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• W2092160553 abstract "COUP-TF-interacting protein 2 (CTIP2; also known as Bcl11b) is a transcription factor that plays key roles in the development of the central nervous and immune systems. CTIP2 is also highly expressed in the developing epidermis, and at lower levels in the dermis and in adult skin. Analyses of mice harboring a germline deletion of CTIP2 revealed that the protein plays critical roles in skin during development, particularly in keratinocyte proliferation and late differentiation events, as well as in the development of the epidermal permeability barrier. At the core of all of these actions is a relatively large network of genes, described herein, that is regulated directly or indirectly by CTIP2. The analysis of conditionally null mice, in which expression of CTIP2 was ablated specifically in epidermal keratinocytes, suggests that CTIP2 functions in both cell and non-cell autonomous contexts to exert regulatory influence over multiple phases of skin development, including barrier establishment. Considered together, our results suggest that CTIP2 functions as a top-level regulator of skin morphogenesis. COUP-TF-interacting protein 2 (CTIP2; also known as Bcl11b) is a transcription factor that plays key roles in the development of the central nervous and immune systems. CTIP2 is also highly expressed in the developing epidermis, and at lower levels in the dermis and in adult skin. Analyses of mice harboring a germline deletion of CTIP2 revealed that the protein plays critical roles in skin during development, particularly in keratinocyte proliferation and late differentiation events, as well as in the development of the epidermal permeability barrier. At the core of all of these actions is a relatively large network of genes, described herein, that is regulated directly or indirectly by CTIP2. The analysis of conditionally null mice, in which expression of CTIP2 was ablated specifically in epidermal keratinocytes, suggests that CTIP2 functions in both cell and non-cell autonomous contexts to exert regulatory influence over multiple phases of skin development, including barrier establishment. Considered together, our results suggest that CTIP2 functions as a top-level regulator of skin morphogenesis. cornified envelope COUP-TF-interacting protein 2 epidermal permeability barrier heparin-binding EGF-like growth factor homologous recombination keratinocyte growth factor phosphate-buffered saline transepidermal water loss wild-type The development of the skin epidermis begins with the commitment of the primitive ectoderm to the keratinocyte cell fate. The subsequent processes of cellular proliferation, stratification, and differentiation result in formation of the multilayered structure of epidermis. During embryonic development, keratinocytes of the innermost layer of the epidermis, the proliferative, basal cell layer, undergo a program of the terminal differentiation, then exit the basal cell layer and migrate upward to the surface of the skin (Byrne et al., 2003Byrne C. Hardman M. Nield K. Covering the limb—formation of the integument.J Anat. 2003; 202: 113-123Crossref PubMed Scopus (69) Google Scholar; Mack et al., 2005Mack J.A. Anand S. Maytin E.V. Proliferation and cornification during development of the mammalian epidermis.Birth Defects Res C Embryo Today. 2005; 75: 314-329Crossref PubMed Scopus (64) Google Scholar). These cells initially differentiate into spinous and then granular cells, and finally into the tough, enucleated cells of the cornified layer, the corneocytes (Byrne et al., 2003Byrne C. Hardman M. Nield K. Covering the limb—formation of the integument.J Anat. 2003; 202: 113-123Crossref PubMed Scopus (69) Google Scholar). The events of epidermal development are orchestrated by the concerted action of a number of transcription factors that regulate both the proliferative capacity and differentiative potential of epidermal keratinocytes (Segre, 2003Segre J. Complex redundancy to build a simple epidermal permeability barrier.Curr Opin Cell Biol. 2003; 15: 776-782Crossref PubMed Scopus (79) Google Scholar, Segre, 2006Segre J.A. Epidermal barrier formation and recovery in skin disorders.J Clin Invest. 