Myosin 18B is an unconventional myosin that has been implicated in tumor progression in humans. In addition, loss-of-function mutations of the MYO18B gene have recently been identified in several patients exhibiting symptoms of nemaline myopathy. In mouse, mutation of Myo18B results in early developmental arrest associated with cardiomyopathy, precluding analysis of its effects on skeletal muscle development. The zebrafish, frozen (fro) mutant was identified as one of a group of immotile mutants in the 1996 Tübingen genetic screen. Mutant embryos display a loss of birefringency in their skeletal muscle, indicative of disrupted sarcomeric organization. Using meiotic mapping, we localized the fro locus to the previously unannotated zebrafish myo18b gene, the product of which shares close to 50% identity with its human ortholog. Transcription of myo18b is restricted to fast-twitch myocytes in the zebrafish embryo; consistent with this, fro mutant embryos exhibit defects specifically in their fast-twitch skeletal muscles. We show that sarcomeric assembly is blocked at an early stage in fro mutants, leading to the disorganized accumulation of actin, myosin, and α-actinin and a complete loss of myofibrillar organization in fast-twitch muscles.

This thesis explores the zebrafish as a model organism to illuminate our understanding of processes that orchestrate the progression of cancer metastasis and myogenesis. First, I describe the successful establishment of a cancer metastasis model in the zebrafish, specifically to study intravasation one of the earliest steps in the metastatic cascade. Fluorescently labeled tumour cell are transplanted into pervitelline space of 2 days post fertilisation (dpf) zebrafish embryos before they are exposed to normoxic or hypoxic conditions, allowing us to study the effect of hypoxia on tumour-induced angiogenesis and metastasis. Hypoxia elicited an enhanced angiogenic response and neovascularisation to the transplanted tumour and escalated the extent of metastasis in the living zebrafish embryo. Loss of function experiments such as vascular endothelial growth factor (VEGF) blockade using a clinically available drug – Sunitinib or VEGF morpholino knockdown attenuated tumour-induced angiogenesis and metastasis. The in vivo metastasis assay in zebrafish delivers numerous unique advantages over conventional in vitro cell-based or biochemical chemotaxis assays. The transparent embryo facilitates the tracking of the entire intravasation process in real time within a living organism, allowing us to explore the dynamic interplay of tumour cells and the environmental cues that drive metastasis. This model can be further employed to discriminate between tumour cells of different metastatic potential and to identify novel factors that present as impetus for the earliest step of the metastatic cascade. Next, I demonstrate the analysis of one class of motility mutants originally identified in the 1996 Tubingen genetic screen that potentially serve as models for human myopathies and dystrophies. Two of these mutants have previously been cloned and shown to encode proteins involved in muscle fibre attachment whilst a third has been found to encode the molecular chaperone Heat shock protein (HSP) 90. I describe the phenotypic and molecular characterization of another of these zebrafish motility mutants, frozen (fro). I present evidence that the fro mutation disrupts the locus encoding the autophagy pathway component Atg10. This analysis implicates Atg10 in the assembly of both skeletal and cardiac muscle fibers suggesting a previously uncharacterized role for the autophagy pathway in this process. Together, my findings illustrate the utility of the zebrafish as a model organism that complements established mammalian and invertebrate models. The fecundity and amenability of the zebrafish to genetic manipulation together with the rapid development and translucency of its embryos combine to provide a powerful system with which to unravel and inform the underlying mechanisms that govern fundamental biological processes and shed light on our understanding of human development and diseases. LIST OF PUBLICATIONS I. Samantha Lin Chiou Lee, Pegah Rouhi, Lasse Jensen, Danfang Zhang, Hong Ji, Giselbert Hauptmann, Philip Ingham and Yihai Cao, Nov 17 2009, Hypoxia-induced pathological angiogenesis mediates tumor cell dissemination, invasion, and metastasis in a zebrafish tumor model, Proc Natl Acad Sci (PNAS) USA,106, 46, 19485-90. II. Pegah Rouhi*, Samantha Lin Chiou Lee*, Ziquan Cao, Eva Hedlund, Lasse Jensen and Yihai Cao, Mar 1 2010, Pathological angiogenesis facilitates tumor cell dissemination and metastasis, Cell Cycle, 9, 5, 913-7, *Co-first author III. Samantha Lin Chiou Lee, Sarah Baxendale, Stephen Moore, Ng Chee Peng, Tan Swee Chuan and Philip Ingham, The Autophagy Pathway component, Atg10, is required for skeletal and cardiac muscle fibre assembly in zebrafish (manuscript).

Clinically detectable metastases represent an ultimate consequence of the metastatic cascade that consists of distinct processes including tumor cell invasion, dissemination, metastatic niche formation, and re-growth into a detectable metastatic mass. Although angiogenesis is known to promote tumor growth, its role in facilitating early events of the metastatic cascade remains poorly understood. We have recently developed a zebrafish tumor model that enables us to study involvement of pathological angiogenesis in tumor invasion, dissemination and metastasis. This non-invasive in vivo model allows detection of single malignant cell dissemination under both normoxia and hypoxia. Further, hypoxia-induced VEGF significantly facilitates tumor cell invasion and dissemination. These findings demonstrate that VEGF-induced pathological angiogenesis is essential for tumor dissemination and further corroborates potentially beneficial effects of clinically ongoing anti-VEGF drugs for the treatment of various malignancies.

Mechanisms underlying pathological angiogenesis in relation to hypoxia in tumor invasion and metastasis remain elusive. Here, we have developed a zebrafish tumor model that allows us to study the role of pathological angiogenesis under normoxia and hypoxia in arbitrating early events of the metastatic cascade at the single cell level. Under normoxia, implantation of a murine T241 fibrosarcoma into the perivitelline cavity of developing embryos of transgenic fli1:EGFP zebrafish did not result in significant dissemination, invasion, and metastasis. In marked contrast, under hypoxia substantial tumor cells disseminated from primary sites, invaded into neighboring tissues, and metastasized to distal parts of the fish body. Similarly, expression of the hypoxia-regulated angiogenic factor, vascular endothelial growth factor (VEGF) to a high level resulted in tumor cell dissemination and metastasis, which correlated with increased tumor neovascularization. Inhibition of VEGF receptor signaling pathways by sunitinib or VEGFR2 morpholinos virtually completely ablated VEGF-induced tumor cell dissemination and metastasis. To the best of our knowledge, hypoxia- and VEGF-induced pathological angiogenesis in promoting tumor dissemination, invasion, and metastasis has not been described perviously at the single cell level. Our findings also shed light on molecular mechanisms of beneficial effects of clinically available anti-VEGF drugs for cancer therapy.