Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Heidelberg University

Induction of the otic placode involves a number of regulatory interactions. Early studies revealed that the induction of this program is initiated by instructive signals from the mesendoderm as well as from the adjacent hindbrain. Further investigations on the molecular level identified in zebrafish Fgf3, Fgf8, Foxi1, Pax8, Dlx3b and Dlx4b genes as key players during the induction phase. Thereafter an increasing number of genes participates in the regulatory interactions finally resulting in a highly structured sensory organ. Based on data from zebrafish we selected medaka genes with presumptive functions during early ear development for an expression analysis. In addition we isolated Foxi1 and Dlx3b gene fragments from embryonic cDNA. Altogether we screened the spatio-temporal distribution of more than 20 representative marker genes for otic development in medaka embryos, with special emphasis on the early phases. Whereas the spatial distribution of these genes is largely conserved between medaka and zebrafish, our comparative analysis revealed several differences, in particular for the timing of expression.

Expression of marker genes during early ear development in medaka.

Medaka (Oryzias latipes) is a small freshwater teleost that provides an excellent developmental genetic model complementary to zebrafish. Our recent mutagenesis screening using medaka identified headfish (hdf) which is characterized by the absence of trunk and tail structures with nearly normal head including the midbrain-hindbrain boundary (MHB). Positional-candidate cloning revealed that the hdf mutation causes a functionally null form of Fgfr1. The fgfr1hdf is thus the first fgf receptor mutant in fish. Although FGF signaling has been implicated in mesoderm induction, mesoderm is induced normally in the fgfr1hdf mutant, but subsequently, mutant embryos fail to maintain the mesoderm, leading to defects in mesoderm derivatives, especially in trunk and tail. Furthermore, we found that morpholino knockdown of medaka fgf8 resulted in a phenotype identical to the fgfr1hdf mutant, suggesting that like its mouse counterpart, Fgf8 is a major ligand for Fgfr1 in medaka early embryogenesis. Intriguingly, Fgf8 and Fgfr1 in zebrafish are also suggested to form a major ligand-receptor pair, but their function is much diverged, as the zebrafish fgfr1 morphant and zebrafish fgf8 mutant acerebellar (ace) only fail to develop the MHB, but develop nearly unaffected trunk and tail. These results provide evidence that teleost fish have evolved divergent functions of Fgf8-Fgfr1 while maintaining the ligand-receptor relationships. Comparative analysis using different fish is thus invaluable for shedding light on evolutionary diversification of gene function.

Mutant analyses reveal different functions of fgfr1 in medaka and zebrafish despite conserved ligand-receptor relationships.

Nodal activity in the left lateral plate mesoderm (LPM) is required to activate left-sided Nodal signaling in the epithalamic region of the zebrafish forebrain. Epithalamic Nodal signaling subsequently determines the laterality of neuroanatomical asymmetries. We show that overactivation of Wnt/Axin1/beta-catenin signaling during late gastrulation leads to bilateral epithalamic expression of Nodal pathway genes independently of LPM Nodal signaling. This is consistent with a model whereby epithalamic Nodal signaling is normally bilaterally repressed, with Nodal signaling from the LPM unilaterally alleviating repression. We suggest that Wnt signaling regulates the establishment of the bilateral repression. We identify a second role for the Wnt pathway in the left/right regulation of LPM Nodal pathway gene expression, and finally, we show that at later stages Axin1 is required for the elaboration of concordant neuroanatomical asymmetries.

Wnt/Axin1/beta-catenin signaling regulates asymmetric nodal activation, elaboration, and concordance of CNS asymmetries.

Adequate fluid loading is the first step of hemodynamic optimization in cardiac patients undergoing surgery. Neither a clinical approach alone nor conventional parameters like central venous pressure (CVP) and pulmonary capillary wedge pressure (PCWP) are thought to be sufficient for recognizing fluid deficiency or overload. The aim of this study was to evaluate the suitability of CVP, PCWP, global end-diastolic volume index (GEDVI), pulse pressure variation (PPV), and stroke volume variation (SVV) for predicting changes in the cardiac index (CI) and stroke volume index (SVI) after sternotomy.

Prediction of volume response under open-chest conditions during coronary artery bypass surgery.

Functional lateralization is a conserved feature of the central nervous system (CNS). However, underlying left-right asymmetries within neural circuitry and the mechanisms by which they develop are poorly described.

Brain asymmetry is encoded at the level of axon terminal morphology.

The centrosome is the cell's microtubule organising centre, an organelle with important roles in cell division, migration and polarity. However, cells can divide and flies can, for a large part of development, develop without them. Many centrosome proteins have been identified but the roles of most are still poorly understood. The centrioles of the centrosome are similar to the basal bodies of cilia, hair-like extensions of many cells that have important roles in cell signalling and development. In a number of human diseases, such Bardet-Biedl syndrome, centrosome/cilium proteins are mutated, leading to polycystic kidney disease, situs inversus, and neurological problems, amongst other symptoms.

Cep70 and Cep131 contribute to ciliogenesis in zebrafish embryos.

