Embryonal rhabdomyosarcoma (ERMS) is a devastating cancer with specific features of muscle differentiation that can result from mutational activation of RAS family members. However, to date, RAS pathway activation has not been reported in a majority of ERMS patients. Here, we have created a zebrafish model of RAS-induced ERMS, in which animals develop externally visible tumors by 10 d of life. Microarray analysis and cross-species comparisons identified two conserved gene signatures found in both zebrafish and human ERMS, one associated with tumor-specific and tissue-restricted gene expression in rhabdomyosarcoma and a second comprising a novel RAS-induced gene signature. Remarkably, our analysis uncovered that RAS pathway activation is exceedingly common in human RMS. We also created a new transgenic coinjection methodology to fluorescently label distinct subpopulations of tumor cells based on muscle differentiation status. In conjunction with fluorescent activated cell sorting, cell transplantation, and limiting dilution analysis, we were able to identify the cancer stem cell in zebrafish ERMS. When coupled with gene expression studies of this cell population, we propose that the zebrafish RMS cancer stem cell shares similar self-renewal programs as those found in activated satellite cells.[Keywords: Zebrafish; rhabdomyosarcoma; RAS; P53; transgenic] Supplemental material is available at http://www.genesdev.org.
Germline mutations of the fumarate hydratase (FH, fumarase) gene are found in the recessive FH deficiency syndrome and in dominantly inherited susceptibility to multiple cutaneous and uterine leiomyomatosis (MCUL). We have previously reported a number of germline FH mutations from MCUL patients. In this study, we report additional FH mutations in MCUL and FH deficiency patients. Mutations can readily be found in about 75% of MCUL cases and most cases of FH deficiency. Some of the more common FH mutations are probably derived from founding individuals. Protein-truncating FH mutations are functionally null alleles. Disease-associated missense FH changes map to highly conserved residues, mostly in or around the enzyme's active site or activation site; we predict that these mutations severely compromise enzyme function. The mutation spectra in FH deficiency and MCUL are similar, although in the latter mutations tend to occur earlier in the gene and, perhaps, are more likely to result in a truncated or absent protein. We have found that not all mutation-carrier parents of FH deficiency children have a strong predisposition to leiomyomata. We have confirmed that renal carcinoma is sometimes part of MCUL, as part of the variant hereditary leiomyomatosis and renal cancer (HLRCC) syndrome, and have shown that these cancers may have either type II papillary or collecting duct morphology. We have found no association between the type or site of FH mutation and any aspect of the MCUL phenotype. Biochemical assay for reduced FH functional activity in the germline of MCUL patients can indicate carriers of FH mutations with high sensitivity and specificity, and can detect reduced FH activity in some patients without detectable FH mutations. We conclude that MCUL is probably a genetically homogeneous tumour predisposition syndrome, primarily resulting from absent or severely reduced fumarase activity, with currently unknown functional consequences for the smooth muscle or kidney cell.
Pancreatitis is caused by inflammatory injury to the exocrine pancreas, from which both humans and animal models appear to recover via regeneration of digestive enzyme-producing acinar cells. This regenerative process involves transient phases of inflammation, metaplasia and redifferentiation, driven by cell-cell interactions between acinar cells, leukocytes and resident fibroblasts. The NFκB signaling pathway is a critical determinant of pancreatic inflammation and metaplasia, whereas a number of developmental signals and transcription factors are devoted to promoting acinar redifferentiation after injury. Imbalances between these pro-inflammatory and pro-differentiation pathways contribute to chronic pancreatitis, characterized by persistent inflammation, fibrosis and acinar dedifferentiation. Loss of acinar cell differentiation also drives pancreatic cancer initiation, providing a mechanistic link between pancreatitis and cancer risk. Unraveling the molecular bases of exocrine regeneration may identify new therapeutic targets for treatment and prevention of both of these deadly diseases.
