IκB Kinase α Is Essential for Development of the Mammalian Cornea and Conjunctiva

Abstract

purpose. To determine the requirement of IκB kinase α (Ikkα) for differentiation of the mammalian cornea and conjunctiva.

methods. Newborn mice or surgically removed embryonic day (E)18 to E19 fetuses of wild-type and Ikkα^{− /−} mice were analyzed by light microscopy and electron microscopy or immunocytochemistry using anti-keratin (K)12, K4, K5, IκB, or nuclear factor (NF)-κB (p50) antibody.

results. In the Ikkα^{− /−} eyes, the epithelium of the cornea and the conjunctiva consisted of poorly differentiated cells with round nuclei. K5 was much stronger in the conjunctiva of the Ikkα^{− /−} mice. Expression of K12 in the cornea and K4 in the conjunctiva was impaired in the Ikkα^{− /−} mice. IκB expression was low in epithelium of the cornea and conjunctiva of the wild type mice but was very strong in that of the Ikkα^{− /−} mice. During normal development of the conjunctiva, nuclear localization of p50 was seen in areas where basal undifferentiated cells give rise to differentiated cell types, marked by expression of cK4. However, in the Ikkα^{− /−} tissues, no nuclear p50 staining was detected.

conclusions. IKKα is specifically required for formation of cornea and conjunctiva. This function may be exerted through an effect on NF-κB activity.

Development of the vertebrate eye involves differentiation of the surface ectoderm into the epithelia of the cornea and conjunctiva. In the mouse, the surface ectoderm surrounding the eye on day 11 of embryonic development (E11), begins to differentiate into the primordial cornea and conjunctiva on E13. ¹ The differentiation of these epithelia is characterized by the appearance of a cuboidal basal cell layer and a squamous surface cell layer. These morphologic changes are accompanied by changes in cytokeratin gene (cK) expression. ² For example, the cK3/cK12 pair is expressed in differentiated corneal epithelial cells, whereas cK4 is specifically expressed in differentiated conjunctiva epithelial cells. ^{3 4 5} Synthesis of these proteins is regulated both temporally and spatially within the developing cornea and conjunctiva. ³ The mechanisms that control the organogenic and cell differentiation processes are not well understood. 

Nuclear factor (NF)-κB/Rel proteins are dimeric transcription factors with activity that is regulated by interaction with IκB inhibitors. ^{6 7} In nonstimulated cells NF-κB proteins are retained in the cytoplasm because IκBs mask their nuclear localization sequence. ⁷ Exposure to proinflammatory stimuli results in rapid phosphorylation, ubiquitinilation, and degradation of the IκB kinases (IKKs). ^{6 7 8} NF-κB dimers translocate to the nucleus and regulate target gene transcription. The protein kinase that phosphorylates IκBs in response to proinflammatory stimuli is a multiprotein complex, the IKK, composed of two catalytic subunits, IKKα and IKKβ, ^{9 10 11 12 13} and a regulatory subunit, IKKγ, or NEMO. ^{14 15} In Drosophila NF-κB/Rel proteins control both innate immune responses and morphogenetic processes. ¹⁶ In mammals, however, there is ample evidence for the involvement of NF-κB/Rel proteins in innate immune responses, but so far clear evidence for their role in morphogenesis has not been found, in that the disruption of single Rel genes has not resulted in obvious morphogenetic defects. ¹⁷ This does not necessarily mean that mammalian NF-κB/Rel proteins are not involved in morphogenesis but may simply reflect their functional redundancy. Indeed, disruption of the genes coding for IKK subunits has resulted in much more dramatic effects on NF-κB activity and function than those caused by disruption of individual NF-κB/Rel genes. ^{18 19 20 21} Loss of IKKβ expression resulted in a major defect in NF-κB activation by numerous proinflammatory stimuli and lethality at midgestation due to profound hepatic apoptosis. ^{20 21} The latter is caused by the absence of activated NF-κB required for prevention of apoptosis induced by tumor necrosis factor (TNF)-α. ^{22 23} Although the loss of IKKα had little effect on NF-κB activation by proinflammatory stimuli, it interfered with morphogenetic events that are involved in limb and skeletal patterning and formation of the epidermis. ^{18 19} Instead of being organized as a stratified epithelium with a fully keratinized outer layer, the epidermis of Ikkα^{− /−} mice, which die perinatally, is fairly uniform, hyperplastic, taut, and completely devoid of a keratinized outer layer. ^{18 19} Because IKKα function is very important for proper differentiation of the epidermis, we examined whether IKKα is also required for formation of other ectodermal derivatives—namely, the cornea and conjunctiva. We found major defects in development and differentiation of these two tissues in Ikkα^{− /−} mice, indicating the importance of this IKK subunit for formation of all outer epithelial tissues derived from the embryonic ectoderm. 

