Identification of novel genes including NAV2 associated with isolated tall stature.

Very tall people attract much attention and represent a clinically and genetically heterogenous group of individuals. Identifying the genetic etiology can provide important insights into the molecular mechanisms regulating linear growth. We studied a three-generation pedigree with five isolated (non-syndromic) tall members and one individual with normal stature by whole exome sequencing; the tallest man had a height of 211 cm. Six heterozygous gene variants predicted as damaging were shared among the four genetically related tall individuals and not present in a family member with normal height. To gain insight into the putative role of these candidate genes in bone growth, we assessed the transcriptome of murine growth plate by microarray and RNA Seq. Two (Ift140, Nav2) of the six genes were well-expressed in the growth plate. Nav2 (p-value 1.91E-62) as well as Ift140 (p-value of 2.98E-06) showed significant downregulation of gene expression between the proliferative and hypertrophic zone, suggesting that these genes may be involved in the regulation of chondrocyte proliferation and/or hypertrophic differentiation. IFT140, NAV2 and SCAF11 have also significantly associated with height in GWAS studies. Pathway and network analysis indicated functional connections between IFT140, NAV2 and SCAF11 and previously associated (tall) stature genes. Knockout of the all-trans retinoic acid responsive gene, neuron navigator 2 NAV2, in Xenopus supports its functional role as a growth promotor. Collectively, our data expand the spectrum of genes with a putative role in tall stature phenotypes and, among other genes, highlight NAV2 as an interesting gene to this phenotype.

FGF-mediated establishment of left-right asymmetry requires Rab7 function in the dorsal mesoderm in Xenopus.

Gastrulation denotes a very important developmental process, which includes significant structural tissue rearrangements and patterning events that shape the emerging vertebrate organism. At the end of gastrulation, the three body axes are spatially defined while the left-right axis still lacks any molecular or morphological polarity. In most vertebrates, this is established during neurulation by a symmetry breaking LR organizer. However, this mesoderm-derived structure depends on proper induction and specification of the mesoderm, which in turn requires involvement of several signaling pathways. Endocytosis and the endosomal machinery offer manifold platforms for intracellular pathway regulation, especially late endosomes claim increasing attention. The late endosomal regulator Rab7 has been linked to mesoderm specification during gastrulation. Distinct axial defects due to compromised dorsal mesoderm development in rab7-deficient Xenopus embryos suggested a requirement of Rab7 for FGF-dependent mesoderm patterning and LR asymmetry. Here we specifically addressed such a role of Rab7, demonstrating a functional requirement for LR organizer development and symmetry breakage. Using different FGF/MAPK pathway components we show that Rab7 participates in dorsal mesoderm patterning. We suggest a hierarchical classification of Rab7 upstream of MAPK-dependent mesoderm specification, most probably at the level of the small GTPase Ras. Thus, this study affords an insight on how the Rab7-regulated endosomal machinery could participate in signal transduction to enable correct mesoderm specification and left-right asymmetry.

Identification of ZBTB26 as a Novel Risk Factor for Congenital Hypothyroidism.

Congenital primary hypothyroidism (CH; OMIM 218700) is characterized by an impaired thyroid development, or dyshormonogenesis, and can lead to intellectual disability and growth retardation if untreated. Most of the children with congenital hypothyroidism present thyroid dysgenesis, a developmental anomaly of the thyroid. Various genes have been associated with thyroid dysgenesis, but all known genes together can only explain a small number of cases. To identify novel genetic causes for congenital hypothyroidism, we performed trio whole-exome sequencing in an affected newborn and his unaffected parents. A predicted damaging de novo missense mutation was identified in the ZBTB26 gene (Zinc Finger A and BTB Domain containing 26). An additional cohort screening of 156 individuals with congenital thyroid dysgenesis identified two additional ZBTB26 gene variants of unknown significance. To study the underlying disease mechanism, morpholino knock-down of zbtb26 in Xenopus laevis was carried out, which demonstrated significantly smaller thyroid anlagen in knock-down animals at tadpole stage. Marker genes expressed in thyroid tissue precursors also indicated a specific reduction in the Xenopus ortholog of human Paired-Box-Protein PAX8, a transcription factor required for thyroid development, which could be rescued by adding zbtb26. Pathway and network analysis indicated network links of ZBTB26 to PAX8 and other genes involved in thyroid genesis and function. GWAS associations of ZBTB26 were found with height. Together, our study added a novel genetic risk factor to the list of genes underlying congenital primary hypothyroidism and provides additional support that de novo mutations, together with inherited variants, might contribute to the genetic susceptibility to CH.

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.

Rab7 is required for mesoderm patterning and gastrulation in Xenopus.

