Tbx2 Mediates Dorsal Patterning and Germ Layer Suppression Through
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Reich et al. BMC Molecular and Cell Biology (2020) 21:39 BMC Molecular and https://doi.org/10.1186/s12860-020-00282-1 Cell Biology RESEARCH ARTICLE Open Access Tbx2 mediates dorsal patterning and germ layer suppression through inhibition of BMP/GDF and Activin/Nodal signaling Shoshana Reich1, Peter Kayastha2, Sushma Teegala2 and Daniel C. Weinstein1,2* Abstract Background: Members of the T-box family of DNA-binding proteins play a prominent role in the differentiation of the three primary germ layers. VegT, Brachyury, and Eomesodermin function as transcriptional activators and, in addition to directly activating the transcription of endoderm- and mesoderm-specific genes, serve as regulators of growth factor signaling during induction of these germ layers. In contrast, the T-box gene, tbx2, is expressed in the embryonic ectoderm, where Tbx2 functions as a transcriptional repressor and inhibits mesendodermal differentiation by the TGFβ ligand Activin. Tbx2 misexpression also promotes dorsal ectodermal fate via inhibition of the BMP branch of the TGFβ signaling network. Results: Here, we report a physical association between Tbx2 and both Smad1 and Smad2, mediators of BMP and Activin/Nodal signaling, respectively. We perform structure/function analysis of Tbx2 to elucidate the roles of both Tbx2-Smad interaction and Tbx2 DNA-binding in germ layer suppression. Conclusion: Our studies demonstrate that Tbx2 associates with intracellular mediators of the Activin/Nodal and BMP/GDF pathways. We identify a novel repressor domain within Tbx2, and have determined that Tbx2 DNA- binding activity is required for repression of TGFβ signaling. Finally, our data also point to overlapping yet distinct mechanisms for Tbx2-mediated repression of Activin/Nodal and BMP/GDF signaling. Keywords: Xenopus, Development, Gastrulation, Ectoderm, Gene regulation, Tbx2, Smads Background skeletal systems, and the epidermis, neural tissue, neural Understanding the development of an embryo from a crest, and cranial sensory placodes derive from ecto- single totipotent cell to a highly differentiated, multicel- derm. The precise coordination of germ layer differenti- lular organism is a foundational concern of developmen- ation is, by definition, crucial for normal embryogenesis. tal biology. In triploblasts, three embryonic germ layers, During vertebrate development, the formation and ectoderm, mesoderm, and endoderm, give rise to virtu- patterning of the three germ layers is a rapid and tightly ally all tissue types. The endodermal germ layer differen- regulated process; studies in the frog Xenopus laevis tiates into pancreas, liver, lung, and other components have been essential for our understanding of these pro- of the digestive and respiratory systems. The mesoder- cesses. An initiating step in development of the germ mal layer gives rise the muscular, circulatory, and layers occurs when VegT, a maternally supplied tran- scription factor, directly initiates an endoderm-specific * Correspondence: [email protected] gene expression program among cells located in the 1The Graduate Center, The City University of New York, New York, NY 10016, vegetal pole [1]. VegT also activates nodal and nodal-re- USA 2Department of Biology, Queens College, The City University of New York, lated gene expression; these transcripts encode proteins Queens, NY 11367, USA that induce cells in the region adjacent to the vegetal © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Reich et al. BMC Molecular and Cell Biology (2020) 21:39 Page 2 of 11 pole, in the so-called marginal zone, to differentiate into transcriptional activators in this context [3, 23]. Our lab mesoderm [2, 3]. In this VegT-centric model of germ has recently shown that Tbx2 functions in the ectoderm layer formation, differentiation in the animal pole is the as a transcriptional repressor during gastrulation [10]. consequence of an absence of both extracellular signal- When tbx2 is knocked down in the presumptive ecto- ing and germ layer-specific transcriptional activation -- derm, ectopic mesendoderm forms; moreover, ectopic VegT is not expressed in this region, and it is far from Tbx2 suppresses mesendoderm in animal cap