The evolution of paired appendages in vertebrates: T-box genes in the zebrafish

The presence of two sets of paired appendages is one of the defining features of jawed vertebrates. We are interested in identifying genetic systems that could have been responsible for the origin of the first set of such appendages, for their subsequent duplication at a different axial level, and/or for the generation of their distinct identities. It has been hypothesized that four genes of the T-box gene family (Tbx2–Tbx5) played important roles in the course of vertebrate limb evolution. To test this idea, we characterized the orthologs of tetrapod limb-expressed T-box genes from a teleost, Danio rerio. Here we report isolation of three of these genes, tbx2, tbx4, and tbx5. We found that their expression patterns are remarkably similar to those of their tetrapod counterparts. In particular, expression of tbx5 and tbx4 is restricted to pectoral and pelvic fin buds, respectively, while tbx2 can be detected at the anterior and posterior margins of the outgrowing fin buds. This, in combination with conserved expression patterns in other tissues, suggests that the last common ancestor of teleosts and tetrapods possessed all four of these limb-expressed T-box genes (Tbx2–Tbx5), and that these genes had already acquired, and have subsequently maintained, their gene-specific functions. Furthermore, this evidence provides molecular support for the notion that teleost pectoral and pelvic fins and tetrapod fore- and hindlimbs, respectively, are homologous structures, as suggested by comparative morphological analyses. Received: 14 July 1999 / Accepted: 4 September 1999     

Too much interference: injection of double-stranded RNA has nonspecific effects in the zebrafish embryo

We have investigated the ability of double-stranded RNA (dsRNA) to inhibit gene expression in a vertebrate, the zebrafish, Danio rerio. Injection of dsRNA corresponding to the T-box gene tbx16/spadetail (spt) into early wild-type embryos caused a rapid and dramatic loss of tbx16/spt mRNA in the blastula. mRNAs from the papc, tbx6, and gata1 genes, which depend on tbx16/spt function for their expression, were reduced, apparently mimicking the spt mutant phenotype. However, mRNAs from a number of genes that are unaffected by the spt mutation, such as beta catenin, stat3, and no tail, were also lost, indicating that the "interference" effect of tbx16/spt dsRNA was not restricted to the endogenous tbx16/spt mRNA. We compared the effects of injecting dsRNA from the zebrafish tbx16/spadetail, nieuwkoid/bozozok, and Brachyury/no tail genes with dsRNA from the bacterial lacZ gene. In each case the embryos displayed a variable syndrome of abnormalities at 12 and 24 h postfertilization. In blind studies, we could not distinguish between the effects of the various dsRNAs. Consistent with a common effect of dsRNA, regardless of sequence, injection of dsRNA from the lacZ gene was likewise effective in strongly reducing tbx16/spt and beta catenin mRNA in the blastula. These findings indicate that, despite published reports, the current methodology of double-stranded RNA interference is not a practical technique for investigating zygotic gene function during early zebrafish development.     

Characterization of the zebrafish tbx16 gene and evolution of the vertebrate T-box family

 We report on a new zebrafish T-box-containing gene, tbx16. It encodes a message that is first detected throughout the blastoderm soon after the initiation of zygotic gene expression. Following gastrulation, expression becomes restricted to paraxial mesoderm and later primarily to the developing tail bud. To gain an evolutionary prospective on the potential function of this gene, we have analyzed its phylogenetic relationships to known T-box genes from other species. Zebrafish tbx16 is likely orthologous to the chicken Tbx6L and Xenopus Xombi/Antipodean/Brat/VegT genes. Our analysis also shows that zebrafish tbx6 and mouse Tbx6 genes are paralogous to zebrafish tbx16. We present evidence which argues, that despite the same name and similar expression, zebrafish tbx6 and mouse Tbx6 genes are not orthologous to each other but instead represent relatively distant paralogs. The expression patterns of all genes are discussed in the light of their evolutionary relationships. Received: 27 November 1997 / Accepted: 27 January 1998     

Neuronal function of Tbx20 conserved from nematodes to vertebrates

The Tbx20 orthologue, mab-9, is required for development of the Caenorhabditis elegans hindgut, whereas several vertebrate Tbx20 genes promote heart development. Here we show that Tbx20 orthologues also have a role in motor neuron development that is conserved between invertebrates and vertebrates. mab-9 mutants exhibit guidance defects in dorsally projecting axons from motor neurons located in the ventral nerve cord. Danio rerio (Zebrafish) tbx20 morphants show defects in the migration patterns of motor neuron soma of the facial and trigeminal motor neuron groups. Human TBX20 is expressed in motor neurons in the developing hindbrain of human embryos and we show that human TBX20 can substitute for zebrafish tbx20 in promoting cranial motor neuron migration. mab-9 is also partially able to rescue the zebrafish migration defect, whereas other vertebrate T-box genes cannot. Conversely we show that the human TBX20 T-box domain can rescue motor neuron defects in C. elegans. These data suggest the functional equivalence of Tbx20 orthologues in regulating the development of specific motor neuron groups. We also demonstrate the functional equivalence of human and C. elegans Tbx20 T-box domains for regulating male tail development in the nematode even though these genes play highly diverged roles in organogenesis.     

