Alternatives to in vivo tests to detect endocrine disrupting chemicals (EDCs) in fish and amphibians--screening for estrogen, androgen and thyroid hormone disruption.

Endocrine disruption is considered a highly relevant hazard for environmental risk assessment of chemicals, plant protection products, biocides and pharmaceuticals. Therefore, screening tests with a focus on interference with estrogen, androgen, and thyroid hormone pathways in fish and amphibians have been developed. However, they use a large number of animals and short-term alternatives to animal tests would be advantageous. Therefore, the status of alternative assays for endocrine disruption in fish and frogs was assessed by a detailed literature analysis. The aim was to (i) determine the strengths and limitations of alternative assays and (ii) present conclusions regarding chemical specificity, sensitivity, and correlation with in vivo data. Data from 1995 to present were collected related to the detection/testing of estrogen-, androgen-, and thyroid-active chemicals in the following test systems: cell lines, primary cells, fish/frog embryos, yeast and cell-free systems. The review shows that the majority of alternative assays measure effects directly mediated by receptor binding or resulting from interference with hormone synthesis. Other mechanisms were rarely analysed. A database was established and used for a quantitative and comparative analysis. For example, a high correlation was observed between cell-free ligand binding and cell-based reporter cell assays, between fish and frog estrogenic data and between fish embryo tests and in vivo reproductive effects. It was concluded that there is a need for a more systematic study of the predictive capacity of alternative tests and ways to reduce inter- and intra-assay variability.

Adult neural stem cell fate is determined by thyroid hormone activation of mitochondrial metabolism.

OBJECTIVE: In the adult brain, neural stem cells (NSCs) located in the subventricular zone (SVZ) produce both neuronal and glial cells. Thyroid hormones (THs) regulate adult NSC differentiation towards a neuronal phenotype, but also have major roles in mitochondrial metabolism. As NSC metabolism relies mainly on glycolysis, whereas mature cells preferentially use oxidative phosphorylation, we studied how THs and mitochondrial metabolism interact on NSC fate determination. METHODS: We used a mitochondrial membrane potential marker in vivo to analyze mitochondrial activity in the different cell types in the SVZ of euthyroid and hypothyroid mice. Using primary adult NSC cultures, we analyzed ROS production, SIRT1 expression, and phosphorylation of DRP1 (a mitochondrial fission mediator) as a function of TH availability. RESULTS: We observed significantly higher mitochondrial activity in cells adopting a neuronal phenotype in vivo in euthyroid mice. However, prolonged hypothyroidism reduced not only neuroblast numbers but also their mitochondrial activity. In vitro studies showed that TH availability favored a neuronal phenotype and that blocking mitochondrial respiration abrogated TH-induced neuronal fate determination. DRP1 phosphorylation was preferentially activated in cells within the neuronal lineage and was stimulated by TH availability. CONCLUSIONS: These results indicate that THs favor NSC fate choice towards a neuronal phenotype in the adult mouse SVZ through effects on mitochondrial metabolism.

Adult neural stem cell cycling in vivo requires thyroid hormone and its alpha receptor.

Thyroid hormones (TH) are essential for brain development. However, information on if and how this key endocrine factor affects adult neurogenesis is fragmentary. We thus investigated the effects of TH on proliferation and apoptosis of stem cells in the subventricular zone (SVZ), as well as on migration of transgene-tagged neuroblasts out of the stem cell niche. Hypothyroidism significantly reduced all three of these processes, inhibiting generation of new cells. To determine the mechanisms relaying TH action in the SVZ, we analyzed which receptor was implicated and whether the effects were played out directly at the level of the stem cell population. The alpha TH receptor (TRalpha), but not TRbeta, was found to be expressed in nestin positive progenitor cells of the SVZ. Further, use of TRalpha mutant mice showed TRalpha to be required to maintain full proliferative activity. Finally, a direct TH transcriptional effect, not mediated through other cell populations, was revealed by targeted gene transfer to stem cells in vivo. Indeed, TH directly modulated transcription from the c-myc promoter reporter construct containing a functional TH response element containing TRE but not from a mutated TRE sequence. We conclude that liganded-TRalpha is critical for neurogenesis in the adult mammalian brain.

Differential thyroid hormone sensitivity of fast cycling progenitors in the neurogenic niches of tadpoles and juvenile frogs.

