Rodney A. Stewart

The kinesin KIF1Bβ acts downstream from EglN3 to induce apoptosis and is a potential 1p36 tumor suppressor

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 KIF1Bbeta acts downstream from EglN3 and is both necessary and sufficient for neuronal apoptosis when NGF becomes limiting. KIF1Bbeta maps to chromosome 1p36.2, which is frequently deleted in neural crest-derived tumors including neuroblastomas. We identified inherited loss-of-function KIF1Bbeta missense mutations in neuroblastomas and pheochromocytomas and an acquired loss-of-function mutation in a medulloblastoma, arguing that KIF1Bbeta is a pathogenic target of these deletions.

Making waves in cancer research: new models in the zebrafish

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.

Phosphatase-dependent and -independent functions of Shp2 in neural crest cells underlie LEOPARD syndrome pathogenesis.

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 mother superior mutation ablates foxd3 activity in neural crest progenitor cells and depletes neural crest derivatives in zebrafish.

The zebrafish mutation mother superior (mosm188) 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 mosm188 mutation on zebrafish chromosome 6 in the vicinity of the foxd3 gene. Furthermore, we found that mosm188 does not complement the sym1/foxd3 mutation, indicating that mosm188 resides within the foxd3 locus. Injection of PAC clones containing the foxd3 gene into mosm188 embryos restored foxd3 expression in NC progenitors and suppressed the mosm188 phenotype. However, sequencing the foxd3 transcribed area in mosm188 embryos did not reveal nucleotide changes segregating with the mosm188 phenotype, implying that the mutation most likely resides outside the foxd3‐coding region. Based on these findings, we propose that the mosm188 mutation perturbs a NC‐specific foxd3 regulatory element. Further analysis of mosm188 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.

A Src-Tks5 Pathway Is Required for Neural Crest Cell Migration during Embryonic Development

In the adult organism, cell migration is required for physiological processes such as angiogenesis and immune surveillance, as well as pathological events such as tumor metastasis. The adaptor protein and Src substrate Tks5 is necessary for cancer cell migration through extracellular matrix in vitro and tumorigenicity in vivo. However, a role for Tks5 during embryonic development, where cell migration is essential, has not been examined. We used morpholinos to reduce Tks5 expression in zebrafish embryos, and observed developmental defects, most prominently in neural crest-derived tissues such as craniofacial structures and pigmentation. The Tks5 morphant phenotype was rescued by expression of mammalian Tks5, but not by a variant of Tks5 in which the Src phosphorylation sites have been mutated. We further evaluated the role of Tks5 in neural crest cells and neural crest-derived tissues and found that loss of Tks5 impaired their ventral migration. Inhibition of Src family kinases also led to abnormal ventral patterning of neural crest cells and their derivatives. We confirmed that these effects were likely to be cell autonomous by shRNA-mediated knockdown of Tks5 in a murine neural crest stem cell line. Tks5 was required for neural crest cell migration in vitro, and both Src and Tks5 were required for the formation of actin-rich structures with similarity to podosomes. Additionally, we observed that neural crest cells formed Src-Tks5-dependent cell protrusions in 3-D culture conditions and in vivo. These results reveal an important and novel role for the Src-Tks5 pathway in neural crest cell migration during embryonic development. Furthermore, our data suggests that this pathway regulates neural crest cell migration through the generation of actin-rich pro-migratory structures, implying that similar mechanisms are used to control cell migration during embryogenesis and cancer metastasis.

Drosophila Src-family kinases function with Csk to regulate cell proliferation and apoptosis

Elevated Src protein levels and activity are associated with the development and progression of a variety of cancers. The consequences of deregulated Src activity have been studied extensively in cell culture; however, the effects of this deregulation in vivo, as well as the mechanisms of Src-induced tumorigenesis, remain poorly understood. In this study, the effect of expressing wild-type and constitutively active Drosophila Src-family kinases (SFKs) in the developing eye was examined. Overexpression of either wild-type Drosophila SFK (Src64 and Src42) is sufficient to induce ectopic proliferation in G1/G0-arrested, uncommitted cells in eye imaginal discs. In addition, both kinases trigger apoptosis in vivo, in a dosage-dependent manner. Constitutively active mutants are hypermorphic as they trigger proliferation and death more potently than their wild-type counterparts. Moreover, SFK-induced proliferation and apoptosis are largely independent events, as blocking ectopic proliferation does not block cell death. Further, DCsk (the Drosophila homolog of the C-terminal Src kinase) phosphorylates and interacts genetically with the wild-type SFKs, but not with the constitutively active mutants in which a conserved C-terminal tyrosine was mutated to phenylalanine, providing the first in vivo evidence that Csk regulates SFKs during development through phosphorylation of their C-terminal tyrosine.

