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WNT ligands induce Ca super(2+) signalling on target cells. PKD1 (polycystin 1) is considered an orphan, atypical G-protein-coupled receptor complexed with TRPP2 (polycystin 2 or PKD2), a Ca super(2+)-permeable ion channel. Inactivating mutations in their genes cause autosomal dominant polycystic kidney disease (ADPKD), one of the most common genetic diseases. Here, we show that WNTs bind to the extracellular domain of PKD1 and induce whole-cell currents and Ca super(2+) influx dependent on TRPP2. Pathogenic PKD1 or PKD2 mutations that abrogate complex formation, compromise cell surface expression of PKD1, or reduce TRPP2 channel activity suppress activation by WNTs. Pkd2 super(-/-) fibroblasts lack WNT-induced Ca super(2+) currents and are unable to polarize during directed cell migration. In Xenopus embryos, pkd1, Dishevelled 2 (dvl2) and wnt9a act within the same pathway to preserve normal tubulogenesis. These data define PKD1 as a WNT (co)receptor and implicate defective WNT/Ca super(2+) signalling as one of the causes of ADPKD.

Xenopus laevis and tropicalis tadpoles display incredible regenerative capacity of their tail. Amaya and colleagues find that tadpole tail amputation induces the production of reactive oxygen species (ROS) to induce cell proliferation and regeneration, through activation of the Wnt/β-catenin and Fgf20 signalling pathways. Understanding the molecular mechanisms that promote successful tissue regeneration is critical for continued advancements in regenerative medicine. Vertebrate amphibian tadpoles of the species Xenopus laevis and Xenopus tropicalis have remarkable abilities to regenerate their tails following amputation 1 , 2 , through the coordinated activity of numerous growth factor signalling pathways, including the Wnt, Fgf, Bmp, Notch and TGF-β pathways 3 , 4 , 5 , 6 . Little is known, however, about the events that act upstream of these signalling pathways following injury. Here, we show that Xenopus tadpole tail amputation induces a sustained production of reactive oxygen species (ROS) during tail regeneration. Lowering ROS levels, using pharmacological or genetic approaches, reduces the level of cell proliferation and impairs tail regeneration. Genetic rescue experiments restored both ROS production and the initiation of the regenerative response. Sustained increased ROS levels are required for Wnt/β-catenin signalling and the activation of one of its main downstream targets, fgf20 (ref.  7 ), which, in turn, is essential for proper tail regeneration. These findings demonstrate that injury-induced ROS production is an important regulator of tissue regeneration.

Neural crest abnormalities 'CHARGE syndrome' is a rare congenital condition characterized by malformations of the craniofacial structures, peripheral nervous system, ears, eyes and heart. It is caused by heterozygous mutations in the gene for CHD7, an ATP-dependent chromatin-remodelling protein. It was postulated 25 years ago that CHARGE syndrome results from the abnormal development of the neural crest. This hypothesis has remained untested, but Bajpai et al . now show that CHD7 is essential for the formation of multipotent migratory neural crest, and is essential for activating the neural crest transcriptional circuitry. In addition, CHD7 is shown to cooperate with another chromatin-remodelling complex, PBAF, to promote neural crest gene expression and cell migration. Heterozygous mutations in the gene encoding CHD7, an ATP-dependent chromatin-remodelling protein, result in CHARGE syndrome — a disorder characterized by malformations of the craniofacial structures, peripheral nervous system, ears, eyes and heart. In humans and Xenopus , CHD7 is now shown to be essential for the formation of multipotent migratory neural crest and for activating the transcriptional circuitry of the neural crest; shedding light on the pathoembryology of CHARGE syndrome. Heterozygous mutations in the gene encoding the CHD (chromodomain helicase DNA-binding domain) member CHD7, an ATP-dependent chromatin remodeller homologous to the Drosophila trithorax-group protein Kismet 1 , 2 , result in a complex constellation of congenital anomalies called CHARGE syndrome, which is a sporadic, autosomal dominant disorder characterized by malformations of the craniofacial structures, peripheral nervous system, ears, eyes and heart 3 , 4 . Although it was postulated 25 years ago that CHARGE syndrome results from the abnormal development of the neural crest, this hypothesis remained untested 5 . Here we show that, in both humans and Xenopus , CHD7 is essential for the formation of multipotent migratory neural crest (NC), a transient cell population that is ectodermal in origin but undergoes a major transcriptional reprogramming event to acquire a remarkably broad differentiation potential and ability to migrate throughout the body, giving rise to craniofacial bones and cartilages, the peripheral nervous system, pigmentation and cardiac structures 6 , 7 . We demonstrate that CHD7 is essential for activation of the NC transcriptional circuitry, including Sox9 , Twist and Slug . In Xenopus embryos, knockdown of Chd7 or overexpression of its catalytically inactive form recapitulates all major features of CHARGE syndrome. In human NC cells CHD7 associates with PBAF (polybromo- and BRG1-associated factor-containing complex) 8 and both remodellers occupy a NC-specific distal SOX9 enhancer 9 and a conserved genomic element located upstream of the TWIST1 gene. Consistently, during embryogenesis CHD7 and PBAF cooperate to promote NC gene expression and cell migration. Our work identifies an evolutionarily conserved role for CHD7 in orchestrating NC gene expression programs, provides insights into the synergistic control of distal elements by chromatin remodellers, illuminates the patho-embryology of CHARGE syndrome, and suggests a broader function for CHD7 in the regulation of cell motility.

