The in situ hybridization uses a labeled complementary RNA strand to localize a specific mRNA sequence in a tissue. This method is widely used to describe the spatial and temporal expression patterns ...of developmentally regulated genes. Here we describe a technique that employs in vitro synthesized RNA tagged with digoxigenin uridine-5'-triphosphate (UTP) to determine expression of genes on whole-mount zebrafish embryos and young larvae. Following hybridization, the localization of the specific transcript is visualized immunohistochemically using an anti-digoxigenin antibody conjugated to alkaline phosphatase that hydrolyzes the 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to 5-bromo-4-chloro-3-indole and inorganic phosphate. 5-Bromo-4-chloro-3-indole can be oxidized by nitro blue tetrazolium (NBT), which forms an insoluble dark blue diformazan precipitate after reduction.This protocol has been used for performing large-scale analyses of the spatial and temporal expression of the zebrafish genome, resulting in the description of more than 8,400 expression patterns that are available at the zebrafish information network (ZFIN.org) in the gene expression section.
Fibroblast growth factors (FGF) are secreted molecules which function through the activation of specific tyrosine kinases receptors, the FGF receptors that transduce the signal by activating ...different pathways including the Ras/MAP kinase and the phospholipase-C gamma pathways. FGFs are involved in the regulation of many developmental processes including patterning, morphogenesis, differentiation, cell proliferation or migration. Such a diverse set of activities requires a tight control of the transduction signal which is achieved through the induction of different feedback inhibitors such as the Sproutys, Sef and MAP kinase phosphatase 3 which are responsible for the attenuation of FGF signals, limiting FGF activities in time and space.
During the course of their classic experiments, Hilde Mangold and Hans Spemann discovered that the dorsal blastopore lip of an amphibian gastrula was able to induce formation of a complete embryonic ...axis when transplanted into the ventral side of a host gastrula embryo. Since then, the inducing activity of the dorsal lip has been known as the Spemann or dorsal organizer. During the past 25 years, studies performed in a variety of species have led to the identification of molecular factors associated with the properties of this tissue. However, none of them is, by itself, able to induce formation of the main body axis from a population of naive pluripotent embryonic cells. Recently, experiments performed using the zebrafish (Danio rerio) revealed that the organizing activities present in the embryo are not restricted to the Spemann organizer but are distributed along the entire blastula/gastrula margin. These organizing activities result from the interaction between two opposing gradients of morphogens, BMP and Nodal, that are the primary signals that trigger the cascade of developmental events leading to the organization of the embryo. These studies mark the end of the era during which developmental biologists saw the Spemann organizer as the core element for the organization of the vertebrate embryonic axis and, instead, provides opportunities for the experimental control of morphogenesis starting with a population of embryonic pluripotent cells that will be instructed using those two morphogen gradients.
Generating properly differentiated embryonic structures in vitro from pluripotent stem cells remains a challenge. Here we show that instruction of aggregates of mouse embryonic stem cells with an ...experimentally engineered morphogen signalling centre, that functions as an organizer, results in the development of embryo-like entities (embryoids). In situ hybridization, immunolabelling, cell tracking and transcriptomic analyses show that these embryoids form the three germ layers through a gastrulation process and that they exhibit a wide range of developmental structures, highly similar to neurula-stage mouse embryos. Embryoids are organized around an axial chordamesoderm, with a dorsal neural plate that displays histological properties similar to the murine embryo neuroepithelium and that folds into a neural tube patterned antero-posteriorly from the posterior midbrain to the tip of the tail. Lateral to the chordamesoderm, embryoids display somitic and intermediate mesoderm, with beating cardiac tissue anteriorly and formation of a vasculature network. Ventrally, embryoids differentiate a primitive gut tube, which is patterned both antero-posteriorly and dorso-ventrally. Altogether, embryoids provide an in vitro model of mammalian embryo that displays extensive development of germ layer derivatives and that promises to be a powerful tool for in vitro studies and disease modelling.
In vertebrates, the animal–vegetal axis is determined during oogenesis and at ovulation, the egg is radially symmetric. In anamniotes, following fertilization, a microtubule-dependent movement leads ...to the displacement of maternal dorsal determinants from the vegetal pole to the future dorsal side of the embryo, providing the initial breaking of radial symmetry Weaver C, Kimelman D (2004) Development 131:3491–3499. These dorsal determinants induce β-catenin nuclear translocation in dorsal cells of the blastula. Previous work in amphibians has shown that secreted Wnt11/5a complexes, regulated by the Wnt antagonist Dkk-1, are required for the initiation of embryonic axis formation Cha et al. (2009) Curr Biol 29:1573–1580. In the current study, we determined that the vegetal maternal dorsal determinant in fish is not the Wnt11/5a complex but the canonical Wnt, Wnt8a. Translation of this mRNA and secretion of the Wnt8a protein result in a dorsal-to-ventral gradient of Wnt stimulation, extending across the entire embryo. This gradient is counterbalanced by two Wnt inhibitors, Sfrp1a and Frzb. These proteins are essential to restrict the activation of the canonical Wnt pathway to the dorsal marginal blastomeres by defining the domain where the Wnt8a activity gradient is above the threshold value necessary for triggering the canonical β-catenin pathway. In summary, this study establishes that the zebrafish maternal dorsal determinant, Wnt8a, is required to localize the primary dorsal center, and that the extent of this domain is defined by the activity of two maternally provided Wnt antagonists, Sfrp1a and Frzb.
