Highlights d prkdc À/À , il2rga À/À zebrafish reared at 37 C engraft a wide array of human cancers d Growth and therapy responses can be dynamically visualized at single-cell resolution d Combination olaparib and temozolomide kill xenografted human rhabdomyosarcoma d Engrafting patient-derived cancers opens new avenues for personalized therapy Authors

MacLeod RJ, Hayes M, Pacheco I. Wnt5a secretion stimulated by the extracellular calcium-sensing receptor inhibits defective

Using zebrafish, we have characterised the function of Protein tyrosine kinase 7 (Ptk7), a transmembrane pseudokinase implicated in Wnt signal transduction during embryonic development and in cancer. Ptk7 is a known regulator of mammalian neural tube closure and Xenopus convergent extension movement. However, conflicting reports have indicated both positive and negative roles for Ptk7 in canonical Wnt/β-catenin signalling. To clarify the function of Ptk7 in vertebrate embryonic patterning and morphogenesis, we generated maternal-zygotic (MZ) ptk7 mutant zebrafish using a zinc-finger nuclease (ZFN) gene targeting approach. Early loss of zebrafish Ptk7 leads to defects in axial convergence and extension, neural tube morphogenesis and loss of planar cell polarity (PCP). Furthermore, during late gastrula and segmentation stages, we observe significant upregulation of β-catenin target gene expression and demonstrate a clear role for Ptk7 in attenuating canonical Wnt/β-catenin activity in vivo. MZptk7 mutants display expanded differentiation of paraxial mesoderm within the tailbud, suggesting an important role for Ptk7 in regulating canonical Wnt-dependent fate specification within posterior stem cell pools post-gastrulation. Furthermore, we demonstrate that a plasma membrane-tethered Ptk7 extracellular fragment is sufficient to rescue both PCP morphogenesis and Wnt/β-catenin patterning defects in MZptk7 mutant embryos. Our results indicate that the extracellular domain of Ptk7 acts as an important regulator of both non-canonical Wnt/PCP and canonical Wnt/β-catenin signalling in multiple vertebrate developmental contexts, with important implications for the upregulated PTK7 expression observed in human cancers.

Scoliosis is a complex genetic disorder of the musculoskeletal system, characterized by three-dimensional rotation of the spine. Curvatures caused by malformed vertebrae (congenital scoliosis/CS) are apparent at birth. Spinal curvatures with no underlying vertebral abnormality (idiopathic scoliosis/IS) most commonly manifest during adolescence. The genetic and biological mechanisms responsible for IS remain poorly understood due largely to limited experimental models. Here we describe zygotic ptk7 (Zptk7) mutant zebrafish, deficient in a critical regulator of Wnt signaling, as the first genetically defined developmental model of IS. We identify a novel sequence variant within a single IS patient that disrupts PTK7 function, consistent with a role for dysregulated Wnt activity in disease pathogenesis. Furthermore, we demonstrate that embryonic loss of gene function in maternal-zygotic ptk7 mutants (MZptk7) leads to vertebral anomalies associated with congenital scoliosis. Our data suggest novel molecular origins of, and genetic links between, congenital and idiopathic forms of disease.

Cancers contain a wide diversity of cell types that are defined by differentiation states, genetic mutations and altered epigenetic programmes that impart functional diversity to individual cells. Elevated tumour cell heterogeneity is linked with progression, therapy resistance and relapse. Yet, imaging of tumour cell heterogeneity and the hallmarks of cancer has been a technical and biological challenge. Here we develop optically clear immune-compromised rag2E450fs (casper) zebrafish for optimized cell transplantation and direct visualization of fluorescently labelled cancer cells at single-cell resolution. Tumour engraftment permits dynamic imaging of neovascularization, niche partitioning of tumour-propagating cells in embryonal rhabdomyosarcoma, emergence of clonal dominance in T-cell acute lymphoblastic leukaemia and tumour evolution resulting in elevated growth and metastasis in BRAFV600E-driven melanoma. Cell transplantation approaches using optically clear immune-compromised zebrafish provide unique opportunities to uncover biology underlying cancer and to dynamically visualize cancer processes at single-cell resolution in vivo.

Axonemes are microtubule-based organelles of crucial importance in the structure and function of eukaryotic cilia and flagella. Despite great progress in understanding how axonemes are assembled, the signals that initiate axoneme outgrowth remain unknown. Here, we identified phosphatidylinositol phosphates (phosphoinositides) as key regulators of early stages of axoneme outgrowth in Drosophila melanogaster spermatogenesis. In a study of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] function in developing Drosophila male germ cells, we depleted PtdIns(4,5)P2 by expression of a potent phosphoinositide phosphatase. Phosphatase expression dramatically inhibited sperm tail formation and perturbed microtubule organization in a manner reversible by co-expression of a PtdIns 4-phosphate 5-kinase. Depletion of PtdIns(4,5)P2 caused increased levels of basal body γ-tubulin and altered the distribution of proteins known to be required for axoneme assembly. Examination of PtdIns(4,5)P2-depleted spermatids by transmission electron microscopy revealed defects in basal body docking to the nuclear envelope, and in axoneme architecture and integrity of the developing flagellar axoneme and axial sheath. Our results provide the first evidence that phosphoinositides act at several steps during flagellar biogenesis, coordinately regulating microtubule and membrane organization. They further suggest that phosphoinositides play evolutionarily conserved roles in flagella and cilia, across phyla and in structurally diverse cell types.

Overall survival rates for pediatric patients with high-risk or relapsed rhabdomyosarcoma (RMS) have not improved significantly since the 1980s. Recent studies have identified a number of targetable vulnerabilities in RMS, but these discoveries have infrequently translated into clinical trials. We propose streamlining the process by which agents are selected for clinical evaluation in RMS. We believe that strong consideration should be given to the development of combination therapies that add biologically targeted agents to conventional cytotoxic drugs. One example of this type of combination is the addition of the WEE1 inhibitor AZD1775 to the conventional cytotoxic chemotherapeutics, vincristine and irinotecan. K E Y W O R D Scancer biology, early-phase clinical trials, genomics, rhabdomyosarcoma

Summary Tumor-propagating cells (TPCs) share self-renewal properties with normal stem cells and drive continued tumor growth. However, mechanisms regulating TPC self-renewal are largely unknown, especially in embryonal rhabdomyosarcoma (ERMS)—a common pediatric cancer of muscle. Here, we used a zebrafish transgenic model of ERMS to identify a role for intracellular NOTCH1 (ICN1) in increasing TPCs by 23-fold. ICN1 expanded TPCs by enabling the de-differentiation of zebrafish ERMS cells into self-renewing myf5+ TPCs, breaking the rigid differentiation hierarchies reported in normal muscle. ICN1 also had conserved roles in regulating human ERMS self-renewal and growth. Mechanistically, ICN1 up-regulated expression of SNAIL1, a transcriptional repressor, to increase TPC number in human ERMS and to block muscle differentiation through suppressing MEF2C, a myogenic differentiation transcription factor. Our data implicate the NOTCH1/SNAI1/MEF2C signaling axis as a major determinant of TPC self-renewal and differentiation in ERMS, raising hope of therapeutically targeting this pathway in the future.