Precise genome editing by homologous recombination.

Simple and efficient methods are presented for creating precise modifications of the zebrafish genome. Edited alleles are generated by homologous recombination between the host genome and double-stranded DNA (dsDNA) donor molecules, stimulated by the induction of double-strand breaks at targeted loci in the host genome. Because several kilobase-long tracts of sequence can be exchanged, multiple genome modifications can be generated simultaneously at a single locus. Methods are described for creating: (1) alleles with simple sequence changes or in-frame additions, (2) knockin/knockout alleles that express a reporter protein from an endogenous locus, and (3) conditional alleles in which exons are flanked by recombinogenic loxP sites. Significantly, our approach to genome editing allows the incorporation of a linked reporter gene into the donor sequences so that successfully edited alleles can be identified by virtue of expression of the reporter. Factors affecting the efficiency of genome editing are discussed, including the finding that dsDNA products of I-SceI meganuclease enzyme digestion are particularly effective as donor molecules for gene-editing events. Reagents and procedures are described for accomplishing efficient genome editing in the zebrafish.

Nuclear physics: quantitative single-cell approaches to nuclear organization and gene expression.

The internal workings of the nucleus remain a mystery. A list of component parts exists, and in many cases their functional roles are known for events such as transcription, RNA processing, or nuclear export. Some of these components exhibit structural features in the nucleus, regions of concentration or bodies that have given rise to the concept of functional compartmentalization--that there are underlying organizational principles to be described. In contrast, a picture is emerging in which transcription appears to drive the assembly of the functional components required for gene expression, drawing from pools of excess factors. Unifying this seemingly dual nature requires a more rigorous approach, one in which components are tracked in time and space and correlated with onset of specific nuclear functions. In this chapter, we anticipate tools that will address these questions and provide the missing kinetics of nuclear function. These tools are based on analyzing the fluctuations inherent in the weak signals of endogenous nuclear processes and determining values for them. In this way, it will be possible eventually to provide a computational model describing the functional relationships of essential components.

Concurrent infection of a cat with cowpox virus and feline parvovirus.

Concurrent infection with cowpox and feline parvovirus was diagnosed in a 5-month-old male European Short Hair cat. Microscopical examination of the facial skin, ears and foot pads revealed multifocal to coalescing, ulcerative to necrotizing dermatitis and panniculitis with ballooning epidermal degeneration and eosinophilic cytoplasmic inclusion bodies. Immunohistochemistry, polymerase chain reaction testing and virus isolation confirmed infection with a strain of cowpox virus similar to that isolated from a cat in Germany 5 years previously. Lymphoid tissues were depleted and there was catarrhal enteritis caused by feline parvovirus as confirmed by immunohistochemistry and in-situ hybridization. This co-infection did not result in a more severe and rapid course of the poxvirus-associated disease.

Diffusion and binding properties investigated by Fluorescence Correlation Spectroscopy (FCS).

During the last years, Fluorescence Correlation Spectroscopy (FCS) has proven to be a powerful tool for basic research in many applications. The combination of a minimal detection volume in the femtoliter range coupled with very high sensitivity extends the possibilities to design sensitive homogeneous tests. In this article we illustrate the analysis of binding processes with FCS based on the changes in diffusion characteristics of GFP upon binding to an antibody. Problems induced by highly heterogeneous samples are discussed and differences of GFP binding to a monoclonal and a polyclonal antibody are shown and analyzed. We stress data processing, limitations and useful approximations in FCS methodology. Basic ideas of data acquisition and processing as well as new developments and applications are presented.

Fluorescence-based assessment of LRP activity: a comparative study.