explants the source of Nodal signaling; thus, neither endodermal exposed to Activin or basic Fibroblast Growth Factor nor mesodermal differentiation ensues, and ectoderm (bFGF) [10]. To date, however, little is known about the forms in the animal pole “by default” [4, 5]. mechanisms through which Tbx2 promotes repressor Recently, however, it has become clear that the sup- activity. pression of inappropriate cell fate also plays a critical The population of cells in the ectoderm develops into role in germ layer determination in the vertebrate em- several distinct tissue types. Ventral ectoderm differenti- bryo [6]. Several proteins have been implicated in ates into epidermis, while the dorsal ectoderm gives rise mesendodermal suppression in the ectoderm. For ex- to neural tissue [24]; cells at the border of these two ample, coco (dand5), a maternally supplied transcript, populations develop into the sensory placodes and encodes an inhibitor of TGFβ signaling [7, 8]. Depletion neural crest [25, 26]. Bone Morphogenetic Protein-4 of Coco in the animal cap explant results in ectopic (BMP-4), expressed throughout the ectoderm, ventra- mesodermal formation [7]. Another protein identified as lizes the ectoderm, which subsequently differentiates a suppressor of mesendoderm is the Foxi-class transcrip- into epidermis [27]. Dorsally, at the initiation of gastru- tion factor Xema/Foxi1e. Knockdown of foxi1e in animal lation, the Spemann organizer secretes BMP antagonists cap explants leads to an increase in the expression of allowing proximal ectodermal cells to adopt a dorsal, mesendodermal markers [9]. Moreover, ectopic expres- neural fate [27]. Tbx2 misexpression also dorsalizes sion of Foxi1e in the marginal zone inhibits mesoderm ectoderm, resulting in neuralization; this effect is accom- development. Foxi1e is a transcriptional activator, and is panied by downregulation of Bone Morphogenetic Pro- thus not expected to directly repress mesendodermal tein (BMP) activity [10]. gene expression; instead, Foxi1e likely stimulates expres- Tbx2 thus appears to suppress signaling through both sion of genes encoding repressor protein(s) that is/are the Activin/Nodal and BMP/GDF branches of the TGFβ responsible for suppressing ectopic mesendoderm in the pathway. In canonical TGFβ signaling, upon ligand bind- animal pole [9]. We recently identified tbx2, encoding ing, the TGFβ type II receptor phosphorylates the TGFβ the repressor T-box protein Tbx2, as one such candidate type I receptor, which propagates the signal and phos- target of Foxi1e [10]. phorylates the carboxyl terminus of the intracellular sig- T-box proteins are an evolutionarily conserved family nal transducers Smad1/5 or Smad2/3, downstream of of transcription factors critical for a number of processes BMP/GDF or Activin/Nodal ligands, respectively [28, during development [11]. The first identified T-box pro- 29]. These receptor-mediated Smads then form a hetero- tein, T, encoded by brachyury, induces mesoderm in the meric complex with Smad4 which translocates to the early mammalian embryo [12]. In the mouse, a homozy- nucleus where it associates with transcription factors to gous loss-of-function mutation in brachyury is embryon- regulate gene expression [28]. ically lethal, highlighting the importance of T-box The discovery that Tbx2 has a global impact on TGFβ proteins in early development [13]. T-box proteins have signaling prompted us to examine more directly the rela- also been implicated in development of craniofacial tis- tionship between Tbx2 and components of the Activin/ sue, liver, heart, and lung [3, 14–17]. Nodal and BMP/GDF networks. Here we report that The T-box proteins are comprised of five subfamilies; Tbx2 physically associates with both Smad1 and Smad2, all contain a highly conserved region of 180–200 amino essential intracellular mediators of BMP and Nodal acids, called the T-box, which confers DNA binding speci- signaling, respectively. Deletion analysis of the Tbx2 ficity [18, 19]. Many T-box proteins have been shown to protein identified the T-box DNA-binding domain as bind the core T-box binding element TCACACCT [19]. sufficient for Tbx2-Smad binding. Structure-function T-box proteins bind to DNA as either monomers or di- analyses suggest a model in which Tbx2 regulates inter- mers, depending