BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.

Melanoma is the most lethal form of skin cancer, and the incidence and mortality rates are rapidly rising. Epidemiologically, high numbers of nevi (moles) are associated with higher risk of melanoma . The majority of melanomas exhibit activating mutations in the serine/threonine kinase BRAF . BRAF mutations may be critical for the initiation of melanoma ; however, the direct role of BRAF in nevi and melanoma has not been tested in an animal model. To directly test the role of activated BRAF in nevus and melanoma development, we have generated transgenic zebrafish expressing the most common BRAF mutant form (V600E) under the control of the melanocyte mitfa promoter. Expression of mutant, but not wild-type, BRAF led to dramatic patches of ectopic melanocytes, which we have termed fish (f)-nevi. Remarkably, in p53-deficient fish, activated BRAF induced formation of melanocyte lesions that rapidly developed into invasive melanomas, which resembled human melanomas and could be serially transplanted. These data provide direct evidence that BRAF activation is sufficient for f-nevus formation, that BRAF activation is among the primary events in melanoma development, and that the p53 and BRAF pathways interact genetically to produce melanoma.

Hematopoietic defects in rps29 mutant zebrafish depend upon p53 activation.

Disruption of ribosomal proteins is associated with hematopoietic phenotypes in cell culture and animal models. Mutations in ribosomal proteins are seen in patients with Diamond Blackfan anemia, a rare congenital disease characterized by red cell aplasia and distinctive craniofacial anomalies. A zebrafish screen uncovered decreased hematopoietic stem cells in embryos with mutations in ribosomal protein rps29. Here, we determined that rps29(-/-) embryos also have red blood cell defects and increased apoptosis in the head. As the p53 pathway has been shown to play a role in other ribosomal protein mutants, we studied the genetic relationship of rps29 and p53. Transcriptional profiling revealed that genes upregulated in the rps29 mutant are enriched for genes upregulated by p53 after irradiation. p53 mutation near completely rescues the rps29 morphological and hematopoietic phenotypes, demonstrating that p53 mediates the effects of rps29 knockdown. We also identified neuronal gene orthopedia protein a (otpa) as one whose expression correlates with rps29 expression, suggesting that levels of expression of some genes are dependent on rps29 levels. Together, our studies demonstrate a role of p53 in mediating the cellular defects associated with rps29 and establish a role for rps29 and p53 in hematopoietic stem cells and red blood cell development.

A non-canonical function of zebrafish telomerase reverse transcriptase is required for developmental hematopoiesis.

Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from "authentic" telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words).

A chemical genetic screen for cell cycle inhibitors in zebrafish embryos.

Chemical genetic screening is an effective strategy to identify compounds that alter a specific biological phenotype. As a complement to cell line screens, multicellular organism screens may reveal additional compounds. The zebrafish embryo is ideal for small molecule studies because of its small size and the ease of waterborne treatment. We first examined a broad range of known cell cycle compounds in embryos using the mitotic marker phospho-histone H3. The majority of the known compounds exhibited the predicted cell cycle effect in embryos. To determine whether we could identify novel compounds, we screened a 16 320-compound library for alterations of pH3. This screen revealed 14 compounds that had not been previously identified as having cell cycle activity despite numerous mitotic screens of the same library with mammalian cell lines. With six of the novel compounds, sensitivity was greater in embryos than cell lines, but activity was still detected in cell lines at higher doses. One compound had activity in zebrafish embryos and cell lines but not in mammalian cell lines. The remaining compounds exhibited activity only in embryos. These findings demonstrate that small molecule screens in zebrafish can identify compounds with novel activity and thus may be useful tools for chemical genetics and drug discovery.

Small molecules that delay S phase suppress a zebrafish bmyb mutant.

Bmyb is a ubiquitously expressed transcription factor involved in cellular proliferation and cancer. Loss of bmyb function in the zebrafish mutant crash&burn (crb) results in decreased cyclin B1 expression, mitotic arrest and genome instability. These phenotypic observations in crb mutants could be attributed to the decreased expression of cyclin B1, a cell-cycle regulatory protein that is responsible for driving cell progression from G2 through mitosis. To identify small molecules that interact with the bmyb pathway, we developed an embryo-based suppressor screening strategy. In 16 weeks we screened a diverse approximately 16,000 compound library, and discovered one previously unknown compound, persynthamide (psy, 1), that suppressed bmyb-dependent mitotic defects. Psy-treated embryos showed an S-phase delay, and knockdown of the cell-cycle checkpoint regulator ataxia telangiectasia--and Rad-related kinase (ATR) abrogated the suppression of crb. The DNA synthesis inhibitors aphidicolin (2) and hydroxyurea (3) also suppressed crb. S-phase inhibition upregulated cyclin B1 mRNA, promoting the progression of cells through mitosis. Our study demonstrates that chemical suppressor screening in zebrafish can identify compounds with cell-cycle activity and can be used to identify pathways that interact with specific cell-cycle phenotypes.

tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors.

TP53 is the most frequently mutated tumor suppressor gene in human cancer, with nearly 50% of all tumors exhibiting a loss-of-function mutation. To further elucidate the genetic pathways involving TP53 and cancer, we have exploited the zebrafish, a powerful vertebrate model system that is amenable to whole-genome forward-genetic analysis and synthetic-lethal screens. Zebrafish lines harboring missense mutations in the tp53 DNA-binding domain were identified by using a target-selected mutagenesis strategy. Homozygous mutant fish from two of these lines were viable and exhibited mutations similar to those found in human cancers (tp53(N168K) and tp53(M214K)). Although homozygous tp53(N168K) mutants were temperature-sensitive and suppressed radiation-induced apoptosis only at 37 degrees C, cells in the tp53(M214K) embryos failed to undergo apoptosis in response to gamma radiation at both 28 and 37 degrees C. Unlike wild-type control embryos, irradiated tp53(M214K) embryos also failed to up-regulate p21 and did not arrest at the G(1)/S checkpoint. Beginning at 8.5 months of age, 28% of tp53(M214K) mutant fish developed malignant peripheral nerve sheath tumors. In addition to providing a model for studying the molecular pathogenic pathways of malignant peripheral nerve sheath tumors, these mutant zebrafish lines provide a unique platform for modifier screens to identify genetic mutations or small molecules that affect tp53-related pathways, including apoptosis, cell-cycle delay, and tumor suppression.

Tsc2 null murine neuroepithelial cells are a model for human tuber giant cells, and show activation of an mTOR pathway.

Cortical tubers are developmental brain malformations in the tuberous sclerosis complex (TSC) that cause epilepsy and autism in TSC patients whose pathogenesis is uncertain. Tsc2 null murine neuroepithelial progenitor (NEP) cells display persistent growth when growth factors are withdrawn, express GFAP at high levels, and have reduced expression of a set of early neuronal lineage markers. Tsc2 null NEP cells exhibit aberrant differentiation into giant cells that express both beta III-tubulin and GFAP and that are morphologically similar to giant cells in human tubers. Tsc2 null giant cells and tuber giant cells have similar transcriptional profiles. Tsc2 null NEP cells express high levels of phosphorylated S6kinase, S6, Stat3, and 4E-BP-1, which is reversed by treatment with rapamycin, an inhibitor of mTOR. We conclude that giant cells in human tubers likely result from a complete loss of TSC2 expression and activation of an mTOR pathway during cortical development.