Molecular regulation of ocular gland development

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Review

Molecular regulation of ocular gland development Isabelle Miletich Centre for Craniofacial and Regenerative Biology, King’s College London, Floor 27, Guy’s Tower, Guy’s Hospital, London SE1 9RT, UK

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Article history: Received 19 September 2017 Received in revised form 1 June 2018 Accepted 24 July 2018 Available online xxx

a b s t r a c t The tear film is produced by two ocular glands, the lacrimal glands, which produce the aqueous component of this film, and the meibomian glands, which secrete the lipidic component that is key to reduce evaporation of the watery film at the surface of the eye. Embryonic development of these exocrine glands has been mostly studied in mice, which also develop Harderian glands, a third type of ocular gland whose role is still not well understood. This review provides an update on the signalling pathways, transcription factors andextracellular matrix components that have been shown to play a role in ocular gland development. © 2018 Published by Elsevier Ltd.

Contents 1. 2.

3.

4. 5.

6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Overview of embryonic development of ocular glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.1. Extra-orbital and intra-orbital lacrimal glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.2. Meibomian glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 2.3. Harderian glands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Molecular control of ocular gland development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1. Role of signalling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1.1. FGF signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2. Bmp signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.3. Wnt signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.4. Ectodysplasin pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.5. Notch signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Role of extracellular matrix components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.1. Heparan sulfate proteoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Role of transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.1. Pax6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.2. SOX family of transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.3. Barx2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Role of micro RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction The anterior surface of the eye is protected by a tear film composed of three layers of distinct composition, an inner mucin layer, an intermediate aqueous layer and an outer lipid layer. Goblet cells distributed within the conjunctival epithelium lining the eye-

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lids produce the inner mucous coating, while two types of ocular glands secrete the two outer layers. In humans, the lacrimal gland, formed of an orbital and a palpebral portion, is a large serous gland found in the upper lateral region of each orbit that produces the aqueous portion of the tear film, releasing its secretions through multiple ducts. The meibomian glands are small specialized sebaceous glands located at the rim (tarsal plate) of the upper and lower eyelids, which produce the oily components (meibum) of the outer lipid layer that prevents tear overflow and evaporation.

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Fig. 1. Timeline of lacrimal gland development in the mouse. The extra-orbital lobe of the lacrimal gland (ELG) initiates at embryonic day (E) 13 as a thickening of the conjunctival epithelium at the temporal side of the eye on the deepest aspect of the conjunctival fold (white arrowhead). By E15, the ELG has grown towards the ear and reached an initial bud stage. Branching morphogenesis starts at E16 and concomitantly, microlumens form proximally in the main epithelial stalk, which will eventually give rise to the main excretory duct of the gland. As branching expands the epithelial tree, tubulogenesis progresses proximal-to-distal in the epithelial tree (black arrow). At E17, the intra-orbital lobe buds ventrally from the main epithelial stalk of the ELG, close to the eye. By then, the ELG has reached its final location, anterior to the ear pinna and adjacent to the parotid salivary gland. The LG carries on expanding until postnatal day 50 (P50).

These glands open onto the cutaneous side of the mucocutaneous junction [1]. Although a host of studies have been conducted to study adult ocular gland homeostasis, understand the genesis of dysfunctions of the tear film leading to dry-eye syndrome and investigate the potential of stem cell-based therapies, the molecular mechanisms controlling the morphogenesis of major ocular glands are still poorly understood. Mice, which have been used to explore the normal and abnormal development of ocular glands, present two specificities with regards to ocular glands compared to humans. The murine lacrimal gland possesses two lobes located at a distance from each other, a small intra-orbital lacrimal gland located in the ventro-temporal region of the orbit below the lower eyelid, and a large extensively branched extra-orbital lobe, of a similar size to the parotid salivary gland, located anterior and adjacent to the parotid gland, itself found anterior to the ear pinna (Fig. 1). In addition to the lacrimal gland and meibomian glands, mice also exhibit a third type of major ocular glands, the paired Harderian glands. Harderian glands are found in vertebrates possessing a nictitating membrane (translucent inner eyelid) and constituthe the largest ocular gland in rodents [2]. It is found in the posterior part of each orbit and produces an oily secretion that also contains melatonin and porphyrins, the role of which is not completely understood in eye homeostasis. 2. Overview of embryonic development of ocular glands 2.1. Extra-orbital and intra-orbital lacrimal glands Of all ocular glands, embryonic development of the ELG is best understood. Development of this gland has been well characterised by using a Pax6-LacZ reporter mouse line displaying LacZ expression in the lens, conjunctival epithelium and developing lacrimal gland epithelium [3]. Following the specification of the LG epithelium, the development of the extra-orbital lobe of the LG (ELG) starts with an induction phase at E (embryonic day) 13 when a single epithelial outgrowth forms in the dorsal region of the conjunctival epithelium at the temporal edge of the eye. Continuous with the corneal epithelium, the conjunctival epithelium forms deep folds around the developing eye and the ELG bud forms on the deepest aspect of the conjunctival fold (Fig. 1). Between E13 and E15, this epithelial bud extends posteriorly in the periorbital mesenchyme towards the ear pinna, presenting a single thick epithelial bud connected to the future conjunctival epithelium via a thin long stalk, which will constitute the principal duct

