Increased retinoic acid levels through ablation of
Cyp26b1 determine the processes of embryonic skin
barrier formation and peridermal development
Junko Okano1, Ulrike Lichti2, Satoru Mamiya3, Maria Aronova4, Guofeng Zhang4, Stuart H. Yuspa2,
Hiroshi Hamada3, Yasuo Sakai5and Maria I. Morasso1,*
1Developmental Skin Biology Section, NIAMS, NIH, Bethesda, MD 20892, USA
2Laboratory of Cancer Biology and Genetics, NCI, NIH, Bethesda, MD 20892, USA
3Developmental Genetics Group, Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan
4Laboratory of Bioengineering and Physical Science, NIBIB, NIH, Bethesda, MD 20892, USA
5Department of Plastic Surgery, Osaka University School of Medicine, Osaka, 565-0871, Japan
*Author for correspondence (email@example.com)
Accepted 5 December 2011
Journal of Cell Science 125, 1827–1836
? 2012. Published by The Company of Biologists Ltd
The process by which the periderm transitions to stratified epidermis with the establishment of the skin barrier is unknown.
Understanding the cellular and molecular processes involved is crucial for the treatment of human pathologies, where abnormal skin
development and barrier dysfunction are associated with hypothermia and perinatal dehydration. For the first time, we demonstrate that
retinoic acid (RA) levels are important for periderm desquamation, embryonic skin differentiation and barrier formation. Although
excess exogenous RA has been known to have teratogenic effects, little is known about the consequences of elevated endogenous
retinoids in skin during embryogenesis. Absence of cytochrome P450, family 26, subfamily b, polypeptide 1 (Cyp26b1), a retinoic-acid-
degrading enzyme, results in aberrant epidermal differentiation and filaggrin expression, defective cornified envelopes and skin barrier
formation, in conjunction with peridermal retention. We show that these alterations are RA dependent because administration of
exogenous RA in vivo and to organotypic skin cultures phenocopy Cyp26b1–/–skin abnormalities. Furthermore, utilizing the Flaky tail
(Ft/Ft) mice, a mouse model for human ichthyosis, characterized by mutations in the filaggrin gene, we establish that proper
differentiation and barrier formation is a prerequisite for periderm sloughing. These results are important in understanding pathologies
associated with abnormal embryonic skin development and barrier dysfunction.
Key words: Retinoic acid, Cyp26b1, Periderm, Skin differentiation, Skin barrier formation, Filaggrin
Retinoic acid (RA) is the most biologically active retinoid, being
also a potent teratogen in rodent and human fetuses, with severe
effects in craniofacial and limb development (Lammer et al.,
1985; Ross et al., 2000). The endogenous level of RA is
determined by the balance of the activity between the enzymes
that synthesize RA (retinaldehyde dehydrogenates) and the
enzymes that degrade RA (CYP26s). RA mediates its activity
through the binding to RA receptors (RARs) (Niederreither and
Dolle ´, 2008; Ross et al., 2000).
Retinoids are well known to influence skin development;
however, contrasting results have been reported between in vivo
and in vitro results (Imakado et al., 1995; Lee et al., 2009; Saitou
et al., 1995; Yuspa et al., 1983; Yuspa and Harris, 1974), and the
specific role of RA during embryonic skin differentiation has not
been determined. Skin functions as a barrier to the physical
environment, preventing dehydration and invasion of pathogenic
microorganisms. During murine embryogenesis, the first barrier
is a transitional superficial layer, termed periderm, that emerges
at approximately embryonic day (E)8.5 (M’Boneko and Merker,
1988). The induction of the keratin-associated proteins (KAP)
complex genes is associated with peridermal development (Cui
et al., 2007), and coincides with the formation of a functional
barrier by the underlying developing stratified epidermis. It has
been proposed that when peridermal cells start to slough off at
differentiated epidermis replace it as a barrier (Hardman et al.,
1999; Segre, 2006). Keratinocytes in the epidermis undergo a
differentiation process that culminates with the formation of the
cornified layers. This process is associated with expression of
differentiation-specific proteins, such as filaggrin (FLG), loricrin
(LOR), S100 proteins, late cornified envelope (LCE) proteins and
the small proline-rich (SPRR) proteins, encoded by genes
clustered in the epidermal differentiation complex (Jackson
et al., 2005; Marshall et al., 2001; Mischke et al., 1996).
Cornified layers consist of corneocytes, which are terminally
differentiated keratinocytes characterized by the cornified
envelope (CE) (Segre, 2006).
Utilizing mouse models with embryonic deletion of cytochrome
P450, family 26, subfamily b, polypeptide 1 (Cyp26b1), which
encodesan RA-degrading enzyme, in vivo administration of RA to
pregnant mice and to organotypic skin cultures, we have
addressed, for the first time, the effects of RA in developing
mammalian skin. We demonstrate that alterations in the
Journal of Cell Science
desquamation of the peridermal layer are concomitant with RA-
dependent abnormal epidermal morphology and differentiation,
and decreased filaggrin expression, resulting in impairment of the
skin barrier formation. Our findings establish the requirement of a
functional barrier for peridermal sloughing, and are validated by
the analysis of the Flaky tail (Ft/Ft) mouse model (Fallon et al.,
2009; Presland et al., 2000; Scharschmidt et al., 2009). In this
model, characterized by filaggrin mutations and consequential
barrier defects, we have determined peridermal retention.
