Toyoizumi R et al. (2005), Xenopus nodal related-1 is indispensable only f...

XB-ART-1115

Int J Dev Biol 2005 Jan 01;498:923-38. doi: 10.1387/ijdb.052008rt.

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Xenopus nodal related-1 is indispensable only for left-right axis determination.

Toyoizumi R , Ogasawara T , Takeuchi S , Mogi K .

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In Xenopus, multiple nodal-related genes are expressed in the organizer region. Among them, only Xenopus nodal related-1 (Xnr-1) is expressed unilaterally in the left lateral plate mesoderm (LPM) at late neurula-early tailbud stage. To elucidate the essential role of Xnr-1 for left-right specification, loss of function experiments using antisense morpholino oligonucleotides (MOs) targeting three different regions of Xnr-1 were performed. Left-side injection of Xnr-1 MO suppresses the left-side specific genes such as Xnr-1, Xenopus antivin (lefty) and Xenopus pitx2 and randomizes cardiac and visceral left-right orientation. In contrast, paraxial bilateral expression of Xnr-1 along the posterior notochord is not affected by the Xnr-1 MO. In embryos injected with the Xnr-1 MO, morphology of dorsal axial structures is normal and dorsal expression of sonic hedgehog and TGF-beta5 is not changed. Right-side injection of Nodal protein, or polyethyleneimine-based gene transfer of Xnr-1 mRNA in the right LPM induces Xnr-1 and pitx2 in the same side and fully (more than 90%) reverses situs of the internal organs. Left-side injection of Nodal protein restores normal left-right orientation in the embryos that were injected with Xnr-1 MO into the left blastomere and would cause randomization of the left-right axis without the Nodal injection. Taken together, unilateral expression of Xnr-1 in the left LPM directs the orientation of the left-right axis by driving the left-specific gene cascade. Knockdown of Xnr-1 function by the MOs suggests that Xnr-1 is indispensable only for the left-right orientation and dispensable for other embryonic axes probably owing to the redundancy in the function of multiple Xnrs.

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Species referenced: Xenopus laevis
Genes referenced: lefty1 nodal nodal1 pitx2 shh tbx2
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	Fig. 1. Morpholino oligonucleotides (MOs) designed for knockdown of Xnr-1 and inhibition of Xnr-1 protein synthesis by the MO. (A) Structure of the Xnr-1 gene and target regions of the MOs. Exons, introns and UTRs are colored in blue, gray and yellow, respectively. Xnr-1 gene has three exons and two introns and the transcript is 1500bp. FAST (forkhead activin signal transducer)-binding sites are marked by red circles (Osada et al., 2000). Xnr-1 MO-1 is hybridized to the translation start codon region. Xnr-1 MO-2 is hybridized to the splicing region at the interface of the first exon and first intron. Xnr-1 MO-3 is hybridized to the 5'-UTR region. Xnr-1 MO-2 and MO-3 are labeled with FITC at their 3'-end. (B) Inhibition of in vitro translation of fluorescently labeled Xnr-1 protein by Xnr-1 MO-1. Fluorescent Lysine was added to the translation reaction of Xnr-1. The molecular weight of the fluorescent product is approximately 50kDa. Lane 1, no supplement of Xnr-1 MO-1; Lane 2, supplement of Xnr-1 MO-1; Lane 3, supplement of Xnr-1 5-mismatch MO-1 (control MO).
