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N. Satah et al.
Ciona eggs (Fig. 3B). In this case, Am(Bb)Bra2 was expressed in the presumptive
endoderm and endodermal strand cells. Whole-mount in situ hybridization with
the probe for the notochord-specific gene revealed differentiation of extra notochord cells in the ventral region of the embryo (Fig. 3B). We obtained the same
result for Am(Bb)Bral. Therefore, BrachYlIry of lower chordate amphioxus is able
to form notochord in tunicate embryos.
Then, we examined PfBra of non-chordate deuterostome hemichordates. We
made a fusion gene construct Ci-fkh/PfBra in which PfBra cDNA was fused with
the Ci-fkh promoter (Fig. 3C). When this construct was electroporated into Ciona
eggs, PfBra was expressed in the presumptive endoderm and endodermal strand
cells. As is evident in Fig. 3C, whole-mount in situ hybridization with the notochord-specific gene probe revealed the formation of extra notochords in the ventral
region of the embryo. Therefore, BrachYlIry of non-chordate hemichordates is able
to form notochord when the gene was expressed in tunicate embryos. As present,
we are examining the potential of the sea urchin ,"'pTa by a fusion gene construct
Ci-fkh/SpTa.
The present results therefore provide convincing evidence for the functional
conservation of Brachyury between non-chordate and chordate deuterostomes. The
results also emphasize an essential role of Brachyury in the occurrence of notochord during the evolution of chordates. Namely, the occurrence of notochord during chordate evolution was not brought about by the appearance of new developmental genes but by an acquisition of a new function of a pre-existing gene.
5 Upstream and Downstream Regulation of
Ascidian Brachyury
The above-mentioned results suggest that comparative studies, between chordates
and non-chordate deuterostomes, of the genetic circuitry of Brachyury, in particular downstream or target genes, may lead to further profound understanding of
molecular developmental mechanisms underlying the notochord formation and thus
the evolution of chordates. At present, we are investigating the upstream and downstream regulation of ascidian Brachyury genes.
5.1 Upstream Control of the Ascidian Brachyury Genes
Both As-T of H. roretzi and Ci-Bra of C. intestinalis are exclusively expressed in
notochord cells (Yasuo and Satoh 1993; Corbo et al. 1997a). How is this restricted
expression of ascidian Brachyury genes controlled? Corbo et al. (1997a) examined
a minimal promoter for the notochord-specific expression of Ci-Bra. As is shown
in Fig. 4, they demonstrated that the 434 bp of a minimal enhancer of Ci-Bra
contains three distinctive regions. From distal to proximal, a negative control region (from -434 to -299) excludes Ci-Bra expression from inappropriate embryonic
N. Satah et al.
Ciona eggs (Fig. 3B). In this case, Am(Bb)Bra2 was expressed in the presumptive
endoderm and endodermal strand cells. Whole-mount in situ hybridization with
the probe for the notochord-specific gene revealed differentiation of extra notochord cells in the ventral region of the embryo (Fig. 3B). We obtained the same
result for Am(Bb)Bral. Therefore, BrachYlIry of lower chordate amphioxus is able
to form notochord in tunicate embryos.
Then, we examined PfBra of non-chordate deuterostome hemichordates. We
made a fusion gene construct Ci-fkh/PfBra in which PfBra cDNA was fused with
the Ci-fkh promoter (Fig. 3C). When this construct was electroporated into Ciona
eggs, PfBra was expressed in the presumptive endoderm and endodermal strand
cells. As is evident in Fig. 3C, whole-mount in situ hybridization with the notochord-specific gene probe revealed the formation of extra notochords in the ventral
region of the embryo. Therefore, BrachYlIry of non-chordate hemichordates is able
to form notochord when the gene was expressed in tunicate embryos. As present,
we are examining the potential of the sea urchin ,"'pTa by a fusion gene construct
Ci-fkh/SpTa.
The present results therefore provide convincing evidence for the functional
conservation of Brachyury between non-chordate and chordate deuterostomes. The
results also emphasize an essential role of Brachyury in the occurrence of notochord during the evolution of chordates. Namely, the occurrence of notochord during chordate evolution was not brought about by the appearance of new developmental genes but by an acquisition of a new function of a pre-existing gene.
5 Upstream and Downstream Regulation of
Ascidian Brachyury
The above-mentioned results suggest that comparative studies, between chordates
and non-chordate deuterostomes, of the genetic circuitry of Brachyury, in particular downstream or target genes, may lead to further profound understanding of
molecular developmental mechanisms underlying the notochord formation and thus
the evolution of chordates. At present, we are investigating the upstream and downstream regulation of ascidian Brachyury genes.
5.1 Upstream Control of the Ascidian Brachyury Genes
Both As-T of H. roretzi and Ci-Bra of C. intestinalis are exclusively expressed in
notochord cells (Yasuo and Satoh 1993; Corbo et al. 1997a). How is this restricted
expression of ascidian Brachyury genes controlled? Corbo et al. (1997a) examined
a minimal promoter for the notochord-specific expression of Ci-Bra. As is shown
in Fig. 4, they demonstrated that the 434 bp of a minimal enhancer of Ci-Bra
contains three distinctive regions. From distal to proximal, a negative control region (from -434 to -299) excludes Ci-Bra expression from inappropriate embryonic