conjunctival epithelium. Since several cell populations can be now identified by their transcriptional profile, it is now possible to investigate the extent to which PAX6 is expressed in these cell clusters or if some cells in these clusters exhibit higher or transient PAX6 expression. Another recent study has reported postnatal modification of PAX6 dosage in the Pax6 Sey−Neu/+ (Pax6 +/− ) mouse model by inhibiting the mitogen-activated protein kinase (MEK) pathway [126]. In that study, pharmacologic MEK inhibition by ocular or systemic routes increased PAX6 protein expression in the basal epithelial layers to normal levels as in wild-type mice, resulting in restoration of corneal anatomy and transparency to a normal phenotype. This result provides evidence for a PAX6-dependent role in epithelial cell specification, even postnatally. 2.3. Does the limbus of AAK patients exhibit developmental defects? Similarities between aniridia syndromes and LSCD may suggest that epithelial defects in AAK are the result of LSC failure. However, since pathological changes in aniridia are also observed in the nerves, inflammatory cells and corneal epithelium early in life, even before the limbus becomes overtly affected [29], it suggests that multiple pathological mechanisms may be involved and may precede and/or promote LSC insufficiency. The exact location and differentiation characteristics of LSCs or their precursors during human development is not well studied at the molecular level, especially the postnatal formation of limbus niche structures which differ from those observed in mice [127]. It has been previously hypothesized that the delayed formation of the limbus could be a cause for the late onset of AAK in aniridia [71]. The ΔN isoform of Tp63 α (ΔNp63 α ) is considered as a LSC marker [128] and as a master inducer needed for the progression from an embryonic ectodermal monolayer epithelium into a dynamic, stratified epithelium found in the adult stage (e.g. in the corneal epithelium and epidermis) [129]. PAX6 expression occurs developmentally before the expression of TP63 [130,131]. In contrast to PAX6 that is widely expressed in eye tissues, TP63 expression is limited to the ocular surface epithelia (conjunctiva, limbus, cornea) and is associated with ocular glands (lacrimal and meibomian) [132,133]. Mutations in the TP63 gene lead to Ectrodactyly-Ectodermal dysplasia-Cleft lip/palate (EEC) L. Latta et al. The Ocular Surface 22 (2021) 245–266 252 and Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) syndromes, two syndromes with numerous tissues affected. These syndromes can also be associated with LSCD [134]. TP63 was described to be equally expressed in the fetal limbal and central cornea [71]. In adult humans, TP63 expression is restricted to the limbus, and positive staining is only detected in central corneal epithelium when the corneal epithelium is regenerating after wounding [71,135]. The occurrence of limbal stem cell dysgenesis shows that stem cells are required for corneal maintenance, however identifying the LSCs themselves is challenging [136]. Two recent single-cell RNA-seq studies led to the identification of glycoprotein hormone subunit alpha 2 (GPHA2) as a novel marker of an outer population of limbal stem cells. GPHA2, is a largely unexplored gene whose function appears to be essential for LSC self-renewal and differentiation [70,107]. GPHA2-overexpressing transgenic animals showed no gross phenotype alterations [137], but it would be interesting to examine the phenotype of these mice and generate a knockout mouse strain. GPHA2 expression was dramatically reduced to barely detectable levels following cultivation of human LSCs and in immunodeficient mice. This suggests that GPHA2 may be regulated by T cells critical for the adaptive immune response, and that may serve as an important contributor to the LSC niche [70,107]. Noticeably, like the putative LSC marker KRT15, GPHA2 is not only expressed by basal limbal epithelial cells (i.e. LSCs), but it is also occasionally detected in limbal supra-basal cells [107], and therefore was also proposed to mark limbal committed or differentiated cells [138]. The processes, however, responsible for creating the limbal niche structure secretion of its basement membrane, as well as association and recruitment of niche cells, are not sufficiently understood. It would be of interest to determine whether GPHA2 expression is altered in AAK patients and/or in animal models of aniridia, to yield further evidence for the loss of LSC function, as is widely believed. In addition, lineage tracing in Pax6 +/− mice enabled the detection of pathogenic mechanisms associated with aniridia and the LSCs in light of developmental processes. For example, Pax6 +/− LSCs in Pax6 +/− ↔ Pax6 +/+ chimeric mice are functional and produce streams of epithelial cells that migrate normally into the cornea, although these progeny are less likely to reach the center of the cornea than wild-type cells [139]. This suggests that dosage deficiency of Pax6 does not preclude normal specification of LSCs in a cell-autonomous manner. It remains unknown, however, if the relatively normal behavior of Pax6 +/− cells in the chimeric mouse limbus represents a non-autonomous ‘rescue’ by secreted protein factors such as GPHA2 released from the wild-type cells. 2.4. Does lens development impact AAK? In contrast to aniridia patients, Pax6 Sey/+ (Pax6 +/− ) mice have a more prominent anterior segment dysplasia and the lens often remains attached to the cornea. The mouse lens is larger in proportion to the rest of the eye, as compared to the human eye. This could result in a more severe lens and corneal phenotype in the mouse compared to the human eye [11]. In the chicken, it was shown that surgical removal of the lens affects multiple eye developmental processes including eye growth, and inhibits normal development of the peripheral retina, ciliary body, iris, and migration of NCC into the cornea [140]. The corneal cell fate of surface ectoderm is stabilized by NCC migrating in the lens peripheral ectoderm. The formed stroma prevents PAX6 downregulation in the corneal epithelium [141]. The developing lens is a key signaling center during eye development. During formation of the lens placode, ligands of Wnt, BMP and retinoic acid secreted from the surface ectoderm play a role in patterning of the optic cup [50,52]. Surgical removal of the lens from developing chicken eyes leads to downregulation of genes associated with retinoic acid, BMP and Wnt signaling in the peripheral retina, including the ciliary body, and some aspects of the lens-deficient phenotype (e.g. microphthalmia) can be recapitulated by inhibiting retinoic acid signaling, or rescued in lens-removed eyes by restoring retinoic acid [142]. Pax6 is essential for early stages of lens induction, possibly through influencing modification and remodeling of chromatin [19,52] as reviewed in Ref. [54]. In addition to its role within the lens lineage, Pax6 also plays a role in the adjacent optic vesicle to trigger lens formation. When Pax6 is knocked out at an early stage in the optic vesicle, the lens does not develop [113]. Consistent with the importance of Pax6 in lens formation, there is evidence that the lens is exquisitely sensitive to the correct Pax6 gene dosage. In Mexican Cavefish Astyanax mexicana, it is the loss of Pax6 expression specifically during lens development, that precipitates lens apoptosis which in turn leads to failure of retinal growth and the loss of anterior segment structures [143]. In mice, less than 80% or more than 120% of normal Pax6 activity is thought to result in lens defects that affect the rest of eye development, even though to adulthood [21]. Moreover, results from experimental inactivation one allele of Pax6 in mice, specifically in the lens or in the optic cup, indicate that Pax6 expression in the lens is necessary for normal development of the anterior chamber [112]. Further evidence of the sensitivity of lens development to Pax6 levels was concluded based on mouse chimera experiments indicating that heterozygous Pax6 Sey−Neu/+ (Pax6 −/+ ) cells do not contribute to the developing embryonic lens, in contrast to their contribution to the other eye tissues [144]. Additionally, in chimeric mice where the lens was wild-type, virtually all other aspects of anterior segment development were restored, including normal iris development, corneal epithelial morphology and limbal function [145,146]. A plausible working model for the developmental defects underlying the development of the aniridia phenotype is that lens signals regulated by the correct Pax6 dosage are required for normal development of other anterior segment structures. Identification of these lens signals orchestrating anterior segment development should therefore be a high priority in eye research, irrespective of their roles in aniridia. An early onset of cataract is prevalent in aniridia [147]. This is likely due to abnormality of the lens epithelium and lens fiber cells, and a thinning of the lens capsule [148–150]. Non-cell-autonomous mechanisms for cataract in aniridia have been also proposed as abnormal zonular fibers are reported to be associated with congenital cataract in aniridia [151,152]. Anterior opacities in the lens and iris remnants in the anterior chamber are sometimes observed in aniridic eyes (Fig. 4), suggesting that the separation of the lens and iris from the cornea may sometimes be arrested in human embryos [4]. Further detailed studies are necessary to investigate if the lens status affects the corneal endothelium. Since the keratopathy is mostly present in the anterior layers of the cornea, it is not obvious whether an incomplete separation of the lens or iris from the cornea could impact AAK. The putative crosstalk between the lens and other developing ocular tissues has been discussed above. Similar to dysfunctional lens epithelial cells, an abnormally thin corneal epithelium in the heterozygous Pax6 +/− mouse [28] may represent developmentally immature epithelial cells (including limbal stem and progenitor cells) incapable to form the fully stratified multiple corneal epithelial layers that normally arise postnatally in the mouse cornea [87]. However, it must be noted that any apparent undifferentiated state may be a secondary consequence of the chronic abrasion and wound-healing physiology of the aniridic cornea [153,154]. 