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Development
2005 Mar 01;1325:1021-34. doi: 10.1242/dev.01641.
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BMP4-dependent expression of Xenopus Grainyhead-like 1 is essential for epidermal differentiation.
Tao J
,
Kuliyev E
,
Wang X
,
Li X
,
Wilanowski T
,
Jane SM
,
Mead PE
,
Cunningham JM
.
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Morphogen-dependent epidermal-specific transacting factors have not been defined in vertebrates. We demonstrate that a member of the grainyhead transcription factor family, Grainyhead-like 1 (XGrhl1) is essential for ectodermal ontogeny in Xenopus laevis. Expression of this factor is restricted to epidermal cells. Moreover, XGrhl1 is regulated by the BMP4 signaling cascade. Disruption of XGrhl1 activity in vivo results in a severe defect in terminal epidermal differentiation, with inhibition of XK81A1 epidermal keratin gene expression, a key target of BMP4 signaling. Furthermore, transcription of the XK81A1 gene is modulated directly by binding of XGRHL1 to a promoter-localized binding motif that is essential for high-level expression. These results establish a novel developmental role for XGrhl1 as a crucial tissue-specific regulator of vertebrate epidermal differentiation.
Fig. 2. Expression of XGrhl1 is restricted to tissues with an epidermal fate. Whole-mount in situ hybridization analysis of XGrhl1 expression in staged embryos. Maternal XGrhl1 transcripts are detected in the animal pole of early cleavage (A), blastula (B-D) and gastrula (E-H) stage embryos. Embryos are in a lateral orientation except for F (dorsal) and G (ventral). Zygotic expression is observed in presumptive epidermis through neurulation (I-L; dorsal orientation), tailbud and swimming tadpole stages (Q and R respectively; lateral orientation, anterior towards the left). Transverse embryonic section at stage 15 (M) demonstrates XGrhl1 expression (blue stain) in the presumptive epidermis, this stain being absent from the neural plate (arrowhead). By stage 21 (P), the neural plate is closed and covered by epidermis. Epidermal keratin (XK81A1) and zygotic XGrhl1 have a similar pattern of expression (N,O; anterior and dorsal orientation, respectively).
Fig. 3. XGrhl1 expression is dependent on the BMP4 signaling pathway. Microinjection of a dominant-negative BMP receptor mutant (tBR) results in a decrease in XGrhl1 and XK81A1 expression with a concomitant increase in transcripts encoding the NCAM neural marker. ODC was used as a control for RNA recovery. Uninjected, uninjected cap; WE, whole embryo. (A) Enforced expression of the BMP4 antagonist noggin represses both XGrhl1 and XK81A1 expression. (B) RT-PCR analysis of animal pole explants at stage 21 injected at the one-cell stage with noggin mRNA. (C) Factors antagonizing BMP4 signaling block XGrhl1 expression in vivo. In situ hybridization analysis (blue) for XGrhl1 in embryos injected with neuralizing [noggin (600 pg), Ngeminin (1 ng)] or epidermal-inducing factors [XVent-2 (400 pg), BMP4 (1 ng)]. Embryos were co-injected with β-galactosidase mRNA (50 pg; stained red) for lineage tracing. Vent-2 and geminin, anteroventral view; noggin, lateral view; BMP4, ventral view. (D) Co-injection of xMad1 rescues XGrhl1 expression in tBR-expressing explants. RT-PCR analysis of animal pole explants injected at the one-cell stage with either tBR alone (2 ng), or tBR with increasing concentrations of xMAD1 encoding RNA. Induction of XLMO2 indicates functional XMad1 transcripts (Mead et al., 2001). (E) Ectopic expression of XGrhl1 does not alter epidermal specification or neuralization in vivo. In situ hybridization for XK81A1 expression (blue) of stage 14 embryos injected at the one- to four-cell stage in the animal pole with XGrhl1-encoding transcripts (4 ng). β-Galactosidase (red stain) was used as a lineage tracer. The upper panels illustrate representative embryos injected in blastomeres with an epidermal fate (lateral orientation); lower panels are representative of blastomeres with a neural fate (anterior orientation). (F) Co-injection of XGrhl1 transcripts does not rescue tBR-induced neuralization. RT-PCR analysis of animal cap explants injected at the one-cell stage with either tBR (2 ng) alone or tBR with XGrhl1 encoding RNA (4 ng).