2006; 116: 1150-1158Crossref PubMed Scopus (345) Google Scholar). These include c-Myc, p63, Klf4, GATA3, the AP-1 transcription factors c-Fos and c-Jun, the Id family of proteins, and others (Segre et al., 1999Segre J.A. Bauer C. Fuchs E. Klf4 is a transcription factor required for establishing the barrier function of the skin.Nat Genet. 1999; 22: 356-360Crossref PubMed Scopus (637) Google Scholar; Langlands et al., 2000Langlands K. Down G.A. Kealey T. Id proteins are dynamically expressed in normal epidermis and dysregulated in squamous cell carcinoma.Cancer Res. 2000; 60: 5929-5933PubMed Google Scholar; Angel et al., 2001Angel P. Szabowski A. Schorpp-Kistner M. Function and regulation of AP-1 subunits in skin physiology and pathology.Oncogene. 2001; 20: 2413-2423Crossref PubMed Scopus (340) Google Scholar; Arnold and Watt, 2001Arnold I. Watt F.M. c-Myc activation in transgenic mouse epidermis results in mobilization of stem cells and differentiation of their progeny.Curr Biol. 2001; 11: 558-568Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar; Shaulian and Karin, 2002Shaulian E. Karin M. AP-1 as a regulator of cell life and death.Nat Cell Biol. 2002; 4: E131-E136Crossref PubMed Scopus (2205) Google Scholar; Koster et al., 2004Koster M.I. Kim S. Mills A.A. DeMayo F.J. Roop D.R. p63 is the molecular switch for initiation of an epithelial stratification program.Genes Dev. 2004; 18: 126-131Crossref PubMed Scopus (552) Google Scholar, Koster et al., 2007Koster M.I. Dai D. Marinari B. Sano Y. Costanzo A. Karin M. et al.p63 induces key target genes required for epidermal morphogenesis.Proc Natl Acad Sci USA. 2007; 104: 3255-3260Crossref PubMed Scopus (191) Google Scholar; de Guzman Strong et al., 2006de Guzman Strong C. Wertz P.W. Wang C. Yang F. Meltzer P.S. Andl T. et al.Lipid defect underlies selective skin barrier impairment of an epidermal-specific deletion of Gata-3.J Cell Biol. 2006; 175: 661-670Crossref PubMed Scopus (67) Google Scholar). The end product of epidermal development is the formation of the epidermal permeability barrier (EPB), which provides a crucial physical and permeability barrier. Formation of the EPB is a stepwise and orderly process (Segre, 2003Segre J. Complex redundancy to build a simple epidermal permeability barrier.Curr Opin Cell Biol. 2003; 15: 776-782Crossref PubMed Scopus (79) Google Scholar, Segre, 2006Segre J.A. Epidermal barrier formation and recovery in skin disorders.J Clin Invest. 2006; 116: 1150-1158Crossref PubMed Scopus (345) Google Scholar). In the granular layer of epidermis, lipids are packaged inside lamellar bodies, and structural proteins, such as keratins, loricrin and filaggrin, are assembled into macrofibrils. The cornified envelope (CE) is formed by deposition of precursor proteins on the inner surface of the plasma membrane (Elias, 2005Elias P.M. Stratum corneum defensive functions: an integrated view.J Invest Dermatol. 2005; 125: 183-200Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). Transglutaminase enzymes then cross-link the CE proteins, creating a tough sac that holds the keratin fibers (Candi et al., 2005Candi E. Schmidt R. Melino G. The cornified envelope: a model of cell death in the skin.Nat Rev Mol Cell Biol. 2005; 6: 328-340Crossref PubMed Scopus (1267) Google Scholar). Finally, cellular organelles, including the nucleus, are degraded and lipids from lamellar bodies are extruded into the intercellular space and onto the CE scaffold, forming a series of extracellular lipid membranes (lamellar bilayers; Candi et al., 2005Candi E. Schmidt R. Melino G. The cornified envelope: a model of cell death in the skin.Nat Rev Mol Cell Biol. 2005; 6: 328-340Crossref PubMed Scopus (1267) Google Scholar). COUP-TF-interacting protein 2 (CTIP2) is a C2H2 zinc finger transcription factor that represses transcription of reporter genes in transiently transfected cells, either by tethering to other promoter-bound transcription factors (Avram et al., 2000Avram D. Fields A. Pretty On Top K. Nevrivy D.J. Ishmael J.E. Leid M. Isolation of a novel family of C(2)H(2) zinc finger proteins implicated in transcriptional repression mediated by chicken ovalbumin upstream promoter transcription factor (COUP-TF) orphan nuclear receptors.J Biol Chem. 2000; 