Na,K-ATPase alpha2 and Ncx4a regulate zebrafish left-right patterning

A conserved molecular cascade involving Nodal signaling that patterns the laterality of the lateral mesoderm in vertebrates has been extensively studied, but processes involved in the initial break of left-right (LR) symmetry are just beginning to be explored. Here we report that Na,K-ATPase alpha2 and Ncx4a function upstream of Nodal signaling to regulate LR patterning in zebrafish. Knocking down Na,K-ATPase alpha2 and Ncx4a activity in dorsal forerunner cells (DFCs), which are precursors of Kupffer's vesicle (KV), is sufficient to disrupt asymmetric gene expression in the lateral plate mesoderm and randomize the placement of internal organs, indicating that the activity of Na,K-ATPase alpha2 and Ncx4a in DFCs/KV is crucial for LR patterning. High-speed videomicroscopy and bead implantation experiments show that KV cilia are immobile and the directional fluid flow in KV is abolished in Na,K-ATPase alpha2 and Ncx4a morphants, suggesting their essential role in KV ciliary function. Furthermore, we found that intracellular Ca(2+) levels are elevated in Na,K-ATPase alpha2 and Ncx4a morphants and that the defects in ciliary motility, KV fluid flow and placement of internal organs induced by their knockdown could be suppressed by inhibiting the activity of Ca(2+)/calmodulin-dependent protein kinase II. Together, our data demonstrate that Na,K-ATPase alpha2 and Ncx4a regulate LR patterning by modulating intracellular calcium levels in KV and by influencing cilia function, revealing a previously unrecognized role for calcium signaling in LR patterning.     

Calcium dynamics integrated into signalling pathways that influence vertebrate axial patterning

Many aspects of animal development including fertilization as well as organ formation and function are dependent upon the dynamic release of calcium (Ca(2+)) ions. Although the controlled release and/or accumulation of Ca(2+) ions has been extensively studied, how the release dynamics produce a specific biological output in embryonic development is less clear. We will briefly summarize Ca(2+) sources, highlight data on endogenous Ca(2+) release in vertebrate embryos relevant to body plan formation and cell movement, and integrate pharmacological and molecular-genetic studies to lend insight into the signalling pathways involved. Finally, based on in vivo imaging in zebrafish genetic mutants, we will put forward the model that distinct Ca(2+) release dynamics lead to antagonism of the developmentally important Wnt/beta-catenin signalling pathway, while sustained Ca(2+) release modulates cell polarization or directed migration.     

β-Catenin 1 and β-catenin 2 play similar and distinct roles in left-right asymmetric development of zebrafish embryos

β-Catenin-mediated canonical Wnt signaling has been found to be required for left-right (LR) asymmetric development. However, the implication of endogenous β-catenin in LR development has not been demonstrated by loss-of-function studies. In zebrafish embryos, two β-catenin genes, β-catenin 1 (ctnnb1) and β-catenin 2 (ctnnb2) are maternally expressed and their zygotic expression occurs in almost all types of tissues, including Kupffer's vesicle (KV), an essential organ that initiates LR development in teleost fish. We demonstrate here that morpholino-mediated knockdown of ctnnb1, ctnnb2, or both, in the whole embryo or specifically in dorsal forerunner cells (DFCs) interrupts normal asymmetry of the heart, liver and pancreas. Global knockdown of ctnnb2 destroys the midline physical and molecular barrier, while global knockdown of ctnnb1 impairs the formation of the midline molecular barrier. Depletion of either gene or both in DFCs/KV leads to poor KV cell proliferation, abnormal cilia formation and disordered KV fluid flow with downregulation of ntl and tbx16 expression. ctnnb1 and ctnnb2 in DFCs/KV differentially regulate the expression of charon, a Nodal antagonist, and spaw, a key Nodal gene for laterality development in zebrafish. Loss of ctnnb1 in DFCs/KV inhibits the expression of charon around KV and of spaw in the posterior lateral plate mesoderm, while ctnnb2 knockdown results in loss of spaw expression in the anterior lateral plate mesoderm with little alteration of charon expression. Taken together, our findings suggest that ctnnb1 and ctnnb2 regulate multiple processes of laterality development in zebrafish embryos through similar and distinct mechanisms.     

