Keiko Ogura

Tbx5 and Tbx4 genes determine the wing/leg identity of limb buds

Much progress has been made in understanding limb development. Most genes are expressed equally and in the same pattern in the fore- and hindlimbs, which nevertheless develop into distinct structures. The T-box genes Tbx5 and Tbx4, on the other hand, are expressed differently in chick wing (Tbx5) and leg (Tbx4) buds. Molecular analysis of the optomotor blind gene, which belongs to the same family of transcription factors, has revealed that this gene is involved in the transdetermination of Drosophila wing and leg imaginal discs. In addition, expression of Tbx5 and Tbx4 correlates well with the identity of ectopic limb buds induced by fibroblast growth factor,,. Thus, it is thought that Tbx5 and Tbx4 might be involved in determining limb identity. Another candidate is the Pitx1 gene, which encodes a bicoid-type homeodomain transcription factor that is expressed in leg buds,. Here we determine the importance of these factors in establishing limb identity.

Tbx5 specifies the left/right ventricles and ventricular septum position during cardiogenesis

Extensive misexpression studies were carried out to explore the roles played by Tbx5, the expression of which is excluded from the right ventricle (RV) during cardiogenesis. When Tbx5 was misexpressed ubiquitously, ventricular septum was not formed, resulting in a single ventricle. In such heart, left ventricle (LV)-specific ANF gene was induced. In search of the putative RV factor(s), we have found that chick Tbx20 is expressed in the RV, showing a complementary fashion to Tbx5. In the Tbx5-misexpressed heart, this gene was repressed. When misexpression was spatially partial, leaving small Tbx5-negative area in the right ventricle, ventricular septum was shifted rightwards, resulting in a small RV with an enlarged LV. Focal expression induced an ectopic boundary of Tbx5-positive and -negative regions in the right ventricle, at which an additional septum was formed. Similar results were obtained from the transient transgenic mice. In such hearts, expression patterns of dHAND and eHAND were changed with definitive cardiac abnormalities. Furthermore, we report that human ANF promoter is synergistically activated by Tbx5, Nkx2.5 and GATA4. This activation was abrogated by Tbx20, implicating the pivotal roles of interactions among these heart-specific factors. Taken together, our data indicate that Tbx5 specifies the identity of LV through tight interactions among several heart-specific factors, and highlight the essential roles of Tbx5 in cardiac development.

Tbx5 and Tbx4 trigger limb initiation through activation of the Wnt/Fgf signaling cascade.

A tight loop between members of the fibroblast growth factor and the Wnt families plays a key role in the initiation of vertebrate limb development. We show for the first time that Tbx5 and Tbx4 are directly involved in this process. When dominant-negative forms of these Tbx genes were misexpressed in the chick prospective limb fields, a limbless phenotype arose with repression of both Wnt and Fgf genes By contrast, when Tbx5 and Tbx4 were misexpressed in the flank, an additional wing-like and an additional leg-like limbs were induced, respectively. This additional limb formation was accompanied by the induction of both Wnt and Fgf genes These results highlight the pivotal roles of Tbx5 and Tbx4 during limb initiation, specification of forelimb/hindlimb and evolution of tetrapod limbs, placing Tbx genes at the center of a highly conserved genetic program.

The prepattern transcription factor Irx2, a target of the FGF8/MAP kinase cascade, is involved in cerebellum formation.

The cerebellum develops from the rhombic lip of the rostral hindbrain and is organized by fibroblast growth factor 8 (FGF8) expressed by the isthmus. Here we report characterization of Irx2, a member of the Iroquois (Iro) and Irx class of homeobox genes, that is expressed in the presumptive cerebellum. When Irx2 is misexpressed with Fgf8a in the chick midbrain, the midbrain develops into cerebellum in conjunction with repression of Otx2 and induction of Gbx2. During this event, signaling by the FGF8 and mitogen-activated protein (MAP) kinase cascade modulates the activity of Irx2 by phosphorylation. Our data identify a link between the isthmic organizer and Irx2, thereby shedding light on the roles of Iro and Irx genes, which are conserved in both vertebrates and invertebrates.

Cloning and chromosome mapping of human and chicken Iroquois (IRX) genes

Three highly homologous homeobox genes (caupolican, araucan and mirror) have been identified in Drosophila. These genes belong to the novel Iroquois complex, which acts as a pre-pattern molecule in Drosophila neurogenesis. Recently several vertebrate Iroquois homologues (Irx) were isolated and found to be involved in pattern formation of various tissues. Here we report cytogenetic mapping of four human and five chicken Iroquois genes by FISH. Our findings revealed that vertebrate Irx genes are clustered at two different loci.