SUMMARY Amplification of the MYCN oncogene in childhood neuroblastoma is often accompanied by mutational activation of ALK (anaplastic lymphoma kinase), suggesting their pathogenic cooperation. We generated a transgenic zebrafish model of neuroblastoma in which MYCN-induced tumors arise from a subpopulation of neuroblasts that migrate into the adrenal medulla analogue following organogenesis. Coexpression of activated ALK with MYCN in this model triples the disease penetrance and markedly accelerates tumor onset. MYCN overexpression induces adrenal sympathetic neuroblast hyperplasia, blocks chromaffin cell differentiation, and ultimately triggers a developmentally-timed apoptotic response in the hyperplastic sympathoadrenal cells. Coexpression of activated ALK with MYCN provides prosurvival signals that block this apoptotic response and allow continued expansion and oncogenic transformation of hyperplastic neuroblasts, thus promoting progression to neuroblastoma.

VHL, NF-1, c-Ret, and Succinate Dehydrogenase Subunits B and D act on a developmental apoptotic pathway that is activated when nerve growth factor (NGF) becomes limiting for neuronal progenitor cells and requires the EglN3 prolyl hydroxylase as a downstream effector. Germline mutations of these genes cause familial pheochromocytoma and other neural crest-derived tumors. Using an unbiased shRNA screen we found that the kinesin KIF1B␤ acts downstream from EglN3 and is both necessary and sufficient for neuronal apoptosis when NGF becomes limiting. KIF1B␤ maps to chromosome 1p36.2, which is frequently deleted in neural crest-derived tumors including neuroblastomas. We identified inherited loss-of-function KIF1B␤ missense mutations in neuroblastomas and pheochromocytomas and an acquired loss-of-function mutation in a medulloblastoma, arguing that KIF1B␤ is a pathogenic target of these deletions.[Keywords: Apoptosis; kinesin; neuroblastoma; pheochromocytoma; prolyl hydroxylase] Supplemental material is available at http://www.genesdev.org.

The vertebrate neural crest is a pluripotent cell population that generates a large variety of cell types, including peripheral neurons, cartilage and pigment cells. Mechanisms that control the patterning of the neural crest toward specific cell fates remain only partially understood. Zebrafish homozygous for the sympathetic mutation 1 (sym1) have defects in a subset of neural crest derivatives, such as peripheral neurons, glia and cartilage, but retain normal numbers of melanocytes. The sym1 mutation is a nucleotide deletion that disrupts the forkhead DNA-binding domain of the foxd3 gene, which encodes a conserved winged-helix transcription factor. We show that sym1 mutants have normal numbers of premigratory neural crest cells, but these cells express reduced levels of snai1b and sox10, implicating foxd3 as an essential regulator of these transcription factors in the premigratory neural crest. The onset of neural crest migration is also delayed in sym1 mutants, and there is a reduction in the number of migratory trunk neural crest cells, particularly along the medial migration pathway. TUNEL analysis revealed aberrant apoptosis localized to the hindbrain neural crest at the 15-somite stage, indicating a critical role for foxd3 in the survival of a subpopulation of neural crest cells. These results show that foxd3 selectively specifies premigratory neural crest cells for a neuronal, glial or cartilage fate, by inducing the expression of lineage-associated transcription factors in these cells and regulating their subsequent migration.

We have previously used mosaic flies to screen for tumour suppressors or negative regulators of cell proliferation. The cellular composition of these flies resembles that of cancer patients who are chimaeric individuals carrying a small number of mutated somatic cells. One of the genes we identified is the large tumour suppressor gene, lats (also known as wts), which encodes a putative serine/threonine kinase. Somatic cells mutant for lats undergo extensive proliferation and form large tumours in many tissues in mosaic adults. Homozygous mutants for various lats alleles display a range of developmental defects including embryonic lethality. Although many tumour suppressors have been identified in Drosophila melanogaster, it is not clear whether these fly genes are directly relevant to tumorigenesis in mammals. Here, we have isolated mammalian homologues of Drosophila lats. Human LATS1 suppresses tumour growth and rescues all developmental defects, including embryonic lethality in flies. In mammalian cells, LATS1 is phosphorylated in a cell-cycle-dependent manner and complexes with CDC2 in early mitosis. LATS1-associated CDC2 has no mitotic cyclin partner and no kinase activity for histone H1. Furthermore, lats mutant cells in Drosophila abnormally accumulate cyclin A. These biochemical observations indicate that LATS is a novel negative regulator of CDC2/cyclin A, a finding supported by genetic data in Drosophila demonstrating that lats specifically interacts with cdc2 and cyclin A.

