Inducible endothelial cell-specific gene expression in transgenic mouse embryos and adult mice

Utilizing both the TET-OFF and TET-ON systems in combination with transcriptional control elements of the Tie-2 gene, we have established a series of transgenic activator and responder mice for TET-regulated endothelial cell-specific transgene expression in double transgenic mouse embryos and in adult mice. TET-regulated expression of LacZ reporter genes could be achieved in virtually all endothelia in mid gestation stage mouse embryos. In contrast in adult mice, using the very same Tie-2 tTA activator mouse strain, we observed striking differences of TET-induced gene expression from various inducible expression constructs in different vascular beds. Non-endothelial expression was never detected. The prominent differences in completeness of TET-induced endothelial expression highlight the still underestimated critical role of the responder mouse lines for uniform TET-induced gene expression in heterogeneous cell populations such as endothelial cells. Interestingly, in double transgenic mice inducibly expressing several different adhesion molecules, no adverse effects were observed even though these proteins were robustly expressed on endothelial cells in adult tissues. These transgenic model systems provide versatile tools for the TET-regulated manipulation of endothelial cell-specific gene expression in the entire embryonic vasculature and distinct vascular beds in adult mice.

What is to be done with surplus embryos? Attitude formation with ambivalence in German fertility patients

Improved pregnancy rates in IVF have led to increasing numbers of surplus embryos which can potentially be used for purposes like donation to another infertile couple or further research. Individuals report high levels of ambivalence concerning the donation of surplus embryos. This study examined which strategies infertile patients use to deal with this ambivalence when asked to evaluate potential dispositions of surplus embryos created during IVF. Guideline-based interviews with fertility patients were audio-recorded and transcribed verbatim. Following the principle of theoretical sampling, eight interviews were analysed by use of Grounded Theory. Analyses focused on processes of individual attitude formation. Strategies for handling ambivalence during attitude formation were identified: the six strategies comprise cognitive and communicative strategies, and were integrated into a model of attitude formation under ambivalence. As ambivalence is a relevant phenomenon in attitude formation within IVF treatment, assessment of ambivalence is strongly recommended in social science studies investigating ethical problems in patient care. In the context of informed consent, there is a need for individual counselling which draws attention to the conflicting values during attitude formation. Counsellors should be aware of the signs of and the strategies to deal with ambivalence.

Estrogen receptor subtype beta2 is involved in neuromast development in zebrafish (Danio rerio) larvae

Estrogens are known to play a role in both reproductive and non-reproductive functions in mammals. Estrogens and their receptors are involved in the development of the central nervous system (brain development, neuronal survival and differentiation) as well as in the development of the peripheral nervous system (sensory-motor behaviors). In order to decipher possible functions of estrogens in early development of the zebrafish sensory system, we investigated the role of estrogen receptor beta(2) (ERbeta(2)) by using a morpholino (MO) approach blocking erbeta(2) RNA translation. We further investigated the development of lateral line organs by cell-specific labeling, which revealed a disrupted development of neuromasts in morphants. The supporting cells developed and migrated normally. Sensory hair cells, however, were absent in morphants' neuromasts. Microarray analysis and subsequent in situ hybridizations indicated an aberrant activation of the Notch signaling pathway in ERbeta(2) morphants. We conclude that signaling via ERbeta(2) is essential for hair cell development and may involve an interaction with the Notch signaling pathway during cell fate decision in the neuromast maturation process.