Adult neurogenesis occurs in neural stem cell (NSC) niches where slow cycling stem cells give rise to faster cycling progenitors. In the adult mouse NSC niche thyroid hormone, T3, and its receptor TRα act as a neurogenic switch promoting progenitor cell cycle completion and neuronal differentiation. Little is known about whether and how T3 controls proliferation of differentially cycling cells during xenopus neurogenesis. To address this question, we first used Sox3 as a marker of stem cell and progenitor populations and then applied pulse-chase EdU/IdU incorporation experiments to identify Sox3-expressing slow cycling (NSC) and fast cycling progenitor cells. We focused on the lateral ventricle of Xenopus laevis and two distinct stages of development: late embryonic development (pre-metamorphic) and juvenile frogs (post-metamorphic). These stages were selected for their relatively stable thyroid hormone availability, either side of the major dynamic phase represented by metamorphosis. TRα expression was found in both pre and post-metamorphic neurogenic regions. However, exogenous T3 treatment only increased proliferation of the fast cycling Sox3+ cell population in post-metamorphic juveniles, having no detectable effect on proliferation in pre-metamorphic tadpoles. We hypothesised that the resistance of proliferative cells to exogenous T3 in pre-metamorphic tadpoles could be related to T3 inactivation by the inactivating Deiodinase 3 enzyme. Expression of dio3 was widespread in the tadpole neurogenic niche, but not in the juvenile neurogenic niche. Use of a T3-reporter transgenic line showed that in juveniles, T3 had a direct transcriptional effect on rapid cycling progenitors. Thus, the fast cycling progenitor cells in the neurogenic niche of tadpoles and juvenile frogs respond differentially to T3 as a function of developmental stage.

Cross-talk between signal transducer and activator of transcription (Stat5) and thyroid hormone receptor-beta 1 (TRbeta1) signaling pathways.

PRL and T3 are involved in antagonistic regulations during various developmental processes in vertebrate species. We have studied cross-talk between transcription factors activated by these signaling pathways, i.e. signal transducer and activator of transcription 5 (Stat5) and thyroid hormone receptor beta1 (TRbeta1). Liganded TRbeta1 in the presence of its heterodimeric partner, retinoid X receptor gamma (RXRgamma), inhibited the PRL-induced Stat5a- and Stat5b-dependent reporter gene expression by up to 60%. This T3-inhibitory effect studied on Stat5 activity was partly reversed by overexpression of a TRbeta1 dominant negative variant mutated within its nuclear localization signal (TR2A). We next showed that TRbeta1 and TR2A in the presence of RXRgamma increased and decreased, respectively, Stat5 localization into the nucleus regardless of hormonal stimulation. Thus, our data suggest that TRbeta1 can be associated with Stat5 in the cytoplasm and may be involved in Stat5 nuclear translocation. In PRL-treated cells overexpressing TRbeta1/RXRgamma, both Stat5 and TRbeta1 were coimmunoprecipitated, indicating physical association of the two transcription factors. In these cells, addition of T3 with ovine (o)PRL decreased the amounts of total and tyrosine-phosphorylated Stat5 in the cytoplasm compared with oPRL-treated cells. In the nucleus, no clear difference was observed on Stat5 DNA-binding after treatment with PRL and T3 vs. PRL alone in TRbeta1/RXRgamma transfected cells. However, antibodies directed against TRbeta1 lowered Stat5-DNA binding and addition of the deacetylase inhibitor trichostatin A (TSA) relieved T3 inhibition on Stat5 transcriptional activity. Thus, we postulated that the negative cross-talk between TR and Stat5 on target genes could involve histone deacetylase recruitment by liganded TRbeta1.

Assignment of the beta-thyroid hormone receptor to 3,5,3'-triiodothyronine-dependent inhibition of transcription from the thyrotropin-releasing hormone promoter in chick hypothalamic neurons.