	Fig. 2. Injection of morpholino oligonucleotides targeting Xnr-1 (Xnr- 1 MOs) induces left-right reversal of the internal organs. (A-D) Lineage tracing of a ventral left blastomere of a Xenopus 4-cell stage embryo. Xnr- 1 MO-2 was injected into the blastomere and the offspring including the left LPM cells which express Xnr-1 mRNA are fluorescently labeled. Upper column, dorsal view of the embryo at stage 16. Lower column, posterior view of the embryo at stage 19. Note that only the left part of the embryos are labeled. (E) Injection of Xnr-1 MO-1 (1mM, 10nl) into the right blastomere at the 4-cell stage did not affect the organ situs (left embryo at stage 42), while injection into the left blastomere at the same dose caused the left-right reversal in both the heart and gut (right embryo). Note that the morphology of the internal organs is normal except for the left-right orientation. Red curved arrows show the direction of blood flow. Yellow arrows indicate the curvature of the gut. (F,G) Stage 44/45 embryos after injecting with Xnr- 1 MO-2 (1mM, 10nl) into the ventral right (F) or ventral left (G) blastomere at the 4-cell stage. The embryo in (F) shows normal situs, while the embryo in G shows the left-right reversal of the heart. (H) Dorsal view of a stage 42 embryo after injecting with 5nl of 1mM Xnr-1 MO-1 into each one of the two dorsal blastomeres at the 4-cell stage. Morphology of the outer figure is normal, suggesting that the redundancy in the function of multiple Xnrs has rescued the embryo from abnormal morphogenesis. (I) Dorsal view of a eight-day tadpole after injecting with Xnr-1 MO-3 into the dorsal left blastomere at the 4-cell stage. Morphology of the dorsal structures is symmetric and normal.
	Fig. 3. Occurrence of left-right reversal after the Xnr-1 MO-1 (A, B, C), 5-bp mismatch MO-1 (D), MO-2 (E), MO-3 (F) and 5-bp mismatch MO- 2 (G) injections at the 4-cell stage. In all the illustrations, the incidence of left-right reversal is shown as a percentage of [number of the embryos showing heart and/or gut left-right reversal]/[number of the embryos examined for organ situs]. (A) The incidence of left-right reversal of the organ situs in the embryos injected with Xnr-1 MO-1 at the 4-cell stage. After the injection into either the dorsal-left or ventral-left blastomere, Xnr-1-deficient embryos showed more than 67% left-right reversal, while the right-side injections at the same dose caused only a low incidence of the reversal. Left-right symmetric phenotypes of the internal organs are rarely observed. (B,C) The incidence of left-right reversal in the case of Xnr-1 MO-1 injection into two left or right blastomeres (B) and two dorsal or ventral blastomeres (C). Five nanoliter of 1mM Xnr-1 MO was injected into each of two blastomeres and the injected embryos showed situs inversus when the Xnr-1 activity was downregulated in the left half of the embryos. The incidence of left-right reversal after injecting into two dorsal or ventral blastomeres was almost the same with the case of the injection into a single left blastomere, suggesting that rightside injection of Xnr-1 MO did not enhance or reduce the effect of the leftside MO injection. (D) Five-bp mismatch Xnr-1 MO-1 (control MO) caused minimal effects on the organ situs, showing that the effect of Xnr- 1 MO-1 is specific to the translation of Xnr-1 mRNA. (E,F) Both Xnr-1 MO- 2 and MO-3 are effective on the organ situs only when they are injected into the left blastomeres. Incidence of the left-right reversal after the leftside injections ranges from 57 to 77%, suggesting that randomization in the orientation of left-right axis has occurred. (G) Five-bp mismatch Xnr- 1 MO-2 did not induce significant left-right reversal, demonstrating that Xnr-1 MO-2 is specifically targeted for Xnr-1 mRNA.
