A dual function of FGF signaling in Xenopus left-right axis formation.

Organ left-right (LR) asymmetry is a conserved vertebrate feature, which is regulated by left-sided activation of Nodal signaling. Nodal asymmetry is established by a leftward fluid-flow generated at the ciliated LR organizer (LRO). Although the role of fibroblast growth factor (FGF) signaling pathways during mesoderm development is conserved, diverging results from different model organisms suggest a non-conserved function in LR asymmetry. Here, we demonstrate that FGF is required during gastrulation in a dual function at consecutive stages of Xenopus embryonic development. In the early gastrula, FGF is necessary for LRO precursor induction, acting in parallel with FGF-mediated mesoderm induction. During late gastrulation, the FGF/Ca2+-branch is required for specification of the flow-sensing lateral LRO cells, a function related to FGF-mediated mesoderm morphogenesis. This second function in addition requires input from the calcium channel Polycystin-2. Thus, analogous to mesoderm development, FGF activity is required in a dual role for laterality specification; namely, for generating and sensing leftward flow. Moreover, our findings in Xenopus demonstrate that FGF functions in LR development share more conserved features across vertebrate species than previously anticipated.

Serotonin and MucXS release by small secretory cells depend on Xpod, a SSC specific marker gene.

Mucus secretion and ciliary motility are hallmarks for muco-ciliary epithelia (MCE). Both, mammalian airways as well as the less complex epidermis of Xenopus embryos show cilia-driven mucus flow to protect the organism against harmful effects by exogenous pathogens or pollutants. Four cell types set up the epidermal MCE in Xenopus. Multi-ciliated cells (MCCs) generate an anterior to posterior flow of mucus. Ion secreting cells (ISCs) are characterized by the expression of ion transporters, presumably to maintain a favorable homeostasis. The largest cell type is represented by goblet cells, which cover most of the epidermis and exhibit secretory properties. Additionally, small secretory cells (SSCs) release mucus, antibiotic compounds, and the monoamine serotonin (5-hydroxytryptamine; 5-HT). We have recently shown that serotonin regulates flow velocity by acting on ciliary beat frequency. Here, we describe the identification and functional characterization of Xenopus polka-dots (Xpod). No homologous genes or proteins were found in other vertebrates, including Xenopus tropicalis. We demonstrate that Xpod serves as an SSC-specific marker, starting to be expressed shortly after SSC specification at neurula stages. Overexpression of a tagged Xpod protein resulted in the localization of secretory granules. Notch signaling induced SSC cell fate, in contrast to its repressing effect on MCC and ISC specification. Xpod loss-of-function revealed that mucus and 5-HT release by SSCs was severely diminished, which impaired the ciliary beating of MCCs. In summary, Xpod specifically marked SSCs and was required for muco-ciliary secretion in Xenopus laevis.

Xenopus, an ideal model organism to study laterality in conjoined twins.

Conjoined twins occur at low frequency in all vertebrates including humans. Many twins fused at the chest or abdomen display a very peculiar laterality defect: while the left twin is normal with respect to asymmetric organ morphogenesis and placement (situs solitus), the organ situs is randomized in right twins. Although this phenomenon has fascinated already some of the founders of experimental embryology in the 19th and early 20th century, such as Dareste, Fol, Warynsky and Spemann, its embryological basis has remained enigmatic. Here we summarize historical experiments and interpretations as well as current models, argue that the frog Xenopus is the only vertebrate model organism to tackle the issue, and outline suitable experiments to address the question of twin laterality in the context of cilia-based symmetry breakage.

The evolution and conservation of left-right patterning mechanisms.

Morphological asymmetry is a common feature of animal body plans, from shell coiling in snails to organ placement in humans. The signaling protein Nodal is key for determining this laterality. Many vertebrates, including humans, use cilia for breaking symmetry during embryonic development: rotating cilia produce a leftward flow of extracellular fluids that induces the asymmetric expression of Nodal. By contrast, Nodal asymmetry can be induced flow-independently in invertebrates. Here, we ask when and why flow evolved. We propose that flow was present at the base of the deuterostomes and that it is required to maintain organ asymmetry in otherwise perfectly bilaterally symmetrical vertebrates.

