Author Correction: Gap junction protein Connexin-43 is a direct transcriptional regulator of N-cadherin in vivo

Correction to: Nature Communications (2018); https://doi.org/10.1038/s41467-018-06368-x, published online 21 September 2018. The original version of this Article contained an error in the spelling of the author Alexandra Schambony, which was incorrectly given as Alexandra Schambon. This has now been corrected in both the PDF and HTML versions of the Article

The deleted in brachydactyly B domain of ROR2 is required for receptor activation by recruitment of Src

The transmembrane receptor 'ROR2' resembles members of the receptor tyrosine kinase family of signalling receptors in sequence but its' signal transduction mechanisms remain enigmatic. This problem has particular importance because mutations in ROR2 are associated with two human skeletal dysmorphology syndromes, recessive Robinow Syndrome (RS) and dominant acting Brachydactyly type B (BDB). Here we show, using a constitutive dimerisation approach, that ROR2 exhibits dimerisation-induced tyrosine kinase activity and the ROR2 C-terminal domain, which is deleted in BDB, is required for recruitment and activation of the non-receptor tyrosine kinase Src. Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity. Eight sites of Src-mediated ROR2 phosphorylation have been identified by mass spectrometry. Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes. These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src

Live Imaging of Xwnt5A-ROR2 Complexes

Secreted molecules of the Wnt family regulate key decisions in embryogenesis and adult tissue homeostasis by activating a complex network of Wnt signaling pathways. Although the different branches of Wnt signaling have been studied for more than 25 years, fluorophore tagged constructs for live cell imaging of Wnt molecules activating the Wnt/β-catenin pathway have become available only recently. We have generated a fluorophore tagged Wnt construct of the Xenopus Wnt5a protein (Xwnt5A) with the enhanced green fluorescent protein (EGFP), Xwnt5A-EGFP. This construct activates non-canonical Wnt pathways in an endocytosis dependent manner and is capable of compensating for the loss of endogenous Xwnt5A in Xenopus embryos. Strikingly, non-canonical Wnt pathway activation was restricted to short-range signaling while an inhibitory effect was observed in transwell cell cultures taken as long-range signaling model sytem. We used our Xwnt5A-EGFP construct to analyze in vivo binding of Wnt5A to its co-receptor ROR2 on the microscopic and on the molecular level. On the microscopic level, Xwnt5A-EGFP clusters in the membrane and recruits ROR2-mCherry to these clusters. Applying dual-colour dual-focus line-scanning fluorescence correlation spectroscopy on dorsal marginal zone explants, we identified membrane tethered Xwnt5A-EGFP molecules binding to ROR2-mCherry molecules. Our data favour a model, in which membrane-tethered Wnt-5A recruits ROR2 to form large ligand/receptor clusters and signals in an endocytosis-dependent manner

Wnt5b–Ryk pathway provides directional signals to regulate gastrulation movement

The soluble ligand Wnt5b repels cells expressing the Ryk (related to tyrosine kinase) receptor, establishing directional motility during gastrulation

Coordination of Cell Polarity during Xenopus Gastrulation

Cell polarity is an essential feature of animal cells contributing to morphogenesis. During Xenopus gastrulation, it is known that chordamesoderm cells are polarized and intercalate each other allowing anterior-posterior elongation of the embryo proper by convergent extension (CE). Although it is well known that the cellular protrusions at both ends of polarized cells exert tractive force for intercalation and that PCP pathway is known to be essential for the cell polarity, little is known about what triggers the cell polarization and what the polarization causes to control intracellular events enabling the intercalation that leads to the CE. In our research, we used EB3 (end-binding 3), a member of +TIPs that bind to the plus end of microtubule (MT), to visualize the intracellular polarity of chordamesoderm cells during CE to investigate the trigger of the establishment of cell polarity. We found that EB3 movement is polarized in chordamesoderm cells and that the notochord-somite tissue boundary plays an essential role in generating the cell polarity. This polarity was generated before the change of cell morphology and the polarized movement of EB3 in chordamesoderm cells was also observed near the boundary between the chordamesoderm tissue and naïve ectoderm tissue or lateral mesoderm tissues induced by a low concentration of nodal mRNA. These suggest that definitive tissue separation established by the distinct levels of nodal signaling is essential for the chordamesodermal cells to acquire mediolateral cell polarity

Sfrp Controls Apicobasal Polarity and Oriented Cell Division in Developing Gut Epithelium

