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The transcription factor AP2 (TFAP2) has an important role in regulating gene expression in both epidermis and neural crest cells. In order to further characterize these functions we have used a hormone inducible TFAP2alpha fusion protein in a Xenopus animal cap assay to identify downstream targets of this factor. The most common pattern comprised genes predominantly expressed in the epidermis. A second group was expressed at high levels in the neural crest, but all of these were also expressed in the epidermis as well as in other tissues in which TFAP2alpha has not been detected, suggesting modular control involving both TFAP2-dependent and TFAP2-independent components. In addition, a few strongly induced genes did not overlap at all in expression pattern with that of TFAP2alpha in the early embryo, and were also activated precociously in the experimentally manipulated ectoderm, and thus likely represent inappropriate regulatory interactions. A final group was identified that were repressed by TFAP2alpha and were expressed in the neural plate. These results provide further support for the importance of TFAP2alpha in ectoderm development, and also highlight the molecular linkage between the epidermis and neural crest in the Xenopus embryo.
Fig. 1. Animal caps isolated from embryos injected with
Wnt3a (300 pg), chordin (3 ng) and Dex-inducible TFAP2α
(GRAP2, 600 pg) were treated from stage 8 until stage 14 with
10 μM Dex (Dex+) or solvent alone (Dex–). Caps from uninjected
embryos (UI) were also collected. RNA isolated at stage 14 was
analyzed by Northern blotting with neural crest (Slug, Sox9),
epidermal (Id2, XK81) and neural plate (Sox2) markers. Neural
crest and epidermal gene expression was induced by
activating TFAP2α, while neural plate was repressed (Sox2).
EF1α probe was used to check RNA integrity, and equal loading
monitored by ethidium bromide staining (18S).
Fig. 2. Induced genes showing predominantly epidermal
expression. Where identity of gene products, when known,
indicated in parentheses. (A) 066j24 (chitobiase); (B) 110o11
(epithelial membrane protein 2); (C) 103p10; (D) 108j09 (Zona
Occludens protein 3 – ZO3); (E) 106h09 (calpain 8); (F) 107p17;
(G) 108o19 (BLIMP-1); (H) 085p16; (I) 107h17 (Id2); (J) sagittal
section of embryo shown in (I).
Fig. 3. Co-induction of epidermal and neural crest gene
expression by attenuated bone morphogenetic proteins (BMP)
and Wnt/beta catenin signaling. Animal caps isolated from
uninjected (UI), chordin RNA injected (250 pg) or chordin and
Wnt3a RNA injected (250 pg, 300 pg, respectively), cultured to
sibling stage 14 and analyzed by Northern blot. Epidermal
gene expression (keratin XK81, and cDNA o66j24, chitobiase;
Chito.) was strongly induced, but to a level less than in
untreated, isolated ectoderm (UI). Neural crest (Slug) was also
strongly induced, while neural plate (Sox2) was reduced. EF1α
probe was used to check RNA integrity, and equal loading
monitored by ethidium bromide staining (18S).
Fig. 4. Neural crest patterns. Four induced genes showing
strong expression in neural crest (indicated by red arrows), in
addition to other sites. (A) 099c17 (55% identity to RTP-801
gene); (B) 097h17 (protocadherin PCNS); (C) 085p13 (Myosin
X); (D) 087l09 (Inca).
Fig. 5. Inappropriately induced genes. (A) 83j13 and (B)
106g05 were two cDNA showing a punctate pattern in embryos
at stage 25 or later. (C) Northern analysis showing that neither
RNA could be detected until after stage 15, and neither was
induced by injection of chordin and Wnt3a RNA, whereas
activation of GRAP2 with Dex yielded strong induction.
Treatment of uninjected animal caps with Dex did not induce
either cDNA detectably. Ethidium bromide staining of 18S rRNA
shown as a control for equal loading.
Fig. 6. Dependence of epidermal genes on TFAP2 activity.
Animal caps from uninjected embryos (UI) or from embryos
injected with RNA encoding dominant negative TFAP2α (DN)
were analyzed by Northern blot hybridization with probes
corresponding to the epidermally expressed mRNA (Fig. 2).
The epidermal keratin XK81 – which is a direct TFAP2 target –
was used as a control. XK81 and five of the mRNA were
significantly reduced (top 6 panels), while the remaining four
showed little if any effect. Ethidium bromide staining of 28S
rRNA shown as a control for equal loading.
Fig. 7. Genes repressed by TFAP2α activation. Three genes
with predominantly neural plate expression selected from a
group of 10 that were strongly reduced in the initial screen. (A)
88m05 (nephrin); (B) 104k22; (C) 78k17, Xenopus laevis
pinhead.
capn8.3 (calpain 8 gene3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior up.
emp2 (epithelial membrane protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, lateral view, anterior up, dorsal right.
tjp2 (tight junction protein 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorsal view, anterior up.
