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Keywords:

  • epidermis;
  • morphogenesis;
  • differentiation;
  • coordinate expression;
  • dermokine;
  • keratinocyte differentiation-associated protein;
  • suprabasin;
  • Krt77

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The mammalian epidermis is the first line of defense against external environmental challenges including dehydration. The epidermis undergoes a highly intricate developmental program in utero, transforming from a simple to a complex stratified epithelium. During this process of stratification and differentiation, epidermal keratinocytes express a defined set of structural proteins, mainly keratins, whose expression is controlled by largely unknown mechanisms. In order to identify novel factors contributing to epidermal morphogenesis, we performed a global transcriptional analysis of the developing mouse epidermis after separating it from the underlying dermis (E12.5–E15.5). Unexpectedly, the recently identified genes encoding secreted peptides dermokine (Dmkn), keratinocyte differentiation-associated protein (krtdap), and suprabasin (Sbsn) as well as a largely uncharacterized embryonic keratin (Krt77), were among the most highly differentially expressed genes. The three genes encoding the secreted proteins form a cluster in an ∼40-Kb locus on human chromosome 19 and the syntenic region on mouse chromosome 7 known as the stratified epithelium secreted peptides complex (SSC). Using whole mount in situ hybridization, we show that these genes show a coordinated spatio-temporal expression pattern during epidermal morphogenesis. The expression of these genes initiates in the nasal epithelium and correlates with the initiation of other epidermal differentiation markers such as K1 and loricrin (Byrne et al. [1994] Development 120:2369–2383), as well as the initiation of barrier formation. Our observations reveal a coordinated mode of expression of the SSC genes as well as the correlation of their initiation in the nasal epithelium with the initiation of barrier formation at this site. Developmental Dynamics 236:913–921, 2007. © 2007 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Epidermal stratification and differentiation during mammalian development are critical for the survival of the animal in the postnatal terrestrial environment (Sengel,1976; Byrne et al.,2003; Segre,2003). In the mouse embryo, the commitment to the transformation of the simple epithelium into a stratified complex epidermis starts as early as embryonic day 7.5 (E7.5) and is completed by E17–E18.5, when the full epidermal barrier is established (Byrne et al.,2003; Koster and Roop,2004; Laurikkala et al.,2006). During this transformation, the forming epidermis expresses the cytokeratin pair 5 and 14 (K5/14) and down-regulates cytokeratins 8 and 18 (K8/18), markers of single-layered simple epithelia (Koster and Roop,2004; and this work). The expression of the transcription factor p63 (TAp63 in particular) precedes that of K14 and regulates its expression. Indeed, p63 is both a master regulator and among the earliest signs of the commitment to epidermal stratification and differentiation (Koster et al.,2004; Koster and Roop,2004; Laurikkala et al.,2006).

The first asymmetric vertical cell divisions in the epidermis give rise to the periderm (∼E9.5). This relatively simple structure is believed to transiently protect the embryo from the amniotic fluid until the functional barrier forms (Byrne et al.,2003). However, it is not until around E14.5–E15.5 that the first morphological signs of a three-tiered stratification and differentiation program are evident (Fig. 1) (Byrne et al.,1994). In this respect, the differentiated keratinocytes of the spinous layer of the epidermis appear above the proliferative basal keratinocytes and show cytokeratin 1 (K1) immunoreactivity, an established marker for the spinous layer in adult life (Histology in Fig. 1B,C; E14.5–E15.5) (Fuchs and Green,1980; Byrne et al.,1994; K1 immunofluorescence not shown). At E15.5 and later, the granular layer keratinocytes arise from the differentiating spinous layer and express the cornified envelope precursor protein loricrin (Histology in Fig. 1B,C; E14.5–E15.5) (Byrne et al,1994; Loricrin immunofluorescence not shown). The final stages of barrier formation begin with the terminal differentiation of the cornified keratinocytes to form the hydrophobic cornified layer (Hardman et al.,1998; Byrne et al.,2003).

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Figure 1. Histological samples of epidermal morphogenesis. A: From E12.5–E14, the epidermis is mainly composed of a single epithelial layer and an overlying periderm (e.g., E13.5 in A). B,C: E14.5–E15.5 morphological stratification and differentiation follows the earlier molecular changes (transcriptional program in this study). The hair follicle placodes and germs are also evident (C, E15.5 asterisk). D: A histological example of enzymatically and mechanically separated epidermis (top) and dermis (bottom) at E14.5 showing the two discrete compartments.

