EGFP Transgenic Zebrafish Research Articles

Embryonic Ethanol Exposure Affects the Early Development, Migration, and Location of Hypocretin/Orexin Neurons in Zebrafish.

Embryonic ethanol (EtOH) exposure is known to increase alcohol drinking later in life and have long-term effects on neurochemical systems in the brain. With zebrafish having marked advantages for elucidating neural mechanisms underlying brain disorders, we recently tested and showed in these fish, similar to rodents, that low-dose embryonic EtOH stimulates voluntary consumption of EtOH while increasing expression of hypocretin/orexin (hcrt) neurons, a neuropeptide that promotes consummatory and reward-related behaviors. The goal of the present study was to characterize how embryonic EtOH affects early development of the hcrt system and produces persistent changes at older ages that may contribute to this increase in EtOH consumption. We utilized live imaging and Imaris software to investigate how low-dose embryonic EtOH (0.5%), administered from 22 to 24hours postfertilization, affects specific properties of hcrt neurons in hcrt:EGFP transgenic zebrafish at different ages. Time-lapse imaging from 24 to 28 hpf showed that embryonic EtOH increased the number of hcrt neurons, reduced the speed, straightness, and displacement of their migratory paths, and altered their direction early in development. At older ages up to 6 dpf, the embryonic EtOH-induced increase in hcrt neurons was persistent, and the neurons became more widely dispersed. These effects of embryonic EtOH were found to be asymmetric, occurring predominantly on the left side of the brain, and at 6 dpf, they resulted in marked changes in the anatomical location of the hcrt neurons, with some detected outside their normal position in the anterior hypothalamus again primarily on the left side. Our findings demonstrate that low-dose embryonic EtOH has diverse, persistent, and asymmetric effects on the early development of hypothalamic hcrt neurons, which lead to abnormalities in their ultimate location that may contribute to behavioral disturbances, including an increase in EtOH consumption.

Generation of a mef2aa:EGFP transgenic zebrafish line that expresses EGFP in muscle cells.

Transgenesis is an important tool for exploring gene expression and function. The myocyte enhancer factor 2a (mef2a) gene encodes a member of the Mef2 protein family that is involved in vertebrate skeletal, cardiac, and smooth muscle development and differentiation during myogenesis. According to studies on human and animal models, mef2a is highly expressed in the heart and somites. To explore the potential of mef2a as a tool for selective labeling of muscle cells in living zebrafish embryos, we constructed a transgene mef2aa:EGFP to induce the expression of green fluorescent protein (GFP) under the control of mef2a promoter. A ~2-kb DNA fragment, upstream of the translational start site of mef2aa, was identified to drive muscle-specific expression of EGFP in zebrafish embryos. Interestingly, the cranial muscles, abductor muscle, and adductor muscle were clearly labeled with EGFP in the established line Tg(mef2aa:EGFP) ntu803 . In addition, we showed that mef2aa mRNA was highly present in adult zebrafish heart, but not the skeleton muscle, whereas it was expressed in both embryonic heart and myotome, suggesting that mef2a is vital to the function of adult heart in vertebrates.

Beneficial roles of melanoma cell adhesion molecule in spinal cord transection recovery in adult zebrafish.

Melanoma cell adhesion molecule (MCAM) is a multifunctional protein involved in miscellaneous processes, including development and tumor angiogenesis. Here, spinal cord transection in adult zebrafish was used to investigate the effects of MCAM on spinal cord injury (SCI) and subsequent recovery. Expression of MCAM mRNA increased and co-localized with motoneurons in the spinal cord after SCI. With MCAM morpholino treatment, inhibition of MCAM retarded both axon regrowth and locomotor recovery in the spinal cord injured zebrafish. Furthermore, MCAM mRNA expression was also observed in fli1a:EGFP transgenic zebrafish, which specifically show labeled blood vessels. Inhibition of MCAM down-regulated the expression of angiogenesis-related factors, such as VEGFR-2, p-p38 and p-AKT, and the inflammatory factors TNF-α, IL-1β and IL-8. Taken together, these data suggest that MCAM may have a beneficial role in the recovery from SCI, via the promotion of neurogenesis and angiogenesis. In the context of adult zebrafish spinal cord injury, we proved that Melanoma cell adhesion molecule (MCAM) is beneficial to the recovery, possibly via mechanisms of angiogenensis and inflammation. MCAM promotes angiogenesis by adjusting VEGFR-2, p-p38 and p-AKT. MCAM affects inflammatory factors such as TNF-α, IL-1β and IL-8. Our results extend the beneficial role of MCAM in the regeneration of central nervous system.