LATS (large tumour suppressor) is a family of conserved tumour suppressors identified in Drosophila and mammals. Here we show that human LATS1 binds to LIMK1 in vitro and in vivo and colocalizes with LIMK1 at the actomyosin contractile ring during cytokinesis. LATS1 inhibits both the phosphorylation of cofilin by LIMK1 and LIMK1-induced cytokinesis defects. Inactivation of LATS1 by antibody microinjection or RNA-mediated interference in cells, or gene knockout in mice, abrogates cytokinesis and increases the percentage of multinucleate cells. Our findings indicate that LATS1 is a novel cytoskeleton regulator that affects cytokinesis by regulating actin polymerization through negative modulation of LIMK1.

The zebrafish mutation mother superior (mos m188 ) leads to a depletion of neural crest (NC) derivatives including the craniofacial cartilage skeleton, the peripheral nervous system (sympathetic neurons, dorsal root ganglia, enteric neurons), and pigment cells. The loss of derivatives is preceded by a reduction in NC-expressed transcription factors, snail1b, sox9b, sox10, and a specific loss of foxd3 expression in NC progenitor cells. We employed genetic linkage analysis and physical mapping to place the mos m188 mutation on zebrafish chromosome 6 in the vicinity of the foxd3 gene. Furthermore, we found that mos m188 does not complement the sym1/foxd3 mutation, indicating that mos m188 resides within the foxd3 locus. Injection of PAC clones containing the foxd3 gene into mos m188 embryos restored foxd3 expression in NC progenitors and suppressed the mos m188 phenotype. However, sequencing the foxd3 transcribed area in mos m188 embryos did not reveal nucleotide changes segregating with the mos m188 phenotype, implying that the mutation most likely resides outside the foxd3-coding region. Based on these findings, we propose that the mos m188 mutation perturbs a NC-specific foxd3 regulatory element. Further analysis of mos m188 mutants and foxd3 morphants revealed that NC cells are initially formed, suggesting that foxd3 function is required to maintain the pool of NC progenitors. Developmental Dynamics 235:3199 -3212, 2006.

SUMMARY The tyrosine phosphatase SHP2 (PTPN11) regulates cellular proliferation, survival, migration and differentiation during development. Germline mutations in PTPN11 cause Noonan and LEOPARD syndromes, which have overlapping clinical features. Paradoxically, Noonan syndrome mutations increase SHP2 phosphatase activity, while LEOPARD syndrome mutants are catalytically impaired, raising the possibility that SHP2 has phosphatase-independent roles. By comparing shp2-deficient zebrafish embryos with those injected with mRNA encoding LEOPARD syndrome point mutations, we identify a phosphatase- and Erk-dependent role for Shp2 in neural crest specification and migration. We also identify an unexpected phosphatase-and Erk-independent function, mediated through its SH2 domains, which is evolutionarily conserved and prevents p53-mediated apoptosis in the brain and neural crest. Our results indicate that previously enigmatic aspects of LEOPARD syndrome pathogenesis can be explained by the combined effects of loss of Shp2 catalytic function and retention of an SH2 domain-mediated role that is essential for neural crest cell survival.

The zebrafish (Danio rerio) has proven to be a powerful vertebrate model system for the genetic analysis of developmental pathways and is only beginning to be exploited as a model for human disease and clinical research. The attributes that have led to the emergence of the zebrafish as a preeminent embryological model, including its capacity for forward and reverse genetic analyses, provides a unique opportunity to uncover novel insights into the molecular genetics of cancer. Some of the advantages of the zebrafish animal model system include fecundity, with each female capable of laying 200-300 eggs per week, external fertilization that permits manipulation of embryos ex utero, and rapid development of optically clear embryos, which allows the direct observation of developing internal organs and tissues in vivo. The zebrafish is amenable to transgenic and both forward and reverse genetic strategies that can be used to identify or generate zebrafish models of different types of cancer and may also present significant advantages for the discovery of tumor suppressor genes that promote tumorigenesis when mutationally inactivated. Importantly, the transparency and accessibility of the zebrafish embryo allows the unprecedented direct analysis of pathologic processes in vivo, including neoplastic cell transformation and tumorigenic progression. Ultimately, high-throughput modifier screens based on zebrafish cancer models can lead to the identification of chemicals or genes involved in the suppression or prevention of the malignant phenotype. The identification of small molecules or gene products through such screens will serve as ideal entry points for novel drug development for the treatment of cancer. This review focuses on the current technology that takes advantage of the zebrafish model system to further our understanding of the genetic basis of cancer and its treatment.