SUMMARYDuring development, morphogenetic processes require a precise coordination of cell differentiation, cell shape changes and, often, cell migration. Yet, how pattern information is used to orchestrate these different processes is still unclear. During lateral line (LL) morphogenesis, a group of cells simultaneously migrate and assemble radially organized cell clusters, termed rosettes, that prefigure LL sensory organs. This process is controlled by Fibroblast growth factor (FGF) signalling, which induces cell fate changes, cell migration and cell shape changes. However, the exact molecular mechanisms induced by FGF activation that mediate these changes on a cellular level are not known. Here, we focus on the mechanisms by which FGFs control apical constriction and rosette assembly. We show that apical constriction in the LL primordium requires the activity of non-muscle myosin. We demonstrate further that shroom3, a well-known regulator of non-muscle myosin activity, is expressed in the LL primordium and that its expression requires FGF signalling. Using gain-and loss-of-function experiments, we demonstrate that Shroom3 is the main organizer of cell shape changes during rosette assembly, probably by coordinating Rho kinase recruitment and non-muscle myosin activation. In order to quantify morphogenesis in the LL primordium in an unbiased manner, we developed a unique trainable 'rosette detector'. We thus propose a model in which Shroom3 drives rosette assembly in the LL downstream of FGF in a Rho kinase-and non-muscle myosindependent manner. In conclusion, we uncovered the first mechanistic link between patterning and morphogenesis during LL sensory organ formation.

Smad-interacting protein 1 (SIP1, also known as ZEB2) represses the transcription of E-cadherin and mediates epithelial-mesenchymal transition in development and tumor metastasis. Due to the lack of human SIP1-specific antibodies, its expression in human tumor tissues has not been studied in detail by immunohistochemistry. Hence, we generated two anti-SIP1 monoclonal antibodies, clones 1C6 and 6E5, with IgG1 and IgG2a isotypes, respectively. The specificity of these antibodies was shown by Western blotting studies using siRNA mediated downregulation of SIP1 and ZEB1 in a human osteosarcoma cell line. In the same context, we also compared them with 5 commercially available SIP1 antibodies. Antibody specificity was further verified in an inducible cell line system by immunofluorescence. By using both antibodies, we evaluated the tissue expression of SIP1 in paraffin-embedded tissue microarrays consisting of 22 normal and 101 tumoral tissues of kidney, colon, stomach, lung, esophagus, uterus, rectum, breast and liver. Interestingly, SIP1 predominantly displayed a cytoplasmic expression, while the nuclear localization of SIP1 was observed in only 6 cases. Strong expression of SIP1 was found in distal tubules of kidney, glandular epithelial cells of stomach and hepatocytes, implicating a co-expression of SIP1 and E-cadherin. Squamous epithelium of the esophagus and surface epithelium of colon and rectum were stained with moderate to weak intensity. Normal uterus, breast and lung tissues remained completely negative. By comparison with their normal tissues, we observed SIP1 overexpression in cancers of the kidney, breast, lung and uterus. However, SIP1 expression was found to be downregulated in tumors from colon, rectum, esophagus, liver and stomach tissues. Finally we did nuclear/cytoplasmic fractionation in 3 carcinoma cell lines and detected SIP1 in both fractions, nucleus being the dominant one. To our best knowledge, this is the first comprehensive immunohistochemical study of the expression of SIP1 in a series of human cancers. Our finding that SIP1 is not exclusively localized to nucleus suggests that the subcellular localization of SIP1 is regulated in normal and tumor tissues. These novel monoclonal antibodies may help elucidate the role of SIP1 in tumor development.

Background: SLIT-ROBO families of proteins mediate axon pathfinding and their expression is not solely confined to nervous system. Aberrant expression of SLIT-ROBO genes was repeatedly shown in a wide variety of cancers, yet data about their collective behavior in hepatocellular carcinoma (HCC) is missing. Hence, we quantified SLIT-ROBO transcripts in HCC cell lines, and in normal and tumor tissues from liver.

Acetylcholinesterase (AChE), an enzyme responsible for degradation of acetylcholine, has been identified as a prognostic marker in liver cancer. Although in vivo Ache tumorigenicity assays in mouse are present, no established liver cancer xenograft model in zebrafish using an ache mutant background exists. Herein, we developed an embryonic zebrafish xenograft model using epithelial (Hep3B) and mesenchymal (SKHep1) liver cancer cell lines in wild-type and achesb55 sibling mutant larvae after characterization of cholinesterase expression and activity in cell lines and zebrafish larvae. The comparison of fluorescent signal reflecting tumor size at 3-days post-injection (dpi) revealed an enhanced tumorigenic potential and a reduced migration capacity in cancer cells injected into homozygous achesb55 mutants when compared with the wild-type. Increased tumor load was confirmed using an ALU based tumor DNA quantification method modified for use in genotyped xenotransplanted zebrafish embryos. Confocal microscopy using the Huh7 cells stably expressing GFP helped identify the distribution of tumor cells in larvae. Our results imply that acetylcholine accumulation in the microenvironment directly or indirectly supports tumor growth in liver cancer. Use of this model system for drug screening studies holds potential in discovering new cholinergic targets for treatment of liver cancers.

Rapidly accumulating literature has proven feasibility of the zebrafish xenograft models in cancer research. Nevertheless, online databases for searching the current zebrafish xenograft literature are in great demand. Herein, we have developed a manually curated database, called ZenoFishDb v1.1 (https://konulab.shinyapps.io/ zenofishdb), based on R Shiny platform aiming to provide searchable information on ever increasing collection of zebrafish studies for cancer cell line transplantation and patient-derived xenografts (PDXs). ZenoFishDb v1.1 user interface contains four modules: DataTable, Visualization, PDX Details, and PDX Charts. The DataTable and Visualization pages represent xenograft study details, including injected cell lines, PDX injections, molecular modifications of cell lines, zebrafish strains, as well as technical aspects of the xenotransplantation procedures in table, bar, and/or pie chart formats. The PDX Details module provides comprehensive information on the patient details in table format and can be searched and visualized. Overall, ZenoFishDb v1.1 enables researchers to effectively search, list, and visualize different technical and biological attributes of zebrafish xenotransplantation studies particularly focusing on the new trends that make use of reporters, RNA interference, overexpression, or mutant gene constructs of transplanted cancer cells, stem cells, and PDXs, as well as distinguished host modifications.

Early and differential diagnosis of hepatocellular carcinoma (HCC) requires sensitive and specific tissue and serum markers. On the other hand, proteins involved in tumorigenesis are extensively modulated on exposure to apoptotic stimuli, including ultraviolet (UVC) irradiation. Hence, we generated monoclonal antibodies by using UVC-irradiated apoptotic cells of an HCC cell line, HUH7, aiming to explore proteins differentially expressed in tumors and apoptosis. We obtained 18 hybridoma clones recognizing protein targets in apoptotic HUH7 cells, and clone 6D5 was chosen for characterization studies because of its strong reactivity in cell-ELISA assay. Subtype of the antibody was IgG3 (kappa). Targets of 6D5 antibody were found to be abundantly expressed in all HCC cell lines except FLC4, which resembles normal hepatocytes. We also observed the secretion of 6D5 ligands by some of the HCC cell lines. Moreover, cellular proteins recognized by the antibody displayed a late upregulation in UVC-induced apoptotic cells. We concluded that 6D5 target proteins are modulated in liver tumorigenesis and apoptotic processes. We therefore propose the validation of our antibody in tissue and serum samples of HCC patients to assess its potential use for the early diagnosis of HCC and to understand the role of 6D5 ligands in liver carcinogenesis.