SUMMARY

Preterm birth is a major global health problem that results in a large number of infant deaths, many of which are attributable to the complications of an immature epidermal permeability barrier (EPB), for which there is currently no effective therapeutic option. The mammalian EPB is formed during development and is essential for survival as it maintains thermoregulation and hydration, and provides a defense against infection. Using transgenic mouse technology, we have demonstrated the importance of claudin (Cldn)-containing tight junctions (TJs) in epidermal differentiation and, in particular, that epidermal suprabasal overexpression of Cldn6 results in an EPB-deficient phenotype that phenocopies the dysfunctional EPB of premature human infants. In this study, we used the same approach to target a Cldn6 tail deletion mutant to the epidermis of mice [involucrin (Inv)-Cldn6-CΔ206 transgenic mice]. The Inv-Cldn6-CΔ206 transgenic mice displayed a developmental delay in EPB formation, as shown by the expression of keratins and Cldns, and by X-Gal penetration assays. Trans-epidermal water loss measurements and immunolocalization studies indicated that the epidermal differentiation program was also perturbed in postnatal Inv-Cldn6-CΔ206 transgenic mice resulting in a delayed maturation. Notably, however, expression/localization of epidermal differentiation and maturation markers, including Cldns, indicated that the transgenic epidermis matured and normalized by postnatal day 10, which is 3 days after the wild-type epidermis. Our results suggest that activation of the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway and Cldn1 phosphorylation are associated with the repair and maturation of the skin barrier processes. These studies provide additional support for the crucial role of Cldns in epidermal differentiation, maturation and the formation of the EPB, and describe a novel animal model for evaluating postnatal epidermal maturation and therapies that may accelerate the process.

INTRODUCTION

The prevalence of preterm birth is widespread with very little understanding of its causes and no unambiguous epidemiological data for predicting its occurrence. Formed in weeks 30 to 33 of pregnancy (Wilson and Maibach, 1980), the protective epidermal permeability barrier (EPB) of the skin is essential for survival as it provides the first line of defense against infection, environmental insult, and the loss of heat and solutes (Baharestani, 2007; Gibson et al., 2006; Shwayder and Akland, 2005; Soll, 2008). In infants born before 32 weeks of pregnancy, severe EPB dysfunction may result in death or long-term complications (Pilling et al., 2008).

The EPB is formed in the later stages of epidermal terminal differentiation, and consists of tight junction (TJ) strands of adjacent cells that associate laterally (Brandner, 2009; Brandner et al., 2002; Langbein et al., 2002; Langbein et al., 2003; Schluter et al., 2007; Turksen and Troy, 2002) and function in sealing intracellular spaces for paracellular diffusion control (Farquhar and Palade, 1963). The selective permeability of the EPB is provided by a family of 23 highly conserved integral membrane proteins known as claudins (Cldns), a relatively recently identified component of TJs (Angelow et al., 2008; Chiba et al., 2008; Findley and Koval, 2009; Krause et al., 2008; Turksen and Troy, 2004; Van Itallie and Anderson, 2006). Heterogeneity within the Cldn family results from distinctly charged amino acid sequences within the first external loop; thus, the specific permeability properties of different epithelia are attributed to their different Cldn compositions (Daugherty et al., 2007; Katoh, 2003; Krause et al., 2008; Turksen and Troy, 2004). Recent studies have clearly demonstrated that Cldn-containing TJs are intricately involved in epidermal differentiation programs, and that TJ function, and thus barrier integrity, is modified in response to Cldn modulation (Arabzadeh et al., 2006; Furuse et al., 2002; Troy et al., 2005b; Troy and Turksen, 2007; Turksen and Troy, 2002). For instance, Cldn1 knockout mice die shortly after birth owing to EPB dysfunction (Furuse et al., 2002). Inv-Cldn6 transgenic mice, in which the involucrin (Inv) promoter targets Cldn6 to the suprabasal layers of the epidermis, also suffer EPB abnormalities with a phenotype mimicking that seen in premature human babies, the severity/lethality of which is dependent upon the level of Cldn6 overexpression (Troy et al., 2005b; Turksen and Troy, 2002). Inefficient membrane targeting of Cldn proteins and a highly proliferative epidermal phenotype – apparently as a result of the unfolded protein response pathway – were observed upon overexpression of a cytoplasmic tail-ablated Cldn6 (Inv-Cldn6-CΔ187) in mice (Arabzadeh et al., 2006). Furthermore, dependent on the level of overexpression, Inv-Cldn6-CΔ196 mice (with half the cytoplasmic tail ablated) (Troy and Turksen, 2007) displayed EPB dysfunction and an aging-related skin barrier defect resulting in an intrinsic propensity for injury, inefficient repair and chronic dermatitis.

Fig. 1.

Inv-Cldn6-CΔ206 transgenic mice. (A) The Inv-Cldn6-CΔ206 mutant was created by truncating the C-terminal cytoplasmic tail domain of Cldn6 after amino acid 206 (Cldn6-CΔ206). (B) The Cldn6 protein sequence is shown; transmembrane-spanning regions are encased within boxes, the CXXC motifs are underlined, and the deletion site is indicated with an arrow. (C) Cldn6-CΔ206 cDNA was expressed in the suprabasal cells of the transgenic mouse epidermis, where TJs are localized, under the control of the 3.7 kb 5′-flanking element of the human involucrin gene. (D) Inv-Cldn6-CΔ206 mice were easily identifiable by their wiry and lackluster coat, a phenotype that was maintained throughout life. (E) TEWL measurements demonstrated an approximately twofold increase in water loss for transgenic neonates, and daily measurements revealed a robust epidermal repair mechanism where normal hydration levels were achieved by 4 days after birth. (F) RT-PCR using primers spanning the Inv exon and Cldn6 confirmed that transgene expression was consistent and could therefore not be used to explain the changes in TEWL; GAPDH controls are shown. (G,H) Evaluation of cornified envelopes, which were characterized by a rigid shape and uniform size, comparable to that of the wild type (G), and the absence of X-Gal penetration (H) confirmed the presence of sufficient neonatal barrier function in Inv-Cldn6-CΔ206 mice.

Fig. 1.

Inv-Cldn6-CΔ206 transgenic mice. (A) The Inv-Cldn6-CΔ206 mutant was created by truncating the C-terminal cytoplasmic tail domain of Cldn6 after amino acid 206 (Cldn6-CΔ206). (B) The Cldn6 protein sequence is shown; transmembrane-spanning regions are encased within boxes, the CXXC motifs are underlined, and the deletion site is indicated with an arrow. (C) Cldn6-CΔ206 cDNA was expressed in the suprabasal cells of the transgenic mouse epidermis, where TJs are localized, under the control of the 3.7 kb 5′-flanking element of the human involucrin gene. (D) Inv-Cldn6-CΔ206 mice were easily identifiable by their wiry and lackluster coat, a phenotype that was maintained throughout life. (E) TEWL measurements demonstrated an approximately twofold increase in water loss for transgenic neonates, and daily measurements revealed a robust epidermal repair mechanism where normal hydration levels were achieved by 4 days after birth. (F) RT-PCR using primers spanning the Inv exon and Cldn6 confirmed that transgene expression was consistent and could therefore not be used to explain the changes in TEWL; GAPDH controls are shown. (G,H) Evaluation of cornified envelopes, which were characterized by a rigid shape and uniform size, comparable to that of the wild type (G), and the absence of X-Gal penetration (H) confirmed the presence of sufficient neonatal barrier function in Inv-Cldn6-CΔ206 mice.

These data provide support for the importance of the cytoplasmic tail portion of Cldn molecules in epidermal differentiation and EPB function. Although relatively constant in length, sequences within the Cldn cytoplasmic tail are divergent but include a number of putative functional domains that are present in many family members. To continue to address functional domain activities, we again used the Inv promoter to target a Cldn6 cytoplasmic tail deletion mutant (Cldn6-CΔ206), to the suprabasal compartment of the epidermis; this deletion encompasses the PDZ-binding domain and a putative protein kinase A (PKA) phosphorylation site. Inv-Cldn6-CΔ206 transgenic mice have a developmental delay in EPB formation, suffer trans-epidermal water loss (TEWL) at birth, and exhibit a perturbed epidermal maturation program manifested by a 3-day lag in the initiation of the normal epidermal thinning process (which occurs at day 10 in the Inv-Cldn6-CΔ206 transgenic mice versus day 7 in the wild-type epidermis). Our data suggest that this process stems from the remodeling of Cldn1 expression in the repairing Inv-Cldn6-CΔ206 transgenic epidermis, and that the first phase of repair requires shedding of phospoCldn1-expressing cells from the differentiated epidermal compartment where extracellular signal-regulated kinase 1/2 (Erk1/2) is activated. These results suggest that although Inv-Cldn6-CΔ206 transgenic mice suffer developmental delays in epidermogenesis, the epidermis undergoes epidermal maturation and repair after birth, normalizing by postnatal day 10, thus providing a model to elucidate the molecular mechanisms by which Cldns regulate the postnatal maturation of the epidermis. This model may also be useful to screen for agents that accelerate formation of a functional barrier, which could provide useful therapeutic options for improvement of the EPB in premature infants.

