Expression screen for Wnt signaling-like phenotypes identifies Fam132b as a novel inhibitor of BMP signaling in Xenopus

by Juliane Melchert
Doctoral thesis
Date of Examination:2013-03-18
Date of issue:2014-03-13
Advisor:Prof. Dr. Tomas Pieler
Referee:Prof. Dr. Tomas Pieler
Referee:Prof. Dr. Ernst A. Wimmer
Persistent Address: http://dx.doi.org/10.53846/goediss-4414

 

 

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Abstract

English

The canonical Wnt signaling pathway is known to regulate multiple developmental events,including development of the digestive tract. In this study, we wanted to systematically analyze the role of the Wnt/β-Catenin signaling pathway during early and late phases of endodermal organogenesis. We generated a set of putative hormone-inducible activators or repressors of the canonical Wnt signaling pathway. Analysis of Wnt target gene expression and axis formation assays revealed that only a subset of these GR-fusion proteins is indeed inducible. These constructs were overexpressed in the endoderm of Xenopus embryos and protein activity was induced before or after specification of endodermal precursor cells. Analysis of pancreatic marker gene expression revealed that activation as well as repression of canonical Wnt signaling, early and late, inhibit exocrine pancreatic development. Expression cloning was used to identify novel regulators of early embryonic patterning.We indentified Fam132b as a factor that induces hyperdorsalization and secondary axis formation in Xenopus embryos. Analysis of Wnt and BMP target gene expression as well as luciferase reporter experiments revealed that Fam132b does not regulate Wnt signaling activity, but antagonizes the BMP signaling pathway. Fam132b contains a conserved C-terminal C1q domain and an N-terminal signal peptide. Overexpression studies in oocytes demonstrate that Fam132b is indeed a secreted factor. Analysis of endogenous target gene expression and promoter reporter studies indicated that Fam132b selectively inhibits BMP and not activin or FGF induced signaling, and that inhibition occurs at the extracellular level. Fam132b strongly interacts with BMP type I receptors, and weakly with BMP4 itself, as demonstrated by CoIP experiments. Fam132b deletion analysis demonstrated that the C1q domain is dispensable for the BMP antagonizing activity. Sequence analysis and axis duplication assays revealed that Fam132b protein sequence and protein function are only weakly conserved in a comparison ofXenopus and other vertebrate species. Fam132b is expressed in the ventral blood islands and later in circulating blood cells. In animal cap explants Fam132b is induced by Etv2/er71, which is known to activate expression of endothelial and hematopoietic genes in this system.Analysis of hematopoietic and vascular marker gene expression in Etv2/er71 expressing animal cap explants using multiplex Nanostring nCounter analysis revealed that Fam132b can enhance endothelial development at the expense of blood cell lineages.
Keywords: Fam132b; BMP antagonist; Xenopus
 
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