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A Xenopus laevis homeobox gene, Xhox3, has been isolated using the homeobox of the Drosophila pair-rule gene even skipped as a hybridization probe. Xhox3 is first transcribed at the midblastula transition; RNA levels peak at the early neurula stage and decrease thereafter. During the early period of Xhox3 expression, the gastrula and neurula stages, transcripts are found in a graded fashion along the anteroposterior (A-P) axis in the mesoderm and are most concentrated at the posterior pole. In the late period of expression, the tailbud and tadpole stage, transcripts are concentrated at the two ends of the embryo: in the anteriornervous system and posteriortail bud. Analysis of Xhox3 expression in experimentally perturbed embryos shows that different A-P fates in the mesoderm are correlated with different levels of Xhox3 expression. Based on these results and those with other frog homeobox genes, we propose a role for homeobox genes in the patterning of the A-P embryonic axis.
Fig. 1. (A) Xhox3 genomic and cDNA plasmid clones. The
black box represents the homeobox. The broken lines show
the position of the different probes used for hybridization
experiments: GP, genomic probe; RP, RNase protection
probe. The Eco RI sites shown in parenthesis are derived
from the cDNA cloning protocol. (B) Amino acid sequence
of the even skipped (Macdonald et al. 1986), Xhox3 and
XhoxlA (Harvey et al. 1986) homeoboxes. The brackets
represent the putative helix 2 and helix 3 domains of the
helix-turn-helix motif (see Laughon & Scott, 1984;
Shepherd et al. 1984; Ptashne, 1986). The arrow indicates
the position of the splice site deduced from a comparison of
the genomic and cDNA clones. The vertical lines depict
similar amino acid residues between homeoboxes.
Fig. 3. Graded distribution of Xhox3 transcripts
along the anteroposterior axis during early
development. The schematic respresentations of
the given stages are shown in the top. Stage 11,
mid-gastrula; Stage 13, late gastrula-early
neurula; Stage 15, mid-neurula; Stage 19, late
neurula. D, dorsal; V, ventral; A, anterior; M,
middle; P, posterior. Note the graded
distribution of Xhox3 transcripts with the highest
levels at the posterior pole. As a control for
RNA recovery and quantification, the same RNA
samples were used to determine the abundance
of EF-Ia mRNA which is a reflection of cell
number (Krieg & Melton, 1989). Ten and one
embryo equivalents were used for the Xhox3 and
EF-Ia probes, respectively. Exposure periods
were 4 hours for EF-Ia and 4 days for Xhox3.
Fig. 4. Xhox3 expression in exogastrulated embryos.
Embryos were induced to exogastrulate by incubation in
high salt. Neurula stage exogastrulae were carefully
dissected into ectodennal, mesodermal and endodermal
parts. RNA was extracted and assayed for the presence of
Xhox3, muscle-specific actin and EF-la transcripts by
RNase protection. Note that muscle-specific (MS)-actin and
Xhox3 are expressed only in mesoderm. Ten, one and three
embryo equivalents were used for the Xhox3, EF-la and
MS-actin probes. Exposure periods were 4 hours for EF-la
and MS-actin and 4 days for Xhox3.
Fig. 5. Localization of Xhox3
â¢J/ transcripts in late embryos. Tailbud
(stage 26) or swimming tadpoles
(stage 36) were dissected along the
dorsoventral and anteroposterior
- axes, and the parts were assayed for
the presence of Xhox3 transcripts
by RNase protection. As controls,
EF-Ia, muscle-specific actin, and
N-CAM probes were used with the
same RNA samples. Note that the
MS-actin reflects the position of the
somites (mesoderm) and N-CAM
that of the CNS (neural tissue). The
N-CAM signal in the most posterior
regions is below the level of
detection in this assay, probably
because of the small size of the
spinal cord in this region. D, dorsal;
C, central; V, ventral. Numbers
refer to the different consecutive
dissected parts along the A-P axis.
Ten embryo equivalents were used
for the Xhox3 and N-CAM probes.
One and three embryo equivalents
were used for the EF-Ia and MSactin
probes, respectively. Exposure
periods ranged from 4 hours {EF-Ia
and MS-actin) to 4 days (Xhox3
and N-CAM).
Fig. 6. In situ localization of Xhox3 transcripts in the brain
of tadpole stage (stage 36) embryos. A sagittal section
hybridized with a 35S-labelled Xhox3 antisense RNA probe
is shown after exposure for one month. Xhox3 transcripts
are found only in a small area of the brain (large arrow).
The white arc around the dorsal half of the eye (small
arrow) is due to the presence of pigment cells in the eye of
heterozygote albino embryos at this late stage. Anterior is
to the left, dorsal is on top. A line drawing of the section
(top panel) is shown to orient the hybridization signal in the
dark field photograph (bottom panel), eg, cement gland;
e, eye; en, endoderm; hb, hind brain; mb, mid brain;
n, notochord.
Fig. 7. Localization of Xhox3 transcripts in feeding larvae.
Stage 45 tadpoles were dissected into five parts along the
A-P axis. RNA was extracted and assayed for the presence
of Xhox3 and EF-la transcripts by RNase protection
protocols. The brain of similarly staged tadpoles was
dissected and processed in the same way. Ten and one
larvae equivalents were used for the Xhox3 and EF-la
probes respectively. Exposure times were as in Fig. 4. A,
anterior; P, posterior; T, total or whole larvae; B, brain.
Fig. 8. Xhox3 expression in u.v. and Li treated embryos.
Embryos were treated with u.v. or Li and allowed to
develop to the neurula stage, u.v.-treated embryos with no
head and little trunk and lithium-treated embryos with little
or no trunk were selected. RNA from these embryos was
assayed by RNase protection assays for the presence of
Xhox3 and EF-la transcripts. Ten and one embryos were
used for the Xhox3 and EF-la probes respectively.
Exposure times were as in Fig. 4.
in control, u.v. and Li treated embryos during early
development. The plotted values were obtained by scanning
densitometry of the products of RNase protection assays.
Developmental periods and N/F stages are shown below
the graph. MBT, midblastula transition; G, gastrulation;
N, neurulation; TB, tailbud and TP, tadpole stages. Full
bars indicate the periods of Xhox3 expression when
transcripts are clearly localized. Broken lines indicate the
extent of the expression periods where localization is not
well defined.