RESULTS

Generation and phenotype of Inv-Cldn6-CΔ206 transgenic mice

We truncated the C-terminal cytoplasmic tail domain of Cldn6 after amino acid 206 (Cldn6-CΔ206), removing 14 amino acids to encompass a region including the PDZ-binding domain (YV) and a putative PKA phosphorylation site (Fig. 1A,B). Cldn6-CΔ206 cDNA was expressed in the suprabasal cells of the transgenic mouse epidermis, where TJs are localized, under the control of the 3.7 kb 5′-flanking element of the human Inv gene (IVL) (Fig. 1C). Founder Inv-Cldn6-CΔ206 transgenic mice (five females) were identified by PCR, and two lines were established with identical phenotypes. Newborn Inv-Cldn6-CΔ206 transgenic mice appeared grossly phenotypically comparable to the wild type. However, as was observed in our Inv-Cldn6, Inv-Cldn6-CΔ187 and Inv-Cldn6-CΔ196 mouse models, with time the Inv-Cldn6-CΔ206 transgenic mice were easily distinguishable from the wild type by coat appearance: smooth and shiny in the wild-type mice versus wiry and lackluster in the transgenic mice, a phenotype that was maintained throughout the life of the mouse (Fig. 1D). Since it does not appear to have any direct relevance to the delayed epidermal maturation and postnatal EPB formation reported here, coat characteristics have not been investigated further.

Although no gross phenotypic abnormalities were apparent at birth, daily dermal phase meter (DPM) measurements (Fig. 1E) demonstrated significant, albeit non-lethal, TEWL in neonatal Inv-Cldn6-CΔ206 transgenic mice. Compared with the wild-type mice (DPM in the range of 95–105), Inv-Cldn6-CΔ206 transgenic neonates expressed DPM values in the range of 175–185. However, DPM levels were already reduced in the transgenic mice at 2 days after birth (~125–135), and by 4 days after birth, DPM values reached wild-type levels (in the range of 100–106). Reverse transcriptase (RT)-PCR with primers spanning the junction of the Inv exon and the Cldn6 sequences in the transgene confirmed that the observed changes in TEWL leading to reversion to a normal or wild-type phenotype are not simply the result of decreased expression of the transgene (Fig. 1F, a GAPDH control is shown). Despite the higher TEWL in transgenic epidermis, the presence of substantial neonatal barrier function was confirmed not only by mouse viability but also by analysis of cornified envelope extracts from Inv-Cldn6-CΔ206 skin, which appeared relatively normal with a uniform size and rigid shape (Fig. 1G), and the absence of X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) penetration (Fig. 1H).

Barrier formation and epidermal differentiation is delayed in the Inv-Cldn6-CΔ206 transgenic epidermis during development

Although non-lethal, the significantly higher TEWL observed in neonatal Inv-Cldn6-CΔ206 transgenic mice suggested aberrations in the epidermal differentiation program during development. X-Gal penetration assays confirmed that, at E15.5 (15.5 days post coitum), Inv-Cldn6-CΔ206 transgenic embryos were delayed in barrier formation as compared with their wild-type counterparts (Fig. 2A), a delay that was sustained at E17.5, when EPB function is normally achieved (Fig. 2B) (Hardman et al., 1998; Troy et al., 2007), and at E18.5 (Fig. 2C, marked with arrows).

To complement these studies, we evaluated transverse histological sections of E15.5 and E17.5 embryo torsos in which the epidermal initiation sites, as well as the dorsal and ventral midlines, were included; because the data were consistent, we report results from E15.5 Inv-Cldn6-CΔ206 transgenic and wild-type mice only. At each site, a somewhat delayed/immature epidermal differentiation program was evident in the transgenic epidermis. For instance, in contrast to the well-established 3–4-cell layered stratum intermedium of the wild-type epidermis (Fig. 3A), the initiation site of the Inv-Cldn6-CΔ206 transgenic epidermis possessed only 2–3 intermediate cell layers. Although the dorsal midline (Fig. 3B) of the developing wild-type epidermis was characterized by a 2–3-cell layer intermediate zone, that of the transgenic epidermis was thinner with only 1–2 layers of intermediate cells evident between the basal and periderm layers. Furthermore, the ventral midline (Fig. 3C) of the wild-type epidermis was characterized by two layers of intermediate cells, whereas that of the transgenic mice was thinner with only one defined intermediate cell layer together with some nascent infiltrating intermediate cells.

Consistent with the histological results, abnormalities in the expression of epidermal differentiation markers and Cldns were present in the developing Inv-Cldn6-CΔ206 embryo (Fig. 4, E15.5 epidermal initiation sites are shown). Although there was no apparent difference in the transgenic and wild-type epidermal basal compartments, as shown by the localization of K14 (Fig. 4A), K5 and K15 (not shown), K1 (Fig. 4B), which is associated with stratifying suprabasal cells (Fuchs and Byrne, 1994), occupied a moderately thinner compartment in the transgenic epidermis corresponding with the thinner intermediate layer that was observed histologically. Cldn1 (Fig. 4C), Cldn6 (Fig. 4D), Cldn11 (Fig. 4E), Cldn12 (Fig. 4F) and Cldn18 (Fig. 4G), which at this developmental time point are all strictly localized to the epidermal suprabasal compartment (Troy et al., 2007), also occupied a comparatively thinner zone of expression in the developing Inv-Cldn6-CΔ206 transgenic epidermis. Similarly, the expression compartment of various structural proteins in the stratum corneum, including involucrin (Fig. 4H), filaggrin (Fig. 4I) and loricrin (Fig. 4J) was reduced in the E15.5 Inv-Cldn6-CΔ206 transgenic epidermis.

Fig. 2.

X-Gal assays demonstrate a developmental delay in EPB formation for Inv-Cldn6-CΔ206 transgenic mice. (A–C) Using age-matched wild-type embryos as a control, X-Gal penetration assays at E15.5 (A), E17.5 (B) and E18.5 (C) confirmed a developmental delay in EPB formation for Inv-Cldn6-CΔ206 transgenic mice. Even at E18.5 there was some evidence that barrier formation had not yet been completed in Inv-Cldn6-CΔ206 transgenic embryos (indicated with arrows).

Fig. 2.

X-Gal assays demonstrate a developmental delay in EPB formation for Inv-Cldn6-CΔ206 transgenic mice. (A–C) Using age-matched wild-type embryos as a control, X-Gal penetration assays at E15.5 (A), E17.5 (B) and E18.5 (C) confirmed a developmental delay in EPB formation for Inv-Cldn6-CΔ206 transgenic mice. Even at E18.5 there was some evidence that barrier formation had not yet been completed in Inv-Cldn6-CΔ206 transgenic embryos (indicated with arrows).

Fig. 3.

Histological evidence confirms EPB formation delays in transgenic mice. Evaluation of transverse histological sections at E15.5 confirmed a somewhat delayed epidermal differentiation in Inv-Cldn6-CΔ206 transgenic mice during development. (A) Transgenic epidermal initiation sites were characterized by 2–3 intermediate cell layers in contrast to the more mature 3–4-cell layered stratum intermedium of the wild type. (B,C) There were only 1–2 layers of intermediate cells at the dorsal midline (B) of the Inv-Cldn6-CΔ206 epidermis, whereas that of the wild type possessed 2–3 cell layers, and the transgenic ventral midline (C) was thinner than that of the wild type, with only one defined intermediate cell layer together with some infiltrating intermediate cells. Bar, 20 μm.

Fig. 3.

Histological evidence confirms EPB formation delays in transgenic mice. Evaluation of transverse histological sections at E15.5 confirmed a somewhat delayed epidermal differentiation in Inv-Cldn6-CΔ206 transgenic mice during development. (A) Transgenic epidermal initiation sites were characterized by 2–3 intermediate cell layers in contrast to the more mature 3–4-cell layered stratum intermedium of the wild type. (B,C) There were only 1–2 layers of intermediate cells at the dorsal midline (B) of the Inv-Cldn6-CΔ206 epidermis, whereas that of the wild type possessed 2–3 cell layers, and the transgenic ventral midline (C) was thinner than that of the wild type, with only one defined intermediate cell layer together with some infiltrating intermediate cells. Bar, 20 μm.

The epidermis matures postnatally in the Inv-Cldn6-CΔ206 transgenic mice

In parallel with the delayed program of epidermal differentiation and EPB formation observed in the developing Inv-Cldn6-CΔ206 transgenic epidermis, postnatal TEWL measurements suggested that the Inv-Cldn6-CΔ206 transgenic epidermis underwent a robust epidermal maturation process after birth. Histological analyses of newborn (Fig. 5A), 2-day-old and 4-day-old skin samples (not shown) revealed that the Inv-Cldn6-CΔ206 transgenic epidermis was comparable to the wild type. However, although by 1 week of age the wild-type epidermis had commenced the normal thinning pattern associated with epidermal maturation, the transgenic epidermis maintained an immature phenotype with many suprabasal cell layers, the prevalent appearance of nuclei in the upper differentiation layers, and a much less compacted granular layer (Fig. 5B). However, by 10 days after birth (Fig. 5C), the Inv-Cldn6-CΔ206 transgenic epidermis had thinned to be morphologically comparable to the wild-type epidermis. Samples from 1-month-old (Fig. 5D) and 3-month-old (not shown) transgenic mice were indistinguishable from wild-type samples.

The histological results suggested that changes occurred in the transgenic epidermis reflecting approximately a 3-day lag in the normal epidermal maturation process, a possibility supported by the expression of epidermal differentiation markers and Cldns. Because results were similar from newborn to 7 days of age, normalized after 10 days, and were maintained throughout life, we report results only from 7- and 10-day-old Inv-Cldn6-CΔ206 transgenic mice compared with 7-day-old wild-type mice. K5 and K15 were restricted to basal cells at all time points in both the wild-type and Inv-Cldn6-CΔ206 epidermis (not shown), but K14 occupied an expanded zone extending into the suprabasal compartment until epidermal maturation was achieved in the 10-day-old Inv-Cldn6-CΔ206 transgenic epidermis (Fig. 6A). K6 and K17, which are keratins associated with a hyperproliferative epidermis (Leigh et al., 1995; McGowan and Coulombe, 1998a; McGowan and Coulombe, 1998b), were not expressed throughout the time analyzed (not shown); however, a broadened K1 (Fig. 6B) expression compartment was seen in the transgenic epidermis early after birth, but normalized by 10 days of age. Similarly, the expression compartments for involucrin (Fig. 6C), filaggrin (Fig. 6D) and loricrin (Fig. 6E) were also expanded, with an obvious packing defect reminiscent of the observed histological abnormalities of the stratum corneum, in the immature Inv-Cldn6-CΔ206 transgenic epidermis until thinning that was comparable to the wild-type epidermis was achieved.

