Abstract
BackgroundMetamorphosis in the bryozoan Bugula neritina (Linne) includes an initial phase of rapid morphological rearrangement followed by a gradual phase of morphogenesis. We hypothesized that the first phase may be independent of de novo synthesis of proteins and, instead, involves post-translational modifications of existing proteins, providing a simple mechanism to quickly initiate metamorphosis. To test our hypothesis, we challenged B. neritina larvae with transcription and translation inhibitors. Furthermore, we employed 2D gel electrophoresis to characterize changes in the phosphoproteome and proteome during early metamorphosis. Differentially expressed proteins were identified by liquid chromatography tandem mass spectrometry and their gene expression patterns were profiled using semi-quantitative real time PCR.ResultsWhen larvae were incubated with transcription and translation inhibitors, metamorphosis initiated through the first phase but did not complete. We found a significant down-regulation of 60 protein spots and the percentage of phosphoprotein spots decreased from 15% in the larval stage to12% during early metamorphosis. Two proteins--the mitochondrial processing peptidase beta subunit and severin--were abundantly expressed and phosphorylated in the larval stage, but down-regulated during metamorphosis. MPPbeta and severin were also down-regulated on the gene expression level.ConclusionsThe initial morphogenetic changes that led to attachment of B. neritina did not depend on de novo protein synthesis, but the subsequent gradual morphogenesis did. This is the first time that the mitochondrial processing peptidase beta subunit or severin have been shown to be down-regulated on both gene and protein expression levels during the metamorphosis of B. neritina. Future studies employing immunohistochemistry to reveal the expression locality of these two proteins during metamorphosis should provide further evidence of the involvement of these two proteins in the morphogenetic rearrangement of B. neritina.
Highlights
Metamorphosis in the bryozoan Bugula neritina (Linne) includes an initial phase of rapid morphological rearrangement followed by a gradual phase of morphogenesis
In the presence of the transcription inhibitor DRB and the translation inhibitor emetine, the swimming larvae of B. neritina attached and initiated metamorphosis up to the preancestrulae stage, but the subsequent phase of metamorphosis was inhibited at most concentrations
We hypothesized that protein phosphorylation status may be important during the first phase of metamorphosis of B. neritina and the initiation of the second phase because 1) we showed that their metamorphosis could initiate, but not complete, without de novo synthesis of proteins, 2) settlement and metamorphosis has been suggested to be necessarily speedy [1] and changes in protein phosphorylation status serve as a rapid molecular switch in many higher organisms [19,20], and 3) in insect and amphibian metamorphosis, protein phosphorylation networks are known to undergird many developmental processes such as apoptosis during tail reabsorption of amphibian tadpoles, innate immunity, and generation of nervous systems [21,22,23]
Summary
Metamorphosis in the bryozoan Bugula neritina (Linne) includes an initial phase of rapid morphological rearrangement followed by a gradual phase of morphogenesis. The first phase completes within minutes and transforms the swimming larva into a sessile, transitory metamorphic stage termed the "preancestrula." The second phase of metamorphosis is more gradual, ranging in duration from 36 to 48 h and includes complete degradation of larval tissues and substantial morphogenesis such as the elongation of the tubular preancestrula, differentiation of the polypide, and complete development of the cystid, resulting in a juvenile or "ancestrula" [5,6]. Whether the two phases of metamorphosis in B. neritina correlate with a shift from independence to dependence on de novo protein synthesis is unknown
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