BACKGROUND: Broad resistance to anticancer drugs is a major cause of failure in cancer treatment. The Lung Resistance-related Protein (LRP) is a protein associated with drug resistance, which is involved in nucleo-cytoplasmic transport and is known to predict a poor response to chemotherapy in acute myeloid leukaemia. The only method allowing the detection of LRP activity is based on radio-labelled daunorubicin incorporation. Our goal was to develop a fluorescence-based assay to analyse LRP function. MATERIALS AND METHODS: We used human colon carcinoma cell lines treated with sodium butyrate (NaB) in order to induce LRP expression. Daunorubicin efflux in isolated nuclei was measured by flow cytometry, the localization and quantification of Daunorubicin analysed by confocal laser scanning microscopy (CLSM) and the diffusion coefficient of this drug estimated by Fluorescence Correlation Spectrometry (FCS). RESULTS: According to the method using [14C] Doxorubicin cells incubated with NaB displayed an efflux of Daunorubicin out of isolated nuclei demonstrated by flow cytometry or CLSM. The FCS method was able to evaluate kinetics of Daunorubicin molecules in nucleus and cytoplasm and showed a higher dispersion of Daunorubicin kinetics with cells previously NaB-treated. This argument is in favour of an increase of nucleo-cytoplasmic exchange. CONCLUSION: Using CLSM we showed that LRP was able to modify anticancer drug repartition in the cells. LRP activity assessment needs either isolated nuclei if flow cytometry is employed, or FCS, and only a few cells may be analysed.

Tetrahydrofolate biosynthesis in plants: molecular and functional characterization of dihydrofolate synthetase and three isoforms of folylpolyglutamate synthetase in Arabidopsis thaliana.

Tetrahydrofolate coenzymes involved in one-carbon (C1) metabolism are polyglutamylated. In organisms that synthesize tetrahydrofolate de novo, dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) catalyze the attachment of glutamate residues to the folate molecule. In this study we isolated cDNAs coding a DHFS and three isoforms of FPGS from Arabidopsis thaliana. The function of each enzyme was demonstrated by complementation of yeast mutants deficient in DHFS or FPGS activity, and by measuring in vitro glutamate incorporation into dihydrofolate or tetrahydrofolate. DHFS is present exclusively in the mitochondria, making this compartment the sole site of synthesis of dihydrofolate in the plant cell. In contrast, FPGS is present as distinct isoforms in the mitochondria, the cytosol, and the chloroplast. Each isoform is encoded by a separate gene, a situation that is unique among eukaryotes. The compartmentation of FPGS isoforms is in agreement with the predominance of gamma-glutamyl-conjugated tetrahydrofolate derivatives and the presence of serine hydroxymethyltransferase and C1-tetrahydrofolate interconverting enzymes in the cytosol, the mitochondria, and the plastids. Thus, the combination of FPGS with these folate-mediated reactions can supply each compartment with the polyglutamylated folate coenzymes required for the reactions of C1 metabolism. Also, the multicompartmentation of FPGS in the plant cell suggests that the transported forms of folate are unconjugated.

Nadine Dobrovolskaïa-Zavadskaïa and the dawn of developmental genetics.

In one of the first genetic screens aimed at identifying induced developmental mutants, Nadine Dobrovolskaïa-Zavadskaïa, working at the Pasteur Laboratory in the 1920s, isolated and characterized a mutation affecting Brachyury, a gene that regulates tail and axial development in the mouse. Dobrovolskaïa-Zavadskaïa's analysis of Brachyury and other mutations affecting tail development were among the earliest attempts to link gene action with a tissue-specific developmental process in a vertebrate. Her analyses of genes that interacted with Brachyury led to the discovery of the t-haplotype chromosome of mouse. After 70 years, Brachyury and the multiple genes with which it interacts continue to occupy a prominent focus in developmental biology research. A goal of this review is to identify the contributions that Dobrovolskaïa-Zavadskaïa made to our current thinking about Brachyury and how she helped to shape the dawn of the field of developmental genetics. BioEssays 23:365-371, 2001.

Developmental expression of the calcium release channels during early neurogenesis of the mouse cerebral cortex.