	Fig. 4. Xnr-1 MO does not change the expression of early axial marker genes. (A) Dorsal expression of sonic hedgehog (shh) in the gastrula embryo (stage 12). Xnr-1 MO-1 injection into two dorsal blastomeres of 4 cell-stage embryos did not change shh expression in the organizer region in the majority of the embryos (n=29/33). (B) The expression of shh in the dorsal axial structure of tailbud embryos was also not changed in all of the embryos that had injected with Xnr-1 MO-1 into a dorsal blastomere at the 4-cell stage (n=35/35). (C) The expression of TGF-Î²5 in somites was not affected by the Xnr-1 MO-1 injection into a dorsal blastomere of 4-cell embryos (n=22/22). (D)The cross section of the larva injected with Xnr-1 MO-3 is observed to be normal except for the left-right reversal. Cross sections were prepared and observed for stage 43-46 larvae injected with Xnr-1 MO-1, 2 or 3 (n=9, 14, 4, respectively). s, spinal cord; n, notochord; p, pronephros; g, gut; l, liver. Scale bar, 0.5 mm.
	Fig. 5. Alternation of the gene expression pattern of Xnr-1, Xenopus antivin (Xatv) and Xenopus pitx2 after injecting with Xnr-1 MO-1 (1 mM, 10 nl) into either left or right one of ventral blastomeres at the 4- cell stage. (A) Tailbud embryo (upper) in which Xnr-1 expression in the left LPM has been abolished after Xnr-1 MO-1 injection into the left blastomere (red arrow). The embryo injected with Xnr-1 MO-1 into the right blastomere (lower) maintains Xnr-1 expression in the left LPM (blue arrow). (B) Right surface of the embryos shown in (A). No ectopic Xnr-1 expression is observed on the right side. (C,D) Tailbud embryos after the Xnr-1 5- mismatch MO-1 injection into the left (upper embryo) or right (lower embryo) blastomere. Xnr-1 expression in the left LPM is normal (C, blue arrows) and no ectopic expression is observed in the right LPM (D). (E) After the Xnr-1 MO-1 injection into the ventral left blastomere, only one embryo kept Xatv expression in the left LPM (blue arrow) and in other embryos the expression was abolished (red arrows). Axial expression of Xatv was maintained in the posterior part of the notochord and hypochord or through the full length of these axial structures (for details, see Table 4). (F) Xnr-1 MO-1 injection into the right blastomere does not interfere with the Xatv expression in the left LPM (blue arrows). (G,H) Expression of Xenopus pitx2 in the embryos injected with Xnr-1 MO-1 into the ventral left (upper embryo) or ventral right blastomere (lower embryo). Pitx2 expression in the left LPM is absent after the left-side injection (G, upper, red arrow), but does not change after the right-side injection (G, lower, blue arrow). (I) The ventral surface of the archenteron roof of a stage 24/25 embryo after Xnr- 1 MO-1 injection into the ventral left blastomere. Bilateral posterior paraxial expression of Xnr-1 is not changed (green arrows; for details, see Table 3), suggesting that this expression domain is maintained by unknown factor(s) other than Xnr-1. (J) A line drawing shows the position of the posterior bilateral expression in the sagittal section (green arrow).
	Fig. 6. Injection of Nodal protein into the neurula embryos. (A,B)Distribution of the Nile Blue solution at stage 20 (A) or at stage 28 (B), after injecting with the mixture of Nodal protein (250Î¼g/ml) and the dye into the right flank at stage 15/16 (midneurula stage). The dye keeps to the periphery of the right side. (C,D) Left (C) or right (D) surface of tailbud embryos after injecting with 5ng of Nodal protein mixed with Nile Blue (vital dye) into the center of the left LPM at the midneurula stage (stage 15/16). Note that the vital dye is observed in the center of the left LPM and does not leak out of the area. (E) Left embryo; Stage 42 embryo shows normal situs of the internal organs after injecting with 5ng of Nodal protein into the left LPM. Right embryo; Contrary to the left-side injection, after injecting with Nodal protein into the right LPM at the same stage, the embryo shows left-right reversal in both the heart and gut. Red curved arrows show the direction of blood flow. Yellow arrows indicate the curvature of the gut.(F) Stage-dependency of the effect of Nodal injection on left-right orientation of the organs. Percentage of the embryos showing left-right reversal after the right-side injection was scored. Nodal injection fully reversed the left-right axis of stage 13-20 neurula embryos, but the early tailbud embryos was not sensitive to the injection. (G) Magnified view of the inverted heart of a 14-day tadpole after Nodal injection at the neurula stage. Except for left-right orientation, morphology of the heart and gill filaments are normal. (H) Dorsal view of the 14-day tadpole shown in G. Dorsal axial structures and the sensory organs are normal.
