Deletion of Fgfr2 in Ductal Basal Epithelium With Tamoxifen Induces Obstructive Meibomian Gland Dysfunction

Abstract

Purpose: Fibroblast growth factor receptor 2 (Fgfr2) is crucial for the homeostasis of meibomian gland (MG). However, the role of Fgfr2 in MG ductal epithelial progenitors remains to be delineated. Herein, we created a new transgenic mouse model with conditional deletion of Fgfr2 from MG ductal progenitors and investigated the cell-specific role in the pathogenesis of obstructive meibomian gland dysfunction.

Methods: Peritoneal injection of tamoxifen (TAM) at 50 µg/gm for three consecutive days was performed to induce conditional deletion of Fgfr2 in two-month-old Krt5Fgfr2^CKO or Krt5Fgfr2^CKO-mTmG mice. Phenotypes of MG after Fgfr2 deletion were monitored by meibography, lipid staining, and immunostaining against keratin-6a in MG whole mounts. Lineage tracing of the Krt5⁺ progenitors of MG and biomarkers for ductal differentiation and proliferation were also examined by immunostainings.

Results: The Krt5Fgfr2^CKO mice developed extensive ductal occlusion and acinar atrophy at day 10 after TAM administration. Robust thickening of ductal epithelium with abnormal differentiation and proliferation of ductal basal meibocytes were observed in the MGs of Krt5Fgfr2^CKO mice. In Krt5Fgfr2^CKO-mTmG mice, the Krt5⁺ progenitors and its progeny were labeled by EGFP after Fgfr2 depletion by TAM with evident expansion of the suprabasal and superficial layers of MG ductal epithelium when compared with the controls.

Conclusions: Our results substantiated the crucial role of Fgfr2 in homeostasis of the MG ductal epithelium. Deletion of Fgfr2 affects the MG ductal basal progenitors by impacting the differentiation of ductal meibocytes and the maintenance of acinar meibocytes, which are likely the underlying pathogenesis of obstructive MGD.

Meibomian glands (MGs) are specialized sebaceous glands located in the inner tarsal plates of the superior and inferior eyelids. Lipids secreted by MGs are known as meibum, which comprises the outermost layer of tear film and helps stabilize the tear film to prevent the evaporation of aqueous tear.¹ Proper MG function is essential for maintaining the homeostasis of ocular surface. Meibomian gland dysfunction (MGD) is a chronic disorder affecting meibomian glands and is commonly associated with obstruction/obliteration of the MG terminal ducts and compromised secretions of meibum. Alterations in the composition and quantity of meibum would result in tear film instability because of the deficiency of tear lipid layer.² MGD has been identified as the leading cause of the evaporative dry eye, which usually induces recurrent dry eye symptoms, such as ocular dryness, burning and foreign body sensation.^3–5 

MGD is generally categorized as either obstructive MGD (OMGD) or age-related MGD (ARMGD) based on their distinct clinical and pathophysiological characteristics.⁶ OMGD is the most common form of MGD, characterized by inspissated ductal orifices, viscous meibum expression and acinar atrophy. MG biopsies of patients with OMGD revealed dilation of the central duct of MG with acinar atrophy and regional “hyperkeratinization” or thickening of the ductal epithelium.⁷ It is commonly believed that MG ductal hyperkeratinization initiates the pathogenesis of orifice plugging, abnormal lipid accumulation, cystic MG dilation with secondary acinar atrophy.^8–10 However, the cellular mechanism responsible for OMGD remains elusive. 

Animal model is paramount for better delineation the pathophysiology and the development of novel therapies for OMGD. Prior strategies for inducing OMGD include physical or chemical blockage of MG orifices via polychlorinated biphenyl¹¹ or 2% epinephrine,⁹ MG occlusion in murine allergic eye disease,^12,13 and genetic deletion of essential enzymes involved in meibum synthesis.¹⁴ Nevertheless, those existing models only resemble partial or selective pathophysiological characteristics of OMGD, and do not replicate human OMGD with high fidelity. In addition, the MG obstruction in these models is generally late-onset and irreversible, which greatly impedes their utility for evaluating the efficacy of novel therapeutics for OMGD. An animal model that can recapitulate the pathogenic manifestations of clinical OMGD is critically needed. 

Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases crucial for the development and homeostasis of ocular tissues including cornea, lacrimal gland, and meibomian gland.^15-19 Our previous studies have demonstrated that conditional Fgfr2 deletion via systemic doxycycline administration in a transgenic mice model could induce severe MG atrophy resembling ARMGD.^19,20 In this study, we further developed a novel OMGD model with close resemblance of human condition via tamoxifen (TAM)-mediated deletion of Fgfr2 in MG ductal basal epithelial progenitors. A triple transgenic model was further developed by incorporating a reporter tracer into the aforementioned Fgfr2^CKO mice to monitor the spatiotemporal lineage of Krt5⁺ MG progenitor cells in the pathogenesis of OMGD. 

