Onjiko RM et al. (2021), Altering metabolite distribution at Xenopus cle...

XB-ART-58196

Genesis 2021 Jun 01;595-6:e23418. doi: 10.1002/dvg.23418.

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Altering metabolite distribution at Xenopus cleavage stages affects left-right gene expression asymmetries.

Onjiko RM , Nemes P , Moody SA .

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The left-right (L-R) axis of most bilateral animals is established during gastrulation when a transient ciliated structure creates a directional flow of signaling molecules that establish asymmetric gene expression in the lateral plate mesoderm. However, in some animals, an earlier differential distribution of molecules and cell division patterns initiate or at least influence L-R patterning. Using single-cell high-resolution mass spectrometry, we previously reported a limited number of small molecule (metabolite) concentration differences between left and right dorsal-animal blastomeres of the eight-cell Xenopus embryo. Herein, we examined whether altering the distribution of some of these molecules influenced early events in L-R patterning. Using lineage tracing, we found that injecting right-enriched metabolites into the left cell caused its descendant cells to disperse in patterns that varied from those in control gastrulae; this did not occur when left-enriched metabolites were injected into the right cell. At later stages, injecting left-enriched metabolites into the right cell perturbed the expression of genes known to: (a) be required for the formation of the gastrocoel roof plate (foxj1); (b) lead to the asymmetric expression of Nodal (dand5/coco); or (c) result from asymmetrical nodal expression (pitx2). Despite these perturbations in gene expression, we did not observe heterotaxy in heart or gut looping at tadpole stages. These studies indicate that altering metabolite distribution at cleavage stages at the concentrations tested in this study impacts the earliest steps of L-R gene expression that then can be compensated for during organogenesis.

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Genes referenced: dand5 fgfr4 foxj1 foxj1.2 nodal pitx2 zic3

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	FIGURE 2 Distribution patterns of FDA-labeled clones in theneural ectoderm at the end of gastrulation (Stages 12–13). (a) At theend of gastrulation, neural ectodermal clones of FDA-injected 16-celldorsal midline blastomeres extended from the animal pole, which isthe future forebrain region, to the posterior, closing blastopore (bl).The patterns were sorted into categories according to the lateralextent of the cells, from narrow (type A) to broad (type D), asdescribed in the Methods. Type E was only observed in embryos inwhich the Rmet cocktail was injected into L-D1; it distinctly did notcontain cells that extended to the blastopore. (b) The frequency of thevarious types of clones observed for control embryos that wereinjected with only FDA, that is, without added metabolites (R-D11, L-D11) or embryos injected with metabolite cocktails at the 8-cell stageand then FDA at the 16-cell stage (Lmet-R-D11, Rmet-L-D11).Samples sizes are in parentheses.
	FIGURE 3 Altering metabolite distributions at cleavage stagesperturbs foxj1 expression at gastrula stages. (a) In control gastrulastage 10.5 embryos, foxj1 expression (purple) was equivalent in lengthon the right (R) and left (L) sides of the embryo. Using a video cameraattached to a stereomicroscope, the midpoint of the pigmented cellsforming the blastopore, was marked (red asterisk), a line was drawnfrom that point toward the animal pole (red line) and the length of thefoxj1 expression domain measured from that line with an eyepiecereticule. (b) Examples of embryos in which the Lmet cocktail wasinjected into the R-D1 blastomere. (c) Lengths of the right and leftdomains of foxj1 expression in each embryo, in reticule units, werecompared by a two-tailed, paired Wilcoxon test (*p < .005). The rightand left domains of foxj1 expression in Lmet-R-D1 embryos weresignificantly different in length when compared by a two-tailed, pairedMann–Whitney test (#p < .0005). Bars indicate SEM and samples sizes are in parentheses.
	FIGURE 4 Altered Lmet concentration in R-D1 perturbs dand5expression in the gastrocoel roof plate (GRP). (a) Expression of dand5in the GRP is typically larger, longer and/or darker on the right (R) sidecompared to the left (L) side (left panel), particularly in controlembryos. In a minority of control cases, it was longer on the left side(left middle), equivalent in length on both sides (right middle) or notexpressed at the stage examined (right panel). Tissue is oriented froma ventral view with posterior (Post) to the top and anterior (Ant) tothe bottom. (b) The frequencies of left–right dand5 expressionpatterns were dramatically different between control embryos andsiblings in which Lmet was injected into R-D1. Samples sizes are in parentheses
	FIGURE 5 Altering metabolite distribution on the right side atcleavage stages perturbs the distribution of pitx2 expression in thelarval lateral plate mesoderm. (a) In an embryo (left images) in whichRmet cocktail was injected into L-D1, pitx2 expression in the lateralplate mesoderm is confined to the left side (arrow). In an embryo(right image) in which Lmet cocktail was injected into R-D1, pitx2expression also was detected in the right lateral plate mesoderm(arrow). We did not observe any Lmet-R-D1 embryo in which pitx2was expressed only in the right lateral plate mesoderm. (b) Thepercentage of embryos in which pitx2 expression was detected on theleft side or on both sides. Samples sizes are in parentheses.

References [+] :

Adams, Early, H+-V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates. 2006, Pubmed, Xenbase