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. Author manuscript; available in PMC: 2026 Aug 4.
Published in final edited form as: Exp Eye Res. 2024 Jan 19;240:109797. doi: 10.1016/j.exer.2024.109797

Expression of Nephronectin in the Descemet’s Membrane of Mouse Corneas during Development and Adult Homeostasis

Matthew Garis 1, Matthew D Meyer 2, Peter Lwigale 1,*
PMCID: PMC13430871  NIHMSID: NIHMS2196606  PMID: 38246333

Abstract

Nephronectin (Npnt) is an extracellular matrix (ECM) protein with pleiotropic functions during organogenesis, disease, and homeostasis. Although the ECM plays a crucial role during development and homeostasis of the adult cornea, little is known about the expression of Npnt in the mammalian cornea. Here, we investigated the expression of Npnt during early embryonic and postnatal development, and in adult mouse corneas. We combined ultrastructural and immunohistochemical analyses to study the early formation of the Descemet’s membrane and how the expression of Npnt relates to key basement membrane proteins. Our section in situ hybridization and immunohistochemical analyses revealed that Npnt mRNA is expressed by the nascent corneal endothelial cells at embryonic day (E) 14.5, whereas the protein is localized in the adjacent extracellular matrix. These expression patterns were maintained in the corneal endothelium and Descemet’s membrane throughout development and in adult corneas. Ultrastructural analysis revealed discontinuous electron dense regions of protein aggregates at E18.5 that was separated from the endothelial layer by an electron lucent space. At birth (postnatal day, P0), the Descemet’s membrane was a single layer, which continuously thickened throughout P4, P8, P10, and P14. Npnt was localized to the Descemet’s membrane by E18.5 and overlapped with Collagens IV and VIII, Laminin, and Perlecan. However, the proteins subsequently shifted and formed distinct layers in the adult cornea, whereby Npnt localized between two Collagen VIII bands and anterior to Collagen IV but overlapped with Laminin and Perlecan. Combined, our results reveal the expression of Npnt in the mouse cornea and define its spatiotemporal localization relative to key basement membrane proteins during the formation of the Descemet’s membrane and in the adult cornea. Understanding the spatiotemporal expression of Npnt is important for future studies to elucidate its function in the mammalian cornea.

Keywords: Nephronectin, Extracellular matrix, Descemet’s membrane, Basement membrane proteins, Cornea, Development

1. Introduction

The extracellular matrix (ECM) plays an important role in regulating cell behavior and fate in response to environmental cues during development, homeostasis, and disease of various tissues and organs including the cornea. Although numerous studies have elucidated the expression and roles of ECM proteins in the cornea, very little is known about Nephronectin (Npnt), a protein that was relatively recently identified in the kidney as an ECM ligand for integrin α8β1 (Brandenberger et al., 2001) and in pre-osteoblast cells as a protein with an epidermal growth factor (EGF)-like repeat and meprin-A5 protein-receptor protein tyrosine phosphatase μ (MAM) domain (Morimura et al., 2001).

Npnt is a 70-90 kDa protein that consists of three functional domains; an N-terminal region with five EGF-like repeats, an Arg-Gly-Asp (RGD) linker region, and a C-terminal MAM domain (Brandenberger et al., 2001; Morimura et al., 2001). Studies in various tissues and cancer research have shown that Npnt has pleiotropic functions through cellular interactions with each of these functional domains. Signaling associated with the EGF-like repeats was reported to induce cell proliferation and differentiation in osteoblasts and dental stem cells (Fang et al., 2010; Kahai et al., 2010; Morimura et al., 2001), and also to activate migration of vascular endothelial cells (Kuek et al., 2016). Cell interactions with the RGD domain via integrin α8β1 were linked to cross-talk between mesenchyme and epithelial cells during kidney development and homeostatsis (Brandenberger et al., 2001; Cheng et al., 2008; Inagi et al., 2017; Miner, 2001; Müller et al., 1997; Müller-Deile et al., 2017; Sato et al., 2009; Zimmerman et al., 2018). Our recent study showed that Npnt signals through integrin α8 to promote periocular neural crest cell migration during chick cornea development (Ma et al., 2022). The MAM domain was shown to bind to heparin sulfate proteoglycans and potentially play a role in Npnt deposition into basement membranes (Sato et al., 2009). Other studies have shown that Npnt plays a role in differentiation of erector pili muscles of mice hair follicles (Fujiwara et al., 2011), limb development in xenopus (Abu-Daya et al., 2011), heart development in zebrafish (Patra et al., 2011), targeting retinal ganglion axons to the superior colliculus region of the brain (Su et al., 2021), and in cancer metastasis (Magnussen et al., 2021).

