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The developmentally important hedgehog (Hh) pathway is activated by binding of Hh to patched (Ptch1), releasing smoothened (Smo) and the downstream transcription factor glioma associated (Gli) from inhibition. The mechanism behind Ptch1-dependent Smo inhibition remains unresolved. We now show that by mixing Ptch1-transfected and Ptch1 small interfering RNA-transfected cells with Gli reporter cells, Ptch1 is capable of non-cell autonomous repression of Smo. The magnitude of this non-cell autonomous repression of Smo activity was comparable to the fusion of Ptch1-transfected cell lines and Gli reporter cell lines, suggesting that it is the predominant mode of action. CHOD-PAP analysis of medium conditioned by Ptch1-transfected cells showed an elevated 3beta-hydroxysteroid content, which we hypothesized to mediate the Smo inhibition. Indeed, the inhibition of 3beta-hydroxysteroid synthesis impaired Ptch1 action on Smo, whereas adding the 3beta-hydroxysteroid (pro-)vitamin D3 to the medium effectively inhibited Gli activity. Vitamin D3 bound to Smo with high affinity in a cyclopamine-sensitive manner. Treating zebrafish embryos with vitamin D3 mimicked the smo(-/-) phenotype, confirming the inhibitory action in vivo. Hh activates its signalling cascade by inhibiting Ptch1-dependent secretion of the 3beta-hydroxysteroid (pro-)vitamin D3. This action not only explains the seemingly contradictory cause of Smith-Lemli-Opitz syndrome (SLOS), but also establishes Hh as a unique morphogen, because binding of Hh on one cell is capable of activating Hh-dependent signalling cascades on other cells.

Deviation from proper muscle development or homeostasis results in various myopathic conditions. Employing genetic as well as chemical intervention, we provide evidence that a tight regulation of Wnt/beta-catenin signaling is essential for muscle fiber growth and maintenance. In zebrafish embryos, gain-of-Wnt/beta-catenin function results in unscheduled muscle progenitor proliferation, leading to slow and fast muscle hypertrophy accompanied by fast muscle degeneration. The effects of Wnt/beta-catenin signaling on fast muscle hypertrophy were rescued by misexpression of Myostatin or p21(CIP/WAF), establishing an in vivo regulation of myofibrillogenesis by Wnt/beta-catenin signaling and Myostatin. Epistatic analyses suggest a possible genetic interaction between Wnt/beta-catenin and Myostatin in regulation of slow and fast twitch muscle myofibrillogenesis.

Previous studies have shown that Wnt signals, relayed through β‐catenin and T‐cell factor 4 (Tcf4), are essential for the induction and maintenance of crypts in mice. We have now generated a tcf4 ( tcf7l2 ) mutant zebrafish by reverse genetics. We first observe a phenotypic defect at 4 weeks post‐fertilization (wpf), leading to death at about 6 wpf. The phenotype comprises a loss of proliferation at the base of the intestinal folds of the middle and distal parts of the intestine. The proximal intestine represents an independent compartment, as it expresses sox2 in the epithelium and barx1 in the surrounding mesenchyme, which are early stomach markers in higher vertebrates. Zebrafish are functionally stomach‐less, but the proximal intestine might share its ontogeny with the mammalian stomach. Rare adult homozygous tcf4 −/− ‘escapers’ show proliferation defects in the gut epithelium, but have no other obvious abnormalities. This study underscores the involvement of Tcf4 in maintaining proliferative self‐renewal in the intestine throughout life.

