Tbx2/3 is an essential mediator within the Brachyury gene network during Ciona notochord development

Development, Jun 2013

Diana S. José-Edwards, Izumi Oda-Ishii, Yutaka Nibu, Anna Di Gregorio

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Tbx2/3 is an essential mediator within the Brachyury gene network during Ciona notochord development

Diana S. Jos-Edwards 0 Izumi Oda-Ishii 0 Yutaka Nibu 0 Anna Di Gregorio 0 0 Department of Cell and Developmental Biology, Weill Medical College of Cornell University , 1300 York Avenue, Box 60, New York, NY 10065 , USA SUMMARY T-box genes are potent regulators of mesoderm development in many metazoans. In chordate embryos, the T-box transcription factor Brachyury (Bra) is required for specification and differentiation of the notochord. In some chordates, including the ascidian Ciona, members of the Tbx2 subfamily of T-box genes are also expressed in this tissue; however, their regulatory relationships with Bra and their contributions to the development of the notochord remain uncharacterized. We determined that the notochord expression of Ciona Tbx2/3 (Ci-Tbx2/3) requires Ci-Bra, and identified a Ci-Tbx2/3 notochord CRM that necessitates multiple Ci-Bra binding sites for its activity. Expression of mutant forms of Ci-Tbx2/3 in the developing notochord revealed a role for this transcription factor primarily in convergent extension. Through microarray screens, we uncovered numerous Ci-Tbx2/3 targets, some of which overlap with known Ci-Bra-downstream notochord genes. Among the Ci-Tbx2/3 notochord targets are evolutionarily conserved genes, including caspases, lineage-specific genes, such as Noto4, and newly identified genes, such as MLKL. This work sheds light on a large section of the notochord regulatory circuitry controlled by T-box factors, and reveals new components of the complement of genes required for the proper formation of this structure. INTRODUCTION T-box (or Tbx) genes comprise an evolutionarily conserved class of transcription factors defined by the presence of a distinctive DNA-binding domain. Members of the Tbx family are crucial regulators of embryogenesis (Naiche et al., 2005), and mutations in T-box genes are linked to several human genetic diseases (Packham and Brook, 2003), further underscoring the importance of Tbx transcription factors. The discovery and functional characterization of Brachyury (Bra), the founding Tbx family member, revealed its involvement in the specification and development of the notochord (Herrmann et al., 1990). The notochord, the defining feature of chordates, provides structural support for developing embryos throughout this phylum (Jiang and Smith, 2007); a basement membrane and a perinotochordal sheath consisting of extracellular matrix (ECM) proteins contribute to its rigidity (Stemple, 2005). Elongation of the chordate body plan is in part accomplished through convergent extension (CE) of notochord cells via mediolateral intercalation (Keller et al., 2000), which requires cells to become motile and polarized, to remodel cell-adhesive contacts and to interact with ECM components. Following intercalation, pressure exerted on the notochordal sheath by the formation and expansion of vacuoles provides the tail with further rigidity (Stemple, 2005). Studies in all chordates examined thus far have shown that Bra is indispensible for notochord fate commitment (Showell et al., 2004). Conversely, few studies have addressed the composition and regulation of the battery of genes required for later notochord morphogenetic events. *These authors contributed equally to this work Authors for correspondence (; ) In addition to the group containing Bra, phylogenetic analyses have categorized vertebrate T-box genes into four further subfamilies, including that of Tbx2, which in mammals has four members, Tbx2, Tbx3, Tbx4 and Tbx5 (Naiche et al., 2005). The paralogs Tbx2 and Tbx3 are important regulators of heart formation (Harrelson et al., 2004), brain morphogenesis (Manning et al., 2006) and cell migration (Fong et al., 2005). In humans, altered levels of these genes have deleterious results: haploinsufficiency caused by mutations in TBX3 plays a role in ulnar-mammary syndrome (UMS) (Packham and Brook, 2003), and recently TBX2 and TBX3 have been found to be amplified or upregulated in different cancers (Lu et al., 2010). These transcriptional regulators can contribute to tumorigenesis through inhibition of apoptosis and by increasing invasiveness through control of cell adhesion genes (Lu et al., 2010). A thorough understanding of the contributions of Tbx2/3 genes to normal development will thus inform studies of these transcription factors in pathological processes. Previous studies have suggested that Tbx2 subfamily members could also contribute to notochord formation. Xenopus and zebrafish notochords both express tbx3, and the zebrafish tbx2b gene is also found in the notochord (Dheen et al., 1999; Takabatake et al., 2000); however, their individual functions and regulatory interactions in this tissue remain incompletely characterized. Impaired Tbx2b activity results in defects in notochord specification, which precludes the study of its involvement in later stages of notochord morphogenesis (Dheen et al., 1999; Fong et al., 2005). Hence, the roles and targets of tb (...truncated)


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Diana S. José-Edwards, Izumi Oda-Ishii, Yutaka Nibu, Anna Di Gregorio. Tbx2/3 is an essential mediator within the Brachyury gene network during Ciona notochord development, Development, 2013, pp. 2422-2433, 140/11, DOI: 10.1242/dev.094227