Thyroid hormone, T3, is essential to the normal development and metabolism of vertebrates. Fine tuning of circulating levels of T3 is critical and involves feedback inhibition of the TRH and TSH genes by T3 at the hypothalamic and hypophyseal levels. However, the molecular basis of T3 inhibition of TRH gene expression in the hypothalamus is not known. The actions of T3 on target gene expression are mediated through nuclear receptor proteins, TR alpha and TR beta. To examine their effects on T3-dependent transcription from the rat TRH promoter, we used a gene transfer technique to express TR alpha and TR beta in cultured embryonic chick hypothalamic cells. Transcription from the TRH promoter construct transfected into these cultures was depressed in the presence of 10(-9) M T3. Cotransfecting TR alpha or TR beta activated transcription from the TRH promoter. However, only TR beta-dependent TRH transcription was differentially modulated by T3. Physiological concentrations of T3 decreased TR beta-dependent TRH transcription 4-fold. Thus, when T3 levels increase, TR beta mediates inhibition of TRH expression, a key step in down-regulating the hypophyseal-thyroid axis. This study demonstrates for the first time a T3-dependent differential regulation of the TRH promoter by TR beta and not TR alpha. Thus, the negative regulation of the TRH promoter in transiently transfected primary embryonic chick hypothalamic neurons provides a useful system for studying the molecular actions of thyroid hormone receptors.

T3-dependent physiological regulation of transcription in the Xenopus tadpole brain studied by polyethylenimine based in vivo gene transfer.

The formulation of cationic polymers of polyethylenimine (PEI) with plasmid DNA has been optimized to deliver genes into the Xenopus tadpole brain in vivo. Using intraventricular microinjections of 1 microl (containing 0.5 to 1 microg DNA) we show that the linear, low molecular weight polymer, 22 kDa PEI was significantly more efficient than a branched 25 kDa polymer. Complexes bearing a slightly positive net charge (formed with a ratio of 6 PEI amines per DNA phosphate) provided the best levels of transfection. Transgene expression was DNA-dose dependent and was maintained over 6 days, the time course of the experiment. Spatial distribution was examined using a beta-galactosidase construct and neurones expressing this transgene were found spread throughout the brain. The possibility of using this technique to evaluate physiological regulations was approached by examining the effects of tri-iodothyronine (T3), on transcription from the mammalian TRH and Krox-24 promoter sequences. Adding physiological concentrations of T3 to the aquarium water significantly reduced transcription from the rat TRH promoter whilst the same treatment increased transcription from a mouse Krox-24 -luciferase construct. Thus, PEI-DNA transfection provides a versatile and easily applied method for following physiological regulations at the transcriptional level in the tadpole brain.

A somatic gene transfer approach using recombinant fusion proteins to map muscle-motoneuron projections in Xenopus spinal cord.

A combination of somatic gene transfer with fusion protein technology has been developed, thus providing an innovative means of mapping muscle-motoneuronal connections in Xenopus tadpole spinal cord. We analyzed whether a neuronal tracer created by the fusion of the LacZ gene to the tetanus toxin C fragment (LacZ-TTC) could be produced from plasmid DNA injected into muscle, and whether it could be released and undergo retrograde transport into motoneurons. Plasmids encoding various fusion protein constructions, with or without a signal peptide, were injected into dorsal or caudal muscles of premetamorphic tadpoles. The marker was produced in the muscle at constantly high levels. At one month post-injection, the fusion protein passed the neuromuscular junction and underwent retrograde transport into motoneurons. Transfer into motoneurons was seen for every animal injected, emphasizing the high reproducibility and efficiency of the process. No uptake of beta-gal protein into motoneurons was observed in the absence of the TTC fragment. Furthermore, no enhancement was obtained by adding a signal peptide. These results provide the first demonstration of the synthesis and transport of a TTC fusion protein produced directly from exogenous DNA in a vertebrate system.

Gene transfer into intact vertebrate embryos.

Intact chick embryos at 40 h incubation were transfected in vivo with chimeric vectors expressing chloramphenicol acetyl transferase (CAT) under different promoter sequences. The cationic lipid, dioctadecylamidoglycyl spermine (DOGS) used as the transfecting agent had no noticeable toxic effects on embryonic development. CAT activity was monitored 48 h post-transfection on homogenates of embryos dissected free of all annexes. Of the various constructs tested, those containing the AP-1 response element linked to CAT (TRE-tk-CAT) gave high expression and consistent enzyme responses within groups. Co-transfection experiments in which embryos were exposed simultaneously to a CAT vector containing the cAMP response element and to a vector expressing the catalytic subunit of protein kinase A showed that the promoters of the introduced genes can be regulated by their respective transacting factors. This method may therefore represent a general tool for introducing genes into intact vertebrate embryos at precise developmental times.

Human longevity is characterised by high thyroid stimulating hormone secretion without altered energy metabolism.