The developmental changes of intracellular calcium release channels of mouse neocortex were studied at the onset of neurogenesis, which occurs between embryonic days E11 and E17. The three main isoforms of the two families of intracellular calcium release channels, namely the inositol trisphosphate receptors (IP3R) and the ryanodine receptors (RyR), were detected by their transcripts in the cerebral hemispheres, as early as stage E11. The major isoforms of each family, IP3R-1 and RyR-2, were found at the protein level by Western blot analysis. Expression of these proteins increases progressively throughout brain development. Their localization in coronal sections of cortex has been observed by immunodetection from E12, and compared to the TuJ1 (anti-class III beta-tubulin antibody) neuronal specific labelling. The expression of both channels is greatly enhanced after E12, and both were seen to be present in most of the proliferative and neuronal cells of the slice. Between E12 and E13, there is a striking transition in the pattern of calcium release elicited by specific agonists of these channels, thimerosal for IP3R and caffeine for RyR. The signals induced by thimerosal were not zone-specific, while the observed calcium release signals induced by caffeine were predominantly restricted out of the ventricular zone. This zone-specific caffeine sensitivity is consistent with the main RyR localization immunodetected at E13. Our results indicate that there is a time lag of several days between the molecular detection of calcium release channels and their functional expression, around the time of neuronal differentiation. Altogether, they provide a molecular basis for analyzing the developmental modulation of calcium signals useful for neurogenesis progression.

Calmodulin and immunophilin are required as functional partners of a ryanodine receptor in ascidian oocytes at fertilization.

Fertilization of oocytes incites numerous changes relying on Ca(2+) signaling. In inseminated ascidian eggs, an increase in the egg surface membrane, monitored by a change in electrical capacitance, is recorded at the onset of meiosis resumption. This membrane addition to the cell surface is controlled by calcium release through a ryanodine receptor (RyR), sensitive to cyclic ADP-ribose. Using confocal microscopy analysis of ascidian oocytes immunostained with anti-RyR antibody, we show here that this calcium channel is asymmetrically located in the vegetal cortical zone. Interestingly, the increase in cell capacitance occurring at fertilization is correlated with a fluorescent signal, imaged by the marker of vesicle trafficking FM 1-43, located close to the RyR region. Two putative partners of RyR, namely an FKBP-like protein and a calmodulin, are identified in these oocyte extracts by detection of enzyme activity and PCR amplification. Both are necessary to sustain ryanodine receptor activity in these oocytes since the membrane insertion triggered by fertilization is inhibited by the FKBP ligand rapamycin and by a calmodulin antagonist peptide. These findings suggest that exocytosis in ascidian eggs is triggered at fertilization by a functional Ca(2+) release unit operating as a complex of several proteins, including a calmodulin and an immunophilin, around the intracellular calcium channel itself.

Cloning and characterization of a new isoform of skeletal muscle triadin.

We have shown that several isoforms of triadin, a protein involved in calcium release process through the ryanodine receptor, are expressed in rat skeletal muscle, and we have cloned two of these isoforms. One is the rat homolog of the 95-kDa triadin identified in rabbit skeletal muscle, and the second one, shorter, is a truncated form of the previous one, but with a new unique COOH-terminal end. We propose to name the two proteins identified here Trisk 95 and Trisk 51. We have produced antibodies specific to each isoform. Using these antibodies, we have shown that the newly identified protein, Trisk 51, is actually expressed in adult rat skeletal muscle and also in rat embryo skeletal muscle. Immunofluorescent labeling of rat skeletal muscle with anti-Trisk 95, anti-Trisk 51, or anti-ryanodine receptor antibodies shows a similar localization of these proteins, in the tissue. Transfection of L6 cells with cDNA of Trisk 51 or Trisk 95 leads to the expression of proteins with the expected molecular weight, identical to those detected in rat skeletal muscle. Both proteins appear during differentiation of satellite cells in myotubes which may indicate the involvement of these two isoforms in the building of a functional calcium release machinery.