A novel serotonin-secreting cell type regulates ciliary motility in the mucociliary epidermis of Xenopus tadpoles.

The embryonic skin of Xenopus tadpoles serves as an experimental model system for mucociliary epithelia (MCE) such as the human airway epithelium. MCEs are characterized by the presence of mucus-secreting goblet and multiciliated cells (MCCs). A third cell type, ion-secreting cells (ISCs), is present in the larval skin as well. Synchronized beating of MCC cilia is required for directional transport of mucus. Here we describe a novel cell type in the Xenopus laevis larval epidermis, characterized by serotonin synthesis and secretion. It is termed small secretory cell (SSC). SSCs are detectable at early tadpole stages, unlike MCCs and ISCs, which are specified at early neurulation. Subcellularly, serotonin was found in large, apically localized vesicle-like structures, which were entirely shed into the surrounding medium. Pharmacological inhibition of serotonin synthesis decreased the velocity of cilia-driven fluid flow across the skin epithelium. This effect was mediated by serotonin type 3 receptor (Htr3), which was expressed in ciliated cells. Knockdown of Htr3 compromised flow velocity by reducing the ciliary motility of MCCs. SSCs thus represent a distinct and novel entity of the frog tadpole MCE, required for ciliary beating and mucus transport across the larval skin. The identification and characterization of SSCs consolidates the value of the Xenopus embryonic skin as a model system for human MCEs, which have been known for serotonin-dependent regulation of ciliary beat frequency.

A secretory cell type develops alongside multiciliated cells, ionocytes and goblet cells, and provides a protective, anti-infective function in the frog embryonic mucociliary epidermis.

The larval epidermis of Xenopus is a bilayered epithelium, which is an excellent model system for the study of the development and function of mucosal and mucociliary epithelia. Goblet cells develop in the outer layer while multiciliated cells and ionocytes sequentially intercalate from the inner to the outer layer. Here, we identify and characterise a fourth cell type, the small secretory cell (SSC). We show that the development of these cells is controlled by the transcription factor Foxa1 and that they intercalate into the outer layer of the epidermis relatively late, at the same time as embryonic hatching. Ultrastructural and molecular characterisation shows that these cells have an abundance of large apical secretory vesicles, which contain highly glycosylated material, positive for binding of the lectin, peanut agglutinin, and an antibody to the carbohydrate epitope, HNK-1. By specifically depleting SSCs, we show that these cells are crucial for protecting the embryo against bacterial infection. Mass spectrometry studies show that SSCs secrete a glycoprotein similar to Otogelin, which may form the structural component of a mucus-like protective layer, over the surface of the embryo, and several potential antimicrobial substances. Our study completes the characterisation of all the epidermal cell types in the early tadpole epidermis and reinforces the suitability of this system for the in vivo study of complex epithelia, including investigation of innate immune defences.

ATP4a is required for development and function of the Xenopus mucociliary epidermis - a potential model to study proton pump inhibitor-associated pneumonia.