Methods

Animal and Tissue Processing

Ikkα ^{− /−} mutant fetuses were generated as described. ¹⁸ Newborn mice or surgically removed E14 and E18 to E19 fetuses were used in all experiments. Eyes of normal and mutant mice were fixed in ice-cold 4% paraformaldehyde in 0.1 M borate buffer (pH 9.5) for 2 hours. After they were embedded in paraffin, the 3-μm coronal sections were processed for hematoxylin-eosin (H&E) staining and indirect immunofluorescence. Samples for IκBα, p50 (NF-κB1), or cK4 immunocytochemistry were embedded in optimal cutting temperature compound (OCT; Tissue Tek, Miles, Elkhart, IN) and frozen in liquid nitrogen, and 10-μm-thick cryostat sections were mounted onto poly-l-lysine coated slides. For electron microscopy, eyes were fixed overnight at 4°C in 5% glutaraldehyde in 0.1 M phosphate buffer for 3 hours, block stained, and embedded in resin after dehydration in a graded alcohol series. The blocks were sectioned semiserially, and 0.7-μm-thick sections were stained with toluidine blue. Ultrathin sections were stained with uranyl acetate and lead citrate and examined by electron microscope (100CX; JEOL; Tokyo, Japan). The animal experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Immunocytochemistry

Slides were dried for 1 hour, rinsed twice in phosphate-buffered saline and incubated with antibodies to cK12, ⁵ K4 (ICN Biomedicals, Costa Mesa, CA), cK5 (BAbCO; Richmond, CA), p50 (H119; Santa Cruz Biotechnology, Santa Cruz, CA), and IκBα (C-21; Santa Cruz) at 4°C for 12 hours. As a negative control, the IκBα antibody was preincubated with 1 μg/ml of the IκBα peptide immunogen (sc-371P; Santa Cruz). Antibody–antigen complexes were visualized by using the avidin-biotin immunoperoxidase method (Vector, Burlingame, CA). After three 5-minute washes in TNT (50 mM Tris-HCl[ pH 7.4], 150 mM NaCl, 0.05% Tween 20), the sections were incubated with 1:50 dilution of rhodamine tyramide in 1× amplification diluent (NEN Life Science Products) and examined with an epifluorescence microscope (Axiovert 35M; Carl Zeiss; Oberkochen, Germany). 