Material and Methods

Mice

All transgenic mice were bred at the Animal Science Research Center of the University of Missouri (Columbia, MO, USA), in accordance with the institutional guidelines on the Care, Welfare and Treatment of Laboratory Animals. The animal protocol was approved by the University of Missouri Institutional Animal Care and Use Committee. All the experiments conformed to the standards in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Mice were housed under standard conditions of temperature and humidity, with a 12-hour light/dark cycle and free access to food and water. The Krt5CreERT2; Rosa^mTmG reporter mice (referred to as Krt5^mTmG mice) was generated by crossing the Krt5CreERT2 mice (JAX stock no. 029155) with the Rosa^mTmG mice (JAX stock#007676) to visualize the Cre recombination in MGs and other ocular surface tissues with TAM induction. The Krt5CreERT2; Fgfr2^fl/fl (referred as Fgfr2^CKO mice, with Fgfr2^fl/fl mice as controls) mice were generated by crossing the Krt5CreERT2 mice with the Fgfr2^fl/fl mice to excise the Fgfr2 gene in Krt5-expressing cells via TAM exposure. Both Fgfr2^CKO and the control mice were injected with TAM in the experiments. To enable the lineage tracing of Krt5⁺ MG stem/progenitor cells, the Krt5CreERT2; Fgfr2^fl/fl; Rosa^mTmG mice (referred as Fgfr2^CKO-mTmG mice) were generated by crossing the Rosa^mTmG reporter mice with the Fgfr2^CKO mice, and the EGFP fluorescence would manifest in Krt5⁺ meibocytes throughout the MG differentiation. Genotyping was confirmed via mouse tail DNA, using the conditions and primers recommended for each JAX mouse line listed above. Both male and female mice with homozygous Fgfr2 and mTmG transgenes were used. 

Tamoxifen Administration

In two-month-old Krt5^mTmG, Fgfr2^CKO or Fgfr2^CKO-mTmG mice, transgene recombination was induced by intraperitoneal administration of TAM (Sigma-Aldrich Corp., St. Louis, MO, USA) dissolved in 10% ethanol and 90% corn oil (Sigma-Aldrich Corp.) at a dosage of 50 µg/gm body weight for three consecutive days. To assess the effect of recombination inducer or dissolving solvents on the MGs, equivalent dose of TAM or corn oil alone were given to the control mice for comparison. 

Meibography and Oil-Red-O (ORO) Staining

For better exposure of the MGs, the upper and lower tarsal plates were excised by removing the periorbital skin and muscles from the eyelids of Fgfr2^CKO mice at each specified time point. Meibographs were taken on the flat-mount of tarsal plates by a Leica M165 FC fluorescence stereomicroscope equipped with a CCD camera. MG whole-mounts and cryosections of tarsal plates were stained with ORO as previously described.²⁰ In general, the whole-mounts or cryosections of tarsal plates were rinsed with PBS, treated with 60% 2-propanol for 30 seconds, stained with ORO solution for 15 minutes, and destained with 60% 2-propanol for three minutes. 

Histology and Immunostaining

Mouse eyelids, eye globes, and lacrimal glands from the reporter mice, Fgfr2^CKO or Fgfr2^CKO-mTmG mice were harvested, fixed in 4% paraformaldehyde overnight, and then processed for either cryosections to examine the expression of EGFP in the reporter mice, or paraffin sections for hematoxylin and eosin (H&E) staining. Immunofluorescence and immunohistochemical staining were performed as previously described.²⁰ For immunofluorescence, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). For immunohistochemistry, tissue sections were treated with 10 mM of sodium citrate buffer at 95°C for 10 minutes for antigen retrieval, followed by incubation in 3% hydrogen peroxide in PBS for 30 minutes to deactivate endogenous peroxidase activity. Sections were then blocked in 5% horse serum in PBST (PBS plus 0.5% Triton X-100) for one hour and incubated overnight at 4°C with primary antibodies against the following antigens: Pan Cytokeratin (Pan-KRT) (1:500; Cell Signaling Technology, Danvers, MA, USA); Keratin 6a (K6a) (1:2000; BioLegend, San Diego, CA, USA); K17 (1:500; Novus Biologicals, Littleton, CO, USA); K14 (1:1000; BioLegend); K10 (1:500; Covance, Durham, NC, USA); Ki67 (1:500; Cell Signaling Technology); p63 (1:1000; Abcam, Cambridge, MA, USA) and PCNA (1:1000; Abcam). After washing with PBS, tissue sections were incubated at room temperature for one hour with corresponding fluorophore-conjugated or biotinylated secondary antibodies from Thermo Fisher Scientific (Waltham, MA, USA) and Vector Laboratories (Burlingame, CA, USA), respectively. An ABC kit (Vector Laboratories) and a DAB (3,3′-′Diaminobenzidine, 10 mg) tablet (Sigma-Aldrich Corp.) were used to visualize the biotinylated probes, and the sections were counterstained with hematoxylin for cell nuclei. 