As an initial step in establishing the role of Npnt in the mammalian cornea, we investigated its expression in the mouse cornea during embryonic and postnatal development, and in adults. We found that that Npnt is expressed by the corneal epithelial and endothelial cells and the protein localized to the corneal basement membranes during development. We also observed that Npnt was transiently expressed in the corneal epithelium, but it persisted in the Descemet’s membrane throughout development and adulthood. Since Npnt localized to the endothelial region of the cornea during early development, we also performed ultrastructural analysis to determine the early formation of the Descemet’s membrane in mice. To better understand its spatial location in the Descemet’s membrane, we characterized the expression of Npnt relative to major basement membrane proteins during formation and maintenance of the cornea endothelium. Collectively, our results illustrate that Npnt is an integral protein in the Descemet’s membrane of the mouse cornea.

2. Materials and Methods

2.1. Animals

Timed pregnant C57BL/6J mice were obtained from Jackson Laboratory (Bar Harbor, ME). Embryos were collected at embryonic day (E) 14.5, E18.5, and eyes from pups at postnatal day (P) 0, P4, P8, P10, P14 and from adults. At least 3 samples were analyzed for each developmental stage. All animal procedures were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee (IACUC) at Rice University (Houston, TX, USA).

2.2. In situ hybridization

Freshly isolated heads or eyes were fixed overnight in modified Carnoy’s fixative (60% ethanol, 30% formaldehyde, 10% glacial acetic acid), dehydrated, embedded in paraffin, and sectioned at 8-12 μm thickness. Riboprobes for mouse Npnt were generated from gene fragments using cDNA pooled from adult kidneys and cloned into TOPO-pCR4 (Invitrogen). Digoxigenin (DIG)-labeled riboprobes were synthesized following the manufactures protocol (DIG Labeling Kit, Roche). Primers used were mouse Npnt Fwd: 5’-TGATGCTGGAAGTACAAGGTGGCC-3’ and mouse Npnt Rev: 5’-TCACATCATCCAATCCAATCTCCC-3’. Sections were hybridized with antisense riboprobe at 63°C overnight. A sense riboprobe was used as negative control. Hybridization was detected using anti-DIG antibody conjugated with alkaline phosphatase (Roche) and color was developed with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT; Sigma). Immediately following color development, sections were fixed in 4% PFA, rinsed in PBS, and mounted in Cytoseal (Thermo). Samples were imaged with a Zeiss Axiocam mounted on a Zeiss Axioskop 2 microscope (Zeiss, Oberkochen, Baden-Wurttemberg, Germany).

2.3. Immunohistochemistry

Embryos or eyes were collected in chilled phosphate buffered saline (PBS) solution then fixed in methanol-acetic acid (MAA, 97% Methanol 3% Acetic Acid) that had been chilled on dry ice. Samples were maintained in MAA fixative at −80°C for two days, then gradually warmed to room temperature over 24 hours. Samples were dehydrated in ethanol series (50%, 70%, 90%, and two washes in 100%), cleared in Histosol (National Diagnostics), embedded in paraffin, and sectioned at 8 μm thickness. Corneal sections were immunostained as described (McKenna et al., 2014) with minor modifications. Briefly, sections were deparaffinized in Histosol, rehydrated through ethanol series (two washes in 100%, 90%, 70%, 50%) and rinsed in PBS. Antigen retrieval was performed in citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 10 minutes. Cooled slides were rinsed in water, PBS, and PBS containing 0.1% Triton-X (PBT), and then incubated in blocking solution (PBT, 0.1% BSA, and 5% heat-inactivated sheep serum).

