As the major proteins of adult keratinocytes, keratins provide biochemical markers for exploring mouse epidermal embryogenesis. Here, we used a modified method of whole-mount in situ hybridization to track skin-specific expression of endogenous keratin mRNAs throughout embryogenesis. To monitor transcriptional regulation, we coupled this with beta-galactosidase expression of a human epidermal keratin promoter-driven transgene. These studies have radically changed our perception of how the program of gene expression becomes established during epidermal development. Specifically, we have discovered that (1) basal keratin (K5 and K14) genes are first detected at E9.5 in a highly regional fashion, and surprisingly as early as the single layered ectodermal stage; (2) the early patterns do not correlate with morphogenesis per se, but rather with regional variations in the embryonic origin of underlying mesenchyme, supporting morphogenetic criteria that early inductive cues are mesenchymal; (3) epidermal keratin genes are expressed in periderm, supporting the notion that this layer arises from ectodermal stratification, even though it is simple epithelial-like in morphology and is subsequently sloughed during development; (4) later embryonic patterns of K5 and K14 gene expression parallel proliferative capacity and not stratification; and (5) K1 and K10 mRNAs are first detected as early as E13.5, and their patterns correlate with differentiation and not stratification. These patterns of epidermal gene expression led us to explore whether potential transcriptional regulators of these genes are expressed similarly. We show that AP2 (but not Sp1) cRNAs hybridize in a pattern similar to, but preceding that of basal keratin cRNAs. Finally, using gene expression in cultured cells, we demonstrate that AP2 has a strong inductive effect on basal keratin expression in a cellular environment that does not normally possess AP2 activity.

REFERENCES

REFERENCES
Angel
P.
,
Hattori
K.
,
Smeal
T.
,
Karin
M.
(
1988
)
The jun proto-oncogene is positively autoregulated by its product, jun/AP-1.
Cell
55
,
875
885
Banks-Schlegel
S. P.
(
1982
)
Keratin alterations during embryonic epidermal differentiation: a presage of adult epidermal maturation.
J. Cell Biol
93
,
551
559
Behrens
J.
,
Lowrick
O.
,
Klein-Hitpass
L.
,
Birchmeier
W.
(
1991
).
The E-cadherin promoter: Functional analysis of a G.C-rich region and an epithelial cell-specific palindromic regulatory element.
Proc. Natl. Acad. Sci. USA
88
,
11495
11499
Buettner
R.
,
Kannan
P.
,
Imhof
A.
,
Bauer
R.
,
Yim
S. O.
,
Glockshuber Van Dyke
R. M. W.
,
Tainsky
M. A.
(
1993
)
An alternatively spliced mRNA from the AP-2 gene encodes a negative regulator of transcriptional activation by AP-2.
Mol. Cell. Biol
13
,
4174
4185
Byrne
C.
,
Fuchs
E.
(
1993
)
Probing keratinocyte and differentiation specificity of the human K5 promoter in vitro and in transgenic animals.
Mol. Cell. Biol
13
,
3176
3190
Candia
A. F.
,
Hu
J.
,
Crosby
J.
,
Lalley
P. A.
,
Noden
D.
,
Nadeau
J. H.
,
Wright
C. V. E.
(
1992
)
Mox-1 and Mox-2 define a novel homeobox gene subfamily and are differentially expressed during early mesodermal patterning in mouse embryos.
Development
116
,
1123
1136
Chomczynski
P.
,
Sacchi
N.
(
1987
)
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Analyt. Biochem
162
,
156
159
Conlan
R. A.
,
Rossant
J.
(
1992
)
Exogenous retinoic acid rapidly induces anterior ectopic expression of murine HOX-2 genes in vitro.
Development
116
,
357
68
Couly
G. F.
,
Coltey
P. M.
,
Le Douarin
N. M.
(
1992
)
The developmental fate of the cephalic mesoderm in quail-chick chimeras.
Development
114
,
1
15
Dale
B. A.
,
Holbrook
K. A.
,
Kimball
J. R.
,
Hoff
M.
,
Sun
T.-T.
(
1985
)
Expression of epidermal keratins and filaggrin during human fetal skin development.
J. Cell Biol
101
,
1257
69
Dhouailly
D.
