Fluorescent in situ hybridization (FISH) provides sensitive detection and visualization of RNA transcripts in tissues and cells with high resolution. We present here a multiplex RNA FISH method using enhanced tyramide signal amplification (TSA) for colocalization analysis of three different transcripts in intact zebrafish brains. To achieve enhancement of fluorescent signals, essential steps of the FISH procedure are optimized including embryo permeability, hybridization efficacy, and fluorogenic TSA-reaction conditions. Critical to this protocol, the enzymatic peroxidase (PO) reactivity is significantly improved by the application of viscosity-increasing polymers, PO accelerators, and highly effective bench-made tyramide substrates. These advancements lead to an optimized TSA-FISH protocol with dramatically increased signal intensity and signal-to-background ratio allowing for visualization of three mRNA transcript patterns simultaneously. The TSA-FISH procedure can be combined with immunofluorescence (IF) to compare mRNA transcript and protein expression patterns.

Apoptosis, differentiation and proliferation have to be tightly balanced and thus regulated to maintain tissue homeostasis. Stress, metabolic cues, genetic variability, infections and physiological host-commensal interactions influence this balance and thus need to be integrated. Therefore, beyond the discrimination between self and non-self (i.e., foreign) also damage inflicted on tissues under sterile conditions is perceived by the immune system due to altered tissue integrity. Growing knowledge of the interaction between the immune system and wounded or more generally altered tissues allows inferring on anti-tumorous immune responses, too. Despite the lack of adaptive immunity, Drosophila mounts solid and versatile innate immune responses that functionally and molecularly share many properties with their vertebrate counterparts. In fact, tissue overgrowth, tissue dysplasia or endogenous danger signaling activate systemic Toll-signaling in the fat body indicating a role for the Drosophila immune system in maintaining tissue homeostasis.Here we characterize systemic and local immune responses towards altered or transformed tissues by using a Drosophila hypertrophy model, which is based on the overexpression of a dominant-active variant of the small GTPase Ras (Ras85DG12V) in salivary glands and wing discs. We characterized the strong induction of hemocyte recruitment to the glands as a consequence of JNK-dependent MMP1-expression and basal membrane degradation. Apart from this cellular immune reaction, transcriptome profiling revealed comprehensive humoral immune responses mounted by the fat body that involved signatures of Toll- and imd-activation. Moreover, a novel tissue-autonomous response that was spatially restricted to the anterior end of the RasV12-expressing salivary gland itself was identified. While multiple immune genes were found to be upregulated in the anterior compartment as detected by RNA sequencing, particular focus was given to the effector peptide Drosomycin (Drs). Overexpression of Drs with RasV12 in the entire gland similar to the inhibition of the JNK-pathway was able to selectively rescue a characteristic set of RasV12-induced phenotypes, which ultimately blocks the recruitment of hemocytes. Thereby, local immune-related responses in RasV12-expressing salivary glands are able to restrict the tissue damage induced by hypertrophic growth.

