It is estimated that ∼2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5-7 of age. We are performing a large-scale insertional mutagenesis screen in the zebrafish with the goal of isolating ∼1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared ∼36,000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000-1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed ∼80,000-150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of insertional mutagen is 1 embryonic lethal mutation per 70-100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used, a virus designated F 5 . We screened an estimated 760 insertions among F 3 progeny from 92 F 2 families and obtained 9 recessive embryonic lethal mutations. Thus, the efficiency of mutagenesis with this viral vector is approximately one-ninth that observed with the chemical mutagen ENU in zebrafish. We have also obtained two dominant mutations, one of which is described here. As expected, mutated genes can be readily identified. So far, genes mutated in four of the nine recessive mutants and one of the two dominant mutants have been cloned. Further improvements to this technology could make large-scale insertional mutagenesis screening and rapid gene cloning accessible to relatively small zebrafish laboratories.

mcl-1 was identified as an "early-induction" gene that increases in expression during the differentiation of ML-1 human myeloblastic leukemia cells. The mcl-1 gene product proved to be a member of the bcl-2 gene family and, like bcl-2, to have the capacity to promote cell viability. The pattern of expression of mcl-1 has now been characterized, the aim being to determine whether increased expression is consistently associated with differentiation-induction and whether expression is also associated with other changes in proliferative state or cell viability. Expression of the mcl-1 mRNA was found to increase rapidly in ML-1 cells exposed to inducers of monocyte/macrophage differentiation (phorbol esters or lymphocyte conditioned medium), but not cells exposed to an inducer of granulocyte differentiation (retinoic acid). Expression also increased rapidly in response to certain cytotoxic agents (colchicine and vinblastine), but did not increase during serum stimulation or growth-arrest in reduced serum. Increased expression of mcl-1 occurred during the initiation of cell differentiation or death and was not inhibited by cycloheximide, in agreement with the designation of mcl-1 as an early-induction gene. Increased transcription contributed to the increase in expression, and turnover of the mcl-1 mRNA was rapid. These findings suggest that mcl-1 may serve as a modulator of cell viability that can undergo rapid upregulation as well as downregulation, with upregulation harbingering the initiation of cell differentiation or death.

No abstract

Proliferation, differentiation, and apoptosis are tightly regulated during hematopoiesis, allowing amplification along specific lineages while preventing excessive proliferation of immature cells. The MCL1 member of the BCL2 family is up-regulated during the induction of monocytic differentiation (ϳ10-fold with 12-O-tetradecanoylphorbol 13-acetate (TPA)). MCL1 has effects similar to those of BCL2, up-regulation promoting viability, but differs from BCL2 in its rapid inducibility and its pattern of expression. Nuclear factors that regulate MCL1 transcription have now been identified, extending the previous demonstration of signal transduction through mitogen-activated protein kinase. A 162-base pair segment of the human MCL1 5-flank was found to direct luciferase reporter activity, allowing ϳ10-fold induction with TPA that was suppressible upon inhibition of the extracellular signal-regulated kinase (ERK) pathway. Serum response factor (SRF), Elk-1, and Sp1 bound to cognate sites within this segment, SRF and Elk-1 acting coordinately to affect both basal activity and TPA inducibility, whereas Sp1 affected basal activity only. Thus, the mechanism of the TPA-induced increase in MCL1 expression seen in myelomonocytic cells at early stages of differentiation involves signal transduction through ERKs and transcriptional activation through SRF/Elk-1. This finding provides a parallel to early response genes (e.g. c-FOS and EGR1) that affect maturation commitment in these cells and therefore suggests a means through which enhancement of cell viability may be linked to the induction of differentiation.

No abstract