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.     

Mechanism of nodal flow: a conserved symmetry breaking event in left-right axis determination

The leftward flow in extraembryonic fluid is critical for the initial determination of the left-right axis of mouse embryos. It is unclear if this is a conserved mechanism among other vertebrates and how the directionality of the flow arises from the motion of cilia. In this paper, we show that rabbit and medakafish embryos also exhibit a leftward fluid flow in their ventral nodes. In all cases, primary monocilia present a clockwise rotational-like motion. Observations of defective ciliary dynamics in mutant mouse embryos support the idea that the posterior tilt of the cilia during rotational-like beating can explain the leftward fluid flow. Moreover, we show that this leftward flow may produce asymmetric distribution of exogenously introduced proteins, suggesting morphogen gradients as a subsequent mechanism of left-right axis determination. Finally, we experimentally and theoretically characterize under which conditions a morphogen gradient can arise from the flow.     

Planar Cell Polarity Enables Posterior Localization of Nodal Cilia and Left-Right Axis Determination during Mouse and Xenopus Embryogenesis

Left-right asymmetry in vertebrates is initiated in an early embryonic structure called the ventral node in human and mouse, and the gastrocoel roof plate (GRP) in the frog. Within these structures, each epithelial cell bears a single motile cilium, and the concerted beating of these cilia produces a leftward fluid flow that is required to initiate left-right asymmetric gene expression. The leftward fluid flow is thought to result from the posterior tilt of the cilia, which protrude from near the posterior portion of each cell''s apical surface. The cells, therefore, display a morphological planar polarization. Planar cell polarity (PCP) is manifested as the coordinated, polarized orientation of cells within epithelial sheets, or as directional cell migration and intercalation during convergent extension. A set of evolutionarily conserved proteins regulates PCP. Here, we provide evidence that vertebrate PCP proteins regulate planar polarity in the mouse ventral node and in the Xenopus gastrocoel roof plate. Asymmetric anterior localization of VANGL1 and PRICKLE2 (PK2) in mouse ventral node cells indicates that these cells are planar polarized by a conserved molecular mechanism. A weakly penetrant Vangl1 mutant phenotype suggests that compromised Vangl1 function may be associated with left-right laterality defects. Stronger functional evidence comes from the Xenopus GRP, where we show that perturbation of VANGL2 protein function disrupts the posterior localization of motile cilia that is required for leftward fluid flow, and causes aberrant expression of the left side-specific gene Nodal. The observation of anterior-posterior PCP in the mouse and in Xenopus embryonic organizers reflects a strong evolutionary conservation of this mechanism that is important for body plan determination.     

Nodal flow and the generation of left-right asymmetry

The establishment of left-right asymmetry in mammals is a good example of how multiple cell biological processes coordinate in the formation of a basic body plan. The leftward movement of fluid at the ventral node, called nodal flow, is the central process in symmetry breaking on the left-right axis. Nodal flow is autonomously generated by the rotation of cilia that are tilted toward the posterior on cells of the ventral node. These cilia are built by transport via the KIF3 motor complex. How nodal flow is interpreted to create left-right asymmetry has been a matter of debate. Recent evidence suggests that the leftward movement of membrane-sheathed particles, called nodal vesicular parcels (NVPs), may result in the activation of the non-canonical Hedgehog signaling pathway, an asymmetric elevation in intracellular Ca(2+) and changes in gene expression.     

Calcium fluxes in dorsal forerunner cells antagonize beta-catenin and alter left-right patterning

Establishment of the left-right axis is essential for normal organ morphogenesis and function. Ca(2+) signaling and cilia function in the zebrafish Kuppfer's Vesicle (KV) have been implicated in laterality. Here we describe an endogenous Ca(2+) release event in the region of the KV precursors (dorsal forerunner cells, DFCs), prior to KV and cilia formation. Manipulation of Ca(2+) release to disrupt this early flux does not impact early DFC specification, but results in altered DFC migration or cohesion in the tailbud at somite stages. This leads to disruption of KV formation followed by bilateral expression of asymmetrical genes, and randomized organ laterality. We identify beta-catenin inhibition as a Ca(2+)-signaling target and demonstrate that localized loss of Ca(2+) within the DFC region or DFC-specific activation of beta-catenin is sufficient to alter laterality in zebrafish. We identify a previously unknown DFC-like cell population in Xenopus and demonstrate a similar Ca(2+)-sensitive stage. As in zebrafish, manipulation of Ca(2+) release results in ectopic nuclear beta-catenin and altered laterality. Overall, our data support a conserved early Ca(2+) requirement in DFC-like cell function in zebrafish and Xenopus.     

