analysis-transforming-growth-factor-family-cleavage-products-secreted

Analysis of Transforming Growth Factor &#223; Family Cleavage Products Secreted Into the Blastocoele of <em>Xenopus laevis</em> Embryos

The two arms of the Transforming Growth Factor &#223; (Tgf&#223;) superfamily, represented by Tgf&#223;/Nodal or Bone morphogenetic protein (Bmp) ligands, ...

University of Utah, School of Medicine

Morphogens are substances that establish a graded distribution and elicit distinct cellular responses in a dose-dependent manner. They function to provide individual cells within a field with positional information, which is interpreted to give rise to spatial patterns. Morphogens can consist of intracellular factors that set up a concentration gradient by diffusion in the cytoplasm. More commonly, morphogens comprise secreted proteins that form an extracellular gradient across a field of cells. Experimental studies and computational analyses have provided support for a number of diverse strategies by which extracellular morphogen gradients are formed. These include free diffusion in the extracellular space, restricted diffusion aided by interactions with heparan sulfate proteoglycans, transport on lipid-containing carriers or transport aided by soluble binding partners. More specialized modes of transport have also been postulated such as transcytosis, in which repeated rounds of secretion, endocytosis, and intracellular trafficking move morphogens through cells rather than around them, or cytonemes, which consist of filopodial extensions from signal-receiving cells that are hypothesized to reach out to morphogen-sending cells. Once the gradient has formed, cells must distinguish small differences in morphogen concentration and store this information even after the gradient has dissipated. This is often achieved by translating ligand concentration into a proportional increase in numbers of activated cell surface receptors that are internalized and continue to signal from endosomal compartments. Ultimately, this leads to activation of one or a few transcription factors that transduce this information into qualitatively distinct gene responses inside the nucleus.

Morphogen gradients in development: from form to function.

ProBMP4 is generated as a latent precursor that is sequentially cleaved at two sites within the prodomain to generate an active ligand. An initial cleavage occurs adjacent to the ligand domain, which generates a non-covalently associated prodomain/ligand complex that is subsequently dissociated by cleavage at an upstream site. An outstanding question is whether the two sites need to be cleaved sequentially and in the correct order to achieve proper control of BMP4 signaling during development. In the current studies, we demonstrate that mice carrying a knock-in point mutation that causes simultaneous rather than sequential cleavage of both prodomain sites show loss of BMP4 function and die during mid-embryogenesis. Levels of mature BMP4 are severely reduced in mutants, although levels of precursor and cleaved prodomain are unchanged compared with wild type. Our biochemical analysis supports a model in which the transient prodomain/ligand complex that forms during sequential cleavage plays an essential role in prodomain-mediated stabilization of the mature ligand until it can acquire protection from degradation by other means. By contrast, simultaneous cleavage causes premature release of the ligand from the prodomain, leading to destabilization of the ligand and loss of signaling in vivo. 

Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice.

Bone morphogenetic proteins 4 and 7 (BMP4 and BMP7) are morphogens that signal as either homodimers or heterodimers to regulate embryonic development and adult homeostasis. BMP4/7 heterodimers exhibit markedly higher signaling activity than either homodimer, but the mechanism underlying the enhanced activity is unknown. BMPs are synthesized as inactive precursors that dimerize and are then cleaved to generate both the bioactive ligand and prodomain fragments, which lack signaling activity. Our study reveals a previously unknown requirement for the BMP4 prodomain in promoting heterodimer activity. We show that BMP4 and BMP7 precursor proteins preferentially or exclusively form heterodimers when coexpressed in vivo. In addition, we show that the BMP4 prodomain is both necessary and sufficient for generation of stable heterodimeric ligands with enhanced activity and can enable homodimers to signal in a context in which they normally lack activity. Our results suggest that intrinsic properties of the BMP4 prodomain contribute to the relative bioactivities of homodimers versus heterodimers in vivo. These findings have clinical implications for the use of BMPs as regenerative agents for the treatment of bone injury and disease. 

The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo.

Primitive erythropoiesis is regulated in a non cell-autonomous fashion across evolution from frogs to mammals. In Xenopus laevis, signals from the overlying ectoderm are required to induce the mesoderm to adopt an erythroid fate. Previous studies in our lab identified the transcription factor GATA2 as a key regulator of this ectodermal signal. To identify GATA2 target genes in the ectoderm required for red blood cell formation in the mesoderm, we used microarray analysis to compare gene expression in ectoderm from GATA2 depleted and wild type embryos. Our analysis identified components of the non-canonical and canonical Wnt pathways as being reciprocally up- and down-regulated downstream of GATA2 in both mesoderm and ectoderm. We show that up-regulation of canonical Wnt signaling during gastrulation blocks commitment to a hematopoietic fate while down-regulation of non-canonical Wnt signaling impairs erythroid differentiation. Our results are consistent with a model in which GATA2 contributes to inhibition of canonical Wnt signaling, thereby permitting progenitors to exit the cell cycle and commit to a hematopoietic fate. Subsequently, activation of non-canonical Wnt signaling plays a later role in enabling these progenitors to differentiate as mature red blood cells.

GATA2 regulates Wnt signaling to promote primitive red blood cell fate.