A genetic screen for modifiers of the lats tumor suppressor gene identifies C-terminal Src kinase as a regulator of cell proliferation in Drosophila

Disrupting mechanisms that control cell proliferation, cell size and apoptosis can cause changes in animal and tissue size and contribute to diseases such as cancer. The LATS family of serine/threonine kinases control tissue size by regulating cell proliferation and function as tumor suppressor genes in both Drosophila and mammals. In order to understand the role of lats in size regulation, we performed a genetic modifier screen in Drosophila to identify components of the lats signaling pathway. Mutations in the Drosophila homolog of C-terminal Src kinase (dcsk) were identified as dominant modifiers of both lats gain-of-function and loss-of-function phenotypes. Homozygous dcsk mutants have enlarged tissue phenotypes similar to lats and FACS and immunohistochemistry analysis of these tissues revealed that dcsk also regulates cell proliferation during development. Animals having mutations in both dcsk and lats display cell overproliferation phenotypes more severe than either mutant alone, demonstrating these genes function together in vivo to regulate cell numbers. Furthermore, homozygous dcsk phenotypes can be partially suppressed by overexpression of lats, indicating that lats is a downstream mediator of dcsk function in vivo. Finally, we show that dCSK phosphorylates LATS in vitro at a conserved C-terminal tyrosine residue, which is critical for normal LATS function in vivo. Taken together, these results demonstrate a role for dCSK in regulating cell numbers during development by inhibiting cell proliferation and suggest that lats is one of the mediators of the dcsk phenotype.

BIM and other BCL-2 family proteins exhibit cross- species conservation of function between zebrafish and mammals

Here we investigate the function of zebrafish Bcl-2 family proteins and demonstrate important conservation of function across zebrafish and mammalian systems. We have isolated a zebrafish ortholog of mammalian BIM and show that it is the most toxic of the zebrafish BH3-only genes examined, sharing this characteristic with the mammalian BIM gene. The zebrafish bad gene shows a complete lack of embryonic lethality, but like mammalian BAD, its pro-apoptotic activity is regulated through phosphorylation of critical serines. We also found that the pattern of mitochondrial dysfunction observed by zebrafish BH3 domain peptides in a mammalian cytochrome c release assay recapitulates the pattern of embryonic lethality induced by the respective mRNA injections in vivo. In contrast to zebrafish Bim, Bid exhibited only weak binding to zebrafish Bcl-2 and moderate-to-weak overall lethality in zebrafish embryos and isolated mitochondria. Given that zebrafish Bcl-2 binds strongly to mammalian BID and BIM peptides and proteins, the protein identified as the zebrafish Bid ortholog has different properties than mammalian BID. Overall, our results demonstrate the high degree of functional conservation between zebrafish and mammalian Bcl-2 family proteins, thus validating the zebrafish as a model system to further dissect the molecular mechanisms that regulate apoptosis in future forward genetic and chemical modifier screens.

Distinct Neuroblastoma-associated Alterations of PHOX2B Impair Sympathetic Neuronal Differentiation in Zebrafish Models

Heterozygous germline mutations and deletions in PHOX2B, a key regulator of autonomic neuron development, predispose to neuroblastoma, a tumor of the peripheral sympathetic nervous system. To gain insight into the oncogenic mechanisms engaged by these changes, we used zebrafish models to study the functional consequences of aberrant PHOX2B expression in the cells of the developing sympathetic nervous system. Allelic deficiency, modeled by phox2b morpholino knockdown, led to a decrease in the terminal differentiation markers th and dbh in sympathetic ganglion cells. The same effect was seen on overexpression of two distinct neuroblastoma-associated frameshift mutations, 676delG and K155X - but not the R100L missense mutation - in the presence of endogenous Phox2b, pointing to their dominant-negative effects. We demonstrate that Phox2b is capable of regulating itself as well as ascl1, and that phox2b deficiency uncouples this autoregulatory mechanism, leading to inhibition of sympathetic neuron differentiation. This effect on terminal differentiation is associated with an increased number of phox2b+, ascl1+, elavl3− cells that respond poorly to retinoic acid. These findings suggest that a reduced dosage of PHOX2B during development, through either a heterozygous deletion or dominant-negative mutation, imposes a block in the differentiation of sympathetic neuronal precursors, resulting in a cell population that is likely to be susceptible to secondary transforming events.

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.