of the adult LG. Following the specification, induction and elongation phases, the ELG enters a phase of branching morphogenesis. The first branching events are observed at E16, when the single initial bud undergoes successive rounds of cleft formation, leading to the formation of multiple terminal end buds, at the distal extremity of each epithelial branch. At E17, as the ELG has reached its final position anterior to the ear pinna and branching morphogenesis rapidly increases its overall size, a single bud develops on the proximal part of the main stalk, close to the conjunctival epithelium to form the intra-orbital lobe of the lacrimal gland (ILG) (Fig. 1). This epithelial bud projects ventrally and remains connected via a short stalk to the main stalk of the ELG, undergoing branching morphogenesis by E17 (Fig. 1). As branching morphogenesis takes place, epithelial cells within the proximal and distal parts of the ELG epithelial tree exhibit distinct molecular markers. Proximally, epithelial branches that will later hollow out into ducts are Keratin19 (K19)-positive, while distally, terminal end buds that will eventually differentiate into secretory acini are K19-negative [4]. From E16 to E18 as the gland matures, Keratin14 (K14)-positive cells and alpha smooth muscle actin (␣Sma)-positive cells are progressively restricted to the basal cell layers of the whole epithelial tree and terminal end buds, respectively. These two basal epithelial cell markers distinguish three distinct cell populations in terminal end buds: K14-positive cells, ␣Sma-positive cells and cells double positive for K14 and ␣Sma. The branching process was analysed in whole E18 ELGs, taking advantage of K19 expression in presumptive ducts. Quantification of bifurcations uncovered that new branches form in two different ways in two distinct locations: either at the tip of developing branches, following the formation of clefts at the distal tip of terminal end buds, or laterally by epithelial budding from established branches. Importantly, this study revealed the branching pattern differed from one ELG to another suggesting the position of epithelial junction is stochastic rather than predetermined [4]. Use of the Fucci reporter mouse line [5] in which cells express green fluorescence in S/G2/M phases of the cell cycle and red fluorescence in G0/G1 phases revealed general unpatterned cell proliferation at the initial bud stage (E15), while afterwards proliferating cells became restricted to the basal and supra-basal cells of terminal end buds [4]. However, although the ELG significantly increases in size throughout embryonic development, the number of proliferating terminal end bud cells halves between E16 and E18, suggesting another mechanism drives the growth of this exocrine gland in addition to cell proliferation. The R26RConfetti reporter [6] driven by the constitutive K14-Cre construct was used to address this question ex vivo [4]. Upon recombina-

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tion, cells expressing either GFP, YFP, RFP or CFP were generated, allowing a clonal analysis of the developing ELG. Single cells of one origin were identified within a cellular domain of a different clonal origin, indicating a possible role of convergent extension in the expansion of the epithelial tree of the ELG. Furthermore, this clonal analysis revealed mesenchymal-to-epithelial transition might also be involved in the growth of the ELG epithelial compartment since i) epithelial cells devoid of any Confetti reporter fluorescence were identified from E15 and ii) mesenchymal cells positive for the epithelial marker E-cadherin were located in the vicinity of terminal end buds. Tubulogenesis, characterised by the hollowing of the epithelial branches was identified as early as E16, in the proximal part of the branched epithelium, corresponding to the main excretory duct and second-order ducts of the ELG [4]. Microlumens were observed at E16, which did contain apoptotic cells, suggesting cell death plays a role in lumen formation in the ELG. Thereafter, microlumens fused and tubulogenesis progressed distally towards the terminal end buds. Interestingly, after lumen formation K19 expression was restricted to ductal luminal cells, exhibiting a complementary expression pattern to Keratin14, expressed in basal ductal cells. The terminal end buds, which represent the LG presumptive secretory acini, do not show a clear lumen nor secretory granules at birth [7]. While clear lumina visible in some endbuds at P (post-natal day) 3 indicate lumen formation has started to take place, the cells lining the lumina only exhibit secretory granules around one week after birth suggesting the LG is by then a functional secretory gland. Myoepithelial cells, which are contractile starfish-like cells that help to expel LG secretions, are identified as early as P4 as cells triple positive for Epcam, Acta2, and K14 [8]. Recent qPCR and immunostaining analysis have revealed Aquaporin5 (Aqp5) –a water channel involved in tear secretion– and Mist1 –a transcription factor regulating the secretory program– are respectively detected at P1 and P4, suggesting acinar cell differentiation occurs around the time lumina are formed in the LG [8]. Notably, mammalian eye development involves the temporary fusion of the upper and lower eyelids, which takes place in mice between E16.5 and P12-P14. Hence the LGs appear to enter a secretory stage several days before eye re-opening. The LG continues to branch and mature during the postnatal period, with growth of the epithelial compartment complete by P50 [4]. In humans, development of the LG initiates with a thickening of the superior conjunctival fornix at O’Rahilly’s stages 19 and 20, accompanied by condensation of the surrounding mesenchyme [9], forming the ‘presumptive glandular area’. At O’Rahilly’s stage 21, epithelial buds form from the presumptive glandular area and invaginate into the underlying mesenchyme, producing a glandular primordium. This period, known as the epithelial bud stage finishes at the end of the embryonic period (O’Rahilly’s stage 23) when lumens develop within the epithelial invaginations, coinciding with the innervation and vascularisation of the glandular tissue. The human LG is morphologically different from the mouse LG at the bud stage, initiating as multiple epithelial bud units, while the mouse LG initiates as one single epithelial bud [8]. The next major event is the division of the glandular tissue in an orbital and palpebral lobe by the aponeurotic expansion of the levator palpebrae superioris muscle around 10 weeks of foetal development. Between 13–14 weeks of foetal development, the lacrimal and zygomatic nerves anastomose within the LG tissue, vascularisation of the area significantly increases and branching of the epithelium dramatically expands the overall size of the glandular tissue [9]. Molecular profiling of human LGs at different stages of development suggests cell lineage markers are conserved across species. In human foetal LGs, AQP5 and MIST1 were strongly expressed in secretory acinar cells, K14- and K5-positive cells were restricted to basal epithelial cells, while ACTA2-positive cells identifying presumptive myoepithelial cells were found around acini [8]. These

