The FOXJ1 target Cfap206 is required for sperm motility, mucociliary clearance of the airways and brain development.

Cilia are complex cellular protrusions consisting of hundreds of proteins. Defects in ciliary structure and function, many of which have not been characterised molecularly, cause ciliopathies: a heterogeneous group of human syndromes. Here, we report on the FOXJ1 target gene Cfap206, orthologues of which so far have only been studied in Chlamydomonas and Tetrahymena In mouse and Xenopus, Cfap206 was co-expressed with and dependent on Foxj1 CFAP206 protein localised to the basal body and to the axoneme of motile cilia. In Xenopus crispant larvae, the ciliary beat frequency of skin multiciliated cells was enhanced and bead transport across the epidermal mucociliary epithelium was reduced. Likewise, Cfap206 knockout mice revealed ciliary phenotypes. Electron tomography of immotile knockout mouse sperm flagella indicated a role in radial spoke formation reminiscent of FAP206 function in Tetrahymena Male infertility, hydrocephalus and impaired mucociliary clearance of the airways in the absence of laterality defects in Cfap206 mutant mice suggests that Cfap206 may represent a candidate for the subgroup of human primary ciliary dyskinesias caused by radial spoke defects.

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.

CFAP43 modulates ciliary beating in mouse and Xenopus.

Malfunctions of motile cilia cause a variety of developmental defects and diseases in humans and animal model organisms. Defects include impaired mucociliary clearance of the airways, sperm immotility, hydrocephalus and organ laterality. Here, we characterize the evolutionary conserved Cfap43 gene by loss-of-function experiments in the mouse and the frog Xenopus laevis. Cfap43 is expressed in tissues carrying motile cilia and acts as a target gene of the transcription factor FOXJ1, which is essential for the induction of motile ciliogenesis. We show that CFAP43, a protein of unknown biochemical function, localizes to the ciliary axoneme. CFAP43 is involved in the regulation of the beating frequency of tracheal cilia and loss of CFAP43 causes severe mucus accumulation in the nasal cavity. Likewise, morphant and crispant frog embryos revealed impaired function of motile cilia of the larval epidermis, a model for airway mucociliary epithelia. CFAP43 participates in the formation of flagellar axonemes during spermatogenesis as mice mutant for Cfap43 display male infertility, consistent with observations in male sterile patients. In addition, mice mutant for Cfap43 display early onset hydrocephalus. Together, these results confirm the role of CFAP43 in the male reproductive tract and pinpoint additional functions in airway epithelia mucus clearance and brain development.

Conserved role of matrix metalloproteases 2 and 9 in promoting the migration of neural crest cells in avian and mammalian embryos.

Neural crest cells (NCCs) are a unique embryonic cell population that initially reside at the dorsal neural tube but later migrate in the embryo and differentiate into multiple types of derivatives. To acquire motility, NCCs undergo epithelial-to-mesenchymal transition and invade the surrounding extracellular matrix (ECM). Matrix metalloproteases (MMPs) are a large family of proteases which regulate migration of various embryonic and adult cells via ECM remodeling. The gelatinase's subgroup of MMPs is the most studied one due to its key role in metastasis. As it is composed of only two proteases, MMP2 and MMP9, it is important to understand whether each is indispensable or redundant in its biological function. Here we explored the role of the gelatinases in executing NCC migration, by determining whether MMP2 and/or MMP9 regulate migration across species in singular, combined, or redundant manners. Chick and mouse embryos were utilized to compare expression and activity of both MMPs using genetic and pharmacological approaches in multiple in vivo and ex vivo assays. Both MMPs were found to be expressed and active in mouse and chick NCCs. Inhibition of each MMP was sufficient to prevent NCC migration in both species. Yet, NCC migration was maintained in MMP2-/- or MMP9-/- mouse mutants due to compensation between the gelatinases, but reciprocal pharmacological inhibition in each mutant prevented NCC migration. This study reveals for the first time that both gelatinases are expressed in avian and mammalian NCCs, and demonstrates their fundamental and conserved role in promoting embryonic cell migration.

CFAP157 is a murine downstream effector of FOXJ1 that is specifically required for flagellum morphogenesis and sperm motility.

Motile cilia move extracellular fluids or mediate cellular motility. Their function is essential for embryonic development, adult tissue homeostasis and reproduction throughout vertebrates. FOXJ1 is a key transcription factor for the formation of motile cilia but its downstream genetic programme is only partially understood. Here, we characterise a novel FOXJ1 target, Cfap157, that is specifically expressed in motile ciliated tissues in mouse and Xenopus in a FOXJ1-dependent manner. CFAP157 protein localises to basal bodies and interacts with tubulin and the centrosomal protein CEP350. Cfap157 knockout mice appear normal but homozygous males are infertile. Spermatozoa display impaired motility and a novel phenotype: Cfap157-deficient sperm exhibit axonemal loops, supernumerary axonemal profiles with ectopic accessory structures, excess cytoplasm and clustered mitochondria in the midpiece regions, and defective axonemes along the flagella. Our study thus demonstrates an essential sperm-specific function for CFAP157 and suggests that this novel FOXJ1 effector is part of a mechanism that acts during spermiogenesis to suppress the formation of supernumerary axonemes and ensures a correct ultrastructure.