The following antibodies were used diluted in blocking solution: Laminin (Ln, 1:200, L9393, Sigma), Collagen IV (Col IV, 1:200, LSL-LB-1403, CosmoBio), Perlecan (Per, 1:50, SC-33707, Santa Cruz), Collagen VIII alpha II (Col VIII, 1:200, F54619, NSJ Bioreagents), and Nephronectin (Npnt, 1:50, AF4298, R&D Systems). Antibody controls were performed with either no primary antibody or isotype controls. The following secondary antibodies (Molecular Probes) were used at 1:200; Alexa 594 goat anti-rabbit IgG, Alexa 488 goat anti-rabbit IgG, Alexa 488 goat anti-rat IgG1, Alexa 488 goat anti-rabbit IgG, Alexa 594 donkey anti-goat IgG. Sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) at 1:50 in blocking solution to label all nuclei. Slides were cover-slipped with Fluoromount-G (Southern Biotech) and imaged with a Zeiss Axiocam mounted on a Zeiss AxioImager 2 fluorescence microscope with ApoTome (Zeiss, Oberkochen, Baden-Wurttemberg, Germany).

2.4. Transmission Electron Microscopy

Samples (N = 3 for each developmental stage) were fixed overnight in Karnovsky’s fixative (Karnovsky, 1964), post-fixed for one hour in 1% osmium tetroxide, dehydrated in a graded series of ethanol, embedded in EMbed 812 epoxy resin (Electron Microscopy Sciences, Hatfield, PA), and heat polymerized. To identify the area of interest, sections of 500 nm thickness were cut using a Leica EM UC7 ultramicrotome, stained with toluidine blue, and examined using a compound light microscope. Ultra-thin sections of 100 nm thickness were cut using a Leica EM UC7 ultramicrotome and stained with saturated methanolic uranyl acetate and Reynold’s lead citrate. Digital electron micrographs were acquired using a JEOL JEM-1230 transmission electron microscope (TEM) operating at 80 kV of accelerating voltage and equipped with a high-contrast polepiece and an AMT NanoSprint15 Mk-II sCMOS camera. Images and measurements were collected using AMT capture engine software.

2.5. Analysis of protein localization

Fluorescent images of corneal sections were analyzed using ImageJ software. Images were minimally processed and resampled to 300 dpi using Adobe Photoshop. Analyses were performed on .zvi files imported into Fiji software (ImageJ). A spline width of 3 pixels was used to enhance signal to noise. To capture the relative protein distribution in the Descemet’s membrane, lines were extended 6.5 μm into the posterior stroma. Plot profiles of fluorescent intensity were measured running perpendicular to the Descemet’s membrane (Fig. 2H). Independent measurements of mean fluorescent intensity (MFI) across these lines were taken from each sample in 0.16 μm increment bins. At least three measurements were performed for each biological sample. Protein localization was calculated as an average of all samples for each timepoint (N = 3 for E18 and P0; N = 4 for P4, P8, P10, P14, and adult). The plots show means and standard errors of the relative distances from endothelial nuclei.

Figure 2. Expression of Npnt and basement membrane proteins during early formation of the Descemet’s membrane at E18.5.

Figure 2.

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(A) Schematic illustrating posterior region of the central cornea that was imaged (red box). (B,B’) Representative TEM images showing the corneal endothelium and discontinuous (asterisks) electron dense protein aggregates (black arrows), separated by an electron lucent region (white arrow). (C-G) Immunohistochemical analysis of cornea sections showing expression of Npnt, Ln, Col VIII, Per, and Col IV. Sections were counterstained with DAPI (blue). (H) Schematic illustrating the strategy for analyzing distances of peak fluorescent intensities of protein bands from the endothelial cell nuclei plotted in (I). Data are presented as mean values +/− SEM, N = 3 biological replicates. Abbreviations: ep, epithelium; st, stroma; en, endothelium. Scale bars: B = 2 μm; B’ = 500 nm; C-G = 10 μm.