Apoptosis, differentiation and proliferation have to be tightly balanced and thus regulated to maintain tissue homeostasis. Stress, metabolic cues, genetic variability, infections and physiological host-commensal interactions influence this balance and thus need to be integrated. Therefore, beyond the discrimination between self and non-self (i.e., foreign) also damage inflicted on tissues under sterile conditions is perceived by the immune system due to altered tissue integrity. Growing knowledge of the interaction between the immune system and wounded or more generally altered tissues allows inferring on anti-tumorous immune responses, too. Despite the lack of adaptive immunity, Drosophila mounts solid and versatile innate immune responses that functionally and molecularly share many properties with their vertebrate counterparts. In fact, tissue overgrowth, tissue dysplasia or endogenous danger signaling activate systemic Toll-signaling in the fat body indicating a role for the Drosophila immune system in maintaining tissue homeostasis.Here we characterize systemic and local immune responses towards altered or transformed tissues by using a Drosophila hypertrophy model, which is based on the overexpression of a dominant-active variant of the small GTPase Ras (Ras85DG12V) in salivary glands and wing discs. We characterized the strong induction of hemocyte recruitment to the glands as a consequence of JNK-dependent MMP1-expression and basal membrane degradation. Apart from this cellular immune reaction, transcriptome profiling revealed comprehensive humoral immune responses mounted by the fat body that involved signatures of Toll- and imd-activation. Moreover, a novel tissue-autonomous response that was spatially restricted to the anterior end of the RasV12-expressing salivary gland itself was identified. While multiple immune genes were found to be upregulated in the anterior compartment as detected by RNA sequencing, particular focus was given to the effector peptide Drosomycin (Drs). Overexpression of Drs with RasV12 in the entire gland similar to the inhibition of the JNK-pathway was able to selectively rescue a characteristic set of RasV12-induced phenotypes, which ultimately blocks the recruitment of hemocytes. Thereby, local immune-related responses in RasV12-expressing salivary glands are able to restrict the tissue damage induced by hypertrophic growth.

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

In situ hybridization (ISH) has become an invaluable tool for the detection of RNA in cells, tissues and organisms. Due to improvements in target and signal amplification and in probe design remarkable progress has been made concerning sensitivity, specificity and resolution of chromogenic and fluorescent ISH (FISH). These advancements allow for exquisite cellular and sub-cellular resolution and for detecting multiple RNA species at a time by multiplexing. In zebrafish (F)ISH non-enzymatic and enzymatic amplification systems have been employed to obtain enhanced signal intensities and signal-to-noise ratios. These amplification strategies include branched DNA-based RNAscope and in situ hybridization chain reaction (HCR) techniques, as well as alkaline phosphatase (AP)- and horseradish peroxidase (PO)-based immunoassays. For practical application, we provide proven multiplex FISH protocols for AP- and PO-based visualization of mRNAs at high resolution. The protocols take advantage of optimized tyramide signal amplification (TSA) conditions of the PO assay and long-lasting high signal-to-noise ratio of the AP reaction, thereby enabling detection of less abundant transcripts.

This volume contains a comprehensive compilation of chromogenic and fluorescent RNA in situ hybridization (ISH) technology in many of its various shades, forms, and applications. The book is organized

We report a dual fluorescent in situ hybridization (FISH) method for direct comparison of cellular distributions of different gene transcripts in the embryonic zebrafish brain and other tissues. After simultaneous hybridization of two differently labeled antisense RNA probes, the different hapten labels are visualized by peroxidase (POD)-mediated deposition of fluorochrome-labeled tyramides and alkaline phosphatase-based Fast Blue or Fast Red chromogenic staining, respectively. Since chromogenic Fast Blue and Fast Red precipitates display red-fluorescent emission, multiplexed visualization of different transcripts is possible by combination with carboxyfluorescein-labeled tyramides, which show emission in the green spectrum. The application of differential reporter enzymes provides advantages over procedures using sequential POD detection. The POD-coupled and AP-coupled antibodies can be mixed together in a single incubation step reducing the required time, spent otherwise for extensive washings and multiple incubations. In addition, removal or inactivation of antibody-POD conjugates as required in sequential POD detection procedures can be omitted. Therefore, potential false-positive detection of co-localization by insufficient inactivation is prevented.

Despite large-scale gene expression profiling studies, it is still often required to precisely characterize the localization of different transcripts in relation to each other to determine unique and overlapping expression sites at a cellular level. We describe here a versatile protocol for simultaneous examination of three unique mRNA expression patterns in Drosophila melanogaster embryos. Three differently labeled antisense RNA probes are hybridized together to the embryos and detected by sequential alkaline phosphatase-based immunohistochemistry. Transcript distributions are revealed by colorimetric enzymatic reactions that permit to highlight each mRNA expression pattern by a differing and contrasting cellular color precipitate. We provide tips and tricks for each critical step helpful for successful application of the tricolor whole-mount in situ hybridization (WISH) method.

