Discovery of germline-related genes in Cephalochordate amphioxus: A genome wide survey using genome annotation and transcriptome data

Abstract

The generation of germline cells is a critical process in the reproduction of multicellular organisms. Studies in animal models have identified a common repertoire of genes that play essential roles in primordial germ cell (PGC) formation. However, comparative studies also indicate that the timing and regulation of this core genetic program vary considerably in different animals, raising the intriguing questions regarding the evolution of PGC developmental mechanisms in metazoans. Cephalochordates (commonly called amphioxus or lancelets) represent one of the invertebrate chordate groups and can provide important information about the evolution of developmental mechanisms in the chordate lineage. In this study, we used genome and transcriptome data to identify germline-related genes in two distantly related cephalochordate species, Branchiostoma floridae and Asymmetron lucayanum. Branchiostoma and Asymmetron diverged more than 120 MYA, and the most conspicuous difference between them is their gonadal morphology. We used important germline developmental genes in several model animals to search the amphioxus genome and transcriptome dataset for conserved homologs. We also annotated the assembled transcriptome data using Gene Ontology (GO) terms to facilitate the discovery of putative genes associated with germ cell development and reproductive functions in amphioxus. We further confirmed the expression of 14 genes in developing oocytes or mature eggs using whole mount in situ hybridization, suggesting their potential functions in amphioxus germ cell development. The results of this global survey provide a useful resource for testing potential functions of candidate germline-related genes in cephalochordates and for investigating differences in gonad developmental mechanisms between Branchiostoma and Asymmetron species.

Asymmetric Distribution of pl10 and bruno2, New Members of a Conserved Core of Early Germline Determinants in Cephalochordates

“…For instance, musashi, piwil2, and genes of the Pumilio domain family do not appear to be associated specifically with PGCs in B. lanceolatum or B. floridae (this study; Yue et al, 2015). A possible exception is pufA, which we found to be concentrated in a PGC-like domain in some cleavage stage embryos (two-cell to morula) in several independent experiments.…”

“…However, pufA is expressed in germ cells in other species, including zebrafish (Kuo et al, 2009) and P. dumerilii (Gazave et al, 2013). Interestingly, a global search of germline and reproduction-associated genes using the transcriptome and genome of Asymmetron lucayanum and B. floridae, respectively, identified a pumilio/puf gene with expression in oocytes (Yue et al, 2015). Similar studies in maturing gonads in B. lanceolatum may also reveal functions for some of our candidates during germline maturation.…”

“…We identified a single piwil2 (piwiA in Kerner et al, 2011) and three piwil1 (piwiB in Kerner et al, 2011) genes in the genomes of B. belcheri and B. floridae. The latter belong to an apparent tandem duplication cluster (not shown and Yue et al, 2015) that appears to be present in all Branchiostoma, as we successfully cloned two of the three paralogs of piwil1 in B. lanceolatum. We also identified an ortholog of piwiX ("piwilike" in Zhang et al, 2013), but have been unable to clone the gene in B. lanceolatum.…”

Wnt evolution and function shuffling in liberal and conservative chordate genomes

“…Different Wnt expression domains have been observed at different stages of germline formation and gonadogenesis in Olfactores, such as Wnt5 in two ascidian species and mouse, Wnt4 and Wnt8 in zebrafish and mouse, Wnt1 and Wnt3 in chick, and Wnt11 in Xenopus (Additional file 1 : Figure S3 and associated references), while no evidence has been found suggesting any specific Wnt expression in cephalochordate primordial germ cells (PGCs) or the germline (this work and [ 46 , 67 , 83 , 84 ]; reviewed in [ 49 ]). Similarly, the mesodermal cardiac-related expression domain of Wnt9 in Olfactores—i.e., the heart endocardium of vertebrates [ 85 ] and the epithelial walls of the vasculature of the colonial ascidian B.schlosseri [ 33 ]—is not paralleled by any Wnt paralog in amphioxus.…”

Dorsal-ventral patterning in amphioxus: current understanding, unresolved issues, and future directions

“…Cortical granules occupy the cortex of the unfertilized egg, and the subcortical cytoplasm in the vegetal third of the egg contains sheets of dense granules that are interleaved with the cisternae of the endoplasmic reticulum (Holland and Holland, 1992;Holland and Holland, 1991). These vegetal structures are associated with numerous ribosomes and mRNAs of germ cell marker genes, such as Vasa, Nanos, Piwil1, Tudor, Pl10, and bruno2 (Dailey et al, 2016;Wu et al, 2011;Yue et al, 2015;Zhang et al, 2013). On the other hand, the transcripts of the following genes are localized in the animal hemisphere in the amphioxus egg: Nodal, SoxB1b, and Tcf (Cattell et al, 2012;Lin et al, 2006;Onai et al, 2010).…”