3. Results

3.1. Expression of Npnt in embryonic, postnatal, and adult mouse corneas

Previous studies reported the expression of Npnt in the developing mouse lens (Brandenberger et al., 2001) and in the limbal stem cell niche and epithelial progenitors in adults (Polisetti et al., 2016). We also observed that Npnt transcripts were upregulated in the stromal keratocytes during chick cornea development (Bi and Lwigale, 2019). Given that periocular neural crest migrate in two waves in chick and as a single wave in mice (Lwigale et al., 2005; Pei and Rhodin, 1970), we sought to define the localization of Npnt mRNA and protein the mouse cornea. Analysis of Npnt expression via section in situ hybridization at E14.5, revealed vivid expression primarily in the region of the nascent corneal endothelial cells, lens epithelium, optic cup (Fig. 1A, arrowheads), and low levels of expression in the periocular mesenchyme (Fig. 1A). By E18.5, Npnt was expressed in the corneal endothelium, eyelid mesenchyme, lens epithelium, and at low levels in the corneal epithelium (Fig. 1B, arrowheads). Expression of Npnt was transient in the corneal epithelium, but it persisted in the corneal endothelium throughout adulthood (Fig. 1C, arrowhead).

Figure 1. Expression of Npnt mRNA and protein in the mouse cornea.

Figure 1.

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Localization of Npnt mRNA and protein was examined by section in situ hybridization (A-C) or immunohistochemistry (D-F). (A-B) During embryonic development, Npnt is strongly expressed in the presumptive corneal endothelium, lens epithelium, optic cup (arrowheads), and it is also present in the periocular mesenchyme at E14.5. By E18.5, Npnt is strongly expressed in the eyelids, corneal endothelium, lens epithelium, and at low levels in the cornea epithelium (arrowheads). (C) In adults, Npnt localizes to the corneal to the endothelium (arrowhead). (D-F) Expression of Npnt protein is observed in the basement membrane of the corneal epithelium, in the region of the presumptive corneal endothelium, and adjacent to the retinal pigment epithelium at E14.5 (arrowheads in D and D’). By E18.5, Npnt localizes to the basement membranes of the corneal epithelium and endothelium, and in the eyelid (arrowheads in E). Strong expression of Npnt persists in the basement membrane of corneal endothelium in adults (arrowhead in F). Abbreviations: co, cornea; oc, optic cup; pm, periocular mesenchyme; ey, eyelid; L, lens; ep, epithelium; st, stroma; en, endothelium. Scale bars: A-C = 50 μm; D-E = 50 μm; D’ = 20 μm; F = 20 μm.

Next, we examined the expression of Npnt protein at similar time points as above. At E14.5, Npnt was localized in the basement membrane of the corneal epithelium, in the region of the presumptive corneal endothelium, and adjacent to the retinal pigment epithelium (Fig. 1D and 1D’, arrowheads). By E18.5 Npnt staining was localized to the basement membranes of the corneal epithelium and endothelium, and in the eyelid mesenchyme (Fig. 1E; arrowheads). Similar to mRNA expression, protein localization of Npnt was transient in the corneal epithelium, although high levels of staining persisted in the Descemet’s membrane throughout adulthood (Fig. 1F, arrowhead).

3.2. Expression of Npnt relative to other basement membrane proteins during early formation of the Descemet’s membrane

In mice, the three cellular layers of the cornea are established at about E15.5, when the corneal endothelium can be distinguished from the overlying stromal mesenchyme via expression of N-cadherin (Reneker et al., 2000). Given that Npnt mRNA and protein only persist in the corneal endothelial cells and Descemet’s membrane respectively, we focused on characterizing its spatiotemporal expression during development and in adults. The Descemet’s membrane was previously reported not to be evident at E18 (Pei and Rhodin, 1970). Therefore, we chose this timepoint as a basis for our analysis. All images were acquired from the central-posterior regions of the corneas (Fig. 2A). Ultrastructual analysis of the matrix region adjacent to the corneal endothelium at E18.5 revealed a discontinuous electron-dense layer of approximately 53.8 ± 2.21 nm in width (Fig. 2B and B’; arrows), which was seperated from the endothelial cell memebrane by an electron-lucent space that measured 58.9 ± 3.16 nm in thickness (Fig. 2B’, white arrow).

