David W. Barnes

The metazoan parasitic blood flukes, Schistosoma spp., infect over 200 million people worldwide and cause extensive human morbidity and mortality. Research strategies for development of anti-schistosomal agents are impeded by the organism’s complex molluscan–mammalian life cycle, which limits experimental approaches and availability of material. We derived long-term continuously proliferative cultures of Schistosoma mansoni sporocysts capable of generating cercariae in vitro . Cultured organisms retained the ability to parasitize the host, and they exhibited developmental regulation of candidate stage-specific genes in the host-free culture system. Evidence for expression of a reverse transcriptase also was found in the cultured organisms, pointing to this activity as a possible mechanistic contributor to the dynamic relationship between the parasite and its hosts. Continuous in vitro propagation of the asexual sporocyst stage allows isolation of clonally derived parasite populations and provides a means to study schistosomal molecular genetics, metabolism, and evasion of host defenses.

The Japanese pufferfish (genus Fugu), which possesses a highly compact genome, is becoming a popular model among those interested in sequencing and mapping the genomes of higher vertebrates. Although genomic libraries have been derived and used to study the molecular biology of Fugu, biological material derived from the living organism is difficult to obtain for laboratories distant from the Asian Pacific. We have established cell cultures from two Fugu species: kusafugu, Fugu niphobles, and torafugu, F. rubripes. Cultures derived from F. niphobles fry and F. rubripes eye have been passaged more than 60 times over the course of one year, representing approximately 180 population doublings. Proliferating cultures were also initiated from F. rubripes brain, liver, fin, spleen, kidney, swimbladder, and muscle. Karyotype analyses indicated that F. rubripes eye-derived cells possessed a chromosome number in the diploid range; F. niphobles fry cells were slightly hyperploid. Flow cytometry confirmed that the relative amounts of DNA present in cultured cells from both Fugu species were similar to that measured in blood cells collected from F. rubripes, and approximately one-seventh of that measured in diploid human cells. Telomerase activity was easily detectable in lysates prepared from F. niphobles fry cells and F. rubripes eye cells, consistent with the notion that these cultures are capable of indefinite proliferation.

Three methods were used in succession to screen a whole adult zebrafish cDNA library for expressed p53-like genes. The sequences of the resultant clones describe an open reading frame 1122 nucleotides in length, with another 43 and 940 bases of 5' and 3' untranslated sequence, respectively. The deduced amino acid sequence of the zebrafish p53 protein is 63% identical to that of trout and 48% identical to that of human p53. Two of the three zebrafish clones overlap to span the entire reported cDNA sequence and are identical in their deduced amino acid sequence over their coincident length. The third clone contains a conservative amino acid change, as well as an inserted amino acid subsequently found to be at the junction of exons 2 and 3, suggestive of alternative splicing in the p53 mRNA for this species. Northern analysis demonstrated a zebrafish p53-related transcript to be present and most abundant in zygotes and early-cleavage embryos less than 1 hour after fertilization, thereafter declining to barely detectable levels at 48 hours. A similar temporal expression was detected for the zebrafish L-myc, known to be present in maternally derived RNA, whereas zebrafish N-myc and the zebrafish homologue of the murine T gene were not detectable prior to the onset of zygotic transcription.

Pluripotent embryonic stem (ES) cell cultures provide an efficient method for genome manipulation with many applications in marine biotechnology. To develop this technology we have been working to derive fish ES cell lines for in vitro studies of embryo cell growth and differentiation and for the generation of transgenic fish. Zebrafish embryonal cell cultures were derived from blastula-stage embryos in LDF medium supplemented with fetal bovine serum, trout serum, trout embryo extract, selenium, insulin, and leukemia inhibitory factor. Cultures derived under these conditions on feeder layers of zebrafish embryonic fibroblasts possessed a diploid karyotype and exhibited an ES-like morphology with elevated levels of alkaline phosphatase enzyme activity. Injection of primary cell cultures derived from embryos of transgenic fish carrying neo produced chimeric fish detected by polymerase chain reaction analysis. Embryo cells cultured on poly-D-lysine substrate in the presence of retinoic acid or Buffalo rat liver cell-conditioned medium (BRL-CM) and a reduced serum concentration differentiated into neuronal cell types exhibiting elevated levels of acetylcholinesterase enzyme activity and expression of neurofilament and glial fibrillary acidic protein.

We are attempting to develop methods for in vitro culture of zebrafish embryonal stem cells. Primary cultures were initiated from wild-type zebrafish early embryos in basal nutrient medium supplemented with insulin, selenite, leukemia inhibitory factor, trout serum, fetal bovine serum, and trout embryo extract. In this medium, melanocytes appeared on the second day of culture. Basic fibroblast growth factor (bFGF) was mitogenic when cells were plated at low densities. bFGF suppressed melanogenesis is a dose-dependent fashion, with maximal effect at 20 ng/mL. Cultures initiated and maintained with bFGF for 24 hours and then incubated without bFGF for as long as 8 days did not contain pigmented cells. Experiments in which bFGF was added or removed at various times after initiation of cultures indicated that maximum sensitivity to bFGF occurred during the first 12 hours of culture. When wild-type cells from cultures without bFGF were injected into albino blastula-stage embryos, melanocytes subsequently developed in host embryos: no melanocytes appeared when cells from cultures with bFGF were injected into albino hosts.