Results

Thorough examination of newborn Ikkα^{− /−} mice revealed that their eyes appeared wide open in the dissection microscope (Figs. 1a 1b ). However, the eyes of Ikkα^{− /−} mice are actually closed, with underdeveloped eyelids. Analysis of Ikkα^{− /−} eyes in the H&E-stained section revealed that a multicellular layer of ectoderm, consisting of immature cells, had migrated off the eyelid surface and adhered to the cornea, thereby forming a continuous sheet of tissue (Fig. 1d , arrowhead) that obliterated the normal anatomic distinction between the eyelid and the cornea, seen in littermate controls (Fig. 1c) . It is likely that the multicellular layer of ectoderm that covers the cornea of Ikkα^{− /−} mice and fuses the cornea to the eyelid is responsible for the open-eye phenotype, by preventing the normal sliding of the upper and lower eyelids on the corneal surface. Examination at a higher magnification revealed a well-organized corneal structure in wild-type eyes. The corneal epithelium was composed of two layers of flat cells of ectodermal origin, and the corneal stroma consisted of several ordered layers of mesenchymal cells (Fig. 1e) . In Ikkα^{− /−} eyes, however, the corneal epithelial layer was poorly organized and was composed of several layers of more rounded cells (Figs. 1f 2b 2c) . The keratocytes of the mutant cornea near the epithelium were more rounded (Fig. 1f , arrows). Another significant difference at this stage involved the developing conjunctiva. The wild-type tissue was composed mostly of flattened differentiated cells (Fig. 1g) , whereas the mutant conjunctiva consisted of cells with rounded nuclei, and no flattened differentiated cells were detected (Fig. 1h) . cK5, which is mostly expressed by the basal, nondifferentiated, cells of the epidermis ² was not expressed in the conjunctiva of newborn wild-type mice, which is composed of differentiated cells (Fig. 1i) . However, the conjunctiva of newborn mutant mice continued to express high levels of cK5 (Fig. 1j) , indicating its nondifferentiation. The lid buds were not closed in the control eye on E14 (Fig. 1k) . At the same stage, the multicellular layer of ectoderm had already migrated from the eyelid surface onto the cornea of the mutant mice (Fig. 1l) . The corneal stroma of the mutant was poorly organized in contrast to the wild-type stroma (Figs. 1k 1l) . 

The aberrant differentiation of the mutant cornea was also evident from electron microscopic examination. The wild-type corneal epithelium consisted of flat cells (Fig. 2a ). The mutant corneal epithelium, however, consisted of rounded cells (Figs. 2b 2c) . The corneal stroma near the epithelium of the mutant was also poorly organized in comparison to the wild-type stroma. 

We examined the state of differentiation of wild-type and mutant tissues using a number of differentiation markers. Antibodies to cK12 and cK4 were used to examine whether the IKKα deficiency affects the maturation of the cornea and conjunctiva, respectively. Robust expression of cK12, a marker for corneal epithelial cells, ⁵ was detected in wild-type tissue, whereas only a weak signal, slightly above background, was detected in a small number of cells in the mutant corneal epithelium (Figs. 3a 3b ). Expression of cK4, a marker for epithelial cells of the conjunctiva, ³ was also markedly reduced in Ikkα^{− /−} mice, whereas a high level of expression was evident in wild-type conjunctiva (Figs. 3c 3d) . 

To determine whether the absence of IKKα had any effect on the regulation of NF-κB activity during development of the cornea and conjunctiva, we examined the subcellular distribution of NF-κB and IκB proteins in these tissues. Staining with antibodies to p50 (NF-κB1) revealed nuclear location of this protein in cells around the eyelid closure of wild-type mice, where basal cells undergo differentiation to form the conjunctival epithelium, which is marked by expression of cK4 (Figs. 4a 4c 4e ). No nuclear localization of p50 or cK4 expression was detected in the same region of the Ikkα^{− /−} conjunctiva (Figs. 4b 4d 4f) . Staining with anti-IκBα antibodies revealed low expression levels in wild-type cornea and conjunctiva (Figs. 5a 5b 5f 5g ), but there were high levels of expression in the same tissues of Ikkα^{− /−} mice (Figs. 5c 5d 5h 5i) . Especially high levels of IκBα were detected in the multicellular layer of ectoderm that covered the mutant cornea and in the conjunctiva. 