Next, we further analyzed the spatial localization of Npnt within the Descemet’s membrane by investigating its expression relative to other major basement membrane proteins including Ln, Per, Col IV, and Col VIII (Halfter et al., 2020; Tamura et al., 1991). At E18.5, all proteins were expressed in the region of the presumptive Descemet’s membrane (Fig. 2C–2G). To define spatial expression of basement membrane proteins relative to each other, we used an analytical scheme (Fig. 2H) as described in the methods section. Peak values of protein expression were represented as distances from the endothelial cell nuclei. This method revealed that Npnt was expressed furthest at 0.64 μm, whereas Col IV was closest to the endothelial nuclei at 0 μm. Both Col VIII and Per were expressed at 0.161 μm, and Ln at 0.32 μm (Fig. 2H). Based on this analysis, Npnt and all basement membrane proteins were either localized intracellularly or adjacent to the endothelial cell layer during the early formation of the Descemet’s membrane.

3.3. Expression of Npnt relative to other basement membrane proteins during early postnatal formation of the Descemet’s membrane

For early postnatal stages, we examined corneas at P0 and P4. Ultrastructural analysis at P0 revealed a continuous electron dense layer that measured 0.12 ± 0.021 μm in thickness (Fig. 3A, arrows). The electron-lucent space adjacent to the endothelial cell membranes remained visible at a thickness of 43.54 ± 1.29 nm (Fig. 3A, asterisk). Protein analysis at this stage indicated that Npnt was expressed at 0.32 μm and remained diffuse up to 4 μm away from the edge of the endothelial nuclei into the Descemet’s membrane and posterior stroma (Fig. 3C and 3M). Expression of Ln remained unaltered at 0.32 μm and overlapped with Col VIII and Col IV, which had both shifted anteriorly and localized at the same distance (Fig. 3D, 3E, 3G and 3M). Expression of Per remained high and tightly associated with the endothelial nuclei at 0 μm (Fig. 3F and 3M).

Figure 3. Expression of Npnt and basement membrane proteins during early postnatal formation of the Descemet’s membrane.

Figure 3.

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(A,B) Representative TEM images showing electron dense regions of the forming Descemet’s membrane at P0 and P4. (C-L) Immunohistochemical analysis of cornea sections showing expression of Npnt, Ln, Col VIII, Per, and Col IV at P0 and P4. Sections were counterstained with DAPI (blue). (M,N) Mean fluorescent intensities showing relative distribution of the proteins at P0 and P4. Data are presented as mean values +/− SEM, N = 3 for P0 and N = 4 for P4 biological replicates. Abbreviations: st, stroma; en, endothelium. Scale bars: A,B = 500 nm; C-L = 10 μm.

By P4, the anterior banded layer (ABL) of the Descemet’s membrane had substantially increased in thickness to 0.241 ± 0.015 μm (Fig. 3B, bracket). Electron-dense lamellae were observed at the most anterior surface of the Descemet’s membrane in a pattern that indicated the formation of distinct bands (Fig. 3B, arrows). By this time, the electron-lucent space was irregularly spaced between the ABL and the endothelial cells. Npnt was expressed at 0 μm and it remained highly diffuse up to 3 μm away from the endothelial nuclei and into the Descemet’s membrane and posterior stroma (Fig. 3H and 3N). The changes observed in the ultrastructual images were recapitulated in the staining for Col VIII and Per, where the expression shifted anteriorly to 1.29 μm and 0.32 μm, respectively (Fig. 3J, 3K, and 3N). In contrast, expression of Ln and Col IV was maintained at 0.32 μm (Fig. 3I, 3L, and 3N).