Zebrafish (Danio rerio) neuromast: promising biological endpoint linking developmental and toxicological studies

Aquatic toxicology is facing the challenge to assess the impact of complex mixtures of compounds on diverse biological endpoints. So far, ecotoxicology focuses mainly on apical endpoints such as growth, lethality and reproduction, but does not consider sublethal toxic effects that may indirectly cause ecological effects. One such sublethal effect is toxicant-induced impairment of neurosensory functions which will affect important behavioural traits of exposed organisms. Here, we critically review the mechanosensory lateral line (LL) system of zebrafish as a model to screen for chemical effects on neurosensory function of fish in particular and vertebrates in general. The LL system consists of so-called neuromasts, composed of centrally located sensory hair cells, and surrounding supporting cells. The function of neuromasts is the detection of water movements that is essential for the fish's ability to detect prey, to escape predator, to socially interact or to show rheotactic behaviour. Recent advances in the study of these organs provided researchers with a broad area of molecular tools for easy and rapid detection of neuromasts dysfunction and/or disturbed development. Further, genes involved in neuromasts differentiation have been identified using auditory/mechanosensory mutants and morphants. A number of environmental toxicants including metals and pharmaceuticals have been shown to affect neuromasts development and/or function. The use of the LL organ for toxicological studies offers the advantage to integrate the available profound knowledge on developmental biology of the neuromasts with the study of chemical toxicity. This combination may provide a powerful tool in environmental risk assessment.

Background pathology of the ovary in a laboratory population of zebrafish Danio rerio

Adult zebrafish Danio rerio originating from one stock used as control animals in a toxicological study were examined histopathologically for the occurrence of spontaneous lesions in the gonads. While no histopathological changes were seen in the testes, the ovaries showed lesions consisting mainly of acute granulomatous inflammation with increased atresia and the presence of egg debris in the ovarian parenchyma and in the oviduct. Since infectious agents could not be detected and the fish were not exposed to toxicants, we consider these lesions as spontaneous alterations of the ovaries.

Characteristics of rod regeneration in a novel zebrafish retinal degeneration model using N-methyl-N-nitrosourea (MNU)

Primary loss of photoreceptors caused by diseases such as retinitis pigmentosa is one of the main causes of blindness worldwide. To study such diseases, rodent models of N-methyl-N-nitrosourea (MNU)-induced retinal degeneration are widely used. As zebrafish (Danio rerio) are a popular model system for visual research that offers persistent retinal neurogenesis throughout the lifetime and retinal regeneration after severe damage, we have established a novel MNU-induced model in this species. Histology with staining for apoptosis (TUNEL), proliferation (PCNA), activated Müller glial cells (GFAP), rods (rhodopsin) and cones (zpr-1) were performed. A characteristic sequence of retinal changes was found. First, apoptosis of rod photoreceptors occurred 3 days after MNU treatment and resulted in a loss of rod cells. Consequently, proliferation started in the inner nuclear layer (INL) with a maximum at day 8, whereas in the outer nuclear layer (ONL) a maximum was observed at day 15. The proliferation in the ONL persisted to the end of the follow-up (3 months), interestingly, without ongoing rod cell death. We demonstrate that rod degeneration is a sufficient trigger for the induction of Müller glial cell activation, even if only a minimal number of rod cells undergo cell death. In conclusion, the use of MNU is a simple and feasible model for rod photoreceptor degeneration in the zebrafish that offers new insights into rod regeneration.

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.

Methylnitrosourea (MNU)-induced retinal degeneration and regeneration in the zebrafish: histological and functional characteristics.

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial world. Animal models are of pivotal importance to study such diseases. In this regard the photoreceptor-specific toxin N-methyl-N-nitrosourea (MNU) has been widely used in rodents to pharmacologically induce retinal degeneration. Previously, we have established a MNU-induced retinal degeneration model in the zebrafish, another popular model system in visual research. A fascinating difference to mammals is the persistent neurogenesis in the adult zebrafish retina and its regeneration after damage. To quantify this observation we have employed visual acuity measurements in the adult zebrafish. Thereby, the optokinetic reflex was used to follow functional changes in non-anesthetized fish. This was supplemented with histology as well as immunohistochemical staining for apoptosis (TUNEL) and proliferation (PCNA) to correlate the developing morphological changes. In summary, apoptosis of photoreceptors occurs three days after MNU treatment, which is followed by a marked reduction of cells in the outer nuclear layer (ONL). Thereafter, proliferation of cells in the inner nuclear layer (INL) and ONL is observed. Herein, we reveal that not only a complete histological but also a functional regeneration occurs over a time course of 30 days. Now we illustrate the methods to quantify and follow up zebrafish retinal de- and regeneration using MNU in a video-format.