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developmental commonalities between murine and human LG development demonstrate the relevance of the mouse model to study human LG development. 2.2. Meibomian glands Eye development in mammals involves the transient closure of upper and lower eyelids. In mice, eyelids close between E15 and E16 and re-open 12–14 days after birth [10,11]. Meibomian gland (MG) development starts at E15, at a time when eyelids are still open. Lef1-positive clusters of epithelial cells slightly protrude from the conjunctival epithelium near the inner surface of the lid margin into the adjacent mesenchyme, forming epithelial placodes [10,12]. At E18, regularly spaced epithelial thickenings are visible within the fused eyelid margin, in the upper and lower lids, developing opposite to each other in both lids [13]. At the same time, mesenchymal condensation takes places within the mesenchyme underlying the placodes. At P0, the epithelium invaginates into the underlying mesenchyme forming small round buds. Subsequently, these epithelial buds further invaginate, forming tubular structures that exhibit the same diameter throughout, from the distal to the proximal end. Branching is detected at P5 and happens throughout the tubular structure, at both the distal and proximal ends. Branching is extensive by P8, with mature MGs present by P14 between eyelash follicles upon eyelid opening [13]. Although MG development initiates before eyelid closure, transient eyelid closure may play a key role in MG development. A study investigating MG development in 18 distinct mouse mutants exhibiting a failure of eyelid closure identified MG are either absent or severely hypoplastic in these mutants [7]. Furthermore, examination of mutants displaying at birth open eye phenotypes with incomplete penetrance revealed the severity of the MG phenotype correlated with the size of the eye opening rather than the genetic condition, supporting an important role of transient eyelid closure in MG development. In humans, MG formation also takes place during the sealedphase of eyelid formation, between weeks 9–12 of gestation [14,15]. MGs develop from the fused epithelium of the upper and lower eyelids as long epithelial chords. These later grow lateral epithelial buds giving rise to the secretory holocrine sebaceous acini connected to the central excretory duct via short ductules. 2.3. Harderian glands The primary Harderian gland (HG) bud forms at E13 on the deepest aspect of the conjunctival fold on the nasal side of the eye. Expression of the Pax6-LacZ construct is absent from the conjunctival fornix (junction between the eyelid and corneal conjunctiva) as well as from the HG bud [3]. By E15, the HG initial bud has further elongated within the mesenchyme of the eye socket, exhibiting a globular epithelial bulge on the distal side connected to the conjunctival epithelium via a thinner epithelial stalk [16]. At E18, the HG appears to be branched and by P14, when the eyelids re-open, the secretory epithelium of the gland is mature. 3. Molecular control of ocular gland development Most of the current knowledge on ocular gland development comes from studies investigating LG formation, as this gland develops under the skin and is readily observable after skin removal. In addition, the LG is amenable to dissection and ex vivo culture [17] and LG development appears to be similar in and ex vivo [4], hence validating the use of this system to further characterise the developmental pathways by mechanical, chemical or genetic manipulations. Here we review the signalling pathways, ECM components and transcription factors that have been shown to be involved in ocular gland development.

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3.1. Role of signalling pathways 3.1.1. FGF signalling At E13.5, Fgf10 is expressed in the periocular mesenchyme, exhibiting a stronger expression ventrally with high concentration of mRNA expression on the nasal and the temporal sides of the eye, areas where the HG and LG development initiates, respectively. Fgf7, a close homolog of Fgf10, is also expressed in the periocular mesenchyme though at lower levels with no distinct upregulation at the sites of ocular gland development [3,16]. While Fgf7−/− mutant mice do not exhibit ocular gland phenotypes [18], Fgf10−/− and Fgf10+/− mice lack both the LG and HG, suggesting a critical role for Fgf10 in the development of both glands [3,16,19]. This phenotype is mirrored in humans by two autosomal-dominant multiple congenital anomaly syndromes caused by Fgf10 mutations that exhibit lacrimal gland aplasia, atresia or hypoplasia: LacrimoAuriculo-Dento-Digital syndrome (LADD, OMIM149730) [20] and Aplasia of the Lacrimal and Salivary Glands (ALSG, OMIM180920 and OMIM103420) [19]. Conversely in mice, exogenous recombinant Fgf10 can induce ectopic LG formation in eye explant culture [3] and transgenic ectopic expression of Fgf10 in the lens epithelium induces the formation of both HG (Pax6 negative) and LG (Pax6 positive)-like structures within the corneal epithelium, demonstrating Fgf10 is necessary and sufficient to induce HG and LG development [16]. Interestingly, while transgenic lines with high levels of Fgf10 expression induce the formation of both LGs and HGs, transgenic lines with lower levels of Fgf10 expression induce only ectopic LG formation, suggesting HGs require higher levels of Fgf10 expression to develop, hence providing a possible mechanism by which one single growth factor initiates the formation of distinct ocular glands. Similarly, ectopic expression of Fgf7 (also known as Keratinocyte Growth Factor) [18] or Fgf3 [21] within the lens epithelium results in the formation of ectopic serous glandular tissue within the corneal epithelium, whereas lens-specific expression of Fgf4 [22] does not. Hence, it appears only Fgf ligands that can bind and activate the Fgfr2IIIb receptor isoform (Fgf10, Fgf7 and Fgf3, but not Fgf4) can induce ectopic ocular gland formation, suggesting the Fgfr2IIIb receptor isoform is involved in ocular gland formation. Further evidence of the involvement of the Fgfr2IIIb receptor has been provided by explant culture of developing eyes and surrounding tissues in the presence of antisense nucleotides to Fgfr2IIIb versus control oligonucleotides, as antisense nucleotides to Fgfr2IIIb specifically inhibit the formation of LG buds [3]. Moreover, tissue-specific deletion of Fgfr2 demonstrates the requirement of the Fgfr2 receptor in the conjunctival epithelium for LG development. Indeed, Le-Cre/+; Fgfr2flox/flox animals, in which the Cre recombinase is driven by a Pax6 ectodermal enhancer expressed in the lens and corneal epithelium prior to LG development (E12) fail to develop LGs [23]. This failure of LG development results from a failure of LG induction as the initial LG bud is missing at E14.5 in Le-Cre/+; Fgfr2flox/flox animals. In addition, the requirement for Fgfr2 is specific to the conjunctival epithelium since Wnt1-Cre/+; Fgfr2flox/flox animals in which Fgfr2 is knocked out within the neural crest-derived mesenchyme present no LG defect. A similar requirement for FGFR2 is observed in human LG development as LADD syndrome can be caused by mutations in the FGFR2 receptor [24]. Altogether, these data indicate Fgf signalling is essential for LG development. Downstream of the Fgfr2 receptor, the Shp2 protein is also key for LG formation. Tissue specific deletion of Shp2 in the conjunctival epithelium in Le-Cre/+; Shp2flox/flox embryos leads to a complete lack of LGs [23,25]. The Tyrosine phosphatase Shp2 promotes ERK phosphorylation and consistent with a role of ERK as a downstream effector of FGF signalling in LG development, phospho-ERK is expressed at the fornix of the temporal conjunctival epithelium at E12 before LG budding, and at later stages of LG development at the tip of the ini-