	Fig. 7. Right-side injection of Nodal protein induces ectopic expression of normally lefthanded genes. After injecting with 5 ng Nodal protein into the left side (A, C, E, G, H) or right side (B, D, F) of embryos at the midneurula stage (stage 15/16), embryos were fixed for wholemount ISH at the tailbud stage. (A) Laterality of Xnr-1 is not changed after the injection of Nodal protein into the left flank. (B) Right-side injection of Nodal protein induces ectopic expression of Xnr-1 in the right LPM (arrows) and the original Xnr-1 expression in the left LPM does not diminish. (C)Ventral view of the archenteron roof. Bilateral Xnr-1 expression at the posterior archenteron roof of the tailbud embryos is not changed after left-side injection of Nodal protein (arrows). (D) Right-side injection of Nodal protein also does not affect the bilateral Xnr- 1 expression (arrows). (E,F) Expression of Xenopus antivin in embryos injected with Nodal protein. Laterality of antivin expression is not changed either by the left (E) or right (F) injection of Nodal. (G,H) Injection of Nodal protein into the right flank induces Xenopus pitx2 expression in the right flank. An embryo in G shows the case of bilateral but left-dominant expression of pitx2 in LPMs (corresponding to Â«L>RÂ» in Fig. 7J), whereas the one in H shows the right-dominant case (correspoinding to Â«L<RÂ» in Fig. 7J). Bilateral expression of pitx2 at the posterior end of the eyes is not changed by the Nodal injection. (I,J) Effects of the injection of Nodal protein into the left (I) or right (J) flank of the midneurula embryos. Laterality of the normally left-handed Xnr-1, Xenopus antivin (Xatv) and Xenopus pitx2 was assessed for the embryos at stage 24-26 (Xnr-1, Xatv) or stage 28-30 (pitx2). When Nodal was injected on the left side, Xnr-1, antivin and pitx2 kept the original left-handed expression. On the other hand, after the right-side injection of Nodal, both Xnr-1 and pitx2 were induced in the right LPM (Xnr-1, 58%, n=25/43; pitx2, 87%, n=34/39), whereas, in 74% of the embryos injected with Nodal (n=29/39), antivin kept the original left-handed expression.
	Fig. 8. Incorporation of Xnr-1 mRNA into the cells of lateral plate mesoderm of the neurula embryo. (A,B) Using polyethyleneimine-based polymer as a carrier, 2ng of the mRNA was injected into the left (A) or right (B) side of the early neurula (stage 13/14) embryo. An embryo in A shows normal situs, while the one in B shows left-right reversal in both the heart and gut. Note that outer shapes of the embryos are normal. Red curved arrows show the direction of blood flow. Green arrows indicate the curvature of the gut. (C,D)The incidence of left-right reversal in the embryos that were injected with the mixture of Xnr-1 mRNA and polyethyleneimine. The left-side injection did not induce significant left-right reversal (C), while the right-side injection induced up to 92% reversal (D). (E,F) Expression of pitx2 after injecting with 2.5ng Xnr-1 mRNA into the left side (E) or right side (F) of the early neurula embryos. An embryo in (E) shows normal left-handed pitx2 expression (ventral view), while the one in (F) shows bilateral pitx2 expression in the LPMs. (G,H) Classification of the expression pattern of pitx2 after the gene transfer of Xnr-1 mRNA. In (G), the left-side injection did not affect the laterality of pitx2 expression in the LPM. In (H), the right-side injection induced pitx2 expression in the right LPM for the majority of the injected embryos.