Primitive hematopoiesis generates red blood cells that deliver oxygen to the developing embryo. Mesodermal cells commit to a primitive blood cell fate during gastrulation and, in order to do so the mesoderm must receive non-cell autonomous signals transmitted from other germ layers. In Xenopus, the transcription factor Gata2 functions in ectodermal cells to generate or transmit the non-cell autonomous signals. Here we have identified Breast Cancer Antiestrogen Resistance 3 (bcar3) as a gene that is induced in ectodermal cells downstream of Gata2. Bcar3 and its binding partner Bcar1 function to transduce integrin signaling, leading to changes in cellular morphology, motility and adhesion. We show that gata2, bcar3 and bcar1 are co-expressed in ventral ectoderm from early gastrula to early tailbud stages. At later stages of development, bcar3 and bcar1 are co-expressed in the spinal cord, notochord, fin mesenchyme and pronephros but each shows additional unique sites of expression. These co-expression and unique expression patterns suggest that Bcar3 and Bcar1 may function together but also independently during Xenopus development. 

Expression pattern of bcar3, a downstream target of Gata2, and its binding partner, bcar1, during Xenopus development.

In Xenopus laevis, bone morphogenetic proteins (Bmps) induce expression of the transcription factor Gata2 during gastrulation, and Gata2 is required in both ectodermal and mesodermal cells to enable mesoderm to commit to a hematopoietic fate. Here, we identify tril as a Gata2 target gene that is required in both ectoderm and mesoderm for primitive hematopoiesis to occur. Tril is a transmembrane protein that functions as a co-receptor for Toll-like receptors to mediate innate immune responses in the adult brain, but developmental roles for this molecule have not been identified. We show that Tril function is required both upstream and downstream of Bmp receptor-mediated Smad1 phosphorylation for induction of Bmp target genes. Mechanistically, Tril triggers degradation of the Bmp inhibitor Smad7. Tril-dependent downregulation of Smad7 relieves repression of endogenous Bmp signaling during gastrulation and this enables mesodermal progenitors to commit to a blood fate. Thus, Tril is a novel component of a Bmp-Gata2 positive-feedback loop that plays an essential role in hematopoietic specification.

Tril targets Smad7 for degradation to allow hematopoietic specification in Xenopus embryos.

The 10th FASEB meeting 'The TGFβ Superfamily: Signaling in Development and Disease' took place in Lisbon, Portugal, in July 2017. As we review here, the findings presented at the meeting highlighted the important contributions of TGFβ family signaling to normal development, adult homeostasis and disease, and also revealed novel mechanisms by which TGFβ signals are transduced.

The TGFβ superfamily in Lisbon: navigating through development and disease.

Toll-like receptor 4 interactor with leucine-rich repeats (Tril) functions as a coreceptor for Toll-like receptors (Tlrs) to mediate innate immune responses in adults. In embryos, Tril signals to promote degradation of the Bmp inhibitor, Smad7, to allow for blood formation. It is not known whether this function requires, or is independent of, Tlrs. In the current studies, we performed a structure-function analysis, which indicated that the fibronectin type III (FN) domain and the intracellular domain of Tril are required to trigger Smad7 degradation in  embryos. Furthermore, we found evidence suggesting that a Tril deletion mutant lacking the FN domain (Tril∆FN) can dominantly inhibit signaling by endogenous Tril when overexpressed. This finding raises the possibility that the FN domain functions to bind endogenous Tril ligands. We also show that Tril cycles between the cell surface and endosomes and that the Tril extracellular domain, as well as cadherin based cell-cell adhesion, are required for cell surface retention, while the intracellular domain is required for internalization in  ectodermal explants. Using a CHO cell aggregation assay, we show that, unlike other transmembrane proteins that contain leucine-rich repeats, Tril is not sufficient to mediate homophilic adhesion.

Fibronectin type III and intracellular domains of Toll-like receptor 4 interactor with leucine-rich repeats (Tril) are required for developmental signaling.

Bone morphogenetic proteins (Bmps) are synthesized as inactive precursors that are cleaved to generate active ligands, along with prodomain fragments that can modulate growth factor activity. Here we provide three protocols that can be used to examine the process of proteolytic activation of Bmps. The first protocol describes how to generate radiolabeled Bmp precursor proteins in Xenopus oocytes and then analyze the time course of precursor cleavage by recombinant enzymes in vitro. The second protocol details how to analyze cleavage of radiolabeled precursor proteins in Xenopus oocytes over time using pulse-chase analysis and autoradiography. This protocol can also be used to analyze folding and cleavage of radiolabeled precursor proteins at steady state. Finally, the third protocol details methods for isolating Bmp cleavage products from the blastocoele of Xenopus embryos and then analyzing them on immunoblots.

Article	Year
Morphogen gradients in development: from form to function. Wiley interdisciplinary reviews. Developmental biology\| PubMed ID: 23801664	2012
Simultaneous rather than ordered cleavage of two sites within the BMP4 prodomain leads to loss of ligand in mice. Development (Cambridge, England)\| PubMed ID: 24993941	2014
The prodomain of BMP4 is necessary and sufficient to generate stable BMP4/7 heterodimers with enhanced bioactivity in vivo. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 25902523	2015
GATA2 regulates Wnt signaling to promote primitive red blood cell fate. Developmental biology\| PubMed ID: 26365900	2015
Expression pattern of bcar3, a downstream target of Gata2, and its binding partner, bcar1, during Xenopus development. Gene expression patterns : GEP\| PubMed ID: 26631802	2016
Tril targets Smad7 for degradation to allow hematopoietic specification in Xenopus embryos. Development (Cambridge, England)\| PubMed ID: 27633996	2016
The TGFβ superfamily in Lisbon: navigating through development and disease. Development (Cambridge, England)\| PubMed ID: 29254990	2017
Fibronectin type III and intracellular domains of Toll-like receptor 4 interactor with leucine-rich repeats (Tril) are required for developmental signaling. Molecular biology of the cell\| PubMed ID: 29298840	2018
Proteolytic Activation of Bmps: Analysis of Cleavage in Xenopus Oocytes and Embryos. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30414129	2019

Jan L. Christian

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