Iris Söll

Hypoxia-Induced Retinal Angiogenesis in Zebrafish as a Model to Study Retinopathy

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.

Bozozok directly represses bmp2b transcription and mediates the earliest dorsoventral asymmetry of bmp2b expression in zebrafish

Formation of the gastrula organizer requires suppression of ventralizing signals and, in fish and frog, the need to counteract the effect of ubiquitously present maternal factors that activate the expression of Bmps. How the balance between dorsalizing and ventralizing factors is shifted towards organizer establishment at late blastula stages is not well understood. Mutations in zebrafish bozozok (boz) cause severe defects in axial mesoderm and anterior neurectoderm and affect organizer formation. The boz gene encodes the homeodomain protein Bozozok/Dharma and its expression in the region of the organizer is activated through β-catenin signaling. Here, we investigate the molecular mechanism by which boz contributes to the establishment of the organizer. We demonstrate that the homeodomain protein Boz acts as a transcriptional repressor in zebrafish: overexpression of an En-Boz fusion protein can rescue the boz phenotype, whereas a VP16-Boz fusion protein acts as an antimorph. Expression analysis of bmp2b indicates that Boz negatively regulates bmp2b in the prospective organizer. We demonstrate that this Boz activity is independent of that of other zygotic genes, because it also occurs when translation of zygotic genes is suppressed by cycloheximide (CHX). We identify two high-affinity binding sites for Boz within the first intron of the bmp2b gene. Deletion of these control elements abolishes Boz-dependent repression of bmp2b in the early blastula. Thus, Boz directly represses bmp2b by binding to control elements in the bmp2b locus. We propose that early transcriptional repression of bmp2b by Boz is one of the first steps toward formation of a stable organizer, whereas the later-acting Bmp antagonists (e.g. Chordin, Noggin) modulate Bmp activity in the gastrula to induce patterning along the dorsoventral axis. Thus, similar to Drosophila Dpp, asymmetry of Bmp expression in zebrafish is initiated at the transcriptional level, and the shape of the gradient and its function as a morphogen are later modulated by post-transcriptional mechanisms.

Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain

BackgroundIn 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.ResultsWe 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.ConclusionsDevelopment 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.

Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems

BackgroundWhole-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.ResultsWe 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.ConclusionSince 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.

The early embryonic zebrafish forebrain is subdivided into molecularly distinct transverse and longitudinal domains

During early developmental stages, the embryonic vertebrate brain is still relatively simple with few morphological landmarks that would indicate subdivisions in the prosencephalic primordium. To better understand the early organization of the rostral brain of a lower vertebrate, we investigated the embryonic development and regionalization of the fore- and midbrain of a small teleost, the zebrafish (Danio rerio). We used regulatory gene expression patterns to trace putative prosomeric domains to the beginning of the pharyngula period, when morphological manifestations of prosomeres are not immediately evident. We directly compared the expression domains of members of the dlx, emx, fgf, hh, lim, nkx, otx, pax, POU, winged helix and wnt regulatory gene families in the rostral brain by means of two-color whole-mount in situ hybridization. This allowed us to define precisely abutting expression borders of neighboring expression domains of different genes. Our analysis shows that the genes examined are expressed in anteroposteriorly and dorsoventrally restricted domains, and share expression borders at stereotypic positions within the fore- and midbrain. The arrangement of the various expression domains identified four major longitudinal subdivisions, which extend in parallel to the bent longitudinal rostral brain axis. Furthermore, we identified a series of eight transverse diencephalic domains which may indicate a prosomeric organization of the rostral zebrafish brain.

Serotonergic Modulation of Locomotion in Zebrafish—Endogenous Release and Synaptic Mechanisms

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.

Nitric oxide permits hypoxia-induced lymphatic perfusion by controlling arterial-lymphatic conduits in zebrafish and glass catfish

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.

Direct binding of Lef1 to sites in the boz promoter may mediate pre-midblastula-transition activation of boz expression.

The Nieuwkoop center provides signals essential for the establishment of the dorsal gastrula organizer in vertebrates. Activation of β‐catenin is one of the events in the Nieuwkoop center that lead to activation of dorsal‐specific genes during blastula and early gastrula stages. Zebrafish bozozok (boz) mutant embryos have severe defects in axial mesoderm and anterior neuroectoderm. The boz gene is activated in the organizer in response to β‐catenin signaling, and Boz protein has been demonstrated to contribute to organizer formation by repression of ventralizing genes, including bmp2b, vega1, and vega2. Here, we investigate the timing and molecular mechanism by which boz expression is activated in the organizer. We demonstrate that boz is already expressed before midblastula transition (MBT). We further identify high‐affinity binding sites for Tcf/Lef1 within the boz promoter region. These sites, together with the finding that β‐catenin induces boz expression, indicate that transcription of boz may be activated directly by β‐catenin/Lef1. We hypothesize that pre‐MBT activation of boz may be important to build up a sufficiently strong antagonizing activity against zygotic ventralizing genes activated immediately post‐MBT. Thus, the early onset of boz expression may be crucial for organizer establishment in the presence of ubiquitous maternal activators of ventralizing genes. Developmental Dynamics, 2003.

Localized expression of urocortin genes in the developing zebrafish brain.

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.

Molecular characterization of prosomeric and intraprosomeric subdivisions of the embryonic zebrafish diencephalon.

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.