Even moderate doses of alcohol cause considerable impairment of motor coordination, an effect that substantially involves potentiation of GABAergic activity at d subunit-containing GABAA receptors (d-GABAARs). Here, we demonstrate that oxytocin selectively attenuates ethanol-induced motor impairment and ethanol-induced increases in GABAergic activity at ...-GABAARs and that this effect does not involve the oxytocin receptor. Specifically, oxytocin (1 mu g i.c.v.) given before ethanol (1.5 g/kg i.p.) attenuated the sedation and ataxia induced by ethanol in the open-field locomotor test, wire-hanging test, and righting-reflex test in male rats. Using two-electrode voltage-clamp electrophysiology in Xenopus oocytes, oxytocin was found to completely block ethanol-enhanced activity at alpha 4 beta 1d and alpha 4 beta 3d recombinant GABAARs. Conversely, ethanol had no effect when applied to alpha 4 beta 1 or alpha 4 beta 3 cells, demonstrating the critical presence of the d subunit in this effect. Oxytocin had no effect on the motor impairment or in vitro effects induced by the ...-selective GABAAR agonist 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol, which binds at a different site on ...-GABAARs than ethanol. Vasopressin, which is a nonapeptide with substantial structural similarity to oxytocin, did not alter ethanol effects at ...-GABAARs. This pattern of results confirms the specificity of the interaction between oxytocin and ethanol at ...-GABAARs. Finally, our in vitro constructs did not express any oxytocin receptors, meaning that the observed interactions occur directly at d-GABAARs. The profound and direct interaction observed between oxytocin and ethanol at the behavioral and cellular level may have relevance for the development of novel therapeutics for alcohol intoxication and dependence. (ProQuest: ... denotes formulae/symbols omitted.)

The RING finger domain protein Uhrf1 is known to have an important role in DNA methylation pattern maintenance through the recruitment of the methyltransferase Dnmt1 to hemimethylated DNA sites: here, Uhrf1 is shown to act as a ubiquitin ligase for H3, an essential step in Dnmt1 recruitment. Linking DNA methylation and replication The RING finger domain protein Uhrf1 has an essential role in maintaining patterns of DNA methylation during replication by recruiting the DNA methyltransferase Dnmt1 to hemi-methylated DNA sites. Here, Makoto Nakanishi and colleagues reproduce maintenance DNA methylation in an in vitro system using Xenopus egg extracts. They show that Uhrf1 is an E3 ubiquitin ligase for histone H3, and that ubiquitination of H3 is required for the recruitment of Dnmt1 to DNA replication sites. Faithful propagation of DNA methylation patterns during DNA replication is critical for maintaining cellular phenotypes of individual differentiated cells 1 , 2 , 3 , 4 , 5 . Although it is well established that Uhrf1 (ubiquitin-like with PHD and ring finger domains 1; also known as Np95 and ICBP90) specifically binds to hemi-methylated DNA through its SRA (SET and RING finger associated) domain and has an essential role in maintenance of DNA methylation by recruiting Dnmt1 to hemi-methylated DNA sites 6 , 7 , 8 , 9 , 10 , the mechanism by which Uhrf1 coordinates the maintenance of DNA methylation and DNA replication is largely unknown. Here we show that Uhrf1-dependent histone H3 ubiquitylation has a prerequisite role in the maintenance DNA methylation. Using Xenopus egg extracts, we successfully reproduce maintenance DNA methylation in vitro . Dnmt1 depletion results in a marked accumulation of Uhrf1-dependent ubiquitylation of histone H3 at lysine 23. Dnmt1 preferentially associates with ubiquitylated H3 in vitro though a region previously identified as a replication foci targeting sequence 11 . The RING finger mutant of Uhrf1 fails to recruit Dnmt1 to DNA replication sites and maintain DNA methylation in mammalian cultured cells. Our findings represent the first evidence, to our knowledge, of the mechanistic link between DNA methylation and DNA replication through histone H3 ubiquitylation.

Casein kinase 1 (CK1) members play key roles in numerous biological processes. They are considered \"rogue\" kinases, because their enzymatic activity appears unregulated. Contrary to this notion, we have identified the DEAD-box RNA helicase DDX3 as a regulator of the Wnt–β-catenin network, where it acts as a regulatory subunit of CK1ε: In a Wnt-dependent manner, DDX3 binds CK1ε and directly stimulates its kinase activity, and promotes phosphorylation of the scaffold protein dishevelled. DDX3 is required for Wnt–β-catenin signaling in mammalian cells and during Xenopus and Caenorhabditis elegans development. The results also suggest that the kinase-stimulatory function extends to other DDX and CK1 members, opening fresh perspectives for one of the longest-studied protein kinase families.