Our results establish a close relationship between stratum
corneum maturation, skin barrier formation, periderm retention
and how alterations in the interplay of these processes might lead
to consequential pathologies of the skin.
The absence of Cyp26b1 leads to aberrant skin
morphology and upregulation of RA-induced genes
Cyp26b1 expression begins in the mesenchyme surrounding hair
follicles at E14.5 and is strongly expressed in the dermis by
E18.5 (Abu-Abed et al., 2002). To evaluate the expression of
Cyp26b1 in skin, we performed real-time PCR and demonstrated
that it is present in both epidermal and dermal fractions of E17.5
skin (Fig. 1A). The expression of Cyp26b1 in the dermis was
five-fold higher than in epidermis. Cyp26a1 and Cyp26c1, other
members of the Cyp26 family, were undetectable in skin (data
not shown). Keratin 1 and vimentin mRNAs were used to
confirm purity of the epidermal and dermal samples, respectively
RA is a small diffusible molecule, and its concentration is
difficult to measure directly in tissues. Because alterations in RA
concentration correlate to Rarb expression, quantification of
Rarb has been used to determine levels of RA (Johannesson et al.,
2009; Noji et al., 1991). We examined Rarb expression and
determined that it was six-fold higher in Cyp26b1–/–than in wild-
type (WT) epidermis and 14-fold higher in the dermis (Fig. 1C).
Histological analysis of E16.5 Cyp26b1–/–epidermis showed
similar morphology to WT epidermis, with nucleated peridermal
cellsonthe surface (Fig. 1D,E).
morphological alterations were evident in Cyp26b1–/–epidermis
(Fig. 1F,G), and by E18.5, cornified layers were remarkably
reduced and hair follicle growth arrested at the germ stage
(Fig. 1H,I). To determine whether there were differences in
proliferation, we performed immunohistochemistry with anti-
Ki67, which showed an increase in proliferative cells in E18.5
basal layer (supplementary material Fig. S1).
Immunofluorescent staining with anti-caspase 3 antibody,
which was used to detect apoptotic cells, showed no major
embryogenesis (data not shown). Light microscopy of the
surface of Cyp26b1–/–skin showed numerous protrusions not
observed in WT skin (Fig. 1J,K).
Keratin 19 (K19), a keratin not normally expressed in stratified
epithelia, is induced by RA in epithelial cell lines and its
expression is dependent on the extracellular RA concentration
(Crowe, 1993). The altered endogenous RA levels linked to the
absence of Cyp26b1, strongly induced K19 in E18.5 Cyp26b1–/–
epidermis (Fig. 1L,M). Glycogen has been detected in the
epidermis of RA-treated chick embryonic skin culture using
periodic acid–Schiff (PAS) staining (Obinata et al., 1991). We
detected strong PAS staining in suprabasal layers of Cyp26b1–/–
skin but not in WT skin (supplementary material Fig. S2).
To establish whether the observed phenotype was attributable
to increased dermal RA levels, we investigated the consequences
of dermal-specific Cyp26b1 deletion. For this purpose, we
Fig. 1. Altered skin morphology and RA-induced genes in Cyp26b1–/–
developing epidermis. (A,B) Relative Cyp26b1 (A), and keratin 1 (K1) and
vimentin (Vim) expression (B) in E17.5 epidermis and dermis, determined by
real-time PCR. Values are means 6 s.d., relative to epidermis (set as 1).
Keratin 1 (K1) and vimentin (Vim) mRNAs were at undetected level in
dermis and epidermis, respectively. (C) Absence of Cyp26b1 leads to
significant Rarb upregulation in epidermis and dermis. Values are means 6
s.d., relative to WT epidermis (set as 1). (D–I) Histology of developing skin
revealed peridermal cells (arrows) in E16.5 WT and Cyp26b1–/–skin, but
altered epidermal thickness and lack of detectable cornified layers at E17.5
(G) and E18.5 in Cyp26b1–/–skin. (J,K) Gross appearance of E18.5 skin.
Cyp26b1–/–dorsal skin surface has protrusions. (L,M) Keratin 19 (brown) is
upregulated in Cyp26b1–/–epidermis. Epi, epidermis; Der, dermis. **P,0.01.
Scale bars; 50 mm (D–I,L,M); 0.2 mm (J,K).
Journal of Cell Science 125 (7)1828
Journal of Cell Science
Hoxb6Cre;Cyp26b1+/–. Cyp26b1f/+littermates were used as
control. Targeting vector strategy for the generation of Cyp26b1f/f
mice and the genotyping results for Hoxb6Cre;Cyp26b1f/–mice are
shown in Fig. 2A and described in the Materials and Methods.
Dermal-specific Hoxb6Cre recombination was confirmed at E14.5,
the embryonic stage that coincided with the onset of Cyp26b1
expression, by crosses with R26R reporter mice (Fig. 2B). The
recombination was still detectable at E18.5 (Fig. 2B). There was no
morphological difference between E18.5 control (Cyp26b1f/+) and
immunohistochemical analysis of keratin 5 (K5) and filaggrin
showed comparable expression patterns (Fig. 2C, bottom panels).
