Tina Beyer

Bicaudal C, a novel regulator of Dvl signaling abutting RNA-processing bodies, controls cilia orientation and leftward flow

Polycystic diseases and left-right (LR) axis malformations are frequently linked to cilia defects. Renal cysts also arise in mice and frogs lacking Bicaudal C (BicC), a conserved RNA-binding protein containing K-homology (KH) domains and a sterile alpha motif (SAM). However, a role for BicC in cilia function has not been demonstrated. Here, we report that targeted inactivation of BicC randomizes left-right (LR) asymmetry by disrupting the planar alignment of motile cilia required for cilia-driven fluid flow. Furthermore, depending on its SAM domain, BicC can uncouple Dvl2 signaling from the canonical Wnt pathway, which has been implicated in antagonizing planar cell polarity (PCP). The SAM domain concentrates BicC in cytoplasmic structures harboring RNA-processing bodies (P-bodies) and Dvl2. These results suggest a model whereby BicC links the orientation of cilia with PCP, possibly by regulating RNA silencing in P-bodies.

The Nodal Inhibitor Coco Is a Critical Target of Leftward Flow in Xenopus

Vertebrate laterality, which is manifested by asymmetrically placed organs [1], depends on asymmetric activation of the Nodal signaling cascade in the left lateral plate mesoderm [2]. In fish, amphibians, and mammals, a cilia-driven leftward flow of extracellular fluid acts upstream of the Nodal cascade [3-6]. The direct target of flow has remained elusive. In Xenopus, flow occurs at the gastrocoel roof plate (GRP) in the dorsal midline of the embryo [4, 7]. The GRP is bordered by a second, bilaterally symmetrical Nodal expression domain [8]. Here we identify the Nodal inhibitor Coco as a critical target of flow. Coco and Xenopus Nodal-related 1 (Xnr1) are coexpressed in the lateralmost ciliated GRP cells. Coco becomes downregulated on the left side of the GRP as a direct readout of flow. Ablation of flow prevented Coco repression, whereas Xnr1 expression was independent of flow. Loss of flow-induced laterality defects were rescued by knockdown of Coco on the left side. Parallel knockdown of Coco and Xnr1 in GRP cells restored laterality defects in flow-impaired embryos, demonstrating that Coco acted through GRP-expressed Xnr1. Coco thus acts as a critical target of flow, suggesting that symmetry is broken by flow-mediated left-asymmetric release of Nodal repression at the midline.

Xenopus, an ideal model system to study vertebrate left-right asymmetry.

Vertebrate organ laterality is manifested by the asymmetric morphogenesis and placement of inner organs. Asymmetric induction of the Nodal signaling cascade in the left lateral plate mesoderm (LPM) precedes and is essential for asymmetric organ morphogenesis. While the Nodal cascade is highly conserved, symmetry breakage is considered to vary between the different classes of the vertebrates. In Xenopus, early determinants at cleavage stages were thought to break symmetry, opposed to cilia‐driven leftward flow in mammals and fish. The main objectives of this review are to emphasize the conserved nature of symmetry breakage, and to demonstrate the power of Xenopus embryology to analyze and manipulate flow. In addition, mutant phenotypes described in other model organisms can easily be mimicked in frog by single or multiple knockdowns in combination with experimental manipulations and flow analysis. Xenopus, therefore, is ideally suited to address the major open questions in the field. Developmental Dynamics 238:1215–1225, 2009.

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), Kupffers 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.

Evolution of leftward flow.

The asymmetric Nodal signaling cascade as a prerequisite for asymmetric body plan specification is conserved among deuterostomes. In this review we argue that symmetry breakage by cilia-driven leftward flow presents an ancestral character of vertebrates, likely the chordate phylum and maybe all deuterostomes. In vertebrates, leftward flow occurs in a transient structure, a monociliated epithelium, which is derived from superficial mesoderm and localizes to the archenteron roof during gastrulation. The chick as an example for the highly derived birds lacks superficial mesoderm and flow. This loss should be secondary, as flow is present from fish and amphibians to mammals.

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.

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.

Connexin26-mediated transfer of laterality cues in Xenopus.

Summary A cilia-driven leftward flow of extracellular fluid breaks bilateral symmetry in the dorsal midline of the neurula stage vertebrate embryo. The left-specific Nodal signaling cascade in the lateral plate mesoderm (LPM) is key to asymmetric morphogenesis and placement of organs during subsequent development. The nature of the initial asymmetric cue(s) as well as the transfer of information from the midline to the left side has remained elusive. Gap junctional communication has been previously involved in Xenopus left-right (LR) development, however a function at cleavage stages was inferred from inhibitor experiments. Here we show by heptanol-mediated block of connexin function that flow stages during neurulation represent the critical time window. Flow in Xenopus occurs at the gastrocoel roof plate (GRP), a ciliated sheath of cells of mesodermal fate transiently positioned within the dorsal epithelial lining of the forming archenteron. We reasoned that endodermal cells immediately adjacent to the GRP are important for transfer of asymmetry. A systematic screen identified two connexin genes, Cx26 and Cx32, which were co-expressed in these lateral endodermal cells. Gain- and loss-of-function experiments pinpointed Cx26 as the critical connexin for LR development, while Cx32 had no effect on laterality. Importantly, GRP morphology, ciliation and flow were not affected in Cx26 morphants. Our results demonstrate a decisive role of Cx26 in the transfer of laterality cues from the GRP to the left LPM, providing a novel access to the identification of the initial asymmetric signal generated by flow.

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/β-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.

Ciliary and non-ciliary expression and function of PACRG during vertebrate development

BackgroundPark2-co-regulated gene (PACRG) is evolutionarily highly conserved from green algae to mammals. In Chlamydomonas and trypanosomes, the PACRG protein associates with flagella. Loss of PACRG results in shortened or absent flagella. In mouse the PACRG protein is required for spermatogenesis. The purpose of the present study was to analyze (1) the expression patterns of PACRG during vertebrate embryogenesis, and (2) whether the PACRG protein was required for left-right (LR) axis specification through cilia-driven leftward flow in Xenopus laevis.MethodsPACRG cDNAs were cloned and expression was analyzed during early embryonic development of Xenopus, mouse, rabbit and zebrafish. Antisense morpholino oligonucleotide (MO) mediated gene knockdown was applied in Xenopus to investigate LR development at the level of tissue morphology, leftward flow and asymmetric marker gene expression, using timelapse videography, scanning electron microscopy (SEM) and whole-mount in situ hybridization. Results were statistically evaluated using Wilcoxon paired and χ2 tests.ResultsPACRG mRNA expression was found in cells and tissues harboring cilia throughout the vertebrates. Highly localized expression was also detected in the brain. During early development, PACRG was specifically localized to epithelia where leftward flow arises, that is, the gastrocoel roof plate (GRP) in Xenopus, the posterior notochord (PNC) in mammals and Kupffer’s vesicle (KV) in zebrafish. Besides its association with ciliary axonemes, subcellular localization of PACRG protein was found around the nucleus and in a spotty pattern in the cytoplasm. A green fluorescent protein (GFP) fusion construct preferentially labeled cilia, rendering PACRG a versatile marker for live imaging. Loss-of-function in the frog resulted dose dependently in LR, neural tube closure and gastrulation defects, representing ciliary and non-ciliary functions of PACRG.ConclusionsThe PACRG protein is a novel essential factor of cilia in Xenopus.