,
Rogers
G. E.
,
Sengel
P.
(
1978
)
The specification of feather and scale protein synthesis in epidermal-dermal recombinations.
Dev. Biol
65
,
58
68
Fisher
C.
,
Holbrook
K.
(
1987
)
Cell surface and cytoskeletal changes associated with epidermal stratification and differentiation in organ cultures of embryonic human skin.
Dev. Biol
119
,
231
241
Franke
W. W.
,
Schmid
E.
,
Schiller
D. L.
,
Winter
S.
,
Jarasch
E. D.
,
Moll
R.
,
Denn
H.
,
Jackson
B. W.
,
Illmensee
K.
(
1981
)
Differentiation-related patterns of expression of proteins of intermediate-size filaments in tissues and cultured cells.
Cold Spring Harbor Symp. Quant. Biol
46
,
431
453
Fuchs
E.
,
Green
H.
(
1980
)
Changes in keratin gene expression during terminal differentiation of the keratinocyte.
Cell
19
,
1033
1042
Fuchs
E.
,
Weber
K.
(
1994
)
Intermediate filaments: structure, dynamics, function, and disease.
Annu. Rev. Biochem
63
,
345
382
Greer
J.
,
Roop
D.
(
1991
)
Loricrin, a major keratinocyte cell envelope protein is expressed late in development.
J. Invest. Dermatol
96
,
553
–.
Hardy
M. H.
(
1992
)
The secret life of the hair follicle.
Trends Genet
8
,
159
66
Heid
H. W.
,
Werner
E.
,
Franke
W. W.
(
1986
)
The complement of native alpha-keratin polypeptides of hair-forming cells: a subset of eight polypeptides that differ from epithelial cytokeratins.
Differentiation
32
,
101
119
Herrmann
B. G.
(
1991
)
Expression pattern of the Brachyury gene in whole-mount TWis/Twismutant embryos.
Development
113
,
913
917
Hohl
D.
,
Lichti
U.
,
Breitkreutz
D.
,
Steinert
P. M.
,
Roop
D. R.
(
1991
)
Transcription of the human loricrin gene in vitro is induced by calcium and cell density and suppressed by retinoic acid.
J. Invest. Dermatol
96
,
414
418
Holbrook
K. A.
,
Smith
L. T.
,
Kaplan
E. D.
,
Minami
S. A.
,
Hebert
G. P.
,
Underwood
R. A.
(
1993
)
Expression of morphogens during humanfollicle development in vivo and a model for studying follicle morphogenesis in vitro.
J. Invest. Dermatol
101
,
39
–.
Hunt
P.
,
Gulisano
M.
,
Cook
M.
,
Sham
M.
,
Faiella
A.
,
Wilkinson
D.
,
Boncinella
E.
,
Krumlauf
R.
(
1991
)
A distinct Hox code for the branchial region of the vertebrate head.
Nature
353
,
861
864
Jackson
B. W.
,
Grund
C.
,
Winter
S.
,
Franke
W. W.
,
Illmensee
K.
(
1981
)
Formation of cytoskeletal elements during mouse embryogenesis II. Epithelial differentiation and intermediate filaments in early postimplantation embryos.
Differentiation
20
,
203
216
Jiang
T.-X.
,
Chuong
C.-M.
(
1992
)
Mechanisms of skin morphogenesis. I. Analysis with antibodies to adhesion molecules tenascin, N-CAM, and integrin.
Dev. Biol
150
,
82
98
Kopan
R.
,
Fuchs
E.
(
1989
)
A new look into an old problem: keratins as tools to investigate determination, morphogenesis, and differentiation in skin.
Genes Dev
3
,
1
15
Kopan
R.
,
Weintraub
H.
(
1992
)
Mouse Notch: Expression in hair follicles correlates with cell fate determination.
J. Cell Biol
121
,
631
641
Leask
A.
,
Byrne
C.
,
Fuchs
E.
(
1991
)
Transcription factor AP2 and its role in epidermal-specific gene expression.
Proc. Natl. Acad. Sci. USA
88
,
7948
7952
Leask
A.
,
Rosenberg
M.
,
Vassar
R.
,
Fuchs
E.
(
1990
)
Regulation of a human epidermal gene: sequences and nuclear factors involved in keratinocyte-specific gene regulation.