2.5. Does anterior chamber and iris malformation impact AAK development? The conditional inactivation of a single Pax6 allele in mice from either the inner layer or the outer pigmented epithelium of the distal optic cup results in a profound iris hypoplasia [51]. The resulting reduction in Pax6 dosage interrupts different stages of iris development: from reduction in the size of the progenitors, to delayed onset of L. Latta et al. The Ocular Surface 22 (2021) 245–266 253 muscle-specific markers and abrogated iris sphincter morphogenesis [51,155]. Indeed several key factors for iris development are reduced in the developing iris of the Pax6 Sey−1Neu/+ mice including: Pitx2, Igf2, Foxc1, TGFb2, Zic2 and BMP4 [156]. These transcription factors and ligands could impact the differentiation of the iris progenitors as well as the migration of NCC that populate the iris stroma and the cornea [157]. The majority (50%–75%) of aniridia patients develop glaucoma most likely as a consequence of abnormal differentiation of the trabecular meshwork and/or complete absence of Schlemm’s canal [158,159]. Notably, conditional haploinsufficiency of Pax6 in the mouse lens and cornea - but not in the developing optic cup layers - disrupted trabecular meshwork and Schlemm’s canal development and resulted in glaucoma [145]. It is currently unclear, however, if this result is due to Pax6 activity in the lens and cornea regulating factors required for the development of the drainage structures, or due to the abnormal morphology of the eye due to adhesion between lens, cornea and iris epithelium in the model [145]. Another unsolved topic is how partial or complete loss of iris and the abnormal differentiation of the drainage structures in aniridia impact AAK progression. It should be considered that the positioning of the anterior chamber angle may be needed for signaling to LSC and their niche to develop correctly, as well as to ensure the proper flow of aqueous humor important to maintain the correct eye pressure and nutrition of the anterior segment structures. In a recent study examining 87 eyes of aniridia patients, 21 of which had a partial iris, it was shown that the partial presence of an iris was strongly associated with a milder degree of AAK [29]. Although this could support a connection between iris or chamber angle development and AAK, the mild keratopathy could also be caused by the common causative mutation itself. 2.6. Are mesenchymal structures (corneal stroma and endothelium) affected during development? A number of clinical studies have reported that the corneal stroma is abnormally thick in almost all cases of aniridia [6,160,161]. Although the causes of a thick stroma in aniridia are not yet clarified (as the corneal endothelium appears to function normally), it has been reported that, during normal human development, the corneal stroma is thicker in utero and progressively thins with increasing gestational age [162]. This has led to the hypothesis that the normal thinning of the corneal stroma in later developmental stages in utero is disrupted in aniridia [29]. A developmental origin for the thickened corneal stroma is supported by the lack of clinical signs of stromal edema and a sufficiently high endothelial cell density in aniridia for maintaining proper stromal hydration. In the epithelia of the developing lens, retina, ciliary body, iris and cornea, PAX6 is expressed at high levels, and this is easily detectable by in situ hybridization, Western blot and immunohistochemistry. In the mesenchymal component of some other ocular tissues, such as the corneal endothelium, corneal stromal keratocytes and trabecular meshwork mesenchyme, low and transient levels of PAX6 (at the limits of detection by the above techniques) have been reproducibly demonstrated during mid-late stages of development [163,164]. These low levels of PAX6 have nevertheless been shown experimentally to represent a cell-autonomous requirement for contribution of cells to the corneal endothelium and stroma, and also play a role in the differentiation of trabecular meshwork [47,163,164]. Although PAX6 is downregulated in the trabecular meshwork of normal adult eyes upon differentiation [163], recent single cell analysis has revealed the presence of PAX6 transcripts in normal limbal corneal keratocytes, corneal stromal keratocytes as well as in corneal stromal stem cells, into adulthood [70]. This correlates with the previous observation of PAX6 expression in a population of stromal stem cells [165]. At the single-cell level in humans, PAX6 mRNA is detected through all developmental stages at a low level [70]. (Fig. 5). Impaired development of the anterior chamber angle, iris and endothelium due to PAX6 haploinsufficiency has been carefully studied and reviewed in a number of reports [28,40,47,155,163], but the impact of PAX6-deficient corneal or limbal stromal cells on AAK remains elusive [166]. PAX6 dosage is self-evidently crucial for normal eye development, so the molecular mechanism by which different tissues require either ‘high’ or ‘low’ levels of Pax6, and how dosage is controlled via regulatory DNA elements, still requires clarification. PAX6 directly interacts with multiple other proteins [167,168] and the presence and stoichiometry of different binding partners in different cell types is expected to modulate PAX6 activity. Hundreds of genes are regulated, directly or indirectly, by PAX6 during eye development [169] and are affected to different degrees by changes in dosage. It is therefore expected that mesenchymal cells with ‘low’ levels of PAX6 will exhibit a different profile of downstream gene expression from epithelial cells with ‘high’ levels. The biological impact of PAX6-heterozygosity for low levels of expression in mesenchymal cells, if any, are unresolved. Taken together, the most parsimonious scenario is that PAX6, expressed at high levels, is functioning cell-autonomously in the optic cup, lens and corneal epithelium progenitors, and that these tissues have a non-autonomous influence on the surrounding anterior segmental neural crest and mesodermal lineages (See Fig. 5, blue arrows). As the Schlemm’s canal lining and iris stroma are formed by mesodermal cells [59] and are absent in Pax6 +/− mice [65,170], it may be possible that transient PAX6 expression in these cells further contributes directly to their formation. 2.7. Do meibomian and lacrimal gland formation impact AAK development? The functions of lacrimal and meibomian glands are essential for the production, stability and function of the tear film. Any factor disturbing the homeostasis of the ocular surface unit may disrupt the stability of the tears, leading to damage of corneal and conjunctival epithelia and possibly impacting LSC function [171]. These supportive glands are derived from the ocular surface ectoderm, but to date there is no evidence regarding the impact of these structures on other anterior structures during eye development. The morphogenic events necessary for the lacrimal gland development in Pax6 Sey/Sey mice are defective [172]. In Pax6 Sey/+ at E19.5, the lacrimal bud becomes visible but its structure remains vestigial [173]. In lacrimal gland organoid models, Pax6 is necessary for expression of the genes encoding the secretion machinery (aquaporins and neurotransmitters) but these analyses were performed with total Pax6 knockout model [174]. In addition, PAX6 is one of the transcription factors necessary to drive explant cultures or induced pluripotent stem cells (iPSCs) into a lacrimal gland cellular fate [173, 175]. The development of the meibomian glands requires proper eyelid closure and eyelid fusion during embryonic development. PAX6 could influence this process since it is expressed at low levels during eyelid development in a complex expression pattern [176,177]. PAX6 is expressed during development in the acinar cells of meibomian glands, although its contribution to development of these glands requires further investigation [178]. Protein composition of the tear film is altered in aniridia, and an elevation in inflammatory cytokine levels has been observed [179]. Meibomian gland dysfunction has also been documented in aniridia patients [180–183]. Still, it is unknown whether tear film and meibomian gland abnormalities arise from developmental defects or whether the function is impaired postnatally due to deficient ocular surface epithelia. AAK may underpin the inflammatory process and act as a possible trigger mechanism for dry eye and meibomian gland dysfunction. Elevated interleukins in the tear film could be also caused by the chronic wound healing state of the corneal epithelium in aniridia or from inflammation in the limbus and corneal stroma [7]. It is also important to keep in mind that the developmental defects observed in mice may not necessarily be mirrored in humans. Longitudinally L. Latta et al. The Ocular Surface 22 (2021) 245–266 260 eyes, the basal side of the Palisades of Vogt is densely populated by corpuscular nerve endings, suggesting that neurotrophic factors may support stem cells not only directly, but indirectly by maintenance of the limbal niche [287,288]. Human corneal-limbal organoids appear to maintain good niche function without innervation in vitro [289], but this does not preclude an in vivo role in niche development or maintenance. In mammals, though not in birds, PAX6 is expressed transiently during early development of the trigeminal ganglion, in cells of both the OpV and mmV-derived components, and this correlates with delayed innervation of sensory structures such as whisker follicles in mutants (unpublished data). Putative links between PAX6 mutation, disruption of limbal-corneal innervation, the degradation of palisade structure and [207,270] deficiency of the limbal niche require further investigation. As immune cell infiltration into the cornea facilitates (and is facilitated by) neovascularization, a deficit of corneal nerves may directly or indirectly play an important role in development of neovascularization in AAK. In experimental models of corneal neovascularization and trigeminal nerve ablation in the mouse, blood vessel invasion was shown to occur only in areas devoid of nerves, where inflammatory leukocytes and macrophage-lineage cells also infiltrated the cornea [290]. Conversely, where the nerve supply remained intact, the cornea remained vessel-free. A loss of antiangiogenic factors was noted in denervated corneas, in particular loss of vascular endothelial growth factor receptor-3 (VEGFR3) expression in the corneal epithelium and loss of pigment epithelium-derived factor (PEDF) expression in the corneal stroma. In a different mouse model of inflammation-induced dry eye disease, corneal nerves were diminished and expressed elevated levels of the proinflammatory neuropeptide SP [291]. Blockade of SP or its neurokinin-1 receptor effectively prevented vascular endothelial cell activation and reduced corneal neovascularization. In addition, VEGF has been shown to mediate corneal repair in abrasion-induced corneal nerve damage models, where VEGF blockade effectively suppressed nerve regeneration [292,293]. The source of VEGF in the corneal abrasion model is infiltrating T-cells and