Discussion

The results illustrate that the IKKα subunit of the IKK complex is involved in formation and differentiation of the outer epithelial layers that provide protection to the mammalian eye. In Ikkα^{− /−} mice, the epithelia of the cornea and conjunctiva consist of cells with round nuclei, and the flattened differentiated cells that are characteristic of the squamous epithelia of wild-type tissues are absent. The abnormal stromal cells were distributed near the epithelium (Figs. 1f 2b 2c) , suggesting the role of corneal epithelium in differentiation of the keratocytes. These cells may not be keratocytes at all but inflammatory cells or cells derived from a population other than the neural crest cells that become keratocytes. Expression of cK12, a marker for differentiated corneal epithelial cells, ⁵ and cK4, a marker for conjunctival epithelial cells, ³ was impaired in Ikkα^{− /−} tissues. By contrast, expression of cK5, a marker for undifferentiated basal epithelial cells ² that is downregulated in differentiated cells, was considerably elevated in the poorly differentiated Ikkα^{− /−} conjunctiva. These changes in epithelial cell differentiation and cK expression correlate with aberrant expression and subcellular localization of IκB and NF-κB proteins. In comparison to wild-type mice, expression of IκBα was also markedly elevated in the cornea and conjunctiva of Ikkα^{− /−} mice. The elevated expression of IKKα could be due to either higher rates of synthesis or lower rates of degradation. Given the role of IKK in induction of IκB phosphorylation and degradation, ²⁴ the latter explanation (i.e., decreased IκB degradation) is more plausible. These findings support the notion that although IKKα is not required for responsiveness to proinflammatory stimuli, it is activated by yet to be defined developmental cues that lead to activation of NF-κB during epithelial differentiation. ^{18 19}  

Some of the developmental and morphogenetic problems observed in IKKα-deficient mice may indeed be due to defects in the NF-κB activation pathway. During normal development of the conjunctiva, nuclear localization of p50 is seen in areas where basal undifferentiated cells give rise to differentiated cell types marked by expression of cK4. However, in the Ikkα^{− /−} tissues, consistent with the increased levels of IKKα, no nuclear p50 staining was detected. In addition, it is possible that several of the cK genes may be directly regulated by NF-κB. There is a κB motif in the promoter of the rabbit cK3 gene, which is specifically expressed in corneal epithelial cells, and this element is necessary for promoter activity. ⁵ In contrast, NF-κB proteins repress the cK5 promoter. ²⁵ cK5 is expressed all over the surface ectoderm of the eye at E11 (data not shown) but is not expressed in the conjunctiva at birth, indicating that also in this tissue cK5 is a marker for nondifferentiated cells. ² Strong expression of both cK5 and IKKα in the conjunctiva of Ikkα^{− /−} mice is consistent with the idea that cK5 is negatively regulated by NF-κB, the developmental activation of which depends on IKKα. 

In summary, the present results demonstrate the importance of IKKα for proper differentiation of the conjunctiva and cornea, two ectodermal derivatives that provide protection to the mammalian eye. Previously, IKKα was found to be essential for proper differentiation of the skin epidermis, the major ectodermally derived organ that provides a physical barrier that protects the inner cell layers of the mammalian body from loss of water and harmful chemical and physical conditions. The anomaly of cornea and conjunctiva development may simply reflect effects of the loss of IKKα on the differentiation of keratinocytes, rather than ocular epithelial cells specifically. Although IKKα is not required for activation of innate immune responses, ²⁶ it plays an important protective function after all. Given the aberrant distribution of NF-κB and IKK proteins in mutant tissues, this function of IKKα may be mediated by NF-κB. 

 Supported by grants from the National Institutes of Health (MK) and a postdoctoral fellowship from the Arthritis Foundation (YH).

 Submitted for publication April 17, 2000; revised July 11, 2000; accepted July 28, 2000.

 Commercial relationships policy: N.

 

Figure 1.