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In adult life, the same program of differentiation is reiterated with basal keratinocytes detaching from the basement membrane and ascending through the differentiation program until they are shed, a process that takes place every two weeks in mice (Sengel,1976). The differentiating epidermal keratinocytes express a large number of epidermal-specific differentiation genes, many of which are clustered on mouse chromosome 3 and the syntenic region on human chromosome 1q21 (Volz et al.,1993; Rothnagel et al.,1994; Mischke et al.,1996). This gene cluster is termed the epidermal differentiation complex (EDC) and harbors genes encoding three families of proteins (Volz et al.,1993; Mischke et al.,1996). The first family, characterized by short tandem peptide repeats in the central region, includes proteins such as loricrin, involucrin, and small proline-rich proteins (Sprr). The second family (the fused group) includes proteins such as profillagrin and trichohyalin with an EF-hand domain (Ca2+-binding domain) in their N-terminal region followed by tandem repeats. The third family is represented by S100 family members also with EF-hand domains (Volz et al.,1993; Rothnagel et al.,1994; Mischke et al.,1996).

Recently, a new stratified epithelium-related gene cluster termed the stratified epithelium secreted peptides complex (SSC) was identified (Matsui et al.,2004; Moffatt et al.,2004). This gene cluster of ∼40 Kb on human chromosome 19 and mouse chromosome 7 was discovered using subtractive hybridization screens, signal-trap assays, and high-throughput in situ hybridization (ISH), and harbors at least three genes encoding secreted peptides, dermokine (Dmkn), keratinocyte differentiation-associated protein (Krtdap), and suprabasin (Sbsn) (Oomizu et al.,2000; Park et al.,2002; Matsui et al.,2004; Moffatt et al.,2004; Tsuchida et al.,2004). Not only are these genes tightly clustered in the genome, but also they are transcribed in the same orientation and their mRNAs are expressed in the suprabasal layers of stratified epithelia concomitant with the appearance of the differentiated layers (Fig. 2D,E) (Oomizu et al.,2000; Park et al.,2002; Matsui et al.,2004; Moffatt et al.,2004; Tsuchida et al.,2004).

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Figure 2. Dmkn, Krtdap, and Sbsn transcripts are highly induced in the suprabasal epidermis during epidermal morphogenesis. A:Dmkn and Krtdap, which are, unlike Sbsn, represented on MOE430A chips, are very highly induced (compared to baseline levels at E12.5) at the mRNA level as early as E13.5 to reach more than 1,900-fold by E15.5. Please note the difference in scale on the Y-axis between this graph and subsequent ones. dpc, days post coitus. B: Semi-quantitative RT-PCR validations of the microarray results, in addition to Sbsn, are highly representative of the microarray outcome (the numbers below). β-actin is a loading control for this and subsequent RT-PCRs. C: qRT-PCR analyses of the three SSC genes also corroborate the microarray as well as the previous RT-PCR results. D–F: Sections of the whole-mount ISH at E15.5 (see Fig. 6) localize the mRNAs for all three genes exclusively to the suprabasal layers of the developing epidermis. Less intense signals were detected above the developing hair follicle placodes (asterisk in E). Dotted line designates the epidermal-dermal junction. Scale bars (D–F) = 40 μm.

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Figure 6. Whole mount ISH for Dmkn, Krtdap, and Sbsn mRNA reveals that these genes are coordinately expressed during epidermal morphogenesis. A–C:Dmkn mRNA is first detected in the nasal epithelium at E14 (shown E14.5, arrow in A and inset), then detected in larger areas of the skin as development progresses (B,C). The signals at the top of the head in B are artifacts due to the holes created in that region to prevent trapping of the probe in the brain during the ISH procedure. D–F:Krtdap mRNA is also first detected in the nasal epithelium at E14.5 (arrow in D, inset), and also at the proximal edge of the whisker pad (perhaps this embryo is ∼2 hr more advanced than that of Dmkn in A); and at E15.5 (E) and E16.5 (F) it is indistinguishable from Dmkn pattern of expression. G–I:Sbsn mRNA expression pattern is identical to those of Dmkn and Krtdap reflecting the coordination of their expression in a spatio-temporal fashion. J,K: Sections of the Dmkn ISH at E15 through the nostril region localizes the mRNA to the stratifying nasal epithelium (arrows). K represents the inset in J. Scale bars = (J) 160 μm, (K) 80 μm.

