INTRODUCTION The developmental roles of the T-box genes have been an area of intense research for many years (Papaioannou and Silver, 1998; Tada and Smith, 2001). Much of our current understanding of the critical role of T-box genes comes from the analysis of phenotypes produced by genetic mutations. Importantly, several TBX genes are causally implicated in human congenital malformations. For example, the cardiovascular defects in DiGeorge syndrome are caused by haploinsufficiency of TBX1 (Merscher et al., 2001); the ulnar- mammary syndrome is a pleiotropic disorder characterized by defects in limb, apocrine gland, tooth and genital development because of haploinsufficiency of TBX3 (Bamshad et al., 1997); and Holt-Oram syndrome, a developmental disorder of upper limb malformation and cardiac septation defects, is associated with haploinsufficiency of TBX5 (Li et al., 1997; Bruneau et al., 2001). The family of T-box transcription factors shares a phylogenetically conserved DNA-binding domain, which is required for specific DNA sequence recognition. The functional activity of these genes are mediated by binding to their cognate regulatory sites within the promoter and enhancer regions of genes, leading to the activation or repression of gene expression depending upon the type of T-box gene and the context within the promoter (Carreira et al., 1998; He et al., 1999; Sinha et al., 2000; Tada and Smith, 2001). Members of this gene family have been shown to have selective patterns of expression and play many key roles in patterning and specifying the development of a variety of tissues (Basson et al., 1999; Merscher et al., 2001; Lamolet et al., 2001; Bruneau et al., 2001; Tada and Smith, 2001). We recently described a novel zebrafish T-box gene, hrT (tbx20 – Zebrafish Information Network), which is expressed in the developing heart and dorsal aorta (Griffin et al., 2000; Ahn et al., 2000). hrT is expressed in the cardiogenic mesoderm of the anterior lateral plate from the beginning of segmentation, and continues to be expressed in the heart field until at least 72 hours (Griffin et al., 2000; Ahn et al., 2000). The onset of hrT expression in the lateral plate is earlier than the first expression of nkx2.5 or tbx5, and coincident with the start of nkx2.7 expression (Lee et al., 1996; Begemann and Ingham, 2000). hrT is thus expressed during all the key stages of cardiac development, which include cardiac cell fate specification, morphogenesis, looping of the heart tube and chamber formation (Srivastavia and Olson, 2000; Yelon et al., 1999; Stainier, 2001). In addition, hrT begins to be expressed in the dorsal aorta at the 15-somite stage, in addition to sites of expression in the hindbrain, eye and at the anal opening (Griffin et al., 2000; Ahn et al., 2000). 5093 Development 129, 5093-5101 (2002) Printed in Great Britain © The Company of Biologists Limited 2002 DEV5029 The recently identified zebrafish T-box gene hrT is expressed in the developing heart and in the endothelial cells forming the dorsal aorta. Orthologs of hrT are expressed in cardiovascular cells from Drosophila to mouse, suggesting that the function of hrT is evolutionarily conserved. The role of hrT in cardiovascular development, however, has not thus far been determined in any animal model. Using morpholino antisense oligonucleotides, we show that zebrafish embryos lacking hrT function have dysmorphic hearts and an absence of blood circulation. Although the early events in heart formation were normal in hrT morphant embryos, subsequently the hearts failed to undergo looping, and late onset defects in chamber morphology and gene expression were observed. In particular, we found that the loss of hrT function led to a dramatic upregulation of tbx5, a gene required for normal heart morphogenesis. Conversely, we show that overexpression of hrT causes a significant downregulation of tbx5, indicating that one key role of hrT is to regulate the levels of tbx5. Secondly, we found that HrT is required to inhibit the expression of the blood lineage markers gata1 and gata2 in the most posterior lateral plate mesoderm. Finally, we show that HrT is required for vasculogenesis in the trunk, leading to similar vascular defects to those observed in midline mutants such as floating head. hrT expression in the vascular progenitors depends upon midline mesoderm, indicating that this expression is one important component of the response to a midline-derived signal during vascular morphogenesis. Key words: Zebrafish, hrT, Cardiogenesis, Dorsal aorta, tbx5, fli1, floating head SUMMARY DEVELOPMENT AND DISEASE hrT is required for cardiovascular development in zebrafish Daniel P. Szeto, Kevin J. P. Griffin and David Kimelman* Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195-7530, USA *Author for correspondence (e-mail: kimelman@u.washington.edu) Accepted 30 July 2002