 

Aberrant differentiation of the cornea and conjunctiva in the absence of IKKα (wild-type [WT] control, left; Ikkα^{− /−} right). Photographs taken through a dissection microscope. (a, b) Low magnification and (c, d) high magnification of the cornea (e, f) and conjunctiva (g, h) and immunodetection of cK5 of the conjunctiva (i, j) from eyes of WT and Ikkα^{− /−} newborn mice. (k, l) H&E staining of paraffin sections of WT and Ikkα^{− /−} eyes on E14. Outlined areas in (c, d) are shown in higher magnification in (e through h). The eyes of Ikkα^{− /−} mice (b) appeared wide open, unlike those of control littermates (a). In the Ikkα^{− /−} eye, a multicellular layer of ectoderm migrated off the eyelid surface onto the cornea (d, arrowhead). The tarsal and bulbar conjunctiva were stuck to each other (d, arrow). The WT (e) corneal epithelium (226) and stroma (∗∗) consisted of ordered layers of differentiated cells. In the mutant (f), the corneal epithelium (∗) and stroma (∗∗) are round and the epithelium is covered by a multicellular epithelial layer, derived from the eyelid (f). Round nucleus of the epithelium (f, arrowhead). (e, f; arrows outside) Junctions of epithelial cells and stromal cells. The mutant conjunctiva contained cells with round nuclei (h) and was missing the flattened differentiated cells that were detected in the WT tissue (g). Expression of cK5, a marker for undifferentiated basal cells, was examined by indirect immunofluorescence (i, j). Although little cK5 was expressed by the WT conjunctiva, high levels of cK5 were expressed in the mutant conjunctiva. The lid buds were not closed in the control eye on E14 (k). On the same stage, the multicellular layer of ectoderm had migrated from the eyelid surface onto the cornea of the mutant mice (l).

Figure 2.

 

Electron micrographs of newborn normal and mutant cornea. (a) Wild-type eyes had a well-organized corneal structure. The corneal epithelium consisted of differentiated cells, and the corneal stroma contained ordered layers of mesenchymal cells. (b, c) The cornea of Ikkα^{− /−} mice consisted of more round cells in both the corneal epithelial and stromal cell layers near the epithelium.

Figure 3.

 

Aberrant expression of cKs in the newborn Ikkα^{− /−} cornea and conjunctiva. Immunohistochemistry of wild-type (WT; a, c) and mutant (b, d) cornea with anti-cK12 (a, b) and conjunctiva with anti-cK4 (c, d). Both cK4 and cK12 were underexpressed in the mutant tissues.

Figure 4.

 

Defective nuclear localization of p50 in Ikkα^{− /−} conjunctiva. Sections of newborn wild-type (WT; a, c, e) and mutant (b, d, f) conjunctiva surrounding the eyelid closure were examined by H&E staining (a, b) or indirect immunofluorescence for expression of cK4 (c, d) or p50 (e, f). Whereas in WT tissue, nuclear p50 was detected in differentiating cells derived from the conjunctival basal layer (arrowhead), in Ikkα^{− /−} conjunctiva, p50 was confined to the cytoplasm. The arrow in (a) points to differentiated conjunctival cells.

Figure 5.

 

Expression of IκBα in wild-type (WT; a, b, f, g) and mutant (c, d, e, h, i, j) cornea and conjunctiva. H&E staining (a, c, f, h) and immunodetection of IκBα (b, d, e, g, i, j) in cornea (a through e) and conjunctiva (f through j) of WT and Ikkα^{− /−} newborn mice. To control for specificity, the anti-IκBα antibody was blocked by incubation with the IκBα peptide immunogen. Low levels of IκBα expression are seen in WT tissues, whereas high levels of IκBα are detected in mutant tissues, especially in the multicellular layer of the ectoderm that migrated off the eyelid surface onto the cornea (d) and in the conjunctiva (i).

The authors thank Winston W. Y. Kao for cK12 antibodies, Mark Ellisman and Thomas Deerinck for assistance with histology and microscopy using a National Institutes of Health–sponsored facility.