Persistence of endocrine disruption in zebrafish (Danio rerio) after discontinued exposure to the androgen 17β-trenbolone.

The aim of the present study was to investigate the effects of the androgenic endocrine disruptor 17β-trenbolone on the sexual development of zebrafish (Danio rerio) with special emphasis on the question of whether adverse outcomes of developmental exposure are reversible or persistent. An exposure scenario including a recovery phase was chosen to assess the potential reversibility of androgenic effects. Zebrafish were exposed to environmentally relevant concentrations of 17β-trenbolone (1 ng/L-30 ng/L) from fertilization until completion of gonad sexual differentiation (60 d posthatch). Thereafter, exposure was either followed by 40 d of recovery in clean water or continued until 100 d posthatch, the age when zebrafish start being able to reproduce. Fish exposed for 100 d to 10 ng/L or 30 ng/L 17β-trenbolone were masculinized at different biological effect levels, as evidenced from a concentration-dependent shift of the sex ratio toward males as well as a significantly increased maturity of testes. Gonad morphological masculinization occurred in parallel with decreased vitellogenin concentrations in both sexes. Changes of brain aromatase (cyp19b) mRNA expression showed no consistent trend with respect to either exposure duration or concentration. Gonad morphological masculinization as well as the decrease of vitellogenin persisted after depuration over 40 d in clean water. This lack of recovery suggests that androgenic effects on sexual development of zebrafish are irreversible.

Reversibility of endocrine disruption in zebrafish (Danio rerio) after discontinued exposure to the estrogen 17α-ethinylestradiol.

The aim of the present study was to investigate the persistence of the feminizing effects of discontinued 17α-ethinylestradiol (EE2) exposure on zebrafish (Danio rerio). An exposure scenario covering the sensitive phase of sexual differentiation, as well as final gonad maturation was chosen to examine the estrogenic effects on sexual development of zebrafish. Two exposure scenarios were compared: continuous exposure to environmentally relevant concentrations (0.1-10 ng/L EE2) up to 100 days post-hatch (dph) and developmental exposure up to 60 dph, followed by 40 days of depuration in clean water. The persistence of effects was investigated at different biological organization levels from mRNA to population-relevant endpoints to cover a broad range of important parameters. EE2 had a strong feminizing and inhibiting effect on the sexual development of zebrafish. Brain aromatase (cyp19b) mRNA expression showed no clear response, but vitellogenin levels were significantly elevated, gonad maturation and body growth were inhibited in both genders, and sex ratios were skewed towards females and undifferentiated individuals. To a large extent, all of these effects were reversed after 40 days of recovery, leading to the conclusion that exposure to the estrogen EE2 results in very strong, but reversible underdevelopment and feminization of zebrafish. The present study is the first to show this reversibility at different levels of organization, which gives better insight into the mechanistic basis of estrogenic effects in zebrafish.

Photopic and scotopic spatiotemporal tuning of adult zebrafish vision.

Sensitivity to spatial and temporal patterns is a fundamental aspect of vision. Herein, we investigated this sensitivity in adult zebrafish for a wide range of spatial (0.014 to 0.511 cycles/degree [c/d]) and temporal frequencies (0.025 to 6 cycles/s) to better understand their visual system. Measurements were performed at photopic (1.8 log cd m(-2)) and scotopic (-4.5 log cd m(-2)) light levels to assess the optokinetic response (OKR). The resulting spatiotemporal contrast sensitivity (CS) functions revealed that the OKR of zebrafish is tuned to spatial frequency and speed but not to temporal frequencies. Thereby, optimal test parameters for CS measurements were identified. At photopic light levels, a spatial frequency of 0.116 ± 0.01 c/d (mean ± SD) and a grating speed of 8.42 ± 2.15 degrees/second (d/s) was ideal; at scotopic light levels, these values were 0.110 ± 0.02 c/d and 5.45 ± 1.31 d/s, respectively. This study allows to better characterize zebrafish mutants with altered vision and to distinguish between defects of rod and cone photoreceptors as measurements were performed under different light conditions.