275: 10315-10322Crossref PubMed Scopus (159) Google Scholar) or by direct, sequence-specific DNA-binding activity (Avram et al., 2002Avram D. Fields A. Senawong T. Topark-Ngarm A. Leid M. COUP-TF (chicken ovalbumin upstream promoter transcription factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding protein.Biochem J. 2002; 368: 555-563Crossref PubMed Scopus (113) Google Scholar; Topark-Ngarm et al., 2006Topark-Ngarm A. Golonzhka O. Peterson V.J. Barrett Jr., B. Martinez B. Crofoot K. et al.CTIP2 associates with the NuRD complex on the promoter of p57KIP2, a newly identified CTIP2 target gene.J Biol Chem. 2006; 281: 32272-32283Crossref PubMed Scopus (81) Google Scholar). The hypothesis that CTIP2 functions as a transcriptional regulator has been supported by transcriptome analyses in human neuroblastoma cells (Topark-Ngarm et al., 2006Topark-Ngarm A. Golonzhka O. Peterson V.J. Barrett Jr., B. Martinez B. Crofoot K. et al.CTIP2 associates with the NuRD complex on the promoter of p57KIP2, a newly identified CTIP2 target gene.J Biol Chem. 2006; 281: 32272-32283Crossref PubMed Scopus (81) Google Scholar), striatal medium spiny neurons (Arlotta et al., 2008Arlotta P. Molyneaux B.J. Jabaudon D. Yoshida Y. Macklis J.D. Ctip2 controls the differentiation of medium spiny neurons and the establishment of the cellular architecture of the striatum.J Neurosci. 2008; 28: 622-632Crossref PubMed Scopus (207) Google Scholar), and mouse thymocytes (Leid M. et al., unpublished data). CTIP2 is expressed early during mouse development as well as in the adult animal, and in both cases, expression is most predominant in the central nervous system, thymus, and skin and other tissues of ectodermal origin (Leid et al., 2004Leid M. Ishmael J.E. Avram D. Shepherd D. Fraulob V. Dolle P. CTIP1 and CTIP2 are differentially expressed during mouse embryogenesis.Gene Expr Patterns. 2004; 4: 733-739Crossref PubMed Scopus (111) Google Scholar; Golonzhka et al., 2007Golonzhka O. Leid M. Indra G. Indra A.K. Expression of COUP-TF-interacting protein 2 (CTIP2) in mouse skin during development and in adulthood.Gene Expr Patterns. 2007; 7: 754-760Crossref PubMed Scopus (30) Google Scholar). Mice null for expression of CTIP2 exhibit perinatal lethality and severe phenotypes in tissues that express the gene. In the central nervous system, CTIP2 is required for proper axonal projection by corticospinal neurons and normal development of striatal medium spiny neurons (Arlotta et al., 2005Arlotta P. Molyneaux B.J. Chen J. Inoue J. Kominami R. Macklis J.D. Neuronal subtype-specific genes that control corticospinal motor neuron development in vivo.Neuron. 2005; 45: 207-221Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar, Arlotta et al., 2008Arlotta P. Molyneaux B.J. Jabaudon D. Yoshida Y. Macklis J.D. Ctip2 controls the differentiation of medium spiny neurons and the establishment of the cellular architecture of the striatum.J Neurosci. 2008; 28: 622-632Crossref PubMed Scopus (207) Google Scholar). Germline disruption of CTIP2 results in an arrest of T-cell development at an immature, CD4−CD8− stage(s), with the complete absence of αβ T cells (Wakabayashi et al., 2003Wakabayashi Y. Watanabe H. Inoue J. Takeda N. Sakata J. Mishima Y. et al.Bcl11b is required for differentiation and survival of alphabeta T lymphocytes.Nat Immunol. 2003; 4: 533-539Crossref PubMed Scopus (278) Google Scholar). Here we report a previously unknown function of CTIP2 in skin development. We show that CTIP2 controls epidermal proliferation/differentiation programs and EPB formation. CTIP2−/− mice exhibit a hypoplastic epidermis, defects in EPB development, and increased transepidermal water loss (TEWL). These defects likely arise from large-scale disruption of gene expression in the mutant skin, including genes encoding structural protein and lipid modifying enzymes, as well as those involved in epidermal proliferation and differentiation, and EPB establishment. In the context of EPB development, CTIP2 was found to function cell autonomously. However, the actions of CTIP2 in epidermal proliferation and early differentiation were found to be non-cell autonomous, most likely arising from CTIP2-dependent regulation of paracrine growth factor(s) expression