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.     

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.     

Inhibition of proteoglycan synthesis eliminates left-right asymmetry in Xenopus laevis cardiac looping

The heart of any vertebrate is formed from an apparently symmetric cardiac tube that loops consistently in the same direction along the left-right axis of the embryo. In the amphibian Xenopus laevis, inhibition of proteoglycan synthesis by p-nitrophenyl-beta-D-xylopyranoside during a narrow period of development from late gastrula to early neurula specifically eliminated the looping of the cardiac tube. Most of the proteoglycans synthesized during this period were heparan sulfate proteoglycans. Treatment with p-nitrophenyl-alpha-D-xylopyranoside, an analogue that does not inhibit proteoglycan synthesis, did not interfere with cardiac looping. The critical period for proteoglycan synthesis was coincident with the migration of cardiac primordia to the ventral midline. The inhibition of cardiac looping was further explored in explants of cardiac primordia and anterioventral ectoderm. In recombinate embryos in which half the embryo, and thus one of the two heart primordia, was treated with p-nitrophenyl-beta-D-xylopyranoside, and the other half was untreated, cardiac looping occurred normally. It is proposed that the left-right axis in Xenopus, as reflected in cardiac looping, is established early in development, and that proteoglycan synthesis is involved in the transduction of left-right axial information to the cardiac primordia during migration.     

Motor protein control of ion flux is an early step in embryonic left-right asymmetry

The invariant left-right asymmetry of animal body plans raises fascinating questions in cell, developmental, evolutionary, and neuro-biology. While intermediate mechanisms (e.g., asymmetric gene expression) have been well-characterized, very early steps remain elusive. Recent studies suggested a candidate for the origins of asymmetry: rotary movement of extracellular morphogens by cilia during gastrulation. This model is intellectually satisfying, because it bootstraps asymmetry from the intrinsic biochemical chirality of cilia. However, conceptual and practical problems remain with this hypothesis, and the genetic data is consistent with a different mechanism. Based on wide-ranging data on ion fluxes and motor protein action in a number of species, a model is proposed whereby laterality is generated much earlier, by asymmetric transport of ions, which results in pH/voltage gradients across the midline. These asymmetries are in turn generated by a new candidate for "step 1": asymmetric localization of electrogenic proteins by cytoplasmic motors.     

terra is a left-right asymmetry gene required for left-right synchronization of the segmentation clock

To establish the vertebrate body plan, it is fundamental to create left-right asymmetry in the lateral-plate mesoderm to correctly position the organs. However, it is also crucial to maintain symmetry between the left and the right sides of the presomitic mesoderm, ensuring the allocation of symmetrical body structures, such as the axial skeleton and skeletal muscles. Here, we show that terra is an early left-sided expressed gene that links left-right patterning with bilateral synchronization of the segmentation clock.     

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.     

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.     

A novel gene encoding a ferredoxin reductase-like protein expressed in the neuroectoderm in Xenopus neurula

In an attempt to elucidate the molecular mechanisms of early neural development in Xenopus laevis, we identified, using a differential display method, several genes that are induced after Concanavalin A treatment in the animal caps prepared from stage 9 blastula. One such gene was found to encode a possible type IIIa membrane protein of 66.2 kDa sharing similarities with several prokaryotic and eukaryotic redox enzymes, hence the putative product was named Nfrl, neurula-specific ferredoxin reductase-like protein. Northern blot analysis confirmed that the expression of the Nfrl gene is up-regulated around the neurula stage, and is much lower in embryos of earlier stages and in adult tissues. The temporally limited expression of this gene implies neurula- and early larva-specific redox reactions of certain substrates, the nature of which remains to be elucidated.     

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.     

Rapid differential transport of Nodal and Lefty on sulfated proteoglycan-rich extracellular matrix regulates left-right asymmetry in Xenopus

The spatiotemporally dynamic distribution of instructive ligands within embryonic tissue, and their feedback antagonists, including inherent stabilities and rates of clearance, are affected by interactions with cell surfaces or extracellular matrix (ECM). Nodal (here, Xnr1 or Nodal1 in Xenopus) and Lefty interact in a cross-regulatory relationship in mesendoderm induction, and are the conserved instructors of left-right (LR) asymmetry in early somitogenesis stage embryos. By expressing Xnr1 and Lefty proproteins that produce mature functional epitope-tagged ligands in vivo, we found that ECM is a principal surface of Nodal and Lefty accumulation. We detected Lefty moving faster than Nodal, with evidence that intact sulfated proteoglycans in the ECM facilitate the remarkable long distance movement of Nodal. We propose that Nodal autoregulation substantially aided by rapid ligand transport underlies the anteriorward shift of Nodal expression in the left LPM (lateral plate mesoderm), and speculate that the higher levels of chondroitin-sulfate proteoglycan (CSPG) in more mature anterior regions provide directional transport cues. Immunodetection and biochemical analysis showed transfer of Lefty from left LPM to right LPM, providing direct evidence that left-side-derived Lefty is a significant influence in ensuring the continued suppression of right-sided expression of Nodal, maintaining unilateral expression of this conserved determinant of asymmetry.     