Proton pump inhibitors (PPIs), which target gastric H(+)/K(+)ATPase (ATP4), are among the most commonly prescribed drugs. PPIs are used to treat ulcers and as a preventative measure against gastroesophageal reflux disease in hospitalized patients. PPI treatment correlates with an increased risk for airway infections, i.e. community- and hospital-acquired pneumonia. The cause for this correlation, however, remains elusive. The Xenopus embryonic epidermis is increasingly being used as a model to study airway-like mucociliary epithelia. Here we use this model to address how ATP4 inhibition may affect epithelial function in human airways. We demonstrate that atp4a knockdown interfered with the generation of cilia-driven extracellular fluid flow. ATP4a and canonical Wnt signaling were required in the epidermis for expression of foxj1, a transcriptional regulator of motile ciliogenesis. The ATP4/Wnt module activated foxj1 downstream of ciliated cell fate specification. In multiciliated cells (MCCs) of the epidermis, ATP4a was also necessary for normal myb expression, apical actin formation, basal body docking and alignment of basal bodies. Furthermore, ATP4-dependent Wnt/ß-catenin signaling in the epidermis was a prerequisite for foxa1-mediated specification of small secretory cells (SSCs). SSCs release serotonin and other substances into the medium, and thereby regulate ciliary beating in MCCs and protect the epithelium against infection. Pharmacological inhibition of ATP4 in the mature mucociliary epithelium also caused a loss of MCCs and led to impaired mucociliary clearance. These data strongly suggest that PPI-associated pneumonia in human patients might, at least in part, be linked to dysfunction of mucociliary epithelia of the airways.

Cilia are required for asymmetric nodal induction in the sea urchin embryo.

BACKGROUND: Left-right (LR) organ asymmetries are a common feature of metazoan animals. In many cases, laterality is established by a conserved asymmetric Nodal signaling cascade during embryogenesis. In most vertebrates, asymmetric nodal induction results from a cilia-driven leftward fluid flow at the left-right organizer (LRO), a ciliated epithelium present during gastrula/neurula stages. Conservation of LRO and flow beyond the vertebrates has not been reported yet. RESULTS: Here we study sea urchin embryos, which use nodal to establish larval LR asymmetry as well. Cilia were found in the archenteron of embryos undergoing gastrulation. Expression of foxj1 and dnah9 suggested that archenteron cilia were motile. Cilia were polarized to the posterior pole of cells, a prerequisite of directed flow. High-speed videography revealed rotating cilia in the archenteron slightly before asymmetric nodal induction. Removal of cilia through brief high salt treatments resulted in aberrant patterns of nodal expression. Our data demonstrate that cilia - like in vertebrates - are required for asymmetric nodal induction in sea urchin embryos. CONCLUSIONS: Based on these results we argue that the anterior archenteron represents a bona fide LRO and propose that cilia-based symmetry breakage is a synapomorphy of the deuterostomes.

Symmetry breakage in the vertebrate embryo: when does it happen and how does it work?

Asymmetric development of the vertebrate embryo has fascinated embryologists for over a century. Much has been learned since the asymmetric Nodal signaling cascade in the left lateral plate mesoderm was detected, and began to be unraveled over the past decade or two. When and how symmetry is initially broken, however, has remained a matter of debate. Two essentially mutually exclusive models prevail. Cilia-driven leftward flow of extracellular fluids occurs in mammalian, fish and amphibian embryos. A great deal of experimental evidence indicates that this flow is indeed required for symmetry breaking. An alternative model has argued, however, that flow simply acts as an amplification step for early asymmetric cues generated by ion flux during the first cleavage divisions. In this review we critically evaluate the experimental basis of both models. Although a number of open questions persist, the available evidence is best compatible with flow-based symmetry breakage as the archetypical mode of symmetry breakage.

Symmetry breakage in the frog Xenopus: role of Rab11 and the ventral-right blastomere.

Vertebrates display asymmetric arrangements of inner organs such as heart and stomach. The Nodal signaling cascade in the left lateral plate mesoderm in all cases directs asymmetric morphogenesis and placement during organogenesis. Mechanisms that lead up to left-asymmetric Nodal induction seem to differ between the vertebrates. Cilia produce a leftward extracellular fluid flow in zebrafish, medaka, mouse, rabbit, and Xenopus embryos during neurulation. In Xenopus, earlier asymmetric cues were described. Some, such as Rab11, apparently act in the zygote. Others were efficiently manipulated in ventral-right cells at the four-cell stage, a lineage presumably independent of the ciliated gastrocoel roof plate (GRP) during neurulation. Here, we show that one- and four-cell manipulations of Rab11 showed equal low efficiencies of left-right disturbances. We also reevaluated the lineage of the GRP. By tracing back future ciliated cells from the gastrula to the four-cell stage, we show that ventral cells contribute to ciliated sensory cells at the border of the GRP. Knockdown of the Nodal inhibitor Coco in the ventral right lineage resulted in embryos with ectopic right-sided Nodal and Pitx2c expression. Together, these experiments support a cilia-based mechanism of symmetry breakage in the frog Xenopus.

