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Patterning along the anterior-posterior axis takes place during gastrulation and early neurulation. Homeobox genes like Otx-2 and members of the Hox family have been implicated in this process. The caudal genes in Drosophila and C. elegans have been shown to determine posterior fates. In vertebrates, the caudal genes begin their expression during gastrulation and they take up a posterior position. By injecting sense and antisense RNA of the Xenopus caudal gene Xcad-2, we have studied a number of regulatory interactions among homeobox genes along the anterior-posterior axis. Initially, the Xcad-2 and Otx-2 genes are mutually repressed and, by late gastrulation, they mark the posterior- or anterior-most domains of the embryo, respectively. During late gastrulation and neurulation, Xcad-2 plays an additional regulatory function in relation to the Hox genes. Hox genes normally expressed anteriorly are repressed by Xcad-2 overexpression while those normally expressed posteriorly exhibit more anterior expression. The results show that the caudal genes are part of a posterior determining network which during early gastrulation functions in the subdivision of the embryo into anteriorhead and trunk domains. Later in gastrulation and neurulation these genes play a role in the patterning of the trunk region.
Fig. 1. Phenotypes induced by Xcad-2
overexpression or partial loss of
function. Xenopus embryos were
injected at the 4-cell stage with Xcad-
2 sense (B,E,H), antisense (C,F,I) or
the ÃH3 dominant negative (D,G)
capped RNA at different
concentrations. A sibling control
uninjected embryo is also shown (A).
The effects on the formation of the
head and the length of the trunk are
shown. The type of RNA (s, sense; as,
antisense; dn, dominant negative) and
the amount injected in ng/embryo are
marked.
Fig. 2. Changes in the pattern of Krox-20 expression as a result of
Xcad-2 overexpression and partial loss of function. Embryos injected
with sense (s), antisense (as) or dominant negative (ÃH3; dn) Xcad-2
were studied during neurulation to determine the effects on the Krox-
20 pattern of expression. (A) Control uninjected embryo. Embryos
injected with 0.2 ng (B) or 0.8 ng (C) of Xcad-2 mRNA exhibit
elimination of Krox-20 expression. Injection of 0.2 ng (D) or 0.4 ng
(E) of antisense Xcad-2 RNA shows the reorganization of the head
region. (F) Embryo injected with 0.8 ng of dominant negative Xcad-2
RNA, which results in a similar phenotype as antisense RNA
injections.
Fig. 3. The regulatory interactions between the Xcad-2 and the Otx-2
genes. Early neurula embryos injected with Xcad-2 constructs were
hybridized with an Otx-2-specific probe (A-C). Xenopus embryos
were either uninjected control (A), injected with sense Xcad-2 RNA
at 1.6 ng (B) or antisense Xcad-2 RNA also at 1.6 ng (C). Gastrulastage
embryos injected with Otx-2 sense RNA were probed for Xcad-
2 expression (D-F). (D) Control uninjected embryo, (E) embryo
injected with 0.8 ng Otx-2 mRNA and (F) embryo injected with 1.6
ng Otx-2 mRNA.
Fig. 4. Dose-response of Hoxd-1 to the gradual increase in the levels
of Xcad-2. Xenopus embryos were injected at the 4-cell stage and
hybridized with an Hoxd-1-specific probe at stage 12. (A) Control
embryo injected with prolactin mRNA. (B) Embryo injected with 0.2
ng of sense Xcad-2 RNA. (C) Injection of 0.8 ng of Xcad-2 results in
the almost complete elimination of Hoxd-1 expression. (D)
Elimination of Hoxd-1 expression was observed in embryos injected
with 1.6 ng of Xcad-2 sense RNA.
Fig. 5. The response of Hoxb-3 and Hoxb-4 to changes in the levels of Xcad-2. Embryos
injected with Xcad-2-derived RNAs were probed either with the Hoxb-3 (A-C) or Hoxb-4 (DF)
probes. Control embryos were injected with prolactin RNA as a control (A) or uninjected
(D). Embryos injected with 0.4 ng sense Xcad-2 RNA are shown for both probes (B,E).
Injections with 0.4 and 0.8 ng antisense Xcad-2 RNA are shown for the Hoxb-3 (C) and
Hoxb-4 (F) hybridizations, respectively.
Fig. 6. The effect of Xcad-2 on more posterior (5¢) Hox genes. The
effect of the injection of Xcad-2 sense and antisense RNA on the
Hoxc-6 (A) and Hoxb-9 (B) genes was studied by determining the
position of their anterior boundaries of expression. The position of
the anterior boundary of expression was determined relative to the
total length of the embryo; the posterior end was determined as 0
length while the head end was determined as 1. Both RNAs were
injected at four different concentrations, 200, 400, 800 and 1600 pg.
Control uninjected and prolactin-injected embryos were used to
determine the normal position of the anterior boundary of expression
of both genes. Embryos injected with prolactin mRNA (C) or 0.2 ng
of sense Xcad-2 RNA (D) are shown to illustrate the anteriorization
of the anterior expression and the up-regulation of the Hoxb-9 gene.
Fig. 7. The interaction between the Otx-2 and the Hox genes.
Injected embryos were hybridized with the Otx-2 (A-C) or Hoxd-1
(D-F) probes. To study the response of the Otx-2 gene embryos were
injected with 1.6 ng of sense Hoxd-1 RNA (B) and 0.8 ng of Hoxb-4
mRNA (C). The response of Hoxd-1 to injection of 1.6 ng of Otx-2
mRNA (E) and 0.2 ng of Hoxb-4 sense RNA (F) is shown.
Fig. 8. Patterning of the anterior-posterior axis as evidenced from
the patterns of expression if Xcad-2, Otx-2 and Hoxd-1. Double in
situ hybridizations were performed at different developmental
stages to study the dynamics of the establishment of the anteriorposterior
axis. The initial subdivision of the embryo into anterior
and posterior domains and their distancing was evidenced from the
analysis of the Otx-2 (turquoise) and Xcad-2 (magenta) of
expression during stages 11 (A), 11.5 (B), 12 (C) and 12.5 (D).
The partial overlap between the Hoxd-1 (magenta) and Xcad-2
(turquoise) was determine at stage 11.5 (E). The identification of
Hoxd-1 as one of the genes expressed in the gap between the
anterior and posterior was determined by studying the patterns of
expression of Otx-2 (magenta) and Hoxd-1 (turquoise) at stage
12.5 (F).
