The ion channel polycystin-2 is required for left-right axis determination in mice

Generation of laterality depends on a pathway which involves the asymmetrically expressed genes nodal, Ebaf, Leftb, and Pitx2. In mouse, node monocilia are required upstream of the nodal cascade. In chick and frog, gap junctions are essential prior to node/organizer formation. It was hypothesized that differential activity of ion channels gives rise to unidirectional transfer through gap junctions, resulting in asymmetric gene expression. PKD2, which if mutated causes autosomal dominant polycystic kidney disease (ADPKD) in humans, encodes the calcium release channel polycystin-2. We have generated a knockout allele of Pkd2 in mouse. In addition to malformations described previously, homozygous mutant embryos showed right pulmonary isomerism, randomization of embryonic turning, heart looping, and abdominal situs. Leftb and nodal were not expressed in the left lateral plate mesoderm (LPM), and Ebaf was absent from floorplate. Pitx2 was bilaterally expressed in posterior LPM but absent anteriorly. Pkd2 was ubiquitously expressed at headfold and early somite stages, with higher levels in floorplate and notochord. The embryonic midline, however, was present, and normal levels of Foxa2 and shh were expressed, suggesting that polycystin-2 acts downstream or in parallel to shh and upstream of the nodal cascade.     

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.     

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.     

Diffusion of nodal signaling activity in the absence of the feedback inhibitor Lefty2.

The role of Lefty2 in left-right patterning was investigated by analysis of mutant mice that lack asymmetric expression of lefty2. These animals exhibited various situs defects including left isomerism. The asymmetric expression of nodal was prolonged and the expression of Pitx2 was upregulated in the mutant embryos. The absence of Lefty2 conferred on Nodal the ability to diffuse over a long distance. Thus, Nodal-responsive genes, including Pitx2, that are normally expressed on the left side were expressed bilaterally in the mutant embryos, even though nodal expression was confined to the left side. These results suggest that Nodal is a long-range signaling molecule but that its range of action is normally limited by the feedback inhibitor Lefty2.     

Gut endoderm is involved in the transfer of left-right asymmetry from the node to the lateral plate mesoderm in the mouse embryo

In the mouse, the initial signals that establish left-right (LR) asymmetry are determined in the node by nodal flow. These signals are then transferred to the lateral plate mesoderm (LPM) through cellular and molecular mechanisms that are not well characterized. We hypothesized that endoderm might play a role in this process because it is tightly apposed to the node and covers the outer surface of the embryo, and, just after nodal flow is established, higher Ca(2+) flux has been reported on the left side near the node, most likely in the endoderm cells. Here we studied the role of endoderm cells in the transfer of the LR asymmetry signal by analyzing mouse Sox17 null mutant embryos, which possess endoderm-specific defects. Sox17(-/-) embryos showed no expression or significantly reduced expression of LR asymmetric genes in the left LPM. In Sox17 mutant endoderm, the localization of connexin proteins on the cell membrane was greatly reduced, resulting in defective gap junction formation, which appeared to be caused by incomplete development of organized epithelial structures. Our findings suggest an essential role of endoderm cells in the signal transfer step from the node to the LPM, possibly using gap junction communication to establish the LR axis of the mouse.     

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.     

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.     

Transforming growth factor β2 is negatively regulated by endogenous retinoic acid during early heart morphogenesis

Vitamin A‐deficient (VAD) quail embryos lack the vitamin A‐active form, retinoic acid (RA) and are characterized by a phenotype that includes a grossly abnormal cardiovascular system that can be rescued by RA. Here we report that the transforming growth factor, TGFβ2 is involved in RA‐regulated cardiovascular development. In VAD embryos TGFβ2 mRNA and protein expression are greatly elevated. The expression of TGFβ receptor II is also elevated in VAD embryos but is normalized by treatment with TGFβ2‐specific antisense oligonucleotides (AS). Administration of this AS or an antibody specific for TGFβ2 to VAD embryos normalizes posterior heart development and vascularization, while the administration of exogenous active TGFβ2 protein to normal quail embryos mimics the excessive TGFβ2 status of VAD embryos and induces VAD cardiovascular phenotype. In VAD embryos pSmad2/3 and pErk1 are not activated, while pErk2 and pcRaf are elevated and pSmad1/5/8 is diminished. We conclude that in the early avian embryo TGFβ2 has a major role in the retinoic acid‐regulated posterior heart morphogenesis for which it does not use Smad2/3 pathways, but may use other signaling pathways. Importantly, we conclude that retinoic acid is a critical negative physiological regulator of the magnitude of TGFβ2 signals during vertebrate heart formation.