Skeletal preparations showed abnormal hindlimb development in
Hoxb6Cre;Cyp26b1f/–mice (supplementary material Fig. S3), a
phenotype that corroborated previously determined effects of RA
excess in limb development (Campbell et al., 2004; Dranse et al.,
2011; Yashiro et al., 2004). These results support that dermal
mice by crosses between
(Fig. 2C,top panels) and
deletion of Cyp26b1 is not a sufficient effector for the development
of the epidermal phenotype observed in Cyp26b1–/–skin.
Loss of Cyp26b1 in developing skin leads to decreased
filaggrin expression and defective skin barrier formation
To determine the molecular effectors and mechanisms involved
in the epidermal phenotype in Cyp26b1–/–skin, we performed
microarray analysis comparing E18.5 WT and Cyp26b1–/–skin.
We found upregulation of genes involved in CE assembly,
particularly genes of the Lce and Sprr families (Fig. 3A). Lce3a,
Lce3b, Lce3c and Lce3f were prominently upregulated, and Lce3c
has been recently shown to be upregulated in response to skin
barrier disruption (de Cid et al., 2009). The retinoic-acid-binding
proteins cellular retinoic acid binding protein II (CrabpII) and
Rarb were upregulated 13.6- and 7.54-fold, respectively.
Interestingly, when CRABPII was increased by RA treatment of
human keratinocytes it was associated with absence of terminal
differentiation (Siegenthaler et al., 1992). Lipid metabolism is
known to be an integral part of barrier formation (Elias, 2005).
LipK, LipM and Alox12b, which encode enzymes with essential
function in lipid metabolism, were upregulated in Cyp26b1–/–
skin (Fig. 3A). The most downregulated genes were keratins
associated with hair follicle morphogenesis, which is consistent
with our finding that hair follicle development arrested at germ
stage in Cyp26b1–/–skin (supplementary material Table S1).
Because expression of many genes involved in CE formation
was altered, we performed skin dye permeability assays and
transepidermal water loss (TEWL) measurements to determine
whether Cyp26b1–/–fetuses developed a functional skin barrier.
Normally, the skin barrier begins to form in the dorsal side of the
embryo at approximately E16, and is identified by absence of dye
penetration (Hardman et al., 1998). Dye penetration occurred
over the whole body in Cyp26b1–/–fetuses from E16.25 to E19,
indicating an impairment of skin barrier formation (Fig. 3B).
TEWL was significantly higher (P,0.01) in Cyp26b1–/–fetuses
at E18.5, demonstrating that the skin barrier was impaired
Transmission electron microscopy (TEM) of E18.5 mutant
epidermis showed that keratohyalin granules, generally seen in
the granular layer in WT skin (Fig. 3C, white arrows) were not
present in the Cyp26b1–/–granular layer. Cornified layers that
were hard to distinguish under the light microscope in the mutant
epidermis were observed by TEM. However, the thickness and
number of Cyp26b1–/–cornified layers was notably reduced, and
intact nuclei were seen underneath the stratum corneum (Fig. 3C,
The atypical cornified layer and disrupted barrier led us to
isolate CEsfrom E18.5
considerably fewer CEs and they had a fragile and irregular
morphology, and many contained nuclei (Fig. 3D; arrow, inset
shown at higher magnification). By contrast, CEs from WT skin
were mostly rigid and had polygonal morphology (Fig. 3D).
To analyze alterations in the epidermal differentiation process,
we examined the expression of differentiation proteins (Fig. 4A–
H). Immunohistochemistry of keratin 5 (K5), keratin 10 (K10),
involucrin and loricrin showed comparable strata expression
pattern in Cyp26b1–/–and WT epidermis (Fig. 4A–F). However,
filaggrin was expressed at lower levels with considerably fewer
and smaller filaggrin granules in the Cyp26b1–/–epidermis
(Fig. 4G,H). Western blot analysis revealed that profilaggrin was
processed to filaggrin similarly in E18.5 and postnatal 3 days WT
skin. There were
Fig. 2. Dermal-specific Cyp26b1 deletion is not sufficient for development
of skin phenotype. (A) Schematic representation of targeting vector and
generation of Cyp26b1f/fmice. H, HincII; N, NsiI; RV, EcoRV; S, SpeI.
(Right) Genotyping of E18.5 Hoxb6Cre;Cyp26b1f/–mice by PCR determined
Cyp26b1 deletion by Cre recombinase in dermis and hindlimb but not in
epidermis and brain. (B) X-gal staining in Hoxb6Cre;R26R revealed dermal-
specific Cre recombination in the skin. Dotted line indicates the border
between the epidermis and the dermis. (C) H&E histological and
immunohistological analysis showing no phenotypic difference between
E18.5 WT and Hoxb6Cre;Cyp26b1f/–epidermis. Fila, filaggrin (green); K5,
keratin 5 (red); DAPI (blue). Scale bars: 50 mm.