Genes Dev
4
,
1985
1998
Leonard
M. W.
,
Lim
C. K.
,
Engel
J. D.
(
1993
)
Expression of the chicken GATA factor family during early erythroid development and differentiation.
Development
119
,
519
531
Lersch
R.
,
Fuchs
E.
(
1988
)
Sequence and expression of a type II keratin, K5, in human epidermal cells.
Mol. Cell. Biol
8
,
486
493
Lersch
R.
,
Stellmach
V.
,
Stocks
C.
,
Giudice
G.
,
Fuchs
E.
(
1989
)
Isolation, sequence, and expression of a human keratin K5 gene: transcriptional regulation of keratins and insights into pairwise control.
Mol. Cell. Biol
9
,
3685
3697
Lynch
M. H.
,
O'Guin
W. M.
,
Hardy
C. M. L.
,
Sun
T.-T.
(
1986
)
Acidic and basic hair/nail (‘hard’) keratins: their colocalization in upper cortical and cuticle cells of the human hair follicle and their relationship to ‘soft’ keratins.
J. Cell Biol
103
,
2593
2606
Magnaldo
T.
,
Bernerd
F.
,
Freedberg
I. W.
,
Ohtsuki
M.
,
Blumenberg
M.
(
1993
)
Transcriptional regulators of expression of K16, the disease-associated keratin.
DNA and Cell Biol
12
,
911
23
M'Boneko
V.
,
Merker
H.-J.
(
1988
)
Development and morphology of the periderm of mouse embryos (days 9–12 of gestation).
Acta. Anat
133
,
325
336
Moser
M.
,
Pscherer
A.
,
Bauer
R.
,
Imhof
A.
,
Seegers
S.
,
Kersher
M.
,
Buettner
R.
(
1993
)
The complete murine cDNA sequence of the transcription factor AP-2.
Nucl. Acids Res
21
,
4844
–.
Mitchell
P. J.
,
Timmons
P. M.
,
Hebert
J. M.
,
Rigby
P. W. J.
,
Tijan
R.
(
1991
)
Transcription factor AP-2 is expressed in neural crest cell lineages during mouse embryogenesis.
Genes Dev
5
,
105
119
Moll
R.
,
Moll
I.
,
Wiest
W.
(
1982
)
Changes in the pattern of cytokeratin polypeptides in epidermis and hair follicles during skin development in human fetuses.
Differentiation
23
,
170
78
Nelson
W. G.
,
Sun
T. T.
(
1983
)
The 50-and 58-kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies.
J. Cell Biol
97
,
244
51
Nichols
D.
(
1981
)
Neural crest formation in the head of the mouse embryo as observed by a new histological technique.
J. Embryol. Exp. Morph
64
,
105
120
Noden
D. M.
(
1988
)
Interactions and fates of avian craniofacial mesenchyme.
Development
103
,
121
140
Powell
B.
,
Crocker
L.
,
Rogers
G.
(
1992
)
Hair follicle differentiation: expression, structure and evolutionary consequences of the hair type II keratin intermediate filament gene family.
Development
114
,
417
433
Richman
J.
,
Tickle
C.
(
1989
)
Epithelia are interchangeable between facial primordia of chick embryos and morphogenesis is controlled by the mesenchyme.
Dev. Biol
136
,
201
210
Roop
D. R.
,
Huitfeldt
H.
,
Kilkenny
A.
,
Yuspa
S. H.
(
1987
)
Regulated expression of differentiation-associated keratins in cultured epidermal cellsdetected by monospecific antibodies to unique peptides of mouse epidermal keratins.
Differentiation
35
,
143
150
Rothnagel
J. A.
,
Greenhalgh
D. A.
,
Gagne
T. A.
,
Longley
M. A.
,
Roop
D. R.
(
1993
)
Identification of a calcium inducible, epidermal-specific regulatory element in the 3-flanking region of the human keratin 1 gene.
J. Invest. Derm
101
,
506
513
Saffer
J. D.
,
Jackson
S. P.
,
Annaraella
M. B.
(
1991
)
Developmental expression of SP1 in the mouse.
Mol. Cell. Biol
11
,
2189
2199
Schweizer
J.