Few studies have included subjects with the propensity to reach old age in good health, with the aim to disentangle mechanisms contributing to staying healthier for longer. The hypothalamic-pituitary-thyroid (HPT) axis maintains circulating levels of thyroid stimulating hormone (TSH) and thyroid hormone (TH) in an inverse relationship. Greater longevity has been associated with higher TSH and lower TH levels, but mechanisms underlying TSH/TH differences and longevity remain unknown. The HPT axis plays a pivotal role in growth, development and energy metabolism. We report that offspring of nonagenarians with at least one nonagenarian sibling have increased TSH secretion but similar bioactivity of TSH and similar TH levels compared to controls. Healthy offspring and spousal controls had similar resting metabolic rate and core body temperature. We propose that pleiotropic effects of the HPT axis may favour longevity without altering energy metabolism.

Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression.

Striated muscle is the only tissue found to be capable of taking up and expressing reporter genes that are transferred in the form of plasmid DNA. Thus, direct gene transfer is a potential method of gene therapy for the primary inherited myopathies. However, results to date have had insufficient and too variable expression to consider using direct gene transfer in human trials. We have determined that much of the variability of expression is due to nonuniform distribution of substances injected into skeletal muscle in vivo, and have developed a model to ameliorate this. Preinjection of muscles with a relatively large volume of hypertonic sucrose improves the distribution of injected substances and results in significantly less variable expression of reporter genes for luciferase or beta-galactosidase; the coefficient of variation for mean luciferase activity was reduced from about 120% to 25%. Expression is not directly proportional to dose, but is more so if the muscles are preinjected with sucrose than not. Expression is higher and less variable if DNA is injected in a larger than a smaller volume. The choice of promoter appears to be particularly important. Luciferase reporter gene expression from the SV40 promoter was transient and low, whereas expression driven by the Rous sarcoma virus (RSV) promoter was high and sustained, such that a 1,000-fold difference in expression could be observed. The mechanism of gene uptake is still unknown, but our findings indicate that fibers damaged by the injection procedure do not take up and express plasmid DNA.

Plasmid DNA is superior to viral vectors for direct gene transfer into adult mouse skeletal muscle.

Direct gene transfer into skeletal muscle offers several therapeutic possibilities. We assessed direct intramuscular injection of recombinant plasmids, adenovirus, or retrovirus in normal or regenerating muscles of mice. The incorporation and expression of reporter genes introduced by any of these three vectors is greater in regenerating than in mature muscle. In regenerating muscle, pure DNA and adenovirus result in equivalent numbers of fibers expressing reporter gene (> 10%), but adenovirus also induces considerable cellular infiltration. In mature muscle, recombinant DNA is better than adenovirus. Retrovirus failed to infect mature muscle fibers and was less effective than plasmid DNA or adenovirus in regenerating muscle. The surprisingly high relative efficiency of pure plasmid DNA suggests that this method will provide a simple, safe and viable alternative for gene therapy involving muscle tissue.

Lipospermine-based gene transfer into the newborn mouse brain is optimized by a low lipospermine/DNA charge ratio.

Nonviral, plasmid-based gene transfer into somatic tissues offers the prospect of various simple and safe therapeutic possibilities as well as applications in fundamental research. Although cationic lipids display efficient transfection activities in many in vitro systems, only low success rates using these vectors in vivo have been reported. We succeeded in defining conditions providing high levels of in vivo transfection in the brains of newborn mice. Our hypothesis was that conditions favorable for in vitro transfection (highly positively charged particles) were unlikely to be appropriate for in vivo conditions. When using the cationic lipid dioctadecylamido glycylspermine (Transfectam, DOGS) with a cytomegalovirus (CMV)-luciferase reporter gene, the best levels of transfection were obtained when using a low ratio of positive charges (supplied by the DOGS) to negative charges (carried by the DNA). Moreover, addition of the neutral lipid dioleoylphosphatidyl ethanolamine (DOPE) significantly enhanced transfection. Expression of the transgene diminished over time, independently of lipopolysaccharide content of the plasmid preparation used. This suggests that either a mitotic population of cells was preferentially transfected, or that promoter silencing was occurring. Histological examination of the spatial distribution of a beta-galactosidase-expressing transgene showed numerous groups of transfected cells both within the striatal parenchyma and in the paraventricular area. Thus, DNA-lipid complexes bearing overall charges close to neutrality open promising possibilities for modulating gene expression in the developing central nervous system and for therapy in the brain.

A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: polyethylenimine.

