Xenopus neurula left-right asymmetry is respeficied by microinjecting TGF-beta5 protein

A variety of TGF-beta-related ligands regulate the left-right asymmetry of vertebrates but the involvement of TGF-betas in left-right specification has not been reported. We assessed whether TGF-beta signaling is involved in the left-right specification of Xenopus post-gastrula embryos by microinjecting Xenopus TGF-beta5 protein into the left or right flank of neurula-tailbud embryos. Injection on the right side of neurulae caused left-right reversal of the internal organs in 93% of the embryos, while injection on the left side caused less than 5% left-right reversal. Expression of Xenopus nodal related-1 (Xnr-1 ), Xenopus antivin and Xenopus Pitx2, which are normally expressed on the left, was unaltered by the left-side injection. In contrast, right-side injection into neurulae induced the expression of these genes predominantly on the right side. Right-side injection into tailbud embryos caused bilateral expression of these handed genes. Time course analysis of asymmetric gene expression revealed that Xnr-1 could be induced by TGF-beta5 at late neurula stage, while antivin and Pitx2 could be induced by TGF-beta5 at the latertail bud stage. Injection of the antisense morpholino oligonucleotide against Xenopus TGF-beta5 into the left dorsal blastomere inhibited the normal left-handed expression of Xnr-1 and Pitx2, and caused the organ reversal in the injected embryos. These results suggest that normal left-right balance of endogenous TGF-beta5 signaling in the neurula embryo may be needed to determine the laterality of the asymmetric genes and to generate the correct left-right axis.     

Subtilisin-like proprotein convertase activity is necessary for left–right axis determination in Xenopus neurula embryos

Signaling by members of TGF-β superfamily requires the activity of a family of site-specific endopeptidases, known as Subtilisin-like proprotein convertases (SPCs), which cleave these ligands into mature, active forms. To explore the role of SPCs in lateral plate mesoderm (LPM) differentiation in Xenopus, two SPC inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK) and hexa-arginine, were injected into the left and right LPM of Xenopus neurulae. Left-side injection caused heart-specific left–right reversal, and this phenotype was rescued by co-injection of mature Nodal protein. In contrast, right-side injection caused left–right reversal of both the heart and gut. Tailbud embryos were less sensitive to SPC inhibitors than neurula embryos. Injection of inhibitors into either side of neurula embryos completely abolished expression of the left-LPM-specific genes, Xnr-1, antivin, and pitx2. SPC1 enzyme (Furin) was injected into the left or right LPM of mid-neurula embryos to determine the effect of enhancing SPC activity. Left-side injection of SPC1 did not cause a significant left–right reversal of the internal organs. However, right-side injection of SPC1 strongly induced the expression of Xnr-1 and pitx2 in the right LPM, and caused 100% left–right reversal of both the heart and gut. These results suggest that moderate level of SPC activity in the right LPM of the neurulae is necessary for proper left–right specification. Taken together, SPC enzymatic activity must be present in both LPMs for expression of the left-handed genes and left–right axis determination of the heart and gut in Xenopus embryos.     

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.     

The role of an endogenous PKA inhibitor, PKIalpha, in organizing left-right axis formation

Protein kinase inhibitor (PKI) is an endogenous inhibitor of cAMP-dependent protein kinase A (PKA). We have found that the alpha-isoform of PKI (PKIalpha) is asymmetrically expressed along the left-right (L-R) axis in chick embryos. At stage 6, PKIalpha is expressed on the right side of the node, and this asymmetric expression continues until stage 7+. After stage 8, PKIalpha expression returns symmetric. Treatment of embryos with antisense PKIalpha oligonucleotides increased the incidence of reversed heart looping. Antisense oligonucleotides also induced ectopic expression of the left-specific genes Nodal and Pitx2, and suppressed the expression of the right-specific gene SnR in the right lateral plate mesoderm. Similarly, treatment with PKA activators forskolin and Sp-cAMPs resulted in both reversed heart looping and bilateral expression of NODAL: Ectopic activin induced PKIalpha on the left side of the node, while ectopic Shh and anti-Shh antibody had no effect on PKIalpha expression. Taken together, these data suggest that PKIalpha induced by an activin-like molecule, through the inhibition of PKA activity, suppresses the Nodal-Pitx2 pathway on the right side of the body.     

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.     