in the dermis, including keratinocyte growth factor (KGF) and/or GM-CSF (Szabowski et al., 2000Szabowski A. Maas-Szabowski N. Andrecht S. Kolbus A. Schorpp-Kistner M. Fusenig N.E. et al.c-Jun and JunB antagonistically control cytokine-regulated mesenchymal-epidermal interaction in skin.Cell. 2000; 103: 745-755Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar; Werner and Smola, 2001Werner S. Smola H. Paracrine regulation of keratinocyte proliferation and differentiation.Trends Cell Biol. 2001; 11: 143-146Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). CTIP2−/− mice were generated by floxing exon 4 of the CTIP2 locus, which encodes ∼75% of the CTIP2 open reading frame and six zinc finger motifs (Figure 1a). The targeting vector used to modify the CTIP2 locus by homologous recombination (HR) is schematically depicted in Figure 1b and c, and HR was confirmed by 5′ and 3′ Southern analyses (Figure 1d and e). Heterozygous mice harboring one wild-type (+) and one mutant CTIP2 allele (-), did not show an overt phenotype, and were viable and fertile. CTIP2+/- mice were bred to generate the CTIP2−/− mouse, and the lack of CTIP2 protein in these mice was confirmed by immunoblotting in skin (Figure 1f). CTIP2−/− mice were born at the expected Mendelian ratio and without obvious skin abnormalities, with the exception of an open eye phenotype (Figure 2a, arrow; compare; Figure S1a and b), as previously described by Kominami and co-workers (Wakabayashi et al., 2003Wakabayashi Y. Watanabe H. Inoue J. Takeda N. Sakata J. Mishima Y. et al.Bcl11b is required for differentiation and survival of alphabeta T lymphocytes.Nat Immunol. 2003; 4: 533-539Crossref PubMed Scopus (278) Google Scholar). However, CTIP2−/− mice did not feed and died within 6–8hours after birth. These phenotypic characteristics, together with severe thymic hypocellularity and complete loss of αβ-T lymphocytes (data not shown), recapitulated previously reported observations of CTIP2-null mice (Wakabayashi et al., 2003Wakabayashi Y. Watanabe H. Inoue J. Takeda N. Sakata J. Mishima Y. et al.Bcl11b is required for differentiation and survival of alphabeta T lymphocytes.Nat Immunol. 2003; 4: 533-539Crossref PubMed Scopus (278) Google Scholar).Figure 2CTIP2 mutant mice exhibit permeability barrier defects and epidermal hypoplasia. (a) X-gal diffusion assay performed on CTIP2+/+, CTIP2−/−, CTIP2L2/L2, and CTIP2ep-/- fetuses at E17.5 and E18.5 as indicated. (b) Transepidermal water loss measurements from dorsal and ventral skin of CTIP2+/+, CTIP2−/−, CTIP2L2/L2, and CTIP2ep-/- mice at E17.5 and E18.5. (Plotted are mean measurements of three independent mice per genotype±SEM). *P<0.05, #not statistically significant. (c) Histology of toluidine blue stained dorsal skin biopsies (4-μm thick section) of CTIP2+/+ and CTIP2−/− mice at E18.5. Marked epidermal hypoplasia was observed in CTIP2−/− mice. D, dermis; B, basal layer; S, spinous layer; G, granular layer; C, cornified layer. Scale bar: 50μm. (d) H&E staining of dorsal skin biopsies (10-μm thick section) of CTIP2L2/L2 and CTIP2ep-/- mice at E18.5. Abbreviations are as in (c). Scale bar: 50μm. (e) Quantitative histomorphometry of CTIP2−/− and CTIP2ep-/- phenotypes. Epidermal (Te) and dermal (Td) thicknesses were measured on eight independent wt (CTIP2+/+) and null (CTIP2−/−) mice, and on six floxed (CTIP2L2/L2) and conditionally null (CTIP2ep-/-) mice. The ratio of Te to Td for the four genotypes is plotted on the ordinate (left side). Bars on the right represent the number of proliferating Ki67+ cells (as a ratio of DAPI+ cells and expressed as percentage of each, corresponding control). The asterisks and hash denotations are as described in (b), and refer to the comparison of CTIP2+/+ and CTIP2−/−, and CTIP2L2/L2 and CTIP2ep-/- mice, respectively. The results depicted in this (a) to (d) are representative of 6–12 additional studies that have been conducted over the course of 2 years using numerous litters of mice of identical genotypes.View Large Image Figure ViewerDownload (PPT) Download .jpg (.12 MB) Help with files Supplementary Figure S1Characterization of the CTIP2 null and conditional mutant mice. (a-b) H & E staining of coronal section of the eye in CTIP2+/+ (b) and