,
Winter
H.
(
1982
)
Keratin polypeptide analysis in fetal and in terminally differentiating newborn mouse epidermis.
Differentiation
22
,
19
24
Sengel
P.
(
1990
)
Pattern formation in skin development.
Int. J. Dev. Biol
32
,
33
50
Smart
I. H. M.
(
1970
)
Variation in plane of cell cleavage during the process of stratification in mouse epidermis.
Br. J. Dermatol
82
,
276
282
Snape
A. M.
,
Jonas
E. A.
,
Sargent
T. D.
(
1990
)
KTF-1, a transcription activator of Xenopus embryonic keratin expression.
Development
109
,
157
165
Snape
A. M.
,
Winning
R. S.
,
Sargent
T. D.
(
1991
)
Transcription factor AP-2 is tissue-specific and is closely related or identical to keratin transcription factor 1 (KTF-1).
Development
113
,
283
293
Stark
H. J.
,
Breitkreutz
D.
,
Limat
A.
,
Ryle
C.-M.
,
Roop
D.
,
Leigh
I.
,
Fusenig
N.
(
1990
)
Keratin 1 and 10 or homologues as regular constituents of inner root sheath and cuticle cells of human hair follicle.
Eur. J. Cell Biol
52
,
359
372
Takahashi
Y.
,
Monsoro-Burq
A.-H.
,
Bontoux
M.
,
Le Douarin
N. M.
(
1992
)
A role for Quox-8 in the establishment of the dorsoventral pattern during vertebrate development.
Proc. Natl. Acad. Sci. USA
89
,
10237
10241
Thorey
I. S.
,
Meneses
J. J.
,
Neznanov
N.
,
Kulesh
D. A.
,
Pedersen
R. A.
,
Oshima
R. G.
(
1993
)
Embryonic expression of human keratin 18 and K18-beta-galactosidase fusion genes in transgenic mice.
Dev. Biol
160
,
519
534
Tyner
A. L.
,
Fuchs
E.
(
1986
)
Evidence for posttranscriptional regulation of the keratins expressed during hyperproliferation and malignant transformation in human epidermis.
J. Cell Biol
103
,
1945
1955
Van Exan
R. J.
,
Hardy
M. F.
(
1980
)
A spatial relationship between innervation and the early differentiation of vibrissa follicles in the embryonic mouse.
J. Anat
131
,
643
656
Van Exan
R. J.
,
Handy
M. F.
(
1984
)
The differentiation of dermis in the laboratory mouse.
Amer. J. Anat
169
,
149
164
Watt
F. M.
,
Keeble
S.
,
Fisher
C.
,
Hudson
D. L.
,
Codd
J.
,
Salisbury
J. R.
(
1989
)
Onset of expression of peanut lectin-binding glycoproteins in correlated with stratification of keratinocytes during human epidermal development in vivo and in vitro.
J. Cell Sci
94
,
355
359
Weiss
L. W.
,
Zelickson
A. S.
(
1975
)
Embryology of the epidermis: ultrastructural aspects. II Period of differentiation in mouse with mammalian comparisons.
Acta Dermatol
55
,
321
329
Wessells
N. K.
(
1963
)
Effects of extra-epithelial factors on the incorporation of thymidine by embryonic epidermis.
Exp. Cell Res
30
,
36
55
Williams
T.
,
Admon
A.
,
Luscher
B.
,
Tjian
R.
(
1988
)
Cloning and expression of AP2, a cell-type-specific transcription factor that activates inducible enhancer elements.
Genes Dev
2
,
1557
1569
Winning
R. S.
,
Shea
L. J.
,
Sargent
T. D.
(
1991
)
Developmental regulation of transcription factor AP-2 during Xenopus laevis embryogenesis.
Nucl. Acids. Res
19
,
3709
3714
Wu
Y.-J.
,
Rheinwald
J. G.
(
1982
)
A new small (40 kd) keratin filament protein made by some cultured human squamous cell carcinomas.
Cell
25
,
627
635
Yoneda
K.
,
Steinert
P.
(
1993
)
Overexpression of human loricrin in transgenic mice produces a normal phenotype.
Proc. Natl. Acad. Sci. USA
90
,
10754
10758
This content is only available via PDF.