3.4. Expression of Npnt relative to other basement membrane proteins during late postnatal formation of the Descemet’s membrane

For late postnatal stages, we examined corneas at P8, P10 and P14. Ultrastructural analysis at P8 indicated that thickness of the ABL had more than doubled to 0.487 ± 0.029 μm (Fig. 4A, bracket). The spatial expression of all the basement membrane proteins changed at this stage. Expression of Npnt was no longer defuse and it was tightly localized as a single band that peaked at 0.968 μm (Fig. 4D and 4S). Ln was expressed in two layers, with an anterior band in the stroma region that appeared discontinuous at 2.58 μm and a posterior band that co-localized with Per at 1.129 μm (Fig. 4E, 4G and 4S). Localization of the Col VIII band shifted anteriorly to 1.774 μm, but it was broader and corresponded with its strong expression in the stroma (Fig. 4F and 4S). Col IV was expressed as a thick band with its highest intensity at 0 μm, but it was also localized in the posterior stroma region at 2.58 μm (Fig. 4H and 4S).

Figure 4. Expression of Npnt and basement membrane proteins during late postnatal formation of the Descemet’s membrane.

Figure 4.

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(A-C) Representative TEM images showing electron dense regions of the forming ABL layer of the Descemet’s membrane (indicated by the brackets) at P8, P10 and P14. (D-R) Immunohistochemical analysis of cornea sections showing expression of Npnt, Ln, Col8a2, Per, and Col4 at P8, P10, and P14. Sections were counterstained with DAPI (blue). (S-U) Mean fluorescent intensities showing relative distribution of the proteins at P8, P10, and P14. Data are presented as mean values +/− SEM, N = 4 biological replicates for each timepoint. Abbreviations: st, stroma; en, endothelium. Scale bars: A-C = 500 nm; D-R = 10 μm.

At P10, the Descemet’s membrane had increased in thickness to 0.629 ± 0.020 μm (Fig. 4B, bracket). Thin strands of electron dense materials appeared to connect the Descemet’s membrane to the stroma and to the endothelial cells (Fig 4B, arrows). Expression of Npnt shifted anteriorly and it co-localized with the most posterior Ln band at 1.129 μm (Fig. 4I, 4J, and 4T). At this time, both Ln bands were continuous but the distance of the second band from the endothelial nuclei was variable and therefore, resolved as two peaks at 2.58 μm and 3.71 μm (Fig. 4T). Col VIII remained broadly expressed with strong staining closer to the endothelial nuclei at 1.61 μm (Fig. 4K and 4T). Per was diffusely expressed with strong staining observed at 2.41 um (Fig. 4L and 4T). Col IV was expressed in two bands for the first time, and they localized at 0.645 μm and 2.258 μm (Fig. 4M and 4T).

At P14, electron dense bands from the stroma appeared to integrate into the anterior surface of the Descemet’s membrane (Fig. 4C, arrows), which was measured at an average thickness of 0.842 ± 0.023 μm (Fig. 4C, bracket). Npnt was expressed as a single band that localized at 2.25 μm (Fig. 4N and 4U). Ln expression was maintained as two bands (Fig. 4O), with a posterior band at 1.29 μm and an anterior band at variable distances represented as peak intensities at 3.54 μm and 4.51 μm (Fig. 4U). Col VIII was expressed at 2.09 μm (Fig. 4P and 4U). Per was diffusely expressed throughout the cornea endothelium and Descemet’s membrane with peak intensity at 0 μm (Fig. 4Q and 4U). Expression of Col IV was maintained as two bands at 1.129 μm and 2.902 μm (Fig. 4R and 4U).

3.5. Expression of Npnt relative to other basement membrane proteins in the Descemet’s membrane of adult corneas

In the adult cornea, the ABL is well constructed as cells transition to depositing the posterior non-banded layer (PNBL), which grows steadily throughout life (Murphy et al., 1984 and Wilson et al., 2020). Given that Npnt is expressed in the adult Descemet’s membrane (Fig. 1H), we investigated its spatial relationship with other basement membrane proteins. Our data indicate that Npnt localizes at 1.725 μm away from the endothelial nuclei (Fig. 5A, 5E, 5I, 5M and 5D, 5H, 5L, 5P). Ln was expressed in a single band that was localized at 1.552 μm and overlapped posteriorly with Npnt (Fig. 5A–5D). Col VIII appeared as two bands that localized at 1.207 μm and 2.328 μm, with the Npnt band in between (Fig. 5E–5H). Per was expressed in a single band that coincided with Ln at 1.552 μm and also overlapped posteriorly with Npnt (Fig. 5I–5L). Col IV was expressed closest to the endothelial cell nuclei at 0.862 μm and localized posterior to the Npnt band (Fig. 5M–5P).

