Leg development in Drosophila has been studied in much detail. However, Drosophila limbs form in the larva as imaginal discs and not during embryogenesis as in most other arthropods. Appendage genes have been analyzed in the spider Cupiennius salei and the beetle Tribolium castaneum. Differences in decapentaplegic expression suggest a different mode of distal morphogen signaling suitable for the specific geometry of growing limb buds. Also, expression of the proximal genes homothorax and extradenticle (exd) is significantly altered: in the spider, exd is restricted to the proximal leg and hth expression extends distally, while in insects, exd is expressed in the entire leg and hth is restricted to proximal parts. This reversal of spatial specificity demonstrates an evolutionary shift, which is nevertheless compatible with a conserved role for this gene pair as instructor of proximal fate. Different expression dynamics of dachshund and Distal-less point to modifications in the regulation of the leg gap gene system. The significance of this finding is discussed in terms of attempts to homologize leg segments in different arthropod classes. Comparison of the expression profiles of H15 and optomotor-blind to the Drosophila patterns suggests modifications also in the dorsal-ventral patterning system of the legs. Together, these results suggest alterations in many components of the leg developmental system, namely proximal-distal and dorsal-ventral patterning, and leg segmentation. Thus, the leg developmental system exhibits a propensity to evolutionary change, which probably forms the basis for the impressive diversity of arthropod leg morphologies (Prpic, 2003).
The Drosophila genes wingless and decapentaplegic comprise the top level of a hierarchical gene cascade involved in proximal-distal (PD) patterning of the legs. It remains unclear, whether this cascade is common to the appendages of all arthropods. Here, wg and dpp are studied in the millipede Glomeris marginata, a representative of the Myriapoda. Glomeris wg (Gm-wg) is expressed along the ventral side of the appendages compatible with functioning during the patterning of both the PD and dorsal-ventral (DV) axes. Gm-wg may also be involved in sensory organ formation in the gnathal appendages by inducing the expression of Distal-less (Dll) and H15 in the organ primordia. Expression of Glomeris dpp (Gm-dpp) is found at the tip of the trunk legs as well as weakly along the dorsal side of the legs in early stages. Taking data from other arthropods into account, these results may be interpreted in favor of a conserved mode of WG/DPP signaling. Apart from the main PD axis, many arthropod appendages have additional branches (e.g., endites). It is debated whether these extra branches develop their PD axis via the same mechanism as the main PD axis, or whether branch-specific mechanisms exist. Gene expression in possible endite homologs in Glomeris argues for the latter alternative. All available data argue in favor of a conserved role of WG/DPP morphogen gradients in guiding the development of the main PD axis. Additional branches in multibranched (multiramous) appendage types apparently do not utilize the WG/DPP signaling system for their PD development. This further supports recent work on crustaceans and insects, that lead to similar conclusions (Prpic, 2004).
In Drosophila, the T-box genes optomotor-blind (omb) and H15 have been implicated in specifying the development of the dorso-ventral (DV) axis of the appendages. Results from the spider Cupiennius salei have suggested that this DV patterning system may be at least partially conserved. This study extends the study of the DV patterning genes omb and H15 to a representative of the Myriapoda in order to add to the existing comparative data set and to gain further insight into the evolution of the DV patterning system in arthropod appendages. The omb gene of the millipede Glomeris marginata is expressed on the dorsal side of all appendages including trunk legs, maxillae, mandibles, and antennae. This is similar to what is known from Drosophila and Cupiennius and suggests that the role of omb in instructing dorsal fates is conserved in arthropods. Interestingly, the lobe-shaped portions of the mouthparts do not express omb, indicating that these are ventral components and thus may be homologous to the endites present in the corresponding appendages in insects. Concerning the H15 gene, two paralogous genes were identified in Glomeris. Both genes are expressed in the sensory organs of the maxilla and antenna, but only Gm-H15-1 is expressed along the ventral side of the trunk legs. The expression is more extensive than in Cupiennius, but less so than in Drosophila. In addition, no ventral expression domain is present in the maxilla, mandible, and antenna. Because of this, the role of H15 in the determination of ventral fate remains unclear (Prpic, 2005).