Imaging of EGFP⁺ Clonal Dynamics in MGs

To track the Krt5⁺ lineage dynamics of MGs during the development of obstructive MGD, Fgfr2^CKO-mTmG mice were administered with TAM for three days and euthanized at seven and 10 days after TAM. Excised tarsal plates were fixed with 4% paraformaldehyde for flat-mounts and EGFP⁺ cells were imaged by a Leica M165 FC fluorescence stereomicroscope (Leica, Wetzlar, Germany). To better delineate the Krt5⁺ epithelial lineage in MG ducts and acini, tarsal plates were further processed for cross and sagittal cryosections. Cell nuclei were stained with DAPI. 

Immunofluorescent Staining of MG Whole-Mount

Whole-mount immunofluorescent staining of MGs has been described previously.²⁰ Tarsal plates were fixed in 4% paraformaldehyde for 30 minutes and rinsed with PBS. Tissue samples were incubated in a blocking solution containing 10% serum in TBST (Tris-buffered saline solution containing 0.15% Tween-20) overnight at room temperature. Tarsal plates were incubated with primary antibody against Krt6a (BioLegend, diluted at 1:500 in blocking solution) for 24 hours on a rocking plate at 4°C. After washing with PBS, the tarsal plates were incubated in Alexa Fluor 488 conjugated secondary antibody overnight at room temperature. Samples were rinsed with TBST and flat-mounted with Mowiol (cat. no. 10849; Sigma-Aldrich). Images were acquired using a Leica TCS SP8 MP inverted spectral confocal microscope. 

Statistical Analysis

To evaluate the severity of acinar atrophy and orifice plugging in the TAM-induced Fgfr2^CKO mice, the MG areas were marked in the ORO-stained meibographs, and the measurements were recorded using NIH Image J software. The counting of the obstructed glands with white plugs were labeled manually and recorded by NIH Image J software. Statistical tests were performed using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). The data were presented as mean ± SD, and analyzed using unpaired Student's t-tests, with P < 0.05 representing statistically significance. 

Results

EGFP Expression in MGs of the Krt5-Reporter Mice

To ascertain the efficiency and tissue-specificity of the Krt5-driven gene deletion system in MGs, we generated the dual recombinase-activated Krt5CreERT2; Rosa^mTmG mice and performed the pulse-chase experiment with TAM induction (Fig. 1A). The longitudinal and spatial expression of EGFP reporter in ocular surface tissues was examined at days 4, 7 and 10 after TAM injection (Fig. 1B). The EGFP fluorescence was visible and specifically expressed within MGs after TAM exposure (Fig. 1C). Four days after the initial TAM injection, EGFP could be readily detected in the peripheral basal meibocytes (Figs. 1D, 1D1). After a seven-day chase, the EGFP was robustly expressed in the suprabasal acinar layer and only scattered among the ductal epithelium (Figs. 1E, 1E1). After chasing for 10 days after TAM, the EGFP fluorescence no longer retained in most acinar meibocytes, whereas EGFP⁺ were abundantly retained in the ductal epithelium (Figs. 1F, 1F1). Interestingly, scattered Krt5⁺EGFP⁺ cells were shown in the myoepithelial and ductal cells in the lacrimal gland (Fig. 1G). Clusters of Krt5⁺EGFP⁺ cells were also found in the basal and differentiated corneal epithelium after TAM induction (Fig. 1H). EGFP was barely expressed in ocular tissues in the absence of TAM induction (Fig. 1I), suggesting minimal leakage of Cre in the Krt5CreERT2 line. These data indicate that the inducible Krt5-driven gene recombination system enables efficient conditional gene deletion in MGs and other ocular surface tissues such as cornea or lacrimal gland. 

Figure 1.

