Analysis of silicatein gene expression and spicule formation in the demosponge Amphimedon queenslandica

The skeletal elements in most sponges are siliceous spicules. These are fabricated into species-specific sizes and shapes. Demosponges, in particular, have specialised cells called sclerocytes that possess the unique ability to synthesise biosilica and these spicules. Underlying the diversity of demosponge spicules morphology is a conserved protein, called silicatein. This thesis aims to investigate the process of spiculogenesis in the different developmental stages of the demosponge Amphimedon queenslandica, and the evolution and developmental expression of the silicatein gene family in relation to spicule formation. A. queenslandica is the only sponge species to have its genome fully sequenced, assembled and annotated, and currently is one of the best models to study sponge development (Srivastava et al. 2010). This species broods embryos year-round, facilitating the access to embryological and larval material (Leys and Degnan 2001). This combination of logistical advantages means that I was able to trace the expression of silicatein genes through A. queenslandica embryonic, larval and postlarval development. Spicule formation starts in the early embryogenesis in A. queenslandica during, gastrulation or the brown stage. Spicule number increases throughout embryonic development until the pre-hatching larval stage, with the emerging larvae having about 1000 spicules. No detectable increase in spicule number was recorded during larval and postlarval development. Spicule number varied remarkably between different individual embryos and larvae of the same stage of development. I initially identified six silicatein β like genes in the genome of A. queenslandica, among which four can be categorised as non-conventional by the absence of the serine in the catalytic triad of the protein. These genes do not have direct orthologues in other sponge species and appear to have evolved by a lineage-specific gene duplication. A comprehensive phylogenetic analysis of this gene family in sponge indicated that silicatein α arose from silicatein β by gene duplication and that silicatein β gene share traits with both cathepsin L and silicatein. Conservation of gene structure and exon length in silicatein and cathepsin L genes suggests that these genes have preserved an ancestral gene structure common to both gene families in both marine and freshwater sponges. Using in situ hybridisation, I demonstrated that silicatein genes are expressed during A. queenslandica early embryonic development, with genes being expressed exclusively in sclerocytes. Analysis of gene expression levels through embryogenesis and metamorphosis, using RNA-Seq performed on a pool of same stage individuals, revealed that all silicatein-like genes are differentially expressed throughout development, and the expression of silicatein genes occurs prior to spicule formation. However, some silicatein-like gene expression levels and spicule number do not appear to be tightly correlated. Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my