neutrophils [292]. Taken together, these studies indicate that corneal nerves, where damaged or accompanied by inflammation or when lost entirely, lose their ability to express angiostatic factors and instead express proinflammatory and regenerative factors, contributing to enhanced corneal neovascularization. These effects can be considered part of the normal wound healing response, and may thus explain why the cornea in AAK, which is in a chronically inflamed wound-like state, is characterized by a deficit of nerves and an abundance of blood vessels. Potential therapeutic approaches supplying deficient factors such as VEGFR3 or PEDF, or blocking factors aiding neovascular growth such as SP or VEGF, warrant further investigation in relevant in vitro and in vivo aniridia models. Given the role of corneal nerves in maintaining avascularity and epithelial integrity, restoration of a healthy corneal nerve population may represent a viable therapeutic approach for AAK. As described above, autologous serum eye drops have been shown to relieve symptoms of neuropathic corneal pain and increase the abundance of corneal nerves in non-aniridia subjects [294]. In subjects with toxic corneal epitheliopathy induced by anti-glaucoma eye drops, a course of autologous serum drops significantly improved corneal sensitivity [295]. In a cohort of thirteen subjects with AAK, autologous serum eye drops instilled over an 8-week period resulted in subjective improvement in keratopathy symptoms and healed corneal epithelial defects in several cases; however, neovascularization and stromal scarring did not significantly improve during the course of treatment [296]. Longer-term treatment, careful patient selection (e.g., based on AAK grade) and/or use of molecules with specific nerve regenerative capacity may be of benefit in AAK. The recently approved recombinant human nerve growth factor (NGF) treatment (Cenegermin) may be of interest in this regard. Indicated for treatment of moderate to severe neurotrophic keratopathy (a rare disease characterized by diminished corneal innervation, non-healing epithelial wounds and corneal ulceration), NGF stimulates corneal epithelial cell growth and survival, aids in the maintenance of limbal stem cell function, promotes tear production and supports corneal re-innervation [297]. In clinical studies, 65–75% of patients receiving an 8-week course of Cenegermin eye drops exhibited complete corneal healing, although relapse occurred in about 20% while eye pain and reduced visual acuity were frequently reported adverse effects [297,298]. Notably, it has also been shown that corneal subbasal nerve density significantly increased following an 8-week course of Cenegermin [299]. Whether these promising effects could be extended to AAK is unknown and would need to be explored; however, use of Cenegermin for conditions other than neurotrophic keratopathy is currently off-label. Here, animal models would be useful for initial investigations. 4. Molecular biology and genetics perspective Due to the many observed PAX6 mutations independently leading to aniridia and the heterogeneous clinical phenotype, it is difficult to associate specific mutations to AAK severity [4]. However, some classes of mutations (selected missense or non-coding mutations) are associated with reduced AAK progression and mild or absent LSCD [29]. Comparative OMICs of such patient samples could help elucidate correlations between specific mutations and degree of AAK severity at the molecular level [125,300]. Nonsense mediated decay (NMD) of mutant mRNA is the most common mechanism of PAX6 protein deficiency, but there is no evidence this is modulated by the position of mutations in the transcript. The effect of missense mutation is difficult to predict but some in vitro studies described the effect of the mutation in different binding domains. It might not be useful to compare different missense mutations with each other since they could have different properties. For PAX6 run-on mutations (with a mutated stop codon), it is thought (but not yet demonstrated) that the mutated PAX6 protein is degraded or not produced. Mutations in regulatory domains of PAX6 (also found as 3′-cis-regulatory region deletions) are interesting since PAX6 reduction could be less pronounced, and these mutations have been related to milder phenotypes [4,206,300]. In rare cases, genes other than PAX6 can putatively cause aniridia [301–303]. Primary culture of patient cells (both epithelial and mesenchymal) should help to identify deregulated genes important for AAK development especially in cases of aniridia with mild AAK, to discriminate from PAX6-dependent expression changes that are not causative of AAK [125]. Recent comprehensive reviews summarize in detail the genetics of congenital aniridia [1,2, 304]. Although the impact of PAX6 levels and splice variants on iris and ciliary body development have been systematically studied [155], we lack such detailed knowledge for LSCs and corneal epithelial cells. Also, PAX6 protein has been described to be located in the nucleus, cytosol or even in secreted form. It must be further evaluated how localization of PAX6 is controlled by post-translational modifications. PAX6 mRNA expression may be not as dramatically altered as at the protein level, and thus mRNA expression and translation of PAX6 need to be studied in detail in cells such as LSCs and differentiated epithelium. Interestingly, it has recently been shown that Pax6 expression is negatively regulated by the microRNAs miR-7 and miR-135, and that protection of this inhibitory mechanism was capable of restoring PAX6 protein levels in isolated pancreatic islets in an aniridia mouse model [305]. It remains to be determined whether a similar regulatory mechanism also occurs in LSCs. Nevertheless, these data, in addition to the putative target miR-204-5p discussed earlier, appear to suggest that RNA-based therapies could represent a potential innovative therapeutic strategy for AAK. 5. Conclusions for further research strategies Nearly all patients with aniridia suffer from AAK [4]. The onset of clinically apparent AAK differs between individuals even with the same mutations. Based on our present level of knowledge, it is not clear if AAK is caused by LSCD while it seems likely that some loss of LSC function L. Latta et al. The Ocular Surface 22 (2021) 245–266 261 occurs, it is not necessary to invoke LSCD as the main cause of the phenotype seen in AAK eyes. Clinical microscopy findings suggest that a minimal degree of keratopathy is likely to exist in all cases of aniridia even before ocular surface changes become visible at the slit lamp. This ‘minimal keratopathy’ includes reduced mechanical touch sensitivity, a deficit of corneal nerves, and increased inflammatory cell presence in the central cornea [4]. LSCD may in fact be a consequence of the early minimal keratopathy in subjects where the specific PAX6 mutation predisposes the cornea to a progressive AAK phenotype. The minimal keratopathy may in turn have a developmental origin. The chronic wound healing pathology of the corneal epithelium in AAK may overwhelm the PAX6 +/− limbal regenerative potential during normal life [153,154]. Essential knowledge, however, is still lacking concerning the critical factors needed to specify and maintain the limbal niche and how these relate to PAX6 expression both prenatally and postnatally; therefore, pathogenic mechanisms at the molecular level are still speculative. Early indications are that PAX6 regulation of and by other genes and factors is complex, and multiple signaling pathways, molecular and cellular mechanisms and feedback loops appear to be active, resulting in the observed AAK phenotypes. Deciphering some of the key pathways and mechanisms involved can provide insights that will be important for future and new therapies targeting AAK, keeping in mind that the complexity itself may provide multiple potential therapeutic targets. Declaration of competing interest No conflicting relationship pertaining to this work exists for any author. Acknowledgments This review is based upon work from the Work Group5 of the COST Action CA18116 (Aniridia: networking to address an unmet medical, scientific, and societal challenge), supported by COST (European Cooperation in Science and Technology). The European Joint Programme on Rare Diseases (EJP RD 2020) is acknowledged for its support to NL, LL, DA, MJC, NS, RAP (AAK-INSIGHT). The work of LL and NS at the Dr. Rolf M. Schwiete Center for Limbal Stem Cell and Congenital Aniridia Research at Saarland University, Germany was supported by the Dr. Rolf M. Schwiete Foundation. References [1] Wawrocka A, Krawczynski MR. The genetics of aniridia - simple things become complicated. J Appl Genet 2018;59:151–9. [2] Lima Cunha D, Arno G, Corton M, Moosajee M. The spectrum of PAX6 mutations and genotype-phenotype correlations in the eye. Genes 2019;10. [3] Gerber S, Alzayady KJ, Burglen L, Br´ emond-Gignac D, Marchesin V, Roche O, et al. Recessive and dominant de novo ITPR1 mutations cause Gillespie syndrome. Am J Hum Genet 2016;98:971–80. [4] Lagali N, Wowra B, Fries FN, Latta L, Moslemani K, Utheim TP, et al. em>PAX6</em>mutational status determines aniridia-associated keratopathy phenotype. Ophthalmology 2020;127:273–5. ≤. [5] Nishida K, Kinoshita S, Ohashi Y, Kuwayama Y, Yamamoto S. Ocular surface abnormalities in aniridia. Am J Ophthalmol 1995;120:368–75. [6] Lagali N, Eden U, Utheim TP, Chen X, Riise R, Dellby A, et al. In vivo morphology of the limbal palisades of vogt correlates with progressive stem cell deficiency in aniridia-related keratopathy. Invest Ophthalmol Vis Sci 2013;54:5333–42. [7] Lagali N, Wowra B, Dobrowolski D, Utheim TP, Fagerholm P, Wylegala E. Stagerelated central corneal epithelial transformation in congenital aniridia-associated keratopathy. Ocul Surf 2018;16:163–72. [8] Freund C, Horsford DJ, McInnes RR. Transcription factor genes and the developing eye: a genetic perspective. Hum Mol Genet 1996:1471–88. 