 

TAM-induced EGFP expression in MG and ocular surface tissues of the reporter mice. (A) Illustration of the inducible fluorescence reporter system in Krt5Rosa^mTmG mice. Prior to TAM exposure, all cells express Tomato (red) fluorescence. Upon TAM induction, Cre recombinase driven by the Krt5 promoter is activated and gain access to the nucleus to delete the Tomato cassette, leading to the expression of EGFP (green) in Krt5-positive cells (and future cell lineages derived from these cells). (B) Experimental scheme. (C) Visible EGFP fluorescence in the MG of the reporter mice after seven days after TAM induction. (D–F, D1–F1) Sagittal and cross sections of MGs from the TAM-injected reporter mice stained with DAPI at indicated time points after the first injection of TAM. (G) Discrete EGFP was detected in ductal and myoepithelial cells of lacrimal gland at day 7 after TAM. (H) EGFP expression in the corneal epithelium on D10 in reporter mice. (I) DAPI staining of MG from reporter mice in the absence of TAM. MG, Meibomian gland; a, acini; d, duct; conj, conjunctiva; Mus: Muscle. Scale bars: 100 µm.

TAM-Induced Fgfr2 Ablation in Krt5⁺ Epithelial Lineage Caused Severe MG Acinar Atrophy and Ductal Obstruction

We have previously reported that Fgfr2 is essential for the homeostasis of meibocytes in adult mice.¹⁵ To further access the temporal profile of Fgfr2 in the basal progenitors of MG, we developed the Krt5CreERT2; Fgfr2^fl/fl mice to investigate the phenotypes of Fgfr2 deletion from Krt5 epithelial lineage by TAM administration (Fig. 2A). At day 10, the TAM-induced Fgfr2^CKO mice developed robust MG orifice plugging and signs of ocular irritations (Figs. 2B, 2C). As shown in meibographs, the MG areas progressively reduced to 93.30%, 65.25% and 12.65% at days 4, 7 and 10 after TAM, respectively, when compared with the TAM-treated control mice. The averaged proportion of plugged glands to total glands peaked at day 10 to 52.55% (Figs. 2D, 2D1). ORO-staining revealed that MG acini initially became atrophic at day 7 with subsequent atrophy/loss of ductules (Figs. 2E–H) and abundant meibum occlusion of the orifices of the MG ducts at day 10 (Figs. 2E1–H1). Of note, these glands with severe acinar atrophy/loss and ductal occlusion underwent efficient and spontaneous recovery at day 17 post-TAM (Figs. 2I, 2I1), suggesting that the Fgfr2 deletion was not sustained after discontinuing TAM induction and the reversible nature of our mouse model. 

Figure 2.

 

MG atrophy and MG orifice plugging shortly after TAM induction in Fgfr2^CKO mice. (A) Experimental scheme to examine the morphological changes of MG. (B, C) Appearance of eye and eyelids on D10. Compared with the control mice, eye squeezing (B) and MG orifice plugs (C) developed in the TAM-injected Cre⁺Fgfr2^CKO mice. (D) The quantitative analysis of MG area at indicated time points based on the ORO-stained meibographs. (D1) The ratio of the plugged glands to total glands in MG from single eyelid on day 10 in Fgfr2^CKO mice. (E–H) The bright- field meibographs showed that MG acini gradually became atrophic from day 4 (F) to day 7 (G). Severe acinar atrophy and white plugs developed in MG on D10 (H). (I) Spontaneous restoration of MG after induced atrophy on D17 in Fgfr2^CKO mice. (E1–I1) Tarsal plates dissected from upper and lower eyelids stained with lipid dye ORO. Abundant lipid-producing acini were shown in control mice (E1). Progressive reduction of lipid accumulation in MGs was noted on day 4 (F1), day 7 (G1), and day 10 (H1) in TAM-induced Fgfr2^CKO mice. Plugs with stagnant lipids were observed in the orifice of upper and lower MGs on day 10. Spontaneous recovery of lipid accumulation in MGs on D17 (I1) in TAM-induced Fgfr2^CKO mice. MGO, Meibomian gland orifice.

Hyper-Differentiation of the Ductal Epithelium was Associated With MG Obstruction

Compared with the TAM-treated Cre-negative control mice (Figs. 3A–C), the terminal segment (near the lid margin) of the central duct bulged in the TAM-induced Fgfr2^CKO mice at day 10 post-TAM (Figs. 3D–F). The glandular orifice of the TAM-treated Fgfr2^CKO mice was sealed with a thin, eosin-positive cornified layer extended from the epidermis as indicated by arrow heads in the H&E staining (Fig. 3D). Immunostaining with the pan-cytokeratin antibody which marks the MG ductal compartment demonstrated the hyper-stratification (acanthosis) of the central ductal epithelium with severe ductular loss in the proximal and distal MG in Fgfr2^CKO after TAM induction (Fig. 3E). The expression of the proliferative marker, Ki67, was enhanced in the basal ductal epithelium of MG in TAM-induced Fgfr2^CKO mice compared with the controls (cf., Figs. 3C, 3F). 

Figure 3.