tial LG epithelial bud. Moreover, phospho-ERK expression is lost at the temporal conjunctival fornix in Le-Cre/+; Fgfr2flox/flox embryos [23]. The mechanism by which Shp2 regulates FGF signalling has been investigated in more detail. Constitutively activated Ras signalling –with Ras being upstream of ERK– only slightly rescues the LG phenotype in Le-Cre/+; Shp2flox/flox . Instead of the complete lack of LG epithelial bud observed in Le-Cre/+; Shp2flox/flox mutants, Le-Cre/+; Shp2flox/flox ; LSL-KrasG12D animals -in which the Le-Cre transgene simultaneously knocks out Shp2 and activates Kras signalling in the conjunctival epithelium- exhibit a small thickening at the fornix of the temporal conjunctival epithelium, associated with phospho-ERK expression [25]. Interestingly, LG development is fully restored after additional deletion of Sprouty2 (Spry2). Spry2 is a target of FGF signalling and as expected, Spry2 expression is lost in Le-Cre/+; Shp2flox/flox mutants. Furthermore, under its phosphorylated form, Spry2 is also an inhibitor of FGF signalling. Shp2 directly interacts with Spry2 [25] and it has been suggested Shp2 may control Spry2 phosphorylation, therefore negatively regulating Spry2 activities, and ultimately promoting FGF signalling. Active FGF signalling appears to stimulate LG bud formation and proliferation of LG epithelial precursors within the conjunctival fornix on the temporal side of the eye and within the LG initial bud. Indeed, Le-Cre/+; Fgfr2flox/flox conditional mutants exhibit a thin presumptive LG epithelium with few proliferating cells at E14.5, while the same area in wild-type control animals shows a newly formed LG outgrowth displaying numerous rapidly dividing cells [23]. It is important to note that in addition to be secreted and act in a paracrine fashion, Fgf10 is actively compartmentalized within the nucleus of Fgf10-producing cells, suggesting Fgf10 is also involved in intracrine signalling. Fgf10 exhibits two nuclear localization signals (NLSs) and strikingly, the LADD syndrome-causing G138E mutation lies within one of these NLS outside the FGF10/FGFR2 interaction domain, suggesting defective binding of FGF10 to FGFR2 is not the cause of the defects observed in this syndrome. Cell transfection studies have shown the G138E mutation impairs the nuclear import of endogenous FGF10, which subsequently becomes hyperglycosylated and fails to progress through the secretory pathway [26]. FGF10 could either play a yet unknown role within the nucleus or the nuclear import of FGF10 could regulate levels of FGF10 available for paracrine signaling. Recent investigations exploring the signalling pathways that control Fgf10 expression in the periocular mesenchyme have identified Fgf signalling in the neural crest is required for expression of Fgf10 in the periocular mesenchyme [27]. The periocular mesenchyme is of neural crest origin and conditional deletion of either both Fgfr1 and Fgfr2, Frs2 (encoding an adaptor protein that directly interacts with activated FGFRs) or Shp2 (encoding a tyrosine phosphatase binding to Frs2 and positively regulating the mitogen-activated protein kinase (MAPK) signalling pathway) in the neural crest using a Wnt1-Cre or Sox10-Cre driver impairs LG budding. Fgf-dependent expression of Fgf10 in the periocular mesenchyme is mediated by the homeodomain transcription factor Alx4, which binds a regulatory element in Alx4 first intron. Interestingly, this Alx4 binding sequence is conserved throughout land animals whose eyes are exposed to air and missing in amphibians that are lacking LGs. Furthermore, Alx4lst−J mutant mice [28,29], in which both the homeodomain and downstream CAR domain are missing, exhibit a sharp reduction in Fgf10 expression in the peri-ocular mesenchyme accompanied by shorter LG buds at E16 displaying increased epithelial cell death, leading to missing LG at P1. Alx4 requirement for LG formation appears to be conserved in humans since MRI imaging of a patient carrying a mutation in ALX4 resulting in the truncation of the homeobox and C-terminal OAR domains [30] revealed a bilateral absence of LGs. It is noteworthy to mention this young patient only exhibited repeated eye infections and irritable eyes, while other congenital LG aplasia have been diagnosed in children after parents noticed an absence of tears

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when crying [31]. These mild symptoms associated with the paucity of reports of LG aplasia in the presence of normally formed salivary glands suggests LG aplasia may not be readily diagnosed in the human population. 3.2. Bmp signalling At the initiation stage of LG development, although Bmp7 is expressed in the peri-orbital mesenchyme, it is markedly absent from the temporal peri-orbital mesenchyme adjacent to the LG primary bud. A discrete patch of mesenchyme strongly expressing Bmp7 is found between the eye and the pinna of the ear, which is later invaded by the LG primary bud and invasion of this Bmp7-expressing mesenchyme appears to coincide with the onset of branching morphogenesis. During epithelial branching, Bmp7 expression is then distinctly increased in the mesenchyme at the tip of the developing branches while it is also sporadically expressed in the epithelium [32]. Bmp7 null mutants show variable LG phenotypes ranging from normal intra and extralobular lobes to severely hypoplastic LGs exhibiting ectopic buds along the primary duct in addition to the proximally located ELG bud, indicating Bmp7 promotes branching morphogenesis and is required for correct lobe distribution. Significantly, Fgf10 expression is unchanged in the LG mesenchyme of Bmp7 null mutants. In vitro culture of isolated LG mesenchyme in the presence of Bmp7 have suggested Bmp7 acts primarily in the LG mesenchyme by stimulating mesenchymal proliferation, aggregation and condensation (by upregulation of cadherin adhesion molecules and Cx43 junctional protein), three mesenchymal cell behaviours that are hallmarks of signalling centres and essential for branching morphogenesis. Interestingly, while whole LG-explant cultures with the BMP inhibitors Noggin and Follistatin exhibited a reduction in epithelial branching similar to Bmp7−/− LGs, they also displayed abnormally large acini compared to the Bmp7−/− LGs, suggesting another Bmp ligand is involved in LG development. The lacrimal gland phenotype of Le-Cre/+; Smad4flox/flox conditional mutants, characterised by a hypoplastic ELG connected to the eye via a short duct, further validates the role of Bmp signalling in LG branching, as Smad4 is a co-Smad essential for Bmp intra-cellular signalling [33]. The forkhead transcription factor Foxc1, expressed in both the mesenchyme and the epithelium of the developing LG, has also been implicated as a mediator of Bmp signalling in LG development. The Foxc1−/− ELG is severely reduced in size with a reduced number of epithelial branches and a shorter duct, while the ILG is absent [34]. Foxc1 is likely to mediate responses to Bmp7 in the LG mesenchyme as in vitro culture of isolated Foxc1−/− LG mesenchyme fails to proliferate and aggregate in the presence of exogenous Bmp7 [34]. Bmp/Smad signalling is also required for induction of MG development. Indeed, inhibition of this pathway using K14-Noggin mice (in which the Bmp antagonist Noggin is secreted by ectodermal cells) or Le-Cre;Smad4fl/fl mice (in which the co-Smad Smad4 is knocked out in the conjunctival epithelium giving rise to MGs) resulted in the differentiation of an ectopic row of pilosebaceous units instead of MGs on the inner edge of the eyelids, a defect known as distichiasis [35,36]. Ectopic hair follicles replacing MGs often grew towards the cornea, rubbing against it and causing irritation. Altogether, these data suggest Bmp signalling is required for the differentiation of MG progenitor cells. Mice haploinsufficient for Foxc2, encoding a Forkhead box transcription factor, also present distichiasis [37] and the key role of this gene in MG development appears conserved in humans, as individuals carrying a mutation in the FOXC2 gene exhibit lymphedema-distichiasis syndrome (OMIM153400) [38]. Distichiasis is also found in Dkk2 null mice, in which Foxc2 expression is lost in the conjunctival ectoderm of eyelids [39], from which can be inferred excessive Wnt signalling suppresses Foxc2 expression leading to distichiasis. Bmp signalling