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In this study, we utilized microsurgical techniques to separate developing mouse skin at 12-hr intervals between E12.5–E15.5 into its epidermal and dermal compartments (e.g., in Fig. 1, E14.5 histology of separated epidermis and dermis). We performed microarray analyses using MOE430A chips from Affymetrix on the transcripts extracted from both compartments and compared their relative expression during morphogenesis. Surprisingly, we found that Dmkn and Krtdap had the two highest differentially expressed transcripts in the developing epidermis (more than 1,900-fold by E15.5), whereas Sbsn is not represented with a probe on this array. Moreover, Krt77, a largely uncharacterized mouse embryonic keratin gene (Hesse et al.,2001,2004; Rogers et al.,2005; Senshu et al.,2005), was also among the highly induced mRNAs during epidermal differentiation (827-fold by E15.5). Using whole mount ISH, we observed a coordinated spatio-temporal expression of the SSC cluster genes that is correlated with the initiation of barrier formation in the nasal epithelium later in epidermal development.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Transcriptional Profiling of Developing Mouse Epidermis

In order to identify novel factors involved in both epidermal and hair follicle morphogenesis, we used microdissection techniques coupled with microarray analyses on the developing epidermis (Fig. 1) (E12.5–E15.5, see Experimental Procedures section). We performed the microarray experiment with three or four biological replicates of epidermis at each time point. We used two commercially available software packages, GeneTraffic and GeneSpring, to normalize and analyze the differentially expressed genes between the time points. We also utilized these programs to perform unsupervised hierarchical clustering analyses of the biological replicates to assess correlation within and between the replicates. Both softwares gave comparable results for clustering and differential expression analyses. The biological replicates correlated closely with each other (under the same branch), and the lists of differentially expressed genes were highly comparable (data not shown). In addition, we fixed E12.5 as a developmental baseline, to which all the subsequent time points were compared. The lists of differentially expressed genes generated by our analyses became larger as the timepoints became more developmentally distant from the baseline (e.g., E12.5 vs. E13.5 compared to E12.5 vs. E15.5). This result reflects the gradual accumulation of transcriptional changes due to the addition of newly differentiated populations of keratinocytes as epidermal morphogenesis progresses. We categorized these lists of differentially expressed genes based on gene ontology.

Dmkn and Krtdap Are the Highest Differentially Expressed Genes on the Microarray

The two most highly differentially expressed transcripts at all time points studied were consistently those of Dmkn and Krtdap (Table 1, Fig. 2A). The transcript levels increased sharply as early as E13.5 (36- to 56-fold), continuing through E14–E14.5 (200 to more than 1,000-fold), and remain high at E15.5 (1,900- to more than 2,400-fold) (Table 1, Fig. 2A). Sbsn, which is located in the same SSC cluster as Dmkn and Krtdap, is not represented by a probe on MOE430A arrays used in our study. The microarray output for Dmkn and Krtdap, as well as Sbsn transcript expression, was assessed and/or validated by semi-quantitative RT-PCR (Fig. 2B) as well as quantitative real time PCR (Fig. 2C). Our results show that Sbsn mRNA also gradually increases, although it lags slightly behind those of Dmkn and Krtdap, during epidermal morphogenesis until E14.5 (Fig. 2B,C). Moreover, as reported elsewhere in several studies, Dmkn, Krtdap, and Sbsn mRNAs are localized exclusively to the suprabasal layers of the developing epidermis, where Sbsn transcripts are more prominent in the developing granular layer (E15.5, Fig. 2D,E) (Oomizu et al.,2000; Park et al.,2002; Matsui et al.,2004; Moffatt et al.,2004; Tsuchida et al.,2004).

Table 1. Dmkn and Krtdap mRNA Expression Kinetics in the Epidermis From E12.5–E15.5a
SSC genes12.513.51414.515.5
  • a

    0, Baseline levels or no change; numbers, fold of baseline (E12.5).

Dmkn0362201,1052,484
Krtdap0562376361,914

Genes Involved in Adhesion, Communication, Transcription, as Well as Enzymes, Are Among the Differentially Expressed Groups of Genes

Our lists of differentially expressed genes during epidermal morphogenesis included genes that are well known to play a role during epidermal keratinocyte differentiation (Table 2, Fig. 3A,B). For example, genes encoding cell–cell communication junctions and adhesion molecules such as desmocollins (Dsc1,3), S100 proteins, and aquaporins (Aqp3), showed prominent changes during epidermal development (Table 2, Fig. 3A). This was also true for genes encoding transcription factors that are known to regulate gene transcription in the epidermis (AP-1 factors, Cebpα/β, Klf4) (Table 2, Fig. 3A). In addition, genes encoding enzymes that cross-link the components of the cornified envelope (Tgm1) and other enzymes and their inhibitors (Serpins) were also differentially expressed (Table 2, Fig. 3B). Finally, our microarray lists uncovered some differentially regulated genes whose function in the developing epidermis is yet to be determined (Rdh12, Calb1) (Table 2, Fig. 3B). We chose a representative subset of these genes and validated their expression by semi-quantitative RT-PCR (Fig. 3C).