ECS (Elongin BC-Cul2/Cul5-SOCS-box protein) ubiquitin ligases recruit substrates to E2 ubiquitin-conjugating enzymes through a SOCS-box protein substrate receptor, an Elongin BC adaptor and a cullin (Cul2 or Cul5) scaffold which interacts with the RING protein. In vitro studies have shown that the conserved amino acid sequence of the cullin box in SOCS-box proteins is required for complex formation and function. However, the in vivo importance of cullin boxes has not been addressed. To explore the biological functions of the cullin box domain of ankyrin repeat and SOCS-box containing protein 11 (d-Asb11), a key mediator of canonical Delta-Notch signaling, we isolated a zebrafish mutant lacking the Cul5 box (Asb11(Cul)). We found that homozygous zebrafish mutants for this allele were defective in Notch signaling as indicated by the impaired expression of Notch target genes. Importantly, asb11(Cul) fish were not capable to degrade the Notch ligand DeltaA during embryogenesis, a process essential for the initiation of Notch signaling during neurogenesis. Accordingly, proper cell fate specification within the neurogenic regions of the zebrafish embryo was impaired. In addition, Asb11(Cul) mRNA was defective in the ability to transactivate a her4::gfp reporter DNA when injected in embryos. Thus, our study reporting the generation and the characterization of a metazoan organism mutant in the conserved cullin binding domain of the SOCS-box demonstrates a hitherto unrecognized importance of the SOCS-box domain for the function of this class of cullin-RING ubiquitin ligases and establishes that the d-Asb11 cullin box is required for both canonical Notch signaling and proper neurogenesis.

Truncation of the tumour suppressor adenomatous polyposis coli (Apc) constitutively activates the Wnt/β-catenin signalling pathway 1 . Apc has a role in development: for example, embryos of mice with truncated Apc do not complete gastrulation 2 . To understand this role more fully, we examined the effect of truncated Apc on zebrafish development. Here we show that, in contrast to mice, zebrafish do complete gastrulation. However, mutant hearts fail to loop and form excessive endocardial cushions. Conversely, overexpression of Apc or Dickkopf 1 (Dkk1), a secreted Wnt inhibitor 3 , blocks cushion formation. In wild-type hearts, nuclear β-catenin, the hallmark of activated canonical Wnt signalling 4 , accumulates only in valve-forming cells, where it can activate a Tcf reporter. In mutant hearts, all cells display nuclear β-catenin and Tcf reporter activity, while valve markers are markedly upregulated. Concomitantly, proliferation and epithelial–mesenchymal transition, normally restricted to endocardial cushions, occur throughout the endocardium. Our findings identify a novel role for Wnt/β-catenin signalling in determining endocardial cell fate.

Patients with a high total nevus count (TNC) merit a totalbody examination, but a simple strategy to identify these high-risk individuals is essentially missing. The aim of this study was to investigate the correlation between the number of melanocytic nevi on both arms and the TNC, and to evaluate patient variables that may have an effect on this association. In this multicenter, cross-sectional study, 2175 patients were examined and the mean number of arm nevi in relation to TNC was calculated. A mean value of fewer than 10 arm nevi was found in patients with TNC lower than 51 and a mean value of greater than 19 arm nevi was scored in patients with TNC greater than 50. These values remained unchanged after adjustment for various patient variables. In relation to TNC greater than 50, the presence of 20 or more arm nevi had specificity and negative predictive values of 95.2 and 89.6%, respectively. The sensitivity was 65.5% in patients younger than 50 years of age and 37.5% in the older age group. The number of arm nevi was significantly higher in individuals with a history of melanoma and in those with a melanoma detected during the study period. The presence of 20 or more nevi on the arms is an independent predictor of a high TNC and risk of melanoma. This sign thus represents a simple and rapid screening tool for either the primary care physician or the dermatologist to help identify high-risk patients.

Background: The clinical variability of cutaneous sarcoidosis (CS) often makes its correct diagnosis challenging. Although traditionally employed for the diagnosis of skin tumors, during the past years dermoscopy also gained increasing interest as an aid in the clinical diagnosis of inflammatory and infectious skin manifestations in general dermatology. Objective: Our purpose was to evaluate the usefulness of dermoscopy in the differential diagnosis of CS. Methods: This was a retrospective analysis of 7 clinical and dermoscopic images of CS that were collected at dermatology clinics in France and Italy between 2005 and 2009. Results: Retrospective dermoscopic evaluation revealed small grouped, translucent orange globular structures associated with linear vessels of variable diameter in all 7 cases. In 5 cases, additional central scar-like areas were seen. Conclusion: Lesions showing dermoscopically translucent yellow to orange globular-like or structureless areas associated with linear vessels should raise the suspicion of a granulomatous skin disease, including CS.