Figure 5. Expression of Npnt and basement membrane proteins in the adult Descemet’s membrane.

Figure 5.

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Immunohistochemical analysis of cornea sections showing expression of Npnt, Ln, Col VIII, Per, and Col IV in adult corneas and their relative distribution in the Descemet’s membrane. Sections were counterstained with DAPI (blue). (A-D) Npnt overlaps with Ln. (E-H) Npnt overlaps with two Col VIII bands. (I-L) Npnt overlaps with Per. (M-P) Npnt overlaps with Col IV. (D,H,L,P) Data are presented as mean values +/− SEM, N = 4 biological replicates. Abbreviations: st, stroma; en, endothelium. Scale bars, 10 μm.

4. Discussion

In this study, we performed a comprehensive analysis of the expression of Npnt in the mouse cornea. We combined our findings with ultrastructural analysis of the formation of the Descemet’s membrane and evaluated the expression of Npnt relative to other basement membrane proteins. Our results demonstrate the earliest expression of Npnt in the posterior cornea and during the formation of the Descemet’s membrane, where it either localizes adjacent to or overlaps with major basement membrane proteins into adulthood. Therefore, suggesting that Npnt has a potential role during development and maintenance of the Descemet’s membrane.

Our results indicate that Npnt mRNA is expressed in the posterior region of the cornea at E14.5 prior to the formation of the corneal endothelium at E15.5 (Reneker et al., 2000). Similarly, Npnt protein is localized to this region of the cornea, suggesting that the nascent corneal endothelial cells secrete Npnt prior to the formation of the Descemet’s membrane. However, we cannot rule out the potential contribution from the lens epithelial cells, which also express Npnt and are proximal to the endothelial layer at this early stage of development. Moreover, Npnt is maintained in the corneal endothelium throughout development as well as in adults, indicating that the endothelial cells are the source of the Npnt protein that is expressed in the developing and adult Descemet’s membrane. We also observed that Npnt is transiently expressed in the corneal epithelium and the protein is localized to the epithelial basement membrane in the peripheral region of the cornea during early stages of development, but in this study, we only focused on the Descemet’s membrane.

To better understand the expression of Npnt in the Descemet’s membrane, we coupled immunohistochemistry with ultrastructural analyses at developmental stages not previously reported in the mouse cornea. Our results indicate so far, the earliest formation of the Descemet’s membrane as dense bands of protein aggregates that form adjacent to the endothelial layer by E18.5. While it is known that the formation of the Descemet’s membrane initiates embryonically in human (Lesueur et al., 1994; Murphy et al., 1984), previous studies in mice suggested that it is not present between E18-E19 (Pei and Rhodin, 1970) and at P2 (Haustein, 1983). The first indication of the Descemet’s membrane was reported between P2-P4 (Smith et al., 2001), and it was also studied at P14 and later stages (Jun et al., 2006).

Our immunohistochemistry results reveal that by E18.5, Npnt overlaps with other basement membrane proteins, and that they are all expressed in the region of the forming ABL with strong expression at close distances from the endothelial cell layer. As the ABL becomes continuous and begins to thicken during the early postnatal stages (P0 and P4), these changes are also reflected in the dynamic expression of the proteins, whereby Npnt appeared as a broad band that overlapped with the four basement membrane proteins. Per shifted anteriorly and overlapped with Npnt, Ln and Col IV, whereas Col VIII was expressed relatively posterior, but it also overlapped with Npnt. These results indicate that Npnt is expressed throughout the ABL during early postnatal stages and that other basement membrane proteins shift within its expression domain.