Heart formative factor(s) is localized in the anterior endoderm of early Xenopus neurula

To elucidate the mechanisms of early heart morphogenesis in Xenopus laevis, we examined the effect of endoderm on heart morphogenesis in the early Xenopus neurula. Explants of anterior ventral (presumptive heart) mesoderm from early neurula were cultured alone or in combination with endoderm dissected from various regions. Heart formation was scored by an original heart index based on morphology. These explant studies revealed that anterior ventral endoderm plays a critical role in heart morphogenesis. Furthermore, we found that it was possible to confer this heart-forming ability on posterior ventral endoderm by the injection of poly(A)⁺ RNA from stage 13 anterior endoderm. These results imply that the heart formative factor(s) is localized in the anterior endoderm of the early neurula and that at least part of this activity is encoded by mRNA(s).     

The zebrafish nodal-related gene southpaw is required for visceral and diencephalic left-right asymmetry

We have identified and characterized a new zebrafish gene, southpaw, that is required for visceral and diencephalic left-right asymmetry. southpaw encodes a new member of the nodal-related class of proteins, a subfamily within the transforming growth factor beta superfamily of secreted factors. southpaw is expressed bilaterally in paraxial mesoderm precursors and then within the left lateral plate mesoderm. At late somite stages, left-sided southpaw expression transiently overlaps the left-sided expression domains of other genes that mark the developing heart, such as lefty2. We have injected morpholinos to block the translation of the southpaw mRNA or to block splicing of the southpaw pre-mRNA. These morpholinos cause a severe disruption of early (cardiac jogging) and late (cardiac looping) aspects of cardiac left-right asymmetry. As the left-right asymmetry of the pancreas is also affected, southpaw appears to regulate left-right asymmetry throughout a large part of the embryo. Consistent with the morphological changes, the left-sided expression domains of downstream genes (cyclops, pitx2, lefty1 and lefty2) are severely downregulated or abolished within the lateral plate mesoderm of Southpaw-deficient embryos. Surprisingly, despite the absence of southpaw expression in the brain, we find that early diencephalic left-right asymmetry also requires Southpaw activity. These observations lead to a model of how visceral organ and brain left-right asymmetry are coordinated during embryogenesis.     

Regulation of left-right asymmetry by thresholds of Pitx2c activity

Although much progress has been made in understanding the molecular mechanisms regulating left-right asymmetry, the final events of asymmetric organ morphogenesis remain poorly understood. The phenotypes of human heterotaxia syndromes, in which organ morphogenesis is uncoupled, have suggested that the early and late events of left-right asymmetry are separable. The Pitx2 homeobox gene plays an important role in the final stages of asymmetry. We have used two new Pitx2 alleles that encode progressively higher levels of Pitx2c in the absence of Pitx2a and Pitx2b, to show that different organs have distinct requirements for Pitx2c dosage. The cardiac atria required low Pitx2c levels, while the duodenum and lungs used higher Pitx2c doses for normal development. As Pitx2c levels were elevated, the duodenum progressed from arrested rotation to randomization, reversal and finally normal morphogenesis. In addition, abnormal duodenal morphogenesis was correlated with bilateral expression of Pitx2c. These data reveal an organ-intrinsic mechanism, dependent upon dosage of Pitx2c, that governs asymmetric organ morphogenesis. They also provide insight into the molecular events that lead to the discordant organ morphogenesis of heterotaxia.     

Dkk3 is required for TGF-beta signaling during Xenopus mesoderm induction

Abstract The Dickkopf (Dkk) family is composed of four main members (Dkk1–4), which typically regulate Wnt/β-catenin signaling. An exception is Dkk3, which does not affect Wnt/β-catenin signaling and whose function is poorly characterized. Here, we describe the Xenopus dkk3 homolog and characterize its expression and function during embryogenesis. Dkk3 is maternally expressed and zygotically in the cement gland, head mesenchyme, and heart. We show that depletion of Dkk3 in Xenopus embryos by Morpholino antisense oligonucleotides induces axial defects as a result of Spemann organizer and mesoderm inhibition. Dkk3 depletion leads to down-regulation of Activin/Nodal signaling by reducing levels of Smad4 protein. Dkk3 overexpression can rescue phenotypic effects resulting from overexpression of the Smad4 ubiquitin ligase Ectodermin. Furthermore, depletion of Dkk3 up-regulates FGF signaling, while Dkk3 overexpression reduces it. These results indicate that Dkk3 modulates FGF and Activin/Nodal signaling to regulate mesoderm induction during early Xenopus development.     