Early embryogenesis requires tightly controlled temporal and spatial coordination of cellular behavior and signaling. Modulations are achieved at multiple levels, from cellular transcription to tissue-scale behavior. Intracellularly, the endolysosomal system emerges as an important regulator at different levels, but in vivo studies are rare. In the frog Xenopus, little is known about the developmental roles of endosomal regulators, or their potential involvement in signaling, especially for late endosomes. Here, we analyzed a hypothesized role of Rab7 in this context, a small GTPase known for its role as a late endosomal regulator. First, rab7 showed strong maternal expression. Following localized zygotic transcript enrichment in the mesodermal ring and neural plate, it was found in tailbud-stage neural ectoderm, notochord, pronephros, eyes and neural crest tissues. Inhibition resulted in strong axis defects caused by a requirement of rab7 for mesodermal patterning and correct gastrulation movements. To test a potential involvement in growth factor signaling, we analyzed early Wnt-dependent processes in the mesoderm. Our results suggest a selective requirement for ligand-induced Wnt activation, implicating a context-dependent role of Rab7.

Identification of Transient Receptor Potential Channel 4-Associated Protein as a Novel Candidate Gene Causing Congenital Primary Hypothyroidism.

BACKGROUND: Congenital primary hypothyroidism (CH) is the most common endocrine disorder in neonates. METHODS: To identify novel genes, we performed whole exome sequencing (WES) in 6 patients with CH due to thyroid dysgenesis (TD). The potential effects of the most relevant variants were analyzed using in silico prediction tools. The most promising candidate gene, transient receptor potential channel 4-associated protein (TRPC4AP), was sequenced in 179 further patients with TD. Expression of TRPC4AP in human thyroid was investigated using RT-PCR. Trpc4ap- functional analysis was performed in Xenopus laevis using Morpholino (MO) antisense oligomers. RESULTS: WES identified a likely damaging mutation in TRPC4AP leading to a de novo stop codon p.Q552*. Targeted sequencing of TRPC4AP demonstrated gene variants with predicted damaging potential in 5 patients resulting each in an amino acid exchange (p.P706S, p.F729L, p.S777C, and p.N229S). We demonstrated that TRPC4AP is expressed in human thyroid gland tissue. Using Xenopus laevis, we showed that the volume of the tadpole thyroid anlage was reduced by 20% in Trpc4ap MO knockdowns compared to controls and by 41% in "Clustered Regularly Interspaced Short Palindromic Repeats"/Cas9-mediated gene knockout experiments. DISCUSSION: A recognized interaction of TRPC4AP and the NF-kappa-B-essential-modulator encoded by IKBKG gene was identified by IPA analysis. IKBKG plays a role in activation of the NF-κB-signaling pathway and regulates genes involved in proliferation and survival of thyrocytes and expression of key enzymes of thyroid hormone synthesis. CONCLUSION: TRPC4AP was identified as a novel candidate gene in TD, but further studies are needed to validate its role in thyroid function.

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.

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.

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.

Wnt Signaling Translocates Lys48-Linked Polyubiquitinated Proteins to the Lysosomal Pathway.

Cellular proteins are degraded in either proteasomes or lysosomes depending on the types of ubiquitin chains that covalently modify them. It is not known whether the choice between these two pathways is physiologically regulated. The Lys48-polyubiquitin chain is the major signal directing proteins for degradation in proteasomes. Here, we report the unexpected finding that canonical Wnt signaling translocates some K48-linked polyubiquitinated proteins to the endolysosomal pathway. Proteasomal target proteins, such as b-catenin, Smad1, and Smad4, were targeted into endolysosomes in a process dependent on GSK3 activity. Relocalization was also dependent on Axin1 and the multivesicular body (MVB) proteins HRS/Vps27 and Vps4. The Wnt-induced accumulation of K48-linked polyubiquitinated proteins in endolysosomal organelles was accompanied by a transient decrease in cellular levels of free mono-ubiquitin, which may contribute to Wnt-regulated stabilization of proteins (Wnt/ STOP). We conclude that Wnt redirects Lys48-polyubiquitinated proteins that are normally degraded in proteasomes to endolysosomes.

Left-right asymmetry: cilia and calcium revisited.

Leftward flow generated by motile cilia is known to underlie left-right asymmetry in vertebrate embryos. A new study now links intraciliary calcium oscillations to cilia motility and the downstream nodal signaling cascade that drives left-sided development.

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.

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.

Presenilin deficiency or lysosomal inhibition enhances Wnt signaling through relocalization of GSK3 to the late-endosomal compartment.

Sustained canonical Wnt signaling requires the inhibition of glycogen synthase kinase 3 (GSK3) activity by sequestration of GSK3 inside multivesicular endosomes (MVEs). Here, we show that Wnt signaling is increased by the lysosomal inhibitor chloroquine, which causes accumulation of MVEs. A similar MVE expansion and increased Wnt responsiveness was found in cells deficient in presenilin, a protein associated with Alzheimer's disease. The Wnt-enhancing effects were entirely dependent on the functional endosomal sorting complex required for transport (ESCRT), which is needed for the formation of intraluminal vesicles in MVEs. We suggest that accumulation of late endosomal structures leads to enhanced canonical Wnt signaling through increased Wnt-receptor/GSK3 sequestration. The decrease in GSK3 cytosolic activity stabilized cytoplasmic GSK3 substrates such as ß-catenin, the microtubule-associated protein Tau, and other proteins. These results underscore the importance of the endosomal pathway in canonical Wnt signaling and reveal a mechanism for regulation of Wnt signaling by presenilin deficiency.

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.

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.

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.

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.