CTIP2−/− (c) mice at E18.5. (c) PCR detection of the CTIP2L- and CTIP2L2 alleles in the epidermis and mesenchyme at E17.5. Note that the L- allele is detected only in the epidermis in the presence of the Cre transgene (+ cre). (d) Histology of toluidine blue stained dorsal skin biopsies of CTIP2+/+ and CTIP2−/− mice at E17.5. Marked epidermal hypoplasia was observed in CTIP2−/− mice. D-dermis, B- basal layer, S-spinous layer, G-granular layer, c-cornified layer (e) H&E staining of dorsal skin biopsies of CTIP2L2/L2 and CTIP2ep−/− mice at E17.5. Abbreviations are as in (a). Scale bar for d and e : 50 μm. (f-g) Nile red staining of CTIP2L2/L2 (f) and CTIP2ep−/− (g) mouse skin at E17.5. E- epidermis, D-dermis. Scale bar in g: 50 μm for f-g. (h) Immunoblot of epidermal skin extracts from CTIP2+/+ and CTIP2−/− mice at E18.5 using indicated antibodies. CTIP2 floxed mice (CTIP2L2/L2; see Figure 1) were crossed with a K14-Cre transgenic mouse (Indra et al., 2000Indra A.K. Li M. Brocard J. Warot X. Bornert J.M. Gerard C. et al.Targeted somatic mutagenesis in mouse epidermis.Horm Res. 2000; 54: 296-300Crossref PubMed Scopus (48) Google Scholar) to obtain a mouse line in which CTIP2 was specifically ablated in keratinocytes (epidermis-specific knockout; CTIP2ep-/-). To confirm excision of the floxed CTIP2 allele in CTIP2ep-/- mice, we separated epidermis and dermis and genotyped both tissues for excised (-) and floxed (L2) alleles. The excised, but not L2, allele was detected in the epidermis of E17.5 CTIP2ep-/- fetuses, whereas only the L2 allele was present in the dermis of these mice (Figure S1c), thus demonstrating that CTIP2 was efficiently and selectively ablated in the keratinocytes of the developing epidermis. In agreement with this result CTIP2 protein was not detected in epidermal extracts from CTIP2ep-/- fetuses by immunoblotting, thus validating the CTIP2ep-/- mouse model (Figure 1g). CTIP2ep-/- mice were born without any apparent abnormalities, fed normally, survived into adulthood, and were fertile. CTIP2 is highly expressed in the mouse ectoderm during development, beginning around embryonic day E10.5 (Golonzhka et al., 2007Golonzhka O. Leid M. Indra G. Indra A.K. Expression of COUP-TF-interacting protein 2 (CTIP2) in mouse skin during development and in adulthood.Gene Expr Patterns. 2007; 7: 754-760Crossref PubMed Scopus (30) Google Scholar). To determine if the perinatal lethality of CTIP2−/− mice was due to defects in the EPB, we investigated barrier function in these mice by performing X-gal permeability assays at E17.5 and E18.5. Control embryos showed very little X-gal staining at either E17.5 or E18.5 (Figure 2a, first and second panel). In contrast, CTIP2−/− fetuses stained strongly with X-gal at E17.5, particularly on the ventral surface and head (Figure 2a). The dorsal surface of CTIP2−/− mice did not take up appreciable amounts of X-gal, signifying that dorsal to ventral barrier establishment had commenced in the mutants, but perhaps in a delayed fashion. Similar studies conducted at E18.5 revealed that EPB function in CTIP2−/− and wild-type (wt) mice was indistinguishable, with the exception of blue staining around the eyelids of the CTIP2−/− mice (Figure 2a, arrow in second panel). To determine if CTIP2 regulates EPB establishment in a cell autonomous manner, we performed X-gal permeability assays on CTIP2L2/L2 (as a control) and CTIP2ep-/- mice at E17.5 and E18.5 (Figure 2a, third and fourth panels). Similar to CTIP2−/− mice, CTIP2ep-/- mice exhibited a delay in barrier establishment. CTIP2ep-/- mice showed extensive blue staining on the ventral aspect of the body, limbs and head at E17.5, and residual X-gal staining at E18.5, whereas control embryos showed very little coloration at either E17.5 or E18.5 (Figure 2a). These results demonstrate that CTIP2, in a cell autonomous manner, regulates EPB formation during fetal development. We also performed TEWL studies in CTIP2−/− mice to assess the rate of water evaporation from the skin at E17.5 and E18.5. At E17.5, TEWL from CTIP2−/− dorsal skin averaged ∼12gm−2h−1, which was about six-fold higher than that of wt controls (Figure 2b, first panel). The rate of water loss on the ventral surface of E17.5 CTIP2−/− mice was approximately