 

Dilation and hyper-differentiation of MG ductal epithelium in the TAM-induced Fgfr2^CKO mice. (A, D) Compared with the controls (A), H&E staining revealed the dilated central duct, atrophic acini and orifice cornification (arrowheads indicate thin cornified layer over MG orifice) of MG on D10 in the TAM-induced Fgfr2^CKO mice (D). (B, E) Compared with the controls (B), immuno-staining of pan-keratin showed the hyper-differentiated ductal epithelium and dilated central duct in MG of the TAM-induced Fgfr2^CKO mice (E). (C, F) Scattered expression of Ki67 was noted in the MG basal acinar, basal ductular and basal conjunctival epithelium in control mice (C), whereas increased expression of Ki67 was observed in the basal ductal and conjunctival epithelium in the TAM-induced Fgfr2^CKO mice (F). (G, J) Compared with the controls (G), immunofluorescent staining with anti-K6a in MG whole-mount showed enlarged proximal duct near the lid margin and severe ductular loss in MG of the TAM-induced Fgfr2^CKO mice (J). (H, K) K17 was expressed in the differentiated ductal epithelium of MG with p63 expressed in basal ductal epithelium in the control mice (H). K17 was robustly expressed in the hyper-differentiated ductal wall with narrowing ductal cavity of the TAM-induced Fgfr2^CKO mice (K). (H1, K1) Compared with the control mice (H1), EdU-positive cells were increased in hyper-differentiated ductal epithelium of the TAM-induced Fgfr2^CKO mice (K1). (I, L) K10 was preferentially expressed in keratinized epidermal and partially expressed in orifice of MG central duct of the control mice (I), but K10 was only expressed only in the lid margin and not in the orifice of the central duct or the ductal epithelium in the TAM-induced Fgfr2^CKO mice (L). MG was outlined by the white dotted line in H1, K1, I, L. HF, hair follicle. Scale bars: 100 µm.

Krt6a is a cytokeratin marker normally expressed in the differentiated ductal epithelium of MG. To determine whether the meibum occlusion was associated with the MG ductal malformation, we performed the immunostaining of K6a in whole-mounts to outline the spatial organization of the MG ductal structures in Krt5Fgfr2^CKO and the control mice. As shown in Fig. 3G, the ductal compartment of MG in the controls comprised of the long, straight central duct and dozens of highly intertwined ductular branches. In the TAM-treated Fgfr2^CKO mice, the central duct was extensively enlarged in the proximal MG near the lid margin, while severe ductular atrophy and loss occurred in the middle and distal MG (Fig. 3J). Similarly, Krt17 is a keratin marker which is expressed in the differentiated ductal epithelium of MG (Fig. 3H). We found that Krt17 was robustly expressed in the suprabasal ductal epithelial cells in the TAM-induced Fgfr2^CKO mice (Fig. 3K), suggesting that the central ductal epithelium of MG was hyper-stratified, which was likely resulted from TAM-induced Fgfr2 deletion. In addition, the ductal basal cells in MG marked by p63 were notably increased in the Fgfr2^CKO mice when compared with the controls. (cf., Figs. 3H, 3K), suggesting a boost of ductal basal cell population in response to the TAM-induced Fgfr2 deletion. Edu is usually accessed to determine the S phase of the cell cycle. Meibocytes proliferation measured by EdU incorporation suggested that Fgfr2 deletion led to increased ductal meibocyte proliferation (Figs. 3H1, 3K1). Interestingly, the conjunctival epithelium was notably more stratified after TAM treatment in the Fgfr2^CKO mice (Figs. 3D–F, 3L) when compared with the Cre-negative controls (Figs. 3A–C, 3I). Krt10 is highly expressed in keratinized epithelium such as lid epidermal and partially expressed in the ductal epithelium of MG orifice in the lid margin, but not expressed in the differentiated ductal epithelial cells of middle and distal MG. Krt10 was used to determine whether the more stratified ductal epithelium of MG is also hyperkeratinized. It is noteworthy that Krt10 was not over-expressed in the TAM-induced Fgfr2^CKO mice when compared with the controls, suggesting that the MG ductal epithelium in our MGD model was not hyperkeratinized as those noted in the epidermal epithelium adjacent to the MG orifice (Figs. 3I, 3L). 

