May 1, 1990;
The Xenopus XIHbox 6 homeo protein, a marker of posterior neural induction, is expressed in proliferating neurons.
XIHbox 6 is an early spatially restricted marker for molecular studies of neural induction. The sequence of the full-length XIHbox 6 protein is reported. An antibody raised against a beta-galactosidase/XIHbox 6 fusion protein was used to analyze the expression of XIHbox 6 proteins during frog embryogenesis. The anterior
border of XIHbox 6 expression lies just posterior
of the hindbrain/spinal cord junction. Immunostaining extends the entire length of the spinal cord. A much weaker transient expression with a similar anterior
border is observed in mesoderm
. Almost all nuclei in the newly closed spinal cord contain XIHbox 6. The number of positive nuclei decreases over the next stages of development, until in later embryos XIHbox 6 is restricted to nuclei of the dividing neuroepithelium, and not the mantle or marginal zones of the spinal cord. When the limb
buds begin to grow, there is a second burst of XIHbox 6 expression in proliferating neurons of the cervical and lumbar enlargements, where nerves arise that supply the limbs. The data suggest that XIHbox 6 expression is spatially and temporally restricted to immature neurons of the spinal cord, before their differentiation into mature neurons.
[+] show captions
hoxb9 ( homeobox B9) gene expression in Xenopus laevis embryo, assayed via immunohistochemistry, NF stage 32, lateral view, anterior left, dorsal up.
Fig. 1. Construction of XlHbox 6//?-galactosidase fusion
proteins. The full-length XlHbox 6 protein is represented as
a box with the homeodomain filled in black. FP1 was made
from a filled-in EcoRl cDNA fragment (Sharpe et al. 1987)
cloned into filled-in SamHI-cut pTRBO. FP2 was made by
cloning a Smal fragment from this same cDNA fragment
into the same recipient vector. As stated in the text, all
immunostaining patterns described in this paper are
identical with antibodies purified on affinity matrices
containing either FP1 or FP2.
Fig. 2. Sequence of the XlHbox 6 homeodomain protein. (A) The nucleotide and deduced amino acid sequence of the
XlHbox 6 homeodomain protein. The 61 amino acid homeodomain is shown in bold and amino acids are numbered in
intervals of 10. The position of a 1.59 kb intron is indicated. Six glutamine residues start at Gln70. (B) Comparison of the
XlHbox 6 homeodomain sequence with those of other genes with Abd-B-like homeodomains. Sequence sources are: Hox-
2.5 (Bogarad et al. 1989); Hox-1.7 (Rubin et al. 1987); Hox-3.2 (Breier et al. 1988); Hox-5.2 (Dolle and Duboule, 1989);
HB4 (Dolecki et al. 1988); AbdB (Celniker et al. 1989); Antp (McGinnis et al. 1984; Scott and Weiner, 1984). A dash
indicates identity with the XlHbox 6 amino acid residue above it. The number in brackets indicates the percentage similarity
allowing for conservative amino acid replacements. The conservative changes allowed in this estimate were: T,S; F,Y; E,D;
Q,N; K,R; L,V,I,M.
Fig. 3. Expression of XlHbox 6 antigens during Xenopus
embryogenesis. Albino embryos were fixed at various stages
and subjected to whole-mount immunostaining with
XlHbox 6 antibodies. (A and A') Lateral and dorsal views
of stage 23-24; lateral views in all subsequent panels.
(B) Stage 26; (C) stage 29; (D) stage 32. Positive staining is
at all times nuclear. The anterior border, which is constant
throughout all of these stages, is marked by an arrowhead.
The apparent staining in the cement gland (eg) and in the
somite nuclei (myo) is artefactual as explained in the text.
Abbreviations: arch, archenteron; endo, endoderm;
eg, cement gland; myo, myotome; LPM, lateral plate
Fig. 4. Expression of XlHbox 6 antigens is limited to
posterior central nervous system. (A) A tailbud embryo
(stage 25) was paraffin sectioned and immunostained for
XlHbox 6 antigens. Black areas in the spinal cord represent
immunostaining. This embryo was curled so that an anterior
section through the brain is at left and a section through the
spinal cord is at right. (B) Expression of XlHbox 6 in a
stage 38 spinal cord. This is a slightly skewed, or oblique,
lateral section. The periodic row of black dots (arrowheads)
represents staining in a very few nuclei at the ependymal
surface of the spinal cord. Abbreviations: ec, ependymal
canal; sc, spinal cord; myo, myotomes; hb, hindbrain;
no, notochord; endo, endoderm; LPM, lateral plate
mesoderm; df, dorsal fin.
Fig. 5. Most neuronal precursor cells express XlHbox 6 in stage 24 spinal cord. Dorsal is uppermost in all panels.
(A) Section at high power magnification immunostained with anti-XlHbox 6 antibodies. (B) Same section counterstained
with Hoechst 33258 viewed under dark field illumination. Note that nearly all nuclei appear to be darkly stained in panel A
and that the Hoechst fluorescence in these nuclei is quenched by the immunostaining reaction product. (C) XlHbox 6
expression in lateral plate mesoderm. Transverse section of stage 25-26 tadpole decorated with XlHbox 6 antibodies and
overstained to show more clearly the less intense staining in the lateral plate mesoderm nuclei. Abbreviations:
ecto, ectoderm; ec, ependymal canal; sc, spinal cord; myo, myotome; LPM, lateral plate mesoderm; endo, endoderm;
arch, archenteron; no, notochord, remains of blastopore.
Fig. 6. Distribution of XlHbox 6 antigens in the spinal cord
of stage 27 tadpole. A transverse section was (A)
immunostained with XlHbox 6 antibodies and (B)
counterstained with Hoechst 33258. At this stage of
development, the spinal cord starts to display a degree of
differentiation into defined layers. Now, not all of the spinal
cord nuclei express XlHbox 6 antigens. The more
peripheral nuclei which probably correspond to
differentiated primary neurons are no longer stained.
Immunostaining is confined to the entire inner layer
(ependymal layer) and a few cells that have migrated
laterally away from this mitotic zone. Note the nuclei that
are much less intensely immunostained in the lateral plate
mesoderm indicated by arrowheads. Abbreviations:
ec, ependymal canal; sc, spinal cord; no, notochord;
endo, endoderm; myo, myotome; ecto, ectoderm;
LPM, lateral plate mesoderm.
Fig. 7. Expression of XlHbox 6 antigens at early
metamorphosis. A stage 49 albino tadpole was
immunostained in whole-mount with anti-XlHbox 6
antibodies and then dissected under clearing solution to
remove the central nervous system and tightly associated
structures. (A) Low power magnification showing two
condensations of intensely stained nuclei in the spinal cord
(CE and LE). The arrow labelled Ant.B shows that the
same anterior border of XlHbox 6 expression (Ant.B.) is
observed as in earlier stages. (B) Dorsal view at higher
power showing the cervical and lumbar condensations in
more detail. Note the sympathetic ganglia which represent a
possible additional site of XlHbox 6 expression. (C) Lateral
view of the same tissue. Note that there are many
immunostained nuclei between CE and LE, and also
posteriorly of LE (arrowheads). Abbreviations;
Ant.B., anterior boundary of XlHbox 6 expression;
hb, hindbrain; sc, spinal cord; myo, myotome;
sg, sympathetic ganglia; no, notochord; CE, cervical
enlargement; LE, lumbar enlargement.