The gene regulatory networks that coordinate the development of the cardiac and pulmonary systems are essential for terrestrial life but poorly understood. The T-box transcription factor Tbx5 is critical for both pulmonary specification and heart development, but how these activities are mechanistically integrated remains unclear. Here using Xenopus and mouse embryos, we establish molecular links between Tbx5 and retinoic acid (RA) signaling in the mesoderm and between RA signaling and sonic hedgehog expression in the endoderm to unveil a conserved RA-Hedgehog-Wnt signaling cascade coordinating cardiopulmonary (CP) development. We demonstrate that Tbx5 directly maintains expression of aldh1a2, the RA-synthesizing enzyme, in the foregut lateral plate mesoderm via an evolutionarily conserved intronic enhancer. Tbx5 promotes posterior second heart field identity in a positive feedback loop with RA, antagonizing a Fgf8-Cyp regulatory module to restrict FGF activity to the anterior. We find that Tbx5/Aldh1a2-dependent RA signaling directly activates shh transcription in the adjacent foregut endoderm through a conserved MACS1 enhancer. Hedgehog signaling coordinates with Tbx5 in the mesoderm to activate expression of wnt2/2b, which induces pulmonary fate in the foregut endoderm. These results provide mechanistic insight into the interrelationship between heart and lung development informing CP evolution and birth defects.

The doublesex and mab-3 related transcription factor 1 ( dmrt1 ) plays a crucial role in metazoan sexual differentiation. This gene, or its paralogs, independently became triggers for sex determination several times, including in the tetraploid African clawed frog Xenopus laevis . To explore functional evolution of this gene, we generated knockout lines of each of two dmrt1 homeologs in X. laevis and an ortholog in the closely related diploid Western clawed frog X. tropicalis . Our findings evidence sex-specific functional evolution following duplication by allotetraploidization in an ancestor of X. laevis . In females, dmrt1 was essential for fertility and oogenesis in the Xenopus ancestor, but this important function was lost (subfunctionalized) in one X. laevis homeolog ( dmrt1.S ) after allotetraploidization. In males – in sharp contrast – dmrt1 was not essential for fertility and spermatogenesis in the Xenopus ancestor, but this essentiality was acquired (neofunctionalized) in the other X. laevis homeolog ( dmrt1.L ) after allotetraploidization. Transcriptomic analysis of the mesonephros/gonad complex during sexual differentiation identifies distinctive patterns of dysregulation in male and female knockouts of dmrt1.L and dmrt1.S relative to same-sex wildtype siblings, including possible autocatalysis of dmrt1.L and activation of the female-determining gene dm-w . Previous work demonstrates that dm-w was recently derived from partial gene duplication of dmrt1.S – a gene that our analysis demonstrates is non-essential in both sexes. Thus, in X. laevis , a developmental system was pushed past a “tipping point” to a novel state where sexual differentiation is now orchestrated by a sex-specific duplicate of a dispensable gene.

Multiciliate cells function prominently in the respiratory system, brain ependyma and female reproductive tract to produce vigorous fluid flow along epithelial surfaces. These specialized cells form during development when epithelial progenitors undergo an unusual form of ciliogenesis, in which they assemble and project hundreds of motile cilia. Notch inhibits multiciliate cell formation in diverse epithelia, but how progenitors overcome lateral inhibition and initiate multiciliate cell differentiation is unknown. Here we identify a coiled-coil protein, termed multicilin, which is regulated by Notch and highly expressed in developing epithelia where multiciliate cells form. Inhibiting multicilin function specifically blocks multiciliate cell formation in Xenopus skin and kidney, whereas ectopic expression induces the differentiation of multiciliate cells in ectopic locations. Multicilin localizes to the nucleus, where it directly activates the expression of genes required for multiciliate cell formation, including foxj1 and genes mediating centriole assembly. Multicilin is also necessary and sufficient to promote multiciliate cell differentiation in mouse airway epithelial cultures. These findings indicate that multicilin initiates multiciliate cell differentiation in diverse tissues, by coordinately promoting the transcriptional changes required for motile ciliogenesis and centriole assembly. Several specialized cell types assemble hundreds of motile cilia to accomplish their function. Kintner and colleagues identify the coiled-coil protein multicilin as an essential regulator of multicilia formation in Xenopus skin and the mammalian kidney. Their data indicate that multicilin activates the transcription of genes required for multicilia formation, including the transcription factor Foxj1.

Loop extrusion by structural maintenance of chromosomes (SMC) complexes has been proposed as a mechanism to organize chromatin in interphase and metaphase. However, the requirements for chromatin organization in these cell cycle phases are different, and it is unknown whether loop extrusion dynamics and the complexes that extrude DNA also differ. Here, we used Xenopus egg extracts to reconstitute and image loop extrusion of single DNA molecules during the cell cycle. We show that loops form in both metaphase and interphase, but with distinct dynamic properties. Condensin extrudes DNA loops non-symmetrically in metaphase, whereas cohesin extrudes loops symmetrically in interphase. Our data show that loop extrusion is a general mechanism underlying DNA organization, with dynamic and structural properties that are biochemically regulated during the cell cycle.