Table 2. Genes Involved in Communication/Adhesion and Transcription as Well as Enzymes and Others Are Differentially Expressed During Epidermal Morphogenesisa
 12.513.51414.515.5
  • a

    0, Baseline levels or no change; −, down-regulation; numbers, fold of baseline (E12.5).

Communication/adhesion     
 Dsc100011130
 Dsc3000410
 Pkp100249
 Jup00025
 Eppk00048
 Evp000313
 Ppl00026
 Cdh400−2−20
 Cdh1300−2−5−10
 Pcdh8000−9−14
 Pcdh10041000
 S100A180000177
 S100A6000433
 S100A140461527
 Aqp3032052162
Transcription factors     
 Klf40571337
 Klf500037
 Klf600034
 Cebpα004810
 Cebpβ034912
 Fos000010
 Jun00203
 Junb00004
 Jdp20−5−5−5−11
 Dlx507131639
 Ovol1003610
 Irx6000−7−43
Enzymes/Miscellaneous     
 Tgm1, K00035
 Tgm1, E000311
 Serpinb5002883
 Serpinb2000054
 Serpinb3c13122767
 Serpinb11000022
 Elov113747186
 Lgals300464559
 Lgals70021061
 Lgals1200005
 Lmna0361218
 Calm4000121,066
 Casp14000055
 p21000314
 Calb10−4−10−9−12
 Rdh120042588
 Vav3014212021
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Figure 3. Graphical representation of selected genes from Tables 2 and 3 show increasing kinetics of expression during epidermal morphogenesis. A: The expression of genes involved in adhesion (Dsc1), communication (Aqp3, S100A18), and transcription (Klf4, Cebpα) is highly up-regulated especially at E15.5 when the first signs of spinous differentiation are evident. B: Transcripts of enzymes for cross-linking the future cornified envelope (Tgm1, E, and K are two polypeptides), enzymes involved in retinoic acid biosynthesis (Rdh12), cytoskeletal regulators (Vav3), and nuclear intermediate filaments (Lmna) are induced during early epidermal differentiation. C: RT-PCR validation of selected genes (A,B) corroborate the levels of induction in the microarray (below).

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Table 3. The Structural Proteins That Are Hallmarks of Epidermal Keratinocytes Differentiation Are Differentially Expressed on the Microarraya
Structural proteins12.513.51414.515.5
  • a

    0, Baseline levels or no change; numbers, fold of baseline (E12.5); -, down-regulation.

Krt100037103
Krt10041157137
Lor0078640
Krt7700042827
Krt6a000026
Krt402950317
Krt80−5−7−60
Krt180−3−3−40
Krt70−2−2−40
Krt17000490
Krt190−2238
Sprr1B0004146
Sprr1A000029
Sprr2A01518100

Krt77 mRNA Is Highly Expressed Among Other Keratins in the Epidermis During Morphogenesis

Since keratins are the hallmarks of epidermal keratinocytes, they were also abundantly differentially expressed during epidermal morphogenesis (Table 3, Fig. 4A). Whereas the transcripts for the simple epithelial keratins such as Krt7,8 and 18 were down-regulated as early as E13.5, those of the spinous layer keratins such as the keratin pair Krt1 and 10 were concomitantly up-regulated (Table 3, Fig. 4A,B). Interestingly, Krt8 and Krt18 mRNA slightly increase again by E15.5 (Fig. 4B). In this respect, K8 expression is maintained in the periderm and is present in a discrete layer of the developing hair follicle by E16.5 (Fig. 4C, and data not shown), while K18 expression is extended to the basal layer as well as the developing hair follicles by E15.5 and decreases in expression again by E16.5 (Fig. 4C, data not shown). These expression data are in accordance with the changes in their transcript levels (Fig. 4A,B). These changes in keratin expression were accompanied by the up-regulation of loricrin mRNA, a granular layer marker and a cornified envelope component, which was increased up to 640-fold by E15.5 (Table 3, Fig. 4A). Moreover, the mucosal type keratin, Krt4, whose expression is undetectable in normal adult epidermis but is up-regulated under abnormal conditions (Virtanen et al.,2000), was also highly up-regulated during epidermal morphogenesis (Table 3, Fig. 4A,B).