Retinoic acid signaling sequentially controls visceral and heart laterality in zebrafish

During zebrafish development, the left-right (LR) asymmetric signals are first established around the Kupffer vesicle (KV), a ciliated organ generating directional fluid flow. Then, LR asymmetry is conveyed and stabilized in the lateral plate mesoderm. Although numerous molecules and signaling pathways are involved in controlling LR asymmetry, mechanistic difference and concordance between different organs during LR patterning are poorly understood. Here we show that RA signaling regulates laterality decisions at two stages in zebrafish. Before the 2-somite stage (2So), inhibition of RA signaling leads to randomized visceral laterality through bilateral expression of nodal/spaw in the lateral plate mesoderm, which is mediated by increases in cilia length and defective directional fluid flow in KV. Fgf8 is required for the regulation of cilia length by RA signaling. Blockage of RA signaling before 2So also leads to mild defects of heart laterality, which become much more severe through perturbation of cardiac bmp4 asymmetry when RA signaling is blocked after 2So. At this stage, visceral laterality and the left-sided Nodal remain unaffected. These findings suggest that RA signaling controls visceral laterality through the left-sided Nodal signal before 2So, and regulates heart laterality through cardiac bmp4 mainly after 2So, first identifying sequential control and concordance of visceral and heart laterality.     

The Rho kinase Rock2b establishes anteroposterior asymmetry of the ciliated Kupffer's vesicle in zebrafish

The vertebrate body plan features a consistent left-right (LR) asymmetry of internal organs. In several vertebrate embryos, motile cilia generate an asymmetric fluid flow that is necessary for normal LR development. However, the mechanisms involved in orienting LR asymmetric flow with previously established anteroposterior (AP) and dorsoventral (DV) axes remain poorly understood. In zebrafish, asymmetric flow is generated in Kupffer's vesicle (KV). The cellular architecture of KV is asymmetric along the AP axis, with more ciliated cells densely packed into the anterior region. Here, we identify a Rho kinase gene, rock2b, which is required for normal AP patterning of KV and subsequent LR development in the embryo. Antisense depletion of rock2b in the whole embryo or specifically in the KV cell lineage perturbed asymmetric gene expression in lateral plate mesoderm and disrupted organ LR asymmetries. Analyses of KV architecture demonstrated that rock2b knockdown altered the AP placement of ciliated cells without affecting cilia number or length. In control embryos, leftward flow across the anterior pole of KV was stronger than rightward flow at the posterior end, correlating with the normal AP asymmetric distribution of ciliated cells. By contrast, rock2b knockdown embryos with AP patterning defects in KV exhibited randomized flow direction and equal flow velocities in the anterior and posterior regions. Live imaging of Tg(dusp6:memGFP)(pt19) transgenic embryos that express GFP in KV cells revealed that rock2b regulates KV cell morphology. Our results suggest a link between AP patterning of the ciliated Kupffer's vesicle and LR patterning of the zebrafish embryo.     

Origin and shaping of the laterality organ in zebrafish

Handedness of the vertebrate body plan critically depends on transient embryonic structures/organs that generate cilia-dependent leftward fluid flow within constrained extracellular environments. Although the function of ciliated organs in laterality determination has been extensively studied, how they are formed during embryogenesis is still poorly understood. Here we show that Kupffer's vesicle (KV), the zebrafish organ of laterality, arises from a surface epithelium previously thought to adopt exclusively extra-embryonic fates. Live multi-photon confocal imaging reveals that surface epithelial cells undergo Nodal/TGFbeta signalling-dependent ingression at the dorsal germ ring margin prior to gastrulation, to give rise to dorsal forerunner cells (DFCs), the precursors of KV. DFCs then migrate attached to the overlying surface epithelium and rearrange into rosette-like epithelial structures at the end of gastrulation. During early somitogenesis, these epithelial rosettes coalesce into a single rosette that differentiates into the KV with a ciliated lumen at its apical centre. Our results provide novel insights into the morphogenetic transformations that shape the laterality organ in zebrafish and suggest a conserved progenitor role of the surface epithelium during laterality organ formation in vertebrates.     

Wnt-5/pipetail functions in vertebrate axis formation as a negative regulator of Wnt/beta-catenin activity

We provide genetic evidence defining a role for noncanonical Wnt function in vertebrate axis formation. In zebrafish, misexpression of Wnt-4, -5, and -11 stimulates calcium (Ca2+) release, defining the Wnt/Ca2+ class. We describe genetic interaction between two Wnt/Ca2+ members, Wnt-5 (pipetail) and Wnt-11 (silberblick), and a reduction of Ca2+ release in Wnt-5/pipetail. Embryos genetically depleted of both maternal and zygotic Wnt-5 product exhibit cell movement defects as well as hyperdorsalization and axis-duplication phenotypes. The dorsalized phenotypes result from increased beta-catenin accumulation and activation of downstream genes. The Wnt-5 loss-of-function defect is consistent with Ca2+ modulation having an antagonistic interaction with Wnt/beta-catenin signaling.     