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being required for Foxc2 expression [36], excessive Wnt signalling may inhibit Bmp signalling in eyelids. 3.3. Wnt signalling Canonical Wnt signalling (CWS) inhibits branching morphogenesis in the LG. Indeed, in vitro culture of whole LGs in the presence of either Wnt3a (a canonical Wnt ligand) or lithium chloride (an inhibitor of the phosphorylation activity of Gsk3-␤ that mimics CWS) leads to a reduction in the number of epithelial branches, while similar cultures supplemented with ␤-catenin antisense morpholinos show an increase in branching of the LG [40]. Strikingly, this increase in branching is associated with an increase in Fgf10 expression levels, suggesting CWS negatively regulates branching by controlling Fgf10 expression in the LG mesenchyme. Moreover, CWS can also inhibit Bmp7-induced cell proliferation in whole LG in in vitro culture. Altogether, these data suggest there is a fine control of cell proliferation within the LG through positive (FGFs and BMPs) and negative (WNTs) regulators. It will be important to determine which cells of the LG activate CWS during epithelial branching to further understand the precise regulation of this mechanism. CWS has been shown to be an important player during MG development, with the Wnt antagonist Dickkopf 4 (Dkk4) having a central role in the modulation of this signalling pathway at early stages of MG formation. Dkk4 is a secreted protein that binds to the Lrp6 Wnt co-receptor and the Kremen transmembrane proteins, resulting in endocytosis of Lrp6, hence inhibiting Wnt signalling. At E15, during the initiation stage of MGs, Dkk4 and its Wnt target Wnt10b are expressed in MG placodes [12]. Skin-specific Dkk4 expression using the K14-Dkk4 transgenic mice [41] did not affect MG initiation, but impaired further growth of these glands. Interestingly, while a full length Dkk4 (FL-Dkk4) was present at E15, increasing levels of cleaved Dkk4 (CL-Dkk4) were identified at later stages and ex vivo eyelid cultures revealed only FL-Dkk4 had an inhibitory effect on MG development, suggesting proteolytic cleavage of Dkk4 modulates Wnt signalling during MG development and more specifically allows active Wnt signalling during the expansion phase of MG development. Consistent with these data, elevation of Lrp6 via lentiviral transfection in ex vivo culture of K14-Dkk4 eyelids rescued MG development [12]. 3.4. Ectodysplasin pathway Prior to LG primary bud formation, Edar (encoding the Ectodysplasin receptor) is expressed in the conjunctival epithelium at the future site of LG initiation, whereas Ectodysplasin (Eda, encoding Edar ligand) is expressed in the adjacent corneal epithelium. Later, Edar expression is found in the LG epithelium [42]. Both the HG and LG show variable phenotypes in the Tabby (Eda) hemyzygote mutant. Tabby hemizygote HG and ELG are either missing or exhibit delayed embryonic development while the ILG is absent [43]. Although the initiation of MG development is not affected in Tabby hemizygotes, since hypoplastic glandular rudiments are formed in their eyelids, MG are however missing in newborn mice, suggesting they fail to differentiate and regress [43]. Consistent with an essential role of the Eda pathway in MG development, mice homozygous mutant for the Edaradd gene (encoding a death domain-containing protein that interacts with Edar) or for the TNF Receptor Associated factor-6 (TRAF6) gene (encoding a cytoplasmic protein upstream of NF-␬B, a downstream effector of Eda-Edar signalling) also lack MGs [44,45]. Importantly, the key role of Eda-Edar signalling in MG development is conserved in humans, as transillumination examination of the MG of 22 patients suffering from hypohydrotic ectodermal dysplasia showed MG defects in 21 of