The effect of RA on developing skin1829
Journal of Cell Science
skin, and conversely, no filaggrin was detected in Cyp26b1–/–
skin (Fig. 4I). By contrast, loricrin was more abundant in
Cyp26b1–/–skin than in E18.5 and postnatal 3 days WT skin
(Fig. 4I). The downregulation of filaggrin expression was
corroborated at the RNA level by real-time PCR (Fig. 4J).
Altogether, our results demonstrate that the absence of
Cyp26b1 during embryogenesis is associated with altered
filaggrin expression accompanied by abnormal CE formation
and skin barrier defects.
Increased RA in epidermis affects peridermal development
We identified that the onset of the epidermal phenotype of
Cyp26b1–/–fetuses correlated with the timing of peridermal
desquamation. Histological analysis revealed that, whereas
Fig. 3. Cyp26b1–/–skin is defective in CE and skin barrier formation.
in E18.5 Cyp26b1–/–skin compared with WT skin. (B) Dye exclusion assay
revealed absence of functional barrier in Cyp26b1–/–fetuses. Transepidermal
water loss (TEWL) measurements showed increased water loss in E18.5
Cyp26b1–/–dorsal skin (**P,0.01). Values are means 6 s.d., relative to control
(set as 1). (C) Transmission electron micrographs of E18.5 dorsal skin revealed
lack of keratohyalin granules in Cyp26b1–/–skin. The number and thickness of
cornified layers (C1–8) was reduced in Cyp26b1–/–skin. (D) CEs isolated from
in shape and many retained nuclei (arrow, and inset at a higher magnification).
Scale bars: 200 nm (upper panels in C); 150 nm (lower panels in C).
Fig. 4. Altered skin differentiation with decreased filaggrin expression in
Cyp26b1–/–epidermis. (A–F) Keratin 5 (K5; red), keratin 10 (K10; green),
involucrin (Inv; green) and loricrin (Lori; green) were detected by
immunohistochemistry in E18.5 WT and Cyp26b1–/–skin. Pan-Krt; pan-
keratin (red). (G,H) Filaggrin (Fila; green) was reduced and distribution was
irregular on Cyp26b1–/–skin, as observed by immunohistochemistry. DAPI
staining is blue. (I) Western blot analysis showed that profilaggrin is
processed to filaggrin in E18.5 WT and in postnatal 3 days (P3) WT skin, but
no filaggrin is detected in Cyp26b1–/–skin. Increased loricrin (Lori) was
detected in E18.5 Cyp26b1–/–skin. (J) Significant filaggrin downregulation
was detected by real-time PCR on Cyp26b1–/–E16.5, E17.5 and E18.5 skin
samples. Values are means 6 s.d., relative to E16.5 WT skin (set as 1);
**P,0.01. Scale bars: 50 mm.
Journal of Cell Science 125 (7)1830
Journal of Cell Science
cornified layers were formed in E17.5 WT skin, peridermal cells
were still present in Cyp26b1–/–skin, with hardly detectable
cornified layers at this stage (Fig. 5A,B). Keratin 6 (K6), a
characterized peridermal marker (Mazzalupo and Coulombe,
2001), was observed on the surface of both WT and Cyp26b1–/–
E16.5 epidermis and was still robustly detected in E17.5
Cyp26b1–/–epidermis (Fig. 5C–F).
upregulated genes in mutant skin, Krtap13, 2310034C09Rik
respectively), were members of the KAP (keratin-associated
proteins) complex (Fig. 5G). KAP genes are clustered on mouse
chromosome 16 and have been shown to be markers of periderm
development (Fig. 5G) (Cui et al., 2007). During normal
embryogenesis, expression of Krtap13 is highest at E16.5 and
markedly decreases by E17.5, timings that coincide with
peridermal desquamation (Takaishi et al., 1998). In situ
hybridization analysis showed that Krtap13 was restricted to
the periderm in E16.5 Cyp26b1–/–skin, as in the WT, but that
substantial Krtap13 expression was still detected in the upper
layers of E17.5 Cyp26b1–/–epidermis (Fig. 5H–K). Real-time
2310057N15Rik were expressed and maintained at high levels
in Cyp26b1–/–skin from E16.5 onwards, whereas expression for
those genes was markedly decreased in E17.5 WT skin (Fig. 5L).
analysisdetermined that themosthighly
Peridermal-specific Krtap13 expression is directly linked
to increased RA levels
Next, we addressed the effect of increased in vivo RA levels on
the epidermal barrier formation and peridermal development, by
feeding RA through gavage to WT pregnant mice with embryos
at E15.4, E16.5 and E18.4 of gestation (Fig. 6A). Defective
barrier formation in RA-treated fetuses became evident by dye
penetration assay, and skin from E18.5 RA-treated fetuses had
fewer CEs that presented the characteristic fragile morphology of
immature CEs with retention of nuclei seen in Cyp26b1–/–fetuses
(Fig. 6B). Histologicalanalysis
substantial differences between E18.5 vehicle- and RA-treated
groups: the RA-treated skin phenocopied the Cyp26b1–/–skin,
with fewer cornified layers and protrusions on the skin surface
(Fig. 6C–F). There was also downregulation of filaggrin
expression in RA-treated E18.5 skin (Fig. 6G,H). This finding
was corroborated by real-time PCR analysis, which revealed
significant (P,0.01) downregulation of filaggrin expression in
RA-treated skin compared with vehicle (oil) alone-treated skin
(Fig. 6I). In addition, RA-treated skin showed expression of K6
(Fig. 6J,K) and upregulated Krtap13 expression, demonstrating
that Krtap13 responds to increased RA levels (Fig. 6L). Krtap13
RA-dependent induction decreased at E17.5, correlating with the
timing of RA administration (no RA administration between
E16.5 and E18.4).
