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Dev Biol
2009 Jan 01;3251:249-62. doi: 10.1016/j.ydbio.2008.10.031.
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Maternal Interferon Regulatory Factor 6 is required for the differentiation of primary superficial epithelia in Danio and Xenopus embryos.
Sabel JL
,
d'Alençon C
,
O'Brien EK
,
Van Otterloo E
,
Lutz K
,
Cuykendall TN
,
Schutte BC
,
Houston DW
,
Cornell RA
.
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Early in the development of animal embryos, superficial cells of the blastula form a distinct lineage and adopt an epithelial morphology. In different animals, the fate of these primary superficial epithelial (PSE) cells varies, and it is unclear whether pathways governing segregation of blastomeres into the PSE lineage are conserved. Mutations in the gene encoding Interferon Regulatory Factor 6 (IRF6) are associated with syndromic and non-syndromic forms of cleft lip and palate, consistent with a role for Irf6 in development of oral epithelia, and mouse Irf6 targeted null mutant embryos display abnormal differentiation of oral epithelia and skin. In Danio rerio (zebrafish) and Xenopus laevis (African clawed frog) embryos, zygotic irf6 transcripts are present in many epithelial tissues including the presumptive PSE cells and maternal irf6 transcripts are present throughout all cells at the blastula stage. Injection of antisense oligonucleotides with ability to disrupt translation of irf6 transcripts caused little or no effect on development. By contrast, injection of RNA encoding a putative dominant negative Irf6 caused epiboly arrest, loss of gene expression characteristic of the EVL, and rupture of the embryo at late gastrula stage. The dominant negative Irf6 disrupted EVL gene expression in a cell autonomous fashion. These results suggest that Irf6 translated in the oocyte or unfertilized egg suffices for early development. Supporting the importance of maternal Irf6, we show that depletion of maternal irf6 transcripts in X. laevis embryos leads to gastrulation defects and rupture of the superficialepithelium. These experiments reveal a conserved role for maternally-encoded Irf6 in differentiation of a simple epithelium in X. laevis and D. rerio. This epithelium constitutes a novel model tissue in which to explore the Irf6 regulatory pathway.
Fig. 5. Expression of irf6 in Xenopus laevis. (A) RT-PCR analysis of irf6 expression at different stages of development. Developmental stages are indicated at the top (Nieuwkoop and Faber, 1956). − RT, negative control reaction without reverse transcriptase. (B–G) Whole mount in situ hybridization for irf6. (B) Stage 10.5, left is vegetal view, arrow indicates expression in the dorsal marginal zone. Right is animal pole view. (C) Stage 11, expression is throughout marginal zone, but beginning to be lost from dorsal midline. (D) Stage 12.5, expression around blastopore, absent from prospective notochord. (E) Stage 18, expression flanking prospective tailbud region, arrow. (F) Stage 28 and (G) stage 30, notable expression in tailbud, arrowheads. (H) Section of animal cap hybridized with an irrelevant control probe (xnr3). (I) Section of animal cap hybridized with an antisense irf6 probe. Diffuse expression is found in both superficial and deep layers. (J) Quantitative RT-PCR of irf6 (green) and esr6e (blue) expression in ectodermal explants; st. 10 (stage 10 whole embryo), DEL (deep layer explant), SE (superficial layer explant).
itln2 (intelectin 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, dorsal view, anteriorleft.
irf6 (interferon regulatory factor 6) gene expression in Xenopus laevis embryos, assayed via in situ hybridization, NF stage 10.5, vegetal/blastoporal view, dorsal up (left) and animal pole view ( right).
irf6 (interferon regulatory factor 6) gene expression in Xenopus laevis embryos, assayed via in situ hybridization, NF stage 11, vegetal/blastoporal view.
irf6 (interferon regulatory factor 6) gene expression in Xenopus laevis embryos, assayed via in situ hybridization, NF stage 12.5, dorso-posterior view, anterior up.
irf6 (interferon regulatory factor 6) gene expression in Xenopus laevis embryos, assayed via in situ hybridization, NF stage 18, posterior view, dorsal up.
irf6 (interferon regulatory factor 6) gene expression in bisected Xenopus laevis embryo, mid-sagittal section, assayed via in situ hybridization, NF stage 10.5, animal hemisphere up.
irf6 (interferon regulatory factor 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27/28, lateral view, anteriorleft, dorsal up.
Fig. 6. Defects in development of superficialepithelium in irf6DBD-injected X. laevis embryos. (A) Animal and, (C) vegetal views of stage 11 uninjected controls (Un). (B) Animal and (D) vegetal views of stage 11 embryos injected with 2Â ng D. rerio irf6DBD mRNA. (E) Uninjected (Un) and (F) irf6DBD-injected stage 14 embryos. Arrow in (F) indicates lesion in the ectoderm. (G) RT-PCR analysis of SE markers, esr6e and grhl3 in control (Un) and irf6DBD-injected embryos and explants. St. 11, whole embryo stage 11; cap, stage 11 animal cap; DEL, deep layer explants; SE, superficial layer explants.
Fig. 7. Maternal Irf6 is required for SE specification in X. laevis. (A) Control (left side) and Irf6-depleted (right side) embryos at stage 12. Upper row is a vegetal view; lower row is an animal view. (B) Control (left embryo) and Irf6-depleted (right four embryos) embryos at stage 24. Arrow indicates epidermal lesion in an Irf6-depleted embryo. (C) Control uninjected embryos. (D) embryos injected with 24 ng irf6 MO at the 1–2 cell stage. (E) Quantitative RT-PCR of esr6e, grhl3, sox11, irf6, sox2 and xbra expression in uninjected embryos (Un) and Irf6-depleted embryos (irf6 MO) at the indicated stages. Graphs represent data from a single PCR run.
Fig. 8. Maternal Irf6 is required for SE differentiation in X. laevis. (A, B) Whole mount in situ hybridization of stage 14, control (A) uninjected (Un) and (B) Irf6-depleted (irf6 MO) embryos for intelectin 2 (intel2). Lateral view is shown, anterior is to the left. Inset shows close up views of the epidermis in a bisected embryo. (C, D) Sections of (C) uninjected and (D) Irf6-depleted stage 13 embryos stained with mAb LP3K. (E, F) Sections of, (E) control and, (F) Irf6-depleted stage 22 embryos stained with Azan. Dotted lines indicate the thickness of the ectoderm; arrows indicate the location of embryonic pigment.
Fig. 9. Specificity of Irf6 depletion in Xenopus. (A) Quantitative RT-PCR of esr6e and grhl3 expression in controls (Un), Irf6-depleted embryos (irf6 MO) and depleted embryos injected with 50 pg irf6.2 mRNA (MO + RNA), or (B) with 250 pg irf6.2 mRNA (MO + RNA). (C– E) Whole mount in situ hybridization for intelectin 2 at stage 14, (C) control uninjected (Un), (D) Irf6-depleted (irf6 MO) and (E) rescued embryos (irf6 MO + irf6 RNA).
itln2 (intelectin 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, lateral view, anteriorleft, and in section (inset)
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