Large-scale data collections of gene expression patterns have been assembled to construct molecular atlases of embryonic and adult vertebrate brains. These open data collections are proving useful as profound resources for developmental, physiological, and functional studies and provide a basis for deciphering the molecular anatomies of vertebrate brains. The interrogation of gene expression data from such resources allows identifying genes with restricted regional patterns and neuronal profiles. The corresponding cDNA/EST clones can be ordered from publicly accessible clone collections strongly facilitating the systematic analysis of gene expression. However, in order to characterize the expression of a large number of genes, it is necessary to develop automated procedures that allow high-throughput analysis of samples. In this chapter, we describe our approach to analyze developmental regulatory genes and neuronal markers in the embryonic zebrafish brain by chromogenic whole-mount in situ hybridization (WISH) using manual and automated procedures. For automation, we make use of the InsituPro robot, which enables automated processing of up to 96 samples in parallel. We provide detailed step-by-step protocols of embryo collection, fixation and permeabilization, cDNA template and RNA probe preparation, manual and automated in situ hybridization, immunohistochemical detection and staining, and mounting and imaging of successfully stained specimen.

During development of the early neural tube, positional information provided by signaling gradients is translated into a grid of transverse and longitudinal transcription factor expression domains. Transcription factor specification codes defining distinct histogenetic domains within this grid are evolutionarily conserved across vertebrates and may reflect an underlying common vertebrate bauplan. When compared to the rich body of comparative gene expression studies of tetrapods, there is considerably less comparative data available for teleost fish. We used sensitive multicolor fluorescent in situ hybridization to generate a detailed map of regulatory gene expression domains in the embryonic zebrafish diencephalon. The high resolution of this technique allowed us to resolve abutting and overlapping gene expression of different transcripts. We found that the relative topography of gene expression patterns in zebrafish was highly similar to those of orthologous genes in tetrapods and consistent with a three‐prosomere organization of the alar and basal diencephalon. Our analysis further demonstrated a conservation of intraprosomeric subdivisions within prosomeres 1, 2, and 3 (p1, p2, and p3). A tripartition of zebrafish p1 was identified reminiscent of precommissural (PcP), juxtacommissural (JcP), and commissural (CoP) pretectal domains of tetrapods. The constructed detailed diencephalic transcription factor gene expression map further identified molecularly distinct thalamic and prethalamic rostral and caudal domains and a prethalamic eminence histogenetic domain in zebrafish. Our comparative gene expression analysis conformed with the idea of a common bauplan for the diencephalon of anamniote and amniote vertebrates from fish to mammals. J. Comp. Neurol. 521:1093–1118, 2013. © 2012 Wiley Periodicals, Inc.

Cellular functions and survival are dependent on a tightly controlled redox potential. Currently, an increasing amount of data supports the concept of local changes in the redox environment and specific redox signaling events controlling cell function. Specific protein thiol groups are the major targets of redox signaling and regulation. Thioredoxins and glutaredoxins catalyze reversible thiol-disulfide exchange reactions and are primary regulators of the protein thiol redox state. Here, we demonstrate that embryonic brain development depends on the enzymatic activity of glutaredoxin 2. Zebrafish with silenced expression of glutaredoxin 2 lost virtually all types of neurons by apoptotic cell death and the ability to develop an axonal scaffold. As demonstrated in zebrafish and in a human cellular model for neuronal differentiation, glutaredoxin 2 controls axonal outgrowth via thiol redox regulation of collapsin response mediator protein 2, a central component of the semaphorin pathway. This study provides an example of a specific thiol redox regulation essential for vertebrate embryonic development.

