Dorsal induction from dorsal vegetal cells in Xenopus occurs after mid-blastula transition

In a screening for activin-responsive genes, we isolated a Xenopus lefty/antivin-related gene, called Xantivin (Xatv). In the animal cap assay, the expression of Xatv was induced by activin signaling, and in the embryo, by nodal-related genes. Overexpression of Xatv in the marginal zone caused suppression of mesoderm formation and gastrulation defects, and inhibited the secondary axis formation induced by Xnr1 and Xactivin, suggesting that Xatv acted as a feedback inhibitor of activin signaling. However, in the animal cap, Xatv failed to antagonize Xnr1 and Xactivin. This result suggested that Xatv has different responses in the marginal zone and in the animal region, and antagonizes to a higher degree activin signaling in the marginal zone.     

Zic3 is involved in the left-right specification of the Xenopus embryo

Establishment of left-right (L-R) asymmetry is fundamental to vertebrate development. Several genes involved in L-R asymmetry have been described. In the Xenopus embryo, Vg1/activin signals are implicated upstream of asymmetric nodal related 1 (Xnr1) and Pitx2 expression in L-R patterning. We report here that Zic3 carries the left-sided signal from the initial activin-like signal to determinative factors such as Pitx2. Overexpression of Zic3 on the right side of the embryo altered the orientation of heart and gut looping, concomitant with disturbed laterality of expression of Xnr1 and Pitx2, both of which are normally expressed in the left lateral plate mesoderm. The results indicate that Zic3 participates in the left-sided signaling upstream of Xnr1 and Pitx2. At early gastrula, Zic3 was expressed not only in presumptive neuroectoderm but also in mesoderm. Correspondingly, overexpression of Zic3 was effective in the L-R specification at the early gastrula stage, as revealed by a hormone-inducible Zic3 construct. The Zic3 expression in the mesoderm is induced by activin (beta) or Vg1, which are also involved in the left-sided signal in L-R specification. These findings suggest that an activin-like signal is a potent upstream activator of Zic3 that establishes the L-R axis. Furthermore, overexpression of the zinc-finger domain of Zic3 on the right side is sufficient to disturb the L-R axis, while overexpression of the N-terminal domain on the left side affects the laterality. These results suggest that Zic3 has at least two functionally important domains that play different roles and provide a molecular basis for human heterotaxy, which is an L-R pattern anomaly caused by a mutation in human ZIC3.     

Rotatin is a novel gene required for axial rotation and left-right specification in mouse embryos

The genetic cascade that governs left-right (L-R) specification is starting to be elucidated. In the mouse, the lateral asymmetry of the body axis is revealed first by the asymmetric expression of nodal, lefty2 and pitx2 in the left lateral plate mesoderm of the neurulating embryo. Here we describe a novel gene, rotatin, essential for the correct expression of the key L-R specification genes nodal, lefty and Pitx2. Embryos deficient in rotatin show also randomized heart looping and delayed neural tube closure, and fail to undergo the critical morphogenetic step of axial rotation. The amino acid sequence deduced from the cDNA is predicted to contain at least three transmembrane domains. Our results show a novel key player in the genetic cascade that determines L-R specification, and suggest a causal link between this process and axial rotation.     

Anteriorward shifting of asymmetric Xnr1 expression and contralateral communication in left-right specification in Xenopus

Transient asymmetric Nodal signaling in the left lateral plate mesoderm (L LPM) during tailbud/early somitogenesis stages is associated in all vertebrates examined with the development of stereotypical left-right (L-R) organ asymmetry. In Xenopus, asymmetric expression of Nodal-related 1 (Xnr1) begins in the posterior L LPM shortly after the initiation of bilateral perinotochordal expression in the posterior tailbud. The L LPM expression domain rapidly shifts forward to cover much of the flank of the embryo before being progressively downregulated, also in a posterior-to-anterior direction. The mechanisms underlying the initiation and propagation of Nodal/Xnr1 expression in the L LPM, and its transient nature, are not well understood. Removing the posterior tailbud domain prevents Xnr1 expression in the L LPM, consistent with the idea that normal embryos respond to a posteriorly derived asymmetrically acting positive inductive signal. The forward propagation of asymmetric Xnr1 expression occurs LPM-autonomously via planar tissue communication. The shifting is prevented by Nodal signaling inhibitors, implicating an underlying requirement for Xnr1-to-Xnr1 induction. It is also unclear how asymmetric Nodal signals are modulated during L-R patterning. Small LPM grafts overexpressing Xnr1 placed into the R LPM of tailbud embryos induced the expression of the normally L-sided genes Xnr1, Xlefty, and XPitx2, and inverted body situs, demonstrating the late-stage plasticity of the LPM. Orthogonal Xnr1 signaling from the LPM strongly induced Xlefty expression in the midline, consistent with recent findings in the mouse and demonstrating for the first time in another species conservation in the mechanism that induces and maintains the midline barrier. Our findings suggest that there is long-range contralateral communication between L and R LPM, involving Xlefty in the midline, over a substantial period of tailbud embryogenesis, and therefore lend further insight into how, and for how long, the midline maintains a L versus R status in the LPM.     