The C. elegans MS blastomere, born at the 7-cell stage of embryogenesis, generates primarily mesodermal cell types, including pharynx cells, body muscles and coelomocytes. A presumptive null mutation in the T-box factor gene tbx-35, a target of the MED-1 and MED-2 divergent GATA factors, was previously found to result in a profound decrease in the production of MS-derived tissues, although the tbx-35- embryonic arrest phenotype was variable. The NK-2 class homeobox gene ceh-51 is a direct target of TBX-35 and at least one other factor, and CEH-51 and TBX-35 share functions. Embryos homozygous for a ceh-51 null mutation arrest as larvae with pharynx and muscle defects, although these tissues appear to be specified correctly. Loss of tbx-35 and ceh-51 together results in a synergistic phenotype resembling loss of med-1 and med-2. Overexpression of ceh-51 causes embryonic arrest and generation of ectopic body muscle and coelomocytes. These data show that TBX-35 and CEH-51 have overlapping function in MS lineage development. As T-box regulators and NK-2 homeodomain factors are both important for heart development in Drosophila and vertebrates, these results suggest that these regulators function in a similar manner in C. elegans to specify a major precursor of mesoderm (Broitman-Maduro, 2009).
T-box transcription factors are critical regulators of early embryonic development. A novel zebrafish T-box transcription factor, hrT (H15-related T box) has been characterized that is a close relative of Drosophila H15 and a recently identified human gene. Drosophila H15 and zebrafish hrT are both expressed early during heart formation, in strong support of previous work postulating that vertebrate and arthropod hearts are homologous structures with conserved regulatory mechanisms. The timing and regulation of zebrafish hrT expression in anterior lateral plate mesoderm suggest a very early role for hrT in the differentiation of the cardiac precursors. hrT is coexpressed with gata4 and nkx2.5 not only in anterior lateral plate mesoderm but also in noncardiac mesoderm adjacent to the tail bud, suggesting that a conserved regulatory pathway links expression of these three genes in cardiac and noncardiac tissues. Finally, hrT expression was examined in pandora mutant embryos, since these have defects in many of the tissues that express hrT, including the heart. hrT expression is much reduced in the early heart fields of pandora mutants, whereas it is ectopically expressed subsequently. Using hrT expression as a marker, a midline patterning defect is described in pandora affecting the anterior hindbrain and associated midline mesendodermal derivatives. The possibility that the cardiac ventricular defect previously described in pandora and the midline defects described in this study are related (Griffin, 2000).
The T-box genes constitute a family of transcriptional regulator genes that have been implicated in a variety of developmental processes ranging from the formation of germ layers to the regionalization of the central nervous system. The cloning and expression pattern of a new T-box gene from zebrafish, which was named tbx20, is described. tbx20 is an ortholog of two other T-box genes isolated from animals of different phyla - H15 of Drosophila melanogaster and tbx-12 of Caenorhabditis elegans, suggesting that the evolutionary origin of this gene predates the divergence between the protostomes and deuterostomes. During development, tbx20 is expressed in embryonic structures of both mesodermal and ectodermal origins, including the heart, cranial motor neurons, and the roof of the dorsal aorta (Ahn, 2000).
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, it was shown that zebrafish embryos lacking hrT function have dysmorphic hearts and an absence of blood circulation. Although the early events in heart formation are normal in hrT morphant embryos, subsequently the hearts fail to undergo looping, and late onset defects in chamber morphology and gene expression are observed. In particular, it was found that the loss of hrT function leads to a dramatic upregulation of tbx5, a gene required for normal heart morphogenesis. Conversely, overexpression of hrT is shown to cause a significant downregulation of tbx5, indicating that one key role of hrT is to regulate the levels of tbx5. It was also 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, it has been shown 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 (Szeto, 2002).
