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We have isolated the Xenopus fork head (fkh) box-containing gene XFKH2. XFKH2 appears to be most similar to the rat liver transcription factor HNF-3 alpha, with 64% amino acid identity throughout the protein and 96% amino acid identity within the fkh box. Dissection experiments demonstrate that XFKH2 is present in the vegetal pole and marginal zone but not the animal pole of gastrulae and in equal levels in the dorsal and ventral halves of both gastrulae and neurulae. By in situ hybridization, we know that the dorsal component of expression is concentrated in the notochord in neurulae. In tailbud embryos, XFKH2 is detected in the foregut, the brain, and in two lines of cells just dorsal and ventral to the notochord, the hypochord and the floor plate of the spinal cord. Finally, although explanted animal caps will turn on XFKH2 autonomously at midneurula stage, activin induces early expression at gastrula stage. The early, growth factor-dependent expression does occur in the presence of the protein synthesis inhibitor cycloheximide, suggesting that XFKH2 expression during gastrula stage may be an immediate early response to mesoderm induction.
FIG. 1. Nuc\eotide sequence and conceptual translation of XFKH2. Amino acids are given in the three~ letter code, the eonserved regions are
underlined, and the sequences corresponding to the PCR primers are italicized. A putative polyadenylation signal is in bold.
FlG. 2. Comparison of the amino acid sequences of HNF-3ct, XFKHl, and the deduced translation of XFKH2. Conserved amino acids are in
black boxes. Also shown are comparisons with fkh, HNF~3,8, and HNF-:ty proteins within the three conserved regions.
FIG. 3. Developmental expression of XFKH2. (A) Northern blot prepared
from 2 ~g of poly(At RNA from ovary (lane 1), stage 12 (lane 2),
stage 18 (lane 3), stage 24 (lane 4), stage 36 (lane 5) embryos, stage 36
embryos that had been LiCI treated or uv-irradiated (lanes 6 and 7,
respectively), and adult liver (lane 8). The blot was probed with
XFKH2 and EFla. (B) Northern blot made from 2.5 embryo equivalents
of total RNA from gastrulae at stages 10 (lane 1), 10.25 (lane 2),
10.5 (lane 3), 11 (lane 4), and 11.5 (lane 5). The blot was probed with
XFKH2 and EFla.
FIG. 4. Spatial distribution of XFKH2 mRNA determined by Northern
analysis of dissected embryos. (A) Embryos were dissected into
animal cap (AC), marginal zone (MZ), or vegetal pole (Vg) pieces at
stages 10.5 and 11.5 or into anterior dorsal (AD), posterior dorsal
(PD), and endoderm at (En) pieces at stage 18. Each lane in the Northern
blot shown has total RNA from 10 dissected pieces. Expression of
(a) XFKH2 and (b) EFla . (B) Stage 10.5, 12, and 18 embryos were
divided into equal dorsal (Do) and ventral (Ve) halves and total RNA
was prepared from 10 di ssected halves per sample. Five whole embryo
(WE) equivalents for each stage were loaded as controL Expression of
(a) XFKH2 and (b) EF1a.
FIG.5 Whole mount in situ hybridization with XFKH2. Embryos were probed with the anti-sense XFKH2 probe with the exception ofF which was
probed with the sense XFKH2. The embryos were fixed at stage 11 (A), stage 18 (B, G, and H), stage 26 (C), and stage 34 (D, E, F, and 1). G is a stage 18
embryo ventralized by uv. H (stage 18) and I (stage 34) were dorsalized by LiCl treatment. In A, Vindicates the ventral side and D the dorsal side. The
notochord is designated nc in B, proctodeum by pd in D, and the floor plate by fp, the hypochord by he, and the foregut near the developing liver
diverticulum by fg in E. All hybridizations were done to albino embryos.
FIG. 6. Expression of XFKH2 in activin-treated animal caps. Animal
caps were explanted from stage 9 embryos and incubated in either
LCMR + 0.5% BSA or LCMR + 0.5% BSA and 50 pM activin. Total
RNA from 2.5 intact embryo equivalents (C) or 10 explants treated
either with control buffer (B) or with buffer plus 50 pM activin (A)
were harvested when intact sibling controls reached stage 11.5, stage
12, stage 14, stage 20, and stage 28. The blot was probed with XFKH2
(a), Xbra (b), and EFla (c).
FIG. 7. Induction of XFKH2 by activin in the presence of cycloheximide.
Lanes 1-4 contain total RNA from 15 explants harvested at
control stage 11 and lane 5 contains total RNA from 2.5 embryo equiva~
lents of stage 11 embryos. The explants were treated with buffer alone
(lane 1), 50 pM activin (lane 2), 50 pM activin and 5 ~g/ml cycloheximide
(lane 3), or 5 ,.,gl ml cycloheximide alone (lane 4). The Northern
was probed with (A) XFKH2, (B) cardiac actin, and (C) EF1a.