two-fold greater than that of wt mice (Figure 2b). Although there was no difference in dorsal water loss between wt and CTIP2−/− mice at E18.5, the ventral surface of the mutants continued to lose water at a greater rate than wt controls (Figure 2d, second panel). The rate of water loss from the dorsal skin of E17.5 CTIP2ep-/- mice was about three-fold higher than that of the control mice (Figure 2b, third panel) but at E18.5 the water loss on the dorsal surface of CTIP2ep-/- mice was not significantly different from controls (Figure 2b, fourth panel). However, TEWL from the ventral side of CTIP2ep-/- mice slightly exceeded that of the control mice at E17.5 and was 2- to 3-fold higher than that of the control mice at E18.5. These findings further demonstrate that CTIP2ep-/- mice exhibit considerably delayed formation of the EPB, recapitulating the cell autonomous barrier phenotype of CTIP2−/− mice. Together, these results indicate that skin barrier establishment is disrupted in the absence of CTIP2, and suggest a previously unreported role of CTIP2 in skin barrier formation. Histological analyses of dorsal skin at E17.5 and E18.5 revealed that CTIP2−/− epidermis was extremely hypoplastic with decreased numbers of differentiating and cornified cell layers, and profound loss of the normal, cornified layer with the typical “basket weave” appearance (Figure 2c; Figure S1d). The number of proliferating, Ki67+ cells in the epidermis was modestly, but significantly reduced CTIP2−/− mice (∼13% reduction relative to wt controls; right panels of Figure 2e), and the thickness of the CTIP2−/− epidermis was approximately 50% that of wt mice, as determined by quantitative histomorphometry (left panels of Figure 2e). All of the epidermal layers were present in the CTIP2ep-/- mice, and both the number of proliferating cells and the epidermal thickness of these conditionally null mice were indistinguishable from that of the corresponding control mice (CTIP2L2/L2; Figure 2e). However, we observed a modest reduction in the number of cornified cell layers in CTIP2ep-/- mice at E17.5 and E18.5 (see Figure 2d; Figure S1e). Reduced epidermal thickness of the CTIP2−/− mutants could be due to alterations in epidermal proliferation and/or differentiation. To test this, we performed immunohistochemical analyses to assess the expression of: (1) a proliferation marker (Ki67; Schluter et al., 1993Schluter C. Duchrow M. Wohlenberg C. Becker M.H. Key G. Flad H.D. et al.The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins.J Cell Biol. 1993; 123: 513-522Crossref PubMed Scopus (650) Google Scholar), (2) a marker of keratinocyte basal cells (keratin 14, K14; Byrne et al., 1994Byrne C. Tainsky M. Fuchs E. Programming gene expression in developing epidermis.Development. 1994; 120: 2369-2383Crossref PubMed Google Scholar), and (3) an early keratinocyte differentiation marker (keratin 10, K10; Fuchs and Green, 1980Fuchs E. Green H. Changes in keratin gene expression during terminal differentiation of the keratinocyte.Cell. 1980; 19: 1033-1042Abstract Full Text PDF PubMed Scopus (821) Google Scholar; Roop et al., 1983Roop D.R. Hawley-Nelson P. Cheng C.K. Yuspa S.H. Keratin gene expression in mouse epidermis and cultured epidermal cells.Proc Natl Acad Sci USA. 1983; 80: 716-720Crossref PubMed Scopus (132) Google Scholar). Reduced numbers of Ki67+ cells (compare Figure 3a and d) and reduced K14 staining (Figure 3b and e) were both observed in the skin of CTIP2−/− mice. Similarly, the expression of the differentiation marker K10 was strongly reduced in CTIP2−/− mice (Figure 3c and f). Immunoblot analyses of skin protein extracts further confirmed the decrease in the expression of K10 and K14, in the mutant fetuses (Figure 3w). These results suggest that reduced epidermal thickness of the CTIP2−/− fetuses may be due to reduced proliferation and/or altered differentiation in the skin of the mutant mice. We performed immunohistochemical analyses of the late epidermal differentiation markers loricrin, involucrin, and filaggrin. The expression levels of involucrin and filaggrin, but not that of loricrin, were reduced in the E18.5 CTIP2−/− skin (compare Figure 3g and j, h and k, i and l, respectively), and this was further confirmed