We further assessed the pathologies of MG with terminal duct obstruction in the Fgfr2^CKO mice. In the healthy MGs of control mice, the central ductal epithelium near the orifice commonly consists of 4 layers of stratified squamous epithelial cells with tiny ductular structures and acinar tissues (Fig. 4A). Cross-sections of the MG near the orifice of the Fgfr2^CKO mice showed that the hyper-stratified ductal epithelium without dilation consisting of 8-10 layers of suprabasal cells along with the obliteration of ductular structures (Fig. 4B). ORO staining showed that the majority of MGs in Fgfr2^CKO mice was enlarged and blocked with lipid contents, or extremely narrowed ductal cavity and thickened ductal wall (Fig. 4D). Co-staining of Ki67 and p63 also suggested an increased proliferation of ductal basal cells in the MGs of the Fgfr2^CKO mice (Figs. 4F, 4H) when compared with the controls (Figs. 4E, 4G). Thus we speculate that the deletion of Fgfr2 in Krt5⁺ ductal lineage enhanced proliferation of basal meibocytes with disruption of normal differentiation. The extensive thickening of ductal epithelium and the narrowing of the proximal ductal cavity due to the abnormal proliferation and differentiation of ductal basal meibocytes are likely attributable for the MG obstruction in the TAM-induced Fgfr2^CKO mice. 

Figure 4.

 

Histopathological changes in MG orifice after Fgfr2 deletion via TAM on day 10. (A, B) H&E staining of MG orifice. Compared with control mice (A), robust thickening and stratification of ductal epithelium and narrowed intraductal space was noted in MGs without plug formation in the Fgfr2^CKO mice (B). (C, D) ORO staining of MG orifice. In the control mice, lipids distributed among the ducts and acini (C). In Fgfr2^CKO mice, MGs with plugs exhibited dilated duct in the terminal segment (near the lid margin) with blockage by excessive lipid content, whereas MGs without plug showed hyper-stratified ductal wall and minimum lipid content (D). (E–H) Immunostainings of Ki67 (red) and p63 (green) in distal MGs. In control mice, Ki67, a proliferation marker, was normally expressed in the p63-positive MG ductal basal epithelium (E, G). Ki67 expression was slightly increased in MG ductal basal epithelium in the Fgfr2^CKO mice (F, H). MG was outlined by the white dotted line. MG, Meibomian gland; d, duct; conj, conjunctiva; HF, hair follicle; epi, epidermis. Scale bars: 100 µm.

Abnormal Differentiation of Krt5⁺ Lineage was Involved in the Hyper-Stratification of the MG Ductal Epithelium

To investigate the potential role of Fgfr2 in MG progenitors contributing to the pathogenesis of MG obstruction, the Krt5CreERT2; Fgfr2^fl/fl; Rosa^mTmG mice were administered with TAM for three days resulting in Fgfr2 deletion and expression of EGFP reporter (Figs. 5A, 5B). Krt5CreERT2; Fgfr2^+/+ Rosa^mTmG mice with TAM administration were used as the controls for comparison. At day 10, EGFP⁺ meibocytes were diffusely distributed among the normal acinar and ductal tissues of MGs in the TAM-treated control mice (Figs. 5C, 5C1). The phenotypes of MG obstruction and acinar atrophy along with EGFP expression were evident at day 10 post TAM induction in the Fgfr2^CKO-mTmG mice (Figs. 5D, 5D1). As shown in meibographs and outlined by EGFP fluorescence in wholemount MG (Fig. 5D1), these mice exhibited similar phenotypes as the Fgfr2^CKO mice including severe ductal obstruction and malformation of MG. At 10 days after TAM, EGFP⁺/Krt5⁺cells were mostly present in the acinar tissues with a few scattered in the suprabasal layers of the ductal epithelium in the homeostatic MGs (Figs. 5E, 5E1, 5G, 5G1) in the control mice. In contrast, EGFP⁺/Krt5⁺lineage cells were notably expanded in the suprabasal and superficial layers of the MG ductal epithelium as well as present in the conjunctival epithelium of the Fgfr2^CKO-mTmG mice (Figs. 5F, 5F1, 5H, 5H1). These findings suggest that the ductal hyper-stratification and OMGD in the Fgfr2^CKO-mTmG mice is induced by the activation of Krt5⁺ lineage in ductal meibocytes with abnormal differentiation after deletion of Fgfr2 by TAM. 

Figure 5.

 

Active differentiation of Krt5⁺ lineage in ductal MG and conjunctival epithelium after TAM-induced Fgfr2 deletion. (A) Krt5CreERT2, Fgfr2^flox/flox mice were crossed with Rosa^mTmG reporter strains to delineate the Krt5⁺ basal meibocytes and its progeny after inducible deletion of Fgfr2 by TAM. (B) Experimental scheme to examine the morphological changes and EGFP expression of Krt5 lineage at day 10. (C–D1) MGs in tarsal plates. Representative images of MGs under bright field (C, D) and with EGFP fluorescence (green) (C1, D1) from the control mice (Krt5^mTmG) (C, C1) and Fgfr2^CKO-mTmG (D, D1) after a 10-day chase. Extensive lipid stagnations (bright spots in D) with MG atrophy and ductal dilation (indicated by arrowheads in D1) were seen in Fgfr2^CKO-mTmG. (E–H1) Expression of EGFP (green) and Tomato (red) in MGs. Sagittal sections (E, F) and cross-sections (G, H) of MGs with corresponding DAPI counterstaining (E1, F1, G1, H1) showed that EGFP was expressed in the acinar and ductal basal cells in control mice, but was expressed in the hyper-stratified ductal epithelium obliterating the MG central ducts of the Fgfr2^CKO-mTmG. White dotted lines indicate MG area and yellow dotted lines indicate duct area. When compared with the controls (E, G), enhanced EGFP expression (arrowheads in F and H) was also noted in the conjunctival epithelium of the Fgfr2^CKO-mTmG mice. MG, Meibomian gland; a, acini; d, duct; conj, conjunctiva; HF, hair follicle. Scale bars: 100 µm.