Response of chemosensitive and chemoresistant leukemic cell lines to drug therapy: simultaneous assessment of proliferation, apoptosis, and necrosis.

BACKGROUND: The balance between cell proliferation and drug-induced cell death by apoptosis or necrosis plays a major role in determining response to chemotherapy. Commonly-used DNA analysis methods cannot study both parameters simultaneously. A new approach described here combines a green fluorescent membrane-intercalating dye (PKH67) with Hoechst 33342 or annexin V and propidium iodide, to allow simultaneous assessment of cell division, cell cycle status, apoptosis, and necrosis, respectively. METHODS: To test this approach, we used cultured K562 leukemic cell lines which are drug-sensitive (K562S) or drug-resistant (K562R) by virtue of whether they lack or exhibit expression, respectively, of the gp-170 (PGP) glycoprotein pump involved in multidrug resistance. RESULTS: We found that: 1) PKH67 fluorescence intensity decreases proportionately to number of cell divisions, 2) labeling with PKH67 does not alter either cell cycle distribution, as assessed by vital DNA staining with Hoechst 33342, or cell growth, and 3) using a simple threshold analysis method suitable for real-time sorting decisions, subpopulations of proliferating cells present at initial levels of >/= 10% can readily be detected after two cell division times, based on decreased PKH67 intensity. Finally, we demonstrated that after treatment of an admixture of K562S and K562R with vincristine, triple-labeling with PKH67, annexin V, and propidium iodide can be used to identify and sort those cells which remain not only viable (nonnecrotic, nonapoptotic) but actively dividing (decreased PKH67 intensity) in the presence of drug. CONCLUSIONS: Although the studies described here were carried out in a model system using cells having known drug resistance phenotypes, we expect that the methods described will be useful in ex vivo studies of clinical leukemic specimens designed to identify the role played by specific chemoresistance proteins and mechanisms in therapeutic outcomes for individual patients.

Maternal and embryonic expression of zebrafish lef1.

Transcription factors of the TCF/LEF family interact with the Wnt signaling pathway to control transcription of downstream genes (Clevers, H., van de Wetering, M., 1997. TCF/LEF factor earn their wings. Trends Genet. 13, 485-489). We were interested in cloning family members which were expressed in zebrafish neural crest, because Wnt signaling modulates specification of neural crest fate (Dorsky, R.I., Moon, R.T., Raible, D.W., 1998. Control of neural crest cell fate by the Wnt signalling pathway. Nature 396, 370-373). We cloned a zebrafish homolog of lef1 and localized its chromosomal position by radiation hybrid mapping. lef1 is expressed in the neural crest as well as the tailbud and developing mesoderm, and is maternally expressed in zebrafish, unlike mouse and Xenopus homologs. In addition, we cloned two tcf3 genes and a homolog of tcf4, neither of which were strongly expressed in premigratory neural crest.

alyron, an insertional mutation affecting early neural crest development in zebrafish.

alyronz12 (aln) is a recessive lethal mutation that affects early stages of neural crest development in the zebrafish. alyron appears to be an insertional mutation as the mutation was generated following microinjection of plasmid DNA into one-cell embryos and the stably integrated transgenic sequences are closely linked to the mutation. The insertion site harbors multiple copies of the plasmid sequence that have experienced complex rearrangements. Host-insert junction fragments have been molecularly cloned and host sequences adjacent to the transgene have been used to map the mutation to the distal arm of linkage group 15. alyron function is required cell-autonomously in the neural crest lineage. alyron mutants have a severe but not complete deficit of premigratory neural crest as judged by reduced expression of several markers associated with early stages of neural crest development. Lack of premigratory neural crest is likely to account for the two most conspicuous characteristics of alyron mutants: the absence of body pigmentation and the inability to affect blood circulation. The neural crest phenotype of alyron mutants resembles that observed in mouse mutants that lack Pax-3 or both Wnt-1 and Wnt-3a function, and expression of the zebrafish homologues of these genes is greatly reduced in the dorsal neural keels of alyron mutants. In contrast, ventral neural keel identity appears unaffected. Given our findings that the mutation is unlinked to pax or wnt genes that have been described in the zebrafish, we propose that alyron is a novel gene function required for the specification and/or proliferative expansion of neural crest progenitors.