Our results also showed that the ABL continued to increase in thickness during the late postnatal stages and reached 0.842 ± 0.023 μm by P14. This is different from the total measurements of the Descemet’s membrane thickness of 1.12 ± 0.22 μm reported at two weeks (Jun et al., 2006). One possibility for this discrepancy is that our measurements were taken from only the mid region of the corneas where the thickness of the Descemet’s membrane appeared uniform. Therefore, exclusion of the peripheral regions where Descemet’s membrane is thicker and uneven (Chaon et al., 2016), may account for the relatively lower values reported in the current study.

At P14, Npnt was expressed as tight band that overlapped with Col VIII. Npnt also localized anterior to Per and between the two bands of Ln and Col IV. These results indicate that expression of Npnt and other basement membrane proteins shift as the Descemet’s membrane continues to thicken. The two bands of Ln and Col IV indicate that they are expressed on both the stromal and endothelial faces of the ABL. Similar patterns of Ln and Col IV expression, referred to as “railroad patterns”, were observed in the infant human Descemet’s membrane (Kabosova et al., 2007). The temporal shifts in protein expression could be in response to various factors that affect basement membranes including, changes in thickness, mechanical properties, rate of protein deposition, or protein degradation by matrix metalloproteases (Khalilgharibi and Mao, 2021).

Ultrastructure of the Descemet’s membrane has been widely studied in adult mice and its reported thickness ranges from 2.5 - 4.19 μm (Hayashi et al., 2002; Jun et al., 2006), and it is comprised of both the ABL and PNBL (Murphy et al., 1984). Our results show that Npnt overlaps with all the basement membrane proteins in the adult cornea. Col VIII is expressed throughout the Descemet’s membrane (de Oliveira and Wilson, 2020; Kabosova et al., 2007), whereas Col IV, Ln, and Per are expressed on the endothelial side (Ljubimov et al., 1995; McKay et al., 2020; Sampaio et al., 2021). The two bands of Col VIII observed in our study may correspond with its expression in both the ABL and PNBL. Based on these observations, we infer that in the adult mice cornea, Npnt it is localized in the PNBL where it overlaps with Ln, Per, and Col IV. Thus, Npnt is likely anchored to the PNBL by Ln and Per such that it is stabilizes anterior to Col IV and between the Col VIII bands that span the entire Descemet’s membrane. Given that Npnt persists in the endothelial cells and Descemet’s membrane throughout adult hood, it is likely to function during the formation and homeostasis of the corneal endothelium. Npnt may also play crucial roles during wound healing and in pathological conditions such as Fuchs dystrophy and bullous keratopathy.

5. Conclusions

In summary, we show that Npnt mRNA is expressed in mouse corneal endothelial cells, whereas the protein localizes to the adjacent ECM during development and throughout adult hood. The expression of Npnt correlates with the dynamic shift of basement membrane proteins during the formation of the ABL. Npnt remains closely associated with the basement membrane proteins in the adult cornea. Thus, Npnt is an integral protein in the Descemet’s membrane of the mouse cornea. Future studies will be necessary to determine the function of Npnt in the mouse cornea during development and in adults.

Highlights.

  • Npnt is expressed early during mouse cornea development, and it is maintained in the corneal endothelium and Descemet’s membrane throughout adult hood.

  • Onset of the formation of the mouse Descemet’s membrane occurs at E18.5 and it appears as discontinuous electron dense regions of protein aggregates that form the anterior banded layer (ABL).

  • Npnt and other corneal basement membrane proteins exhibit dynamic expression patterns as the ABL of the Descemet’s membrane continuously thickens.

  • In the adult cornea, Npnt is localized between two Coll VIII bands, anterior to Coll IV, and it overlaps with Laminin and Perlecan.

Acknowledgements

This study was supported by a grant from the National Eye Institute EY031381 (PYL). We would also like to thank members of the Lwigale Lab for the insightful discussions and suggestions on this project.

Footnotes

Declaration of completing interests

The authors declare no competing interests.

Data availability

Data will be made available upon request.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data will be made available upon request.

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