Mutations in T-box genes are the cause of several congenital diseases and are implicated in cancer. Tbx20-null mice exhibit severely hypoplastic hearts and express Tbx2, which is normally restricted to outflow tract and atrioventricular canal, throughout the heart. Tbx20 mutant hearts closely resemble those seen in mice overexpressing Tbx2 in myocardium, suggesting that upregulation of Tbx2 can largely account for the cardiac phenotype in Tbx20-null mice. Evidence is provided that Tbx2 is a direct target for repression by Tbx20 in developing heart. Tbx2 directly binds to the Nmyc1 promoter in developing heart and can repress expression of the Nmyc1 promoter in transient transfection studies. Repression of Nmyc1 (N-myc) by aberrantly regulated Tbx2 can account in part for the observed cardiac hypoplasia in Tbx20 mutants. Nmyc1 is required for growth and development of multiple organs, including the heart, and overexpression of Nmyc1 is associated with childhood tumors. Despite its clinical relevance, the factors that regulate Nmyc1 expression during development are unknown. These data present a paradigm by which T-box proteins regulate regional differences in Nmyc1 expression and proliferation to effect organ morphogenesis. A model is presented whereby Tbx2 directly represses Nmyc1 in outflow tract and atrioventricular canal of the developing heart, resulting in relatively low proliferation. In chamber myocardium, Tbx20 represses Tbx2, preventing repression of Nmyc1 and resulting in relatively high proliferation. In addition to its role in regulating regional proliferation, Tbx20 regulates expression of a number of genes that specify regional identity within the heart, thereby coordinating these two important aspects of organ development (Cai, 2005).
To elucidate the function of the T-box transcription factor Tbx20 in mammalian development, a graded loss-of-function series was generated by transgenic RNA interference in entirely embryonic stem cell-derived mouse embryos. Complete Tbx20 knockdown results in defects in heart formation, including hypoplasia of the outflow tract and right ventricle, which derive from the anterior heart field (AHF), and decrease in the expression of Nkx2-5 and Mef2c, transcription factors required for AHF formation. A mild knockdown led to persistent truncus arteriosus (unseptated outflow tract) and hypoplastic right ventricle, entities similar to human congenital heart defects; this demonstrates a critical requirement for Tbx20 in valve formation. Finally, an intermediate knockdown revealed a role for Tbx20 in motoneuron development, specifically in the regulation of the transcription factors Isl2 and Hb9, which are important for terminal differentiation of motoneurons. Tbx20 can activate promoters/enhancers of several genes in cultured cells, including the Mef2c AHF enhancer and the Nkx2-5 cardiac enhancer. The Mef2c AHF enhancer relies on Isl1- and Gata-binding sites. A similar Isl1 binding site has been identified in the Nkx2-5 AHF enhancer, which in transgenic mouse embryos is essential for activity in a large part of the heart, including the outflow tract. Tbx20 synergizes with Isl1 and Gata4 to activate both the Mef2c and Nkx2-5 enhancers, thus providing a unifying mechanism for gene activation by Tbx20 in the AHF. It is thus concluded that Tbx20 is positioned at a critical node in transcription factor networks required for heart and motoneuron development where it dose-dependently regulates gene expression (Takeuchi, 2005).