Epithelial tubular morphogenesis leading to alteration of organ shape has important physiological consequences. However, little is known regarding the mechanisms that govern epithelial tube morphogenesis. Here, we show that inactivation of Sfrp1 and Sfrp2 leads to reduction in fore-stomach length in mouse embryos, which is enhanced in the presence of the Sfrp5 mutation. In the mono-cell layer of fore-stomach epithelium, cell division is normally oriented along the cephalocaudal axis; in contrast, orientation diverges in the Sfrps-deficient fore-stomach. Cell growth and apoptosis are not affected in the Sfrps-deficient fore-stomach epithelium. Similarly, cell division orientation in fore-stomach epithelium diverges as a result of inactivation of either Stbm/Vangl2, an Fz/PCP component, or Wnt5a. These observations indicate that the oriented cell division, which is controlled by the Fz/PCP pathway, is one of essential components in fore-stomach morphogenesis. Additionally, the small intestine epithelium of Sfrps compound mutants fails to maintain proper apicobasal polarity; the defect was also observed in Wnt5a-inactivated small intestine. In relation to these findings, Sfrp1 physically interacts with Wnt5a and inhibits Wnt5a signaling. We propose that Sfrp regulation of Wnt5a signaling controls oriented cell division and apicobasal polarity in the epithelium of developing gut

Ror2 Enhances Polarity and Directional Migration of Primordial Germ Cells

The trafficking of primordial germ cells (PGCs) across multiple embryonic structures to the nascent gonads ensures the transmission of genetic information to the next generation through the gametes, yet our understanding of the mechanisms underlying PGC migration remains incomplete. Here we identify a role for the receptor tyrosine kinase-like protein Ror2 in PGC development. In a Ror2 mouse mutant we isolated in a genetic screen, PGC migration and survival are dysregulated, resulting in a diminished number of PGCs in the embryonic gonad. A similar phenotype in Wnt5a mutants suggests that Wnt5a acts as a ligand to Ror2 in PGCs, although we do not find evidence that WNT5A functions as a PGC chemoattractant. We show that cultured PGCs undergo polarization, elongation, and reorientation in response to the chemotactic factor SCF (secreted KitL), whereas Ror2 PGCs are deficient in these SCF-induced responses. In the embryo, migratory PGCs exhibit a similar elongated geometry, whereas their counterparts in Ror2 mutants are round. The protein distribution of ROR2 within PGCs is asymmetric, both in vitro and in vivo; however, this asymmetry is lost in Ror2 mutants. Together these results indicate that Ror2 acts autonomously to permit the polarized response of PGCs to KitL. We propose a model by which Wnt5a potentiates PGC chemotaxis toward secreted KitL by redistribution of Ror2 within the cell

Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein

Citation: Londono-Renteria, B., Troupin, A., Conway, M. J., Vesely, D., Ledizet, M., Roundy, C. M., . . . Colpitts, T. M. (2015). Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein. Plos Pathogens, 11(10), 23. doi:10.1371/journal.ppat.1005202Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were >= 5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses

Novel markers for differentiation of lobular and ductal invasive breast carcinomas by laser microdissection and microarray analysis

BACKGROUND: Invasive ductal and lobular carcinomas (IDC and ILC) are the most common histological types of breast cancer. Clinical follow-up data and metastatic patterns suggest that the development and progression of these tumors are different. The aim of our study was to identify gene expression profiles of IDC and ILC in relation to normal breast epithelial cells. METHODS: We examined 30 samples (normal ductal and lobular cells from 10 patients, IDC cells from 5 patients, ILC cells from 5 patients) microdissected from cryosections of ten mastectomy specimens from postmenopausal patients. Fifty nanograms of total RNA were amplified and labeled by PCR and in vitro transcription. Samples were analysed upon Affymetrix U133 Plus 2.0 Arrays. The expression of seven differentially expressed genes (CDH1, EMP1, DDR1, DVL1, KRT5, KRT6, KRT17) was verified by immunohistochemistry on tissue microarrays. Expression of ASPN mRNA was validated by in situ hybridization on frozen sections, and CTHRC1, ASPN and COL3A1 were tested by PCR. RESULTS: Using GCOS pairwise comparison algorithm and rank products we have identified 84 named genes common to ILC versus normal cell types, 74 named genes common to IDC versus normal cell types, 78 named genes differentially expressed between normal ductal and lobular cells, and 28 named genes between IDC and ILC. Genes distinguishing between IDC and ILC are involved in epithelial-mesenchymal transition, TGF-beta and Wnt signaling. These changes were present in both tumor types but appeared to be more prominent in ILC. Immunohistochemistry for several novel markers (EMP1, DVL1, DDR1) distinguished large sets of IDC from ILC. CONCLUSION: IDC and ILC can be differentiated both at the gene and protein levels. In this study we report two candidate genes, asporin (ASPN) and collagen triple helix repeat containing 1 (CTHRC1) which might be significant in breast carcinogenesis. Besides E-cadherin, the proteins validated on tissue microarrays (EMP1, DVL1, DDR1) may represent novel immunohistochemical markers helpful in distinguishing between IDC and ILC. Further studies with larger sets of patients are needed to verify the gene expression profiles of various histological types of breast cancer in order to determine molecular subclassifications, prognosis and the optimum treatment strategies