Heritable skin diseases represent a broad spectrum of clinical manifestations due to mutations in ∼500 different genes. A number of model systems have been developed to advance our understanding of ...the pathomechanisms of genodermatoses. Zebrafish (Danio rerio), a freshwater vertebrate, has a well-characterized genome, the expression of which can be easily manipulated. The larvae develop rapidly, with all major organs having developed by 5–6 days post-fertilization, including the skin, consisting of the epidermis comprising two cell layers and separated from the dermal collagenous matrix by a basement membrane. This perspective highlights the morphological and ultrastructural features of zebrafish skin, in the context of cutaneous gene expression. These observations suggest that zebrafish provide a useful model system to study the molecular aspects of skin development, as well as the pathogenesis and treatment of select heritable skin diseases.
The origin of resident (noninflammatory) macrophages in vertebrate tissues is still poorly understood. In the zebrafish embryo, we recently described a specific lineage of early macrophages that ...differentiate in the yolk sac before the onset of blood circulation. We now show that these early macrophages spread in the whole cephalic mesenchyme, and from there invade epithelial tissues: epidermis, retina, and brain—especially the optic tectum. In the panther mutant, which lacks a functional fms (M-CSF receptor) gene, early macrophages differentiate and behave apparently normally in the yolk sac, but then fail to invade embryonic tissues. Our video recordings then document for the first time the behavior of macrophages in the invaded tissues, revealing the striking propensity of early macrophages in epidermis and brain to wander restlessly among epithelial cells. This unexpected behavior suggests that tissue macrophages may be constantly “patrolling” for immune and possibly also developmental and trophic surveillance. At 60 h post-fertilization, all macrophages in the brain and retina undergo a specific phenotypic transformation, into “early (amoeboid) microglia”: they become more highly endocytic, they down-regulate the L-plastin gene, and abruptly start expressing high levels of apolipoprotein E, a well-known neurotrophic lipid carrier.
The in situ hybridization (ISH) technique allows the sites of expression of particular genes to be detected. This protocol describes ISH of digoxigenin-labeled antisense RNA probes to whole-mount ...zebrafish embryos. In our method, PCR-amplified sequence of a gene of interest is used as a template for the synthesis of an antisense RNA probe, which is labeled with digoxigenin-linked nucleotides. Embryos are fixed and permeabilized before being soaked in the digoxigenin-labeled probe. We use conditions that favor specific hybridization to complementary mRNA sequences in the tissue(s) expressing the corresponding gene. After washing away excess probe, hybrids are detected by immunohistochemistry using an alkaline phosphatase-conjugated antibody against digoxigenin and a chromogenic substrate. The whole procedure takes only 3 days and, because ISH conditions are the same for each probe tested, allows high throughput analysis of zebrafish gene expression during embryogenesis.
Asymmetrically distributed cytoplasmic determinants collectively termed germ plasm have been shown to play an essential role in the development of primordial germ cells (PGCs). Here, we report the ...identification of a nanos-like (nanos1) gene, which is expressed in the germ plasm and in the PGCs of the zebrafish. We find that several mechanisms act in concert to restrict the activity of Nanos1 to the germ cells including RNA localization and control over the stability and translatability of the RNA. Reducing the level of Nanos1 in zebrafish embryos revealed an essential role for the protein in ensuring proper migration and survival of PGCs in this vertebrate model organism.
Despite the fundamental importance of patterning along the dorsal-ventral (DV) and anterior-posterior (AP) axes during embryogenesis, uncertainty exists in the orientation of these axes for the ...mesoderm. Here we examine the origin and formation of the zebrafish kidney, a ventrolateral mesoderm derivative, and show that AP patterning of the non-axial mesoderm occurs across the classic gastrula stage DV axis while DV patterning aligns along the animal-vegetal pole. We find that BMP signalling acts early to establish broad anterior and posterior territories in the non-axial mesoderm while retinoic acid (RA) functions later, but also across the classic DV axis. Our data support a model in which RA on the dorsal side of the embryo induces anterior kidney fates while posterior kidney progenitors are protected ventrally by the RA-catabolizing enzyme Cyp26a1. This work clarifies our understanding of vertebrate axis orientation and establishes a new paradigm for how the kidney and other mesodermal derivatives arise during embryogenesis.
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