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these individuals, ranging from complete absence of MGs to coarsening of the acini [46]. The phenotype of Tabby MGs resembles the one of K14-Dkk4 MGs, since the initiation phase of MG development is not affected in both mouse lines. Interestingly, expression of Dkk4, Lrp6, Wnt10b and Fzd10 is severely reduced in Tabby mice [12] and further analysis of the Lrp6 promoter region revealed the presence of binding sites for NF-␬B, a downstream effector of Eda-Edar signalling, hence placing the Eda pathway upstream of canonical Wnt signalling. Furthermore, MG defects in ex vivo cultures of Tabby eyelids could be rescued by Lrp6 elevation via lentiviral transfection [12]. 3.5. Notch signalling Microarray analysis comparing gene expression in adult versus E16 embryonic ELGs revealed genes of the Notch pathway, including Notch1 and Notch3 –encoding Notch receptors– and Jagged1 (Jag1) –encoding a Notch ligand–, were significantly upregulated at the onset of branching morphogenesis [47]. A subsequent transcriptomics analysis investigating expression levels of Notch pathway elements further identified enrichment of Notch2, Jag1 and Notch signalling target gene Hey1 at E18 [4]. E15 ELG explant cultures treated with the gamma-secretase inhibitor DAPT, which inhibits the intracellular cleavage of Notch receptors and therefore suppresses Notch activation, exhibited an increase in the number of terminal end buds together with a decrease in terminal end bud size, suggesting an inhibitory role of Notch signalling on epithelial branching [47]. Conditional deletion of Notch1 using the Notch1flox/flox mice [48] treated with Ad-CMV-iCre adenoviruses constitutively expressing Cre recombinase, confirmed the results obtained by pharmacological blockade of Notch signalling and pointed at an inhibitory role of the Notch1 receptor on ELG branching. Notch1 being present both in the epithelium and the mesenchyme of branching ELGs [47], whether this branching inhibition takes place through the regulation of ECM remodelling or the control of cell cycle exit and differentiation of epithelial progenitors remains to be investigated. An independent study found that in addition to increased branching, DAPT-mediated inhibition of Notch signalling also caused apoptosis of supra-basal terminal end bud cells leading to hollow terminal end buds [4]. Strikingly, K19-positive cells identifying presumptive ducts were absent in ELG epithelial branches, lumen formation failed to occur and ˛Smapositive cells were ectopically detected in basal cell layers of both terminal end buds and ducts, hinting towards a positive role of Notch signalling in ELG maturation and more specifically in the determination of ductal identity, by promoting expression of K19 in all ductal cells, stimulating lumen formation and inhibiting ˛Sma expression in basal ductal cells. 4. Role of extracellular matrix components 4.1. Heparan sulfate proteoglycans Heparan sulfate proteoglycans (HS-PGs) are glycoproteins present on the cell surface that interact with FGF ligands and their receptors, acting as co-receptors to facilitate Fgf/Fgfr interaction and stabilise Fgf/Fgfr complexes. Heparan sulfates are linear chains of glycosaminoglycan molecules that are modified by sulfotransferase enzymes, which generate significant diversity within the composition of heparan sulfates. Enzymes involved in this process are the N-deacetylase/N-sulfotransferases Ndst1 and Ndst2. Strikingly, Ndst1 is differentially expressed throughout the epithelium of the LG initial bud with a stronger expression at the distal tip of the developing LG initial bud. Furthermore, Ndst1 expression pattern in the LG epithelium correlates with an enrichment in sulphated heparan sulfate in the distal tip of the LG, reveal-

ing a differential regulation of heparan sulfate sulfation in the endbud and the stalk of the LG initial epithelial bud [23]. Conditional knockout of Ndst1 in Le-Cre/+; Ndst1flox/flox results in either hypoplastic or missing LGs, while Le-Cre/+; Ndst1flox/flox ; Ndst2−/− double mutants exhibit a complete absence of LG. Furthermore, in explant culture of Le-Cre/+; Ndst1flox/flox ; Ndst2−/− eye rudiments supplemented with Fgf10, both endogenous and ectopic LGs fail to develop, demonstrating Ndst genes are essential for Fgf10-induced LG formation. LACE (Ligand And Carbohydrate Engagement) assays based on the in situ binding of recombinant Fgf/Fgfr2b-Fc complexes to LG endogenous heparan sulfate –the complexes being later detected with antibodies directed against the IgG-Fc domain tag fused with Fgfr2b– have revealed an essential role of heparin sulfate N-sulfation in Fgf10/Fgfr2b signalling. In wild-type LGs, both Fgf10/Fgfr2b-Fc and Fgf7/Fgfr2b-Fc complexes are specifically detected at the distal tip of the initial bud of developing LGs in addition to the surrounding mesenchymal cells. In Le-Cre/+; Ndst1flox/flox embryos the staining is lost in the LG distal tip epithelium, but not in the surrounding mesenchyme [23]. In conclusion, Ndst enzymes specifically produced by the LG distal tip epithelial cells catalyse N-sulfation of HSPG found at the surface of these distal tip cells. N-sulfated HSPG can then interact with Fgf10/Fgfr2b at the surface of the tip cells, potentiating the ligand/receptor interaction, leading to the activation of Fgf signalling in distal tip cells. Simultaneously, restriction of N-sulfation of HSPG to distal tip epithelial cells also provides a mechanism promoting directional outgrowth of the LG bud. Current data hence point at HSPG N-sulfation as a critical regulator of Fgf/Fgfr2 signalling -a key signalling pathway for LG development- therefore warranting further research to understand upstream signalling controlling the expression of N-sulfation modification enzymes. N-sulfation of heparan sulfate is a pre-requisite for other heparan sulfate modifications such as 2-O-sulfation and 6-O-sulfation. Interestingly, both 2-O and 6-O sulfation of heparan sulfate, two modifications respectively done by the Hs2st and Hs6st1/2 sulfotransferases, appear to contribute to Fgf signalling. Hs2st and Hs6st1/2 are expressed throughout the LG initial bud. While genetic ablation of either Hs2st, Hs6st1, or Hs6st2 shows no LG phenotype, ablation of Hs6st1/2 leads to hypoplastic or missing LGs and ablation of the three sulfotransferases completely abrogates LG development [49]. The Hs2st/Hs6st double mutant LG primordium fails to form cell surface Fgf10/Fgfr2b-Fc complexes in LACE essays and expresses neither phospho-ERK (a downstream effector of Fgf signalling) nor Erm (a downstream target of Fgf signalling), which strongly suggests heparan sulfate O-sulfation is essential for Ffg10/Fgfr2b interaction at the surface of the LG initial bud cells and subsequently initiation of FGF signalling in LG bud cells. By interacting with Fgf ligands, glycosaminoglycans also limit the diffusion of Fgfs within the extracellular space and stabilise Fgfs in the mesenchyme. Indeed, mesenchymal depletion of heparan sulfate and chondroitin sulfate –two major components of glycosaminoglycans– leads to the dispersion of Fgf10, rendering it too dilute to induce Fgf signalling in the presumptive LG epithelium and subsequent budding of the LG epithelium. Genetic deletion of the UDP-glucose dehydrogenase (Ugdh), the single enzyme responsible for processing UDP-glucose into glycosaminoglycan precursors, leads to a complete absence of LG [50]. While Fgf10 expression and Fgf10 protein levels appear to be unchanged in Wnt1-Cre/+; Ugdhflox/flox mutants, Fgf10 diffusion is increased and phospho-ERK and Erm (a downstream target of Fgf signalling) fail to be expressed in the presumptive LG epithelium. Rescue of LG budding by constitutive activation of Ras (a downstream mediator of FGF signalling) in the presumptive LG epithelium of mutants lacking mesenchymal Ugdh suggests a loss of epithelial Fgf signalling is responsible for the absence of LGs in mesenchymal knockouts of Ugdh.