The RNA-binding protein bicaudal C regulates polycystin 2 in the kidney by antagonizing miR-17 activity.

The RNA-binding protein Bicaudal C is an important regulator of embryonic development in C. elegans, Drosophila and Xenopus. In mouse, bicaudal C (Bicc1) mutants are characterized by the formation of fluid-filled cysts in the kidney and by expansion of epithelial ducts in liver and pancreas. This phenotype is reminiscent of human forms of polycystic kidney disease (PKD). Here, we now provide data that Bicc1 functions by modulating the expression of polycystin 2 (Pkd2), a member of the transient receptor potential (TRP) superfamily. Molecular analyses demonstrate that Bicc1 acts as a post-transcriptional regulator upstream of Pkd2. It regulates the stability of Pkd2 mRNA and its translation efficiency. Bicc1 antagonized the repressive activity of the miR-17 microRNA family on the 3'UTR of Pkd2 mRNA. This was substantiated in Xenopus, in which the pronephric defects of bicc1 knockdowns were rescued by reducing miR-17 activity. At the cellular level, Bicc1 protein is localized to cytoplasmic foci that are positive for the P-body markers GW182 and HEDLs. Based on these data, we propose that the kidney phenotype in Bicc1(-/-) mutant mice is caused by dysregulation of a microRNA-based translational control mechanism.

Linking early determinants and cilia-driven leftward flow in left-right axis specification of Xenopus laevis: a theoretical approach.

In vertebrates, laterality - the asymmetric placement of the viscera including organs of the gastrointestinal system, heart and lungs - is under the genetic control of a conserved signaling pathway in the left lateral plate mesoderm (LPM). A key feature of this pathway, shared by embryos of all non-avian vertebrate classes analyzed to date (e.g. fish, amphibia and mammals) is the formation of a transitory midline epithelial structure. Remarkably, the motility of cilia projecting from this epithelium produce a leftward-directed movement of extracellular liquid. This leftward flow precedes any sign of asymmetry in gene expression. Numerous analyses have shown that this leftward flow is not only necessary, but indeed sufficient to direct laterality. Interestingly, however, cilia-independent mechanisms acting much earlier in development in the frog Xenopus have been reported during the earliest cleavage stages, a period before any major zygotic gene transcription. The relationship between these two distinct mechanisms is not understood. In this review we present the conserved and critical steps of Xenopus LR axis formation. Next, we address the basic question of how an early asymmetric activity might contribute to, feed into, or regulate the conserved cilia-dependent pathway. Finally, we discuss the possibility that Spemann's organizer is itself polarized in the left-right dimension. In attempting to reconcile the sufficiency of the cilia-dependent pathway with potential earlier-acting asymmetries, we offer a general practical experimental checklist for the Xenopus community working on the process of left-right determination. This approach indicates areas where work still needs to be done to clarify the relationship between early determinants and cilia-driven leftward flow.

Expression and cilia associated localization of Histone deacetylases 6 in Xenopus.

Histone deacetylases (HDACs) are key posttranslational modulators of the proteome. We show that expression of histone deacetylase 6 ( hdac6 ) is dynamic and appears in a tissue specific manner throughout embryonic development of the frog Xenopus laevis . Interestingly, hdac6 transcripts often associate with ciliated tissues, like the left-right organizer at neurula stage or the pronephros. In the embryonic skin, Hdac6 protein localizes to the cilia base, suggesting a functional link.

dmrt2 and myf5 Link Early Somitogenesis to Left-Right Axis Determination in Xenopus laevis.