The genetic hierarchies guiding lineage specification and morphogenesis of the mammalian embryonic heart are poorly understood. It has been shown by gene targeting that murine T-box transcription factor Tbx20 plays a central role in these pathways, and has important activities in both cardiac development and adult function. Loss of Tbx20 results in death of embryos at mid-gestation with grossly abnormal heart morphogenesis. Underlying these disturbances is a severely compromised cardiac transcriptional program, defects in the molecular pre-pattern, reduced expansion of cardiac progenitors and a block to chamber differentiation. Notably, Tbx20-null embryos show ectopic activation of Tbx2 across the whole heart myogenic field. Tbx2 encodes a transcriptional repressor normally expressed in non-chamber myocardium, and in the atrioventricular canal it has been proposed to inhibit chamber-specific gene expression through competition with positive factor Tbx5. These data demonstrate a repressive activity for Tbx20 and place it upstream of Tbx2 in the cardiac genetic program. Thus, hierarchical, repressive interactions between Tbx20 and other T-box genes and factors underlie the primary lineage split into chamber and non-chamber myocardium in the forming heart, an early event upon which all subsequent morphogenesis depends. Additional roles for Tbx20 in adult heart integrity and contractile function were revealed by in-vivo cardiac functional analysis of Tbx20 heterozygous mutant mice. These data suggest that mutations in human cardiac transcription factor genes, possibly including TBX20, underlie both congenital heart disease and adult cardiomyopathies (Stennard, 2005).
Tbx20, a member of the T-box family of transcriptional regulators, shows evolutionary conserved expression in the developing heart. In the mouse, Tbx20 is expressed in the cardiac crescent, then in the endocardium and myocardium of the linear and looped heart tube before it is restricted to the atrioventricular canal and outflow tract in the multi-chambered heart. Tbx20 is required for progression from the linear heart tube to a multi-chambered heart. Mice carrying a targeted mutation of Tbx20 show early embryonic lethality due to hemodynamic failure. A linear heart tube with normal anteroposterior patterning is established in the mutant. The tube does not elongate, indicating a defect in recruitment of mesenchyme from the secondary heart field, even though markers of the secondary heart field are not affected. Furthermore, dorsoventral patterning of the tube, formation of working myocardium, looping, and further differentiation and morphogenesis fail. Instead, Tbx2, Bmp2 and vinexin alpha (Sh3d4), genes normally restricted to regions of primary myocardium and lining endocardium, are ectopically expressed in the linear heart tube of Tbx20 mutant embryos. Because Tbx2 is both necessary and sufficient to repress chamber differentiation Tbx20 may ensure progression to a multi-chambered heart by repressing Tbx2 in the myocardial precursor cells of the linear heart tube destined to form the chambers (Singh, 2005).
The regulation of vertebrate eye development requires the activity of many transcription factors. The T-box factor Tbx12 is necessary for normal development of the retina. Tbx12 is expressed during early stages of retinal development in multiple species of vertebrate embryos. mRNAs encoding wild type and mutant forms of Tbx12 were injected into Xenopus embryos. The Tbx12 injected embryos exhibit multiple defects in eye development including reduced eye size and disruption of normal retinal laminar organization. Tbx12 appears to function as a repressor of transcription during eye development. These results indicate that Tbx12 activity is required for the proper generation and organization of retinal cells in the vertebrate eye (Carson, 2004).
Little is known about the molecular mechanisms involved with the initial specifications of the cardiac mesoderm. In order to identify potential regulatory factors that play important roles in early heart specification, the chick H15-related T-box gene was isolated and its expression pattern was analzyed during early development. The chick Tbx20 gene was found to be highly homologous to human, mouse, and zebrafish hrT/Tbx20. Its expression is initially detected in the posterior lateral mesoderm, after which it expands to the anterior and is intensively co-expressed with a cardiogenic gene, Nkx2.5, in the anterior lateral mesoderm (Iio, 2001).