The lefty-related factor Xatv acts as a feedback inhibitor of nodal signaling in mesoderm induction and L-R axis development in xenopus

In mouse, lefty genes play critical roles in the left-right (L-R) axis determination pathway. Here, we characterize the Xenopus lefty-related factor antivin (Xatv). Xatv expression is first observed in the marginal zone early during gastrulation, later becoming restricted to axial tissues. During tailbud stages, axial expression resolves to the neural tube floorplate, hypochord, and (transiently) the notochord anlage, and is joined by dynamic expression in the left lateral plate mesoderm (LPM) and left dorsal endoderm. An emerging paradigm in embryonic patterning is that secreted antagonists regulate the activity of intercellular signaling factors, thereby modulating cell fate specification. Xatv expression is rapidly induced by dorsoanterior-type mesoderm inducers such as activin or Xnr2. Xatv is not an inducer itself, but antagonizes both Xnr2 and activin. Together with its expression pattern, this suggests that Xatv functions during gastrulation in a negative feedback loop with Xnrs to affect the amount and/or character of mesoderm induced. Our data also provide insights into the way that lefty/nodal signals interact in the initiation of differential L-R morphogenesis. Right-sided misexpression of Xnr1 (endogenously expressed in the left LPM) induces bilateral Xatv expression. Left-sided Xatv overexpression suppresses Xnr1/XPitx2 expression in the left LPM, and leads to severely disturbed visceral asymmetry, suggesting that active 'left' signals are critical for L-R axis determination in frog embryos. We propose that the induction of lefty/Xatv in the left LPM by nodal/Xnr1 provides an efficient self-regulating mechanism to downregulate nodal/Xnr1 expression and ensure a transient 'left' signal within the embryo.     

Different spatial distribution of mRNAs for activin receptors (type IIA and IIB) and follistatin in developing embryos of Xenopus laevis

Spatial distribution of mRNAs for activin receptors and follistatin was studied by Northern blot hybridization using RNAs from different parts of dissected Xenopus embryos. mRNAs of two activin receptors (type IIA and IIB) occurred uniformly in pre-gastrular embryos, but occurred in larger amounts in ectoderm (in gastrulae), neural plate (in neurulae) and anterior (head) regions (in tailbud embryos) than in other embryonic regions. By contrast, follistatin mRNA appeared almost exclusively in the dorsal mesoderm including invaginating organizer region at the gastrula stage, in notochord and in dorsal ectoderm at the neurula stage, then in anterior part at the tailbud stage. The localized patterns of the distribution of these mRNAs may be due to the regionally different zygotic expression of genes in embryos at later stages. From the relatively widespread pattern of distribution of their mRNAs, we assume that both type IIA and type IIB activin receptors have broad functions in ectodermal and neural differentiation. On the other hand, follistatin mRNA showed quite a restricted pattern of expression, and therefore, we assume that follistatin may have functions more specifically related to the sites of expression of its mRNA. Thus, follistatin may be involved in the differentiation of notochord itself and/or directly be responsible for organizer functions such as neural induction and subsequent differentiation of induced neural tissues at the gastrula and later stages.     

XCR2, one of three Xenopus EGF-CFC genes, has a distinct role in the regulation of left-right patterning

Members of the EGF-CFC family facilitate signaling by a subset of TGFbeta superfamily ligands that includes the nodal-related factors and GDF1/VG1. Studies in mouse, zebrafish, and chick point to an essential role for EGF-CFC proteins in the action of nodal/GDF1 signals in the early establishment of the mesendoderm and later visceral left-right patterning. Antisense knockdown of the only known frog EGF-CFC factor (FRL1), however, has argued against an essential role for this factor in nodal/GDF1 signaling. To address this apparent paradox, we have identified two additional Xenopus EGF-CFC family members. The three Xenopus EGF-CFC factors show distinct patterns of expression. We have examined the role of XCR2, the only Xenopus EGF-CFC factor expressed in post-gastrula embryos, in embryogenesis. Antisense morpholino oligonucleotide-mediated depletion of XCR2 disrupts left-right asymmetry of the heart and gut. Although XCR2 is expressed bilaterally at neurula stage, XCR2 is required on the left side, but not the right side, for normal left-right patterning. Left-side expression of XNR1 in the lateral plate mesoderm depends on XCR2, whereas posterior bilateral expression of XNR1 does not, suggesting that distinct mechanisms maintain XNR1 expression in different regions of neurula-tailbud embryos. Ectopic XCR2 on the right side initiates premature right-side expression of XNR1 and XATV, and can reverse visceral patterning. This activity of XCR2 depends on its co-receptor function. These observations indicate that XCR2 has a crucial limiting role in maintaining a bistable asymmetry in nodal family signaling across the left-right axis.     

Adrenergic neurotransmitters and calcium ionophore-induced situs inversus viscerum in Xenopus laevis embryos

Xenopus laevis embryos at the blastula–early tail bud stage were exposed to norepinephrine or octopamine dissolved in culture saline until they reached the larval stage. The left–right asymmetry of the heart and gut was then examined. We found that these adrenergic neurotransmitters induced situs inversus in the heart and/or gut in up to 35% of tested neurula embryos. Norepinephrine-induced situs inversus was blocked by the α-1 adrenergic antagonist prazosin. Furthermore, A23187, a calcium ionophore, also increased the incidence of situs inversus up to 54% when late-neurula embryos were exposed to the solution. A23187 treatment initiated before neural groove formation was less effective. The incidence of situs inversus induced by these reagents decreased towards the control level (2.2%, 25 untreated embryos out of 1127 embryos in total) in embryos past the stage of neural tube closure. In the present experiments we obtained 22 gut-only situs inversus embryos having an inverted gut and a normal heart. In contrast, such embryos were not observed among the 1127 untreated embryos. An adrenergic signal mediated by an increase in intracellular free calcium may be involved in the asymmetrical visceral morphogenesis of Xenopus embryos.     