5 Spec No. [9] Hill RE, Favor J, Hogan BL, Ton CC, Saunders GF, Hanson IM, et al. Mouse Small eye results from mutations in a paired-like homeobox-containing gene. Nature 1992;355:750. [10] Lyon MF, Phillips RJ, Fisher G. Dose-response curves for radiation-induced gene mutations in mouse oocytes and their interpretation. Mutat Res 1979;63:161–73. [11] Glaser T, Lane J, Housman D. A mouse model of the aniridia-Wilms tumor deletion syndrome. Science 1990;250:823–7. [12] Theiler K, Varnum DS, Stevens LC. Development of Dickie’s small eye: an early lethal mutation in the house mouse. Anat Embryol 1980;161:115–20. [13] Thaung C, West K, Clark BJ, McKie L, Morgan JE, Arnold K, et al. Novel ENUinduced eye mutations in the mouse: models for human eye disease. Hum Mol Genet 2002;11:755–67. [14] Lyon MF, Bogani D, Boyd Y, Guillot P, Favor J. Further genetic analysis of two autosomal dominant mouse eye defects, Ccw and Pax6(coop). Mol Vis 2000;6: 199–203. [15] Favor J, Peters H, Hermann T, Schmahl W, Chatterjee B, Neuh¨ auser-Klaus A, et al. Molecular characterization of Pax6(2Neu) through Pax6(10Neu): an extension of the Pax6 allelic series and the identification of two possible hypomorph alleles in the mouse Mus musculus. Genetics 2001;159:1689–700. [16] Graw J, L¨ oster J, Puk O, Münster D, Haubst N, Soewarto D, et al. Three novel Pax6 alleles in the mouse leading to the same small-eye phenotype caused by different consequences at target promoters. Invest Ophthalmol Vis Sci 2005;46: 4671–83. [17] Simpson TI, Pratt T, Mason JO, Price DJ. Normal ventral telencephalic expression of Pax6 is required for normal development of thalamocortical axons in embryonic mice. Neural Dev 2009;4:19. [18] St-Onge L, Sosa-Pineda B, Chowdhury K, Mansouri A, Gruss P. Pax6 is required for differentiation of glucagon-producing alpha-cells in mouse pancreas. Nature 1997;387:406–9. [19] Ashery-Padan R, Marquardt T, Zhou X, Gruss P. Pax6 activity in the lens primordium is required for lens formation and for correct placement of a single retina in the eye. Genes Dev 2000;14:2701–11. [20] Voskresenskaya A, Pozdeyeva N, Batkov Y, Vasilyeva T, Marakhonov A, West RA, et al. Morphometric analysis of the lens in human aniridia and mouse Small eye. Exp Eye Res 2021;203:108371. [21] Dor` a N, Ou J, Kucerova R, Parisi I, West JD, Collinson JM. PAX6 dosage effects on corneal development, growth, and wound healing. Dev Dynam : an official publication of the American Association of Anatomists 2008;237:1295–306. [22] Schedl A, Ross A, Lee M, Engelkamp D, Rashbass P, van Heyningen V, et al. Influence of PAX6 gene dosage on development: overexpression causes severe eye abnormalities. Cell 1996;86:71–82. [23] Ramaesh T, Ramaesh K, Martin Collinson J, Chanas SA, Dhillon B, West JD. Developmental and cellular factors underlying corneal epithelial dysgenesis in the Pax6+/−mouse model of aniridia. Exp Eye Res 2005;81:224–35. [24] Davis J, Duncan MK, Robison Jr WG, Piatigorsky J. Requirement for Pax6 in corneal morphogenesis: a role in adhesion. J Cell Sci 2003;116:2157–67. [25] Hickmott JW, Gunawardane U, Jensen K, Korecki AJ, Simpson EM. Epistasis between Pax6(Sey) and genetic background reinforces the value of defined hybrid mouse models for therapeutic trials. Gene Ther 2018;25:524–37. [26] Davis-Silberman N, Ashery-Padan R. Iris development in vertebrates; genetic and molecular considerations. Brain Res 2008;1192:17–28. [27] Weh E, Reis LM, Happ HC, Levin AV, Wheeler PG, David KL, et al. Whole exome sequence analysis of Peters anomaly. Hum Genet 2014;133:1497–511. [28] Ramaesh T, Collinson JM, Ramaesh K, Kaufman MH, West JD, Dhillon B. Corneal abnormalities in Pax6 +/−small eye mice mimic human aniridia-related keratopathy. Invest Ophthalmol Vis Sci 2003;44:1871–8. [29] Lagali N, Wowra B, Fries FN, Latta L, Moslemani K, Utheim TP, et al. Early phenotypic features of aniridia-associated keratopathy and association with PAX6 coding mutations. Ocul Surf 2020;18:130–40. [30] Wang GM, Prasov L, Al-Hasani H, Marrs CER, Tolia S, Wiinikka-Buesser L, et al. Phenotypic variation in a four-generation family with aniridia carrying a novel PAX6 mutation. J Ophthalmol 2018:5978293. 2018. [31] Halder G, Callaerts P, Gehring WJ. New perspectives on eye evolution. Curr Opin Genet Dev 1995;5:602–9. [32] Chow RL, Altmann CR, Lang RA, Hemmati-Brivanlou A. Pax6 induces ectopic eyes in a vertebrate. Development 1999;126:4213–22. [33] Walther C, Gruss P. Pax-6, a murine paired box gene, is expressed in the developing CNS. Development 1991;113:1435–49. [34] Sander M, Neubüser A, Kalamaras J, Ee HC, Martin GR, German MS. Genetic analysis reveals that PAX6 is required for normal transcription of pancreatic hormone genes and islet development. Genes Dev 1997;11:1662–73. [35] Gosmain Y, Marthinet E, Cheyssac C, Gu´ erardel A, Mamin A, Katz LS, et al. Pax6 controls the expression of critical genes involved in pancreatic α cell differentiation and function. J Biol Chem 2010;285:33381–93. [36] Cvekl A, Callaerts P. PAX6: 25th anniversary and more to learn. Exp Eye Res 2017;156:10–21. [37] Ding J, Gao Y, Zhao J, Yan H, Guo S-y, Zhang Q-x, et al. Pax6 haploinsufficiency causes abnormal metabolic homeostasis by down-regulating glucagon-like peptide 1 in mice. Endocrinology 2009;150:2136–44. [38] Sisodiya SM, Free SL, Williamson KA, Mitchell TN, Willis C, Stevens JM, et al. PAX6 haploinsufficiency causes cerebral malformation and olfactory dysfunction in humans. Nat Genet 2001;28:214–6. [39] Berntsson SG, Kristoffersson A, Daniilidou M, Dahl N, Ekstr¨ om C, Semnic R, et al. Aniridia with PAX6 mutations and narcolepsy. J Sleep Res 2020;29:e12982. [40] Shaham O, Menuchin Y, Farhy C, Ashery-Padan R. Pax6: a multi-level regulator of ocular development. Prog Retin Eye Res 2012;31:351–76. [41] Dor` a NJ, Manuel M, Kleinjan D-J, Price DJ, Collinson JM, Hill RE, et al. A conditional Pax6 depletion study with no morphological effect on the adult mouse corneal epithelium. BMC Res Notes 2018;11:705. [42] Zuber ME, Gestri G, Viczian AS, Barsacchi G, Harris WA. Specification of the vertebrate eye by a network of eye field transcription factors. Development 2003; 130:5155–67. [43] Grindley JC, Davidson DR, Hill RE. The role of Pax-6 in eye and nasal development. Development 1995;121:1433–42. L. Latta et al. The Ocular Surface 22 (2021) 245–266 262 [44] Williams AL, Bohnsack BL. Neural crest derivatives in ocular development: discerning the eye of the storm. Birth Defects Res C Embryo Today 2015;105: 87–95. [45] Jasrapuria-Agrawal S, Lwigale PY. Chapter 10 - neural crest cells in ocular development. In: Trainor PA, editor. Neural crest cells. Boston: Academic Press; 2014. p. 189–203. [46] Lwigale PY. Chapter four - corneal development: different cells from a common progenitor. In: Hejtmancik JF, Nickerson JM, editors. Progress in molecular biology and translational science. Academic Press; 2015. p. 43–59. [47] Cvekl A, Tamm ER. Anterior eye development and ocular mesenchyme: new insights from mouse models and human diseases. Bioessays 2004;26:374–86. [48] Fuhrmann S. Eye morphogenesis and patterning of the optic vesicle. Curr Top Dev Biol 2010;93:61–84. [49] Adler R, Canto-Soler MV. Molecular mechanisms of optic vesicle development: complexities, ambiguities and controversies. Dev Biol 2007;305:1–13. [50] Carpenter AC, Smith AN, Wagner H, Cohen-Tayar Y, Rao S, Wallace V, et al. Wnt ligands from the embryonic surface ectoderm regulate ’bimetallic strip’ optic cup morphogenesis in mouse. Development 2015;142:972–82. [51] Davis-Silberman N, Kalich T, Oron-Karni V, Marquardt T, Kroeber M, Tamm ER, et al. Genetic dissection of Pax6 dosage requirements in the developing mouse eye. Hum Mol Genet 2005;14:2265–76. [52] Steinfeld J, Steinfeld I, Coronato N, Hampel M-L, Layer PG, Araki M, et al. RPE specification in the chick is mediated by surface ectoderm-derived BMP and Wnt signalling. Development 2013;140:4959–69. [53] Collinson JM, Hill RE, West JD. Different roles for Pax6 in the optic vesicle and facial epithelium mediate early morphogenesis of the murine eye. Development 2000;127:945–56. [54] Cvekl A, Ashery-Padan R. The cellular and molecular mechanisms of vertebrate lens development. Development 2014;141:4432–47. [55] Aota S-i, Nakajima N, Sakamoto R, Watanabe S, Ibaraki N, Okazaki K. Pax6 autoregulation mediated by direct interaction of Pax6 protein with the head surface ectoderm-specific enhancer of the mouse Pax6 gene. Dev Biol 2003;257: 1–13. [56] Kamachi Y, Uchikawa M, Collignon J, Lovell-Badge R, Kondoh H. Involvement of Sox1, 2 and 3 in the early and subsequent molecular events of lens induction. Development 1998;125:2521–32. [57] Shaham O, Smith AN, Robinson ML, Taketo MM, Lang RA, Ashery-Padan R. Pax6 is essential for lens fiber cell differentiation. Development 2009;136:2567–78. [58] Cvekl A, Zhang X. Signaling and gene regulatory networks in mammalian lens development. Trends Genet 2017;33:677–702. [59] Gage PJ, Rhoades W, Prucka SK, Hjalt T. Fate maps of neural crest and mesoderm in the mammalian eye. Invest Ophthalmol Vis Sci 2005;46:4200–8. [60] Trainor PA, Tam PP. Cranial paraxial mesoderm and neural crest cells of the mouse embryo: co-distribution in the craniofacial mesenchyme but distinct segregation in branchial arches. Development 1995;121:2569–82. [61] Yoshida S, Shimmura S, Nagoshi N, Fukuda K, Matsuzaki Y, Okano H, et al. Isolation of multipotent neural crest-derived stem cells from the adult mouse cornea. Stem Cell 2006;24:2714–22. [62] Murphy C, Alvarado J, Juster R, Maglio M. Prenatal and postnatal cellularity of the human corneal endothelium. A quantitative histologic study. Invest Ophthalmol Vis Sci 1984;25:312–22. [63] Johnston MC, Noden DM, Hazelton RD, Coulombre JL, Coulombre AJ. Origins of avian ocular and periocular tissues. Exp Eye Res 1979;29:27–43. [64] Noden D. Periocular mesenchyme: neural crest and mesodermal interactions. Duane’s fundations of clinical ophthalmology 1993;1:1–23. [65] Kanakubo S, Nomura T, Yamamura K-i, Miyazaki J-i, Tamai M, Osumi N. Abnormal migration and distribution of neural crest cells in Pax6 heterozygous mutant eye, a model for human eye diseases. Gene Cell 2006;11:919–33. [66] Akula M, Park JW, West-Mays JA. Relationship between neural crest cell specification and rare ocular diseases. J Neurosci Res 2019;97:7–15. [67] Smith RS, Zabaleta A, Savinova OV, John SWM. The mouse anterior chamber angle and trabecular meshwork develop without cell death. BMC Dev Biol 2001; 1:3. [68] Rem´ e C, Lalive d’Epinay S. Periods of development of the normal human chamber angle. Doc Ophthalmol 1981;51:241–68. [69] McMenamin PG. A morphological study of the inner surface of the anterior chamber angle in pre and postnatal human eyes. Curr Eye Res 1989;8:727–39. [70] Collin J, Queen R, Zerti D, Bojic S, Dorgau B, Moyse N, et al. A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells Ocul Surf. 2021. [71] Davies SB, Chui J, Madigan MC, Provis JM, Wakefield D, Di Girolamo N. Stem cell activity in the developing human cornea. Stem Cell 2009;27:2781–92. [72] Sevel D, Isaacs R. A re-evaluation of corneal development. Trans Am Ophthalmol Soc 1988;86:178–207. [73] Kubilus JK, Zapater i Morales C, Linsenmayer TF. The corneal epithelial barrier and its developmental role in isolating corneal epithelial and conjunctival cells from one AnotherDevelopment of the corneal-conjunctival barrier. Invest Ophthalmol Vis Sci 2017;58:1665–72. [74] Wolosin JM, Schütte M, Zieske JD, Budak MT. Changes in connexin43 in early ocular surface development. Curr Eye Res 2002;24:430–8. [75] Wolosin JM, Budak MT, Akinci MA. Ocular surface epithelial and stem cell development. Int J Dev Biol 2004;48:981–91. [76] Kruse FE, Chen JJ, Tsai RJ, Tseng SC. Conjunctival transdifferentiation is due to the incomplete removal of limbal basal epithelium. Invest Ophthalmol Vis Sci 1990;31:1903–13. [77] Wei ZG, Wu RL, Lavker RM, Sun TT. In vitro growth and differentiation of rabbit bulbar, fornix, and palpebral conjunctival epithelia. Implications on conjunctival epithelial transdifferentiation and stem cells. Invest Ophthalmol Vis Sci 1993;34: 1814–28. [78] Wei ZG, Sun TT, Lavker RM. Rabbit conjunctival and corneal epithelial cells belong to two separate lineages. Invest Ophthalmol Vis Sci 1996;37:523–33. [79] Davis JA, Reed RR. Role of olf-1 and pax-6 transcription factors in neurodevelopment. J Neurosci 1996;16:5082–94. [80] Koroma BM, Yang JM, Sundin OH. The Pax-6 homeobox gene is expressed throughout the corneal and conjunctival epithelia. Invest Ophthalmol Vis Sci 1997;38:108–20. [81] Pellegrini G, Golisano O, Paterna P, Lambiase A, Bonini S, Rama P, et al. Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J Cell Biol 1999;145:769–82. [82] Wei ZG, Lin T, Sun TT, Lavker RM. Clonal analysis of the in vivo differentiation potential of keratinocytes. Invest Ophthalmol Vis Sci 1997;38:753–61. [83] Marko CK, Menon BB, Chen G, Whitsett JA, Clevers H, Gipson IK. Spdef null mice lack conjunctival goblet cells and provide a model of dry eye. Am J Pathol 2013; 183:35–48. [84] Sellheyer K, Spitznas M. Ultrastructural observations on the development of the human conjunctival epithelium. Graefes Arch Clin Exp Ophthalmol 1988;226: 489–99. [85] Miyashita K, Azuma N, Hida T. Morphological and histochemical studies of goblet cells in developing human conjunctiva. Jpn J Ophthalmol 1992;36:169–74. [86] Ríos JD, Forde K, Diebold Y, Lightman J, Zieske JD, Dartt DA. Development of conjunctival goblet cells and their neuroreceptor subtype expression. Invest Ophthalmol Vis Sci 2000;41:2127–37. [87] Swamynathan SK. Ocular surface development and gene expression. Journal of Ophthalmology 2013:22. 2013. [88] Gage PJ, Kuang C, Zacharias AL. The homeodomain transcription factor PITX2 is required for specifying correct cell fates and establishing angiogenic privilege in the developing cornea. Dev Dynam 2014;243:1391–400. [89] Gage PJ, Qian M, Wu D, Rosenberg KI. The canonical Wnt signaling antagonist DKK2 is an essential effector of PITX2 function during normal eye development. Dev Biol 2008;317:310–24. [90] Chung EH, Bukusoglu G, Zieske JD. Localization of corneal epithelial stem cells in the developing rat. Invest Ophthalmol Vis Sci 1992;33:2199–206. [91] Zieske JD. Corneal development associated with eyelid opening. Int J Dev Biol 2004;48:903–11. [92] Tanifuji-Terai N, Terai K, Hayashi Y, Chikama T-i, Kao WW-Y. Expression of keratin 12 and maturation of corneal epithelium during development and postnatal growth. Invest Ophthalmol Vis Sci 2006;47:545–51. [93] Liu JJ, Kao WW, Wilson SE. Corneal epithelium-specific mouse keratin K12 promoter. Exp Eye Res 1999;68:295–301. [94] Chaloin-Dufau C, Dhouailly D, Sun T-T. Appearance of the keratin pair K3/K12 during embryonic and adult corneal epithelial differentiation in the chick and in the rabbit. Cell Differ Dev 1990;32:97–108. [95] Liu C-Y, Zhu G, Westerhausen-larson A, Converse R, Candace WCK, Sun T-T, et al. Cornea-specific expression of K12 keratin during mouse development. Curr Eye Res 1993;12:963–74. [96] Kurpakus MA, Maniaci MT, Esco M. Expression of keratins K12, K4 and K14 during development of ocular surface epithelium. Curr Eye Res 1994;13:805–14. [97] Chen Z, de Paiva CS, Luo L, Kretzer FL, Pflugfelder SC, Li D-Q. Characterization of putative stem cell phenotype in human limbal epithelia. Stem Cell 2004;22: 355–66. [98] Pajoohesh-Ganji A, Ghosh SP, Stepp MA. Regional distribution of α 9β1 integrin within the limbus of the mouse ocular surface. Dev Dynam 2004;230:518–28. [99] Kalha S, Shrestha B, Sanz Navarro M, Jones KB, Klein OD, Michon F. Bmi1+ progenitor cell dynamics in murine cornea during homeostasis and wound healing. Stem Cell 2018;36:562–73. [100] Echevarria TJ, Di Girolamo N. Tissue-regenerating, vision-restoring corneal epithelial stem cells. Stem Cell Reviews and Reports 2011;7:256–68. [101] Collinson JM, Morris L, Reid AI, Ramaesh T, Keighren MA, Flockhart JH, et al. Clonal analysis of patterns of growth, stem cell activity, and cell movement during the development and maintenance of the murine corneal epithelium. Dev Dynam 2002;224:432–40. [102] Douvaras P, Webb S, Whitaker DA, Dor` a N, Hill RE, Dorin JR, et al. Rare corneal clones in mice suggest an age-related decrease of stem cell activity and support the limbal epithelial stem cell hypothesis. Stem Cell Res 2012;8:109–19. [103] Amitai-Lange A, Altshuler A, Bubley J, Dbayat N, Tiosano B, Shalom-Feuerstein R. Lineage tracing of stem and progenitor cells of the murine corneal epithelium. Stem Cell 2015;33:230–9. [104] Di Girolamo N, Bobba S, Raviraj V, Delic NC, Slapetova I, Nicovich PR, et al. Tracing the fate of limbal epithelial progenitor cells in the murine cornea. Stem Cell 2015;33:157–69. [105] Richardson A, Lobo EP, Delic NC, Myerscough MR, Lyons JG, Wakefield D, et al. Keratin-14-positive precursor cells spawn a population of migratory corneal epithelia that maintain tissue mass throughout life. Stem cell reports 2017;9: 1081–96. [106] Nasser W, Amitai-Lange A, Soteriou D, Hanna R, Tiosano B, Fuchs Y, et al. Corneal-committed cells restore the stem cell pool and tissue boundary following injury. Cell Rep 2018;22:323–31. [107] Altshuler A, Amitai-Lange A, Tarazi N, Dey S, Strinkovsky L, Hadad-Porat S, et al. Discrete limbal epithelial stem cell populations mediate corneal homeostasis and wound healing. Cell Stem Cell 2021;28:1248–61. e8. L. Latta et al. The Ocular Surface 22 (2021) 245–266 263 [108] Bhattacharya S, Mukherjee A, Pisano S, Altshuler A, Nasser W, Dey S, et al. Soft limbal niche maintains stem cell compartmentalization and function through YAP. bioRxiv 2021:2021. 05.25.445490. [109] McKenna CC, Lwigale PY. Innervation of the mouse cornea during development. Invest Ophthalmol Vis Sci 2011;52:30–5. [110] Lwigale PY. Embryonic origin of avian corneal sensory nerves. Dev Biol 2001;239: 323–37. [111] Reneker LW, Silversides DW, Xu L, Overbeek PA. Formation of corneal endothelium is essential for anterior segment development - a transgenic mouse model of anterior segment dysgenesis. Development 2000;127:533–42. [112] Fujimura N. WNT/β-Catenin signaling in vertebrate eye development. Frontiers in Cell and Developmental Biology 2016;4. [113] Kawakami A, Kimura-Kawakami M, Nomura T, Fujisawa H. Distributions of PAX6 and PAX7 proteins suggest their involvement in both early and late phases of chick brain development. Mech Dev 1997;66:119–30. [114] Nishina S, Kohsaka S, Yamaguchi Y, Handa H, Kawakami A, Fujisawa H, et al. PAX6 expression in the developing human eye. Br J Ophthalmol 1999;83:723–7. [115] Pei YF, Rhodin JAG. The prenatal development of the mouse eye. Anat Rec 1970; 168:105–25. [116] Wulle KG, Lerche W. Electron microscopic observations of the early development of the human corneal endothelium and descemet’s membrane. Ophthalmologica 1969;157:451–61. [117] Feneck EM, Lewis PN, Meek KM. Identification of a primary stroma and novel endothelial cell projections in the developing human cornea. Invest Ophthalmol Vis Sci 2020;61:5. [118] O’Rahilly R. The prenatal development of the human eye. Exp Eye Res 1975;21: 93–112. [119] Peces-Pe˜ na MD, de la Cuadra-Blanco C, Vicente A, M´ erida-Velasco JR. Development of the ciliary body: morphological changes in the distal portion of the optic cup in the human. Cells Tissues Organs 2013;198:149–59. [120] Bard JBL, Hay ED, Meller SM. Formation of the endothelium of the avian cornea: a study of cell movement in vivo. Dev Biol 1975;42:334–61. [121] Renard G, Hirsch M, Savoldelli M, Pouliquen Y. The development of the iridocorneal angle in the chick embryo. Albrecht Von Graefes Arch Klin Exp Ophthalmol 1980;212:135–42. [122] Tsai PS, Evans JE, Green KM, Sullivan RM, Schaumberg DA, Richards SM, et al. Proteomic analysis of human meibomian gland secretions. Br J Ophthalmol 2006; 90:372–7. [123] Kumar S, Duester G. Retinoic acid signaling in perioptic mesenchyme represses Wnt signaling via induction of Pitx2 and Dkk2. Dev Biol 2010;340:67–74. [124] Enwright JF, Grainger RM. Altered retinoid signaling in the heads of small eye mouse embryos. Dev Biol 2000;221:10–22. [125] Latta L, Ludwig N, Krammes L, Stachon T, Fries FN, Mukwaya A, et al. Abnormal neovascular and proliferative conjunctival phenotype in limbal stem cell deficiency is associated with altered microRNA and gene expression modulated by PAX6 mutational status in congenital aniridia. Ocul Surf 2020. [126] Rabiee B, Anwar KN, Shen X, Putra I, Liu M, Jung R, et al. Gene dosage manipulation alleviates manifestations of hereditary <em>PAX6</em> haploinsufficiency in mice. Sci Transl Med 2020;12:eaaz4894. [127] Majo F, Rochat A, Nicolas M, Jaoude GA, Barrandon Y. Oligopotent stem cells are distributed throughout the mammalian ocular surface. Nature 2008;456:250–4. [128] Pellegrini G, Dellambra E, Golisano O, Martinelli E, Fantozzi I, Bondanza S, et al. p63 identifies keratinocyte stem cells. Proc Natl Acad Sci U S A 2001;98:3156–61. [129] Shalom-Feuerstein R, Lena AM, Zhou H, De La Forest Divonne S, Van Bokhoven H, Candi E, et al. ΔNp63 is an ectodermal gatekeeper of epidermal morphogenesis. Cell Death Differ 2011;18:887–96. [130] Li G, Xu F, Zhu J, Krawczyk M, Zhang Y, Yuan J, et al. Transcription Factor PAX6 (Paired Box 6) controls limbal stem cell lineage in development and disease. J Biol Chem 2015;290:20448–54. [131] Bhattacharya S, Serror L, Nir E, Dhiraj D, Altshuler A, Khreish M, et al. SOX2 regulates P63 and stem/progenitor cell state in the corneal epithelium. Stem Cell 2019;37:417–29. [132] Garg A, Zhang X. Lacrimal gland development: from signaling interactions to regenerative medicine. Dev Dynam 2017;246:970–80. [133] Garza-Leon M, Le´ on-Cach´ on RBR, Villafuerte-de la Cruz R, Martínez-Trevi˜ no DA. Infrared meibography and molecular assessment of p63 gene mutations in a Mexican patient with EEC syndrome. Arch Soc Esp Oftalmol 2018;93:562–6. [134] Di Iorio E, Kaye SB, Ponzin D, Barbaro V, Ferrari S, B¨ ohm E, et al. Limbal stem cell deficiency and ocular phenotype in ectrodactyly-ectodermal dysplasia-clefting syndrome caused by p63 mutations. Ophthalmology 2012;119:74–83. [135] Di Iorio E, Barbaro V, Ruzza A, Ponzin D, Pellegrini G, De Luca M. Isoforms of DeltaNp63 and the migration of ocular limbal cells in human corneal regeneration. Proc Natl Acad Sci U S A 2005;102:9523–8. [136] Guo ZH, Zhang W, Jia YYS, Liu QX, Li ZF, Lin JS. An insight into the difficulties in the discovery of specific biomarkers of limbal stem cells. Int J Mol Sci 2018;19: 1982. [137] Okada SL, Ellsworth JL, Durnam