Deviation from proper muscle development or homeostasis results in various myopathic conditions. Employing genetic as well as chemical intervention, we provide evidence that a tight regulation of Wnt/[beta]-catenin signaling is essential for muscle fiber growth and maintenance. In zebrafish embryos, gain-of-Wnt/[beta]-catenin function results in unscheduled muscle progenitor proliferation, leading to slow and fast muscle hypertrophy accompanied by fast muscle degeneration. The effects of Wnt/[beta]-catenin signaling on fast muscle hypertrophy were rescued by misexpression of Myostatin or p21.sup.CIP/WAF, establishing an in vivo regulation of myofibrillogenesis by Wnt/[beta]-catenin signaling and Myostatin. Epistatic analyses suggest a possible genetic interaction between Wnt/[beta]-catenin and Myostatin in regulation of slow and fast twitch muscle myofibrillogenesis.

The process of lateral inhibition in the canonical Delta-Notch pathway is crucial for cell-fate determination. d-Asb11, a regulator of neural progenitors in zebrafish, is an essential mediator of lateral inhibition through ubiquitylation of Delta A. In canonical Delta–Notch signalling, expression of Delta activates Notch in neighbouring cells, leading to downregulation of Delta in these cells 1 . This process of lateral inhibition results in selection of either Delta-signalling cells or Notch-signalling cells. Here we show that d-Asb11 is an important mediator of this lateral inhibition. In zebrafish embryos, morpholino oligonucleotide (MO)-mediated knockdown of d-Asb11 caused repression of specific Delta–Notch elements and their transcriptional targets, whereas these were induced when d-Asb11 was misexpressed. d-Asb11 also activated legitimate Notch reporters cell-non-autonomously in vitro and in vivo when co-expressed with a Notch reporter. However, it repressed Notch reporters when expressed in Delta-expressing cells. Consistent with these results, d-Asb11 was able to specifically ubiquitylate and degrade DeltaA both in vitro and in vivo . We conclude that d-Asb11 is a component in the regulation of Delta–Notch signalling, important in fine-tuning the lateral inhibition gradients between DeltaA and Notch through a cell non-autonomous mechanism.

Korišćenje dermoskopije povećava sposobnost kliničara za diferentovanje različitih tipova melanocitnih nevusa i povećava mogućnost otkrivanja melanoma u najranijem stadijumu. Različiti dermoskopski tipovi nevusa mogu se prepoznati na osnovu strukturnih karakteristika, odnosno na osnovu količine i distribucije pigmenta. Većina osoba sa multiplim nevusima ima predominatni nevusni tip. Moguća povezanost predominatnog melanocitnog nevusa sa uzrastom i/ili fototipom kože omogućila bi lakše prepoznavanje dermoskopskih obrazaca nevusa koji su nuobičajeni za uzrast i/ili fototip kože pacijenta, što bi omogućilo prepoznavanje ranih melanoma. Cilj istraživanja bio je utvrditi korelaciju dermoskopskih obrazaca, pigmentne distribucije i boje melanocitnih nevusa sa starošću i fototipom kože ispitanika. U istraživanje je uključeno 400 pacijenta Klinike za kožne i polne bolesti u Nišu, u periodu 2008 god. do 2012 god. Za svaki nevus određivan je strukturni obrazac, distribucija pigmenta i boja. Povezanost dermoskopskih karakteristika analizirana je u funkciji starosti i fototipa kože ispitanika. Pregledano je ukupno 6 680 stečenih melanocitnih nevusa. Globularni obrazac je bio najpredominatniji u najmlađoj starosnoj grupi, dok retikularni obrazac pokazuje značano povećanu zastupljenost među starijom populacijom.Centralna hiper/hipopigmentacija pokazale su značajne razlike u zastupljenosti u zavisnosti od fototipa kože. Predominatnost dermoskopskih obrazaca melanocitnih nevusa varira od starosti ispitanika, dok boja nevusa i pigmentna distribucija zavise od fototipa kože.