T-box binding protein type two (TBX2) is an immediate early gene target in retinoic-acid-treated B16 murine melanoma cells

Retinoic acid induces growth arrest and differentiation in B16 mouse melanoma cells. Using gene arrays, we identified several early response genes whose expression is altered by retinoic acid. One of the genes, tbx2, is a member of T-box nuclear binding proteins that are important morphogens in developing embryos. Increased TBX2 mRNA is seen within 2 h after addition of retinoic acid to B16 cells. The effect of retinoic acid on gene expression is direct since it does not require any new protein synthesis. We identified a degenerate retinoic acid response element (RARE) between -186 and -163 in the promoter region of the tbx2 gene. A synthetic oligonucleotide spanning this region was able to drive increased expression of a luciferase reporter gene in response to retinoic acid; however, this induction was lost when a point mutation was introduced into the RARE. This oligonucleotide also specifically bound RAR in nuclear extracts from B16 cells. TBX2 expression and its induction by retinoic acid was also observed in normal human and nonmalignant mouse melanocytes.     

The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans

The van gogh (vgo) mutant in zebrafish is characterized by defects in the ear, pharyngeal arches and associated structures such as the thymus. We show that vgo is caused by a mutation in tbx1, a member of the large family of T-box genes. tbx1 has been recently suggested to be a major contributor to the cardiovascular defects in DiGeorge deletion syndrome (DGS) in humans, a syndrome in which several neural crest derivatives are affected in the pharyngeal arches. Using cell transplantation studies, we demonstrate that vgo/tbx1 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest-derived cartilages secondarily. Furthermore, we provide evidence for regulatory interactions between vgo/tbx1 and edn1 and hand2, genes that are implicated in the control of pharyngeal arch development and in the etiology of DGS.     

Evolutionary implications of morphogenesis and molecular patterning of the blind gut in the planarian Schmidtea polychroa

The formation of a through-gut was a key innovation in the evolution of metazoans. There is still controversy regarding the origin of the anus and how it may have been either gained or lost during evolution in different bilaterian taxa. Thus, the study of groups with a blind gut is of great importance for understanding the evolution of this organ system. Here, we describe the morphogenesis and molecular patterning of the blind gut in the sexual triclad Schmidtea polychroa. We identify and analyze the expression of goosecoid, commonly associated with the foregut, and the GATA, ParaHox and T-box genes, members of which commonly are associated with gut regionalization. We show that GATA456a is expressed in the blind gut of triclads, while GATA456b is localized in dorsal parenchymal cells. Goosecoid is expressed in the central nervous system, and the unique ParaHox gene identified, Xlox, is detected in association with the nervous system. We have not isolated any brachyury gene in the T-box complement of S. polychroa, which consists of one tbx1/10, three tbx2/3 and one tbx20. Furthermore, the absence of genes like brachyury and caudal is also present in other groups of Platyhelminthes. This study suggests that GATA456, in combination with foxA, is a gut-specific patterning mechanism conserved in the triclad S. polychroa, while the conserved gut-associated expression of foregut, midgut and hindgut markers is absent. Based on these data and the deviations in spiral cleavage found in more basal flatworms, we propose that the lack of an anus is an innovation of Platyhelminthes. This may be associated with loss of gut gene expression or even gene loss.     

HrT is required for cardiovascular development in zebrafish

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.     

Combinatorial gene regulation by Bmp and Wnt in zebrafish posterior mesoderm formation

Combinatorial signaling is an important mechanism that allows the embryo to utilize overlapping signaling pathways to specify different territories. In zebrafish, the Wnt and Bmp pathways interact to regulate the formation of the posterior body. In order to understand how this works mechanistically, we have identified tbx6 as a posterior mesodermal gene activated by both of these signaling pathways. We isolated a genomic fragment from the tbx6 gene that recapitulates the endogenous tbx6 expression, and used this to ask how the Bmp and Wnt signaling pathways combine to regulate gene expression. We find that the tbx6 promoter utilizes distinct domains to integrate the signaling inputs from each pathway, including multiple Tcf/LEF sites and a novel Bmp-response element. Surprisingly, we found that overexpression of either signaling pathway can activate the tbx6 promoter and the endogenous gene, whereas inputs from both pathways are required for the normal pattern of expression. These results demonstrate that both Bmp and Wnt are present at submaximal levels, which allows the pathways to function combinatorially. We present a model in which overlapping Wnt and Bmp signals in the ventrolateral region activate the expression of tbx6 and other posterior mesodermal genes, leading to the formation of posterior structures.     

Tbx5 and Tbx20 act synergistically to control vertebrate heart morphogenesis

Members of the T-box family of proteins play a fundamental role in patterning the developing vertebrate heart; however, the precise cellular requirements for any one family member and the mechanism by which individual T-box genes function remains largely unknown. In this study, we have investigated the cellular and molecular relationship between two T-box genes, Tbx5 and Tbx20. We demonstrate that blocking Tbx5 or Tbx20 produces phenotypes that display a high degree of similarity, as judged by overall gross morphology, molecular marker analysis and cardiac physiology, implying that the two genes are required for and have non-redundant functions in early heart development. In addition, we demonstrate that although co-expressed, Tbx5 and Tbx20 are not dependent on the expression of one another, but rather have a synergistic role during early heart development. Consistent with this proposal, we show that TBX5 and TBX20 can physically interact and map the interaction domains, and we show a cellular interaction for the two proteins in cardiac development, thus providing the first evidence for direct interaction between members of the T-box gene family.