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.     

A role for BMP signalling in heart looping morphogenesis in Xenopus

The heart develops from a linear tubular precursor, which loops to the right and undergoes terminal differentiation to form the multichambered heart. Heart looping is the earliest manifestation of left-right asymmetry and determines the eventual heart situs. The signalling processes that impart laterality to the unlooped heart tube and thus allow the developing organ to interpret the left-right axis of the embryo are poorly understood. Recent experiments in zebrafish led to the suggestion that bone morphogenetic protein 4 (BMP4) may impart laterality to the developing heart tube. Here we show that in Xenopus, as in zebrafish, BMP4 is expressed predominantly on the left of the linear heart tube. Furthermore we demonstrate that ectopic expression of Xenopus nodal-related protein 1 (Xnr1) RNA affects BMP4 expression in the heart, linking asymmetric BMP4 expression to the left-right axis. We show that transgenic embryos overexpressing BMP4 bilaterally in the heart tube tend towards a randomisation of heart situs in an otherwise intact left-right axis. Additionally, inhibition of BMP signalling by expressing noggin or a truncated, dominant negative BMP receptor prevents heart looping but allows the initial events of chamber specification and anteroposterior morphogenesis to occur. Thus in Xenopus asymmetric BMP4 expression links heart development to the left-right axis, by being both controlled by Xnr1 expression and necessary for heart looping morphogenesis.     

Genetic steps to organ laterality in zebrafish

All internal organs are asymmetric along the left-right axis. Here we report a genetic screen to discover mutations which perturb organ laterality. Our particular focus is upon whether, and how, organs are linked to each other as they achieve their laterally asymmetric positions. We generated mutations by ENU mutagenesis and examined F3 progeny using a cocktail of probes that reveal early primordia of heart, gut, liver and pancreas. From the 750 genomes examined, we isolated seven recessive mutations which affect the earliest left-right positioning of one or all of the organs. None of these mutations caused discernable defects elsewhere in the embryo at the stages examined. This is in contrast to those mutations we reported previously (Chen et al., 1997) which, along with left-right abnormalities, cause marked perturbation in gastrulation, body form or midline structures. We find that the mutations can be classified on the basis of whether they perturb relationships among organ laterality. In Class 1 mutations, none of the organs manifest any left-right asymmetry. The heart does not jog to the left and normally leftpredominant BMP4 in the early heart tube remains symmetric. The gut tends to remain midline. There frequently is a remarkable bilateral duplication of liver and pancreas. Embryos with Class 2 mutations have organotypic asymmetry but, in any given embryo, organ positions can be normal, reversed or randomized. Class 3 reveals a hitherto unsuspected gene that selectively affects laterality of heart. We find that visceral organ positions are predicted by the direction of the preceding cardiac jog. We interpret this as suggesting that normally there is linkage between cardiac and visceral organ laterality. Class 1 mutations, we suggest, effectively remove the global laterality signals, with the consequence that organ positions are effectively symmetrical. Embryos with Class 2 mutations do manifest linkage among organs, but it may be reversed, suggesting that the global signals may be present but incorrectly orientated in some of the embryos. That laterality decisions of organs may be independently perturbed, as in the Class 3 mutation, indicates that there are distinctive pathways for reception and organotypic interpretation of the global signals.     

The formation and positioning of cilia in Ciona intestinalis embryos in relation to the generation and evolution of chordate left-right asymmetry

In the early mouse embryo monocilia on the ventral node rotate to generate a leftward flow of fluid. This nodal flow is essential for the left-sided expression of nodal and pitx2, and for subsequent asymmetric organ patterning. Equivalent left fluid flow has been identified in other vertebrates, including Xenopus and zebrafish, indicating it is an ancient vertebrate mechanism. Asymmetric nodal and Pitx expression have also been identified in several invertebrates, including the vertebrates' nearest relatives, the urochordates. However whether cilia regulate this asymmetric gene expression remains unknown, and previous studies in urochordates have not identified any cilia prior to the larval stage, when asymmetry is already long established. Here we use Scanning and Transmission Electron Microscopy and immunofluorescence to investigate cilia in the urochordate Ciona intestinalis. We show that single cilia are transiently present on each ectoderm cell of the late neurula/early tailbud stage embryo, a time point just before onset of asymmetric nodal expression. Mapping the position of each cilium on these cells shows they are posteriorly positioned, something also described for mouse node cilia. The C. intestinalis cilia have a 9+0 ring ultrastructure, however we find no evidence of structures associated with motility such as dynein arms, radial spokes or nexin. Furthermore the 9+0 ring structure becomes disorganised immediately after the cilia have exited the cell, indicative of cilia which are not capable of motility. Our results indicate that although cilia are present prior to molecular asymmetries, they are not motile and hence cannot be operating in the same way as the flow-generating cilia of the vertebrate node. We conclude that the cilia may have a role in the development of C. intestinalis left-right asymmetry but that this would have to be in a sensory capacity, perhaps as mechanosensors as hypothesised in two-cilia physical models of vertebrate cilia-driven asymmetry.     