Nonviral gene transfer into the central nervous system (CNS) offers the prospect of providing safe therapies for neurological disorders and manipulating gene expression for studying neuronal function. However, results reported so far have been disappointing. We show that the cationic polymer polyethylenimine (PEI) provides unprecedentedly high levels of transgene expression in the mature mouse brain. Three different preparations of PEI (25-, 50-, and 800-kD) were compared for their transfection efficiencies in the brains of adult mice. The highest levels of transfection were obtained with the 25-kD polymer. With this preparation, DNA/PEI complexes bearing mean ionic charge ratios closest to neutrality gave the best results. Under such conditions, and using a cytomegalovirus (CMV)-luciferase construction, we obtained up to 0.4 10(6) RLU/microgram DNA (equivalent to 0.4 ng of luciferase), which is close to the values obtained using PEI to transfect neuronal cultures and the more easily transfected newborn mouse brain (10(6) RLU/microgram DNA). Widespread expression (over 6 mm3) of marker (luciferase) or functional genes (bcl2) was obtained in neurons and glia after injection into the cerebral cortex, hippocampus, and hypothalamus. Transgene expression was found more than 3 months post-injection in cortical neurons. No morbidity was observed with any of the preparations used. Thus, PEI, a low-toxicity vector, appears to have potential for fundamental research and genetic therapy of the brain.

Use of heterologous DNA-based gene transfer to follow physiological, T3-dependent regulation of myosin heavy chain genes in Xenopus tadpoles.

In vivo gene transfer and RNase protection assay were used to follow thyroid hormone (T3)-dependent regulation of myosin heavy chain (myHC) genes in Xenopus tadpole dorsal muscle. One embryonic and one adult myHC form were measured by each approach. RNase protection assay showed that T3 decreased expression of endogenous embryonic mRNA (E3), but increased adult (A7) transcripts. Gene transfer showed that T3 exerted transcriptional effects on mammalian embryonic and adult myHc promoters injected into the same muscle. The kinetics and profiles of the transcriptional responses were superimposable on endogenous responses. The results strengthen the use of in vivo approaches for determining the roles of transcription factors and cis-regulatory sequences in integrated contexts.

Nuclear corepressor and silencing mediator of retinoic and thyroid hormone receptors corepressor expression is incompatible with T(3)-dependent TRH regulation.

Ligand-independent repression by thyroid hormone (T(3)) receptors on positive T(3)-responsive genes requires corepressor proteins. However, the role of corepressors in regulating genes such as hypothalamic TRH, which are under negative control by T(3), is largely unknown. We examined the expression of mRNAs encoding the corepressors NCoR (nuclear corepressor) and SMRT (silencing mediator of retinoic and thyroid hormone receptors) in the TRH-producing paraventricular nucleus of the mouse hypothalamus. Further, we carried out in vivo functional studies by overexpression of both corepressors. Three lines of evidence show that NCoR and SMRT expression is incompatible with physiological regulation of TRH. First, Northern blotting revealed TRH and NCoR mRNA expressions to be inversely correlated during postnatal development and as a function of thyroid status. Second, in situ hybridization showed that NCoR and SMRT mRNA expression profiles in the paraventricular nucleus were distinct from that of TRH mRNA. Third, over-expression of full length NCoR and SMRT in the hypothalamus abolished T(3)-dependent repression of TRH-luciferase. However, over-expression of NCoR or SMRT did not affect either T(3)-independent activation of TRH-luciferase transcription, or transcription from a positively regulated T(3)-response element. We conclude that T(3) -dependent feedback on TRH expression is unlikely to involve the corepressors NCoR or SMRT.

Thyroid status co-regulates thyroid hormone receptor and co-modulator genes specifically in the hypothalamus.

Regulation of Thyrotropin Releasing Hormone (TRH) transcription in the hypothalamus represents the central control point of thyroid function. To examine the expression of potential TRH regulatory components, we simultaneously amplified, by semi-quantitative multiplex PCR system, nine key genes from < or = 100 ng total RNA from two brain areas (hypothalamus and cortex) under different thyroid states. Expression of TR1 and TR2 isoforms, key elements in TRH regulation, was modified by thyroid status in the hypothalamus but not in the cortex. Similarly, hypothyroidism increased specifically hypothalamic levels of three co-modulator genes. This study provides the first demonstration of tissue specific co-regulation of a set of genes by thyroid status within a defined brain area.

GABAA and GABAB receptors on porcine pars intermedia cells in primary culture: functional role in modulating peptide release.