Discussion

Obstructive meibomian gland dysfunction is a chronic and refractory ocular surface morbidity with dry eye symptoms due to extensive plugging of MG orifices and unstable tear film.^4,21–23 The pathophysiology of OMGD has not been well understood due to the lack of appropriate animal models and relevant research tools. As described above, we have devised a novel mouse model of OMGD with robust ductal obstruction and acinar atrophy shortly after genetic deletion of Fgfr2 in Krt5⁺ MG progenitor cells. This inducible Krt5Fgfr2^CKO-TAM transgenic mouse model of OMGD is readily reversible upon discontinuing TAM induction. It closely recapitulates the phenotypic and anatomical features of clinical OMGD and can serve as an ideal model for further investigating the pathophysiology of OMGD or related novel therapeutic strategies. Most importantly, our model demonstrates the crucial and cell-specific role of Fgfr2 signaling in basal progenitors of MG. Absence of Fgfr2 in Krt5⁺ MG progenitor cells would induce abnormal differentiation with hyper-stratification of the ductal meibocytes and disrupt the self-renewal of acinar meibocytes, reconfirming the essential role of Fgfr2 for modulating MG homeostasis and differentiation. Leveraging the tools of lineage tracing and conditional gene deletion, we unequivocally correlated the abnormal differentiation of Krt5⁺ MG ductal progenitor cells with the pathogenesis of OMGD. Our results also reconfirm that Fgfr2 is the key regulator of Krt5⁺ basal progenitors in MG homeostasis. 

Our current model presents a gradual development of OMGD leading to eventual extensive acinar atrophy, hyper-stratification of ductal epithelium and orifice occlusion in MG. We propose that thickening of the ductal epithelium with attenuated ductal cavity as the underlying cause of MG obstruction in this model. The MG ductal pathologies observed by us are similar to several previously reported models of OMGD including hairless mice fed with a lipid-limited diet²⁴ and mice with deletion of β-epithelial Na⁺ channel.²⁵ However, the mechanism regarding MG obstruction remains elusive in those prior models. As evidenced by our studies, the obstruction likely originated from orifice hypercornification, ductal thickening, and altered meibum secretion secondary to the loss of Fgfr2 in Krt5⁺ MG progenitor lineage. 

Fgfr2 signaling is essential for epithelial homeostasis regeneration in multiple self-renewing tissues including cornea, lung, mammary gland, and salivary gland.^{15,16,26–31} We previously have reported that Fgfr2 is required for the homeostasis and restoration of adult MG.^19,20 In this study, we further elucidate the cell-specific role of Fgfr2 in Krt5⁺ basal progenitors of MG. We found that depletion of Fgfr2 from Krt5⁺ basal epithelium by TAM promotes the abnormal differentiation of ductal epithelial cells and lead to ductal hyper-stratification. Previous label-retaining or lineage tracing studies have identified that Krt5-positive progenitor cells are critical for the MG homeostasis and renewal.³² The authors propose that under homeostatic conditions, there are two potential stem cell populations in MG with Krt5+/Krt6-/PPARγ+/Sox9+ giving rise to the acinar meibocytes and Krt5+/Krt6+/PPARγ- giving rise to the ductal epithelium. Alternatively, it is also possible that either lineage can give rise to the other cell lineage interchangeably with both being bipotent stem cell populations.³² Of note, our data suggests that of Krt5⁺ lineage would undergo terminal differentiation robustly and contribute to ductal hyper-stratification and MG obstruction in response to TAM-induced Fgfr2 deletion. We speculate that deletion of Fgfr2 by TAM in Krt5+ basal progenitor lineage might exacerbate the abnormal proliferation of Krt5+ ductal progeny and perturb subsequent differentiation of these Krt5+ basal progenitors responsible for acinar homeostasis, therefore leading to ductal hyper-stratification and acinar atrophy during the pathogenesis of OMGD (Fig. 6). Fgfr2 might play important and complex roles in regulating the ductal progenitor functions following injury. Whether and how Fgfr2 regulates the injury-induced plasticity of ductal progenitors awaits further investigations. 