Smad5 is essential for left-right asymmetry in mice

Left-right (L-R) asymmetry of the vertebrate body plan is established from an originally morphologically symmetric embryo. Recent studies have implicated several TGF-beta family signaling proteins (i.e., nodal, lefty-1, lefty-2, activin receptor type IIB, and Smad2) in L-R axis determination in the mouse. However, the genetic pathways underlying L-R patterning are still unclear. Smad5 is a downstream component in the TGF-beta family signaling cascade, and lack of Smad5 results in embryonic lethality between E9.5 and E11.5. In this report, we demonstrate that Smad5 mutant embryos have defects in heart looping and embryonic turning which are the first signs of L-R asymmetry in mice. To gain more insights into the molecular basis of the laterality defects in the Smad5-deficient embryos, we examined the expression of lefty-1, lefty-2, nodal, and Pitx2 since the asymmetric expression of these genes always closely correlates with the direction of heart looping and embryonic turning. In the absence of Smad5, lefty-1 was expressed at very low or undetectable levels, while nodal, lefty-2, and Pitx2 were expressed bilaterally. These data suggest that Smad5 is upstream of lefty-1, nodal, and lefty-2, and as a consequence also of Pitx2, and Smad5 is essential for L-R axis determination.     

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.     

A gap junction connexin is required in the vertebrate left-right organizer

Early patterning of vertebrate embryos involves the generation of asymmetric signals across the left-right (L-R) axis that position and are required for the proper function of internal organs. This patterning is directed by a conserved nodal/lefty signaling cascade on the left side of the embryo, thought to be asymmetrically directed by ciliary beating that generates a leftward fluid flow in the mammalian node and in Kupffer's vesicle (KV), the related structure in zebrafish. Following morpholino knockdown of Cx43.4, asymmetric gene expression and global organ distribution are randomized, consistent with the expression of Cx43.4 in KV. Randomization is recapitulated in mosaic embryos in which Cx43.4 is depleted preferentially in KV cells, showing that Cx43.4 is specifically required in KV for proper L-R axis formation. The mechanistic basis for the laterality anomalies in Cx43.4-deficient embryos is a primary morphogenesis defect during lumen formation in KV. Additionally, the role of Cx43.4 appears to be conserved given that its ortholog, human Cx45, is able to functionally compensate for zebrafish Cx43.4 during L-R patterning. This is the first report linking connexin function in the ciliated, node-like cells of KV with normal L-R axis development.     

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.     

Isolation and characterization of a Japanese flounder clonal line, reversed, which exhibits reversal of metamorphic left-right asymmetry.

We have isolated a clonal line reversed (rev) of homozygous Japanese flounder through gynogenesis. The homozygous offspring gynogenetically produced from rev exhibited reversal of organization of the metamorphic L/R asymmetry such as the direction of eye-migration at a high frequency (20-30%). The molecular analysis using a left-specific marker pitx2 revealed that the embryonic L/R axis was ambiguously established: in more than half of rev embryos, pitx2 was expressed bilaterally in the lateral plate mesoderm (LPM). Previous studies in other animals demonstrated that ectopic pitx2 expression in the LPM could cause laterality defects of the visceral organs. Likewise, our results using rev imply that bilateral pitx2 expression could lead to randomization of the visceral organs. Coincidence of ectopic pitx2 expression and reversal of the direction of eye-migration in the population of rev offspring suggests that the rev locus is critical in specification of both the metamorphic and the visceral L/R asymmetries. However, reversal of the sidedness of the orientation of the visceral organs was not always accompanied by reversal of the direction of metamorphic eye-migration, suggesting that different mechanisms should be involved downstream of the rev locus in directing these two phases of asymmetric morphogenesis in the Japanese flounder.     

Early embryo orientation by direction of the cardinal point in natural clutches of two ranidae species inhabiting geographically distant regions

The azimuth (the least angle with the north-south direction) of the first cleavage furrow and anteroposterior axes of neurula was measured on projections of photographs of natural clutches. The azimuth distribution of the craniocaudal axis of Rana dalmatina neurulae in clutches from southern Italy and the first cleavage furrows of R. arvalis embryos in clutches from central Russia proved identical. Both craniocaudal axes and first cleavage furrows were mostly oriented from west to east. The azimuth distribution of the craniocaudal axis of R. arvalis neurulae in clutches subjected to repeated cold shock proved to be random. The causes and mechanisms of predominant orientation of the embryos in natural clutches of frogs are discussed. We propose that magnetic sensitivity is acquired by cytoskeleton elements, most likely microtubules, during reorganization in the course of normal development or due to experimental influences.