In parallel with changes in epidermal markers, the suprabasal-specific Cldns, Cldn6 (Fig. 7A), Cldn11 (Fig. 7B), Cldn12 (Fig. 7C) and Cldn18 (Fig. 7D) were observed in the expanded suprabasal region of the immature Inv-Cldn6-CΔ206 transgenic epidermis, and immunostaining demonstrated a loss in membranous localization. Each of these Cldns was normalized, with a strictly membranous localization, upon epidermal maturation to a phenotype that was comparable to the wild type. Cldns not normally expressed in the epidermis (e.g. Cldn2, Cldn3 and Cldn5) (Turksen and Troy, 2002) were not observed in either the wild-type or Inv-Cldn6-CΔ206 transgenic epidermis at any time evaluated (not shown).

We also compared the expression of markers previously reported to be modulated during the epidermal maturation process in the newborn, 4-, 7- and 10-day-old transgenic mice compared with the wild-type epidermis. Changes in the overall surface pH of the epidermis contribute to the activation of enzymes involved in lipid processing for EPB function, and the plasma membrane Na+/H+ exchanger 1 (NHE1) has been implicated in this process (Behne et al., 2003; Fluhr et al., 2004a; Fluhr et al., 2004b; Hachem et al., 2005). Consistent with earlier reports (Behne et al., 2003), we found that NHE1 was localized in a ‘punctate’ pattern at the cell membrane of the basal and suprabasal compartments of the newborn wild-type epidermis, and as epidermal maturation progressed, NHE1 was gradually downregulated with localization restricted to the basal compartment by 7 days after birth (Fig. 8A–D, right column). By contrast, NHE1 was persistently upregulated and associated with both the basal and suprabasal cell compartments of the Inv-Cldn6-CΔ206 transgenic epidermis from birth to 7 days of age; downregulation and strict basal cell association was not observed until postnatal day 10 (Fig. 8A–D, left column). Immunoblot (Fig. 8E) and RT-PCR (Fig. 8F) analyses supported these findings. Similarly, aquaporins (AQPs), which are small integral membrane proteins that selectively transport water across cell membranes, and key lipid processing enzymes [β-glucocerebrosidase (β-celerase) and acid sphingomyelinase (αSMase)] that are important in EPB homeostasis (Behne et al., 2003; Hara-Chikuma and Verkman, 2008) were also modulated in the Inv-Cldn6-CΔ206 transgenic epidermis versus the wild-type epidermis, as shown by immunoblot (Fig. 8E) and RT-PCR (Fig. 8F) analyses.

Fig. 4.

The localization of epidermal differentiation markers and Cldns is altered in the delayed EPB of the Inv-Cldn6-CΔ206 transgenic epidermis. Immunofluorescence was used to evaluate various markers of epidermal differentiation and Cldns during development in Inv-Cldn6-CΔ206 epidermal initiation sites compared with wild type sites (E15.5 is shown). Although there was no difference in K14 (A) expression (e.g. the basal compartment), markers of the epidermal suprabasal zone occupied a moderately thinner expression compartment in the developing Inv-Cldn6-CΔ206 transgenic epidermis as shown by the expression of K1 (B), Cldn1 (C), Cldn6 (D), Cldn11 (E), Cldn12 (F) and Cldn18 (G). In addition, involucrin (H), filaggrin (I) and loricrin (J) expression compartments were also reduced in the developing Inv-Cldn6-CΔ206 transgenic epidermis. Bar, 20 μm.

Fig. 4.

The localization of epidermal differentiation markers and Cldns is altered in the delayed EPB of the Inv-Cldn6-CΔ206 transgenic epidermis. Immunofluorescence was used to evaluate various markers of epidermal differentiation and Cldns during development in Inv-Cldn6-CΔ206 epidermal initiation sites compared with wild type sites (E15.5 is shown). Although there was no difference in K14 (A) expression (e.g. the basal compartment), markers of the epidermal suprabasal zone occupied a moderately thinner expression compartment in the developing Inv-Cldn6-CΔ206 transgenic epidermis as shown by the expression of K1 (B), Cldn1 (C), Cldn6 (D), Cldn11 (E), Cldn12 (F) and Cldn18 (G). In addition, involucrin (H), filaggrin (I) and loricrin (J) expression compartments were also reduced in the developing Inv-Cldn6-CΔ206 transgenic epidermis. Bar, 20 μm.

Changes in Cldn1, phosphoCldn1 and Erk1/2 expression profiles delineate repair and maturation processes in the Inv-Cldn6-CΔ206 transgenic epidermis

The relatively rapid postnatal normalization of TEWL in Inv-Cldn6-CΔ206 transgenic mice, together with the morphological and marker expression changes observed, suggested a robust maturational or repair process. Previous studies, including our own (see above), have demonstrated the importance of Cldn1 in epidermal morphogenesis, differentiation, and EPB formation and repair. In particular, the expression of Cldn1 is unique compared with other Cldns and undergoes a maturation switch, from a strictly suprabasal association to being localized to cell-cell borders in all the living layers of the epidermis, coinciding with the acquisition of barrier function during epidermogenesis (Troy et al., 2007). This normal expression profile is maintained throughout life except in response to acute injury and in tumorigenesis, where basal layer association is lost, and Cldn1-null epithelial cells are progressively more frequent in the lower suprabasal compartment (Arabzadeh et al., 2007; Arabzadeh et al., 2008). In comparison to the wild-type basal to suprabasal localization of Cldn1 (Fig. 9A–D, right column), Cldn1-null epidermal cells were observed in the basal and lower suprabasal layers of the Inv-Cldn6-CΔ206 transgenic epidermis from 4–7 days after birth; as anticipated based on normalization of TEWL and morphology, Cldn1 localization was normalized in samples from 10-day-old mice (Fig. 9A–D, left column). Although no differences were detected in mRNA levels (Fig. 9F), immunoblotting confirmed decreased Cldn1 protein levels (Fig. 9E) in samples of 2-day-old Inv-Cldn6-CΔ206 transgenic epidermis. Given the evidence suggesting that the phosphorylation of different Cldns is involved in either the strengthening or weakening of TJs, and in parallel barrier function (D’Souza et al., 2007; Findley and Koval, 2009; Ikari et al., 2008), we next asked whether expression of the phosphorylated form of Cldn1 (phosphoCldn1) was altered in transgenic versus wild-type epidermis. PhosphoCldn1 was not observed in the wild-type epidermis at any of the time points assayed (data not shown). However, phosphoCldn1 was localized to cell-cell borders in the upper suprabasal zone of the newborn Inv-Cldn6-CΔ206 transgenic epidermis until epidermal repair was achieved at postnatal day 10 (Fig. 10A–D). Concomitantly, expression of Erk1/2 followed the same distribution pattern of phosphoCldn1 (Fig. 10E–H) with a considerable amount of Erk1/2 localized to the cell membrane of differentiating cells in the newborn, 4-, 7- and 10-day-old Inv-Cldn6-CΔ206 transgenic epidermis.

Fig. 5.

Histological abnormalities in the Inv-Cldn6-CΔ206 transgenic epidermis after birth. (A) Histological analyses of skin samples revealed that, at newborn stages, the Inv-Cldn6-CΔ206 epidermis was comparable to the wild type. (B) However, it was evident that, after 7 days, the transgenic epidermis did not undergo the normal process of maturation/thinning that was observed in the wild-type epidermis and was characterized by an increased number of suprabasal cell layers, the prevalent appearance of nuclei in the upper differentiation layers, and a much less compacted granular layer. However, representing a 3-day lag behind the wild type, the transgenic epidermis thinned to be morphologically comparable to the wild type by 10 days after birth (C), a phenotype that was maintained after 1 month (D) and throughout life. Bar, 20 μm.

Fig. 5.

Histological abnormalities in the Inv-Cldn6-CΔ206 transgenic epidermis after birth. (A) Histological analyses of skin samples revealed that, at newborn stages, the Inv-Cldn6-CΔ206 epidermis was comparable to the wild type. (B) However, it was evident that, after 7 days, the transgenic epidermis did not undergo the normal process of maturation/thinning that was observed in the wild-type epidermis and was characterized by an increased number of suprabasal cell layers, the prevalent appearance of nuclei in the upper differentiation layers, and a much less compacted granular layer. However, representing a 3-day lag behind the wild type, the transgenic epidermis thinned to be morphologically comparable to the wild type by 10 days after birth (C), a phenotype that was maintained after 1 month (D) and throughout life. Bar, 20 μm.

Fig. 6.

Perturbation of markers of epidermal differentiation are normalized by postnatal day 10 in the Inv-Cldn6-CΔ206 transgenic epidermis. Changes in the epidermal differentiation program of the Inv-Cldn6-CΔ206 transgenic epidermis were evaluated by immunofluorescence and compared with their age-matched wild-type counterparts (samples from 7 and 10-day-old transgenic mice are shown and compared with the mature wild-type epidermis at 7 days). (A) In the transgenic epidermis, K14 occupied an expanded zone extending into the suprabasal compartment until epidermal maturation was achieved. (B) K1 was first observed in an expanded zone of expression, but normalized by 10 days of age in the Inv-Cldn6-CΔ206 transgenic epidermis. (C–E) Similarly, broadened expression compartments and obvious packing defects for involucrin (C), filaggrin (D) and loricrin (E) characterized the immature Inv-Cldn6-CΔ206 transgenic epidermis until thinning comparable to the wild type was achieved. Bar, 20 μm.

Fig. 6.