Wnt-signalling is involved in a number of biological processes in the course of embryonic development, cell fate determination, proliferation, stem cell maintenance and oncogenesis. Wnt ligands are secreted glycoproteins and the number of Wnt isoforms varies between five in nematodes and 27 in fish. The highly conserved group of Wnt7 genes has been found to signal via at least three Wnt-signalling pathways dependent on the developmental context. These ligands have been identified as important regulators in a number of processes ranging from formation of bones, lungs, kidneys, reproductive organs and placenta to vasculogenesis and synaptogenesis in the brain. The importance of Wnt7 function is underscored by their implication in disease syndromes in man. Unlike the single Wnt7a and Wnt7b mammalian genes we find that the zebrafish genome contains two paralogues genes for each Wnt7 ligand. Here, we compare these four Wnt7 genes evolutionarily and analyse their expression during the first two days of embryonic development. We find Wnt7 genes mainly expressed in a number of CNS structures at developmental stages at which patterning and neural specification takes place. The timely and spatially overlapping as well as complementary gene expression suggests diverse as well as redundant involvements during brain development.

All four zebrafish Wnt7 genes are expressed during early brain development.

The constantly growing number of genetic tools rapidly increases possibilities for various screens in different model organisms and calls for new methods facilitating screen performance. In particular, screening procedures involving fixation and staining of samples are difficult to perform at a genome-wide scale. The time-consuming task to generate these samples makes such screens less attractive. Here, we describe the use of multi-well filter plates for high throughput labellings of different Drosophila organs and zebrafish embryos. Our inexpensive vacuum-assisted staining protocol minimises the risk of sample loss, reduces the amount of staining reagents and drastically decreases labour and repetitive work. The simple handling of the system and the commercial availability of its components makes this method easily applicable to every laboratory.

"Vacuum-assisted staining": a simple and efficient method for screening in Drosophila.

The habenular neural circuit is attracting increasing attention from researchers in fields as diverse as neuroscience, medicine, behavior, development, and evolution. Recent studies have revealed that this part of the limbic system in the dorsal diencephalon is involved in reward, addiction, and other behaviors and its impairment is associated with various neurological conditions and diseases. Since the initial description of the dorsal diencephalic conduction system (DDC) with the habenulae in its center at the end of the nineteenth century, increasingly sophisticated techniques have resolved much of its anatomy and have shown that these pathways relay information from different parts of the forebrain to the tegmentum, midbrain, and hindbrain. The first part of this review gives a brief historical overview on how the improving experimental approaches have allowed the stepwise uncovering much of the architecture of the habenula circuit as we know it today. Our brain distributes tasks differentially between left and right and it has become a paradigm that this functional lateralization is a universal feature of vertebrates. Moreover, task dependent differential brain activities have been linked to anatomical differences across the left-right axis in humans. A good way to further explore this fundamental issue will be to study the functional consequences of subtle changes in neural network formation, which requires that we fully understand DDC system development. As the habenular circuit is evolutionarily highly conserved, researchers have the option to perform such difficult experiments in more experimentally amenable vertebrate systems. Indeed, research in the last decade has shown that the zebrafish is well suited for the study of DDC system development and the phenomenon of functional lateralization. We will critically discuss the advantages of the zebrafish model, available techniques, and others that are needed to fully understand habenular circuit development.

Habenula circuit development: past, present, and future.

Most young researchers leaving the safe haven of postdoctoral life for the unchartered territory of a PI position are confronted with a multitude of new tasks. For those using fish as their favorite model system, this often includes the daunting task of setting up and running a fish facility. New PIs are expected to know everything about this, and are even asked in job interviews about the precise details of the facility they anticipate using. Consulting other laboratories, talking to experienced facility managers, and sifting through books and web material will certainly help but can be enormously time consuming and at times frustrating. You will likely receive conflicting advice or encounter information that is overcomplicated and out of date. In this report, we summarize our collective experience of five different fish facilities, and outline what we consider to be the essential steps for setting up and running a successful facility. We also include valuable tips and tricks such as an optimized protocol for brine shrimp hatching. Our aim is to help researchers spend less time worrying about the technical aspects of their facility so that they can focus on their main job--research.

Don't be afraid to set up your fish facility.

The vertebrate brain is an immensely complex structure, which exhibits numerous morphological and functional asymmetries. The best described brain asymmetries are found in the diencephalic epithalamus, where the habenulae and the dorso-laterally adjacent pineal complex are lateralized in many species. Research in the past decade has shed light on the establishment of the laterality of these structures as well as their asymmetry per se. In particular work in zebrafish (Danio rerio) has substantially contributed to our understanding, which genetic pathways are involved in these processes. The Wnt/beta-catenin pathway has turned out to play a pivotal role in the regulation of brain laterality and asymmetry and acts reiteratively during embryonic development.

The Wnt/beta-catenin signaling pathway establishes neuroanatomical asymmetries and their laterality.