Background Whole-mount in situ hybridization (WISH) is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. To obtain an idea whether a novel gene might be involved in specification of a distinct brain subdivision, nucleus or neuronal lineage, it is often useful to correlate its expression with that of a known regional or neuronal marker gene. Two-color fluorescent in situ hybridization (FISH) can be used to compare different transcript distributions at cellular resolution. Conventional two-color FISH protocols require two separate rounds of horseradish peroxidase (POD)-based transcript detection, which involves tyramide signal amplification (TSA) and inactivation of the first applied antibody-enzyme conjugate before the second detection round. Results We show here that the alkaline phosphatase (AP) substrates Fast Red and Fast Blue can be used for chromogenic as well as fluorescent visualization of transcripts. To achieve high signal intensities we optimized embryo permeabilization properties by hydrogen peroxide treatment and hybridization conditions by application of the viscosity-increasing polymer dextran sulfate. The obtained signal enhancement allowed us to develop a sensitive two-color FISH protocol by combining AP and POD reporter systems. We show that the combination of AP-Fast Blue and POD-TSA-carboxyfluorescein (FAM) detection provides a powerful tool for simultaneous fluorescent visualization of two different transcripts in the zebrafish brain. The application of different detection systems allowed for a one-step antibody detection procedure for visualization of transcripts, which significantly reduced working steps and hands-on time shortening the protocol by one day. Inactivation of the first applied reporter enzyme became unnecessary, so that false-positive detection of co-localization by insufficient inactivation, a problem of conventional two-color FISH, could be eliminated. Conclusion Since POD activity is rather quickly quenched by substrate excess, less abundant transcripts can often not be efficiently visualized even when applying TSA. The use of AP-Fast Blue fluorescent detection may provide a helpful alternative for fluorescent transcript visualization, as the AP reaction can proceed for extended times with a high signal-to-noise ratio. Our protocol thus provides a novel alternative for comparison of two different gene expression patterns in the embryonic zebrafish brain at a cellular level. The principles of our method were developed for use in zebrafish but may be easily included in whole-mount FISH protocols of other model organisms.

BACKGROUND:In recent years, mapping of overlapping and abutting regulatory gene expression domains by chromogenic two-color in situ hybridization has helped define molecular subdivisions of the developing vertebrate brain and shed light on its basic organization. Despite the benefits of this technique, visualization of overlapping transcript distributions by differently colored precipitates remains difficult because of masking of lighter signals by darker color precipitates and lack of three-dimensional visualization properties. Fluorescent detection of transcript distributions may be able to solve these issues. However, despite the use of signal amplification systems for increasing sensitivity, fluorescent detection in whole-mounts suffers from rapid quenching of peroxidase (POD) activity compared to alkaline phosphatase chromogenic reactions. Thus, less strongly expressed genes cannot be efficiently detected.RESULTS:We developed an optimized procedure for fluorescent detection of transcript distribution in whole-mount zebrafish embryos using tyramide signal amplification (TSA). Conditions for hybridization and POD-TSA reaction were optimized by the application of the viscosity-increasing polymer dextran sulfate and the use of the substituted phenol compounds 4-iodophenol and vanillin as enhancers of POD activity. In combination with highly effective bench-made tyramide substrates, these improvements resulted in dramatically increased signal-to-noise ratios. The strongly enhanced signal intensities permitted fluorescent visualization of less abundant transcripts of tissue-specific regulatory genes. When performing multicolor fluorescent in situ hybridization (FISH) experiments, the highly sensitive POD reaction conditions required effective POD inactivation after each detection cycle by glycine-hydrochloric acid treatment. This optimized FISH procedure permitted the simultaneous fluorescent visualization of up to three unique transcripts in different colors in whole-mount zebrafish embryos.CONCLUSIONS:Development of a multicolor FISH procedure allowed the comparison of transcript gene expression domains in the embryonic zebrafish brain to a cellular level. Likewise, this method should be applicable for mRNA colocalization studies in any other tissues or organs. The key optimization steps of this method for use in zebrafish can easily be implemented in whole-mount FISH protocols of other organisms. Moreover, our improved reaction conditions may be beneficial in any application that relies on a TSA/POD-mediated detection system, such as immunocytochemical or immunohistochemical methods.