Perturbation of markers of epidermal differentiation are normalized by postnatal day 10 in the Inv-Cldn6-CΔ206 transgenic epidermis. Changes in the epidermal differentiation program of the Inv-Cldn6-CΔ206 transgenic epidermis were evaluated by immunofluorescence and compared with their age-matched wild-type counterparts (samples from 7 and 10-day-old transgenic mice are shown and compared with the mature wild-type epidermis at 7 days). (A) In the transgenic epidermis, K14 occupied an expanded zone extending into the suprabasal compartment until epidermal maturation was achieved. (B) K1 was first observed in an expanded zone of expression, but normalized by 10 days of age in the Inv-Cldn6-CΔ206 transgenic epidermis. (C–E) Similarly, broadened expression compartments and obvious packing defects for involucrin (C), filaggrin (D) and loricrin (E) characterized the immature Inv-Cldn6-CΔ206 transgenic epidermis until thinning comparable to the wild type was achieved. Bar, 20 μm.

Fig. 7.

Evaluation of Cldns in the delayed EPB of Inv-Cldn6-CΔ206 transgenic mice. The expression/localization of Cldns was also perturbed in the immature Inv-Cldn6-CΔ206 epidermis (7- and 10-day-old transgenic samples are shown and compared with the mature wild type samples at 7 days). (A–D) Cldn6 (A), Cldn11 (B), Cldn12 (C) and Cldn18 (D) were observed in an expanded zone early on, demonstrating a loss in membranous localization, and were normalized by 10 days after birth. Bar, 20 μm.

Fig. 7.

Evaluation of Cldns in the delayed EPB of Inv-Cldn6-CΔ206 transgenic mice. The expression/localization of Cldns was also perturbed in the immature Inv-Cldn6-CΔ206 epidermis (7- and 10-day-old transgenic samples are shown and compared with the mature wild type samples at 7 days). (A–D) Cldn6 (A), Cldn11 (B), Cldn12 (C) and Cldn18 (D) were observed in an expanded zone early on, demonstrating a loss in membranous localization, and were normalized by 10 days after birth. Bar, 20 μm.

DISCUSSION

In this study, we describe the generation of a novel animal model for evaluating developmental delays in EPB formation and the postnatal epidermal maturation processes that are analogous to those observed in the dysfunctional barrier phenotype of human premature babies. Using skin penetration assays and immunohistochemistry to evaluate the expression and localization of classical markers of epidermal differentiation and maturation, as well as the Cldns and signaling molecules that are involved in EPB formation, we demonstrated that Inv-Cldn6-CΔ206 transgenic mice suffered a developmental delay in epidermal differentiation and EPB formation leading to significant TEWL at birth, despite sufficient neonatal barrier formation (the presence of a cornified envelope) and function (the absence of X-Gal penetration) for survival. Postnatal TEWL measurements, along with changes observed in the expression and localization of keratins and Cldns, suggested that the Inv-Cldn6-CΔ206 transgenic epidermis underwent a robust epidermal maturation process after birth to become indistinguishable from the wild type. Although the molecular mechanisms underlying the delayed maturation and repair of the epidermis and EPB in these transgenic mice have not yet been delineated, our data indicate that the Inv-Cldn6-CΔ206 mice constitute an attractive model from a therapeutics point of view, i.e. for the identification of lead compounds for accelerated repair of the often life-threatening permeability barrier defects in premature human infants.

Many studies have described the developmental formation of the EPB (Byrne and Hardman, 2005; Hardman et al., 1999; Hardman et al., 1998; Turksen and Troy, 2002) and it is well recognized that a disruption or delay in its formation before birth may have severe consequences to the survival of the organism (Cartlidge, 2000; Elias, 2005; Mack et al., 2005; Williams et al., 1998). We described previously that perturbations of Cldn6 expression levels in the suprabasal compartment of the epidermis – its endogenous site –result in epidermal differentiation abnormalities and EPB dysfunction (Troy et al., 2005b; Turksen and Troy, 2002). However, depending on the level of expression, and whether normal or mutant forms of Cldn6 are expressed, the severity of the phenotype varies (Arabzadeh et al., 2006; Troy et al., 2005b; Troy and Turksen, 2007; Turksen and Troy, 2002). For example, severe EPB dysfunction manifested in extreme TEWL and neonatal lethality occurs when native Cldn6 is expressed at high levels (Turksen and Troy, 2002), whereas lower levels of expression result in less severe EPB dysfunction and postnatal normalization (Troy et al., 2005b). Overexpression of a mutant form of Cldn6 lacking its entire tail domain (Inv-Cldn6-CΔ187 mice) does not appear to manifest in any prenatal epidermal developmental defects, but an abnormal, postnatal, lifelong epidermal hyperproliferation is observed (Arabzadeh et al., 2006). High overexpression of a different mutant lacking only the C-terminal half of the tail domain of Cldn6 (Inv-Cldn6-CΔ196 mice) (Troy and Turksen, 2007) results in a lethal barrier dysfunction with marked hyperproliferative squamous invaginations/cysts replacing hair follicles, while lower-level expression manifests in an aging-related skin barrier defect resulting in an intrinsic propensity for injury, inefficient repair and chronic dermatitis. We now show that transgenic mice expressing a mutant Cldn6 with a shorter tail deletion (removing the PDZ domain and a putative PKA phosphorylation site) possess a distinct developmental defect in epidermal differentiation resulting in EPB formation delays and that a robust repair response occurs for postnatal epidermal maturation. It is notable that formation of a skin barrier with functional TEWL characteristics that are indistinguishable from the wild type occurred more rapidly than, or prior to, complete morphological maturation of the epidermis in the postnatal Cldn6-CΔ206 mice, indicating an ability to disconnect aspects of the two processes, an observation that is interesting from a developmental standpoint but that may also be therapeutically important (see below).

Fig. 8.

Modulation in NHE1 expression/localization. (A–D) Localization of NHE1 was evaluated in skin samples from newborn (A), 4-day-old (B), 7-day-old (C) and 10-day-old (D) Inv-Cldn6-CΔ206 transgenic mice (left column) compared with wild-type mice (right column). In the Inv-Cldn6-CΔ206 transgenic epidermis, NHE1 was upregulated and associated with both the basal and suprabasal cell compartments from newborn to 7 days after birth; downregulation and strict basal cell association was not observed until postnatal day 10. Bar, 20 μm. These data were supported by immunoblot (E) and RT-PCR (F) analyses. Modulations in mRNA or protein levels of AQP1, β-celerase and αSMase are also shown relative to GAPDH controls.

Fig. 8.

Modulation in NHE1 expression/localization. (A–D) Localization of NHE1 was evaluated in skin samples from newborn (A), 4-day-old (B), 7-day-old (C) and 10-day-old (D) Inv-Cldn6-CΔ206 transgenic mice (left column) compared with wild-type mice (right column). In the Inv-Cldn6-CΔ206 transgenic epidermis, NHE1 was upregulated and associated with both the basal and suprabasal cell compartments from newborn to 7 days after birth; downregulation and strict basal cell association was not observed until postnatal day 10. Bar, 20 μm. These data were supported by immunoblot (E) and RT-PCR (F) analyses. Modulations in mRNA or protein levels of AQP1, β-celerase and αSMase are also shown relative to GAPDH controls.

The mechanisms by which the expression of Cldn6-CΔ206 results in a developmental delay in EPB formation and postnatal epidermal maturation and repair are not yet known, but our observations support the need for Cldn homeostasis in a bona fide epidermal differentiation program and in epidermal repair. We have previously demonstrated that there is a defined Cldn expression profile in the epidermis and that changes in epidermal differentiation elicit concomitant modifications in the Cldn expression profile and vice versa (Troy et al., 2005b; Turksen and Troy, 2002; Turksen and Troy, 2004). Upon injury, or in response to differentiation abnormalities, the spatial expression of the suprabasal Cldns (Cldn6, Cldn11, Cldn12 and Cldn18) generally expands or shrinks to encompass all the cells of the perturbed suprabasal zone, with some concomitant loss in cell membrane association (Arabzadeh et al., 2006; Arabzadeh et al., 2007; Arabzadeh et al., 2008; Troy et al., 2005b; Turksen and Troy, 2002). This was also true in the immature Inv-Cldn6-CΔ206 epidermis: as the epidermis matured, by postnatal day 10, the localization and expression of the Cldns normalized to a strictly membranous association in a suprabasal zone that was comparable in thickness to that of the wild type. However, Cldn1 undergoes more dramatic alterations in response to epidermal homeostasis dysregulation. In the developing epidermis, Cldn1 is first restricted to the stratifying layers and matures to occupy the basal layer upon the completion of barrier formation at E17.5 (Troy and Turksen, 2007). However, in response to TPA (12-O-tetradecanoyl-phorbol-13-acetate)-induced injury and the loss of cell polarity that is seen in tumorigenesis, Cldn1 expression is downregulated in both the basal layer and immediate suprabasal layers of the epidermis (Arabzadeh et al., 2007; Arabzadeh et al., 2008). These changes are also observed in the intrinsic aging process of the Inv-Cldn6-CΔ196 transgenic epidermis and in the delayed epidermal maturation that we now report in Inv-Cldn6-CΔ206 transgenic mice. Notably, Cldn1 expression normalized with the normalization of epidermal differentiation markers and epidermal maturation (see below).

Fig. 9.

Changes in Cldn1 expression and localization in the Inv-Cldn6-CΔ206 transgenic epidermis. Unlike the normal wild type (right column) basal to suprabasal localization of Cldn1, the Inv-Cldn6-CΔ206 transgenic epidermis (left column) showed abnormalities. (A–C) Although localization was normal at newborn stages (A), Cldn1-null epidermal cells were observed in the basal and lower suprabasal layers from 4 days (B) to 7 days (C) after birth, and were normalized by 10 days after birth (D). Bar, 20 μm. (E–F) Immunoblotting confirmed the changes in Cldn1 protein level (E); whereas RT-PCR indicated that there were no differences at the mRNA level (F).