The vertebrate left-right axis is specified during neurulation by events occurring in a transient ciliated epithelium termed left-right organizer (LRO), which is made up of two distinct cell types. In the axial midline, central LRO (cLRO) cells project motile monocilia and generate a leftward fluid flow, which represents the mechanism of symmetry breakage. This directional fluid flow is perceived by laterally positioned sensory LRO (sLRO) cells, which harbor non-motile cilia. In sLRO cells on the left side, flow-induced signaling triggers post-transcriptional repression of the multi-pathway antagonist dand5. Subsequently, the co-expressed Tgf-ß growth factor Nodal1 is released from Dand5-mediated repression to induce left-sided gene expression. Interestingly, Xenopus sLRO cells have somitic fate, suggesting a connection between LR determination and somitogenesis. Here, we show that doublesex and mab3-related transcription factor 2 (Dmrt2), known to be involved in vertebrate somitogenesis, is required for LRO ciliogenesis and sLRO specification. In dmrt2 morphants, misexpression of the myogenic transcription factors tbx6 and myf5 at early gastrula stages preceded the misspecification of sLRO cells at neurula stages. myf5 morphant tadpoles also showed LR defects due to a failure of sLRO development. The gain of myf5 function reintroduced sLRO cells in dmrt2 morphants, demonstrating that paraxial patterning and somitogenesis are functionally linked to LR axis formation in Xenopus.

Bicc1 and Dicer regulate left-right patterning through post-transcriptional control of the Nodal inhibitor Dand5.

Rotating cilia at the vertebrate left-right organizer (LRO) generate an asymmetric leftward flow, which is sensed by cells at the left LRO margin. Ciliary activity of the calcium channel Pkd2 is crucial for flow sensing. How this flow signal is further processed and relayed to the laterality-determining Nodal cascade in the left lateral plate mesoderm (LPM) is largely unknown. We previously showed that flow down-regulates mRNA expression of the Nodal inhibitor Dand5 in left sensory cells. De-repression of the co-expressed Nodal, complexed with the TGFß growth factor Gdf3, drives LPM Nodal cascade induction. Here, we show that post-transcriptional repression of dand5 is a central process in symmetry breaking of Xenopus, zebrafish and mouse. The RNA binding protein Bicc1 was identified as a post-transcriptional regulator of dand5 and gdf3 via their 3'-UTRs. Two distinct Bicc1 functions on dand5 mRNA were observed at pre- and post-flow stages, affecting mRNA stability or flow induced translational inhibition, respectively. To repress dand5, Bicc1 co-operates with Dicer1, placing both proteins in the process of flow sensing. Intriguingly, Bicc1 mediated translational repression of a dand5 3'-UTR mRNA reporter was responsive to pkd2, suggesting that a flow induced Pkd2 signal triggers Bicc1 mediated dand5 inhibition during symmetry breakage.

Flow on the right side of the gastrocoel roof plate is dispensable for symmetry breakage in the frog Xenopus laevis.

Leftward flow of extracellular fluid breaks the bilateral symmetry of most vertebrate embryos, manifested by the ensuing asymmetric induction of Nodal signaling in the left lateral plate mesoderm (LPM). Flow is generated by rotational beating of polarized monocilia at the posterior notochord (PNC; mammals), Kupffer's vesicle (KV; teleost fish) and the gastrocoel roof plate (GRP; amphibians). To manipulate flow in a defined way we cloned dynein heavy chain genes dnah5, 9 and 11 in Xenopus. dnah9 expression was closely related to motile cilia from neurulation onwards. Morphant tadpoles showed impaired epidermal ciliary beating. Leftward flow at the GRP was absent, resulting in embryos with loss of asymmetric marker gene expression. Remarkably, unilateral knockdown on the right side of the GRP did not affect laterality, while left-sided ablation of flow abolished marker gene expression. Thus, flow was required exclusively on the left side of the GRP to break symmetry in the frog. Our data suggest that the substrate of flow is generated within the GRP and not at its margin, disqualifying Nodal as a candidate morphogen.