Delta-mediated specification of midline cell fates in zebrafish embryos.

BACKGROUND: Fate mapping studies have shown that progenitor cells of three vertebrate embryonic midline structures - the floorplate in the ventral neural tube, the notochord and the dorsal endoderm - occupy a common region prior to gastrulation. This common region of origin raises the possibility that interactions between midline progenitor cells are important for their specification prior to germ layer formation. RESULTS: One of four known zebrafish homologues of the Drosophila melanogaster cell-cell signaling gene Delta, deltaA (dlA), is expressed in the developing midline, where progenitor cells of the ectodermal floorplate, mesodermal notochord and dorsal endoderm lie close together before they occupy different germ layers. We used a reverse genetic strategy to isolate a missense mutation of dlA, dlAdx2, which coordinately disrupts the development of floorplate, notochord and dorsal endoderm. The dlAdx2 mutant embryos had reduced numbers of floorplate and hypochord cells; these cells lie above and beneath the notochord, respectively. In addition, mutant embryos had excess notochord cells. Expression of a dominant-negative form of Delta protein driven by mRNA microinjection produced a similar effect. In contrast, overexpression of dlA had the opposite effect: fewer trunk notochord cells and excess floorplate and hypochord cells. CONCLUSION: Our results indicate that Delta signaling is important for the specification of midline cells. The results are most consistent with the hypothesis that developmentally equivalent midline progenitor cells require Delta-mediated signaling prior to germ layer formation in order to be specified as floorplate, notochord or hypochord.

Expression of c-ret in the zebrafish embryo: potential roles in motoneuronal development.

We have isolated and characterized the zebrafish ortholog of c-ret, a gene essential for renal organogenesis and enteric nervous system development in mammals. During zebrafish embryogenesis c-ret transcripts are expressed in a number of tissues including spinal motoneurons, pronephric ducts, cranial ganglia, pharyngeal arches, and the enteric nervous system. We have examined in detail the expression of c-ret during the development of identified spinal primary motoneurons. c-ret expression is regulated in a cell type-specific manner among the three primary motoneurons. c-ret is expressed at its highest levels in caudal primary (CaP) motoneurons and transcripts can be detected shortly before the expression of the CaP-specific gene, islet2. We suggest that c-ret may play a role in specifying CaP cell identity. c-ret is expressed at low levels in the other primary motoneurons and also in a subset of secondary motoneurons, suggesting that it may also play a broader role in motoneuronal survival or maintenance.

tbx6, a Brachyury-related gene expressed by ventral mesendodermal precursors in the zebrafish embryo.

Classical embryology experiments have indicated the existence of dorsal-type and ventral-type mesoderms that arise as a consequence of mesoderm induction during vertebrate development. Here we report that the zebrafish tbx6 gene, a member of the Brachyury-related T-box family of genes, is exclusively expressed by ventral mesendoderm. Three observations link the expression of tbx6 to ventral mesoderm specification. First, the gene is initially expressed at the onset of gastrulation within a ventrolateral subpopulation of cells that express the pan-mesodermal gene, no tail (Brachyury). Second, the mesoderm-inducing factors activin and bFGF activate tbx6 expression in animal caps. Third, dorsalization of the mesendodermal precursor population following exposure of embryos to lithium ions causes down-regulation of tbx6 transcription. tbx6 is expressed transiently in the involuting derivatives of the ventral mesendoderm, which give rise to nonaxial mesodermal tissues; its expression is extinguished as tissue differentiation progresses. Transcription of tbx6 commences about an hour after initiation of expression of the pan-mesendodermal gene no tail and the organizer gene goosecoid. The dependence of tbx6 expression on no tail activity was examined in no tail mutant embryos. The activation of tbx6 transcription in ventral mesoderm does not depend on no tail gene activity. However, no tail appears to contribute to the maintenance of normal levels of tbx6 transcription and may be required for tbx6 transcription in the developing tail.