Fig. 9.

Changes in Cldn1 expression and localization in the Inv-Cldn6-CΔ206 transgenic epidermis. Unlike the normal wild type (right column) basal to suprabasal localization of Cldn1, the Inv-Cldn6-CΔ206 transgenic epidermis (left column) showed abnormalities. (A–C) Although localization was normal at newborn stages (A), Cldn1-null epidermal cells were observed in the basal and lower suprabasal layers from 4 days (B) to 7 days (C) after birth, and were normalized by 10 days after birth (D). Bar, 20 μm. (E–F) Immunoblotting confirmed the changes in Cldn1 protein level (E); whereas RT-PCR indicated that there were no differences at the mRNA level (F).

Fig. 10.

The localization of phosphoCldn1 and Erk1/2 in the epidermis of Inv-Cldn6-CΔ206 transgenic mice. (A–H) Skin samples from newborn (A,E), 4-day-old (B,F), 7-day-old (C,G) and 10 day-old (D,H) Inv-Cldn6-CΔ206 transgenic mice were used to evaluate the localization of phosphoCldn1 (left column) and Erk1/2 (right column). From newborn to 7 days of age, phosphoCldn1 was localized to cell-cell borders in the upper suprabasal zone of the Inv-Cldn6-CΔ206 transgenic epidermis. Correspondingly, Erk1/2 localization followed the same pattern with a considerable distribution at the cell membrane of differentiating cells. Bar, 20 μm.

Fig. 10.

The localization of phosphoCldn1 and Erk1/2 in the epidermis of Inv-Cldn6-CΔ206 transgenic mice. (A–H) Skin samples from newborn (A,E), 4-day-old (B,F), 7-day-old (C,G) and 10 day-old (D,H) Inv-Cldn6-CΔ206 transgenic mice were used to evaluate the localization of phosphoCldn1 (left column) and Erk1/2 (right column). From newborn to 7 days of age, phosphoCldn1 was localized to cell-cell borders in the upper suprabasal zone of the Inv-Cldn6-CΔ206 transgenic epidermis. Correspondingly, Erk1/2 localization followed the same pattern with a considerable distribution at the cell membrane of differentiating cells. Bar, 20 μm.

Although Cldns demonstrate amino acid similarity among family members, the cytoplasmic tails of Cldns are divergent in sequence and possess a number of sites that provide clues about their structure-function relationships in epidermal differentiation, including a PDZ binding sequence (YV) and potential phosphorylation sites (Fujibe et al., 2004; Simard et al., 2006). Proteins with a PDZ domain such as the membrane-associated guanylate kinase (MAGUK) family proteins [zonula occludens (ZO)-1, ZO-2 and ZO-3] (Itoh et al., 1999), as well as the recently identified multi-PDZ domain scaffolding proteins PATJ (protein associated to tight junctions) (Lemmers et al., 2002) and multi-PDZ domain protein 1 (MUPP-1) (Hamazaki et al., 2002), selectively recognize and bind to this sequence (for a review see Gonzalez-Mariscal et al., 2003). However, it seems likely that as yet unidentified novel molecules interact with Cldns in regulating gene expression and epidermal differentiation. Post-translational modifications within the tail domain, including phosphorylation and palmitoylation, are also thought to regulate Cldn activities, including their targeting to the membrane and their insertion into TJs to regulate paracellular permeability (Simard et al., 2006). Phosphorylation of a number of Cldns has been demonstrated to be required for their assembly into TJs [e.g. for Cldn1 and Cldn4 (Banan et al., 2005), and for Cldn16 (Ikari et al., 2008)] but, to date, most Cldns have not been subjected to exhaustive analysis. Our observation of the association of phosphoCldn1 with the differentiated layers of the immature Inv-Cldn6-CΔ206 transgenic epidermis points towards the potential role of Cldn1 phosphorylation in the process of epidermal maturation and EPB repair.

Although the precise sequence of events in the maturation of the skin barrier is not well understood, the notion that exposure to air after birth functions to initiate and accelerate the maturation and repair of the skin barrier has been suggested (Hanley et al., 1997; Williams et al., 1998). The fetal and neonatal anomalies with spontaneous and apparently complete epidermal maturation and barrier repair that we observe in postnatal Inv-Cldn6-CΔ206 transgenic mice support the view that exposure to air induces an intrinsic repair and maturation pathway. Other support for this hypothesis comes from a number of studies; for example, exposing embryonic immature epidermis to air (Williams et al., 1998), lifting skin-equivalent cultures to the air-liquid interface (Fartasch and Ponec, 1994; Komuves et al., 1999), and inducing injury to the EPB by tape stripping (Ahn et al., 1999; Ahn et al., 2001) each result in a robust EPB repair and epidermal maturation response. Although collectively these studies do not provide a mechanism for the observed intrinsic repair/maturation process in the epidermis, our studies suggest that the first phase of repair is the shedding of defective differentiation layers, which we identified as phospoCldn1-expressing cells with co-expression of high levels of Erk1/2. Considering the important role of Erk1/2 in epithelial differentiation (Hobbs et al., 2004; Taupin and Podolsky, 1999; Yu et al., 2007), as well as in the regulation of Cldn expression (Lipschutz et al., 2005), our data support a role for Erk1/2 in the initiation or progression of the epidermal repair and maturation that was observed in Inv-Cldn6-CΔ206 transgenic mice. Concomitantly, initiation probably also involves the well-known phenomena of cross-talk between the differentiating and the basal compartments of the epidermis, which has been demonstrated to tightly regulate the epidermal differentiation program and is responsible for the normal maintenance program of the epidermis (Prowse et al., 1999; Troy et al., 2005b); evidence for this comes from our reported observations regarding the remodeling of Cldn1 expression in the repairing Inv-Cldn6-CΔ206 transgenic epidermis. Further analyses are required to understand how ‘air exposure’ activates the Erk1/2 pathway and the observed Cldn1 phosphorylation process.

Although a variety of maternal and fetal diseases and conditions can lead to premature birth, the reasons underlying the quite dramatic increases in premature birth, in not only underdeveloped but also developed countries, over the last decade are unknown (Darmstadt et al., 2008; Lang and Iams, 2009; Yeaney et al., 2009). Complications owing to comprised skin barrier function (e.g. poor temperature regulation and dehydration) in premature babies are among the primary causes of neonatal sepsis and mortality (Rutter, 1996; Rutter, 2000; Saiman, 2006). Given the importance of skin barrier function in the health of premature babies and the fact that intrinsic risk factors of sepsis include compromised portals of entry for pathogens (Saiman, 2006), there is a surprisingly significant lack of successful approaches/strategies that have been designed specifically to accelerate the postnatal maturation of the epidermis (McIntire and Leveno, 2008; Shapiro-Mendoza et al., 2008; Shapiro-Mendoza et al., 2006; Tyson et al., 2008). Strategies for reducing the rate of sepsis are currently focused on limiting the spread of infection through aseptic clinical techniques and the use of antibiotics, which are increasingly becoming ineffective (Saiman, 2006). In addition, the topical application of relatively low-cost emollients, especially in underdeveloped countries, has proven to have some benefit, although whether this is from provision of a mechanical barrier or from induction of biological responses to some of the ingredients, or both, is not clear (Darmstadt et al., 2008). Understanding these mechanisms, as well as screening for potential novel therapeutics to accelerate postnatal epidermal maturation, have been hampered by the lack of suitable in vivo models. The capacity of the Inv-Cldn6-CΔ206 transgenic epidermis to undergo a postnatal repair response and acquire a mature epidermis by 10 days after birth makes this transgenic model an excellent tool for investigating not only the molecular changes taking place during this maturation period, but also for screening for novel therapeutics to accelerate this process and improve the health of human premature infants.

METHODS

Generation of Inv-Cldn6-CΔ206 transgenic mice

Inv-Cldn6-CΔ206 mice were generated by truncating the cytoplasmic tail domain of Cldn6 after amino acid 206 (Cldn6-CΔ206) and then subcloning Cldn6-CΔ206 into the NotI site of the Inv cassette (H3700-pL2) by our previously utilized strategy (Arabzadeh et al., 2006; Troy and Turksen, 2007; Turksen and Troy, 2002). Purified DNA was injected into ova collected from superovulated CD1 mice at the Transgenic Mouse Facility of the Ottawa Hospital Research Institute (OHRI), as described previously (Arabzadeh et al., 2006; Troy and Turksen, 2007; Turksen and Troy, 2002). The presence of the transgene was confirmed by PCR using genomic DNA and specific primers. Photography of age-matched wild-type and Inv-Cldn6-CΔ206 transgenic mice was performed using a Nikon Coolpix 950 digital camera (Nikon) and image processing was performed with Adobe Photoshop version 7.0 (Adobe Systems). All research was carried out in accordance with the principles and guidelines of the Canadian Council on Animal Care, and the policies of the OHRI Animal Care Committee.

