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Expression of the Xenopus homolog of the mammalian transcription factor AP-2alpha (XAP-2) is activated throughout the animal hemisphere shortly after the midblastula transition, and becomes restricted to prospective epidermis by the end of gastrulation, under the control of BMP signal modulation. Elevated expression in the future neural crest region begins at this time. Ectopic expression of XAP-2 can restore transcription of epidermal genes in neuralized ectoderm, both in ectodermal explants and in the intact embryo. Likewise, loss of XAP-2 function, accomplished by injection of antisense oligonucleotides or by overexpression of antimorphic XAP-2 derivatives, leads to loss of epidermal and gain of neural gene expression. These treatments also result in gastrulation failure. Thus, AP-2 is a critical regulator of ectodermal determination that is required for normal epidermal development and morphogenesis in the frog embryo.
FIG. 1. Early expression of XAP-2. Whole mount in situ hybridizations
at stage 10.25 (A) and stage 12.5–13 (B) to antisense XAP-2
probe, stained with BM-purple. In panel A, the arrowhead indicates
hybridization to a chordin probe to mark the dorsal lip (magentaphos
stain). At the beginning of gastrulation, XAP-2 RNA was
found throughout the ectoderm, then becomes excluded from the
neural plate by the end of gastrulation, at which time high level
expression commences in the cranial neural crest region.
FIG. 2. BMP dependence of XAP-2 expression. (A) Effect of
chordin expression. Fertilized eggs were injected with 250 pg of
RNA encoding chordin, then ectodermal explants were dissected at
stage 7/8 and cultured until sibling embryos reached late gastrula
(st12.5). RNA was extracted and probed by Northern blot using
cDNAs for XAP-2, Zic-r1, or EF1 as a control. Chordin injection
inhibited XAP-2 and simultaneously induced expression of the
neural plate marker Zic-r1. (B) Requirement for protein synthesis.
Embryos were cultured in calcium/magnesium-free medium beginning
at early cleavage stage (16/32-cell), and vitelline envelopes
removed to facilitate continuous dissociation and dispersion, a
procedure which blocks cell-cell communication (Sargent et al.,
1986). Dispersed cells were divided into four pools. Protein synthesis
was inhibited in two of the pools by treatment in 10 g/ml
cycloheximide beginning at the equivalent of stage 7/8 (CHX ).
After 15 min, 50 ng/ml recombinant human BMP4 protein (Research
Diagnostics, Inc., Flanders, NJ) was added to two pools
(BMP ), Ca2 and Mg2 restored to 1 mM each, and the cells
cultured until stage 11. RNA was prepared and analyzed by
Northern blot, using probes for XAP-2, Dlx3, Msx1, and EF1 as a
control for general inhibition of protein synthesis by the CHX
treatment. XAP-2 RNA induction by BMP was inhibited by CHX
treatment, indicating indirect activation by this signaling pathway.
FIG. 3. Rescue of keratin expression in neuralized ectoderm by
XAP-2. Fertilized eggs were injected with 250 pg RNA encoding
chordin, with or without 100 pg RNA encoding XAP-2. Ectodermal
explants were isolated at stage 7–8 and cultured until sibling
embryos reached stage 13, then samples were processed for Northern
blot analysis. Probes were for type I (XK81) and type II (XK76)
embryo-specific epidermal keratins, two epidermal homeodomain
genes, Dlx5 and Msx1, and neural-plate (Zic-r1) or EF1 . Chordin
strongly inhibited all epidermal gene expression. The keratin genes
and Dlx5 were partially restored by addition of XAP-2 RNA. Msx1,
however, was not activated, nor was repression of Zic-r1 observed.
Note that under these conditions endogenous XAP-2 expression
was inhibited, as shown in Fig. 2A and B.
FIG. 4. The XK81 gene is a direct target for XAP-2. (A) Fertilized
eggs were injected with a mixture of RNAs encoding chordin (500
pg) and glucocorticoid-inducible GRXAP-2 (1 ng). Ectodermal explants
were isolated at stage 7–8, and divided into four groups. Two
groups were treated with 10 g/ml cycloheximide (CHX ) to
inhibit protein synthesis. After 15 min, two groups (DEX ) were
treated with 10 M dexamethasone (DEX) to activate the accumulated
GRXAP-2 fusion protein. Controls were treated with an
equivalent volume of the DEX solvent (ethanol) alone. After
culturing to stage 12 equivalent, RNA was isolated and processed
for Northern blot analysis. (A) Hybridization to the XK81 keratin
probe revealed that the GRXAP-2 fusion protein responded as
expected to the DEX administration, resulting in partial recovery of
XK81 expression (DEX vs. DEX-), and that this was not prevented
by cycloheximide (CHX vs. CHX-). Note that different exposures
are shown for XK81 and EF1 probes. (B) Since cycloheximide
treatment has a general inhibitory effect on RNA synthesis in
Xenopus embryos, phosphorimage analysis was performed to normalize
the XK81 expression data to that of the control, EF1 , and
the results shown in histogram format. Based on this analysis,
induction of XK81 by XAP-2 appeared to have a similar magnitude
in the presence or absence of cycloheximide. (C, D) Loss of
competence during gastrulation. Fertilized eggs were injected with
a mixture of chordin and GRXAP-2 RNAs as in panel A. Ectodermal
explants were isolated at stage 7/8, and subsets transferred to
medium containing 10 m DEX at the stages indicated, from stage
8.5 through stage 13. Following culture to the equivalent of stage
20 (to minimize the temporal variable in DEX exposure), RNA was
isolated for Northern blot analysis. Hybridization with an XK81
keratin probe revealed strong recovery of keratin expression when
DEX was added immediately (stage 8.5; note that the uninjected
lane was loaded with a 1/10 dilution to facilitate exposure), but that
this response rapidly declined for subsequent DEX additions.
Hybridization with control EF1 probe showed essentially no
differences, and hybridization with a XAP-2 probe indicated that the injected GRXAP-2 RNA (endogenous XAP-2 hybridization not
visible at this exposure) was not differentially affected by the DEX
treatment. Phosphorimager quantification of Northern blot data
are shown in panel D.
FIG. 6. Loss-of-function analysis; intact embryos. Fertilized eggs
were injected with either (A) 600 pg of ASO281, (B) a mixture of 600
pg ASO281 and 100 pg of XAP-2* RNA, or (C) 600 pg ASO281 with
50 pg each XK81 and XK76, and cultured to stage 20. Control,
uninjected embryos are shown in panel D. ASO281 treatment
resulted in extensive gastrulation failure in 29/29 of the embryos,
with pigmented ectoderm collapsed into a small sac. Co-injection
of XAP-2* RNA rescued essentially normal development in about
12/31 of embryos, partially rescued another 9/31, and had little
effect on the remainder. An example of each is shown in panel B.
Keratin mRNA injection had little if effect; 24/27 were essentially
identical to embryos injected with ASO281 alone, although the
remaining three showed some a more moderate phenotype. One of
the three partially normal embryos is shown in panel C (embryo on
the right).