DM, Haugen HS, Holloway JL, Kelley ML, et al. A glycoprotein hormone expressed in corticotrophs exhibits unique binding properties on thyroid-stimulating hormone receptor. Mol Endocrinol 2006;20: 414–25. [138] Dou S, Wang Q, Qi X, Zhang B, Jiang H, Chen S, et al. Molecular identity of human limbal heterogeneity involved in corneal homeostasis and privilege. Ocul Surf 2021;21:206–20. [139] Collinson JM, Chanas SA, Hill RE, West JD. Corneal development, limbal stem cell function, and corneal epithelial cell migration in the Pax6 +/−mouse. Invest Ophthalmol Vis Sci 2004;45:1101–8. [140] Beebe DC, Coats JM. The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye. Dev Biol 2000;220:424–31. [141] Collomb E, Yang Y, Foriel S, Cadau S, Pearton DJ, Dhouailly D. The corneal epithelium and lens develop independently from a common pool of precursors. Dev Dynam 2013;242:401–13. [142] Smith JN, Walker HM, Thompson H, Collinson JM, Vargesson N, Erskine L. Lensregulated retinoic acid signalling controls expansion of the developing eye. Development 2018;145:dev167171. [143] Yamamoto Y, Jeffery WR. Central role for the lens in cave fish eye degeneration. Science 2000;289:631–3. [144] Collinson JM, Quinn JC, Buchanan MA, Kaufman MH, Wedden SE, West JD, et al. Primary defects in the lens underlie complex anterior segment abnormalities of the <em>Pax6</em>heterozygous eye. Proc Natl Acad Sci Unit States Am 2001;98:9688–93. [145] Kroeber M, Davis N, Holzmann S, Kritzenberger M, Shelah-Goraly M, Ofri R, et al. Reduced expression of Pax6 in lens and cornea of mutant mice leads to failure of chamber angle development and juvenile glaucoma. Hum Mol Genet 2010;19: 3332–42. [146] Klimova L, Kozmik Z. Stage-dependent requirement of neuroretinal Pax6 for lens and retina development. Development 2014;141:1292–302. [147] Shiple D, Finklea B, Lauderdale JD, Netland PA. Keratopathy, cataract, and dry eye in a survey of aniridia subjects. Clin Ophthalmol 2015;9:291–5. [148] Schneider S, Osher RH, Burk SE, Lutz TB, Montione R. Thinning of the anterior capsule associated with congenital aniridia. J Cataract Refract Surg 2003;29: 523–5. [149] Martinez G, de Iongh RU. The lens epithelium in ocular health and disease. Int J Biochem Cell Biol 2010;42:1945–63. [150] Ed´ en U, Lagali N, Dellby A, Utheim TP, Riise R, Chen X, et al. Cataract development in Norwegian patients with congenital aniridia. Acta Ophthalmol 2014;92:e165–7. [151] Wolf M, Zabel B, Lorenz B, Blankenagel A, Ghorbani MB, Schwenn O, et al. [Molecular genetic study of the PAX6 gene in aniridia patients]. Ophthalmologe 1998;95:828–30. [152] Tornqvist K. Aniridia: sight-threatening and hard to cure. Acta Ophthalmol 2008; 86:704–5. [153] Ou J, Lowes C, Collinson JM. Cytoskeletal and cell adhesion defects in wounded and Pax6+/- corneal epithelia. Invest Ophthalmol Vis Sci 2010;51:1415–23. [154] Ou J, Walczysko P, Kucerova R, Rajnicek AM, McCaig CD, Zhao M, et al. Chronic wound state exacerbated by oxidative stress in Pax6+/- aniridia-related keratopathy. J Pathol 2008;215:421–30. [155] Davis N, Yoffe C, Raviv S, Antes R, Berger J, Holzmann S, et al. Pax6 dosage requirements in iris and ciliary body differentiation. Dev Biol 2009;333:132–42. [156] Wang X, Shan X, Gregory-Evans CY. A mouse model of aniridia reveals the in vivo downstream targets of Pax6 driving iris and ciliary body development in the eye. Biochim Biophys Acta (BBA) - Mol Basis Dis 2017;1863:60–7. [157] Ittner LM, Wurdak H, Schwerdtfeger K, Kunz T, Ille F, Leveen P, et al. Compound developmental eye disorders following inactivation of TGFβ signaling in neuralcrest stem cells. J Biol 2005;4:11. [158] Nelson LB, Spaeth GL, Nowinski TS, Margo CE, Jackson L. Aniridia. A review. Survey of Ophthalmology. 1984;28:621–42. [159] Br´ emond-Gignac D. Le glaucome dans l’aniridie. J Fr Ophtalmol 2007;30:196–9. [160] Brandt JD, Casuso LA, Budenz DL. Markedly increased central corneal thickness: an unrecognized finding in congenital aniridia. Am J Ophthalmol 2004;137: 348–50. [161] Whitson JT, Liang C, Godfrey DG, Petroll WM, Cavanagh HD, Patel D, et al. Central corneal thickness in patients with congenital aniridia. Eye Contact Lens 2005;31:221–4. [162] Kirwan C, O’Keefe M, Fitzsimon S. Central corneal thickness and corneal diameter in premature infants. Acta Ophthalmol Scand 2005;83:751–3. [163] Baulmann DC, Ohlmann A, Flügel-Koch C, Goswami S, Cvekl A, Tamm ER. Pax6 heterozygous eyes show defects in chamber angle differentiation that are associated with a wide spectrum of other anterior eye segment abnormalities. Mech Dev 2002;118:3–17. [164] Collinson JM, Quinn JC, Hill RE, West JD. The roles of Pax6 in the cornea, retina, and olfactory epithelium of the developing mouse embryo. Dev Biol 2003;255: 303–12. [165] Funderburgh ML, Du Y, Mann MM, SundarRaj N, Funderburgh JL. PAX6 expression identifies progenitor cells for corneal keratocytes. Faseb J 2005;19: 1371–3. [166] Cvekl A, Tamm ER. Anterior eye development and ocular mesenchyme: new insights from mouse models and human diseases. Bioessays : news and reviews in molecular, cellular and developmental biology 2004;26:374–86. [167] Kamachi Y, Uchikawa M, Tanouchi A, Sekido R, Kondoh H. Pax6 and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens development. Genes Dev 2001;15:1272–86. [168] Cooper ST, Hanson IM. A screen for proteins that interact with PAX6: C-terminal mutations disrupt interaction with HOMER3, DNCL1 and TRIM11. BMC Genet 2005;6:43. [169] Chauhan BK, Reed NA, Zhang W, Duncan MK, Kilimann MW, Cvekl A. Identification of genes downstream of Pax6 in the mouse lens using cDNA microarrays. J Biol Chem 2002;277:11539–48. [170] Matsuo T, Osumi-Yamashita N, Noji S, Ohuchi H, Koyama E, Myokai F, et al. A mutation in the Pax-6 gene in rat small eye is associated with impaired migration of midbrain crest cells. Nat Genet 1993;3:299–304. [171] Aragona P, Rolando M. Towards a dynamic customised therapy for ocular surface dysfunctions. Br J Ophthalmol 2013;97:955. L. Latta et al. The Ocular Surface 22 (2021) 245–266 264 [172] Hogan BLM, Horsburgh G, Cohen J, Hetherington CM, Fisher G, Lyon MF. em>Small eyes</em>(<em>Sey</em>): a homozygous lethal mutation on chromosome 2 which affects the differentiation of both lens and nasal placodes in the mouse. J Embryol Exp Morphol 1986;97:95–110. [173] Makarenkova HP, Ito M, Govindarajan V, Faber SC, Sun L, McMahon G, et al. FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development. Development 2000;127:2563–72. [174] Bannier-H´ elaou¨ et M, Post Y, Korving J, Trani Bustos M, Gehart H, Begthel H, et al. Exploring the human lacrimal gland using organoids and single-cell sequencing. Cell Stem Cell 2021. [175] Hirayama M, Ko SBH, Kawakita T, Akiyama T, Goparaju SK, Soma A, et al. Identification of transcription factors that promote the differentiation of human pluripotent stem cells into lacrimal gland epithelium-like cells. npj Aging and Mechanisms of Disease 2017;3:1. [176] Wang J, Call M, Mongan M, Kao WW-Y, Xia Y. Meibomian gland morphogenesis requires developmental eyelid closure and lid fusion. Ocul Surf 2017;15:704–12. [177] Shi F, Fan Y, Zhang L, Meng L, Zhi H, Hu H, et al. The expression of Pax6 variants is subject to posttranscriptional regulation in the developing mouse eyelid. PloS One 2013;8:e53919. [178] Call M, Fischesser K, Lunn MO, Kao WW. A unique lineage gives rise to the meibomian gland. Mol Vis 2016;22:168–76. [179] Landsend ECS, Utheim ØA, Pedersen HR, Aass HCD, Lagali N, Dartt DA, et al. The level of inflammatory tear cytokines is elevated in congenital aniridia and associated with meibomian gland dysfunction. Invest Ophthalmol Vis Sci 2018; 59:2197–204. [180] Jastaneiah S, Al-Rajhi AA. Association of aniridia and dry eyes. Ophthalmology 2005;112:1535–40. [181] Landsend ECS, Pedersen HR, Utheim ØA, Xiao J, Adil MY, Tashbayev B, et al. Meibomian gland dysfunction and keratopathy are associated with dry eye disease in aniridia. Br J Ophthalmol 2019;103:119–24. [182] Ihnatko R, Ed´ en U, Lagali N, Dellby A, Fagerholm P. Analysis of protein composition and protein expression in the tear fluid of patients with congenital aniridia. Journal of Proteomics 2013;94:78–88. [183] Landsend ECS, Utheim OA, Pedersen HR, Aass HCD, Lagali N, Dartt DA, et al. The level of inflammatory tear cytokines is elevated in congenital aniridia and associated with meibomian gland dysfunction. Invest Ophthalmol Vis Sci 2018; 59:2197–204. [184] Bonnar E, Logan P, Eustace P. Absent meibomian glands: a marker for EEC syndrome. Eye 1996;10:355–61. [185] Townsend WM. The limbal palisades of Vogt. Trans Am Ophthalmol Soc 1991;89: 721–56. [186] Shortt AJ, Secker GA, Munro PM, Khaw PT, Tuft SJ, Daniels JT. Characterization of the limbal epithelial stem cell niche: novel imaging techniques permit in vivo observation and targeted biopsy of limbal epithelial stem cells. Stem Cell 2007; 25:1402–9. [187] Li W, Hayashida Y, Chen Y-T, Tseng SCG. Niche regulation of corneal epithelial stem cells at the limbus. Cell Res 2007;17:26–36. [188] Shanmuganathan VA, Foster T, Kulkarni BB, Hopkinson A, Gray T, Powe DG, et al. Morphological characteristics of the limbal epithelial crypt. Br J Ophthalmol 2007;91:514–9. [189] Cotsarelis G, Cheng SZ, Dong G, Sun TT, Lavker RM. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell 1989;57:201–9. [190] Sun TT, Tseng SC, Lavker RM. Location of corneal epithelial stem cells. Nature 2010;463:E10–1. discussion E1. [191] Daniels JT, Dart JKG, Tuft SJ, Khaw PT. Corneal stem cells in review. Wound Repair Regen 2001;9:483–94. [192] Farrelly O, Suzuki-Horiuchi Y, Brewster M, Kuri P, Huang S, Rice G, et al. Twophoton live imaging of single corneal stem cells reveals compartmentalized organization of the limbal niche. Cell Stem Cell 2021;28:1233–47. e4. [193] Tseng SC. Regulation and clinical implications of corneal epithelial stem cells. Mol Biol Rep 1996;23:47–58. [194] Dua HS, Azuara-Blanco A. Limbal stem cells of the corneal epithelium. Surv Ophthalmol 2000;44:415–25. [195] Puangsricharern V, Tseng SC. Cytologic evidence of corneal diseases with limbal stem cell deficiency. Ophthalmology 1995;102:1476–85. [196] Li W, Chen YT, Hayashida Y, Blanco G, Kheirkah A, He H, et al. Down-regulation of Pax6 is associated with abnormal differentiation of corneal epithelial cells in severe ocular surface diseases. J Pathol 2008;214:114–22. [197] Dziasko MA, Daniels JT. Anatomical features and cell-cell interactions in the human limbal epithelial stem cell niche. Ocul Surf 2016;14:322–30. [198] Dua HS, Miri A, Alomar T, Yeung AM, Said DG. The role of limbal stem cells in corneal epithelial maintenance: testing the dogma. Ophthalmology 2009;116: 856–63. [199] Huang AJ, Tseng SC. Corneal epithelial wound healing in the absence of limbal epithelium. Invest Ophthalmol Vis Sci 1991;32:96–105. [200] Blanpain C, Fuchs E. Stem cell plasticity. Plasticity of epithelial stem cells in tissue regeneration. Science 2014;344:1242281. [201] Rama P, Matuska S, Paganoni G, Spinelli A, De Luca M, Pellegrini G. Limbal stemcell therapy and long-term corneal regeneration. N Engl J Med 2010;363:147–55. [202] Kolli S, Ahmad S, Lako M, Figueiredo F. Successful clinical implementation of corneal epithelial stem cell therapy for treatment of unilateral limbal stem cell deficiency. Stem Cell 2010;28:597–610. [203] Ouyang H, Xue Y, Lin Y, Zhang X, Xi L, Patel S, et al. WNT7A and PAX6 define corneal epithelium homeostasis and pathogenesis. Nature 2014;511:358–61. [204] Le Q, Deng SX, Xu J. In vivo confocal microscopy of congenital aniridia-associated keratopathy. Eye 2013;27:763–6. [205] Schl¨ otzer-Schrehardt U, Latta L, Gießl A, Zenkel M, Fries FN, K¨ asmann-Kellner B, et al. Dysfunction of the limbal epithelial stem cell niche in aniridia-associated keratopathy. Ocul Surf 2021;21:160–73. [206] Voskresenskaya A, Pozdeyeva N, Vasilyeva T, Batkov Y, Shipunov A, Gagloev B, et al. Clinical and morphological manifestations of aniridia-associated keratopathy on anterior segment optical coherence tomography and in vivo confocal microscopy. Ocul Surf 2017;15:759–69. [207] Lagali N, Ed´ en U, Utheim TP, Chen X, Riise R, Dellby A, et al. In vivo morphology of the limbal palisades of vogt correlates with progressive stem cell deficiency in aniridia-related keratopathy. Invest Ophthalmol Vis Sci 2013;54:5333–42. [208] Chen YT, Chen FY, Vijmasi T, Stephens DN, Gallup M, McNamara NA. Pax6 downregulation mediates abnormal lineage commitment of the ocular surface epithelium in aqueous-deficient dry eye disease. PloS One 2013;8:e77286. [209] McNamara NA, Gallup M, Porco TC. Establishing PAX6 as a biomarker to detect early loss of ocular phenotype in human patients with Sjogren’s syndrome. Invest Ophthalmol Vis Sci 2014;55:7079–84. [210] Ljubimov AV, Saghizadeh M. Progress in corneal wound healing. Prog Retin Eye Res 2015;49:17–45. [211] Swamynathan SK, Katz JP, Kaestner KH, Ashery-Padan R, Crawford MA, Piatigorsky J. Conditional deletion of the mouse Klf4 gene results in corneal epithelial fragility, stromal edema, and loss of conjunctival goblet cells. Mol Cell Biol 2007;27:182–94. [212] Tiwari A, Loughner CL, Swamynathan S, Swamynathan SK. KLF4 plays an essential role in corneal epithelial homeostasis by promoting epithelial cell fate and suppressing epithelial-mesenchymal transition. Invest Ophthalmol Vis Sci 2017;58:2785–95. [213] Reis LM, Tyler RC, Volkmann Kloss BA, Schilter KF, Levin AV, Lowry RB, et al. PITX2 and FOXC1 spectrum of mutations in ocular syndromes. Eur J Hum Genet 2012;20:1224–33. [214] Samant M, Chauhan BK, Lathrop KL, Nischal KK. Congenital aniridia: etiology, manifestations and management. Expet Rev Ophthalmol 2016;11:135–44. [215] Leiper LJ, Walczysko P, Kucerova R, Ou J, Shanley LJ, Lawson D, et al. The roles of calcium signaling and ERK1/2 phosphorylation in a Pax6+/- mouse model of epithelial wound-healing delay. BMC Biol 2006;4:27. [216] Harmon RM, Simpson CL, Johnson JL, Koetsier JL, Dubash AD, Najor NA, et al. Desmoglein-1/Erbin interaction suppresses ERK activation to support epidermal differentiation. J Clin Invest 2013;123:1556–70. [217] Pajoohesh-Ganji A, Pal-Ghosh S, Tadvalkar G, Stepp MA. Corneal goblet cells and their niche: implications for corneal stem cell deficiency. Stem Cell 2012;30: 2032–43. [218] Ramos T, Scott D, Ahmad S. An update on ocular surface epithelial stem cells: cornea and conjunctiva. Stem Cell Int 2015;2015:601731. [219] Bertolin M, Breda C, Ferrari S, Van Acker SI, Zakaria N, Di Iorio E, et al. Optimized protocol for regeneration of the conjunctival epithelium using the cell suspension technique. Cornea 2019;38:469–79. [220] Roux Isabelle P, Romain D, Jean-Paul C, Jieqiong Q, Huiqing Z, et al. Modeling of aniridia-related keratopathy by CRISPR/Cas9 genome editing of human limbal epithelial cells and rescue by recombinant PAX6 protein. Stem Cell 2018. [221] Douvaras P, Mort RL, Edwards D, Ramaesh K, Dhillon B, Morley SD, et al. Increased corneal epithelial turnover contributes to abnormal homeostasis in the Pax6(+/-) mouse model of aniridia. PloS One 2013;8:e71117. [222] Barbaro V, Nasti AA, Del Vecchio C, Ferrari S, Migliorati A, Raffa P, et al. Correction of mutant p63 in EEC syndrome using siRNA mediated allele-specific silencing restores defective stem cell function. Stem Cell 2016;34:1588–600. [223] Walczysko P, Rajnicek AM, Collinson JM. Contact-mediated control of radial migration of corneal epithelial cells. Mol Vis 2016;22:990–1004. [224] Azar DT. Corneal angiogenic privilege: angiogenic and antiangiogenic factors in corneal avascularity, vasculogenesis, and wound healing (an American Ophthalmological Society thesis). Trans Am Ophthalmol Soc 2006;104:264–302. [225] Ambati BK, Nozaki M, Singh N, Takeda A, Jani PD, Suthar T, et al. Corneal avascularity is due to soluble VEGF receptor-1. Nature 2006;443:993–7. [226] Shalom-Feuerstein R, Serror L, De La Forest Divonne S, Petit I, Aberdam E, Camargo L, et al. Pluripotent stem cell model reveals essential roles for miR-450b5p and miR-184 in embryonic corneal lineage specification. Stem Cell 2012;30: 898–909. [227] Ryan DG, Oliveira-Fernandes M, Lavker RM. MicroRNAs of the mammalian eye display distinct and overlapping tissue specificity. Mol Vis 2006;12:1175–84. [228] Park JK, Peng H, Yang W, Katsnelson J, Volpert O, Lavker RM. miR-184 exhibits angiostatic properties via regulation of Akt and VEGF signaling pathways. Faseb J 2017;31:256–65. [229] Gao J, Wang Y, Zhao X, Chen P, Xie L. MicroRNA-204-5p-Mediated regulation of SIRT1 contributes to the delay of epithelial cell cycle traversal in diabetic corneas. Invest Ophthalmol Vis Sci 2015;56:1493–504. [230] Lu Y, Tai PWL, Ai J, Gessler DJ, Su Q, Yao X, et al. Transcriptome profiling of neovascularized corneas reveals miR-204 as a multi-target biotherapy deliverable by rAAVs. Mol Ther Nucleic Acids 2018;10:349–60. [231] Shiels A. TRPM3_miR-204: a complex locus for eye development and disease. Hum Genom 2020;14:7. [232] Shaham O, Gueta K, Mor E, Oren-Giladi P, Grinberg D, Xie Q, et al. Pax6 regulates gene expression in the vertebrate lens through miR-204. PLoS Genet 2013;9: e1003357. [233] Xie Q, Yang Y, Huang J, Ninkovic J, Walcher T, Wolf L, et al. Pax6 interactions with chromatin and identification of its novel direct target genes in lens and forebrain. PloS One 2013;8:e54507. L. Latta et al. The Ocular Surface 22 (2021) 245–266 265 [234] Xie Q, Ung D, Khafizov K, Fiser A, Cvekl A. Gene regulation by PAX6: structuralfunctional correlations of missense mutants and transcriptional control of Trpm3/ miR-204. Mol Vis 2014;20:270–82. [235] Conte I, Carrella S, Avellino R, Karali M, Marco-Ferreres R, Bovolenta P, et al. miR-204 is required for lens and retinal development via <em>Meis2</em> targeting. Proc Natl Acad Sci Unit States Am 2010;107:15491–6. [236] Mukwaya A, Jensen L, Peebo B, Lagali N. MicroRNAs in the cornea: role and implications for treatment of corneal neovascularization. Ocul Surf 2019;17: 400–11. [237] Lapid-Gortzak R, Santana NTY, Nieuwendaal CP, Mourits MP, van der Meulen IJE. Topical bevacizumab treatment in aniridia. Int Ophthalmol 2018;38: 1741–6. [238] Nowak JA, Polak L, Pasolli HA, Fuchs E. Hair follicle stem cells are specified and function in early skin morphogenesis. Cell Stem Cell 2008;3:33–43. [239] Rompolas P, Greco V. Stem cell dynamics in the hair follicle niche. Semin Cell Dev Biol 2014;25–26:34–42. [240] Fujiwara H, Ferreira M, Donati G, Marciano DK, Linton JM, Sato Y, et al. The basement membrane of hair follicle stem cells is a muscle cell niche. Cell 2011; 144:577–89. [241] Pinnamaneni N, Funderburgh JL. Concise review: stem cells in the corneal stroma. Stem Cell 2012;30:1059–63. [242] Pearton DJ, Yang Y, Dhouailly D. Transdifferentiation of corneal epithelium into epidermis occurs by means of a multistep process triggered by dermal developmental signals. Proc Natl Acad Sci U S A 2005;102:3714–9. [243] Xie H-T, Chen S-Y, Li G-G, Tseng SCG. Limbal epithelial stem/progenitor cells attract stromal niche cells by SDF-1/CXCR4 signaling to prevent differentiation. Stem Cell 2011;29:1874–85. [244] Gur-Cohen S, Yang H, Baksh SC, Miao Y, Levorse J, Kataru RP, et al. Stem cell–driven lymphatic remodeling coordinates tissue regeneration. Science 2019; 366:1218–25. [245] Kolega J, Manabe M, Sun T-T. Basement membrane heterogeneity and variation in corneal epithelial differentiation. Differentiation 1989;42:54–63. [246] Ljubimov AV, Burgeson RE, Butkowski RJ, Michael AF, Sun TT, Kenney MC. Human corneal basement membrane heterogeneity: topographical differences in the expression of type IV collagen and laminin isoforms. Lab Invest 1995;72: 461–73. [247] Polisetti N, Zenkel M, Menzel-Severing J, Kruse FE, Schlotzer-Schrehardt U. Cell adhesion molecules and stem cell-niche-interactions in the limbal stem cell niche. Stem Cell 2016;34:203–19. [248] Higa K, Kato N, Yoshida S, Ogawa Y, Shimazaki J, Tsubota K, et al. Aquaporin 1positive stromal niche-like cells directly interact with N-cadherin-positive clusters in the basal limbal epithelium. Stem Cell Res 2013;10:147–55. [249] Dziasko MA, Armer HE, Levis HJ, Shortt AJ, Tuft S, Daniels JT. Localisation of epithelial cells capable of holoclone formation in vitro and direct interaction with stromal cells in the native human limbal crypt. PloS One 2014;9:e94283. [250] Polisetti N, Zenkel M, Menzel-Severing J, Kruse FE, Schl¨ otzer-Schrehardt U. Cell adhesion molecules and stem cell-niche-interactions in the limbal stem cell niche. Stem Cell 2016;34:203–19. [251] Higa K, Shimmura S, Miyashita H, Kato N, Ogawa Y, Kawakita T, et al. N-cadherin in the maintenance of human corneal limbal epithelial progenitor cells in vitro. Invest Ophthalmol Vis Sci 2009;50:4640–5. [252] Dziasko MA, Tuft SJ, Daniels JT. Limbal melanocytes support limbal epithelial stem cells in 2D and 3D microenvironments. Exp Eye Res 2015;138:70–9. [253] Funderburgh JL, Funderburgh ML, Du Y. Stem cells in the limbal stroma. Ocul Surf 2016;14:113–20. [254] Raviv S, Bharti K, Rencus-Lazar S, Cohen-Tayar Y, Schyr R, Evantal N, et al. PAX6 regulates melanogenesis in the retinal pigmented epithelium through feedforward regulatory interactions with MITF. PLoS Genet 2014;10. e1004360-e. [255] Nakatsu MN, Gonz´ alez S, Mei H, Deng SX. Human limbal mesenchymal cells support the growth of human corneal epithelial stem/progenitor cells. Invest Ophthalmol Vis Sci 2014;55:6953–9. [256] Gouveia RM, Lepert G, Gupta S, Mohan RR, Paterson C, Connon CJ. Assessment of corneal substrate biomechanics and its effect on epithelial stem cell maintenance and differentiation. Nat Commun 2019;10:1496. [257] Kang YG, Shin JW, Park SH, Oh M-J, Park HS, Shin J-W, et al. Effects of flowinduced shear stress on limbal epithelial