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Figure 4. Well-established and novel structural proteins are highly expressed during epidermal morphogenesis. A: A graph showing the kinetics of mRNA expression of the well-studied keratin pair, Krt1 and 10, and the cornified envelope protein, loricrin, as well as the embryonic keratins, Krt6a and Krt77, during epidermal differentiation. Note the high expression levels of Krt77 mRNA compared to the others by E15.5. A: RT-PCR data reflect data from the microarray (below). C: Indirect immunofluorescence of K8 and K18 shows their persistence in the periderm (E15.5) and the appearance of K18 in the basal layer and hair follicle (see text). DAPI nuclear counterstain is in blue. The dotted line represents epidermal-dermal junction. Scale bars (C) = 60 μm.

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Notably, the most highly expressed keratin was a largely uncharacterized type II basic embryonic type keratin (Hesse et al.,2001,2004; Rogers et al.,2005; Senshu et al.,2005). This keratin is known by several different names in the literature including keratin complex 2, basic, gene 39 (Kb39), Krt 77, K1-embryonic, and mouse K1b (mK1b) (Hesse et al.,2001,2004; Rogers et al.,2005). Krt77 mRNA, according to the new keratin nomenclature (Schweizer et al.,2006), increased steeply at E14.5 (42-fold) and reached 827-fold at E15.5, higher than any other keratin or structural protein transcript on our lists (Table 3, Fig. 4A). Krt6a mRNA, another mouse embryonic type keratin gene, also increased sharply by E15.5 (26-fold) (Table 3, Fig. 4A). The differential expression of Krt77, Krt1, and Krt4 transcripts were validated by RT-PCR (Fig. 4B).

Krt77 mRNA Is Expressed in the Suprabasal Layers of the Developing Epidermis

The human orthologue of Krt77, KRT77, or K1b is expressed at both the mRNA and protein levels exclusively in the luminal cells of the sweat gland ducts of adult human skin (Langbein et al.,2005). Therefore, we wished to localize the expression of Krt77 mRNA during epidermal morphogenesis. Using whole mount ISH, we did not detect Krt77 mRNA at E14.5 (Fig. 5A). However, at E15 we detected Krt77 mRNA in the region surrounding the vibrissae follicles in the whisker pad, behind the pinnae of the ears, around a developing specialized hair follicle in the snout region, in the skin under the forelimbs, and in the developing palmar skin (Fig. 5B,E). Some of these initiation sites of expression were distinct from those reported for Krt1, especially the absence of signals from the nasal region and the vibrissae follicles hair canals, arguing against the cross-reactivity of the Krt77 probe with Krt1 mRNA (Byrne et al.,1994). By E15.5, Krt77 mRNA could be detected in a wider area of the lateral skin, limbs, and tail (Fig. 5C). By E16.5, Krt77 mRNA expression covered essentially the whole embryo including the entire palmar skin (Fig. 5D,F). Sections from these E16.5 embryos revealed that Krt77 mRNA was exclusively and prominently expressed in the developing suprabasal layers of the epidermis (Fig. 5G).

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Figure 5. Whole mount ISH of using a probe against Krt77 mRNA shows expression in the suprabasal layers of the differentiating epidermis during morphogenesis. A: No signal is detected at E14.5 (even in the nasal epithelium where Krt1 and loricrin mRNAs are expressed). B: At E15, Krt77 mRNA is detected in the whisker pad (also distinct from Krt1 mRNA expression (see Results section) (Byrne et al.,1994), behind the ear, in some specialized hair follicles around the eye, in the palmar elevations (E), and some skin areas (armpit region). C,D,F: By E15.5–E16.5, Krt77 transcripts are detected in wider areas of the skin until covering the entire body even the palmar skin (F). Plantar skin shows the same kinetics of expression but at later stages than palmar skin (not shown). G: Sections of whole mount ISH at E16.5 localize Krt77 mRNA exclusively to the developing suprabasal layers of the epidermis. Dotted line, epidermal-dermal junction. Scale bars (G) = 40 μm.