Telomerase Is Essential for Zebrafish Heart Regeneration.

After myocardial infarction in humans, lost cardiomyocytes are replaced by an irreversible fibrotic scar. In contrast, zebrafish hearts efficiently regenerate after injury. Complete regeneration of the zebrafish heart is driven by the strong proliferation response of its cardiomyocytes to injury. Here we show that, after cardiac injury in zebrafish, telomerase becomes hyperactivated, and telomeres elongate transiently, preceding a peak of cardiomyocyte proliferation and full organ recovery. Using a telomerase-mutant zebrafish model, we found that telomerase loss drastically decreases cardiomyocyte proliferation and fibrotic tissue regression after cryoinjury and that cardiac function does not recover. The impaired cardiomyocyte proliferation response is accompanied by the absence of cardiomyocytes with long telomeres and an increased proportion of cardiomyocytes showing DNA damage and senescence characteristics. These findings demonstrate the importance of telomerase function in heart regeneration and highlight the potential of telomerase therapy as a means of stimulating cell proliferation upon myocardial infarction.

The Epicardium in the Embryonic and Adult Zebrafish

The epicardium is the mesothelial outer layer of the vertebrate heart. It plays an important role during cardiac development by, among other functions, nourishing the underlying myocardium, contributing to cardiac fibroblasts and giving rise to the coronary vasculature. The epicardium also exerts key functions during injury responses in the adult and contributes to cardiac repair. In this article, we review current knowledge on the cellular and molecular mechanisms underlying epicardium formation in the zebrafish, a teleost fish, which is rapidly gaining status as an animal model in cardiovascular research, and compare it with the mechanisms described in other vertebrate models. We moreover describe the expression patterns of a subset of available zebrafish Wilms' tumor 1 transgenic reporter lines and discuss their specificity, applicability and limitations in the study of epicardium formation.

Report of the Second European Zebrafish Principal Investigator Meeting in Karlsruhe, Germany, March 21-24, 2012.

The second European Zebrafish Principal Investigator (PI) Meeting was held in March, 2012, in Karlsruhe, Germany. It brought together PIs from all over Europe who work with fish models such as zebrafish and medaka to discuss their latest results, as well as to resolve strategic issues faced by this research community. Scientific discussion ranged from the development of new technologies for working with fish models to progress in various fields of research such as injury and repair, disease models, and cell polarity and dynamics. This meeting also marked the establishment of the European Zebrafish Resource Centre (EZRC) at Karlsruhe that in the future will serve as an important focus and community resource for zebrafish- and medaka-based research.

Pan-epicardial lineage tracing reveals that epicardium derived cells give rise to myofibroblasts and perivascular cells during zebrafish heart regeneration.

Myocardial infarction (MI) leads to a severe loss of cardiomyocytes, which in mammals are replaced by scar tissue. Epicardial derived cells (EPDCs) have been reported to differentiate into cardiomyocytes during development, and proposed to have cardiomyogenic potential in the adult heart. However, mouse MI models reveal little if any contribution of EPDCs to myocardium. In contrast to adult mammals, teleosts possess a high myocardial regenerative capacity. To test if this advantage relates to the properties of their epicardium, we studied the fate of EPDCs in cryoinjured zebrafish hearts. To avoid the limitations of genetic labelling, which might trace only a subpopulation of EPDCs, we used cell transplantation to track all EPDCs during regeneration. EPDCs migrated to the injured myocardium, where they differentiated into myofibroblasts and perivascular fibroblasts. However, we did not detect any differentiation of EPDCs nor any other non-cardiomyocyte population into cardiomyocytes, even in a context of impaired cardiomyocyte proliferation. Our results support a model in which the epicardium promotes myocardial regeneration by forming a cellular scaffold, and suggests that it might induce cardiomyocyte proliferation and contribute to neoangiogenesis in a paracrine manner.