TGF-beta signaling is required for multiple processes during Xenopus tail regeneration

Xenopus tadpoles can fully regenerate all major tissue types following tail amputation. TGF-β signaling plays essential roles in growth, repair, specification, and differentiation of tissues throughout development and adulthood. We examined the localization of key components of the TGF-β signaling pathway during regeneration and characterized the effects of loss of TGF-β signaling on multiple regenerative events. Phosphorylated Smad2 (p-Smad2) is initially restricted to the p63+ basal layer of the regenerative epithelium shortly after amputation, and is later found in multiple tissue types in the regeneration bud. TGF-β ligands are also upregulated throughout regeneration. Treatment of amputated tails with SB-431542, a specific and reversible inhibitor of TGF-β signaling, blocks tail regeneration at multiple points. Inhibition of TGF-β signaling immediately following tail amputation reversibly prevents formation of a wound epithelium over the future regeneration bud. Even brief inhibition immediately following amputation is sufficient, however, to irreversibly block the establishment of structures and cell types that characterize regenerating tissue and to prevent the proper activation of BMP and ERK signaling pathways. Inhibition of TGF-β signaling after regeneration has already commenced blocks cell proliferation in the regeneration bud. These data reveal several spatially and temporally distinct roles for TGF-β signaling during regeneration: (1) wound epithelium formation, (2) establishment of regeneration bud structures and signaling cascades, and (3) regulation of cell proliferation.     

Overexpression of the Xenopus tight-junction protein claudin causes randomization of the left-right body axis

This study presents Xenopus claudin (Xcla), a tight-junction protein that is abundantly expressed in eggs and neuroectodermal precursors during early development. It was isolated via a differential screen for mRNAs enriched in microsomes in the Xenopus blastula. The Xcla protein contains four transmembrane domains and a carboxy-terminal cytoplasmic region with a putative PDZ-binding site. We show that this PDZ-binding site of Xcla is critical for its correct localization on the cell membrane and that a truncated form leads to delocalization of the tight-junction protein ZO-1. Overexpression of Xcla causes changes in the cell adhesion properties of blastomeres and leads to visceral situs randomization. The results suggest that left-right axial patterning is very sensitive to changes in regulation of cell-cell interactions and implicate a tight-junction protein in the determination of left-right asymmetry.     

Pitx2 regulates cardiac left-right asymmetry by patterning second cardiac lineage-derived myocardium

Current models of left-right asymmetry hold that an early asymmetric signal is generated at the node and transduced to lateral plate mesoderm in a linear signal transduction cascade through the function of the Nodal signaling molecule. The Pitx2 homeobox gene functions at the final stages of this cascade to direct asymmetric morphogenesis of selected organs including the heart. We previously showed that Pitx2 regulated an asymmetric pathway that was independent of cardiac looping suggesting a second asymmetric cardiac pathway. It has been proposed that in the cardiac outflow tract Pitx2 functions in both cardiac neural crest, as a target of canonical Wnt-signaling, and in the mesoderm-derived cardiac second lineage. We used fate mapping, conditional loss of function, and chimera analysis in mice to investigate the role of Pitx2 in outflow tract morphogenesis. Our findings reveal that Pitx2 is dispensable in the cardiac neural crest but functions in second lineage myocardium revealing that this cardiac progenitor field is patterned asymmetrically.     

Bbs8, together with the planar cell polarity protein Vangl2, is required to establish left-right asymmetry in zebrafish

Laterality defects such as situs inversus are not uncommonly encountered in humans, either in isolation or as part of another syndrome, but can have devastating developmental consequences. The events that break symmetry during early embryogenesis are highly conserved amongst vertebrates and involve the establishment of unidirectional flow by cilia within an organising centre such as the node in mammals or Kupffer's vesicle (KV) in teleosts. Disruption of this flow can lead to the failure to successfully establish left-right asymmetry. The correct apical-posterior cellular position of each node/KV cilium is critical for its optimal radial movement which serves to sweep fluid (and morphogens) in the same direction as its neighbours. Planar cell polarity (PCP) is an important conserved process that governs ciliary position and posterior tilt; however the underlying mechanism by which this occurs remains unclear. Here we show that Bbs8, a ciliary/basal body protein important for intraciliary/flagellar transport and the core PCP protein Vangl2 interact and are required for establishment and maintenance of left-right asymmetry during early embryogenesis in zebrafish. We discovered that loss of bbs8 and vangl2 results in laterality defects due to cilia disruption at the KV. We showed that perturbation of cell polarity following abrogation of vangl2 causes nuclear mislocalisation, implying defective centrosome/basal body migration and apical docking. Moreover, upon loss of bbs8 and vangl2, we observed defective actin organisation. These data suggest that bbs8 and vangl2 act synergistically on cell polarization to establish and maintain the appropriate length and number of cilia in the KV and thereby facilitate correct LR asymmetry.     

De novo formation of left-right asymmetry by posterior tilt of nodal cilia

In the developing mouse embryo, leftward fluid flow on the ventral side of the node determines left–right (L-R) asymmetry. However, the mechanism by which the rotational movement of node cilia can generate a unidirectional flow remains hypothetical. Here we have addressed this question by motion and morphological analyses of the node cilia and by fluid dynamic model experiments. We found that the cilia stand, not perpendicular to the node surface, but tilted posteriorly. We further confirmed that such posterior tilt can produce leftward flow in model experiments. These results strongly suggest that L-R asymmetry is not the descendant of pre-existing L-R asymmetry within each cell but is generated de novo by combining three sources of spatial information: antero-posterior and dorso-ventral axes, and the chirality of ciliary movement.