Left-right patterning of the mouse lateral plate requires nodal produced in the node

Initial determination of left-right (L-R) polarity in mammalian embryos takes place in the node. However, it is not known how asymmetric signals are generated in the node and transferred to the lateral plate mesoderm (LPM). Mice homozygous for a hypomorphic Nodal allele (Nodal(neo)) were generated and found to exhibit L-R defects, including right isomerism. Although the mutant embryos express Nodal at gastrulation stages, the subsequent expression of this gene in the node and left LPM is lost. A transgene that conferred Nodal expression specifically in the node rescued the L-R defects of the Nodal(neo/neo) embryos. Conversely, ectopic expression of the Nodal inhibitor Lefty2 in the node of Nodal(neo/+) embryos resulted in a phenotype similar to that of the Nodal(neo/neo) mutant. These results indicate that Nodal produced in the node is required for expression of Nodal and other left side-specific genes in the LPM.     

Flounder and fugu have a single lefty gene that covers the functions of lefty1 and lefty2 of zebrafish during L-R patterning

The lefty gene encodes a member of the TGF-beta superfamily that regulates L-R axis formation during embryogenesis via antagonistic activity against Nodal, another TGF-beta superfamily member. Both mouse and zebrafish have two lefty genes, lefty1 and lefty2. Interestingly, the expression domains of mouse and zebrafish lefty are different from one another. At present, the orthology and functional diversity of the mouse and zebrafish lefty genes are not clear. Here, we report that flounder and two fugu species, Takifugu and Tetraodon, have a single lefty gene in their genomes. In addition, we provide evidence that the mouse lefty genes were duplicated on a single chromosome but the zebrafish lefty genes arose from a whole-genome duplication that occurred early in the divergence of ray-finned fishes. These independent origins likely explain the difference in the expression domains of the mouse and zebrafish lefty gene pairs. Furthermore, we found that the duplication corresponding to the zebrafish lefty2 gene was lost from the fugu genome, suggesting that loss of lefty2 in the fugu/flounder lineage occurred after its divergence from the zebrafish lineage. During L-R patterning, the single lefty gene of flounder covers two expression domains, the left side of the dorsal diencephalon and the left LPM, which are regulated separately by lefty1 and lefty2 in zebrafish. We infer that the lefty genes of the ray-finned fishes and mammals underwent independent gene duplication events that resulted in independent regulation of lefty expression.     

Xenopus Brachyury regulates mesodermal expression of Zic3, a gene controlling left-right asymmetry

The Brachyury gene has a critical role in the formation of posterior mesoderm and notochord in vertebrate development. A recent study showed that Brachyury is also responsible for the formation of the left-right (L-R) axis in mouse and zebrafish. However, the role of Brachyury in L-R axis specification is still elusive. Here, it is demonstrated that Brachyury is involved in L-R specification of the Xenopus laevis embryo and regulates expression of Zic3, which controls the L-R specification process. Overexpression of Xenopus Brachyury (Xbra) and dominant-negative type Xbra (Xbra-EnR) altered the orientation of heart and gut looping, concomitant with disturbed laterality of nodal-related 1 (Xnr1) and Pitx2 expression, both of which are normally expressed in the left lateral plate mesoderm. Furthermore, activation of inducible type Xbra (Xbra-GR) induces Zic3 expression within 20 min. These results suggest that a role of Brachyury in L-R specification may be the direct regulation of Zic3 expression.     