stem cell growth and enrichment. PloS One 2014;9. e93023-e. [258] Vicente A, Bystr¨ om B, Lindstr¨ om M, Stenevi U, Pedrosa Domell¨ of F. Aniridiarelated keratopathy: structural changes in naïve and transplanted corneal buttons. PloS One 2018;13:e0198822. [259] Mort RL, Bentley AJ, Martin FL, Collinson JM, Douvaras P, Hill RE, et al. Effects of aberrant Pax6 gene dosage on mouse corneal pathophysiology and corneal epithelial homeostasis. PloS One 2011;6:e28895. [260] Ramaesh T, Ramaesh K, Leask R, Springbett A, Riley SC, Dhillon B, et al. Increased apoptosis and abnormal wound-healing responses in the heterozygous Pax6 +/−mouse cornea. Invest Ophthalmol Vis Sci 2006;47:1911–7. [261] Espana EM, Kawakita T, Romano A, Di Pascuale M, Smiddy R, Liu C-y, et al. Stromal niche controls the plasticity of limbal and corneal epithelial differentiation in a rabbit model of recombined tissue. Invest Ophthalmol Vis Sci 2003;44:5130–5. [262] Zhang Y, Yeh L-K, Zhang S, Call M, Yuan Y, Yasunaga M, et al. Wnt/β-catenin signaling modulates corneal epithelium stratification via inhibition of Bmp4 during mouse development. Development (Cambridge, England) 2015;142: 3383–93. [263] Vicente A, Bystr¨ om B, Pedrosa Domell¨ of F. Altered signaling pathways in aniridiarelated KeratopathySignaling pathways in aniridia-related keratopathy. Invest Ophthalmol Vis Sci 2018;59:5531–41. [264] Bausili MM, Alvarez de Toledo J, Barraquer RI, Michael R, Tresserra F, de la Paz MF. Histopathology findings of corneal buttons in congenital aniridia patients. Ophthalmic Res 2016;56:202–6. [265] Planella S, de la Paz MF, de Toledo JA. Lamellar and penetrating keratoplasty in congenital aniridia. In: Parekh M, Poli B, Ferrari S, Teofili C, Ponzin D, editors. Aniridia: recent developments in scientific and clinical research. Cham: Springer International Publishing; 2015. p. 63–74. [266] Prochiantz A, Di Nardo AA. Homeoprotein signaling in the developing and adult nervous system. Neuron 2015;85:911–25. [267] Lesaffre B, Joliot A, Prochiantz A, Volovitch M. Direct non-cell autonomous Pax6 activity regulates eye development in the zebrafish. Neural Dev 2007;2:2. [268] Wei ZG, Cotsarelis G, Sun TT, Lavker RM. Label-retaining cells are preferentially located in fornical epithelium: implications on conjunctival epithelial homeostasis. Invest Ophthalmol Vis Sci 1995;36:236–46. [269] Latta L, Nordstr¨ om K, Stachon T, Langenbucher A, Fries FN, Szentm´ ary N, et al. Expression of retinoic acid signaling components ADH7 and ALDH1A1 is reduced in aniridia limbal epithelial cells and a siRNA primary cell based aniridia model. Exp Eye Res 2019;179:8–17. [270] Ed´ en U, Fagerholm P, Danyali R, Lagali N. Pathologic epithelial and anterior corneal nerve morphology in early-stage congenital aniridic keratopathy. Ophthalmology 2012;119:1803–10. [271] Tsai RJ, Tseng SC. Effect of stromal inflammation on the outcome of limbal transplantation for corneal surface reconstruction. Cornea 1995;14:439–49. [272] Hertsenberg AJ, Shojaati G, Funderburgh ML, Mann MM, Du Y, Funderburgh JL. Corneal stromal stem cells reduce corneal scarring by mediating neutrophil infiltration after wounding. PloS One 2017;12:e0171712. [273] Shojaati G, Khandaker I, Funderburgh ML, Mann MM, Basu R, Stolz DB, et al. Mesenchymal stem cells reduce corneal fibrosis and inflammation via extracellular vesicle-mediated delivery of miRNA. Stem cells translational medicine 2019;8:1192–201. [274] Calonge M, P´ erez I, Galindo S, Nieto-Miguel T, L´ opez-Paniagua M, Fern´ andez I, et al. A proof-of-concept clinical trial using mesenchymal stem cells for the treatment of corneal epithelial stem cell deficiency. Transl Res 2019;206:18–40. [275] Li Z, Burns AR, Rumbaut RE, Smith CW. gamma delta T cells are necessary for platelet and neutrophil accumulation in limbal vessels and efficient epithelial repair after corneal abrasion. Am J Pathol 2007;171:838–45. [276] Mastropasqua L, Nubile M, Lanzini M, Carpineto P, Ciancaglini M, Pannellini T, et al. Epithelial dendritic cell distribution in normal and inflamed human cornea: in vivo confocal microscopy study. Am J Ophthalmol 2006;142. 736-44.e2. [277] Mayer WJ, Mackert MJ, Kranebitter N, Messmer EM, Grüterich M, Kampik A, et al. Distribution of antigen presenting cells in the human cornea: correlation of in vivo confocal microscopy and immunohistochemistry in different pathologic entities. Curr Eye Res 2012;37:1012–8. [278] Notara M, Shortt AJ, O’Callaghan AR, Daniels JT. The impact of age on the physical and cellular properties of the human limbal stem cell niche. Age (Dordr). 2013;35:289–300. [279] Mastropasqua L, Massaro-Giordano G, Nubile M, Sacchetti M. Understanding the pathogenesis of neurotrophic keratitis: the role of corneal nerves. J Cell Physiol 2017;232:717–24. [280] Müller LJ, Marfurt CF, Kruse F, Tervo TMT. Corneal nerves: structure, contents and function. Exp Eye Res 2003;76:521–42. [281] Shaheen BS, Bakir M, Jain S. Corneal nerves in health and disease. Surv Ophthalmol 2014;59:263–85. [282] Nishida T, Chikama T-i, Morishige N, Yanai R, Yamada N, Saito J. Persistent epithelial defects due to neurotrophic keratopathy treated with a substance pderived peptide and insulin-like growth factor 1. Jpn J Ophthalmol 2007;51: 442–7. [283] Gao N, Lee P, Yu F-S. Intraepithelial dendritic cells and sensory nerves are structurally associated and functional interdependent in the cornea. Sci Rep 2016; 6:36414. [284] Perry VH, Brown MC, Gordon S. The macrophage response to central and peripheral nerve injury. A possible role for macrophages in regeneration. J Exp Med 1987;165:1218–23. [285] Seyed-Razavi Y, Chinnery HR, McMenamin PG. A novel association between resident tissue macrophages and nerves in the peripheral stroma of the murine cornea. Invest Ophthalmol Vis Sci 2014;55:1313–20. [286] Ihnatko R, Eden U, Fagerholm P, Lagali N. Congenital aniridia and the ocular surface. Ocul Surf 2016;14:196–206. [287] Lawrenson JG, Ruskell GL. The structure of corpuscular nerve endings in the limbal conjunctiva of the human eye. J Anat 1991;177:75–84. [288] Ueno H, Ferrari G, Hattori T, Saban DR, Katikireddy KR, Chauhan SK, et al. Dependence of corneal stem/progenitor cells on ocular surface innervation. Invest Ophthalmol Vis Sci 2012;53:867–72. [289] Higa K, Higuchi J, Kimoto R, Miyashita H, Shimazaki J, Tsubota K, et al. Human corneal limbal organoids maintaining limbal stem cell niche function. Stem Cell Res 2020;49:102012. [290] Ferrari G, Hajrasouliha AR, Sadrai Z, Ueno H, Chauhan SK, Dana R. Nerves and neovessels inhibit each other in the cornea. Invest Ophthalmol Vis Sci 2013;54: 813–20. [291] Liu L, Dana R, Yin J. Sensory neurons directly promote angiogenesis in response to inflammation via substance P signaling. Faseb J 2020;34:6229–43. [292] Li Z, Burns AR, Han L, Rumbaut RE, Smith CW. IL-17 and VEGF are necessary for efficient corneal nerve regeneration. Am J Pathol 2011;178:1106–16. L. Latta et al. The Ocular Surface 22 (2021) 245–266 266 [293] Yu CQ, Zhang M, Matis KI, Kim C, Rosenblatt MI. Vascular endothelial growth factor mediates corneal nerve repair. Invest Ophthalmol Vis Sci 2008;49:3870–8. [294] Aggarwal S, Colon C, Kheirkhah A, Hamrah P. Efficacy of autologous serum tears for treatment of neuropathic corneal pain. Ocul Surf 2019;17:532–9. [295] Yoon CH, Lee HJ, Park HY, Kim H, Kim MK, Jeoung JW, et al. Effects of topical autologous serum on the ocular surface in patients with toxic corneal epitheliopathy induced by anti-glaucoma drugs. Int Ophthalmol 2020;40:547–52. [296] L´ opez-García JS, Rivas L, García-Lozano I, Murube J. Autologous serum eyedrops in the treatment of aniridic keratopathy. Ophthalmology 2008;115:262–7. [297] Sheha H, Tighe S, Hashem O, Hayashida Y. Update on Cenegermin eye drops in the treatment of neurotrophic keratitis. Clin Ophthalmol 2019;13:1973–80. [298] Pflugfelder SC, Massaro-Giordano M, Perez VL, Hamrah P, Deng SX, Espandar L, et al. Topical recombinant human nerve growth factor (Cenegermin) for neurotrophic keratopathy: a multicenter randomized vehicle-controlled pivotal trial. Ophthalmology 2020;127:14–26. [299] Mastropasqua L, Lanzini M, Dua HS, D’ Uffizi A, Di Nicola M, Calienno R, et al. In vivo evaluation of corneal nerves and epithelial healing after treatment with recombinant nerve growth factor for neurotrophic keratopathy. Am J Ophthalmol 2020;217:278–86. [300] Vasilyeva TA, Marakhonov AV, Voskresenskaya AA, Kadyshev VV, K¨ asmannKellner B, Sukhanova NV, et al. Analysis of genotype-phenotype correlations in PAX6-associated aniridia. J Med Genet 2020. [301] Law SK, Sami M, Piri N, Coleman AL, Caprioli J. Asymmetric phenotype of Axenfeld-Rieger anomaly and aniridia associated with a novel PITX2 mutation. Mol Vis 2011;17:1231–8. [302] Ito YA, Footz TK, Berry FB, Mirzayans F, Yu M, Khan AO, et al. Severe molecular defects of a novel FOXC1 W152G mutation result in aniridia. Invest Ophthalmol Vis Sci 2009;50:3573–9. [303] Ansari M, Rainger J, Hanson IM, Williamson KA, Sharkey F, Harewood L, et al. Genetic analysis of ‘PAX6-negative’ individuals with aniridia or Gillespie syndrome. PloS One 2016;11:e0153757. [304] Landsend ES, Utheim Ø A, Pedersen HR, Lagali N, Baraas RC, Utheim TP. The genetics of congenital aniridia-a guide for the ophthalmologist. Surv Ophthalmol 2018;63:105–13. [305] Yongblah K, Alford SC, Ryan BC, Chow RL, Howard PL. Protecting Pax6 3’ UTR from MicroRNA-7 partially restores PAX6 in islets from an aniridia mouse model. Mol Ther Nucleic Acids 2018;13:144–53. [306] Macdonald R, Wilson SW. Distribution of Pax6 protein during eye development suggests discrete roles in proliferative and differentiated visual cells. Dev Gene Evol 1997;206:363–9. [307] Ranaei Pirmardan E, Soheili Z-S, Samiei S, Ahmadieh H, Mowla SJ, Naseri M, et al. In vivo evaluation of PAX6 overexpression and NMDA cytotoxicity to stimulate proliferation in the mouse retina. Sci Rep 2018;8:17700. [308] Bard JB, Hay ED. The behavior of fibroblasts from the developing avian cornea. Morphology and movement in situ and in vitro. J Cell Biol 1975;67:400–18. [309] Ramaesh K, Ramaesh T, Dutton GN, Dhillon B. Evolving concepts on the pathogenic mechanisms of aniridia related keratopathy. Int J Biochem Cell Biol 2005;37:547–57. [310] Latta L, Viestenz A, Stachon T, Colanesi S, Szentm´ ary N, Seitz B, et al. Human aniridia limbal epithelial cells lack expression of keratins K3 and K12. Exp Eye Res 2018;167:100–9. [311] Sivak JM, Mohan R, Rinehart WB, Xu P-X, Maas RL, Fini ME. Pax-6 expression and activity are induced in the reepithelializing cornea and control activity of the transcriptional promoter for matrix metalloproteinase gelatinase B. Dev Biol 2000;222:41–54. [312] Leiper LJ, Ou J, Walczysko P, Kucerova R, Lavery DN, West JD, et al. Control of patterns of corneal innervation by Pax6. Invest Ophthalmol Vis Sci 2009;50: 1122–8. L. Latta et al.