Using another approach to address the effects of RA on skin
development, we performed studies on skin organotypic cultures
Fig. 5. Increased RA levels affect peridermal desquamation.
(A,B) Histological analysis revealed peridermal cells in E17.5 Cyp26b1–/–
skin (B, black arrows). (C–F) Keratin 6 (K6; brown), was detected in the
uppermost layer of E17.5 Cyp26b1–/–skin. Counterstaining was performed
with Hematoxylin (blue). (G) Top panel; microarray results of expression of
peridermal-associated genes in E18.5 Cyp26b1–/–skin. Bottom panel: KAP
cluster location on mouse chromosome 16. Highly upregulated KAP genes are
highlighted in red. (H–K) In situ hybridization (red grains) analysis showed
expression of Krtap13 in WT and Cyp26b1–/–peridermal layer at E16.5 (H,I),
with persistent expression in Cyp26b1–/–E17.5 (K) epidermis. Scale bars:
50 mm. (L) Real-time PCR showed marked increase in relative expression of
Krtap13, 2310034C09Rik and 2310057N15Rik in E17.5 Cyp26b1–/–skin.
Values are means 6 s.d.; the expression level of each gene is given relative to
that in E16.5 WT skin (set as 1); **P,0.01.
The effect of RA on developing skin1831
Journal of Cell Science
with back skin of E15.5 embryos. A functional skin barrier was
established 48 hours after initiation of the culture in the control
group (O’Shaughnessy et al., 2007), revealed by the dye Lucifer
Yellow, in the upper layers of the stratum corneum (Fig. 7A).
However, dye diffused through all epidermal layers in RA-treated
organotypic cultures and was present in the dermis and
hypodermis (Fig. 7B). Filaggrin expression was detected in
control skin but not in RA-treated skin (Fig. 7C,D). Real-time
PCR revealed that Krtap13 was upregulated 15.7-fold in RA-
treated skin explants compared with control skin explants, with
Rarb expression also upregulated (35.5-fold; Fig. 7E). By
contrast, Krtap13 was not induced in RA-treated primary
mouse keratinocytes overexpressing Rarb with RA treatment
(data not shown). These results support our findings in
Cyp26b1–/–mice and in WT mice administered RA in vivo,
establishing a link between elevated levels of RA, altered
filaggrin expression, disruption of barrier formation and periderm
retention, evidenced by upregulated Krtap13 and sustained
expression of K6. These results also establish a direct
association between Krtap13 expression and the concomitant
downregulation of filaggrin expression.
Finally, we examined Ft/Ft fetuses, which are homozygous for
a frameshift mutation in the filaggrin gene (Fallon et al., 2009)
(Fig. 8A), to address the link between decreased filaggrin, barrier
defects and periderm desquamation. Ft/Ft mice skin barrier
defects after birth have been reported (Moniaga et al., 2010;
Scharschmidt et al., 2009), but we demonstrated barrier
dysfunction during Ft/Ft fetal development with a dye
exclusion assay (Fig. 8B). C57B6 (B6) mice were used as
control because Ft/Ft mice were backcrossed onto the B6
background (Moniaga et al., 2010; Scharschmidt et al., 2009)
(Fig. 8B). We corroborated that filaggrin was not detectable in
Ft/Ft skin, although it was present in suprabasal layer in B6 skin
Fig. 6. RA excess in WT embryos phenocopies the
epidermal defects of Cyp26b1–/–mouse embryos.
(A) Timetable for RA administration to WT pregnant mice
(red arrows) and for skin sample isolation (black
arrowheads). (B) Dye exclusion assay revealed lack of
barrier function in RA-treated fetuses collected at E16.8
and E18.5 (ii and iv). CEs isolated at E18.5 (iv) from RA-
treated fetuses were irregular in shape and retained nuclei
(arrow and inset at a higher magnification). (C–F) RA-
treated fetal skin phenocopied Cyp26b1–/–skin at E18.5
(iv) both at the histological level and in gross appearance.
(G,H) Immunohistochemical analysis showed that
filaggrin (Fila; green) expression was downregulated in
RA-treated skin (bottom panel). K5, keratin 5 (red).
(I) Downregulation of filaggrin expression observed in
E16.8, E17.5 and E18.5 Cyp26b1–/–skin and in RA-treated
embryos was also corroborated by real-time PCR. Values
are means 6 s.d. relative to that in E16.8 WT skin (set as
1). (J–K) K6 (brown) was positive in RA-treated skin.
Counterstaining was performed with Hematoxylin (blue).
(L) Real-time PCR showed persistent expression of
Krtap13 at E17.5 and significant Krtap13 upregulation 2
hours after RA administration at E18.4; **P,0.01. Scale
bars: 50 mm (C,D,G,H). 0.2 mm (E,F).