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Dmkn, Krtdap, and Sbsn Are Coordinately Expressed During Epidermal Morphogenesis

Since Dmkn and Krtdap had the two most highly expressed transcripts on our microarray, we were interested in determining their expression kinetics during epidermal morphogenesis. Therefore, we used whole mount ISH to analyze the expression of Dmkn, Krtdap, and Sbsn during development. Intriguingly, we detected the mRNA of all three genes in the nasal region as early as E14 and E14.5 (data not shown, and Fig. 6A,D,G, arrows and insets). Sections from this region revealed that the mRNA is localized to the suprabasal layers of the nasal epithelium within the nostril (e.g., Dmkn, Fig. 6J,K). A control sense probe for each of these transcripts showed no signal at this time point or subsequent ones (data not shown). By E15.5, the mRNA signals for all three genes covered almost the entire body excluding the scalp region, which is covered by E16.5 (Fig. 6B,C,E,F,H,I). It is noteworthy that the whole mount expression patterns of Dmkn, Krtdap, and Sbsn are virtually indistinguishable at all time points studied.

The Initiation of Expression of SSC Genes Correlates With Barrier Induction in the Nasal Epithelium

During our analysis of the expression patterns of Dmkn, Krtdap, and Sbsn, we noted that the initiating site of mRNA expression for these genes is the nasal epithelium (Figs. 6A,D,G,J,K, 7A). Moreover, by E15 the mRNA for these SSC genes is detected in the three developing hair follicles around the eye and near the mouth (Fig. 7A). Interestingly, these sites of expression correspond to the sites of initiation of barrier formation in the mouse embryo, which occurs ∼4 days later ∼E17.5 (Fig. 7B) (Hardman et al.,1998).

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Figure 7. The initiation sites of SSC gene expression coincide with the initiation sites of barrier formation. A: The initiation sites for SSC gene mRNA expression, e.g., Dmkn, are indicated in the specialized hair follicles around the eye and the nasal region in the inset (arrows). B: Using the well-established β-galactosidase barrier assay, these same initiation sites of SSC expression are also initiation sites of barrier formation and remain devoid of a blue signal (arrows).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The transformation of the mammalian surface ectoderm from a simple epithelium to a complex epidermis is not completely understood. There is evidence correlating heterogeneity in the underlying mesenchyme and gene expression in the ectoderm, suggesting that the mesenchyme could provide the earliest cues for the transformation of the epidermis as early as E8.5 (Sengel,1986). Early markers of commitment to this complex transformation, along with periderm formation, include the transcription factor TAp63 and its downstream target Krt14 (Koster and Roop,2004; Lechler and Fuchs,2005; Laurikkala et al.,2006). The subsequent stratification and differentiation events that lead to the formation of the spinous layer ∼E15.5 are other hallmarks during epidermal morphogenesis (Byrne et al.,1994; Koster and Roop,2004; Lechler and Fuchs,2005; Laurikkala et al.,2006).

In this study, we were interested in identifying novel factors expressed around the time of epidermal morphogenesis and differentiation, E12.5–E15.5, during the formation of the suprabasal layers of the mature epidermis. The clean separation of developing epidermal and mesenchymal compartments as early as E12.5 resulted in a reliable list of differentially expressed transcripts during epidermal morphogenesis (e.g., E14.5 in Fig. 1D). The abundance of well-studied proteins in the developing and mature epidermis, ranging from structural, enzymatic, communication, transcriptional, and others, provided strong internal controls, lending validity to the technical procedures as well as computational analysis methods (Tables 1–3; Figs. 2–4). The RT-PCR validations of selected genes also corroborate the differential gene expression obtained from the microarray analyses (Figs. 2B,C. 3C, 4B).

Intriguingly, the newly identified genes of the SSC cluster encoding secreted proteins, Dmkn and Krtdap, were the most highly differentially expressed genes as early as E13.5, and as high as 2,400-fold by E15.5 (Table 1, Fig. 2A–C). The third gene in the SSC cluster, Sbsn, which is not represented on the MOE430A array used in our study, was also gradually and highly induced by E15.5 as determined by RT-PCR and qRT-PCR (Fig. 2B,C). The expression of these genes in the epidermis as well as other stratified epithelia has been shown to be maintained in adult tissues (Oomizu et al.,2000; Park et al.,2002; Matsui et al.,2004; Moffatt et al.,2004; Tsuchida et al.,2004). Since these proteins are secreted and their mRNAs are expressed exclusively in the suprabasal layers of the developing epidermis (Fig. 2D–F), and that their expression levels surpass any other induced transcripts in the epidermis, are all indications that they play a significant role during early epidermal differentiation (Matsui et al.,2004; Moffatt et al.,2004). Applying the same line of reasoning to the embryonic keratin Krt77 (827-fold by E15.5 and also expressed in the suprabasal epidermis), this Krt1 homolog represents a novel marker for early epidermal differentiation. The difference in the initiation sites of expression of Krt77 (which is not expressed in the nasal epithelium) compared to Krt1 suggests a complementary role of Krt77 to that of Krt1 during epidermal morphogenesis (Fig. 5B).

