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N. Satoh et al.
We first examined whether overexpression of As-T can induce notochord formation without cell-cell interaction (Yasuo and Satoh 1998). Synthetic As-T mRNA
was injected into fertilized eggs, and blastomeres were dissociated as soon as the
embryos reached the 32-cell stage. When the isolated blastomeres were cultured for
about 12 hrs, they differentiated into notochord celis, suggesting that overexpression
of As-T induces notochord cells without cell-cell interaction at the 32-cell stage.
Then, we examined whether a mis-expression of As-T induces an ectopic differentiation of notochord cells in blastomeres of non-notochord lineage. The mis-expression of As-T induced an ectopic differentiation of notochord cells in at least
endoderm and nerve cord precursor cells (Yasuo and Satoh 1998). These results
strongly suggest that the ascidian Brachyury exerts a master control over the formation of notochord.
4 Functional Conservation of
Deuterostome Brachyury Genes
As described above, chordate as well as non-chordate deuterostomes contain
Brachyury genes, and the ascidian Brachyury exerts a master control over the formation of notochord. Therefore, an important question to be answered is on functional conservation among deuterostome Brachyury genes; namely, whether or not
non-chordate deuterostome (hemichordate and echinoderm) Brachyury genes have
a potential to induce notochord cells in chordate (asci dian) embryos. To determine
this question, G. Satoh et al. (unpublished) took advantage of a Ci-fkh cassette
described below (kindly provided from Mike Levine). Ci-fkh is/ork head/HNF-3f3
gene of C. intestinalis, which is expressed in the endoderm, endodermal strand,
notochord and nerve cord of the tailbud embryo (Corbo et al. 1997b). When a fusion gene construct Ci-tkh/lacZ was electroporated into Ciona fertilized eggs, the
reporter gene is eventually expressed in the endoderm, endodermal strand, notochord and nerve cord, exactly reflecting the spatial expression pattern of Ci-fkh.
We made a fusion gene construct Ci-fkh/Ci-Bra in which Ci-Bra cDNA was under
the control of the Ci-fkh promoter (Fig. 3A). When this construct was electro po rated
into Ciona fertilized eggs, Ci-Bra was ectopically expressed in cells of the endoderm, endodermal strand, and nerve cord of the tailbud embryos. This caused an
ectopic differentiation of notochord cells in the presumptive endoderm and endodermal strand cells, which was visualized by in situ hybridization with probes of
notochord-specific genes (Fig. 3A). Because of vacuolation of notochord cells, CiBra-overexpressing embryos swelled up in their ventral portion (Fig. 3A). Thus, an
ectopic expression of Ci-Bra induces extra notochords in Ciona tailbud embryos, as
in the case of ectopic expression of As-Tin H alocynthia embryos (Yasuo and Satoh
1998).
To examine the potential of the amphioxus Am(Bb)Bra2 to induce notochord
cells in ascidian embryos, a fusion gene construct Ci-fkh/Arn(Bb)Bral in which
Am(Bb)Bra2 cDNA was fused with the Ci-fkh promoter, was electroporated into
N. Satoh et al.
We first examined whether overexpression of As-T can induce notochord formation without cell-cell interaction (Yasuo and Satoh 1998). Synthetic As-T mRNA
was injected into fertilized eggs, and blastomeres were dissociated as soon as the
embryos reached the 32-cell stage. When the isolated blastomeres were cultured for
about 12 hrs, they differentiated into notochord celis, suggesting that overexpression
of As-T induces notochord cells without cell-cell interaction at the 32-cell stage.
Then, we examined whether a mis-expression of As-T induces an ectopic differentiation of notochord cells in blastomeres of non-notochord lineage. The mis-expression of As-T induced an ectopic differentiation of notochord cells in at least
endoderm and nerve cord precursor cells (Yasuo and Satoh 1998). These results
strongly suggest that the ascidian Brachyury exerts a master control over the formation of notochord.
4 Functional Conservation of
Deuterostome Brachyury Genes
As described above, chordate as well as non-chordate deuterostomes contain
Brachyury genes, and the ascidian Brachyury exerts a master control over the formation of notochord. Therefore, an important question to be answered is on functional conservation among deuterostome Brachyury genes; namely, whether or not
non-chordate deuterostome (hemichordate and echinoderm) Brachyury genes have
a potential to induce notochord cells in chordate (asci dian) embryos. To determine
this question, G. Satoh et al. (unpublished) took advantage of a Ci-fkh cassette
described below (kindly provided from Mike Levine). Ci-fkh is/ork head/HNF-3f3
gene of C. intestinalis, which is expressed in the endoderm, endodermal strand,
notochord and nerve cord of the tailbud embryo (Corbo et al. 1997b). When a fusion gene construct Ci-tkh/lacZ was electroporated into Ciona fertilized eggs, the
reporter gene is eventually expressed in the endoderm, endodermal strand, notochord and nerve cord, exactly reflecting the spatial expression pattern of Ci-fkh.
We made a fusion gene construct Ci-fkh/Ci-Bra in which Ci-Bra cDNA was under
the control of the Ci-fkh promoter (Fig. 3A). When this construct was electro po rated
into Ciona fertilized eggs, Ci-Bra was ectopically expressed in cells of the endoderm, endodermal strand, and nerve cord of the tailbud embryos. This caused an
ectopic differentiation of notochord cells in the presumptive endoderm and endodermal strand cells, which was visualized by in situ hybridization with probes of
notochord-specific genes (Fig. 3A). Because of vacuolation of notochord cells, CiBra-overexpressing embryos swelled up in their ventral portion (Fig. 3A). Thus, an
ectopic expression of Ci-Bra induces extra notochords in Ciona tailbud embryos, as
in the case of ectopic expression of As-Tin H alocynthia embryos (Yasuo and Satoh
1998).
To examine the potential of the amphioxus Am(Bb)Bra2 to induce notochord
cells in ascidian embryos, a fusion gene construct Ci-fkh/Arn(Bb)Bral in which
Am(Bb)Bra2 cDNA was fused with the Ci-fkh promoter, was electroporated into