Right-elevated expression of charon is regulated by fluid flow in medaka Kupffer's vesicle

Recent studies have revealed that a cilium-generated liquid flow in the node has a crucial role in the establishment of the left-right (LR) axis in the mouse. In fish, Kupffer's vesicle (KV), a teleost-specific spherical organ attached to the tail region, is known to have an equivalent role to the mouse node during LR axis formation. However, at present, there has been no report of an asymmetric gene expressed in KV under the control of fluid flow. Here we report the earliest asymmetric gene in teleost KV, medaka charon, and its regulation. Charon is a member of the Cerberus/DAN family of proteins, first identified in zebrafish. Although zebrafish charon was reported to be symmetrically expressed in KV, medaka charon displays asymmetric expression with more intense expression on the right side. This asymmetric expression was found to be regulated by KV flow because symmetric and up-regulated charon expression was observed in flow-defective embryos with immotile cilia or disrupted KV. Taken together, medaka charon is a reliable gene marker for LR asymmetry in KV and thus, will be useful for the analysis of the early steps downstream of the fluid flow.     

Regional cell shape changes control form and function of Kupffer's vesicle in the zebrafish embryo

Cilia-generated fluid flow in an 'organ of asymmetry' is critical for establishing the left-right body axis in several vertebrate embryos. However, the cell biology underlying how motile cilia produce coordinated flow and asymmetric signals is not well defined. In the zebrafish organ of asymmetry-called Kupffer's vesicle (KV)-ciliated cells are asymmetrically positioned along the anterior-posterior axis such that more cilia are placed in the anterior region. We previously demonstrated that Rho kinase 2b (Rock2b) is required for anteroposterior asymmetry and fluid flow in KV, but it remained unclear how the distribution of ciliated cells becomes asymmetric during KV development. Here, we identify a morphogenetic process we refer to as 'KV remodeling' that transforms initial symmetry in KV architecture into anteroposterior asymmetry. Live imaging of KV cells revealed region-specific cell shape changes that mediate tight packing of ciliated cells into the anterior pole. Mathematical modeling indicated that different interfacial tensions in anterior and posterior KV cells are involved in KV remodeling. Interfering with non-muscle myosin II (referred to as Myosin II) activity, which modulates cellular interfacial tensions and is regulated by Rock proteins, disrupted KV cell shape changes and the anteroposterior distribution of KV cilia. Similar defects were observed in Rock2b depleted embryos. Furthermore, inhibiting Myosin II at specific stages of KV development perturbed asymmetric flow and left-right asymmetry. These results indicate that regional cell shape changes control the development of anteroposterior asymmetry in KV, which is necessary to generate coordinated asymmetric fluid flow and left-right patterning of the embryo.     

Seson, a novel zinc finger protein, controls cilia integrity for the LR patterning during zebrafish embryogenesis

In zebrafish embryos, bilateral symmetry is broken by asymmetric nodal flow generated in Kupffer’s vesicle (KV), the transient cilia-rich organ, analogous to the mouse node. Asymmetric nodal flow induces the asymmetric expression of several genes, which are critical for the determination of correct LR body patterning. seson encoding three consecutive C₂H₂ zinc finger protein is predominantly expressed in the cilia-rich organs including KV. Inhibition of its function by the injection of a seson-specific MO inhibited the left-side biased expression of spaw, and resulted in randomization of the heart, gut looping and brain laterality. Disruption of the LR patterning in seson morphants appeared to be due to severe cilia defects in KV. Seson function was also required for ciliogenesis in other tissues such as the pronephros and olfactory organs. Collectively, our data suggest that Seson has critical roles in ciliogenesis and LR body axis patterning.     

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.     

