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We present an evolutionary approach to dissecting conserved developmental mechanisms. We reason that important mechanisms for making the bodyplan will act early, to generate the major features of the body and that they will be conserved in evolution across many metazoa, and thus, that they will be available in very different animals. This led to our specific approach of microarrays to screen for very early conserved developmental regulators in parallel in an insect, Drosophila and a vertebrate, Xenopus. We screened for the earliest conserved targets of the ectopically expressed hox gene Hoxc6/Antennapedia in both species and followed these targets up, using in situ hybridization, in the Xenopus system. The results indicate that relatively few of the early Hox target genes are conserved: these are mainly involved in the specification of the antero-posterior body axis and in gastrulation.
Fig. 1. Drosophila Antennapedia and vertebrate Hox6 genes are considered to be orthologous. The figure below shows the single
Drosophila Hox cluster (with its split indicated) and the four primary orthologous Hox clusters in a tetrapod vertebrate. The orthology of
Antennapedia and the Hox6 genes is indicated by the same (light brown) color. Above are the axial domains of action of the different
Hox genes. Indicated by different colors, in Drosophila (left) and a human fetus (right). Note that Antennapedia and Hox 6 genes have
homolgous action domains (anterior thorax) in fly and human.
Fig. 2. The predicted Xl Hoxc6 and Antennapedia homeodomains show 91% sequence identity at the amino acid level. The figure
shows homologous sequences in the two homeoproteins, indicated by colors. Red indicates homologous sequences within the
homeodomain and hexapeptide. The other colors indicate homology in other parts of the homeoprotein. The labeling shows
Antennapedia in Dorosophila melanogaster (ANTP DROMELA) and XlHbox1 (the oldname for Hoxc6) in Xenopus laevis (XlHbox1
XENLA).
Fig. 3. Functional homology between Xl Hoxc6 and Antennapedia. The figure shows: the phenotype obtained by overexpressing
Antennapedia in Drosophila (above). Head segments are transformed to thorax, segment T2. Early XlHoxc6 ectopic expression in
Drosophila gives the same phenotype as Antennapedia ectopic expression but Hoxa7 ectopic expression does not (not shown). The
figure also shows the phenotype obtained by overexpressing XlHoxc6 in Xenopus (below). Head development is repressed and the
thorax is enlarged.
Fig. 4 Heterochrony between Xenopus and Drosophila and plan of the experiment. The figure shows relative stages for ectopic hox
expression, mRNA harvesting and gastrulation in Xenopus and Drosophila. Gb and Ge: beginning and end of gastrulation. Ho1, Ho6:
the times when Hox1 and Hox6 expression start. Nb, Ne: the beginning and end of ectopic expression under the nullo promoter in
Drosophila. Ha: time point for harvesting total mRNA. mRNA: injection of Xlhoxc6 mRNA into Xenopus. T = 0: start of development:
zygote stage. The figure shows that the relationship between Hox expression and gastrulation is different in Xenopus (where Hox
expression begins during gastrualtion) and Drosophila (where Hox expression begins before gastrulation). The latest time point for
harvesting Drosophila mRNA (16 h: end of embryogenesis) is not shown (it is far right, off the figure).
Fig. 5. Numbers of Hoxc6 and Antennapedia targets. This figure shows numbers of targets at three developmental stages each in
Drosophila and Xenopus and numbers of targets that overlap at different stages. Each disc represents targets obtained at a specific
developmental stage, as indicated. Numbers of non-overlapping targets and numbers of overlapping targets (between stages) are as
indicated. PS, probe sets. Top left: Upregulated targets in Drosophila. Top right: Downregulated targets in Drosophila. Bottom left:
Upregulated targets in Xenopus. Bottom right: Downregulated targets in Xenopus.
Fig. 6. Confirmation of Xenopus conserved
targets. Eight Xenopus targets
were confirmed as being regulated
by Hoxc6, as shown. In situ
hybridizations of control embryos
(left in each pair) and of embryos
ectopically expressing Hoxc6 (right in
each pair). We used probes detecting
Xenopus probesets that are also
conservedly regulated in Drosophila.
These probes detected upregulation
(all except Otx2) or downregulation
(Otx2). Hoxc6 wild type expression is
shown to indicate the normal Hoxc6
expression domain. Shown are stage
12 embryos from the vegetal side
(most) or dorsal side (probes
MGC82057, LOC495834, and Otx2).
The name of each Xenopus target
gene is at the top of the right hand
picture in each pair.
ccng1 (cyclin G1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, vegetal view, dorsal up.
herpud1 (homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, dorsal vegetal view, anterior up.
nfe2 (nuclear factor, erythroid 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 12, dorsal vegetal view, anterior up.