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 is misexpressed ubiquitously, ventricular septum is not formed, resulting in a single ventricle. In such a heart, left ventricle (LV)-specific ANF gene is induced. In search of the putative RV factor(s), chick Tbx20 was found to be expressed in the RV, showing a complementary fashion to Tbx5. In the Tbx5-misexpressed heart, this gene is repressed. When misexpression is spatially partial, leaving small Tbx5-negative area in the right ventricle, ventricular septum is shifted rightwards, resulting in a small RV with an enlarged LV. Focal expression induces an ectopic boundary of Tbx5-positive and -negative regions in the right ventricle, at which an additional septum is formed. Similar results were obtained from the transient transgenic mice. In such hearts, expression patterns of dHAND and eHAND are changed with definitive cardiac abnormalities. Furthermore, human ANF promoter is synergistically activated by Tbx5, Nkx2.5 and GATA4. This activation is abrogated by Tbx20, implicating the pivotal roles of interactions among these heart-specific factors. Taken together, these 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 (Takeuchi, 2003).
T-box transcription factors contain a novel type of DNA-binding domain, the T-box domain, and are encoded by an ancient gene family. Four T-box genes, omb, Trg, org-1, and H15, have been identified in Drosophila, whereas in mammals the T-box gene family has expanded, and 12 human T-box genes have been isolated. A new human T-box gene, TBX20, and its mouse homologue Tbx20, have been identified that are more closely related to the Drosophila H15 gene than to any known vertebrate gene. H15 expression in leg imaginal discs correlates with commitment to a ventral fate, implicating this gene in early patterning events. TBX20 is expressed in the fetal heart, eye, and limb, and during embryogenesis in the mouse, Tbx20 is expressed in the developing heart, eye, ventral neural tube, and limbs, indicating a possible role in regulating development of these tissues. The TBX20 gene maps to chromosome 7p14-p15. An association between TBX20 and loci for retinitis pigmentosa, RP9, and blepharophimosis syndrome, BPES, have been excluded (Meins, 2000).
T-box genes encode transcription factors that regulate many developmental processes. A novel mouse T-box gene, Tbx12, has been cloned. Tbx12 is the vertebrate homologue of the Drosophila H15 gene and the Caenorhabditis elegans tbx-12 gene. Tbx12 is expressed in extraembryonic tissues such as the amnion and allantois. In the embryo, Tbx12 is strongly expressed in the neural retina and the heart (Carson, 2000).
T-box genes constitute a conserved multi-gene family with important roles in many developmental processes. The cloning and expression analysis is described of a novel mouse T-box gene, Tbx20. Expression is prominent in the extraembryonic mesoderm, in the developing heart, the eye anlage and motor neurons of hindbrain and spinal cord (Kraus, 2001).
Tbx20 is a member of the T-box transcription factor family expressed in the forming hearts of vertebrate and invertebrate embryos. This study reports analysis of Tbx20 expression during murine cardiac development and assessment of DNA-binding and transcriptional properties of Tbx20 isoforms. Tbx20 was expressed in myocardium and endocardium, including high levels in endocardial cushions. cDNAs generated by alternative splicing encode at least four Tbx20 isoforms, and Tbx20a uniquely carries strong transactivation and transrepression domains in its C terminus. Isoforms with an intact T-box bind specifically to DNA sites resembling the consensus brachyury half site, although with less avidity compared with the related factor, Tbx5. Tbx20 physically interacts with cardiac transcription factors Nkx2-5, GATA4, and GATA5, collaborating to synergistically activate cardiac gene expression. Among cardiac GATA factors, there was preferential synergy with GATA5, implicated in endocardial differentiation. In Xenopus embryos, enforced expression of Tbx20a, but not Tbx20b, leads to induction of mesodermal and endodermal lineage markers as well as cell migration, indicating that the long Tbx20a isoform uniquely bears functional domains that can alter gene expression and developmental behaviour in an in vivo context. It is proposed that Tbx20 plays an integrated role in the ancient myogenic program of the heart, and has been additionally coopted during evolution of vertebrates for endocardial cushion development (Stennard, 2003).