Cilia-driven leftward flow determines laterality in Xenopus.

Determination of the vertebrate left-right body axis during embryogenesis results in asymmetric development and placement of most inner organs. Although the asymmetric Nodal cascade is conserved in all vertebrates, the mechanism of symmetry breakage has remained controversial. In mammalian and fish embryos, a cilia-driven leftward flow of extracellular fluid is required for initiation of the Nodal cascade. This flow is localized at the posterior notochord ("node") and Kupffer's vesicle, respectively. In frog and chick embryos, however, molecular asymmetries are required earlier, from cleavage stages through gastrulation. The validity of a cilia-based mechanism for all vertebrates therefore has been questioned. Here we show that a cilia-driven leftward flow precedes asymmetric nodal expression in the frog Xenopus. Motile monocilia emerged on the gastrocoel roof plate during neurulation and lengthened and polarized from an initially central position to the posterior pole of cells. Concomitantly, a robust leftward fluid flow developed from stage 15 onward, significantly before asymmetric nodal transcription started in the left-lateral-plate mesoderm at stage 19. Injection of 1.5% methylcellulose into the archenteron prevented leftward flow and resulted in laterality defects, demonstrating that the flow itself was required for asymmetric gene expression and organ placement.

An Early Function of Polycystin-2 for Left-Right Organizer Induction in Xenopus.

Nodal signaling controls asymmetric organ placement during vertebrate embryogenesis. Nodal is induced by a leftward fluid flow at the ciliated left-right organizer (LRO). The mechanism of flow sensing, however, has remained elusive. pkd2 encodes the calcium channel Polycystin-2, which is required for kidney development and laterality, and may act in flow perception. Here, we have studied the role of Polycystin-2 in Xenopus and show that pkd2 is indispensable for left-right (LR) asymmetry. Knockdown of pkd2 prevented left-asymmetric nodal cascade induction in the lateral plate mesoderm. Defects were due to failure of LRO specification, morphogenesis, and, consequently, absence of leftward flow. Polycystin-2 synergizes with the unconventional nodal-type signaling molecule Xnr3 to induce the LRO precursor tissue before gastrulation, upstream of symmetry breakage. Our data uncover an unknown function of pkd2 in LR axis formation, which we propose represents an ancient role of Polycystin-2 during LRO induction in lower vertebrates.

A Conserved Role of the Unconventional Myosin 1d in Laterality Determination.

Anatomical and functional asymmetries are widespread in the animal kingdom [1, 2]. In vertebrates, many visceral organs are asymmetrically placed [3]. In snails, shells and inner organs coil asymmetrically, and in Drosophila, genitalia and hindgut undergo a chiral rotation during development. The evolutionary origin of these asymmetries remains an open question [1]. Nodal signaling is widely used [4], and many, but not all, vertebrates use cilia for symmetry breaking [5]. In Drosophila, which lacks both cilia and Nodal, the unconventional myosin ID (myo1d) gene controls dextral rotation of chiral organs [6, 7]. Here, we studied the role of myo1d in left-right (LR) axis formation in Xenopus. Morpholino oligomer-mediated myo1d downregulation affected organ placement in >50% of morphant tadpoles. Induction of the left-asymmetric Nodal cascade was aberrant in >70% of cases. Expression of the flow-target gene dand5 was compromised, as was flow itself, due to shorter, fewer, and non-polarized cilia at the LR organizer. Additional phenotypes pinpointed Wnt/planar cell polarity signaling and suggested that myo1d, like in Drosophila [8], acted in the context of the planar cell polarity pathway. Indeed, convergent extension of gastrula explant cultures was inhibited in myo1d morphants, and the ATF2 reporter gene for non-canonical Wnt signaling was downregulated. Finally, genetic interference experiments demonstrated a functional interaction between the core planar cell polarity signaling gene vangl2 and myo1d in LR axis formation. Thus, our data identified myo1d as a common denominator of arthropod and chordate asymmetry, in agreement with a monophyletic origin of animal asymmetry.

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.