Generation of mt:egfp transgenic zebrafish biosensor for the detection of aquatic zinc and cadmium.

Zebrafish embryo toxicity testing has become a popular method for detecting environmental pollutions. However, the present research showed that zebrafish embryos exhibited no visible paramorphia, malformation, or mortality when exposed to heavy metals in a range above environmental standard limits, indicating that zebrafish embryos are an imprecise model for monitoring environmental heavy metals concentrations above regulatory limits. Aiming to obtain a biosensor for aquatic heavy metals, a metal-sensitive vector including zebrafish metallothionein (MT) promoter and enhanced green fluorescent protein (EGFP) was reconstructed and microinjected into 1-cell stage zebrafish embryos. The authors obtained an mt:egfp transgenic zebrafish line sensitive to aquatic zinc and cadmium. A quantitative experiment showed that zinc and cadmium treatment significantly induced the expression of EGFP in a dose- and time-dependent manner. In particular, EGFP messenger RNA levels increased remarkably when exposed to heavy metals above the standard limits. The results suggest that the transgenic zebrafish is a highly sensitive biosensor for detecting environmental levels of zinc and cadmium. Environ Toxicol Chem 2016;35:2066-2073. © 2016 SETAC.

CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish.

Here we present a protocol for the conversion of eGFP-transgenic zebrafish lines into lines expressing Gal4 from the same locus. This conversion allows the in-depth analysis of the former eGFP-expressing cell population; with the Gal4-upstream activating sequence (UAS) system, diverse UAS transgenes can be transactivated. Site-specific targeting of the gene encoding eGFP is achieved using the clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. A single-guide RNA (sgRNA) that targets eGFP is injected into embryos together with a donor vector containing an optimized version of Gal4 (KalTA4) to trigger integration of the donor into the targeted eGFP genomic location. To enable screening for successful integration events, injection is performed in a UAS:RFP transgenic background; fish showing mosaic eGFP-to-RFP conversion are raised to adulthood. The progeny of these adult fish are then screened for stable germline transmission, and converted progeny are used to generate stable lines. We have been able to generate two stably converted transgenic lines within 4 months.

Different Mechanisms Regulate Expression of Zebrafish Myelin Protein Zero (P0) in Myelinating Oligodendrocytes and Its Induction following Axonal Injury

Zebrafish CNS axons regenerate robustly following injury; it is thought that CNS oligodendrocytes contribute to this response by expressing growth-promoting molecules. We characterized the mpz gene, which encodes myelin protein zero and is up-regulated in oligodendroglia following axonal injury. The 2.5-kb mpz mRNA is expressed from a single TATA box promoter. Four independent Tg(mpz:egfp) transgenic zebrafish lines, in which GFP was expressed under the mpz promoter and 10 kb of genomic 5'-flanking sequence, showed transgene expression in CNS oligodendrocytes from larval development through adulthood. Following optic nerve crush injury, the mpz:egfp transgene was strongly up-regulated in oligodendrocytes along the regenerating retinotectal projection, mirroring up-regulation of endogenous mpz mRNA. GFP-expressing oligodendroglia were significantly more abundant in the regenerating optic pathway, resulting from both transgene induction in oligodendroglial precursors and the birth of new cells. Up-regulation of the mpz:egfp transgene was not dependent on axonal regeneration, suggesting that the primary signal may be axonal loss, debris, or microglial infiltration. Deletion experiments indicated that an oligodendroglial enhancer located in the region from -6 to -10 kb with respect to the mpz transcriptional start site is dissociable from the cis-regulatory element mediating the mpz transcriptional response to axonal injury, which is located between -1 and -4 kb. These data show that different mechanisms regulate expression of zebrafish mpz in myelinating oligodendrocytes and its induction following axonal injury. The underlying molecular events could potentially be exploited to enhance axonal repair following mammalian CNS injury. The transgenic lines and cis-regulatory constructs reported here will facilitate identification of the relevant signaling pathways.

