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. 2013 Jan;133(1):68-77.
doi: 10.1038/jid.2012.269. Epub 2012 Aug 30.

Interferon regulatory factor 6 promotes differentiation of the periderm by activating expression of Grainyhead-like 3

Gabriel de la Garza  1 , Jack Robert Schleiffarth, Martine Dunnwald, Anuj Mankad, Jason L Weirather, Gregory Bonde, Stephen Butcher, Tamer A Mansour, Youssef A Kousa, Cindy F Fukazawa, Douglas W Houston, J Robert Manak, Brian C Schutte, Daniel S Wagner, Robert A Cornell
Affiliations

Interferon regulatory factor 6 promotes differentiation of the periderm by activating expression of Grainyhead-like 3

Gabriel de la Garza et al. J Invest Dermatol. 2013 Jan.

Erratum in

  • J Invest Dermatol. 2013 Mar;133(3):859

Abstract

IFN regulatory factor 6 (IRF6) is a transcription factor that, in mammals, is required for the differentiation of skin, breast epithelium, and oral epithelium. However, the transcriptional targets that mediate these effects are currently unknown. In zebrafish and frog embryos, Irf6 is necessary for differentiation of the embryonic superficial epithelium, or periderm. Here we use microarrays to identify genes that are expressed in the zebrafish periderm and whose expression is inhibited by a dominant-negative variant of Irf6 (dnIrf6). These methods identify Grainyhead-like 3 (Grhl3), an ancient regulator of the epidermal permeability barrier, as acting downstream of Irf6. In human keratinocytes, IRF6 binds conserved elements near the GRHL3 [corrected] promoter. We show that one of these elements has enhancer activity in human keratinocytes and zebrafish periderm, suggesting that Irf6 directly stimulates Grhl3 expression in these tissues. Simultaneous inhibition of grhl1 and grhl3 disrupts periderm differentiation in zebrafish, and, intriguingly, forced grhl3 expression restores periderm markers in both zebrafish injected with dnIrf6 and frog embryos depleted of Irf6. Finally, in Irf6-deficient mouse embryos, Grhl3 expression in the periderm and oral epithelium is virtually absent. These results indicate that Grhl3 is a key effector of Irf6 in periderm differentiation.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Microarray analysis of zebrafish periderm
a,c Schematic representation of methods used to create the periderm-enriched (a) and the dnIrf6-inhibited (c) profile. b,d, Pie charts showing genes whose expression is b 3× higher in FACS-sorted GFP-positive versus GFP-negative cells (c) or significantly reduced (>1.7 fold) in dnIrf6-injected compared to lacZ-injected embryos (d). e Venn diagram portraying the overlap of sets in b and d. f–y Animal-pole view of mid-gastrula stage embryos, uninjected or injected with dnIrf6, and processed to reveal expression of the indicated gene. All are expressed in periderm and all except y (epcam) are strongly reduced in dnIrf6-injected embryos. Scale bar: f, 100 μM. z qRT-PCR for genes whose expression is reduced by dnIrf6 and significantly elevated (with respect to dnIrf6 alone) by co-injection with Irf6 (p <0.05).
Figure 2
Figure 2. Enhancer activity of DNA elements near GRHL3
a Positions of Irf6 binding peaks in human keratinocytes from a ChIP-SEQ experiment (Botti et al. 2011), and construction of luciferase and GFP reporter vectors with these peaks. b Results of luciferase reporter assay. c,f,g,h Transient (c,f,g) and stable (h) transgenic zebrafish embryos of the indicated constructs with GFP expression in the periderm. d Schematic representation of a conserved element within GRHL3 intron 15 (gray box), which contains a conserved Irf6 response element (IRF6-RE). Position-weighted matrix for IRF6-RE defined in vitro (Little et al. 2009) and in vivo (Botti et al. 2011). e Electromobility shift assay with human IRF6DBD and a labeled probe that matches 25 base pairs of the hGrhl3 intron 15 that encompass the putative Irf6RE. Scale bars: c, 100 μm; f, 200 μm.
Figure 3
Figure 3. Knockdown of grhl family members results in loss of periderm markers
a,b Animal-pole view of 8hpf embryos injected mosaically with a, biotinylated dextran mixed with lacZ or b, dnXgrhl1 mRNA. Embryos were fixed at shield stage and processed to reveal krt4 expression (purple) and biotin (brown). Periderm cells inheriting lacZ (brown nuclei) express krt4 variably, like periderm cells in uninjected embryos, while those inheriting dnXGrhl1 (brown nuclei) lack krt4. c–j Embryos injected with control MO (c,e,g,i), or grhl1 and grhl3 MOs (d,f,h,j), fixed at 8 hpf, and processed to reveal expression of the indicated gene, or allowed to develop until 10 hpf (i–j), at which time control MO-injected embryo have normal morphology (i), and grhl1/grhl3 MO-injected embryos have ruptured (j). Scale bars: a, 50 μm; a inset 10 μm; c, 100 μm; i, 100 μm.
Figure 4
Figure 4. Injection of grhl3 restores periderm markers in dnIrf6-injected embryos
A–I Images of live (a–f) and DAPI-stained sections of (g–i) embryos injected with the indicated RNA. Insets, magnified images of the boxed area. g–i The doming of the yolk apparent in lacz-injected embyros (g), is lost in dnIrf6-injected embryos (h), and partially restored in dnIrf6-and-grhl3 -injected embryos (i). j qRT-PCR analysis of mRNA levels. capn9, p=0.03, cldnE, p=0.003, ovol1, p=0.048.k–m 5-hpf embryos processed for capn9 expression. n qRT-PCR analysis of levels of the periderm marker xk81a1, and the deep blastomere marker sox11, in Xenopus embryos derived from oocytes injected with control MO or irf6 MO and injected as zygotes with the XGrhl3 mRNA, as indicated. By ANOVA analysis, xk81a1, p< 0.0002, sox11, p< 0.03. Error bars, standard deviation. Scale bars: a, 200 μm; g, 100 μm.
Figure 5
Figure 5. Expression of Grhl3 in Irf6-deficient mice
a qRT-PCR analysis of Grhl3 levels in epidermis harvested at the indicated stage: e14.5, 2 replicates; e17.5, 3 replicates; p<0.03. b–e Anti-Grhl3 immunofluorescence on the indicated tissue at the indcates stages. In the epidermis of sibling control embryos (b), Grhl3 immunoreactivity (IR) is prominent in all epidermal and peridermal nucleii. In mutant embyros (c), Grhl3 IR is diffuse, non-nuclear, and weak to undetectable in the periderm. In the oral epithelium of control siblings (d), Grhl3 IR is strong in in all nuclei, and strongest in the oral periderm. In mutant embryos (e), Grhl3 IR is highly reduced. Scale bar: b, 50 μm.
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