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Abstract Muscle-based movement is a hallmark of animal biology, but the evolutionary origins of myocytes are unknown. Although believed to lack muscles, sponges (Porifera) are capable of coordinated whole-body contractions that purge debris from internal water canals. This behavior has been observed for decades, but their contractile tissues remain uncharacterized with respect to their ultrastructure, regulation, and development. We examine the spongeEphydatia muelleriand find tissue-wide organization of a contractile module composed of actin, striated-muscle myosin II, and transgelin, and that contractions are regulated by the release of internal Ca²⁺stores upstream of the myosin-light-chain-kinase (MLCK) pathway. The development of this contractile module appears to involve myocardin-related transcription factor (MRTF) as part of an environmentally inducible transcriptional complex that also functions in muscle development, plasticity, and regeneration. As an actin-regulated force-sensor, MRTF-activity offers a mechanism for how the contractile tissues that line water canals can dynamically remodel in response to flow and can re-form normally from stem-cells in the absence of the intrinsic spatial cues typical of animal embryogenesis. We conclude that the contractile module of sponge tissues shares elements of homology with contractile tissues in other animals, including muscles, indicating descent from a common, multifunctional tissue in the animal stem-lineage. 

The discovery that sponges (Porifera) can fully regenerate from aggregates of dissociated cells launched them as one of the earliest experimental models to study the evolution of cell adhesion and allorecognition in animals. This process depends on an extracellular glycoprotein complex called the Aggregation Factor (AF), which is composed of proteins thought to be unique to sponges. We used quantitative proteomics to identify additional AF components and interacting proteins in the classical model,Clathria prolifera, and compared them to proteins involved in cell interactions in Bilateria. Our results confirm MAFp3/p4 proteins as the primary components of the AF but implicate related proteins with calx-beta and wreath domains as additional components. Using AlphaFold, we unveiled close structural similarities of AF components to protein domains in other animals, previously masked by the mutational decay of sequence similarity. The wreath domain, believed to be unique to the AF, was predicted to contain a central beta-sandwich of the same organization as the vWFD domain (also found in extracellular, gel-forming glycoproteins in other animals). Additionally, many copurified proteins share a conserved C-terminus, containing divergent immunoglobulin (Ig) and Fn3 domains predicted to serve as an AF–interaction interface. One of these proteins, MAF-associated protein 1, resembles Ig superfamily cell adhesion molecules and we hypothesize that it may function to link the AF to the surface of cells. Our results highlight the existence of an ancient toolkit of conserved protein domains regulating cell–cell and cell–extracellular matrix protein interactions in all animals, and likely reflect a common origin of cell adhesion and allorecognition. 

This protocol is used to visualize bacteria in and around gemmule-hatched freshwater sponges using a Eubacteria FISH probe. 

This HCR-FISH protocol using probes and amplifiers from Molecular Instruments is intended for gemmule-hatched freshwater sponges grown in 35 mm coverslip bottom cell-culture dishes with a 10 mm inner-well diameter. 

This protocol is intended for the preparation of gemmule-hatched freshwater sponges for imaging with an inverted scanning confocal microscope. 

This is a basic protocol for growing freshwater sponges from gemmules in the laboratory. We specifically developed this protocol for working with Ephydatia muelleri, but have used it for other species as well. This protocol is good for cleaning gemmules, and removing contaminating protists, fungi, and bacteria.