A primary culture of porcine pars intermedia cells with particularly high yields has been developed. The cells, grown in monolayers, secrete the pro-opiomelanocortin-derived peptide alpha-melanocyte-stimulating hormone over several weeks. The patch-clamp technique has been used to demonstrate the presence of gamma-aminobutyrateA (GABAA) receptors on the cells. GABA or the selective GABAA receptor agonist isoguvacine produced a depolarizing increase in chloride conductance that desensitized rapidly. The response was antagonized by bicuculline and by the aminopyridazine derivative of GABA (SR 95103), a novel GABAA receptor antagonist. The effects of specific agonists for each receptor were tested on peptide release from cells maintained in a perfusion system. Isoguvacine (10 microM) potentiated Ba2+-evoked release of alpha-melanocyte-stimulating hormone, whereas (-)-baclofen (50 microM) decreased both basal and stimulated hormone release. This negative effect on peptide secretion was reproduced when GABA (50 microM) was perfused in the presence of bicuculline (10 microM) to block GABAA receptor activation. The possible mechanisms underlying these GABAA and GABAB effects on stimulus-secretion coupling in this neuroendocrine model are discussed.

GABAB receptors negatively regulate transcription in cerebellar granular neurons through cyclic AMP responsive element binding protein-dependent mechanisms.

GABAB receptors affect short-term signalling in various cell types. However, nothing is known about possible long-term effects on transcription. To analyse such effects in the CNS, we studied GABAB receptor-mediated gene regulation in primary cultures of cerebellar granule neurons. Transcription was followed using a chloramphenicol acetyl transferase reporter gene driven by the minimal cyclic AMP-responsive element (TGACGTCA). Transcription was stimulated by activation of both the cyclic AMP (forskolin: 5 x 10(-6) M) and the Ca2+ dependent (KCl: 30 mM) pathways (-)-Baclofen (10(-6) M to 10(-4) M), a specific GABAB receptor agonist, reduced by 50-70% the transcriptional stimulation evoked by both forskolin and KCl, whereas isoguvacine, a GABAA receptor agonist, was without effect. Moreover, the GABAB antagonist CGP 35348 abrogated the inhibitory effects of both GABA and baclofen, indicating that GABAB receptors were specifically implicated in this response. Measurements of cyclic AMP levels suggested that (-) baclofen inhibits forskolin-initiated transcription by reducing cyclic AMP production. Direct transcriptional activation, via the cyclic AMP pathway, by overexpression of the catalytic subunit of the cyclic AMP-dependent protein kinase, was not significantly altered by (-) baclofen. This indicates again that (-) baclofen-dependent inhibitory mechanisms operate upstream of cyclic AMP-dependent protein kinase at the level of second messenger formation. Further, we used a yeast transcriptional activator GAL4-cyclic AMP-responsive element binding protein to analyse whether GABAB receptor-mediated inhibition of cyclic AMP-responsive element transcription implicated the transacting factor cyclic AMP-responsive element binding protein. We show that the negative effects of (-) baclofen implicate this transcription factor and this holds good for both the forskolin and KCl-stimulated pathways. The results indicate that GABAB receptors negatively regulate cyclic AMP-responsive element binding protein-mediated transcription in the CNS.

Temporal and spatial expression of lipospermine-compacted genes transferred into chick embryos in vivo.

We have optimized a lipospermine-based transfection method for introducing genes into intact vertebrate embryos in vivo. The method employs small amounts of the cationic lipid Transfectam (DOGS), in a concentrated (40 mM) ethanolic solution, to compact and to transfer exogenous genes into chick embryos during the early stages of development (< 36 h of incubation). Plasmid vectors containing the reporter gene luciferase were used to follow the time course of expression. Luciferase activity was detected as early as 12 h post-transfection and was highest at this time. Enzyme activity then decreased over the next two days and was usually undetectable by 72-h post-transfection. To follow the spatial expression of the exogenous genes, a Rous sarcoma virus (RSV)-beta-galactosidase vector was used. When the transfection complex was applied externally around the developing embryo, the main site of expression was the cardiac tissue. Expression could be targeted to the nervous system by micro-injecting the DNA/DOGS (DNA/dioctadecylamidoglycylspermine) complex into the developing brain. The results show that reporter genes can be efficiently expressed in both the developing central nervous system and heart. This raises the possibility that lipospermines can be used to transfer functional genes into embryos during defined periods of development and also to deliver genes in other species and in other in vivo contexts.