Fig. 7. RA excess in organotypic skin cultures phenocopies the epidermal
defects of Cyp26b1–/–mouse embryos. (A,B) Lucifer Yellow dye (LY;
green) assays applied onto skin explants showed penetration of the dye into
the hypodermis in RA-treated skin. (C,D) Filaggrin (red) was detected in 48-
hour control cultures but not in RA-treated skin explants. (E) Krtap13 and
Rarb were significantly upregulated in RA-treated skin. Values are means 6
s.d., relative to control skin (set as 1). **P,0.01. Scale bars: 50 mm.
Journal of Cell Science 125 (7) 1832
Journal of Cell Science
(Fig. 8C). Based on the results of our Cyp26b1–/–model, we
hypothesized that the barrier defects in Ft/Ft mice would be
accompanied by alterations in peridermal development. We
addressed this by assaying the expression of K6 and Krtap13 in
Ft/Ft mice, and determined that both of these markers were
upregulated at E17.75 (Fig. 8C,D). Altogether, these results show
a link between impaired barrier function and periderm retention.
We performed a comprehensive analysis of the effects of
high endogenous RA concentration during embryonic skin
development that leads to aberrant CE formation, with lack of
skin barrier function linked to altered periderm development
(Fig. 9), and we demonstrate that they are interrelated processes.
The absence of Cyp26b1 in developing skin leads to an
aberrant epidermal phenotype
Here we show that in developing Cyp26b1–/–skin barrier
formation is defective and cornified layers are markedly
The normal skin development requires controlled epidermal
stratification and CE formation. Abnormal CE assembly linked to
defective barrier acquisition is seen in mouse models with
targeted ablation of Arnt and loricrin, triple targeted deletion of
involucrin, periplakin and envoplakin, and transgenic Ets1
overexpression in the suprabasal layer (Geng et al., 2006; Koch
et al., 2000; Nagarajan et al., 2010; Sevilla et al., 2007).
However, the phenotype of the CEs in these knockout mouse
models is less severe than in Cyp26b1–/–mice, suggesting that
RA potentially regulates an encompassing group of effectors and
structural proteins required for CE assembly. Other genes, which
are part of the epidermal differentiation complex, that are also
involved in CE assembly are Lce and Sprr. These genes, in
particular Lce, have been recently shown to be upregulated in
response to skin barrier disruption (de Cid et al., 2009).
Furthermore, Bergboer and collaborators showed that normal
skin barrier function correlates with LCE groups 1, 2, 5 and 6,
whereas expression of LCE3 genes is linked to barrier repair after
injury or inflammation (Bergboer et al., 2011). These findings, in
conjunction with the specific and prominent upregulation of
expression of the Lce3 group in the Cyp26b1–/–skin, suggest a
mechanistic link between barrier disruption and inflammation in
this mouse model that correlate with the systematic effects of
increased RA levels. In particular, in dominant-negative Rar
mice, an RA-deficiency model, there is a loss of epidermal
barrier function attributed to disruption of the lipid layer
(Imakado et al., 1995). Altogether, the published results and
our present findings establish that specific temporal and spatial
RA levels are required for proper skin differentiation during
We also show that absence of Cyp26b1 and exogenous RA
administration correlate with decreased filaggrin expression.
Filaggrin is produced during late epidermal differentiation as a
large polypeptide precursor that is cleaved, as keratinocytes
terminally differentiate, to produce corneocytes in the stratum
corneum. RA is known to regulate the conversion of profilaggrin
to filaggrin in human keratinocytes (Asselineau et al., 1990), and
the human filaggrin promoter has retinoic acid response elements
(RAREs), which function to suppress the promoter activity in the
presence of RA (Presland et al., 2001; Sandilands et al., 2009).
These results and our findings showing decreased filaggrin
expression in Cyp26b1–/–skin and RA-treated fetuses, strengthen
the connection between excess RA inhibiting profilaggrin, and
therefore filaggrin expression, which is also supported by our
results on RA-treated organotypic cultures. This phenomenon is
specific to filaggrin because loricrin, another established late
differentiation marker, was upregulated in Cyp26b1–/–skin.
Increased loricrin is also observed in Ft/Ft mice, which have
analogous mutations to human filaggrin mutations (Fallon et al.,
2009; Presland et al., 2000), and which have been reported to
Fig. 8. Loss of filaggrin causes skin barrier defects accompanied by
peridermal retention. (A) Genotyping as described by Fallon et al. (Fallon
et al., 2009) showed a 678 bp band for C57BL/6 (B6) and a 559 bp band for
Ft/Ft mice. M, molecular mass marker; bp, base pair. (B) Dye assay revealed
a skin barrier defect in Ft/Ft fetuses. (C) In Ft/Ft E17.75 fetal skin, filaggrin
protein was not detected (top panels). However, the peridermal marker K6
was detected in the superficial layer (bottom panels). (D) Krtap13 was
significantly upregulated in E17.75 Ft/Ft fetal skin. Values are means 6 s.d.
relative to that in B6 skin (set as 1); **P,0.01.
