Tadjuidje E et al. (2016), Nodal signalling in Xenopus: the role of Xnr5 i...

XB-ART-52315

Open Biol 2016 Aug 01;68:. doi: 10.1098/rsob.150187.

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Nodal signalling in Xenopus: the role of Xnr5 in left/right asymmetry and heart development.

Tadjuidje E , Kofron M , Mir A , Wylie C , Heasman J , Cha SW .

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Nodal class TGF-β signalling molecules play essential roles in establishing the vertebrate body plan. In all vertebrates, nodal family members have specific waves of expression required for tissue specification and axis formation. In Xenopus laevis, six nodal genes are expressed before gastrulation, raising the question of whether they have specific roles or act redundantly with each other. Here, we examine the role of Xnr5. We find it acts at the late blastula stage as a mesoderm inducer and repressor of ectodermal gene expression, a role it shares with Vg1. However, unlike Vg1, Xnr5 depletion reduces the expression of the nodal family member xnr1 at the gastrula stage. It is also required for left/right laterality by controlling the expression of the laterality genes xnr1, antivin (lefty) and pitx2 at the tailbud stage. In Xnr5-depleted embryos, the heart field is established normally, but symmetrical reduction in Xnr5 levels causes a severely stunted midline heart, first evidenced by a reduction in cardiac troponin mRNA levels, while left-sided reduction leads to randomization of the left/right axis. This work identifies Xnr5 as the earliest step in the signalling pathway establishing normal heart laterality in Xenopus.