BMP signaling through ACVRI is required for left-right patterning in the early mouse embryo

Vertebrate organisms are characterized by dorsal-ventral and left-right asymmetry. The process that establishes left-right asymmetry during vertebrate development involves bone morphogenetic protein (BMP)-dependent signaling, but the molecular details of this signaling pathway remain poorly defined. This study tests the role of the BMP type I receptor ACVRI in establishing left-right asymmetry in chimeric mouse embryos. Mouse embryonic stem (ES) cells with a homozygous deletion at Acvr1 were used to generate chimeric embryos. Chimeric embryos were rescued from the gastrulation defect of Acvr1 null embryos but exhibited abnormal heart looping and embryonic turning. High mutant contribution chimeras expressed left-side markers such as nodal bilaterally in the lateral plate mesoderm (LPM), indicating that loss of ACVRI signaling leads to left isomerism. Expression of lefty1 was absent in the midline of chimeric embryos, but shh, a midline marker, was expressed normally, suggesting that, despite formation of midline, its barrier function was abolished. High-contribution chimeras also lacked asymmetric expression of nodal in the node. These data suggest that ACVRI signaling negatively regulates left-side determinants such as nodal and positively regulates lefty1. These functions maintain the midline, restrict expression of left-side markers, and are required for left-right pattern formation during embryogenesis in the mouse.     

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

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.     

BMP inhibition by DAN in Hensen's node is a critical step for the establishment of left-right asymmetry in the chick embryo

During left-right (L-R) axis formation, Nodal is expressed in the node and has a central role in the transfer of L-R information in the vertebrate embryo. Bone morphogenetic protein (BMP) signaling also has an important role for maintenance of gene expression around the node. Several members of the Cerberus/Dan family act on L-R patterning by regulating activity of the transforming growth factor-β (TGF-β) family. We demonstrate here that chicken Dan plays a critical role in L-R axis formation. Chicken Dan is expressed in the left side of the node shortly after left-handed Shh expression and before the appearance of asymmetrically expressed genes in the lateral plate mesoderm (LPM). In vitro experiments revealed that DAN inhibited BMP signaling but not NODAL signaling. SHH had a positive regulatory effect on Dan expression while BMP4 had a negative effect. Using overexpression and RNA interference-mediated knockdown strategies, we demonstrate that Dan is indispensable for Nodal expression in the LPM and for Lefty-1 expression in the notochord. In the perinodal region, expression of Dan and Nodal was independent of each other. Nodal up-regulation by DAN required NODAL signaling, suggesting that DAN might act synergistically with NODAL. Our data indicate that Dan plays an essential role in the establishment of the L-R axis by inhibiting BMP signaling around the node.     

More than 95% reversal of left-right axis induced by right-sided hypodermic microinjection of activin into Xenopus neurula embryos

In recent years, genes that show left-right (L-R) asymmetric expression patterns have been identified one after another in vertebrate gastrula-neurula embryos. However, we still have little information about when the irreversible L-R specification is established in vertebrate embryos. In this report, we show that almost 100% of the embryos develop to be L-R-inverted larvae after microinjection of activin molecules into the right lateral hypodermic space of Xenopus neurula embryos. After right-side injection of 10-250 pg activin protein, both early neurulae just after gastrulation movement (stage 13-14) and late neurulae just before neural tube closure (stage 17-18) showed almost 100% reversal of the heart and gut L-R axes. At higher doses of activin, more than 90% of the L-R-inverted embryos showed L-R reversal of both heart and gut. The survival ratio of the right-injected 4-day embryos was 90% on average. In the left-injected embryos, the occurrence of L-R inversion was less than 2% as observed in normal untreated siblings (1.7%). When the same amount of activin (1-50 pg) was microinjected into both sides of neurula embryos, the incidence of L-R inversion was reduced to 58%. The injection of activin along the dorsal midline in the trunk region also randomized the visceral L-R axis. Injection of activin into the right side changed normal left-handed expression of Xnr-1 to right-handed or bilateral expression. In contrast, left-handed expression of Pitx2 was switched to the right side by right activin injection. This is the first report of a method that achieves complete inversion of the visceral L-R axis by treatment of embryos at the neurula stage. Activin not only acts on the neurulae to cancel the original L-R specification up to the late neurula stage, but also rebuilds a new L-R axis whose left side coincides with the injection side. It is suggested that the left and right halves of neurulae have equal potential for L-R differentiation.