The coordinated spatial and temporal expression patterns of the SSC genes are striking and remarkable (Fig. 6A–I). As mentioned earlier, these genes are clustered in a relatively small region of the genome (∼40 Kb on human chromosome 19 and mouse chromosome 7) and they are transcribed in the same orientation (Matsui et al.,2004; Moffatt et al.,2004). Collectively, these observations suggest an interesting mode of coordinated transcriptional regulation in the differentiating epidermis that is worthy of further investigation. We analyzed the upstream regions of each of the SSC genes as well as of Krt1, Krt10, and Lor focusing on the transcription factors that showed differential expression on our microarray (Table 2). We found a significant number of consensus binding sites for Cebpα, AP-1 factors (mainly c-Fos and c-Jun), and AP-2 factors (mainly AP-2α) within ∼1Kb of the upstream regions of these genes. Combinations of these factors have been shown to be essential for regulating the expression of Krt1, Krt10, Lor, and involucrin (Maytin et al.,1999; Zhu et al.,1999; Jang and Steinert,2002; Eckert et al.,2004). In this regard, in vitro experiments on keratinocytes (human or mouse) using differentiating agents such as calcium and phorbol esters (TPA) have shown that the induction of differentiation markers (including Krt1/10, Lor, Dmkn, Krtdap, Sbsn) operate through transcriptional regulators of the AP and Cebp families (Hennings et al.,1980; Maytin et al.,1999; Zhu et al.,1999; Jang and Steinert,2002; Eckert et al.,2004). We reproduced these findings for Dmkn and Sbsn using Calcium and TPA treatment of primary mouse epidermal keratinocytes (data not shown). Therefore, Cebpα, AP-1, and AP-2 factors are potential candidates for the regulation of expression of SSC genes, in addition to the aforementioned structural proteins.

Of particular interest is the initiation of mRNA expression of the SSC genes in the nasal epithelium (E13.5–E14), which is reminiscent of mRNA expression initiation sites for Krt1 and Lor as early as E13.5, indicating that the nasal epithelium is the earliest site in the epidermis that undergoes stratified differentiation (Figs. 6A,D,G,J,K, 7A) (Byrne et al.,1994). Not only is the nasal epithelium perhaps the earliest epidermal differentiation site, but it is also likely to be the earliest to acquire a barrier ∼4 days later during epidermal development (Fig. 7B) (Hardman et al.,1998). The expression of the late constituents of the epidermal cornified envelope was previously correlated with barrier initiation, albeit several days later in development than our study (Marshall et al.,2001). We believe that studying the early kinetics of differentiation of the nasal epithelium will shed more light on the links between stratification, differentiation, and barrier formation in the epidermis and perhaps the role of mesenchymal cues during these processes.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

Microarray Analysis

Dorsal skin was dissected from C57BL/6J embryos at 12-hr intervals starting at E12.5 (12.5 days post coitus, day of plug considered 0.5 days [d]; digit as well as whisker pad follicle formation were used as staging criteria) until E15.5 (refer to Fig. 1 for histology of selected stages), in DMEM with antibiotics and antimycotics (Invitrogen, Carlsbad, CA). The dissected skin was incubated in a 2:1 mixture of 2.5% trypsin (without EDTA) (Invitrogen) and filtered 8% pancreatin (Sigma-Aldrich, St Louis, MO) for 5 min at room temperature followed by 30–40 min at 4°C. The epidermis was carefully and cleanly separated from the dermis, and each tissue was resuspended and frozen in RLT buffer (Qiagen, Valencia, CA). Total RNA was isolated from the epidermal and dermal tissues using the RNeasy® Minikit according to the manufacturer's instructions (Qiagen). Triplicate to quadruplicate RNA samples (each biological sample often represented a single embryo) were amplified once and labeled for hybridization on microarray chips (MOE430A) using the Affymetrix reagents and protocols (Affymetrix Inc., Santa Clara, CA). The data output was normalized and analyzed using both GeneSpring GX 7.0 (Agilent Technologies Inc., Palo Alto, CA) and GeneTraffic™ (Iobion Informatics, La Jolla, CA) commercial software packages, which gave comparable results. The earliest time point, E12.5, was set as a baseline for comparison of subsequent stages. The P value cutoff was set to 0.05 and the significant fold difference was considered two-fold higher or lower than baseline.