RAS family members are among the most frequently mutated oncogenes in human cancers. Given the utility of zebrafish in both chemical and genetic screens, developing RAS-induced cancer models will make large-scale screens possible to understand further the molecular mechanisms underlying malignancy. We developed a heat shock-inducible Cre/Lox-mediated transgenic approach in which activated human kRASG12D can be conditionally induced within transgenic animals by heat shock treatment. Specifically, double transgenic fish Tg(B-actin-LoxP-EGFP-LoxP-kRASG12D; hsp70-Cre) developed four types of tumors and hyperplasia after heat shock of whole zebrafish embryos, including rhabdomyosarcoma, myeloproliferative disorder, intestinal hyperplasia, and malignant peripheral nerve sheath tumor. Using ex vivo heat shock and transplantation of whole kidney marrow cells from double transgenic animals, we were able to generate specifically kRASG12D-induced myeloproliferative disorder in recipient fish. This heat shock-inducible recombination approach allowed for the generation of multiple types of RAS-induced tumors and hyperplasia without characterizing tissue-specific promoters. Moreover, these tumors and hyperplasia closely resemble human diseases at both the morphologic and molecular levels.myeloproliferative disorder ͉ RAS ͉ rhabdomyosarcoma ͉ intestine ͉ malignant peripheral nerve sheath tumor R AS genes encode a family of 21-kDa proteins that switch between inactive GDP-bound (RAS-GDP) and active GTPbound (RAS-GTP) conformations. Once in its activated GTPbound form, RAS interacts with downstream effectors to modulate diverse cellular responses, including proliferation, differentiation, and survival (1). Point mutations within RAS family members often occur at codon 12, 13, or 61 (2), which abolish RAS-GTP hydrolysis and lead to constitutive activation of downstream signaling pathways. These activating mutations are common in human malignancies; for example, Ͼ90% of pancreatic adenocarcinomas, 50% of colorectal cancers, 25-50% of lung cancers, 5-35% of rhabdomyosarcomas (RMS), and 25-50% of myeloid leukemia have mutational activation of RAS family members. Among the three different human RAS genes (H-, N-,
Defining the genetic pathways essential for hematopoietic stem cell (HSC) development remains a fundamental goal impacting stem cell biology and regenerative medicine. To genetically dissect HSC emergence in the aorta-gonadmesonephros (AGM) region, we screened a collection of insertional zebrafish mutant lines for expression of the HSC marker, c-myb. Nine essential genes were identified, which were subsequently binned into categories representing their proximity to HSC induction. Using overexpression and loss-of-function studies in zebrafish, we ordered these signaling pathways with respect to each other and to the Vegf, Notch, and Runx programs. Overexpression of vegf and notch is sufficient to induce HSCs in the tbx16 mutant, despite a lack of axial vascular organization. Although embryos deficient for artery specification, such as the phospholipase C gamma-1 (plc␥1) mutant, fail to specify HSCs, overexpression of notch or IntroductionSpecification of definitive hematopoietic stem cells (HSCs) capable of generating the blood cell lineages is a vertebrate-specific process that occurs in the aorta-gonad-mesonephros (AGM) region of the developing embryo. 1 In the mouse, HSCs are located near the ventral endothelium of the dorsal aorta on embryonic day (E) 10, approximately the same time that HSC activity is present in the AGM region. [2][3][4] The proto-oncogene c-myb and the transcription factor runx1 are both excellent markers of these emerging HSCs and are essential for mammalian HSC development. 5 Runx1 is thought to operate very early in HSC specification as the mouse knockout lacks intraaortic hematopoietic clusters. 4 Based on analysis of a point mutant, c-Myb is thought to act in concert with p300 to regulate the proliferation and differentiation of HSCs. 6 Similarly, zebrafish have an AGM-like region in the ventral wall of the dorsal aorta also marked by c-myb and runx1 expression. 5,[7][8][9][10][11] Lineage tracing experiments have shown cells exiting this region and ultimately seeding the kidney and thymus, the sites of definitive hematopoiesis in the zebrafish. 12-14 Consistent with the mouse knockout data, morpholino knockdown of runx1 translation in zebrafish results in the loss of c-myb cells in the aortic region as well as a loss of definitive marker expression in the thymus. 8 Although many studies have focused on the molecular events important for HSC emergence from the AGM, very little is understood about the signals essential for specifying these cells. 15 Some progress has been made through the analysis of several zebrafish mutants. In zebrafish, definitive HSCs are located between and in the axial vasculature, 14 which is developmentally derived from intermediate mesoderm. Mutants with defects in vascular formation and patterning show deficiencies in HSC specification. For example, the cloche (clo) mutant lacks endothelium and thus a vasculature, has no AGM HSCs and does not undergo definitive hematopoiesis. 11 Loss of vasculature organization, such as that in the spadetail (spt) mutant ha...
The zebrafish has emerged as a powerful genetic model of cancer, but has been limited by the use of stable transgenic approaches to induce disease. Here, a co-injection strategy is described that capitalizes on both the numbers of embryos that can be microinjected and the ability of transgenes to segregate together and exert synergistic effects in forming tumors. Using this mosaic transgenic approach, gene pathways involved in tumor initiation and radiation sensitivity have been identified.
