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First published online February 10, 2005
doi: 10.1242/10.1242/dev.01641

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BMP4-dependent expression of Xenopus Grainyhead-like 1 is essential for epidermal differentiation

¹ Department of Hematology/Oncology, St Jude Children's Research Hospital, Memphis, TN 38105, USA
² Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38105, USA
³ Department of Pathology, St Jude Children's Research Hospital, Memphis, TN 38105, USA
⁴ Rotary Bone Marrow Laboratory, Melbourne, Australia
⁵ Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN 38105, USA

View larger version (63K): [in a new window]   Fig. 1. Expression of XGrhl1 is restricted to the ectodermal layer. (A) RT-PCR analysis of XGrhl1 expression in staged embryos during development (n=5). Ornithine decarboxylase (ODC) gene expression was used as a control for RNA concentration. -RT, no reverse transcriptase; -, no RNA. (B) XGrhl1 expression is restricted to the superficial epidermal layer of the developing embryo. RT-PCR analysis of XGrhl1 and developmental gene expression in different ectodermal tissues at stage 11 of Xenopus development.

View larger version (71K): [in a new window]   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).

View larger version (45K): [in a new window]   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 ß-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). ß-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).

View larger version (62K): [in a new window]   Fig. 4. XGrhl1 is downstream of the BMP4 receptor, and can modulate endogenous BMP4-responsive targets. (A) Dissociated animal cap assays were performed as indicated in the schematic. Dispersed animal pole cells were incubated in increasing doses of recombinant human BMP4 (hrBMP4) (B,C) or one-cell embryos were injected with XGrhl1 mRNA (D), allowed to develop to stage 9, and animal pole explants were dissected, dispersed and allowed to re-aggregate. Aggregates were allowed to mature until stage 18, harvested, RNA prepared and assayed by semi-quantitative RT-PCR. (B) Exposure of dissociated ectodermal cells to hrBMP4 results in an increase in epidermal-specific gene expression, including XGrhl1 and XK81A1. (C) XGrhl1 is not an immediate early response gene. Dissociated caps were incubated in BMP4 in the presence/absence of the protein synthesis inhibitor cycloheximide (10 µg/ml; CHX). (D) Ectopic expression of XGrhl1 in dispersed cap cells results in upregulation of epidermal-specific gene expression.

View larger version (75K): [in a new window]   Fig. 5. Expression of a dominant-negative mutant of XGrhl1 (227XGrhl1) results in global defects in epidermal differentiation. (A) Defective epidermal structures observed in tadpoles (arrowheads) in 227XGrhl1-injected (227) 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 with 227XGrhl1 transcripts. (C) Abnormal accumulation of pigment vesicles in 227XGrhl1-expressing epidermis (white arrowhead). (D) Transverse section through head structures of 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 227XGrhl1 RNA affected cells (right).

View larger version (56K): [in a new window]   Fig. 6. Inhibition of XGrhl1 activity or expression blocks XK81A1 keratin expression in vivo. (A) Expression of 227XGrhl1 blocks endogenous XK81A1 expression specifically. Embryos were injected in one animal blastomere at the four-cell stage with XGrhl1 (2 ng) and/or 227XGrhl1 (1 ng)-encoding transcripts. In situ hybridization for XK81A1 expression (blue stain) was performed on stage 14 embryos. ß-Galactosidase, a lineage tracer, stained red. The broken white lines delineate areas of 227XGrhl1 expression. (B) 227XGrhl1-encoding transcripts (1 ng) do not block expression of other BMP4 signaling pathway components. The product of a factor chimera [227XGrhl1 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 ß-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 the 227XGrhl1-expressing mutants in Fig. 5.

View larger version (23K): [in a new window]   Fig. 7. XGrhl1 modulates XK81A1 keratin promoter activity specifically. (A) A region upstream of the XK81A1 transcriptional start site (red) has significant homology with a Drosophila GRH consensus sequence. Green type indicates a previously defined XAP-2-binding sequence. Mutation of a 4 bp motif (blue line; M2) blocks XGRHL1 binding. (B) Loss of the -200 XGRHL1-binding motif results in a significant defect in XK81A1 promoter activity. Whole embryo reporter assays were performed using XK81A1 keratin promoter sequences linked in cis to the firefly luciferase gene. All values were standardized to the full-length wild-type promoter sequence (arbitrary value of 1). KP487, previously defined XK81A1 promoter; M157E, deletion of previously defined AP-2-binding motif; M2, mutation of the putative XGRHL1-binding site; M2M157E, AP-2/XGRHL1 double mutant; -113KP487, deletion of promoter region with epidermal-specific activity. (C) Expression of 227XGrhl1 blocks XK81A1 promoter activity. The full-length XK81A1 luciferase reporter construct (KP487), a XK81A1 reporter construct in which the XGRHL1-binding site was mutated (M2), or a luciferase only control (pGL3) was co-injected into animal pole blastomeres at the four-cell stage with or without 227XGrhl1-encoding transcripts. Luciferase reporter assays were performed as described in B. All values were standardized with respect to the full-length wild-type promoter sequence (arbitrary value of 1).

View larger version (14K): [in a new window]   Fig. 8. XGrhl1, with XAP-2, modulates BMP4-dependent epidermal structural gene expression. BMP4-dependent phosphorylation of XMad1 induces IER gene expression directly (unbroken arrow). After an unknown number of intermediary steps (broken arrow), these factors activate Dlx3, Dlx5, AP-2 and XGrhl1 gene expression. Blue arrows indicate newly identified tissue-specific components of the BMP4-signaling cascade. XGRHL1 and AP-2, cooperatively, activate structural gene expression directly (blue and green unbroken arrows). The molecular mechanism(s) by which Dlx3/Dlx5 facilitates gene activation in this context are currently unclear (broken arrow). XGrhl1 also stimulates AP-2 and Dlx3/Dlx5 gene expression (broken red arrows). For simplicity, all BMP4-mediated events are not shown.

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