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Interestingly, the morphogen gradients Fgfs form with the extracellular matrix dictates distinct epithelial behaviours in the developing LG epithelium. This is illustrated by in vitro culture of isolated LG epithelium in the presence of different Fgf ligands. While Fgf10 causes the elongation of the LG primordial bud, Fgf7 induces the formation of epithelial branches [51]. Strikingly, mutation of the single amino acid that differs in the heparan sulfatebinding domain of Fgf10 and Fgf7, by replacing the arginin178 of Fgf10 by a valine as found in the corresponding position in Fgf7, converts Fgf10 into a Fgf7 mimic, which induces LG epithelial branching. These data strongly suggest Fgf diffusion and the affinity of Fgf ligands for the Fgfr2b receptor, both dependent on heparan sulfate-binding affinity, induce specific epithelial cellular responses. 5. Role of transcription factors 5.1. Pax6 Pax6, a transcription factor that exhibits both a paired and a homeodomain DNA binding motifs, is expressed in the conjunctival epithelium but not in the periocular mesenchyme [52]. As shown by in situ hybridisation [52] and a Pax6-LacZ reporter mouse [3], at E12.5, prior to any visible sign of LG development, Pax6 is expressed in the lens epithelium and the future conjunctival epithelium. One day later at E13.5, Pax6 expression is restricted to the corneal and conjunctival epithelium including the conjunctival fornix from which the LG epithelial outgrowth will emerge. Thereafter, Pax6 remains expressed throughout LG development. Pax6 requirement for normal lacrimal gland development has been demonstrated by studying Small eye (Sey) mutants, which exhibit a point mutation in Pax6 resulting in truncation of the protein prior to the homeobox [53]. Heterozygous Sey mutants show a complete absence of LG initial bud at E15.5. However, at E19.5, when the LG has normally reached its final position and undergone extensive branching, Sey+/− mutants display a small hypoplastic ELG close to the temporal side of the eye, indicating the inductive phases of LG development are severely delayed in the absence of Pax6. Importantly, Pax6 does not regulate mesenchymal Fgf10 expression as the Fgf10 pattern is unchanged in Sey+/− mutant [3], which suggests Pax6 acts as a LG competence factor in the conjunctival epithelium. However, considering Pax6 is expressed throughout the conjunctival epithelium and the dorsal conjunctival epithelium is unable to form ectopic LG buds in the presence of Fgf10 [3], it is likely other factors than Pax6 are necessary for LG formation. 5.2. SOX family of transcription factors Sox9 and Sox10, two members of the SOX group E, are involved in LG formation [54]. At E14.5, Sox9 is expressed in the conjunctival epithelium and throughout the budding LG and HG, but thereafter Sox9 expression is switched off in the conjunctival epithelium while it is maintained in the developing ocular glands. Sox10 is also expressed in the LG initial bud, however Sox10 expression is restricted to the epithelial cells at the tip of the bud. Conditional knockout of Sox9 in the conjunctival epithelium, lacrimal and meibomian gland epithelia using the Le-Cre transgene leads to a complete absence of LG and HG at birth, whereas MGs are fewer and less developed in the upper and lower eyelids. Conditional knockout of Sox10 using the same Le-Cre transgene shows a milder LG phenotype: the primary bud is shorter, at later stages of development the LG is small and poorly branched and eventually both myoepithelial and secretory acinar cells fail to form. At embryonic stages, Sox10 expression is lost in Le-Cre; Sox9flox/flox and Le-Cre; Fgfr2flox/flox mutants and Sox9 expression is reduced in

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Le-Cre; Fgfr2flox/flox mutants, indicating i) FGF signalling positively regulates Sox9 expression and ii) Sox10 is a downstream target of Sox9 during LG formation. Sox9 expression also appears to be essential for activation of Fgf signalling in the LG bud as levels of phospho-ERK are markedly decreased in Le-Cre; Sox9flox/flox LG. As Sox9 expression is required for LG bud expression of Hs3st3b1 and Hs3st3a1 –encoding heparin-synthesizing enzymes– it has been proposed Sox9 might promote FGF signalling by regulating ECM components and more specifically by controlling the synthesis of heparan sulfate that are required for Fgf/Fgfr interaction and to limit the extracellular diffusion of Fgf ligands. Recent studies using single-cell sequencing at E16 and P14 have suggested Sox10+ cells are precursors of both acinar and myoepithelial cells as Sox10 can be found co-expressed with either acinar (Mist1) or myoepithelial (p63, Keratin14 and Keratin5) markers at P4 [8]. The same study has also revealed by genetic lineage-tracing using the Keratin5-CreERT2; Rosa26mTmG that basal Keratin5+ cells behave as unipotent epithelial progenitor cells that contribute to adult ducts. Runx1-3 genes, expressed in the LG developing epithelium are likely to positively regulate this keratin5-expressing progenitor cells as LGs cultured in the presence of siRNA targeted against Runx1-3 exhibit a dramatic decrease in epithelial branching concomitant with a decrease in the number of keratin5+ progenitor cells [55]. 5.3. Barx2 Barx2 is expressed in the conjunctival epithelium prior to ocular gland initiation and thereafter in the LG, HG and MG epithelium throughout their development [56]. Barx2 homozygous mutants show defects in the elongation of the primary bud and poorly branched ELG while ectopic buds form in the proximal region of the ELG duct. Barx2-/- MGs are also poorly developed and irregularly spaced. Meanwhile, HGs are the most affected ocular glands in the Barx2-/- mutants, since the HG epithelium is absent [56]. Barx2-/isolated LG buds are unable to elongate towards Fgf10 beads, indicating Barx2 is required in LG epithelial cells to receive or transduce Fgf signals. Barx2 positively regulates expression of Matrix metalloproteinases (Mmp) 2, 3 and 9, Mmp2 and Mmp9 being expressed both in the LG epithelium and mesenchyme, while Mmp3 expression is restricted to the mesenchyme. MMPs have been involved in the degradation and remodelling of the ECM, and as such can release not only FGFs but also Fgf-binding heparan sulfate, therefore influencing FGF diffusion. Furthermore, MMPs can regulate Fgf signalling by direct cleavage of FGF receptors [57]. Hence Barx2 could promote Fgf signalling either by acting on Fgf receptors in LG epithelial cells or by regulating MMP secretion by LG bud cells within the extra-cellular space. 6. Role of micro RNAs Evidence of an essential role of the microRNAs miR-205 in LG development has recently been published [58]. MicroRNAs (miRNAs) are small, non-coding RNAs of around 22 nucleotides that regulate gene expression post-transcriptionally by targeting complementary RNAs. MiR-205 is an intergenic miRNA expressed at E14 in the skin and the epithelium of the developing craniofacial glands including the LGs, MGs and salivary submandibular and parotid glands as shown by in situ hybridisation and a LacZ reporter mouse line [59]. Global deletion of miR-205 revealed amongst these craniofacial glands, miR-205 plays a unique and critical role in LG initiation as miR-205-/- mice exhibited missing LGs in 50% of cases and smaller, poorly extended LGs in 25% of cases [58], while MGs were present, although occasionally enlarged, with retained lipidic content. Double heterozygous miR-205+/−; Fgf10+/− exhibited a