RNA isolation and RT-PCR

Skin samples dissected from the mid-dorsal region of transgenic and wild-type mice were frozen in liquid nitrogen, and then homogenized in Trizol (Invitrogen) reagent for total RNA isolation according to the instructions of the manufacturer. After DNase (Invitrogen) treatment, first-strand cDNA was synthesized using random hexamers (Applied Biosystems) and 1 μg of RNA. PCR analysis was then performed, as decribed previously (Troy et al., 2005a) using the following specific primers: Inv exon-Cldn6 [~350 bp; FP: 5′-CTGCCTCAGCCTTACTGTGAG-3′ (KT323), RP: 5′-CCAACAGTGAGTCATACAC-3′ (KT1526)], GAPDH [167 bp; FP: 5′-CAGTATGACTCCACTCACGG-3′ (KT841), RP: 5′-GTGAAGACACCAGTAGACTCC-3′ (KT842)], NHE1 [498 bp; FP: 5′-GAGATCCACACACAGTTC-3′ (KT1515), RP: 5′-TACTGTCAGGTAGTTGGTG-3′ (KT1516)], αSMase [567 bp; FP: 5′-AGACTGGAGAGGTCCTTA-3′ (KT1537), RP: 5′-GTCCCAGTGTAGATCAGTAA-3′ (KT1538)], β-celerase [512 bp; FP: 5′-TACTTTAGGAGAGACACACC-3′ (KT1539), RP: 5′-GGTAAGTGTGAATGGAGTAG-3′ (KT1540)] and Cldn1 [644 bp; FP: 5′-AAAGAGCCATGGCCAACGC-3′ (KT1315), RP: 5′-TCACACATAGTCTTTCCCACTAG-3′ (KT1316)]. RT-PCR products, relative to a housekeeping control (GAPDH), were separated on ethidium bromide-containing agarose gels, visualized by ultraviolet light, and images were acquired using AlphaEaseFC software version 4.0 (Alpha Innotech Corporation).

Skin permeability assays

X-gal penetration assay

Freshly dissected Inv-Cldn6-CΔ206 transgenic embryos (E15.5, E17.5 and E18.5; the embryonic age was estimated based on the appearance of the vaginal plug at E0.5) and euthanized neonates, along with their age-matched wild-type counterparts, were rinsed in PBS and immersed in X-Gal reaction mix at pH 4.5 [100 mM NaPO4, 1.3 mM MgCl2, 3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6 and 1 mg/ml X-Gal], as described previously (Hardman et al., 1998; Turksen and Troy, 2002; Turksen and Troy, 2003). Following an overnight room temperature incubation, specimens were fixed with formalin and images were acquired using a Nikon Coolpix 950 digital camera followed by processing with Adobe Photoshop version 7.0.

Trans-epidermal water loss (TEWL) measurements

A dermal phase meter (DPM) (Nova Technology Corporation) was used to measure the dorsal and ventral impedance/TEWL of Inv-Cldn6-CΔ206 transgenic and wild-type mice, as described previously (Troy et al., 2005b; Turksen and Troy, 2002), at various postnatal time points from birth to the emergence of coat hairs, at which time measurements are no longer feasible owing to improper contact between the meter probe and the skin. Higher DPM values measured over time translate to reduced barrier integrity, illustrated digitally by EDWINA (Nova Technology Corporation) and Excel (Microsoft) software programs.

Cornified envelope extracts

Purified cornified envelope extracts were prepared from CD1 and Inv-Cldn6-CΔ206 transgenic mice by immersion of dorsal skin samples into hot extraction buffer (0.1M Tris-Hcl, pH 8.5, 2% SDS, 20 mM dithiothreitol, 5 mM EDTA), followed by a 15-minute incubation at 95°C and gentle centrifugation, as described previously (Hohl et al., 1991; Troy and Turksen, 2005; Turksen and Troy, 2002).

Sample collection, histology and immunolocalization

Sample preparation

Freshly dissected skin samples (~1 cm2) and whole embryos were collected from wild-type and Inv-Cldn6-CΔ206 transgenic mice at various embryonic and postnatal time points (E15.5, E17.5, E18.5, newborn, and 2 days, 4 days, 6 days, 7 days, 8 days, 10 days, 12 days, 2 weeks, 3 weeks, 1 month and 3 months of age).

Paraffin sections and histology

Following an overnight fixation in Bouin’s solution (75% saturated picric acid, 20% formaldehyde, 5% glacial acetic acid), skin samples were dehydrated by a graded series of ethanol washes (from 30% to 100%) and embedded in paraffin. Sections (5 μm) were mounted onto Superfrost/Plus slides (Fisher Scientific), and were dewaxed using toluene and rehydrated in a reverse series of ethanol washes to water. Following antigen unmasking and washes in PBS, sections were either stained with hematoxylin and eosin (H&E), as described previously (Troy et al., 2005c), or used for immunohistochemistry (see below).

Immunohistochemistry

Non-specific antibody binding (10% goat serum, 0.8% BSA, 1% gelatin in PBS) was blocked, followed by incubation for 1 hour in antibodies that were diluted appropriately with incubation buffer (1% goat serum, 0.8% BSA, 1% gelatin in PBS) (Troy et al., 2005c). Antibodies against the following antigens were evaluated: K15 (1:100; rabbit #UC55), K5 (1:100; rabbit #5054), K14 (1:100; rabbit #199), K1 (1:100; rabbit #UC81), K6 (1:200; BabCO), K17 (1:500; a gift from Dr Pierre Coulombe, Johns Hopkins University School of Medicine, Baltimore, MD), involucrin (1:100; BabCO), filaggrin (1:100; BabCO), loricrin (1:100; rabbit #UC84), Cldn1 (6:100; Zymed Laboratories), Cldn2 (1:200; Zymed Laboratories), Cldn3 (1:50; Zymed Laboratories), Cldn5 (1:100; Zymed Laboratories), Cldn6 (1:100; chicken #3677), Cldn11 (1:100; chicken #3680), Cldn12 (1:100; chicken #5186), Cldn18 (1:100; rabbit #A9953), NHE1 [3:100; Chemicon-AB3031 (incubation was overnight at 4°C)], phosphoCldn1 [1:100; a custom antibody generated from rabbit #2827 against the tail domain of the mouse phosphoCldn1 sequence (Ac-C-Ahx-PYPKP[pT]PSSGKDY-amide), 21st Century Biochemicals] and Erk1/2 (1:100; StressMarq Biosciences Inc.). After incubations in wash buffer (0.8% BSA, 1% gelatin in PBS), FITC-conjugated secondary antibodies against mouse, rabbit and chicken (1:50; Jackson ImmunoResearch) were diluted in incubation buffer and used for 1-hour incubations at room temperature. Following the final washes, skin samples were mounted with Mowiol 4–88 (Calbiochem), containing 2.5% 1,4 diazobicyclo-[2,2,2]-octane (DABCO; Sigma), for observation with a Zeiss Axioplan 2 fluorescence microscope equipped with an AxioCam camera and Axio Vision 2.05 software (Carl Zeiss). Digital photography was presented with Adobe Photoshop version 7.0.

Protein Isolation and Immunoblotting

Freshly dissected back skin samples (0.4 grams) were homogenized in SDS extraction buffer [62.5 mM Tris, pH 6.8, 25% glycerol, 2% SDS and 2% β-mercaptoethanol, with pepstatin A and a complete mini protease inhibitor cocktail (Roche Diagnostics) tablet] followed by high-speed centrifugation. The supernatant containing the proteins was collected and assayed for protein concentration. Proteins were incubated at room temperature for 30 minutes in sample reducing buffer (62.5 mM Tris, pH 6.8, 6 M urea, 25% glycerol, 2% SDS, 0.1% Bromophenol Blue and 2% β-mercaptoethanol), boiled for 5 minutes (samples were not boiled for analysis of NHE1), and centrifuged at high speed for 10 minutes before 5–50 μg samples were separated on 7.5–12% SDS-PAGE gels, transferred to nitrocellulose and incubated in blocking buffer [5% skimmed milk in TBS/0.1% Tween-20 (TBS-T)] for 1 hour at room temperature. Appropriately diluted (5% skimmed milk in TBS-T) antibodies were used for overnight incubations at 4°C. The following antibodies were used: NHE1 (91 kDa, 1:1000; Chemicon), AQP1 (28 kDa, 1:1000; Alpha Diagnostic), Cldn1 (23 kDa, 1:1000; Zymed Laboratories) and GAPDH (36 kDa, 1:10,000; Abcam). After washing in TBS-T, blots were incubated for 1 hour at room temperature in HRP-conjugated secondary antibodies against rabbit or mouse (1:20,000; Amersham Biosciences), then diluted in 5% skimmed milk/TBS-T. Following washes in TBS-T, the Immobilon western blotting detection system (Millipore) was used for detection, and expression levels were normalized to a housekeeping control (GAPDH) and visualized on Kodak BioMax XAR film (Kodak). Films were digitally scanned and images were processed with Adobe Photoshop version 7.0.

Acknowledgements

This work is dedicated to Dr George E. Palade for his discovery of tight junctions with Marilyn G. Farquhar in 1963. We are indebted to Dr Jane E. Aubin (University of Toronto) for her tireless efforts and vigorous dedication to every aspect of our studies. We would also like to thank Dr Pierre Coulombe (Johns Hopkins) for his gift of the anti-K17 antibodies, and StressMarq Biosciences Inc. for their gift of the Erk1/2 antibodies. We acknowledge Mrs Adriana Gambarotta and Mr Pierre Bradley of the animal care facility of the OHRI for their great efforts, and Zaida J. Ticas (MLT) (University of Ottawa) who prepared our histological and paraffin-embedded sections. We also acknowledge Dr Vivian Siegel for her editorial insights. K.T. would like to acknowledge Paul Newman for Cool Hand Luke, eggs and ‘What we got here is…failure to communicate’. This work was sponsored by a research grant from the Canadian Institutes of Health Research (# MOP 84464).

AUTHOR CONTRIBUTIONS

A.E. and N.L. were responsible for all the revisions requested by the reviewers and performed the immunoflourescence experiments for the re-confirmation and presentation of the data. T.-C.T. was involved in each aspect of the work presented herein, including acquisition and presentation of data, the generation of reagents, and the drafting/revising of the manuscript. A.A. was responsible for the photography of embryo-stage immunohistochemical results and was involved in the presentation of the data. E.A. worked very closely with K.T. and T.-C.T. to perform the initial experiments and make the observations described before any revisions were made. K.T. made substantial intellectual contributions to the conception, design, analysis and interpretation of the data, in addition to revising the manuscript for intellectual content, and gave final approval of the version to be published.