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Species referenced: Xenopus laevis
Genes referenced: fgf3 fgf8 foxi1 foxi2 gdf1 lefty mapk1 meis3 msgn1 nodal nodal1 nodal5.2 nodal5.4 odc1 pitx2 smad2 tbxt tek vegfa
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	Figure 1. xnr5 mRNA splicing is reduced by a splice blocking morpholino. (a) Real-time PCR (RT-PCR) analysis of the temporal expression of xnr5 and xnr1 mRNA in sibling embryos at the developmental stages shown. Both xnr5 and xnr1 are expressed at late blastula to mid-gastrula stage. xnr1 mRNA (but not xnr5) is also expressed at late neurula to early tailbud. (b) The xnr5 splice donor MO (Xnr5-MO) targets a conserved exon/intron boundary in all four copies of the xnr5 gene. (c) Gel-based PCR using random hexamer primed cDNAs. Vegetal injection of increasing doses (27, 53 and 107 ng) of the MO reduced splicing of Xnr5 RNA in a dose-responsive fashion, as testified by the progressive appearance of additional PCR products (unspliced) in Xnr5-MO samples. The appearance of the unspliced product correlates with decreases in the mature (spliced) product. odc is used as RNA loading control.
	Figure 2. Xnr5 is a mesoderm induction and ectoderm repression signal. (a) Control, (b) Xnr5-depleted and (c) Xnr5-depleted embryos injected with 0.6pg of xnr5 RNA. Xnr5-depleted embryos (b) usually develop with a slight gastrulation delay (depicted by the delay in blastopore closure: (b) compared with (a)) which is rescued by reintroduction of xnr5 RNA (20/20 cases). (d) Wild-type (upper) and Xnr5-depleted (lower) embryos at early tailbud stage. No morphological alteration of Xnr5-depleted embryos compared with controls is noticed through this stage. (e) Western blot analysis showing phosphorylated Smad2 levels in control and Xnr5-depleted gastrulae (Xnr5MO), and embryos injected with both MO (Xnr5MO) and 0.6pg xnr5 mRNA. (f) Western blot analysis showing phosphorylated Erk in equatorial explants of control, Xnr5-depleted and Xnr5-depleted embryos injected with xnr5 mRNA at the four-cell stage, at early gastrula stages. (g) RT-PCR analysis of the expression of mesodermal markers, xbra, mespo and fgf3 mRNAs, in equatorial explants from control (wt Eq), Xnr5-depleted (Xnr5MO Eq) and Xnr5-depleted embryos injected with xnr5 mRNA at the four-cell stage (Xnr5MO + 0.6 pgRNA). (h) Design of the Nieuwkoop assay used to produce the animal caps (Ac) analysed by RT-PCR in (i) and (j). (i) Mesodermal markers, fgf3 and fgf8 are induced in wild-type animal caps by wild-type vegetal masses (wt Vm), reduced with Xnr5-depleted vegetal masses (Xnr5−Vm), and rescued by co-culture with Xnr5-depleted vegetal masses injected with 6pg xnr5 mRNA (Xnr5−Vm + 6 pg); (j) ectodermal markers (foxi1E and sizzled) are upregulated as a result of Xnr5 depletion. (k–n) Whole mount in situ hybridization of foxi1E mRNA in (k,m) bisected control and (l,n) Xnr5-depleted embryos. Panels (m) and (n) represent the magnification of boxed areas in (k) and (l), respectively. (o) RT-PCR analysis of the expression of ectodermal genes, foxi1E and maternal foxi2 in small animal cap explants from control (uninjected), Xnr5-depleted (Xnr5MO) and Xnr5-depleted embryos injected with 0.6 pg of xnr5 mRNA at the four-cell stage (Xnr5MO/RNA). The diagram shows the area (red box) of the animal cap explants for the experiment. AN, animal cells; MZ, marginal zone; EN, endodermal cells.
	Figure 3. Xnr5 and Vg1 share roles in mesoderm induction and Foxi1E inhibition. (a) Western blot analysis of control, Xnr5−, Vg1− and Vg1−/Xnr5− equatorial explants at the early gastrula stage for dp-Erk and pSmad2; double depletion of both Xnr5 and Vg1 (lanes 4 and 8) causes a more severe reduction in dp-Erk and pSmad2 than single depletion. (b,c) RT-PCR analysis of the expression of mesodermal markers xbra, mespo and fgf3 (b) and ectodermal marker foxi1E (c) in such explants; double depletion (Xnr5−/Vg1−) has a more severe effect on both mesodermal (decrease) and ectodermal (increase) markers. (d) Xnr5 depletion and Vg1 depletion have opposite effects on the expression of xnr1 mRNA at the early (stage 10) and mid-gastrula stage (stage 11).
	Figure 4. Xnr5 is required for the later expression of xnr1 mRNA. (a) RT-PCR (left panel) and in situ hybridization (right panel) analyses of xnr1 mRNA in anterior/posterior bisected embryos; xnr1 mRNA is only detected in posterior explants of controls embryos at neurula stage (stage 17), and this strong expression of xnr1 is severely reduced in Xnr5-depleted (Xnr5−) explants. (b) RT-PCR analysis of the expression of xnr1 and antivin mRNA in whole embryos at the early tailbud stage (stage 22); their expression is reduced in Xnr5-depleted embryos and partially rescued by reintroduction of 0.6 and 6 pg of xnr5 mRNA. (c) RT-PCR analysis of whole embryos (we) or left or right half embryos showing the enrichment of xnr1 and antivin mRNA on the left side of controls (wt left) at early tailbud stage (stage 22); the high levels of expression are lost in Xnr5-depleted embryos (Xnr5− left). (d–f) In situ hybridization analysis of xnr1 (d), antivin (e) and pitx2 (f) mRNA expressions at tailbud stage. xnr1 as well as antivin and pitx2 transcripts are detected in the left (but not right) lateral plate mesoderm in wild-type embryos (wt); the left-sided expression of these transcripts is lost in Xnr5-depleted embryos (Xnr5-); note that the expression of pitx2 in the cement gland (arrow) is not reduced. (g–j) Wild-type (g) or tadpole injected Xnr5MO in all four cells (h) in two right blastomeres (i) or two left blastomeres (j) at four-cell stage; injection of Xnr5MO in all 4 cells ((h) compared with (g)) causes an incomplete gut looping, often with a small and midline linear heart (arrow-head in (g–j)); injecting the same total dose of MO into the two right cells (i) causes no obvious effect, while injecting into the two left cells (j) causes an inversion of gut and heart looping (note the position of the gall-bladder, just below the arrow-head, is inverted in (j) compared with (g)).
	Figure 5. Xnr5 depletion causes heart dysmorphogenesis. (a–e) RT-PCR analysis of the expression of cardiac markers nkx2.5 (a), c-actin (b) and c-troponin (e) and vascular markers vegf (c) and tie2 (d) in wild-type (wt Eq) Xnr5-depleted (Xnr5Mo) Xnr5-depleted injected with 0.6 pg of xnr5 mRNA (Xnr5Mo + RNA) and equatorial explants injected with increasing doses of xnr5 mRNA (xnr5 RNA 0.6 pg, 6 pg and 60 pg), dissected at late blastula stage and cultured to the tailbud stage (stage 33). Xnr5 depletion causes a reduction of the expression of nkx2.5, c-actin, vegf and tie2 whose levels are re-established by the reintroduction of xnr5 mRNA; overexpression of xnr5 has a mild induction effect only on tie2 expression. cardiac troponin (e) is not expressed in wild-type explants (wt Eq) but is dose-dependently induced by xnr5 mRNA. RT-PCR analysis showing that overexpressing xnr1 mRNA (1 pg, 10 pg and 100 pg) does not induce c-troponin in equatorial explants. (f) Whole mount in situ hybridization of 10 pg of xnr1 mRNA injected embryos and 0.6 pg of xnr5 mRNA injected embryos for nkx2.5 and cardiac troponin (tnni3). (g–m) In situ hybridization analysis of nkx2.5 (g), c-actin (h), vegf (i,l) and c-troponin (j,k,m) in control (wt), Xnr5-depleted (Xnr5-) or embryos injected with a mismatched version of Xnr5Mo (MMMO) containing 5 base exchange. The expression of early heart field markers nkx2.5 (g, stage 26) and c-actin (h, stage 33) is unchanged in Xnr5-depleted embryos when compared with controls. The same observation is true for the expression of vegf at stage 32 (i) and stage 41 (l). The expression domain of c-troponin is reduced in Xnr5-depleted embryos at stage 33 (j) and stage 41 (k); a mismatched version of Xnr5MO has no effect on the expression of c-troponin ((m) compared with wt in (k)).
	Figure 6. CRISPR/Cas9 mediated Xnr5 knockout embryos lost asymmetric xnr1 expression. (a) Schematic description of CRISRP/Cas9 mediated NHEJ event on xnr5 genes and clonal sequencing analysis of xnr5 mRNA at stage 9 embryos. (b) In situ hybridization analysis of xnr1, meis3 mRNA expression at tailbud stage. xnr1 transcripts are detected in the left (but not right) lateral plate mesoderm in Cas9-injected embryos (Cas9 only); the left-sided expression of these transcripts is lost in Xnr5 gRNA/Cas9-injected embryos (Xnr5 CRISPR); note that the expression of meis3 is not altered. (c) Xnr5 CRISPR mutants developed heterotaxia (including situs inversus).

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