Hydra Mesoglea Proteome Identifies Thrombospondin as a Conserved Component Active in Head Organizer Restriction

Mark Lommel,Derek N Woolfson,Andrew R Thomson,Jennifer Strompen,Kane Puglisi,Gnana Prakash Balasubramanian,Elena D Christofidou,Andrew L Hellewell,Josephine C Adams,Markus Hartl,Thomas W Holstein,Aimee L Boyle,Suat Özbek

doi:10.1038/s41598-018-30035-2

Abstract

Thrombospondins (TSPs) are multidomain glycoproteins with complex matricellular functions in tissue homeostasis and remodeling. We describe a novel role of TSP as a Wnt signaling target in the basal eumetazoan Hydra. Proteome analysis identified Hydra magnipapillata TSP (HmTSP) as a major component of the cnidarian mesoglea. In general, the domain organization of cnidarian TSPs is related to the pentameric TSPs of bilaterians, and in phylogenetic analyses cnidarian TSPs formed a separate clade of high sequence diversity. HmTSP expression in polyps was restricted to the hypostomal tip and tentacle bases that harbor Wnt-regulated organizer tissues. In the hypostome, HmTSP- and Wnt3-expressing cells were identical or in close vicinity to each other, and regions of ectopic tentacle formation induced by pharmacological β-Catenin activation (Alsterpaullone) corresponded to foci of HmTSP expression. Chromatin immunoprecipitation (ChIP) confirmed binding of Hydra TCF to conserved elements in the HmTSP promotor region. Accordingly, β-Catenin knockdown by siRNAs reduced normal HmTSP expression at the head organizer. In contrast, knockdown of HmTSP expression led to increased numbers of ectopic organizers in Alsterpaullone-treated animals, indicating a negative regulatory function. Our data suggest an unexpected role for HmTSP as a feedback inhibitor of Wnt signaling during Hydra body axis patterning and maintenance.

Highlights

Biochemistry and molecular cloning methods as components of the mesoglea[7,8,9,10]
Analysis in silico of the H. magnipapillata genome-predicted proteome for homologues of the major ECM proteins and adhesion receptors of vertebrates raised the possibility that the mesoglea might include additional conserved ECM components[11,12]
The identification of Hydra magnipapillata TSP (HmTSP) was substantiated by BLAST analyses of the H. magnipapillata genome and transcriptome

Summary

Introduction

Biochemistry and molecular cloning methods as components of the mesoglea[7,8,9,10]. Hydra mesoglea is a relevant and important model to understand fundamental properties and roles of ECM that may be conserved between basal metazoans and mammals. Analysis in silico of the H. magnipapillata genome-predicted proteome for homologues of the major ECM proteins and adhesion receptors of vertebrates raised the possibility that the mesoglea might include additional conserved ECM components[11,12]. By undertaking the first proteomic study of isolated Hydra ECM, we identified Hydra thrombospondin as a prominent novel component of the mesoglea. Thrombospondins are matri-cellular, calcium-binding extracellular glycoproteins that have multi-faceted roles in cell-ECM interactions and cell signaling and are present both in basement membranes and connective tissue ECM15. We show that Hydra TSP shares domains and oligomerization properties with bilaterian pentameric TSPs and exerts an unexpected role in Hydra body patterning as a negative feedback regulator of Wnt/β-Catenin-dependent organizer formation

Methods

Results

Conclusion