The corticotropin‐releasing hormone (CRH) family consists of four paralogous genes, CRH and urocortins (UCNs) 1, 2, and 3. In a previous study, we analyzed CRH in the teleost model organism zebrafish and its transcript distribution in the embryonic brain. Here, we describe full‐length cDNAs encoding urotensin 1 (UTS1), the teleost UCN1 ortholog, and UCN3 of zebrafish. Major expression sites of uts1 in adult zebrafish are the caudal neurosecretory system and brain. By using RT‐PCR analysis, we show that uts1 mRNA is also present in ovary, maternally contributed to the embryo, and expressed throughout embryonic development. Expression of ucn3 mRNA was detected in a range of adult tissues and during developmental stages from 24 hours post fertilization onward. Analysis of spatial transcript distributions by whole‐mount in situ hybridization revealed limited forebrain expression of uts1 and ucn3 during early development. Small numbers of uts1 ‐synthesizing neurons were found in subpallium, hypothalamus, and posterior diencephalon, whereas ucn3 ‐positive cells were restricted to telencephalon and retina. The brainstem was the main site of uts1 and ucn3 synthesis in the embryonic brain. uts1 Expression was confined to the midbrain tegmentum; distinct hindbrain cell groups, including locus coeruleus and Mauthner neurons; and the spinal cord. ucn3 Expression was localized to the optic tectum, serotonergic raphe, and distinct rhombomeric cell clusters. The prominent expression of uts1 and ucn3 in brainstem is consistent with proposed roles of CRH‐related peptides in stress‐induced modulation of locomotor activity through monoaminergic brainstem neuromodulatory systems. J. Comp. Neurol. 518:2978–2995, 2010. © 2010 Wiley‐Liss, Inc.

Mechanisms underlying pathological angiogenesis in relation to hypoxia in tumor invasion and metastasis remain elusive. Here, we have developed a zebrafish tumor model that allows us to study the role of pathological angiogenesis under normoxia and hypoxia in arbitrating early events of the metastatic cascade at the single cell level. Under normoxia, implantation of a murine T241 fibrosarcoma into the perivitelline cavity of developing embryos of transgenic fli1:EGFP zebrafish did not result in significant dissemination, invasion, and metastasis. In marked contrast, under hypoxia substantial tumor cells disseminated from primary sites, invaded into neighboring tissues, and metastasized to distal parts of the fish body. Similarly, expression of the hypoxia-regulated angiogenic factor, vascular endothelial growth factor (VEGF) to a high level resulted in tumor cell dissemination and metastasis, which correlated with increased tumor neovascularization. Inhibition of VEGF receptor signaling pathways by sunitinib or VEGFR2 morpholinos virtually completely ablated VEGF-induced tumor cell dissemination and metastasis. To the best of our knowledge, hypoxia- and VEGF-induced pathological angiogenesis in promoting tumor dissemination, invasion, and metastasis has not been described perviously at the single cell level. Our findings also shed light on molecular mechanisms of beneficial effects of clinically available anti-VEGF drugs for cancer therapy.