H,K-ATPase protein localization and Kir4.1 function reveal concordance of three axes during early determination of left-right asymmetry

Consistent laterality is a fascinating problem, and study of the Xenopus embryo has led to molecular characterization of extremely early steps in left-right patterning: bioelectrical signals produced by ion pumps functioning upstream of asymmetric gene expression. Here, we reveal a number of novel aspects of the H+/K+-ATPase module in chick and frog embryos. Maternal H+/K+-ATPase subunits are asymmetrically localized along the left-right, dorso-ventral, and animal-vegetal axes during the first cleavage stages, in a process dependent on cytoskeletal organization. Using a reporter domain fused to molecular motors, we show that the cytoskeleton of the early frog embryo can provide asymmetric, directional information for subcellular transport along all three axes. Moreover, we show that the Kir4.1 potassium channel, while symmetrically expressed in a dynamic fashion during early cleavages, is required for normal LR asymmetry of frog embryos. Thus, Kir4.1 is an ideal candidate for the K+ ion exit path needed to allow the electroneutral H+/K+-ATPase to generate voltage gradients. In the chick embryo, we show that H+/K+-ATPase and Kir4.1 are expressed in the primitive streak, and that the known requirement for H+/K+-ATPase function in chick asymmetry does not function through effects on the circumferential expression pattern of Connexin43. These data provide details crucial for the mechanistic modeling of the physiological events linking subcellular processes to large-scale patterning and suggest a model where the early cytoskeleton sets up asymmetric ion flux along the left-right axis as a system of planar polarity functioning orthogonal to the apical-basal polarity of the early blastomeres.     

The splanchnic mesodermal plate directs spleen and pancreatic laterality, and is regulated by Bapx1/Nkx3.2

The mechanism by which left-right (LR) information is interpreted by organ primordia during asymmetric morphogenesis is largely unknown. We show that spleen and pancreatic laterality is dependent on a specialised, columnar mesodermal-derived cell layer referred to here as the splanchnic mesodermal plate (SMP). At early embryonic stages, the SMP is bilateral, surrounding the midline-located stomach and dorsal pancreatic bud. Under control of the LR asymmetry pathway, the left SMP is maintained and grows laterally. Mice carrying the dominant hemimelia (Dh) mutation lack the SMP. Significantly, the mice are asplenic and the pancreas remains positioned along the embryonic midline. In the absence of Fgf10 expression, the spleno-pancreatic mesenchyme and surrounding SMP grow laterally but contain no endodermal component, showing that leftward growth is autonomous and independent of endoderm. In the Bapx1(-/-) mutants, the SMP is defective. Normally, the SMP is a source for both Fgf9 and Fgf10; however, in the Bapx1 mutant, Fgf10 expression is downregulated and the dorsal pancreas remains at the midline. We conclude that the SMP is an organiser responsible for the leftward growth of the spleno-pancreatic region and that Bapx1 regulates SMP functions required for pancreatic laterality.     

Serotonin signaling is a very early step in patterning of the left-right axis in chick and frog embryos