Fig. 9. Schematic summary of the findings of this study.
The effect of RA on developing skin1833
Journal of Cell Science
have skin barrier defects (Moniaga et al., 2010; Scharschmidt
et al., 2009). Loss-of-function mutations in the filaggrin gene
have been identified as a cause for ichthyosis vulgaris and atopic
dermatitis (Palmer et al., 2006; Sandilands et al., 2009). These
published results and the results presented here, validate the
effect of RA on the linked processes of filaggrin expression and
development of a functional skin barrier.
The role of RA in peridermal development and skin
The mechanism(s) through which periderm transitions to the
stratified epidermis or the pathways and effectors that determine
the timing of periderm sloughing have not been established.
However, recent reports have demonstrated the correlation
between peridermal phenotype and the expression of genes
clustered in the KAP complex in mice (Cui et al., 2007). The
genes in this complex have been very well characterized with
respect to genomic arrangement and expression patterns in
human hair (Rogers et al., 2002). Of these clustered genes
Krtap13, 2310034C09Rik and 2310057N15Rik, which are
specifically expressed in mouse periderm, were the most
upregulated genes in Cyp26b1–/–skin.
desquamation of periderm by E15.5, and Krtap13, as well as
other genes in the KAP complex, are not detected at E16.5 (Cui
et al., 2007). Therefore, the temporal and spatial expression of
Krtap13 at E16.5 appears to play a key role in peridermal
development, and peridermal retention and/or sloughing are
influenced by the stage of the epidermal differentiation process.
Although, using in vivo and in vitro models, we demonstrate
that KAP genes respond to RA, the molecular mechanism
connecting the expression of KAP-complex genes and RA
signaling is at present unknown. We searched a 90 kb genomic
region harboring the KAP complex and found several RARE-like
and half-RARE sequences, and the mechanistic significance of
these sites will be the focus of future research. Although RARE
consensus sequences are regarded as direct repeats (DR) of 59-
RGKTCA-39 separated by one, two or five nucleotides (Umesono
et al., 1991), chromatin immunoprecipitation analysis on mouse
embryonic fibroblasts recently determined that the majority of
RA target genes contain anomalously spaced DRs rather than
consensus DRs, suggesting more complexity in RA signaling
(Delacroix et al., 2010). Taking these facts into consideration, it
remains a challenge to determine the functionality of these
regulatory elements in the KAP complex. That both exogenous
RA and elevated endogenous RA, caused by the absence of
Cyp26b1, induced KAP complex genes supports the idea that
upregulated expression of these genes is a direct response to RA
and that regulation of the KAP complex is intrinsic to periderm
It has been proposed that the development of differentiated
stratified epidermis occurs concomitantly with the desquamation
of the overlying peridermal cells (Segre, 2006). However, there is
no evidence to support that periderm is required for epidermal
stratification or alternatively that proper barrier formation by the
underlying stratifying epidermis
desquamation of the periderm.
We demonstrate that in Ft/Ft fetuses, which have a
homozygous frameshift filaggrin mutation (Fallon et al., 2009)
(Scharschmidt et al., 2009), have periderm retention, as
mice, thereis premature
is necessaryfor timely
assessed by K6 and Krtap13 upregulation. These results, in
conjunction with our RA-dependent findings, strongly suggest
that formation of a functional barrier in the developing epidermis
is a requisite for a proper temporal peridermal sloughing during
embryogenesis. Further studies will be designed to analyze the
relationship between periderm desquamation, barrier dysfunction
and pathologies of the skin such as ichthyosis and ichthyosis-like
conditions and whether RA metabolism during fetal development
can contribute to the pathology.
Materials and Methods
Cyp26b1+/–mice were described previously (Yashiro et al., 2004). Mice carrying
the floxed Cyp26b1 allele (Cyp26b1f/f) were generated as described in Fig. 2 and
below. The Hoxb6Cre mice were kindly provided by Susan Mackem (Lowe et al.,
2000). R26R reporter mice were purchased from Jackson Laboratories (Soriano,
1999). Flaky tail (Ft) mice were kindly provided by John Sundberg (Presland et al.,
2000). Embryonic skin samples were photographed using an Olympus SZX9
microscope with a SPOT Pursuit USB Camera (SPOT Imaging Solutions).
Targeting vector map and the strategy of generating Cyp26b1flox/flox mice
The targeting vector consisted of a subcloned 22 kb SpeI fragment with one loxP
site inserted at the NsiI site of the intron 4 and an FRT-neo-FRT-loxP cassette
inserted at the HincII site in the 39 untranslated region of Cyp26b1 in a pMCI-
DTpA plasmid. All the DNA fragments for generating the vector were obtained as
previously described (Uehara et al., 2009; Yashiro et al., 2004). The targeting
vector was linearized with SalI before introduction into R1 ES cells by
electroporation. Homologous recombination of a neo allele was confirmed by
Southern blotting. Genomic DNA was digested with EcoRV, and the resulting
fragments were hybridized with the 39 probe indicating the 27.5 kb band from the
WT allele and the 14 kb one from a neo allele (data not shown). To create a flox
allele, a PGK-neo cassette was removed by crossing CAG-FLPe Deleter mice
(Kanki et al., 2006).
