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

The Lef/Tcf factor Tcf3 is expressed throughout the developing vertebrate central nervous system (CNS), but its function and transcriptional targets are uncharacterized. Tcf3 is thought to mediate canonical Wnt signaling, which functions in CNS patterning, proliferation and neurogenesis. In this study, we examine Tcf3 function in the zebrafish spinal cord, and find that this factor does not play a general role in patterning, but is required for the proper expression of Dbx genes in intermediate progenitors. In addition, we show that Tcf3 is required to inhibit premature neurogenesis in spinal progenitors by repressing sox4a, a known mediator of spinal neurogenesis. Both of these functions are mediated by Tcf3 independently of canonical Wnt signaling. Together, our data indicate a novel mechanism for the regulation of neurogenesis by Tcf3-mediated repression.

Tcf3 inhibits spinal cord neurogenesis by regulating sox4a expression.

Neural progenitor cells must be maintained during development in order to produce the full complement of neuronal and glial derivatives. While molecular pathways have been identified that inhibit progenitor differentiation, it is unclear whether the progenitor state itself is actively maintained. In this study, we have investigated the role of Tcf7l1 (formerly named Tcf3) in maintaining spinal progenitor characteristics and allowing the continued production of neurons and glia following primary neurogenesis. We find that spinal cord progenitor markers are progressively lost in embryos lacking Tcf7l1, and that the number of proliferative progenitors decreases accordingly. Furthermore, we show that the production of both neuronal and glial secondary derivatives of the pMN progenitor pool requires Tcf7l1. Together, these results indicate that Tcf7l1 plays an important role in spinal cord progenitor maintenance, indicating that this core function is conserved throughout multiple epithelial cell populations.

Tcf7l1 is required for spinal cord progenitor maintenance.

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.

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.

Proteolytic Activation of Bmps: Analysis of Cleavage in Xenopus Oocytes and Embryos.

BMP7/BMP2 or BMP7/BMP4 heterodimers are more active than homodimers in vitro, but it is not known whether these heterodimers signal in vivo. To test this, we generated knock in mice carrying a mutation () that prevents proteolytic activation of the dimerized BMP7 precursor protein. This mutation eliminates the function of BMP7 homodimers and all other BMPs that normally heterodimerize with BMP7. While  null homozygotes are live born,  homozygotes are embryonic lethal and have broadly reduced BMP activity. Furthermore, compound heterozygotes carrying the  allele together with a null allele of  or  die during embryogenesis with defects in ventral body wall closure and/or the heart. Co-immunoprecipitation assays confirm that endogenous BMP4/7 heterodimers exist. Thus, BMP7 functions predominantly as a heterodimer with BMP2 or BMP4 during mammalian development, which may explain why mutations in either  or  lead to a similar spectrum of congenital defects in humans.

Article	Year
Tcf3 inhibits spinal cord neurogenesis by regulating sox4a expression. Development (Cambridge, England)\| PubMed ID: 19176587	2009
Tcf7l1 is required for spinal cord progenitor maintenance. Developmental dynamics : an official publication of the American Association of Anatomists\| PubMed ID: 21932308	2011
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
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
BMP7 functions predominantly as a heterodimer with BMP2 or BMP4 during mammalian embryogenesis. eLife\| PubMed ID: 31566563	2019

Hyung-Seok Kim

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