Figure 6.

 

Schematic hypothesis of Krt5⁺ lineage contribute to the pathogenesis of OMGD. Disruption of Fgfr2 by TAM in Krt5⁺ basal progenitor lineage might exacerbate the abnormal differentiation of Krt5⁺ ductal progeny and suppress the related differentiation of acinar progenitors, thereby leading to ductal hyper-stratification and acinar atrophy in the pathogenesis of OMGD.

The ductal phenotypes of the Krt5-driven Fgfr2^CKO mice were different from those in the K14-driven Fgfr2^CKO mice in our previous reports.^19,20 Our previous Fgfr2 deletion in K14-expressing cells affects both basal (undifferentiated) cells and differentiated meibocytes, whereas the current Krt5Fgfr2^CKO model enables us to examine the effect of Fgfr2 deletion primarily in the basal cell renewal of MG. The MG ductal epithelium was not atrophic but became thickened in mice with Krt5-driven Fgfr2 ablation in the current report. We surmise that the distinct pathogenic changes in MG ductal system between the two models might be related to the differential expression of Krt14 and Krt5 in MG (See Supplementary Fig. S1). Although OMGD is with more male preponderance clinically, the functional significance of Fgfr2 deletion in OMGD needs further exploration. As the pathogenic mechanisms for OMGD remain unsettled and MG is sensitive to the modulation by sex hormones, systemic administration of tamoxifen in our current model may have led to the generalized inhibition of estrogen receptors with preferential up-regulation by androgens, probably the underlying cause of more prevalent OMGD in male patients. 

In addition, the biological effects of TAM might also exacerbate the ductal thickening of our Krt5-TAM model. TAM is a selective estrogen receptor (ER) modulator commonly used to impede tumor growth for breast cancer.^33–35 While TAM only serves as a generic inducer in animal studies, the potential impact of TAM on meibomian glands or sex hormones has not been investigated previously. Pharmacological studies have reported that high-dose tamoxifen decelerates lipid accumulation and regulates inflammation in liver with reduction of adipocyte area in white adipose tissues.^36,37 Whether the phenotype of MGs after TAM induction in our study is mediated directly by the deletion of Fgfr2 or indirectly via modulating ER or by interference with inflammation awaits further investigation. The potential existence of some off-target effects derived from TAM induction cannot be excluded in our current model. Because TAM exposure could enhance the ductal hyper-stratification and obstruction in MGD, our findings would caution the potential risk of developing OMGD in patients under TAM treatment for breast cancer or menstrual disorder. 

To date, the mechanism of OMGD is not well understood. Multiple factors including inflammation, abnormal lipid metabolism, and external stimuli including physical and chemical exposures have been associated with the development of MG obstruction.^38,39 Previous studies have demonstrated that inflammation with accumulated neutrophils could induce MG obstruction and orifice plugging.^12,13 However, inflammation is not always present in human OMGD and some animal models. MG obstruction and hyperkeratinazation could be induced by other primary causes such as barrier breakup of the MG duct without inflammatory reactions.⁴⁰ Because we did not observe evident inflammatory infiltration in MGs after TAM induction in our current model, we speculate that inflammation might not be a major cause for the development of OMGD induced by TAM. Additionally, the ductal obstruction in our model is unlikely resulting from the defective meibum metabolism associated with the deficient essential enzymes in lipid synthesis.^41–43 

There are some limitations in this study. The biological ramifications of TAM on meibomian gland and other ocular surface tissues needs to be further delineated. As TAM is a regulator of sex hormones and was only used for the first three days in this study, the impact of long-term TAM on MG should be carefully analyzed between different sexes in future studies. 

In summary, our studies have elucidated the cell-specific role of Fgfr2 in the homeostasis of MG basal progenitors via lineage tracing and genetic deletion. We have developed a novel animal model that well simulates clinical OMGD with MG ductal obstruction and extensive acinar atrophy via TAM-induced Fgfr2 deletion in Krt5⁺ progenitor lineage, suggesting the potential role of Fgfr2 in regulating the plasticity of ductal progenitors. Our OMGD model is also reversible and could serve as a reproducible model for investigating the pathophysiology and therapeutic approaches for OMGD. 

Acknowledgments

Supported by the NEI grant EY029106 (to LWR and AJWH) from the National Institutes of Health and Guangdong Basic and Applied Basic Research Foundation (2022A1515111180) and the National Natural Science Foundation of China No. 92368205 and 82401209. 

Disclosure: X. Yang, None; X. Zhong, None; H. Lin, None; A.J.W. Huang, None; L.W. Reneker, None