Serotonin (5-HT) plays an important role in shaping the activity of the spinal networks underlying locomotion in many vertebrate preparations. At larval stages in zebrafish, 5-HT does not change the frequency of spontaneous swimming; and it only decreases the quiescent period between consecutive swimming episodes. However, it is not known whether 5-HT exerts similar actions on the locomotor network at later developmental stages. For this, the effect of 5-HT on the fictive locomotor pattern of juvenile and adult zebrafish was analyzed. Bath-application of 5-HT (1–20 μm) reduced the frequency of the NMDA-induced locomotor rhythm. Blocking removal from the synaptic cleft with the reuptake inhibitor citalopram had similar effects, suggesting that endogenous serotonin is modulating the locomotor pattern. One target for this modulation was the mid-cycle inhibition during locomotion because the IPSPs recorded in spinal neurons during the hyperpolarized phase were increased both in amplitude and occurrence by 5-HT. Similar results were obtained for IPSCs recorded in spinal neurons clamped at the reversal potential of excitatory currents (0 mV). 5-HT also slows down the rising phase of the excitatory drive recorded in spinal cord neurons when glycinergic inhibition is blocked. These results suggest that the decrease in the locomotor burst frequency induced by 5-HT is mediated by a potentiation of mid-cycle inhibition combined with a delayed onset of the subsequent depolarization.

The blood and lymphatic vasculatures are structurally and functionally coupled in controlling tissue perfusion, extracellular interstitial fluids, and immune surveillance. Little is known, however, about the molecular mechanisms that underlie the regulation of bloodlymphatic vessel connections and lymphatic perfusion. Here we show in the adult zebrafish and glass catfish ( Kryptopterus bicirrhis ) that blood-lymphatic conduits directly connect arterial vessels to the lymphatic system. Under hypoxic conditions, arterial-lymphatic conduits (ALCs) became highly dilated and linearized by NO-induced vascular relaxation, which led to blood perfusion into the lymphatic system. NO blockage almost completely abrogated hypoxia-induced ALC relaxation and lymphatic perfusion. These findings uncover mechanisms underlying hypoxia-induced oxygen compensation by perfusion of existing lymphatics in fish. Our results might also imply that the hypoxia-induced NO pathway contributes to development of progression of pathologies, including promotion of lymphatic metastasis by modulating arterial-lymphatic conduits, in the mammalian system.

Mechanistic understanding and defining novel therapeutic targets of diabetic retinopathy and age-related macular degeneration (AMD) have been hampered by a lack of appropriate adult animal models. Here we describe a simple and highly reproducible adult fli-EGFP transgenic zebrafish model to study retinal angiogenesis. The retinal vasculature in the adult zebrafish is highly organized and hypoxia-induced neovascularization occurs in a predictable area of capillary plexuses. New retinal vessels and vascular sprouts can be accurately measured and quantified. Orally active anti-VEGF agents including sunitinib and ZM323881 effectively block hypoxia-induced retinal neovascularization. Intriguingly, blockage of the Notch signaling pathway by the inhibitor DAPT under hypoxia, results in a high density of arterial sprouting in all optical arteries. The Notch suppression-induced arterial sprouting is dependent on tissue hypoxia. However, in the presence of DAPT substantial endothelial tip cell formation was detected only in optic capillary plexuses under normoxia. These findings suggest that hypoxia shifts the vascular targets of Notch inhibitors. Our findings for the first time show a clinically relevant retinal angiogenesis model in adult zebrafish, which might serve as a platform for studying mechanisms of retinal angiogenesis, for defining novel therapeutic targets, and for screening of novel antiangiogenic drugs.

Mammalian liver-X-receptors (LXRs) are transcription factors activated by oxysterols. They play an essential role in lipid and glucose metabolism. We have cloned the open reading frame of zebrafish lxr and describe its genomic organization. Zebrafish lxr encodes a 50-kDa protein with high sequence similarity to mammalian LXRalpha. In transfection assays, the encoded protein showed transcriptional activity in response to LXR-ligands. Treatment of adult zebrafish with the synthetic LXR ligand, GW3965, induced expression of genes involved in hepatic cholesterol and lipid pathways. Using qPCR and in situ hybridization, we found ubiquitous expression of lxr mRNA during the first 24 hr of development, followed by more restricted expression, particularly to the liver at 3dpf and the liver and intestine at 4dpf. In adult fish, all examined organs expressed lxr. In addition to a metabolic role of lxr, the temporal expression pattern suggests a developmental role in, e.g., the liver and CNS.