Xenopus: An Undervalued Model Organism to Study and Model Human Genetic Disease.

The function of normal and defective candidate genes for human genetic diseases, which are rapidly being identified in large numbers by human geneticists and the biomedical community at large, will be best studied in relevant and predictive model organisms that allow high-speed verification, analysis of underlying developmental, cellular and molecular mechanisms, and establishment of disease models to test therapeutic options. We describe and discuss the pros and cons of the frog Xenopus, which has been extensively used to uncover developmental mechanisms in the past, but which is being underutilized as a biomedical model. We argue that Xenopus complements the more commonly used mouse and zebrafish as a time- and cost-efficient animal model to study human disease alleles and mechanisms.

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.

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.

Embryonic exposure to propylthiouracil disrupts left-right patterning in Xenopus embryos.

Antithyroid medications are the preferred therapy for the treatment of Graves' disease during pregnancy. Propylthiouracil (PTU) is favored over methimazole (MMI) due to potential teratogenic concerns with MMI. This study was to determine the teratogenic potential of MMI and PTU using a validated Xenopus tropicalis embryo model. Embryos were exposed to 1 mM PTU (EC(50)=0.88 mM), 1 mM MMI, or vehicle control (water) from stages 2 to 45. Treated embryos were examined for gross morphological defects, ciliary function, and gene expression by in situ hybridization. Exposure to PTU, but not MMI, led to cardiac and gut looping defects and shortening along the anterior-posterior axis. PTU exposure during gastrulation (stage 8-12.5) was identified as the critical period of exposure leading to left-right (LR) patterning defects. Abnormal cilia polarization, abnormal cilia-driven leftward flow at the gastrocoel roof plate (GRP), and aberrant expression of both Coco and Pitx2c were associated with abnormal LR symmetry observed following PTU exposure. PTU is teratogenic during late blastula, gastrulation, and neurulation; whereas MMI is not. PTU alters ciliary-driven flow and disrupts the normal genetic program involved in LR axis determination. These studies have important implications for women taking PTU during early pregnancy.

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.

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.

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.

Discovery of a genetic module essential for assigning left-right asymmetry in humans and ancestral vertebrates.

The vertebrate left-right axis is specified during embryogenesis by a transient organ: the left-right organizer (LRO). Species including fish, amphibians, rodents and humans deploy motile cilia in the LRO to break bilateral symmetry, while reptiles, birds, even-toed mammals and cetaceans are believed to have LROs without motile cilia. We searched for genes whose loss during vertebrate evolution follows this pattern and identified five genes encoding extracellular proteins, including a putative protease with hitherto unknown functions that we named ciliated left-right organizer metallopeptide (CIROP). Here, we show that CIROP is specifically expressed in ciliated LROs. In zebrafish and Xenopus, CIROP is required solely on the left side, downstream of the leftward flow, but upstream of DAND5, the first asymmetrically expressed gene. We further ascertained 21 human patients with loss-of-function CIROP mutations presenting with recessive situs anomalies. Our findings posit the existence of an ancestral genetic module that has twice disappeared during vertebrate evolution but remains essential for distinguishing left from right in humans.

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.

The highly conserved FOXJ1 target CFAP161 is dispensable for motile ciliary function in mouse and Xenopus.

Cilia are protrusions of the cell surface and composed of hundreds of proteins many of which are evolutionary and functionally well conserved. In cells assembling motile cilia the expression of numerous ciliary components is under the control of the transcription factor FOXJ1. Here, we analyse the evolutionary conserved FOXJ1 target CFAP161 in Xenopus and mouse. In both species Cfap161 expression correlates with the presence of motile cilia and depends on FOXJ1. Tagged CFAP161 localises to the basal bodies of multiciliated cells of the Xenopus larval epidermis, and in mice CFAP161 protein localises to the axoneme. Surprisingly, disruption of the Cfap161 gene in both species did not lead to motile cilia-related phenotypes, which contrasts with the conserved expression in cells carrying motile cilia and high sequence conservation. In mice mutation of Cfap161 stabilised the mutant mRNA making genetic compensation triggered by mRNA decay unlikely. However, genes related to microtubules and cilia, microtubule motor activity and inner dyneins were dysregulated, which might buffer the Cfap161 mutation.