Molecular asymmetry in the 8-cell stage Xenopus tropicalis embryo described by single blastomere transcript sequencing

Abstract

Correct development of the vertebrate body plan requires the early definition of two asymmetric, perpendicular axes. The first axis is established during oocyte maturation, and the second is established by symmetry breaking shortly after fertilization. The physical processes generating the second asymmetric, or dorsal–ventral, axis are well understood, but the specific molecular determinants, presumed to be maternal gene products, are poorly characterized. Whilst enrichment of maternal mRNAs at the animal and vegetal poles in both the oocyte and the early embryo has been studied, little is known about the distribution of maternal mRNAs along either the dorsal–ventral or left–right axes during the early cleavage stages. Here we report an unbiased analysis of the distribution of maternal mRNA on all axes of the Xenopus tropicalis 8-cell stage embryo, based on sequencing of single blastomeres whose positions within the embryo are known. Analysis of pooled data from complete sets of blastomeres from four embryos has identified 908 mRNAs enriched in either the animal or vegetal blastomeres, of which 793 are not previously reported as enriched. In contrast, we find no evidence for asymmetric distribution along either the dorsal–ventral or left–right axes. We confirm that animal pole enrichment is on average distinctly lower than vegetal pole enrichment, and that considerable variation is found between reported enrichment levels in different studies. We use publicly available data to show that there is a significant association between genes with human disease annotation and enrichment at the animal pole. Mutations in the human ortholog of the most animally enriched novel gene, Slc35d1, are causative for Schneckenbecken dysplasia, and we show that a similar phenotype is produced by depletion of the orthologous protein in Xenopus embryos.

High-throughput analysis reveals novel maternal germline RNAs crucial for primordial germ cell preservation and proper migration

“…1C). The data support and extend previous analyses that show both a greater complexity and fold enrichment of RNAs at the vegetal pole in comparison to the animal pole (Cuykendall and Houston, 2010;De Domenico et al, 2015). As expected, mRNAs known to be localized at the vegetal pole (nanos1, dazl, ddx25/ deadsouth) and animal pole [dand5 (coco) and slc18a2 (vmat2)] were identified as well as novel mRNAs.…”

“…S1A,B). 12/17 RNAs represented novel germline components, while xpat, sybu, otx1, tob2 and efnb1 were confirmed as previously described (Cuykendall and Houston, 2010;De Domenico et al, 2015).…”

“…S1B). Recently, another group analyzed transcript localization in the 8-cell X. tropicalis embryo by RNA-seq (De Domenico et al, 2015). Comparison of the vegetal/ animal blastomeres revealed that 27 of our filtered 198 transcripts remain vegetally enriched after both fertilization and cortical rotation have occurred (De Domenico et al, 2015).…”

“…1D, Table S1). Of the 198 transcripts identified, 38 have been shown to be vegetally localized, with 23 of them being specifically associated with germ plasm (Cuykendall and Houston, 2010;Claussen et al, 2015;De Domenico et al, 2015). Transcripts were considered localized at the animal pole if they were at least 10-fold enriched compared with the vegetal pole.…”

Spemann organizer transcriptome induction by early beta-catenin, Wnt, Nodal, and Siamois signals in Xenopus laevis

“…The full molecular composition of the cytoplasmic determinant and the mechanism by which β-catenin is stabilized remain unknown although the participation of maternal Wnt11 and Dvl proteins, perhaps in combination with the formation of multivesicular bodies, has been proposed (22,38). It is likely that the elusive cytoplasmic determinant is protein in nature because recent RNA-seq analyses of early cleavage stages (eight-cell) of X. laevis or X. tropicalis embryos have not detected any asymmetrically expressed mRNAs along the D-V axis (39,40). The early β-catenin stabilization, in combination with zygotic Siamois and Nodal signaling, induces Spemann organizer formation at the gastrula stage (11).…”