Identification of direct T-box target genes in the developing zebrafish mesoderm

Garnett, Aaron T.; Han, Tina M.; Gilchrist, Michael J.; Smith, James C.; Eisen, Michael B.; Wardle, Fiona C.; Amacher, Sharon L.

doi:10.1242/dev.024703

Cited by 49 publications

(91 citation statements)

References 94 publications

(100 reference statements)

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“…Our results indicate that CoBra has a highly similar motif to that determined in the mouse Bra-homolog, called T (Fig. 4) (28,(34)(35)(36). Moreover, our results indicate that the T-box DNA recognition sequence is strongly conserved, both across a wide range of T-box classes [including Eomes (37), Tbx1, Tbx4, and Tbx2] and also across different organisms (Fig.…”

Section: Resultssupporting

confidence: 64%

Early evolution of the T-box transcription factor family

Sebé-Pedrós

Ariza-Cosano

Weirauch

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Developmental transcription factors are key players in animal multicellularity, being members of the T-box family that are among the most important. Until recently, T-box transcription factors were thought to be exclusively present in metazoans. Here, we report the presence of T-box genes in several nonmetazoan lineages, including ichthyosporeans, filastereans, and fungi. Our data confirm that Brachyury is the most ancient member of the T-box family and establish that the T-box family diversified at the onset of Metazoa. Moreover, we demonstrate functional conservation of a homolog of Brachyury of the protist Capsaspora owczarzaki in Xenopus laevis. By comparing the molecular phenotype of C. owczarzaki Brachyury with that of homologs of early branching metazoans, we define a clear difference between unicellular holozoan and metazoan Brachyury homologs, suggesting that the specificity of Brachyury emerged at the origin of Metazoa. Experimental determination of the binding preferences of the C. owczarzaki Brachyury results in a similar motif to that of metazoan Brachyury and other T-box classes. This finding suggests that functional specificity between different T-box classes is likely achieved by interaction with alternative cofactors, as opposed to differences in binding specificity.origin multicellularity | premetazoan evolution | subfunctionalization | Porifera | Holozoa T ranscriptional regulation is a central aspect of animal development. Thus, deciphering the early evolution of metazoan transcription factors is vital for achieving a better understanding of the origin of animals. The T-box family of genes is among the most important developmental transcription factors present in Metazoa. This family is characterized by an evolutionary conserved DNA-binding domain of 180-200 amino acids, known as the T-box domain (1-3). Brachyury is the founding and best-characterized member of the T-box family, with well-established roles in blastopore specification, mesoderm differentiation and, in chordates, notochord formation (4-6). It has been hypothesized that the ancestral role of Brachyury was primarily that of blastopore determination and gastrulation (5, 7).Other T-box classes include Tbx4/5, Tbx6, Tbx2/3, Eomes, and Tbx1/15/20. With only a few exceptions (8), all classes of T-box genes are widespread among bilaterian animals, with a handful being identified and studied in nonbilaterian metazoans, such as cnidarians (5, 9), ctenophores (7, 10), and sponges (11-14). T-box genes were initially thought to be specific to metazoans (13, 15), but two recent studies revealed the presence of T-box genes in nonmetazoan lineages (14, 16), including the unicellular filose amoeba Capsaspora owczarzaki, a close relative of animals, and the chytrid fungus Spizellomyces punctatus. T-box genes were not identified in any other sequenced eukaryote, suggesting that T-box genes were secondarily lost in choanoflagellates (both in unicellular and colonial species) and most fungi. Interestingly, one of the T-box genes identified in C. ow...

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Section: Resultssupporting

confidence: 64%

Early evolution of the T-box transcription factor family

Sebé-Pedrós

Ariza-Cosano

Weirauch

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…For instance, pcdh8 and her1 were expanded anteriorly, which was expected because they are Tbx16 target genes (Fig. 2B,C) (Garnett et al, 2009;Yamamoto et al, 1998). Interestingly, sox2 and ntl were reduced in the progenitor zone (Fig.…”

Section: Tbx16 Advances Cells Toward Muscle Differentiationmentioning

confidence: 75%

“…Tbx16 is thought to act directly as a transcriptional activator and has been shown to be involved in activating the transcription of a large number of target genes (Garnett et al, 2009). We investigated whether the repression of ntl and sox2 that we observed was a result of a repressive function of Tbx16 or of Tbx16-driven activation of a repressor.…”