A ryanodine-sensitive calcium store in ascidian eggs monitored by whole-cell patch-clamp recordings.

Using whole cell patch clamp recordings on unfertilized eggs of the ascidian Ciona intestinalis, we are able to detect ryanodine receptors within the oocytes. Our approach is based on measurements of the voltage-activated inward calcium currents. Two types of Ca2+ currents have been described on the oocyte membrane of Ciona: a low threshold slowly activating current, and a high threshold faster one. We show here that caffeine induces a decrease in the intensity of the Ca2+ currents, when applied either externally or internally from the mouth of a patch pipette. Caffeine application mimics fertilization which transiently decreases the high threshold Ca2+ current density during density during the first meiotic cycle. Ryanodine (> 1 nM) has an effect similar to caffeine. This partial decrease in Ca2+ current density elicited by caffeine or ryanodine is prevented by intracellular application of the calcium chelator BAPTA, then imputable to calcium release. In summary, the depolarization-induced Ca2+ current intensity allows monitoring of an intracellular calcium store which is sensitive to low concentrations of ryanodine in Ciona oocytes. Further identification of a ryanodine receptor was obtained by immunological staining with antibodies against mammalian skeletal muscle ryanodine receptor. Ryanodine receptors were asymmetrically localized in the cortex of Ciona eggs. We discuss the methodological relevance of our patch-clamp approach, in connection with the possible biological role of such a ryanodine receptor in the early stages of development.

The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation.

The zebrafish cloche mutation affects both the endothelial and hematopoietic lineages at a very early stage (Stainier, D. Y. R., Weinstein, B. M., Detrich, H. W., Zon, L. I. and Fishman, M. C. (1995). Development 121, 3141-3150). The most striking vascular phenotype is the absence of endocardial cells from the heart. Microscopic examination of mutant embryos reveals the presence of endothelial-like cells in the lower trunk and tail regions while head vessels appear to be missing, indicating a molecular diversification of the endothelial lineage. Cell transplantation experiments show that cloche acts cell-autonomously within the endothelial lineage. To analyze further the role of cloche in regulating endothelial cell differentiation, we have examined the expression of flk-1 and tie, two receptor tyrosine kinase genes expressed early and sequentially in the endothelial lineage. In wild-type fish, flk-1-positive cells are found throughout the embryo and differentiate to form the nascent vasculature. In cloche mutants, flk-1-positive cells are found only in the lower trunk and tail regions, and this expression is delayed as compared to wild-type. Unlike the flk-1-positive cells in wild-type embryos, those in cloche mutants do not go on to express tie, suggesting that their differentiation is halted at an early stage. We also find that the cloche mutation is not linked to flk-1. These data indicate that cloche affects the differentiation of all endothelial cells and that it acts at a very early stage, either by directly regulating flk-1 expression or by controlling the differentiation of cells that normally develop to express flk-1. cloche mutants also have a blood deficit and their hematopoietic tissues show no expression of the hematopoietic transcription factor genes GATA-1 or GATA-2 at early stages. Because the appearance of distinct levels of flk-1 expression is delayed in cloche mutants, we examined GATA-1 expression at late embryonic stages and found some blood cell differentiation that appears to be limited to the region lined by the flk-1-expressing cells. The spatial restriction of blood in the ventroposterior-most region of cloche mutant embryos may be indicative of a ventral source of signal(s) controlling hematopoietic differentiation. In addition, the restricted colocalization of blood and endothelium in cloche mutants suggests that important interactions occur between these two lineages during normal development.