QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00477

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jaubert, J. Articles by Segre, J. A. Search for Related Content PubMed PubMed Citation Articles by Jaubert, J. Articles by Segre, J. A.

Ectopic expression of Kruppel like factor 4 (Klf4) accelerates formation of the epidermal permeability barrier

National Human Genome Research Institute, NIH, 49 Convent Drive, Bethesda, MD 20892, USA

View larger version (34K): [in a new window]   Fig. 1. Characterization of the K5-tTA and TRE-Klf4 transgenic mice. (A) Diagram of the K5-tTA and TRE-lacZ transgenes. (B) The patterned expression of the driver K5-tTA transgene revealed by crossing with the tester TRE-lacZ line is essentially identical to the endogenous K5 expression with lateral expression at E12.5, spreading ventrally and then dorsally to cover almost the entire surface of the embryo by E14.5. (C) Diagram of the TRE-Klf4 construct used for in vitro studies and microinjection. A unique BamHI and a Myc tag were inserted into the Klf4 cDNA by PCR. This fragment was inserted into a vector containing TRE, CMV minimal promoter, and a ß-globin poly(A)+ sequence. (D) Anti-KLF4 and anti-Myc immunohistochemistry of frozen sections of wild-type (wt) and K5-Klf4 DT (DT) epidermis. The endogenous KLF4 is expressed in suprabasal cells and the Myc-tagged ectopic protein is expressed in basal cells. The dotted line indicates the basement membrane; the bracket, basal layer. Representative data from line 2 K5-Klf4 DT is shown. (E) Northern blot analysis of mRNAs isolated from pools of skins of K5-Klf4 DTs at E10.5, E11.5, E12.5, E13.5 and E14.5. Embryos hybridized with a Klf4 cDNA probe, revealing that the ectopic Klf4 transcript is expressed as early as E10.5. The endogenous Klf4 is 3.5 kb and the transgenic Klf4 is 2.5 kb in the K5-Klf4 DTs. G3PDH probe is a control. Data is from line 10 K5-Klf4 DT.

View larger version (41K): [in a new window]   Fig. 2. Allelic series of morphology and expression levels in three lines of TRE-Klf4 mice that were crossed with K5-tTA mice. (A) Gross morphology and skeletal preparations of wild-type and the three lines of K5-Klf4 DTs. (B) Northern blot analysis of mRNAs isolated from E16.5 embryonic skin from the line indicated and hybridized with a Klf4 cDNA probe. The endogenous Klf4 is 3.5 kb and the ectopic Klf4 is at 2.5 kb in the K5-Klf4 DTs. Lines 6 and 10 show a faint band at 3 kb that may be an alternatively spliced form of the endogenous Klf4 gene. G3PDH probe is a control and signals are quantified using Phosphorimager. (C) Western blot analysis of proteins isolated from E16.5 embryonic skin from the line indicated and hybridized with anti-KLF4 polyclonal antibody. The endogenous KLF4 protein is 60 kDa and the ectopic KLF4 protein has a slightly higher molecular mass because of the N-terminal tag. Hybridization with p84 antibody, that recognizes an 84 kDa nuclear matrix protein, is a control.

View larger version (36K): [in a new window]   Fig. 3. Skin barrier acquisition is accelerated in the K5-Klf4 DTs lines. Barrier-dependent dye exclusion assays on three lines of K5-Klf4 DTs at the ages indicated.

View larger version (118K): [in a new window]   Fig. 4. Histology of line 2 K5-Klf4 DT epidermis during development. Control and line 2 K5-Klf4 DT epidermis at the ages indicated were fixed in paraformaldehyde, embedded in paraffin, sectioned and stained with Hematoxylin and Eosin. The K5-Klf4 DT epidermis has accelerated differentiation with normal histology. Similar results were obtained for all three lines except that lines 6 and 10 had more advanced granular and SC layers at E15.5 than line 2. The dotted line indicates the basement membrane.

View larger version (132K): [in a new window]   Fig. 5. Epidermal differentiation markers are expressed earlier in development with normal patterns in K5-Klf4 DTs. Control wild-type and line 2 K5-Klf4 DT epidermis at the age indicated were fixed in paraformaldehyde, embedded in paraffin and sections were stained with epidermal differentiation markers: keratin 14 (K14; basal layer), keratin 1 (K1; spinous layers), loricrin (lor) and filaggrin (fil; granular layers). All of the markers show the normal restriction of expression, but the K5-Klf4 DTs express the terminal differentiation markers loricrin and filaggrin earlier in development. The dotted line indicates the basement membrane.

View larger version (82K): [in a new window]   Fig. 6. Immunohistochemistry for BrdU to visualize the number of mitotically active cells in the epidermis. Samples at low magnification (A,C) and high magnification (B,D) of E16.5 wild-type (A,B) and K5-Klf4 DTs (C,D) reveal a decrease in proliferation in the transgenic mice. The dotted line indicates the basement membrane.

View larger version (46K): [in a new window]   Fig. 7. Structural elements necessary for full barrier acquisition in K5-Klf4 DTs. Profilaggrin, filaggrin, involucrin and loricrin expression in E16.5 epidermis of wild-type and K5-Klf4 DTs. (A) Profilaggrin processed to filaggrin is not detected in wild-type epidermis until after the permeability change (E17.5), but is already processed in all of the E16.5 K5-Klf4 DTs. (B) Involucrin levels do not change during barrier acquisition and there is no difference between the wild-type and K5-Klf4 DTs. (C) Loricrin levels are slightly decreased in K5-Klf4 DT lines 2 and 6. (D,E) Ponceau S stainings; p84 expression was used as a control.

View larger version (43K): [in a new window]   Fig. 8. Cornified envelopes at the stage and genotype specified. Fully mature cornified envelopes are present in E15.5 K5-Klf4 DTs.