Generation and characterization of gsuα:EGFP transgenic zebrafish for evaluating endocrine-disrupting effects

The glycoprotein subunit α (gsuα) gene encodes the shared α subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone β (Fshβ), luteinizing hormone β (Lhβ) and thyroid stimulating hormone β (Tshβ). In our current study, we identified and characterized the promoter region of zebrafish gsuα and generated a stable gsuα:EGFP transgenic line, which recapitulated the endogenous gsuα expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsuα promoters. Our results also demonstrated that the expression patterns of the gsuα:EGFP transgene were all identical to those expression patterns of the endogenous gsuα expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KClO4, dexamethasone (Dex), β-estradiol and progesterone. Thus, this gsuα:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsuα-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well.

The vas::egfp transgenic zebrafish: A practical model for studies on the molecular mechanisms by which environmental estrogens affect gonadal sex differentiation

The vas::egfp transgenic zebrafish (Danio rerio) could significantly enhance studies on the mechanisms by which environmental estrogens disrupt sexual differentiation, because the developing gonad can be visualized during early life via fluorescence detection. There are methodological challenges regarding dissecting out the gonads in early-life-stage fish, however, and transgene responses to estrogen exposure have not been tested. The authors exposed vas::egfp transgenic zebrafish and their wild-type siblings to the model estrogen 17α-ethinylestradiol (EE2; 0.62 ng/L and 3.33 ng/L measured concentrations) during sexual development (20-60 d posthatch) and used enhanced green fluorescent protein (egfp) fluorescence to identify and dissect single gonads (at 40 d posthatch) to provide sufficient RNA for individual gene expression analyses, retaining the remaining gonad in the body cavity for histological analyses of sex and stage of development. Genotyping confirmed that all transgenic control fish were phenotypically egfp positive (showed green fluorescence). Interestingly, however, in a few transgenic fish exposed to EE2, no phenotypic egfp signal was seen, most notably for the 3.33 ng/L EE2 exposure. It was subsequently found that gonadal vasa expression was reduced by this concentration of EE2. Hepatic vitellogenin expression demonstrated that the vas::egfp and wild-type lines responded to estrogen with an equivalent sensitivity. The authors conclude that the vas::egfp zebrafish provides an enhanced and practical system for mechanistic studies on the effects of environmentally relevant concentrations of estrogens on gonad development.

A Promoter Fragment of the sycp1 Gene Is Sufficient to Drive Transgene Expression in Male and Female Meiotic Germ Cells in Zebrafish1

The synaptonemal complex protein 1 (Sycp1) is required for the formation of crossovers that occurs during the meiotic prophase. The tissue and cell-specific expression pattern of the Sycp1 protein have been studied in mammals and fish, but data on the corresponding transcript remain scarce. In this report, we described for the first time in zebrafish the tissue- and cell-specific expression pattern of the sycp1 gene. In ovary, the expression of the sycp1 transcript was restricted to the early primary oocytes. In testis, the sycp1 transcript was observed in primary spermatocytes in agreement with a previous report describing the localization of the Sycp1 protein in those cells. Unexpectedly, sycp1 transcript expression remained high in spermatids. To gain insight on the genomic region responsible for the sycp1 gene expression pattern, we generated four independent Dr_sycp1:eGFP transgenic zebrafish lines carrying the -1482/+338 gene fragment fused to the enhanced green fluorescent protein reporter gene. We demonstrate that this promoter fragment contains the information required for the cell-specific expression of the endogenous sycp1 gene in males and in females. However, the GFP protein and its associated fluorescence persist in spermatozoa and maturing oocytes. The Dr_sycp1:eGFP zebrafish lines have the potential to be valuable models to trace meiosis onset in zebrafish and constitute the first transgenic lines expressing the GFP reporter protein only in the male meiotic and postmeiotic cells in fish.

Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish.

Cautious use of fli1a:EGFP transgenic zebrafish in vascular research

Integration of exogenous sequence into an intact genome may cause some artificial phenotype or unspecific observations. We noticed that there is unspecific vascular expression when using fli1a:EGFP transgenic embryos for whole-mount in situ hybridization (WISH) experiments. We therefore tested whether the residual vector sequence contained in the fli1a:EGFP transgene or the integration of transgene into the genome may cause this expression ‘noise’ and/or deregulation of gene expression at a genome-wide level. RNA probes were synthesized using two different methods, i.e. vector-based and PCR-based. The vector-based dnmt3 probe showed unspecific vascular expression in fli1a:EGFP embryos, but not in wildtype embryos, by WISH. Moreover, we also found that compared to that in wildtype, there were alterations in gene expression at whole-genome level in the fli1a:EGFP embryos. Our finding that the vector sequence contained in the fli1a:EGFP genome causes unspecific vascular expression by WISH and the genome-wide expression profiling is altered in fli1a:EGFP embryos strongly argue that extra caution should be taken for data interpretation when using transgenics, such as fli1a:EGFP, in developmental biology studies.

Use of TSHβ:EGFP transgenic zebrafish as a rapid in vivo model for assessing thyroid-disrupting chemicals

Accumulating evidence indicates that a wide range of chemicals have the ability to interfere with the hypothalamic–pituitary–thyroid (HPT) axis. Novel endpoints should be evaluated in addition to existing methods in order to effectively assess the effects of these chemicals on the HPT axis. Thyroid-stimulating hormone subunit β (TSHβ) plays central regulatory roles in the HPT system. We identified the regulatory region that determines the expression level of zebrafish TSHβ in the anterior pituitary. In the transgenic zebrafish with EGFP driven by the TSHβ promoter, the similar responsive patterns between the expression levels of TSHβ:EGFP and endogenous TSHβ mRNA in the pituitary are observed following treatments with goitrogen chemicals and exogenous thyroid hormones (THs). These results suggest that the TSHβ:EGFP transgenic reporter zebrafish may be a useful alternative in vivo model for the assessment of chemicals interfering with the HPT system.

Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line

BackgroundBecause of the structural and molecular similarities between the two systems, the lateral line, a fish and amphibian specific sensory organ, has been widely used in zebrafish as a model to study the development/biology of neuroepithelia of the inner ear. Both organs have hair cells, which are the mechanoreceptor cells, and supporting cells providing other functions to the epithelium. In most vertebrates (excluding mammals), supporting cells comprise a pool of progenitors that replace damaged or dead hair cells. However, the lack of regenerative capacity in mammals is the single leading cause for acquired hearing disorders in humans.ResultsIn an effort to understand the regenerative process of hair cells in fish, we characterized and cloned an egfp transgenic stable fish line that trapped tnks1bp1, a highly conserved gene that has been implicated in the maintenance of telomeres' length. We then used this Tg(tnks1bp1:EGFP) line in a FACsorting strategy combined with microarrays to identify new molecular markers for supporting cells.ConclusionsWe present a Tg(tnks1bp1:EGFP) stable transgenic line, which we used to establish a transcriptional profile of supporting cells in the zebrafish lateral line. Therefore we are providing a new set of markers specific for supporting cells as well as candidates for functional analysis of this important cell type. This will prove to be a valuable tool for the study of regeneration in the lateral line of zebrafish in particular and for regeneration of neuroepithelia in general.