BACKGROUND: Consistent left-right (LR) asymmetry is a fascinating problem in developmental and evolutionary biology. Conservation of early LR patterning steps among vertebrates as well as involvement of nonprotein small-molecule messengers are very poorly understood. Serotonin (5-HT) is a key neurotransmitter with crucial roles in physiology and cognition. We tested the hypothesis that LR patterning required prenervous serotonin signaling and characterized the 5-HT pathway in chick and frog embryos. RESULTS: A pharmacological screen implicated endogenous signaling through receptors R3 and R4 and the activity of monoamine oxidase (MAO) in the establishment of correct sidedness of asymmetric gene expression and of the viscera in Xenopus embryos. HPLC and immunohistochemistry analysis indicates that Xenopus eggs contain a maternal supply of serotonin that is progressively degraded during cleavage stages. Serotonin's dynamic localization in frog embryos requires gap junctional communication and H,K-ATPase function. Microinjection of loss- and gain-of-function constructs into the right ventral blastomere randomizes asymmetry. In chick embryos, R3 and R4 activity is upstream of the asymmetry of Sonic hedgehog expression. MAO is asymmetrically expressed in the node. CONCLUSIONS: Serotonin is present in very early chick and frog embryos. 5-HT pathway function is required for normal asymmetry and is upstream of asymmetric gene expression. The microinjection data reveal asymmetry existing in frog embryos by the 4-cell stage and suggest novel intracellular 5-HT mechanisms. These functional and localization data identify a novel role for the neurotransmitter serotonin and implicate prenervous serotonergic signaling as an obligate aspect of very early left-right patterning conserved to two vertebrate species.     

Serotonin signaling is required for Wnt-dependent GRP specification and leftward flow in Xenopus

In vertebrates, most inner organs are asymmetrically arranged with respect to the main body axis [1]. Symmetry breakage in fish, amphibian, and mammalian embryos depends on cilia-driven leftward flow of extracellular fluid during neurulation [2-5]. Flow induces the asymmetric nodal cascade that governs asymmetric organ morphogenesis and placement [1, 6, 7]. In the frog Xenopus, an alternative laterality-generating mechanism involving asymmetric localization of serotonin at the 32-cell stage has been proposed [8]. However, no functional linkage between this early localization and flow at neurula stage has emerged. Here, we report that serotonin signaling is required for specification of the superficial mesoderm (SM), which gives rise to the ciliated gastrocoel roof plate (GRP) where flow occurs [5, 9]. Flow and asymmetry were lost in embryos in which serotonin signaling was downregulated. Serotonin, which we found uniformly distributed along the main body axes in the early embryo, was required for Wnt signaling, which provides the instructive signal to specify the GRP. Importantly, serotonin was required for Wnt-induced double-axis formation as well. Our data confirm flow as primary mechanism of symmetry breakage and suggest a general role of serotonin as competence factor for Wnt signaling during axis formation in Xenopus.     

Left-right function of dmrt2 genes is not conserved between zebrafish and mouse

Background

Members of the Dmrt family, generally associated with sex determination, were shown to be involved in several other functions during embryonic development. Dmrt2 has been studied in the context of zebrafish development where, due to a duplication event, two paralog genes dmrt2a and dmrt2b are present. Both zebrafish dmrt2a/terra and dmrt2b are important to regulate left-right patterning in the lateral plate mesoderm. In addition, dmrt2a/terra is necessary for symmetric somite formation while dmrt2b regulates somite differentiation impacting on slow muscle development. One dmrt2 gene is also expressed in the mouse embryo, where it is necessary for somite differentiation but with an impact on axial skeleton development. However, nothing was known about its role during left-right patterning in the lateral plate mesoderm or in the symmetric synchronization of somite formation.

Methodology/Principal Findings

Using a dmrt2 mutant mouse line, we show that this gene is not involved in symmetric somite formation and does not regulate the laterality pathway that controls left-right asymmetric organ positioning. We reveal that dmrt2a/terra is present in the zebrafish laterality organ, the Kupffer''s vesicle, while its homologue is excluded from the mouse equivalent structure, the node. On the basis of evolutionary sub-functionalization and neo-functionalization theories we discuss this absence of functional conservation.

Conclusions/Significance

Our results show that the role of dmrt2 gene is not conserved during zebrafish and mouse embryonic development.     

Left-right asymmetry in the sea urchin embryo is regulated by nodal signaling on the right side

The asymmetric positioning of internal organs on the left or right side of the body is highly conserved in vertebrates and relies on a Nodal signaling pathway acting on the left side of the embryo. Whether the same pathway also regulates left-right asymmetry in invertebrates and what is the evolutionary origin of the mechanisms controlling left-right determination are not known. Here, we show that nodal regulates left-right asymmetry in the sea urchin but that, intriguingly, its expression is reversed compared to vertebrates. Nodal signals emitted from the right side of the larva prevent the right coelomic pouch from forming the imaginal rudiment. Inhibition of Nodal signaling after gastrulation causes formation of an ectopic rudiment on the right side, leading to twinned urchins after metamorphosis. In contrast, ectopic activation of the pathway prevents formation of the rudiment. Our results show that the mechanisms responsible for left-right determination are conserved within basal deuterostomes.