De Domenico E et al. (2015), Molecular asymmetry in the 8-cell stage Xenopus...

XB-ART-50883

Dev Biol 2015 Dec 15;4082:252-68. doi: 10.1016/j.ydbio.2015.06.010.

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

De Domenico E , Owens ND , Grant IM , Gomes-Faria R , Gilchrist MJ .

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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.

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Species referenced: Xenopus tropicalis
Genes referenced: dvl2 dvl3 slc35d1 vegt wnt11b
GO keywords: axis specification
???displayArticle.morpholinos??? slc35d1 MO1

???displayArticle.disOnts??? schneckenbecken dysplasia
???displayArticle.omims??? SCHNECKENBECKEN DYSPLASIA; SHNKND
Phenotypes: Xtr Wt + slc35d1 MO (fig.4.b) [+]

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	Fig. 1. Cortical rotation and the segregation of maternal gene products in the cleavage stage embryo. A. Orientation of early cell divisions. The first cleavage plane is determined by the sperm entry point (SEP) and the animal–vegetal (A–V) axis. The second cleavage is orthogonal to the first but still contains the A–V axis; dorsal blastomeres are defined in the hemisphere opposite the SEP. The third cleavage is slightly above the equator. B. Displacement of maternal gene products. The egg contains vegetally localized maternal gene products. A microtubule network is set up at fertilization and the outer layer, or cortex, of the single cell embryo rotates to displace the vegetal cortical region away the SEP. Maternal gene products are displaced by movement with the cortex or by vesicle trafficking into the presumptive dorsal hemisphere, and are further segregated by subsequent cell divisions.
	Fig. 2. A. Disassembling and sequencing the 8-cell stage embryo. Photograph of the intact 8-cell embryo, and during microsurgical disassembly; note lighter dorsal/animal blastomeres. Sequencing of single blastomeres from a single embryo, showing layout of displayed data for known vegetally enriched gene, VegT. B. Dye tracing experiment to confirm correct identification of dorsal–ventral axis at the 4-cell, and by extension at the 8-cell, stage. The dorsal/animal quadrant is identified by more lightly pigmented blastomeres.
	Fig. 4. Variation in enrichment within and between studies. A. Variation of fold-change with expression level at animal and vegetal poles in this study. Scatter plot of mean expression level at the enriched pole against fold change measured between the poles for the 908 enriched genes in the pooled embryo analysis. Points are color coded according to the numbers of individual embryos in which significant asymmetric expression was detected in the single embryo analysis, see Key. Genes are clearly segregated according to the consistency of detection at the single embryo level. The much greater fold-change for vegetal pole enrichment is clearly visible (note the log scale). Genes enriched in our data and previously published are highlighted (black circles), and the most enriched novel mRNAs are indicated. B. Distribution of fold-changes at either pole for individual embryos. C. Agreement of measured fold-changes between the pooled total mRNA data and the polyA+ mRNA data of Embryo 1: Spearman correlation=0.94. D. Correlation of gene enrichment between this study and earlier work. Scatter plots of enrichment fold-change on the animal–vegetal axis for genes in this study previously reported as enriched in other studies. Left panel, blastomere data (Grant et al., 2014), right panel, oocyte data (Cuykendall and Houston, 2010). Full circles: genes enriched in present study with FDR<0.05; open circles: genes not found enriched, with FDR>0.05, fold-change in our data is simple ratio of vegetal/animal pole reads.
	Fig. 5. Exploration of morpholino knockdown of Slc35d1. A. In vitro validation of Slc35d1-MO blocking translation of HA tagged Slc35d1 protein. B. Body axis shortening phenotype of Slc35d1-MO injected embryos, with approximate dosage effect up to 30 ng injected MO, at stage 33. Penetrance of the phenotype was at least 94% over all concentrations (see Table 3). C. Comparison of uninjected and 30 ng Slc35d1-MO injected embryos under Alcian Blue staining suggests major loss of skeletal development. D. Expression profile of Slc35d1 shows significant levels of polyA+ mRNA maintained into gastrulation ( Collart et al., 2014).
	Fig. 6. Distribution of gene expression for candidate dorsalizing factors in the 8-cell embryo. A. Expression of maternal Wnt11b shows substantial animal–vegetal asymmetry, without any consistent dorsal–ventral segregation, and, specifically, little difference between dorsal and ventral blastomeres, in either the PolyA+ or total mRNA prepared libraries. B. Expression of maternal Disheveled is relatively uniform throughout the early cleavage stage embryo, suggesting that these mRNAs were not concentrated at the vegetal pole of the oocyte or the fertilized egg.

References [+] :

Betley, A ubiquitous and conserved signal for RNA localization in chordates. 2002, Pubmed, Xenbase