SUMMARYThe size of the pancreas is determined by intrinsic factors, such as the number of progenitor cells, and by extrinsic signals that control the fate and proliferation of those progenitors. Both the exocrine and endocrine compartments of the pancreas undergo dramatic expansion after birth and are capable of at least partial regeneration following injury. Whether the expansion of these lineages relies on similar mechanisms is unknown. Although we have shown that the Wnt signaling component β-catenin is selectively required in mouse embryos for the generation of exocrine acinar cells, this protein has been ascribed various functions in the postnatal pancreas, including proliferation and regeneration of islet as well as acinar cells. To address whether β-catenin remains important for the maintenance and expansion of mature acinar cells, we have established a system to follow the behavior and fate of β-catenin-deficient cells during postnatal growth and regeneration in mice. We find that β-catenin is continuously required for the establishment and maintenance of acinar cell mass, extending from embryonic specification through juvenile and adult self-renewal and regeneration. This requirement is not shared with islet cells, which proliferate and function normally in the absence of β-catenin. These results make distinct predictions for the relative role of Wnt–β-catenin signaling in the etiology of human endocrine and exocrine disease. We suggest that loss of Wnt–β-catenin activity is unlikely to drive islet dysfunction, as occurs in type 2 diabetes, but that β-catenin is likely to promote human acinar cell proliferation following injury, and might therefore contribute to the resolution of acute or chronic pancreatitis.
The spliceosome cycle consists of assembly, catalysis, and recycling phases. Recycling of postspliceosomal U4 and U6 small nuclear ribonucleoproteins (snRNPs) requires p110/SART3, a general splicing factor. In this article, we report that the zebrafish earl grey (egy) mutation maps in the p110 gene and results in a phenotype characterized by thymus hypoplasia, other organ-specific defects, and death by 7 to 8 days postfertilization. U4/U6 snRNPs were disrupted in egy mutant embryos, demonstrating the importance of p110 for U4/U6 snRNP recycling in vivo. Surprisingly, expression profiling of the egy mutant revealed an extensive network of coordinately up-regulated components of the spliceosome cycle, providing a mechanism compensating for the recycling defect. Together, our data demonstrate that a mutation in a general splicing factor can lead to distinct defects in organ development and cause disease.small nuclear RNA ͉ small nuclear ribonucleoprotein ͉ splicing ͉ genetic screen ͉ thymus M essenger RNA splicing requires the ordered assembly of the spliceosome from Ͼ100 protein components and five small nuclear RNAs (snRNAs): U1, U2, U4, U5, and U6 (reviewed in refs. 1-3). After splicing catalysis and mRNA release, the spliceosome disassembles, and its components undergo a recycling phase, which still is poorly understood. In humans, recycling of postspliceosomal U4 and U6 small nuclear ribonucleoproteins (snRNPs) to functional U4/U6 snRNPs requires in vitro p110/SART3, a general splicing factor referred to as p110 in the present article (4, 5). In addition, p110 functions in recycling of the U4atac/U6atac snRNP (6). Characteristically, p110 associates only transiently with the U6 and U4/U6 snRNPs but is absent from the U4/U6.U5 tri-snRNP and spliceosomes.The domain structure of the human p110 protein is composed of at least seven tetratricopeptide repeats (TPR) in the Nterminal half, followed by two RNA recognition motifs (RRMs) in the C-terminal half, as well as a stretch of 10 highly conserved amino acids at the C terminus (C10 domain). The N-terminal TPR domain functions in interaction with the U4/U6 snRNPspecific 90K protein, the RRMs are important for U6 snRNA binding, and the conserved C10 domain is critical for interacting with the U6-specific LSm proteins (5,7,8). Thus, multiple contacts mediate the interaction between p110 and the U4 and U6 components.This p110 domain organization is conserved in many other eukaryotes, including Caenorhabditis elegans, Arabidopsis thaliana, Schizosaccharomyces pombe, and Drosophila melanogaster (5). The Saccharomyces cerevisiae Prp24 protein, although functionally related to human p110, is an exception in that it lacks the entire N-terminal half with the TPR domain (9).Here we use the zebrafish system to study the system-wide role and in vivo function of p110. We describe the phenotype of a zebrafish mutant, called earl grey (egy), that originated from a genetic screen for mutants of T cell and thymus development. Surprisingly, the embryonically lethal mutation was mapp...
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