Semi-Quantitative RT-PCR and Quantitative Real Time PCR

Reverse transcription was carried out using Oligo (dT) primer and SuperScript™ III (Invitrogen) according to the manufacturer's instructions. PCR was performed using PfuUltra™ Hotstart PCR Master Mix (Stratagene, La Jolla, CA) in a Peltier Thermal Cycler (MJ Research, Inc, Waltham, MA). The different cDNAs from each of the time points were equalized using β-actin primers as an internal control. Aliquots of each PCR reaction were taken at 25, 30, and 35 cycles, run on a 1% agarose gel, and photographed using a Kodak camera. Quantitative real time PCR (qRT-PCR) was performed on an ABI 7300 (Applied Biosystems, Foster City, CA). Primers were designed according to ABI guidelines and all reactions were performed using ABI Sybr Master Mix, 300-nM primers, 200 ng cDNA at the following consecutive steps: (1) 50°C for 2 min, (2) 95°C for 10 min, (3) 95°C for 15 s, (4) 60°C for 1 min, repeat steps 3 and 4 for 40 cycles. The samples were run in quadruplicate and normalized to an internal control (Tubulin) using the accompanying software. The primers used and aplicon sizes are provided in Table S1 (see Supplemental Table S1, which can be viewed at www.interscience.wiley.com/jpages/1058-8388/suppmat).

Whole Mount In Situ Hybridization

The templates for the in situ hybridization probes were amplified by PCR from the E15.5 epidermal cDNA stock (see RT-PCR section; the primers used and probe length are provided in Supplementary Table S1). The PCR products were cloned into pCRII dual promoter (T7 and SP6) vector (Invitrogen) and standard procedures were followed for the preparation of DIG-labeled cRNA (Roche Applied Science, Indianapolis, IN) antisense (AS) and control sense (S) probes. In situ hybridization was performed on different embryonic stages during epidermal morphogenesis as per detailed published protocols (Wilkinson,1998). The embryos were photographed using an HRC Axiocam fitted onto an SV Stemi stereomicroscope (Carl Zeiss, Thornwood, NY). The stained embryos were post-fixed in 4% PFA, embedded in Tissue-Tek® OCT compound (Fisher Scientific, Hampton, NH), sectioned, and mounted for histological photography.

Immunofluorescence Staining

Frozen sections of whole embryos were fixed in 4% PFA at ambient temperature followed by acetone at −20°C. The sections were washed in 1×PBS, blocked in 10% of the appropriate serum, and incubated with the primary antibody overnight at 4°C. After washing in 1×PBS, 594 Alexafluor® conjugated secondary antibodies (Molecular Probes, Invitrogen) were applied and the signal was visualized using an HRC Axiocam fitted onto an Axioplan2 fluorescence microscope (Carl Zeiss). Guinea pig antibodies against K8 (1:2,000) and K18 (1:1,000) were kindly provided by Dr. Lutz Langbein.

Barrier Assay and In Silico Promoter Analysis

The barrier assay monitors endogenous skin β-galactosidase activity. The X-Gal substrate can penetrate the skin only where the barrier has not formed yet, and therefore, a blue color develops. This assay was performed as described previously (Hardman et al.,1998). For the analysis of the upstream regions of SSC cluster genes as well as Krt1/10, we used the VISTA website for comparative genomics (http://genome.lbl.gov/vista/index.shtml) (Loots et al.,2002), in addition to the AliBaba2.1 tool on the Gene-Regulation website (http://www.gene-regulation.com/pub/programs/alibaba2/index.html?).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

We thank Dr. Yonghui Zhang from the Columbia Genome Center for providing advice on the microarray analysis softwares. We thank Mr. Ming Zhang for excellent technical assistance. We also thank all the members of the Christiano and Jahoda laboratories for helpful assistance and discussion. This work was supported by a grant from the Kirsch Foundation to A.M.C. C.A.B.J. is grateful to the BBSRC for funding (grant Number G18988).

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  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES
  9. Supporting Information

The Supplementary Material referred to in this article can be viewed at www.interscience.wiley.com/jpages/1058-8388/suppmat .

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