REFERENCES

Ahn
SK
,
Hwang
SM
,
Jiang
SJ
,
Choi
EH
,
Lee
SH
(
1999
).
The changes of epidermal calcium gradient and transitional cells after prolonged occlusion following tape stripping in the murine epidermis
.
J Invest Dermatol
.
113
,
189
195
.
Ahn
SK
,
Jiang
SJ
,
Hwang
SM
,
Choi
EH
,
Lee
JS
,
Lee
SH
(
2001
).
Functional and structural changes of the epidermal barrier induced by various types of insults in hairless mice
.
Arch Dermatol Res
.
293
,
308
318
.
Angelow
S
,
Ahlstrom
R
,
Yu
AS
(
2008
).
Biology of Claudins
.
Am J Physiol Renal Physiol
.
295
,
F867
F876
.
Arabzadeh
A
,
Troy
TC
,
Turksen
K
(
2006
).
Role of the cldn6 cytoplasmic tail domain in membrane targeting and epidermal differentiation in vivo
.
Mol Cell Biol
.
26
,
5876
5887
.
Arabzadeh
A
,
Troy
TC
,
Turksen
K
(
2007
).
Changes in the distribution pattern of Claudin tight junction proteins during the progression of mouse skin tumorigenesis
.
BMC Cancer
7
,
196
.
Arabzadeh
A
,
Troy
TC
,
Turksen
K
(
2008
).
Claudin expression modulations reflect an injury response in the murine epidermis
.
J Invest Dermatol
.
128
,
237
240
.
Baharestani
MM
(
2007
).
An overview of neonatal and pediatric wound care knowledge and considerations
.
Ostomy Wound Manage
.
53
,
34
36
,
38
,
40
, passim.
Banan
A
,
Zhang
LJ
,
Shaikh
M
,
Fields
JZ
,
Choudhary
S
,
Forsyth
CB
,
Farhadi
A
,
Keshavarzian
A
(
2005
).
theta Isoform of protein kinase C alters barrier function in intestinal epithelium through modulation of distinct claudin isotypes: a novel mechanism for regulation of permeability
.
J Pharmacol Exp Ther
.
313
,
962
982
.
Behne
MJ
,
Barry
NP
,
Hanson
KM
,
Aronchik
I
,
Clegg
RW
,
Gratton
E
,
Feingold
K
,
Holleran
WM
,
Elias
PM
,
Mauro
TM
(
2003
).
Neonatal development of the stratum corneum pH gradient: localization and mechanisms leading to emergence of optimal barrier function
.
J Invest Dermatol
.
120
,
998
1006
.
Brandner
JM
(
2009
).
Tight junctions and tight junction proteins in mammalian epidermis
.
Eur J Pharm Biopharm
.
72
,
289
294
.
Brandner
JM
,
Kief
S
,
Grund
C
,
Rendl
M
,
Houdek
P
,
Kuhn
C
,
Tschachler
E
,
Franke
WW
,
Moll
I
(
2002
).
Organization and formation of the tight junction system in human epidermis and cultured keratinocytes
.
Eur J Cell Biol
.
81
,
253
263
.
Byrne
C
,
Hardman
MJ
(
2005
).
Whole-mount assays for gene induction and barrier formation in the developing epidermis
.
Methods Mol Biol
.
289
,
127
136
.
Cartlidge
P
(
2000
).
The epidermal barrier
.
Semin Neonatol
.
5
,
273
280
.
Chiba
H
,
Osanai
M
,
Murata
M
,
Kojima
T
,
Sawada
N
(
2008
).
Transmembrane proteins of tight junctions
.
Biochim Biophys Acta
1778
,
588
600
.
Darmstadt
GL
,
Saha
SK
,
Ahmed
AS
,
Ahmed
S
,
Chowdhury
MA
,
Law
PA
,
Rosenberg
RE
,
Black
RE
,
Santosham
M
(
2008
).
Effect of skin barrier therapy on neonatal mortality rates in preterm infants in Bangladesh: a randomized, controlled, clinical trial
.
Pediatrics
121
,
522
529
.
Daugherty
BL
,
Ward
C
,
Smith
T
,
Ritzenthaler
JD
,
Koval
M
(
2007
).
Regulation of heterotypic claudin compatibility
.
J Biol Chem
.
282
,
30005
30013
.
D’Souza
T
,
Indig
FE
,
Morin
PJ
(
2007
).
Phosphorylation of claudin-4 by PKCepsilon regulates tight junction barrier function in ovarian cancer cells
.
Exp Cell Res
.
313
,
3364
3375
.
Elias
PM
(
2005
).
Stratum corneum defensive functions: an integrated view
.
J Invest Dermatol
.
125
,
183
200
.
Farquhar
MG
,
Palade
GE
(
1963
).
Junctional complexes in various epithelia
.
J Cell Biol
.
17
,
375
409
.
Fartasch
M
,
Ponec
M
(
1994
).
Improved barrier structure formation in air-exposed human keratinocyte culture systems
.
J Invest Dermatol
.
102
,
366
374
.
Findley
MK
,
Koval
M
(
2009
).
Regulation and roles for claudin-family tight junction proteins
.
IUBMB Life
61
,
431
437
.
Fluhr
JW
,
Behne
MJ
,
Brown
BE
,
Moskowitz
DG
,
Selden
C
,
Mao-Qiang
M
,
Mauro
TM
,
Elias
PM
,
Feingold
KR
(
2004a
).
Stratum corneum acidification in neonatal skin: secretory phospholipase A2 and the sodium/hydrogen antiporter-1 acidify neonatal rat stratum corneum
.
J Invest Dermatol
.
122
,
320
329
.
Fluhr
JW
,
Mao-Qiang
M
,
Brown
BE
,
Hachem
JP
,
Moskowitz
DG
,
Demerjian
M
,
Haftek
M
,
Serre
G
,
Crumrine
D
,
Mauro
TM
, et al. 
(
2004b
).
Functional consequences of a neutral pH in neonatal rat stratum corneum
.
J Invest Dermatol
.
123
,
140
151
.
Fuchs
E
,
Byrne
C
(
1994
).
The epidermis: rising to the surface
.
Curr Opin Genet Dev
.
4
,
725
736
.
Fujibe
M
,
Chiba
H
,
Kojima
T
,
Soma
T
,
Wada
T
,
Yamashita
T
,
Sawada
N
(
2004
).
Thr203 of claudin-1, a putative phosphorylation site for MAP kinase, is required to promote the barrier function of tight junctions
.
Exp Cell Res
.
295
,
36
47
.
Furuse
M
,
Hata
M
,
Furuse
K
,
Yoshida
Y
,
Haratake
A
,
Sugitani
Y
,
Noda
T
,
Kubo
A
,
Tsukita
S
(
2002
).
Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice
.
J Cell Biol
.
156
,
1099
1111
.
Gibson
A
,
Carney
S
,
Wales
JK
(
2006
).
Growth and the premature baby
.
Horm. Res
.
65
Suppl. 3
,
75
81
.
Gonzalez-Mariscal
L
,
Betanzos
A
,
Nava
P
,
Jaramillo
BE
(
2003
).
Tight junction proteins
.
Prog Biophys Mol Biol
.
81
,
1
44
.
Hachem
JP
,
Behne
M
,
Aronchik
I
,
Demerjian
M
,
Feingold
KR
,
Elias
PM
,
Mauro
TM
(
2005
).
Extracellular pH Controls NHE1 expression in epidermis and keratinocytes: implications for barrier repair
.
J Invest Dermatol
.
125
,
790
797
.
Hamazaki
Y
,
Itoh
M
,
Sasaki
H
,
Furuse
M
,
Tsukita
S
(
2002
).
Multi-PDZ domain protein 1 (MUPP1) is concentrated at tight junctions through its possible interaction with claudin-1 and junctional adhesion molecule
.
J Biol Chem
.
277
,
455
461
.
Hanley
K
,
Jiang
Y
,
Elias
PM
,
Feingold
KR
,
Williams
ML
(
1997
).
Acceleration of barrier ontogenesis in vitro through air exposure
.
Pediatr Res
.
41
,
293
299
.
Hara-Chikuma
M
,
Verkman
AS
(
2008
).
Roles of aquaporin-3 in the epidermis
.
J Invest Dermatol
.
128
,
2145
2151
.
Hardman
MJ
,
Sisi
P
,
Banbury
DN
,
Byrne
C
(
1998
).
Patterned acquisition of skin barrier function during development
.
Development
125
,
1541
1552
.
Hardman
MJ
,
Moore
L
,
Ferguson
MW
,
Byrne
C
(
1999
).
Barrier formation in the human fetus is patterned
.
J Invest Dermatol
.
113
,
1106
1113
.
Hobbs
RM
,
Silva-Vargas
V
,
Groves
R
,
Watt
FM
(
2004
).
Expression of activated MEK1 in differentiating epidermal cells is sufficient to generate hyperproliferative and inflammatory skin lesions
.
J Invest Dermatol
.
123
,
503
515
.
Hohl
D
,
Mehrel
T
,
Lichti
U
,
Turner
ML
,
Roop
DR
,
Steinert
PM
(
1991
).
Characterization of human loricrin. Structure and function of a new class of epidermal cell envelope proteins
.
J Biol Chem
.
266
,
6626
6636
.
Ikari
A
,
Ito
M
,
Okude
C
,
Sawada
H
,
Harada
H
,
Degawa
M
,
Sakai
H
,
Takahashi
T
,
Sugatani
J
,
Miwa
M
(
2008
).
Claudin-16 is directly phosphorylated by protein kinase A independently of a vasodilator-stimulated phosphoprotein-mediated pathway
.
J Cell Physiol
.
214
,
221
229
.
Itoh
M
,
Furuse
M
,
Morita
K
,
Kubota
K
,
Saitou
M
,
Tsukita
S
(
1999
).
Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins
.
J Cell Biol
.
147
,
1351
1363
.
Katoh
M
(
2003
).