Fig. 5. Expression of a dominant-negative mutant of XGrhl1 (δ227XGrhl1) results in global defects in epidermal differentiation. (A) Defective epidermal structures observed in tadpoles (arrowheads) in δ227XGrhl1-injected (δ227) but not wild-type (wt) embryos. All embryos illustrated are stage 40 and were injected in one animal pole blastomere at the eight-cell stage. (B) Defects in trunk and tail structures observed in embryos injected withδ 227XGrhl1 transcripts. (C) Abnormal accumulation of pigment vesicles in δ227XGrhl1-expressing epidermis (white arrowhead). (D) Transverse section through head structures ofδ 227XGrhl1-injected tadpole. Left panels show normal epidermal structure with discrete outer epithelial (OEL) and inner sensorial layers (ISL). A marked increase in the thickness and disorganization of the epidermis is observed in magnified cross-section of injected regions (right). Note the persistence of yolk sac platelets (arrows) and embryonic pigment granules (arrowhead), large round nuclei and prominent nucleoli of OEL. (E,F) Transverse sections through embryonic trunk (E) and fin (F). Middle panels shows a low-power magnification through regions. Key structures are indicated: DNT, dorsal neural tube; SM, somite; NC, notochord; pnt, pronephric duct. Side panels at higher magnification show differences between normal bi-layer (left) and δ227XGrhl1 RNA affected cells (right).
Fig. 6. Inhibition of XGrhl1 activity or expression blocks XK81A1 keratin expression in vivo. (A) Expression of δ227XGrhl1 blocks endogenous XK81A1 expression specifically. Embryos were injected in one animal blastomere at the four-cell stage with XGrhl1 (2 ng) and/or δ227XGrhl1 (1 ng)-encoding transcripts. In situ hybridization for XK81A1 expression (blue stain) was performed on stage 14 embryos. β-Galactosidase, a lineage tracer, stained red. The broken white lines delineate areas of δ227XGrhl1 expression. (B) δ227XGrhl1-encoding transcripts (1 ng) do not block expression of other BMP4 signaling pathway components. The product of a factor chimera [δ227XGrhl1 sequences linked in frame with enhanced green fluorescent protein cDNA (EGFP)] was detected in nuclei of transfected cells (extreme right panel), consistent with appropriate nuclear localization. (C) Injection of a XGrhl1-targeted MO blocks endogenous XK81A1 keratin gene expression specifically. Embryos were injected into one animal blastomere at the four-cell stage with XGrhl1MO±M-XGr (a MO-resistant XGRhl1- expressing RNA transcript) (upper left). In situ hybridization for XK81A1 expression (blue stain) and a β-galactosidase lineage tracer (red) was performed on stage 14 embryos. The XGrhl1-MO mediated block in XK81A1 gene expression is rescued partially by co-expression of M-XGr mRNA (lower left). Coincident blue and red staining is indicated (arrowheads). A control morpholino (CMO, upper right) or M-XGr alone (lower right) failed to affect normal development. (D) Injection of XGrhl1-targeted MO induces an epidermal defect in maturing tadpole specifically. Defects in head and trunk structures representative of those observed in embryos injected with XGrhl1-specific MO in one animal pole blastomere at the eight-cell stage are shown. Epidermal and pigment changes in head and trunk are observed in XGrhl1MO-injected embryos (when compared with CMO-injected embryos) that are identical to those seen with theδ 227XGrhl1-expressing mutants in Fig. 5.
