Suppression of apoptosis by bcl-2 overexpression in lymphoid cells of transgenic zebrafish

Abstract

The zebrafish is an attractive vertebrate model for genetic studies of development, apoptosis, and cancer. Here we describe a transgenic zebrafish line in which T-and B-lymphoid cells express a fusion transgene that encodes the zebrafish bcl-2 protein fused to the enhanced green fluorescence protein (EGFP). Targeting EGFP-bcl-2 to the developing thymocytes of transgenic fish resulted in a 2.5-fold increase in thymocyte numbers and a 1.8-fold increase in GFPlabeled B cells in the kidney marrow. Fluorescent microscopic analysis of living rag2-EGFP-bcl-2 transgenic fish showed that their thymocytes were resistant to irradiation-and dexamethasoneinduced apoptosis, when compared with control rag2-GFP transgenic zebrafish. To test the ability of bcl-2 to block irradiation-induced apoptosis in malignant cells, we compared the responsiveness of Mycinduced leukemias with and without EGFP-bcl-2 expression in living transgenic zebrafish. T-cell leukemias induced by the rag2-EGFP-Myc transgene were ablated by irradiation, whereas leukemias in double transgenic fish expressing both Myc and EGFP-bcl-2 were resistant to irradiation-induced apoptotic cell death. The forward genetic capacity of the zebrafish model system and the ability to monitor GFP-positive thymocytes in vivo make this an ideal transgenic line for modifier screens designed to identify genetic mutations or small molecules that modify bcl-2-mediated antiapoptotic pathways. ( IntroductionApoptosis is a critical mechanism for regulating homeostasis and tissue remodeling during development. Thus, it is not surprising that many of the molecular components and pathways regulating this process have been conserved throughout evolution. [1][2][3][4] Apoptosis is also essential for hematopoietic cell development in vertebrates. 5,6 For example, regulated programmed cell death is critical for the maintenance of hematopoietic stem cell numbers 7,8 and for normal T-lymphocyte development. [9][10][11][12] Failure of lymphoid cells to undergo cell death can contribute to oncogenic transformation, and in fact, the suppression of apoptosis through key mutations in one or more regulatory pathways is often a critical component of malignant transformation. [13][14][15][16] Although many of the molecular pathways that regulate apoptosis are shared among vertebrate and nonvertebrate organisms, evolution has led to increased complexity in the pathways regulating programmed cell death in higher organisms. Unlike worms and flies, vertebrates have multiple antiapoptotic and proapoptotic Bcl-2 family members. 17 Thus, it is not surprising that loss-of-function studies in mice have demonstrated tissue-specific roles for many of these proteins, and in other cases have suggested redundant functions due to the expression of more than one family member. 9,10,[18][19][20][21][22][23] Given the remarkable complexity associated with the control of apoptosis in vertebrates, new insights may be gained from studying cell death pathways in vertebrate forward-genetic model systems, such as the zebr...

Delineation of the cell-extrinsic apoptosis pathway in the zebrafish

“…An incomplete rescue is not surprising, given that high levels of activated caspase-8 in mammalian cells circumvent the requirement for the intrinsic pathway. Coinjection of zBlp1 or zBlp2 (a homolog of Bcl-2 38 ) with zDL1a or zDL1b markedly attenuated the short-tail phenotype and prevented notochord and ICM apoptosis (Figures 6c, 7a and Figure 2a). Similarly, MO-mediated inhibition of zebrafish Mcl-1 homologs sensitized normally resistant embryonic tissues to zDL1b-induced apoptosis (Kratz et al, accompanying manuscript).…”

Bax, Bcl2, and p53 Differentially Regulate Neomycin- and Gentamicin-Induced Hair Cell Death in the Zebrafish Lateral Line

“…The zebrafish Bcl2 coding sequence, fused to the 3′ end of EGFP, was kindly provided by Dr. A. T. Look (Langenau et al 2005). This fusion gene was PCR-amplified with primers containing the appropriate attB sites for cloning into the Gateway middle entry vector (see Kwan et al 2007).…”

Catch of the day: zebrafish as a human cancer model

“…The examination of molecular pathways activated in response to c-myc overexpression closely resembled the most common and most aggressive subclass of human leukemia, which is characterized by the coexpression of tal2 and lmo2 markers . In a separate study, the same group showed that irradiation or dexamethasone-induced apoptosis of fish leukemic cells is dependent on bcl-2 gene function that also operates in mammalian forms of leukemia (Langenau et al, 2005b). Two independent studies used a similar approach to induce different subtypes of leukemia mediated by overexpression of the aberrant TEL-AML1 fusion protein using the ubiquitous b-actin promoter or overexpression of Notch using the rag2 promoter (Sabaawy et al, 2006;Chen et al, 2007).…”