CLDN23 gene, frequently down-regulated in intestinal-type gastric cancer, is a novel member of CLAUDIN gene family
.
Int J Mol Med
.
11
,
683
669
.
Komuves
LG
,
Hanley
K
,
Jiang
Y
,
Katagiri
C
,
Elias
PM
,
Williams
ML
,
Feingold
KR
(
1999
).
Induction of selected lipid metabolic enzymes and differentiation-linked structural proteins by air exposure in fetal rat skin explants
.
J Invest Dermatol
.
112
,
303
309
.
Krause
G
,
Winkler
L
,
Mueller
SL
,
Haseloff
RF
,
Piontek
J
,
Blasig
IE
(
2008
).
Structure and function of claudins
.
Biochim Biophys Acta
1778
,
631
645
.
Lang
CT
,
Iams
JD
(
2009
).
Goals and strategies for prevention of preterm birth: an obstetric perspective
.
Pediatr Clin North Am
.
56
,
537
563
.
Langbein
L
,
Grund
C
,
Kuhn
C
,
Praetzel
S
,
Kartenbeck
J
,
Brandner
JM
,
Moll
I
,
Franke
WW
(
2002
).
Tight junctions and compositionally related junctional structures in mammalian stratified epithelia and cell cultures derived therefrom
.
Eur J Cell Biol
.
81
,
419
435
.
Langbein
L
,
Pape
UF
,
Grund
C
,
Kuhn
C
,
Praetzel
S
,
Moll
I
,
Moll
R
,
Franke
WW
(
2003
).
Tight junction-related structures in the absence of a lumen: occludin, claudins and tight junction plaque proteins in densely packed cell formations of stratified epithelia and squamous cell carcinomas
.
Eur J Cell Biol
.
82
,
385
400
.
Leigh
IM
,
Navsaria
H
,
Purkis
PE
,
McKay
IA
,
Bowden
PE
,
Riddle
PN
(
1995
).
Keratins (K16 and K17) as markers of keratinocyte hyperproliferation in psoriasis in vivo and in vitro
.
Br J Dermatol
.
133
,
501
511
.
Lemmers
C
,
Medina
E
,
Delgrossi
MH
,
Michel
D
,
Arsanto
JP
,
Le Bivic
A
(
2002
).
hINADl/PATJ, a homolog of discs lost, interacts with crumbs and localizes to tight junctions in human epithelial cells
.
J Biol Chem
.
277
,
25408
25415
.
Lipschutz
JH
,
Li
S
,
Arisco
A
,
Balkovetz
DF
(
2005
).
Extracellular signal-regulated kinases 1/2 control claudin-2 expression in Madin-Darby canine kidney strain I and II cells
.
J Biol Chem
.
280
,
3780
3788
.
Mack
JA
,
Anand
S
,
Maytin
EV
(
2005
).
Proliferation and cornification during development of the mammalian epidermis
.
Birth Defects Res C Embryo Today
75
,
314
329
.
McGowan
K
,
Coulombe
PA
(
1998a
).
The wound repair-associated keratins 6, 16, and 17. Insights into the role of intermediate filaments in specifying keratinocyte cytoarchitecture
.
Subcell Biochem
.
31
,
173
204
.
McGowan
KM
,
Coulombe
PA
(
1998b
).
Onset of keratin 17 expression coincides with the definition of major epithelial lineages during skin development
.
J Cell Biol
.
143
,
469
486
.
McIntire
DD
,
Leveno
KJ
(
2008
).
Neonatal mortality and morbidity rates in late preterm births compared with births at term
.
Obstet Gynecol
.
111
,
35
41
.
Pilling
EL
,
Elder
CJ
,
Gibson
AT
(
2008
).
Growth patterns in the growth-retarded premature infant
.
Best Pract Res Clin Endocrinol Metab
.
22
,
447
462
.
Prowse
DM
,
Lee
D
,
Weiner
L
,
Jiang
N
,
Magro
CM
,
Baden
HP
,
Brissette
JL
(
1999
).
Ectopic expression of the nude gene induces hyperproliferation and defects in differentiation: implications for the self-renewal of cutaneous epithelia
.
Dev Biol
.
212
,
54
67
.
Rutter
N
(
1996
).
The immature skin
.
Eur. J. Pediatr
.
155
Suppl. 2
,
S18
S20
.
Rutter
N
(
2000
).
Clinical consequences of an immature barrier
.
Semin Neonatol
.
5
,
281
287
.
Saiman
L
(
2006
).
Strategies for prevention of nosocomial sepsis in the neonatal intensive care unit
.
Curr Opin Pediatr
.
18
,
101
106
.
Schluter
H
,
Moll
I
,
Wolburg
H
,
Franke
WW
(
2007
).
The different structures containing tight junction proteins in epidermal and other stratified epithelial cells, including squamous cell metaplasia
.
Eur J Cell Biol
.
86
,
645
655
.
Shapiro-Mendoza
CK
,
Tomashek
KM
,
Kotelchuck
M
,
Barfield
W
,
Weiss
J
,
Evans
S
(
2006
).
Risk factors for neonatal morbidity and mortality among “healthy,” late preterm newborns
.
Semin Perinatol
.
30
,
54
60
.
Shapiro-Mendoza
CK
,
Tomashek
KM
,
Kotelchuck
M
,
Barfield
W
,
Nannini
A
,
Weiss
J
,
Declercq
E
(
2008
).
Effect of late-preterm birth and maternal medical conditions on newborn morbidity risk
.
Pediatrics
121
,
e223
e232
.
Shwayder
T
,
Akland
T
(
2005
).
Neonatal skin barrier: structure, function, and disorders
.
Dermatol Ther
.
18
,
87
103
.
Simard
A
,
Di Pietro
E
,
Young
CR
,
Plaza
S
,
Ryan
AK
(
2006
).
Alterations in heart looping induced by overexpression of the tight junction protein Claudin-1 are dependent on its C-terminal cytoplasmic tail
.
Mech Dev
.
123
,
210
227
.
Soll
RF
(
2008
).
Heat loss prevention in neonates
.
J. Perinatol
.
28
Suppl. 1
,
S57
S59
.
Taupin
D
,
Podolsky
DK
(
1999
).
Mitogen-activated protein kinase activation regulates intestinal epithelial differentiation
.
Gastroenterology
116
,
1072
1080
.
Troy
TC
,
Turksen
K
(
2005
).
Commitment of embryonic stem cells to an epidermal cell fate and differentiation in vitro
.
Dev Dyn
.
232
,
293
300
.
Troy
TC
,
Turksen
K
(
2007
).
The targeted overexpression of a Claudin mutant in the epidermis of transgenic mice elicits striking epidermal and hair follicle abnormalities
.
Mol Biotechnol
.
36
,
166
174
.
Troy
TC
,
Cheung
RM
,
Turksen
K
(
2005a
).
Epidermal cell analysis by RT-PCR
.
Methods Mol Biol
.
289
,
121
126
.
Troy
TC
,
Rahbar
R
,
Arabzadeh
A
,
Cheung
RM
,
Turksen
K
(
2005b
).
Delayed epidermal permeability barrier formation and hair follicle aberrations in Inv-Cldn6 mice
.
Mech Dev
.
122
,
805
819
.
Troy
TC
,
Rahbar
R
,
Diker
B
,
Turksen
K
(
2005c
).
Immunolocalization in the epidermis
.
Methods Mol Biol
.
289
,
113
120
.
Troy
TC
,
Li
Y
,
O’Malley
L
,
Turksen
K
(
2007
).
The temporal and spatial expression of Claudins in epidermal development and the accelerated program of epidermal differentiation in K14-CaSR transgenic mice
.
Gene Expr Patterns
7
,
423
430
.
Turksen
K
,
Troy
TC
(
2002
).
Permeability barrier dysfunction in transgenic mice overexpressing claudin 6
.
Development
129
,
1775
1784
.
Turksen
K
,
Troy
TC
(
2003
).
Overexpression of the calcium sensing receptor accelerates epidermal differentiation and permeability barrier formation in vivo
.
Mech Dev
.
120
,
733
744
.
Turksen
K
,
Troy
TC
(
2004
).
Barriers built on claudins
.
J Cell Sci
.
117
,
2435
2447
.
Tyson
JE
,
Parikh
NA
,
Langer
J
,
Green
C
,
Higgins
RD
(
2008
).
Intensive care for extreme prematurity-moving beyond gestational age
.
N Engl J Med
.
358
,
1672
1681
.
Van Itallie
CM
,
Anderson
JM
(
2006
).
Claudins and epithelial paracellular transport
.
Annu Rev Physiol
.
68
,
403
429
.
Williams
ML
,
Hanley
K
,
Elias
PM
,
Feingold
KR
(
1998
).
Ontogeny of the epidermal permeability barrier
.
J Invest Dermatol Symp Proc
.
3
,
75
79
.
Wilson
DR
,
Maibach
HI
(
1980
).
Transepidermal water loss in vivo. Premature and term infants
.
Biol Neonate
37
,
180
185
.
Yeaney
NK
,
Murdoch
EM
,
Lees
CC
(
2009
).
The extremely premature neonate: anticipating and managing care
.
BMJ
338
,
b2325
.
Yu
XJ
,
Li
CY
,
Dai
HY
,
Cai
DX
,
Wang
KY
,
Xu
YH
,
Chen
LM
,
Zhou
CL
(
2007
).
Expression and localization of the activated mitogen-activated protein kinase in lesional psoriatic skin
.
Exp Mol Pathol
.
83
,
413
418
.

COMPETING INTERESTS

The authors declare no competing financial interests.