Our long term goal is to understand how Bmp and Tgf-/nodal signaling is regulated in development and disease. Imbalances in Bmp and Tgf-/nodal signaling lead to birth defects and cancers, and yet knowledge of how the equilibrium between these pathways is maintained is incomplete. We identified Tril as a protein that coordinately regulates Bmp and nodal signaling and propose that it does so through a novel mechanism involving Toll-like receptors (Tlrs). Tlrs play evolutionarily conserved roles in mediating innate immune responses. The proposed studies will provide the first demonstration that Tlr signaling is also required for early patterning in vertebrates, and will set the stage for future studies that ask whether Tril or downstream signaling components play a protective and/or causal role in congenital anomalies and disease. Our published studies show that Tril triggers degradation of Smad7 to enhance Bmp signaling during gastrulation in Xenopus, and our new data show that Tril simultaneously inhibits nodal signaling. Our data support the hypothesis that Tril serves as a co-receptor for Tlrs to initiate a signaling cascade in which Irak kinases activate Pellino2. Pellino2 then stimulates Rnf12- mediated degradation of Smad7 to enhance Bmp signaling, and Nedd4L-mediated degradation of nodal signaling components to inhibit nodal signaling. We will test this hypothesis as follows (1) Determine how Tril regulates Nedd4l to inhibit nodal signaling. We will compare the kinetics of nodal pathway inactivation, analyze steady state levels of nodal receptors and ask whether Nedd4l is sufficient to rescue nodal signaling in control and Tril deficient Xenopus embryos and mouse embryonic fibroblasts (MEFs). We will test whether Tril prevents proteosomal degradation and/or stimulates post-translational modification of Nedd4l. (2) Determine whether and how Tril regulates Rnf12 to mediate degradation of Smad7. We will test whether the ability of Tril to trigger degradation of Smad7 is impaired in embryos or in MEFs in which expression of Rnf12 is knocked down, and conversely, whether Rnf12-mediated degradation of Smad7 is impaired in Tril morphants and in tril mutant MEFs. We will test whether steady state levels, subcellular localization and/or post-translational modification of Rnf1 is altered in Tril morphant embryos and tril mutant MEFs. (3) Determine whether Tril activates a Tlr-signaling cascade to regulate Bmp and nodal signaling. We will down-regulate the function of Tlr3, Tlr4, Iraks and Pellino2 in embryos and in MEFs and ask whether Bmp signaling is impaired and nodal signaling is enhanced, and we will upregulate the function of each protein and ask whether this rescues Bmp and nodal signaling in Tril morphant embryos and tril mutant MEFs. We will test whether Tril is required to regulate Pellino2 phosphorylation, subcellular localization and/or binding to Smad7 and Nedd4l, and will use rescue assays to determine if phosphorylation of Pellino2 and/or its ubiquitin ligase activity are required for function during embryogenesis.

Public Health Relevance

We are studying crosstalk between the Toll-like receptor (Tlr) and Transforming growth factor- (Tgf-) family signaling pathways. Tlr signaling is best known for its role in regulating immune responses to bacterial and viral infections, while Tgf- signaling plays critical roles in shaping how embryos form. Imbalances in either pathway lead to birth defects, cancers, and rampant inflammation and thus, understanding how the two pathways intersect to maintain an equilibrium is an important step toward preventing or treating these pathologies.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Research Project (R01)
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Development - 2 Study Section (DEV2)
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Mukhopadhyay, Mahua
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University of Utah
Internal Medicine/Medicine
Schools of Medicine
Salt Lake City
United States
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Kim, Hyung-Seok; McKnite, Autumn; Xie, Yuanyuan et al. (2018) Fibronectin type III and intracellular domains of Toll-like receptor 4 interactor with leucine-rich repeats (Tril) are required for developmental signaling. Mol Biol Cell 29:523-531
Christian, Jan L; Heldin, Carl-Henrik (2017) The TGF? superfamily in Lisbon: navigating through development and disease. Development 144:4476-4480
Green, Yangsook Song; Kwon, Sunjong; Christian, Jan L (2016) Expression pattern of bcar3, a downstream target of Gata2, and its binding partner, bcar1, during Xenopus development. Gene Expr Patterns 20:55-62
Green, Yangsook Song; Kwon, Sunjong; Mimoto, Mizuho S et al. (2016) Tril targets Smad7 for degradation to allow hematopoietic specification in Xenopus embryos. Development 143:4016-4026
Mimoto, Mizuho S; Kwon, Sunjong; Green, Yangsook Song et al. (2015) GATA2 regulates Wnt signaling to promote primitive red blood cell fate. Dev Biol 407:1-11
Mimoto, Mizuho S; Christian, Jan L (2012) Friend of GATA (FOG) interacts with the nucleosome remodeling and deacetylase complex (NuRD) to support primitive erythropoiesis in Xenopus laevis. PLoS One 7:e29882
Christian, Jan L (2012) Morphogen gradients in development: from form to function. Wiley Interdiscip Rev Dev Biol 1:3-15
Mimoto, Mizuho S; Christian, Jan L (2011) Manipulation of gene function in Xenopus laevis. Methods Mol Biol 770:55-75