by immunoblotting (Figure 3w). Immunohistochemical analyses of the CTIP2ep-/- skin revealed no changes in K14 expression (compare Figure 3m and r) and a slight reduction in the expression of K10 relative to control skin (compare Figure 3n and s). Involucrin and filaggrin expression levels were slightly reduced in the upper, terminally differentiating cell layers in the mutants (white arrows in Figure 3o and t and p and u). However, loricrin levels were comparable in wt and mutant skin (Figure 3q and v). Our data indicates that CTIP2−/− mice exhibit defects in keratinocytic proliferation, early differentiation, terminal differentiation, and EPB formation. CTIP2ep-/- mutants on the other hand did not demonstrate severe epidermal proliferation or early differentiation defects. However, CTIP2ep-/- mice did exhibit impaired terminal differentiation and EPB establishment, as was observed in the CTIP2−/− mice. This suggests a cell autonomous function of CTIP2 in controlling the terminal differentiation events, and non-cell autonomous influence of CTIP2 on proliferation and early differentiation of keratinocytes. We performed RT-qPCR analyses to assess expression levels of several genes implicated in the control of early- and late-differentiation events in the epidermis, as well as genes involved in barrier establishment. Interestingly, although expression of Id2 and p57 was upregulated (or derepressed) in the mutant skin, consistent with the previously demonstrated repressor activity of CTIP2 (Senawong et al., 2003Senawong T. Peterson V.J. Avram D. Shepherd D.M. Frye R.A. Minucci S. et al.Involvement of the histone deacetylase SIRT1 in chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting protein 2-mediated transcriptional repression.J Biol Chem. 2003; 278: 43041-43050Crossref PubMed Scopus (111) Google Scholar; Topark-Ngarm et al., 2006Topark-Ngarm A. Golonzhka O. Peterson V.J. Barrett Jr., B. Martinez B. Crofoot K. et al.CTIP2 associates with the NuRD complex on the promoter of p57KIP2, a newly identified CTIP2 target gene.J Biol Chem. 2006; 281: 32272-32283Crossref PubMed Scopus (81) Google Scholar) expressions of both c-Myc and p63 were downregulated in the mutants at E18.5 (Figure 3x), suggesting that CTIP2 may directly or indirectly activate expression of the latter two genes. RT-qPCR analyses for genes encoding structural proteins and transcription factors involved in late terminal differentiation and barrier formation, such as transglutaminase-1 (Tgase1), GATA3, Klf4, and caspase-14 revealed that all were downregulated at both E17.5 and E18.5 in the mutants (Figure 3x and data not shown). We also observed a significant downregulation of c-Fos, but not c-Jun, suggesting that altered gene expression in CTIP2 mutant skin may be mediated, at least in part, by specific members of AP-1 family of transcription factors (Shaulian and Karin, 2002Shaulian E. Karin M. AP-1 as a regulator of cell life and death.Nat Cell Biol. 2002; 4: E131-E136Crossref PubMed Scopus (2205) Google Scholar; Zenz and Wagner, 2006Zenz R. Wagner E.F. Jun signalling in the epidermis: From developmental defects to psoriasis and skin tumors.Int J Biochem Cell Biol. 2006; 38: 1043-1049Crossref PubMed Scopus (130) Google Scholar). Altogether, our data suggest that CTIP2 regulates expression of a subset of the keratinocytic genes encoding transcription factors and other proteins implicated in the epidermal homeostasis and EPB formation. To determine if the observed barrier defect in the CTIP2−/− mutants was due to the altered distribution of polar and neutral lipids, we analyzed the surface lipid distribution of mutant and wt epidermis by Nile Red staining. These studies revealed that neutral lipids formed a yellow-colored and dense, continuous ribbon along the top of the cornified layer in control fetuses but these lipids were unevenly distributed along the cornified layer of th" @default.
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• W2092160553 date "2009-06-01" @default.
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• W2092160553 title "Dual Role of COUP-TF-Interacting Protein 2 in Epidermal Homeostasis and Permeability Barrier Formation" @default.
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