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phenotype similar to miR-205-/-, suggesting miR-205 interferes with Fgf10 signalling and impairs LG development. While Fgf10 signalling is currently the only pathway involved in the initiation of LG development, a number of target genes are upregulated in the absence of miR-205. Hence, further work is required to identify whether another yet unidentified pathway is negatively regulated by miR-205. 7. Conclusion Dry eye disease (DED) is one of the most frequently encountered ocular morbidities with a high prevalence in the elderly population. DED currently affects between 5 and 50% of the general population [60] and this number is expected to increase as the aged population grows. DED occurs when there is either insufficient tear production to maintain the aqueous layer, or the lipid layer is deficient allowing the eye to dry due to increased evaporation, or a combination of both. Hence, DED results from dysfunctions of either the LG or MG, or both ocular gland types. The continued deciphering of embryonic signaling involved in the determination of distinct cellular lineages during LG and MG development will undoubtedly inform the current efforts to regenerate ocular glandular tissue. This review however highlights the paucity of molecular information on MG gland development compared to LG development. As MGs increasingly appear to play a central role in the etiology of DED [61], efforts should be made to better understand how cellular lineages are established in these holocrine glands. Finally, LG development shares a number of similarities with other branching organ -such as the salivary glands- in terms of developmental processes and molecular events taking place during organogenesis. Future work should therefore address what makes the uniqueness of the lacrimal gland, and more specifically identify the molecular determinants leading to a lacrimal secretory function. References [1] E. Knop, N. Knop, T. Millar, H. Obata, D.A. Sullivan, The international workshop on meibomian gland dysfunction: report of the subcommittee on anatomy, physiology, and pathophysiology of the meibomian gland, Invest. Ophthalmol. Vis. Sci. 52 (2011) 1938–1978. [2] A.P. Payne, The harderian gland: a tercentennial review, J. Anat. 185 (Pt. 1) (1994) 1–49. [3] H.P. Makarenkova, M. Ito, V. Govindarajan, S.C. Faber, L. Sun, G. Mcmahon, P.A. Overbeek, R.A. Lang, FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development, Development 127 (2000) 2563–2572. [4] A. Kuony, F. Michon, Epithelial markers aSMA, Krt14, and Krt19 unveil elements of murine lacrimal gland morphogenesis and maturation, Front. Physiol. 8 (2017) 739. [5] A. Sakaue-Sawano, H. Kurokawa, T. Morimura, A. Hanyu, H. Hama, H. Osawa, S. Kashiwagi, K. Fukami, T. Miyata, H. Miyoshi, T. Imamura, M. Ogawa, H. Masai, A. Miyawaki, Visualizing spatiotemporal dynamics of multicellular cell-cycle progression, Cell 132 (2008) 487–498. [6] H.J. Snippert, L.G. Van Der Flier, T. Sato, J.H. Van Es, M. Van Den Born, C. Kroon-Veenboer, N. Barker, A.M. Klein, J. Van Rheenen, B.D. Simons, H. Clevers, Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells, Cell 143 (2010) 134–144. [7] Y.L. Wang, Y. Tan, Y. Satoh, K. Ono, Morphological changes of myoepithelial cells of mouse lacrimal glands during postnatal development, Histol. Histopathol. 10 (1995) 821–827. [8] D.T. Farmer, S. Nathan, J.K. Finley, K. Shengyang Yu, E. Emmerson, L.E. Byrnes, J.B. Sneddon, M.T. Mcmanus, A.D. Tward, S.M. Knox, Defining epithelial cell dynamics and lineage relationships in the developing lacrimal gland, Development 144 (2017) 2517–2528. [9] C. De La Cuadra-Blanco, M.D. Peces-Pena, J.R. Merida-Velasco, Morphogenesis of the human lacrimal gland, J. Anat. 203 (2003) 531–536. [10] G.S. Findlater, R.D. Mcdougall, M.H. Kaufman, Eyelid development, fusion and subsequent reopening in the mouse, J. Anat. 183 (Pt. 1) (1993) 121–129. [11] M.J. Harris, M.J. Mcleod, Eyelid growth and fusion in fetal mice. A scanning electron microscope study, Anat. Embryol. (Berl.) 164 (1982) 207–220. [12] J. Sima, Y. Piao, Y. Chen, D. Schlessinger, Molecular dynamics of Dkk4 modulates Wnt action and regulates meibomian gland development, Development 143 (2016) 4723–4735. [13] C.J. Nien, S. Massei, G. Lin, H. Liu, J.R. Paugh, C.Y. Liu, W.W. Kao, D.J. Brown, J.V. Jester, The development of meibomian glands in mice, Mol. Vis. 16 (2010) 1132–1140.

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