The T-box transcription factors play critical roles in embryonic development including cell type specification, tissue patterning, and morphogenesis. Several T-box genes are expressed in the heart and are regulators of cardiac development. At the earliest stages of heart development, two of these genes, Tbx5 and Tbx20, are co-expressed in the heart-forming region but then become differentially expressed as heart morphogenesis progresses. Although Tbx5 and Tbx20 belong to the same gene family and share a highly conserved DNA-binding domain, their transcriptional activities are distinct. The C-terminal region of the Tbx5 protein is a transcriptional activator, while the C terminus of Tbx20 can repress transcription. Tbx5, but not Tbx20, activates a cardiac-specific promoter (atrial natriuretic factor (ANF)) alone and synergistically with other transcription factors. In contrast, Tbx20 represses ANF promoter activity and also inhibits the activation mediated by Tbx5. Of the two T-box binding consensus sequences in the promoter of ANF, only T-box binding element 1 (TBE1) is required for the synergistic activation of ANF by Tbx5 and GATA4, but TBE2 is required for repression by Tbx20. To elucidate upstream signaling pathways that regulate Tbx5 and Tbx20 expression, recombinant bone morphogenetic protein-2 was added to cardiogenic explants from chick embryos. Using real time reverse transcription-PCR, it was demonstrated that Tbx20, but not Tbx5, is induced by bone morphogenetic protein-2. Collectively these data demonstrate clear differences in both the expression and function of two related transcription factors and suggest that the modulation of cardiac gene expression can occur as a result of combinatorial regulatory interactions of T-box proteins (Plageman, 2004).
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, the cellular and molecular relationship between two T-box genes, Tbx5 and Tbx20, was investigated. 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, 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, it is shown that TBX5 and TBX20 can physically interact, the interaction domains were mapped, and a cellular interaction was shown for the two proteins in cardiac development, thus providing the first evidence for direct interaction between members of the T-box gene family (Brown, 2005).
The cardiac conduction system comprises a specialized tract of electrically coupled cardiomyocytes responsible for impulse propagation through the heart. Abnormalities in cardiac conduction are responsible for numerous forms of cardiac arrhythmias, but relatively little is known about the gene regulatory mechanisms that control the formation of the conduction system. This study demonstrates that a distal enhancer for the connexin 30.2 (Cx30.2, also known as Gjd3) gene, which encodes a gap junction protein required for normal atrioventricular (AV) delay in mice, is necessary and sufficient to direct expression to the developing AV conduction system (AVCS). Moreover, this enhancer requires Tbx5 and Gata4 for proper expression in the conduction system, and Gata4+/- mice have short PR intervals indicative of accelerated AV conduction. These results implicate Gata4 in conduction system function and provide a clearer understanding of the transcriptional pathways that impact normal AV delay (Munshi, 2009).
TBX20 has been shown to be essential for vertebrate heart development. Mutations within the TBX20 coding region are associated with human congenital heart disease, and the loss of Tbx20 in a wide variety of model systems leads to cardiac defects and eventually heart failure. Despite the crucial role of TBX20 in a range of cardiac cellular processes, the signal transduction pathways that act upstream of Tbx20 remain unknown. This study identified and characterized a conserved 334 bp Tbx20 cardiac regulatory element that is directly activated by the BMP/SMAD1 signaling pathway. This element is both necessary and sufficient to drive cardiac-specific expression of Tbx20 in Xenopus, and blocking SMAD1 signaling in vivo specifically abolishes transcription of Tbx20, but not that of other cardiac factors, such as Tbx5 and MHC, in the developing heart. Activation of Tbx20 by SMAD1 is mediated by a set of novel, non-canonical, high-affinity SMAD-binding sites located within this regulatory element, and phospho-SMAD1 directly binds a non-canonical SMAD1 site in vivo. Finally, it was shown that these non-canonical sites are necessary and sufficient for Tbx20 expression in Xenopus, and that reporter constructs containing these sites are expressed in a cardiac-specific manner in zebrafish and mouse. Collectively, these findings define Tbx20 as a direct transcriptional target of the BMP/SMAD1 signaling pathway during cardiac maturation (Mandel 2010).
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