Ca(2+)/calmodulin-dependent protein kinase II is stimulated by Wnt and Frizzled homologs and promotes ventral cell fates in Xenopus

Wnt ligands working through Frizzled receptors have a differential ability to stimulate release of intracellular calcium (Ca(2+)) and activation of protein kinase C (PKC). Since targets of this Ca(2+) release could play a role in Wnt signaling, we first tested the hypothesis that Ca(2+)/calmodulin-dependent protein kinase II (CamKII) is activated by some Wnt and Frizzled homologs. We report that Wnt and Frizzled homologs that activate Ca(2+) release and PKC also activate CamKII activity in Xenopus embryos, while Wnt and Frizzled homologs that activate beta-catenin function do not. This activation occurs within 10 min after receptor activation in a pertussis toxin-sensitive manner, concomitant with autophosphorylation of endogenous CamKII. Based on data that Wnt-5A and Wnt-11 are present maternally in Xenopus eggs, and activate CamKII, we then tested the hypothesis that CamKII participates in axis formation in the early embryo. Measurements of endogenous CamKII activity from dorsal and ventral regions of embryos revealed elevated activity on the prospective ventral side, which was suppressed by a dominant negative Xwnt-11. If this spatial bias in CamKII activity were involved in promoting ventral cell fate one might predict that elevating CamKII activity on the dorsal side would inhibit dorsal cell fates, while reducing CamKII activity on the ventral side would promote dorsal cell fates. Results obtained by expression of CamKII mutants were consistent with this prediction, revealing that CamKII contributes to a ventral cell fate.     

Non-canonical Wnt signaling in Xenopus: regulation of axis formation and gastrulation

Wnt proteins form a family of secreted glycoproteins that are involved in different developmental processes such as differentiation, proliferation, cell migration and cell polarity. To exert its function, Wnt proteins activate different intracellular signaling cascades. Whereas the canonical, Wnt/beta-catenin pathway is well characterized, less is known about the function of non-canonical Wnt pathways in vertebrates. I here summarize recent findings implicating important roles for Wnt/Ca(2+) and Wnt/JNK signaling during different aspects of early Xenopus laevis development, namely axis formation and gastrulation movements.     

Pkd1l1 complexes with Pkd2 on motile cilia and functions to establish the left-right axis

The internal organs of vertebrates show distinctive left-right asymmetry. Leftward extracellular fluid flow at the node (nodal flow), which is generated by the rotational movement of node cilia, is essential for left-right patterning in the mouse and other vertebrates. However, the identity of the pathways by which nodal flow is interpreted remains controversial as the molecular sensors of this process are unknown. In the current study, we show that the medaka left-right mutant abecobe (abc) is defective for left-right asymmetric expression of southpaw, lefty and charon, but not for nodal flow. We identify the abc gene as pkd1l1, the expression of which is confined to Kupffer's vesicle (KV, an organ equivalent to the node). Pkd1l1 can interact and interdependently colocalize with Pkd2 at the cilia in KV. We further demonstrate that all KV cilia contain Pkd1l1 and Pkd2 and left-right dynein, and that they are motile. These results suggest that Pkd1l1 and Pkd2 form a complex that functions as the nodal flow sensor in the motile cilia of the medaka KV. We propose a new model for the role of cilia in left-right patterning in which the KV cilia have a dual function: to generate nodal flow and to interpret it through Pkd1l1-Pkd2 complexes.     

Small heat shock proteins are necessary for heart migration and laterality determination in zebrafish

Small heat shock proteins (sHsps) regulate cellular functions not only under stress, but also during normal development, when they are expressed in organ-specific patterns. Here we demonstrate that two small heat shock proteins expressed in embryonic zebrafish heart, hspb7 and hspb12, have roles in the development of left–right asymmetry. In zebrafish, laterality is determined by the motility of cilia in Kupffer's vesicle (KV), where hspb7 is expressed; knockdown of hspb7 causes laterality defects by disrupting the motility of these cilia. In embryos with reduced hspb7, the axonemes of KV cilia have a 9+0 structure, while control embyros have a predominately 9+2 structure. Reduction of either hspb7 or hspb12 alters the expression pattern of genes that propagate the signals that establish left–right asymmetry: the nodal-related gene southpaw (spaw) in the lateral plate mesoderm, and its downstream targets pitx2, lefty1 and lefty2. Partial depletion of hspb7 causes concordant heart, brain and visceral laterality defects, indicating that loss of KV cilia motility leads to coordinated but randomized laterality. Reducing hspb12 leads to similar alterations in the expression of downstream laterality genes, but at a lower penetrance. Simultaneous reduction of hspb7 and hspb12 randomizes heart, brain and visceral laterality, suggesting that these two genes have partially redundant functions in the establishment of left–right asymmetry. In addition, both hspb7 and hspb12 are expressed in the precardiac mesoderm and in the yolk syncytial layer, which supports the migration and fusion of mesodermal cardiac precursors. In embryos in which the reduction of hspb7 or hspb12 was limited to the yolk, migration defects predominated, suggesting that the yolk expression of these genes rather than heart expression is responsible for the migration defects.