121 Identification, validation and mechanisms of action of novel therapeutic compounds modulating angiogenesis

Abnormal angiogenesis is a signature feature in over 70 health conditions and is critical for tumour growth and metastasis. Therefore, discovering novel therapeutic compounds, that stimulate or abrogate this biological process would be, potentially, clinically invaluable. The zebrafish has emerged as an important model for studying vascular development and angiogenesis. Advantages over other models include rapid embryonic development and optical clarity of the embryos. The small body size of the zebrafish embryo and accessibility of embryonic developmental structures within water make zebrafish embryos suitable for high through-put drug screens. We performed an angiogenesis chemical genetic screen with two compound libraries (LOPAC and Spectrum Collection) using Flk1:EGFP (fluorescently labelled endothelial cells) transgenic zebrafish embryos. The compound bioactivity was measured by numbers of the zebrafish trunk intersegmental vessel (ISV) number. The screens were performed in 96 well plates and a total of ten antiangiogenic compounds and three proangiogenic compounds were identified. A CFI-funded high throughput screening facility will be use for identification of more lead compounds. The goal of the current study was to validate the potential therapeutic efficacy of selected compounds and investigate their molecular mechanisms. Retinoic acid p-hydroxyanilide (4HPR; Fenretinide) was identified as a pro-angiogenic compound. At 4 μM, 4HPR promoted ISV development with increased ISV numbers (ISV = 32.82 ± 0.98; control ISV = 27.58 ± 0.76; P < 0.05). In HUVEC cell proliferation assay, 4HPR stimulated human endothelial cell proliferation at low molar concentrations (0.06-0.12 μM). The effects of this compound on physiological angiogenesis are currently being tested on larvae and adult zebrafish fin regeneration models. Indirubin-3-oxime (IO) was identified as a very potent antiangiogenic compound, which inhibited zebrafish ISV development in a dose-dependent manner (Fig. 1). At 16 μM, IO almost completely inhibited ISV development. In tissue regeneration models, IO inhibited larval and adult zebrafish fin regeneration. The efficacy of IO in cancer therapy will be further studied in a zebrafish cancer metastasis model. Possible molecular mechanisms responsible for IO and 4HPR action such as VEGF and notch signaling will be studied. Using the zebrafish chemical genetic screen, we demonstrated that indirubin-3-oxime is a potent angiogenic inhibitor which inhibits zebrafish angiogenesis and fin regeneration. IO may prove to be effective as an anti-cancer drug. Retinoic acid p-hydroxyanilide was identified as a pro-angiogenic effector, which may be clinically effective in promoting tissue regeneration and in treatment of myocardial infarction.

In vivo alternative assessment of the chemicals that interfere with anterior pituitary POMC expression and interrenal steroidogenesis in POMC: EGFP transgenic zebrafish

Adrenocorticotropin (ACTH) has been considered a classic adrenocorticotropic hormone and the key pituitary-derived peptide controlling steroidogenesis in the adult adrenal. ACTH is encoded by the propiomelanocortin (POMC) gene, and its active form is mainly synthesized and processed from the POMC-encoded multihormone precursor in the anterior pituitary. The ACTH level has always been precisely controlled in the signaling cascade of the hypothalamo–pituitary–adrenal (HPA) axis due to its central role. The purpose of this study was to investigate whether the transgenic zebrafish line with EGFP driven by the POMC promoter can be used as a surrogate marker to detect the interference effects on anterior pituitary POMC expression caused by chemicals in teleost. The Tg (POMC:EGFP) fish treated for 4 days with the known adrenergic agents, dexamethasone (Dex) or aminoglutethimide (AG), exhibited altered levels of EGFP and POMC expression in the anterior domain of pituitary corticotrophs. Whole-mount in situ hybridization revealed impaired patterns of expression of the zebrafish ftz-fl gene (ff1b), a key molecular marker for early interrenal development. Next, several chemicals and six commonly used organophosphorus compounds (OPs) were tested for their effects on anterior pituitary POMC expression and early interrenal development. Our preliminary screening analyses indicated that simazine and 3,3′,4,4′5-pentachlorobiphenyl (PCB126) could interfere with anterior pituitary POMC expression and interrenal development in fish. In summary, our results demonstrated that the Tg ( POMC:EGFP) zebrafish line might be employed as a specific and reproductive in vivo assessment model for the effects of endocrine disruption on HPA signaling.