Wnt/β-catenin signaling plays an important role in embryonic development and adult tissue homeostasis. When Wnt ligands bind to the receptor complex, LRP5/6 coreceptors are activated by phosphorylation and concomitantly endocytosed. In vertebrates, Wnt ligands induce caveolin-dependent endocytosis of LRP6 to relay signal downstream, whereas antagonists such as Dickkopf promote clathrin-dependent endocytosis, leading to inhibition. However, little is known about how LRP6 is directed to different internalization mechanisms, and how caveolin-dependent endocytosis is mediated. In an RNAi screen, we identified the Rab GTPase RAB8B as being required for Wnt/β-catenin signaling. RAB8B depletion reduces LRP6 activity, β-catenin accumulation, and induction of Wnt target genes, whereas RAB8B overexpression promotes LRP6 activity and internalization and rescues inhibition of caveolar endocytosis. In Xenopus laevis and Danio rerio, RAB8B morphants show lower Wnt activity during embryonic development. Our results implicate RAB8B as an essential evolutionary conserved component of Wnt/β-catenin signaling through regulation of LRP6 activity and endocytosis.

RAB8B is required for activity and caveolar endocytosis of LRP6.

The conserved habenular neural circuit relays cognitive information from the forebrain into the ventral mid- and hindbrain. In zebrafish, the bilaterally formed habenulae in the dorsal diencephalon are made up of the asymmetric dorsal and symmetric ventral habenular nuclei, which are homologous to the medial and lateral nuclei respectively, in mammals. These structures have been implicated in various behaviors related to the serotonergic/dopaminergic neurotransmitter system. The dorsal habenulae develop adjacent to the medially positioned pineal complex. Their precursors differentiate into two main neuronal subpopulations which differ in size across brain hemispheres as signals from left-sided parapineal cells influence their differentiation program. Unlike the dorsal habenulae and despite their importance, the ventral habenulae have been poorly studied. It is not known which genetic programs underlie their development and why they are formed symmetrically, unlike the dorsal habenulae. A main reason for this lack of knowledge is that the vHb origin has remained elusive to date.

The ventral habenulae of zebrafish develop in prosomere 2 dependent on Tcf7l2 function.

The analysis of genes in evolutionarily distant but morphologically similar species is of major importance to unravel the changes in genomes over millions of years, which led to gene silencing and functional diversification. We report the analysis of Wnt8a gene expression in the medakafish and provide a detailed comparison to other vertebrates. In all teleosts analyzed there are two paralogous Wnt8a copies. These show largely overlapping expression in the early developing zebrafish embryo, an evolutionarily distant relative of medaka. In contrast to zebrafish, we find that both maternal and zygotic expression of particularly one Wnt8a paralog has diverged in medaka. While Wnt8a1 expression is mostly conserved at early embryonic stages, the expression of Wnt8a2 differs markedly. In addition, both genes are distinctly expressed during organogenesis unlike the zebrafish homologs, which may hint at the emergence of functional diversification of Wnt8a ligands during evolution.

Divergent Wnt8a gene expression in teleosts.

Left-right asymmetries are most likely a universal feature of bilaterian nervous systems and may serve to increase neural capacity by specializing equivalent structures on left and right sides for distinct roles. However, little is known about how asymmetries are encoded within vertebrate neural circuits and how lateralization influences processing of information in the brain. Consequently, it remains unclear the extent to which lateralization of the nervous system is important for normal cognitive and other brain functions and whether defects in lateralization contribute to neurological deficits. Here we show that sensory responses to light and odor are lateralized in larval zebrafish habenulae and that loss of brain asymmetry leads to concomitant loss of responsiveness to either visual or olfactory stimuli. We find that in wild-type zebrafish, most habenular neurons responding to light are present on the left, whereas neurons responding to odor are more frequent on the right. Manipulations that reverse the direction of brain asymmetry reverse the functional properties of habenular neurons, whereas manipulations that generate either double-left- or double-right-sided brains lead to loss of habenular responsiveness to either odor or light, respectively. Our results indicate that loss of brain lateralization has significant consequences upon sensory processing and circuit function.

Left-right asymmetry is required for the habenulae to respond to both visual and olfactory stimuli.

Although left-right asymmetries are common features of nervous systems, their developmental bases are largely unknown. In the zebrafish epithalamus, dorsal habenular neurons adopt medial (dHbm) and lateral (dHbl) subnuclear character at very different frequencies on the left and right sides. The left-sided parapineal promotes the elaboration of dHbl character in the left habenula, albeit by an unknown mechanism. Likewise, the genetic pathways acting within habenular neurons to control their asymmetric differentiated character are unknown.

Matthias Carl

Medical Faculty Mannheim,
Department of Cell and Molecular Biology,
Medical Faculty Mannheim, Department of Cell and Molecular Biology

Heidelberg University

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

Matthias Carl

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Medical Faculty Mannheim,Department of Cell and Molecular Biology,Medical Faculty Mannheim, Department of Cell and Molecular Biology

Heidelberg University

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

Medical Faculty Mannheim,
Department of Cell and Molecular Biology,
Medical Faculty Mannheim, Department of Cell and Molecular Biology