Section: Tbx16 Advances Cells Toward Muscle Differentiationmentioning

confidence: 93%

Wnt signaling andtbx16form a bistable switch to commit bipotential progenitors to mesoderm

et al. 2015

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Anterior to posterior growth of the vertebrate body is fueled by a posteriorly located population of bipotential neuro-mesodermal progenitor cells. These progenitors have a limited rate of proliferation and their maintenance is crucial for completion of the anterior-posterior axis. How they leave the progenitor state and commit to differentiation is largely unknown, in part because widespread modulation of factors essential for this process causes organism-wide effects. Using a novel assay, we show that zebrafish Tbx16 (Spadetail) is capable of advancing mesodermal differentiation cell-autonomously. Tbx16 locks cells into the mesodermal state by not only activating downstream mesodermal genes, but also by repressing bipotential progenitor genes, in part through a direct repression of sox2. We demonstrate that tbx16 is activated as cells move from an intermediate Wnt environment to a high Wnt environment, and show that Wnt signaling activates the tbx16 promoter. Importantly, high-level Wnt signaling is able to accelerate mesodermal differentiation cellautonomously, just as we observe with Tbx16. Finally, because our assay for mesodermal commitment is quantitative we are able to show that the acceleration of mesodermal differentiation is surprisingly incomplete, implicating a potential separation of cell movement and differentiation during this process. Together, our data suggest a model in which high levels of Wnt signaling induce a transition to mesoderm by directly activating tbx16, which in turn acts to irreversibly flip a bistable switch, leading to maintenance of the mesodermal fate and repression of the bipotential progenitor state, even as cells leave the initial highWnt environment.

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“…A gene was annotated as a target if an enriched probe fell within Ϫ9 kb and ϩ3 kb (12 kb) of the TSS. This approach successfully identified all known direct targets of Ntl (dld, tbx6, and wnt8) except wnt3a, which was not represented on our microarrays (8,52). Further information on validation of our approach can be found in SI Text.…”

Section: Ntl Binds Mesodermally Expressed Genes Involved In Transcripmentioning

confidence: 90%

A gene regulatory network directed by zebrafish No tail accounts for its roles in mesoderm formation

Morley

Lachani

Keefe

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Using chromatin immunoprecipitation combined with genomic microarrays we have identified targets of No tail (Ntl), a zebrafish Brachyury ortholog that plays a central role in mesoderm formation. We show that Ntl regulates a downstream network of other transcription factors and identify an in vivo Ntl binding site that resembles the consensus T-box binding site (TBS) previously identified by in vitro studies. We show that the notochord-expressed gene floating head ( flh) is a direct transcriptional target of Ntl and that a combination of TBSs in the flh upstream region are required for Ntl-directed expression. Using our genome-scale data we have assembled a preliminary gene regulatory network that begins to describe mesoderm formation and patterning in the early zebrafish embryo.brachyury ͉ chromatin immunopreciptitation ͉ microarray E mbryonic development proceeds through a series of progressively more restricted cell states in which sets of state-specific genes are expressed in a finely controlled temporal and spatial order. This coordination of gene expression is brought about by the integration of signaling inputs and binding of transcription factors at cis-regulatory modules (CRMs) associated with target genes, and can be described by a gene regulatory network (GRN). Such GRNs have been useful in understanding how development proceeds in the early lineages of sea urchins and ascidians (1, 2). However, although GRNs using data from single gene studies have been used to describe aspects of Xenopus mesendoderm and lamprey neural crest development (3, 4) a systematic approach to building a GRN by genome-scale analysis has not yet been used to describe early cell fate commitment in developing vertebrate embryos. Understanding how transcriptional regulation drives cell fate commitment in vertebrates is essential not only in understanding their development, but also for informing future efforts to recapitulate cell restriction to different tissue lineages for stem cell-based replacement therapies (5). We have thus set out to assemble a GRN that can describe vertebrate mesoderm development using zebrafish as a model system to identify targets of a key transcriptional regulator, No tail (Ntl).Ntl is a zebrafish ortholog of Brachyury, a T-domain transcription factor that is expressed as an early response to mesoderm induction and plays a central role in mesoderm development in all vertebrates. For instance, studies in mice, Xenopus, and zebrafish reveal that Brachyury orthologs influence many aspects of mesoderm specification and patterning, being required for formation of the notochord and posterior somites, for normal cell movements during gastrulation and tail outgrowth, and for establishment of left-right asymmetry (reviewed in refs. 6, 7).Here, we use chromatin immunoprecipitation combined with zebrafish genomic microarrays (ChIP-chip) to survey binding of Ntl at promoter regions. We show that Ntl binds the promoters of transcription factors implicated in posterior identity, muscle specification, cell movements, ...

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Identification of direct T-box target genes in the developing zebrafish mesoderm

Cited by 49 publications

References 94 publications

Early evolution of the T-box transcription factor family

Early evolution of the T-box transcription factor family

Wnt signaling andtbx16form a bistable switch to commit bipotential progenitors to mesoderm

A gene regulatory network directed by zebrafish No tail accounts for its roles in mesoderm formation

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