Early Development of Forebrain Gonadotrophin‐Releasing Hormone (GnRH) Neurones and the Role of GnRH as an Autocrine Migration Factor

Normal migration of the gonadotrophin-releasing hormone (GnRH) neurones during early development, from the olfactory region to the hypothalamus, is crucial for reproductive development in all vertebrates. The establishment of the GnRH system includes tangential migration of GnRH perikarya as well as extension of GnRH fibres to various areas of the central nervous system (CNS). The exact spatio-temporal nature of this process, as well as the factors governing it, are not fully understood. We studied the development of the GnRH system and the effects of GnRH knockdown using a newly developed GnRH3:EGFP transgenic zebrafish line. We found that enhanced green fluorescent protein is specifically and robustly expressed in GnRH3 neurones and fibres. GnRH3 fibres in zebrafish began to extend as early as 26 h post-fertilisation and by 4-5 days post-fertilisation had developed into an extensive network reaching the optic tract, telencephalon, hypothalamus, midbrain tegmentum and hindbrain. GnRH3 fibres also innervated the retina and projected into the trunk via the spinal cord. GnRH3 perikarya were observed migrating along their own fibres from the olfactory region to the preoptic area (POA) via the terminal nerve ganglion and the ventral telencephalon. GnRH3 cells were also observed in the trigeminal ganglion. The establishment of the GnRH3 fibre network was disrupted by morpholino-modified antisense oligonucleotides directed against GnRH3 causing abnormal fibre development and pathfinding, as well as anomalous GnRH3 perikarya localisation. These findings support the hypothesis that GnRH3 neurones migrate from the olfactory region to the POA and caudal hypothalamus. Novel data regarding the early development of the GnRH3 fibre network in the CNS and beyond are described. Moreover we show, in vivo, that GnRH3 is an important factor regulating GnRH3 fibre pathfinding and neurone localisation in an autocrine fashion.

Modulation of angiogenesis by dithiolethione-modified NSAIDs and valproic acid.

Angiogenesis involves multiple signaling pathways that must be considered when developing agents to modulate pathological angiogenesis. Because both cyclooxygenase inhibitors and dithioles have demonstrated anti-angiogenic properties, we investigated the activities of a new class of anti-inflammatory drugs containing dithiolethione moieties (S-NSAIDs) and S-valproate. Anti-angiogenic activities of S-NSAIDS, S-valproate, and the respective parent compounds were assessed using umbilical vein endothelial cells, muscle and tumor tissue explant angiogenesis assays, and developmental angiogenesis in Fli:EGFP transgenic zebrafish embryos. Dithiolethione derivatives of diclofenac, valproate, and sulindac inhibited endothelial cell proliferation and induced Ser(78) phosphorylation of hsp27, a known molecular target of anti-angiogenic signaling. The parent drugs lacked this activity, but dithiolethiones were active at comparable concentrations. Although dithiolethiones can potentially release hydrogen sulphide, NaSH did not reproduce some activities of the S-NSAIDs, indicating that the dithioles regulate angiogenesis through mechanisms other than release of H(2)S. In contrast to the parent drugs, S-NSAIDs, S-valproate, NaSH, and dithiolethiones were potent inhibitors of angiogenic responses in muscle and HT29 tumor explants assessed by 3-dimensional collagen matrix assays. Dithiolethiones and valproic acid were also potent inhibitors of developmental angiogenesis in zebrafish embryos, but the S-NSAIDs, remarkably, lacked this activity. S-NSAIDs and S-valproate have potent anti-angiogenic activities mediated by their dithiole moieties. The novel properties of S-NSAIDs and S-valproate to inhibit pathological versus developmental angiogenesis suggest that these agents may have a role in cancer treatment.