The genotype of a flox and/or a null allele was determined by polymerase chain
reaction (PCR) analysis of genomic DNA with three primers: P1, 59-AAG-
TACACCTGGCAGACATG-39; P2, 59-CCTGTCCCATATTTATTCACTGAC-
39; P3, 59-CTCCTCTTAAAGCTTCTCTA-39.
P2 and P3 will amplify a 400 bp product from a flox allele. P1 and P3 will
amplify a 500 bp product from a null allele but not a 4 kb product from a flox allele
by the following cycling conditions: 95˚C for 2 minutes, 38 cycles of 95˚C for
30 seconds, 58˚C for 50 seconds and 72˚C for 30 seconds, followed by 72˚C for
8 minutes. All reactions included 1.5 mM MgCl2, 0.2 mM dNTPs, 0.3 mM P1,
P2 and P3.
Skin was removed from E18.5 fetuses and fixed in ethanol for 3 days and then the
solution was replaced with acetone. Samples were incubated in 0.02% Alcian Blue,
0.01% Alizarin Red (Sigma) in 75% ethanol at 37˚C for 5 days, and then in 1%
potassium hydroxide for 2 days.
Histology, immunohistochemistry and western blot analysis
For histological analysis, embryos or embryonic skin samples were fixed in 4%
paraformaldehyde, embedded in paraffin and sectioned (10 mm) for staining with
Hematoxylin and Eosin or Periodic acid–Schiff (PAS) reagent. PAS staining was
performed according to the method of Obinata et al. (Obinata et al., 1991).
The antibodies used for western blotting and immunohistochemistry are
described in supplementary material Table S2. For immunofluorescence, the
sections were incubated with primary antibodies overnight at 4˚C and then with
fluorescent secondary antibodies. Sections were mounted with DAPI and
immunohistochemistry, the sections were incubated with primary antibodies
using the Vectastain ABC kit (Vector Laboratories) using diaminobenzidine as the
enzyme substrate. Mouse-on-Mouse (MOM) detection kit and antigen unmasking
solutions (Vector Laboratories) were used if applicable. Images were acquired
using a Zeiss axio scope a1 with a AxioCam MRc camera (Zeiss). Western blot
procedures were as described by Hwang et al. (Hwang et al., 2011). The blots were
probed with primary antibodies and HRP-conjugated secondary antibodies. ECL
(Amersham Pharmacia Biotech) reagent was used for detection.
X-gal staining and in situ hybridization
X-gal staining was performed with 1 mg/ml X-gal on frozen sections (30 mm).
Radioactive in situ hybridization on paraffin sections was carried out according
to Morasso et al. (Morasso et al., 2010). Krtap13 (NM010671) DNA probe was
purchased from Thermo Scientific.
Journal of Cell Science 125 (7)1834
Journal of Cell Science
Transmission electron microscopy (TEM)
Ultrathin sections were counter-stained with uranyl acetate and lead citrate and
examined with a Tecnai TF30 transmission electron microscope.
Barrier function assay and extraction of cornified envelopes
Dye penetration assays were performed as previously described (Hardman et al.,
(Courage+Khazaka, Ko ¨ln, Germany). Unpaired two-tailed Student’s t-test was
used to assess significance of the data. CEs were prepared according to Morasso
et al. (Morasso et al., 1996).
was measuredusinga Tewameter
RNA isolation, real-time PCR and microarray
Fetal skin, epidermis and dermis were collected and kept in Trizol (Invitrogen)
until RNA isolation using an RNeasy kit (Qiagen). Real-time PCR was performed
in duplicate using the iQTM SYBR Green Supermix (Bio-Rad). The primer
sequences for real-time PCR are summarized in supplementary material Table S3.
Individual gene expression was normalized against the RPLP0 housekeeping gene.
Two-tailed Student’s t-test was used to assess significance of the data. Microarray
analysis, including data processing, was performed by the National Institutes of
Health NIDDK Genomics Core Facility (Chattopadhyay et al., 2009) on
independent samples of E18.5 WT and Cyp26b1–/–embryos (n53 each).
RA treatment in pregnant mice
All-trans RA (Sigma) was administered to pregnant CD-1 mice (50 mg/kg of body
weight) in corn oil by oral gavage (Okano et al., 2007). Control pregnant mice
received corn oil only.
Organotypic skin culture was performed as previously described (Kashiwagi et al.,
1997; O’Shaughnessy et al., 2007). RA (Sigma) was added at 2.5 mM in DMSO;
the control group was administered DMSO only. 30 ml of 1 mM Lucifer Yellow
(Sigma) was applied to skin samples and incubated at 37˚C for 1 hour before
fixation with 4% paraformaldehyde.
We thank J. Sundberg for providing the Flaky tail mice; M. Uehara
for helpful discussions; C. Levy for technical assistance; K. Zaal of
the NIAMS Light Imaging Core Facility; R. Leapman of the NIBIB;
J. Segre for the loan of the Tewameter.
This study was supported by an Intramural Research Program of the
National Institute of Arthritis and Musculoskeletal and Skin
Diseases, National Institutes of Health. Deposited in PMC for
release after 12 months.
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