In situ hybridization screen in zebrafish for the selection of genes encoding secreted proteins

Crosier, Philip S.; Bardsley, Anne; Horsfield, Julia A.; Krassowska, Anna K.; LaVallie, Edward R.; Collins-Racie, Lisa A.; Postlethwait, John H.; Yan, Yi‐Lin; McCoy, John; Crosier, Kathryn E.

doi:10.1002/dvdy.1218

Cited by 19 publications

(10 citation statements)

References 53 publications

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“…Cathepsin L encodes a secreted gene product [40] and is a putative zebrafish hatching enzyme that is expressed at high levels in the hatching gland ([37] and figure 6B). The release of hatching enzymes was measured using Z-Phe-Arg-7-amido-4-methylcoumarin as a substrate for cat L, although it is also cleaved by other proteolytic enzymes.…”

Section: Resultsmentioning

confidence: 99%

Regulation of Zebrafish Hatching by Tetraspanin cd63

et al. 2011

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Tetraspanins cause the clustering of membrane proteins into a level of organisation essential for cellular function. Given the importance and complicated nature of this mechanism, we attempted a novel approach to identify the function of a single component in a biologically relevant context. A morpholino knockdown strategy was used to investigate the role of cd63, a membrane protein associated with intracellular transport and a melanoma marker, in embryonic zebrafish. By using three separate morpholinos targeting cd63, we were able to identify a specific phenotype. Strikingly, morphant fish failed to hatch due to the lack of secreted proteolytic enzymes required for chorion-softening. The morphology of the hatching gland at both the cellular and intracellular levels was disorganised, suggesting a role for cd63 in the functioning of this organ. This work identifies a specific role for cd63 in the zebrafish embryo and provides evidence for the suitability of zebrafish as a model system for the investigation of tetraspanin enriched microdomains.

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Section: Resultsmentioning

confidence: 99%

Regulation of Zebrafish Hatching by Tetraspanin cd63

et al. 2011

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“…However, this was restricted to the rostral (anterior) population, with the caudal (posterior) population significantly depleted with spi1.tel-jak2a CML. The spi1.tel-jak2a CML and CMV.tel-jak2a CML embryos also showed a decrease in cells expressing lysozyme (lyz), a marker for mature myeloid cells derived from the rostral compartment 44 ( Figure 2H,J,O), although cells expressing mmp9 45 were increased with spi1.tel-jak2a CML ( Figure 2M,P), compared to uninjected embryos ( Figure 2F and Figure 2K, respectively). In contrast, expression of the ALL-derived fusion did not alter the number of lyz+ ( Figure 2G,I) or mmp9+ ( Figure 2L,P) cells, and only CMV.tel-jak2a ALL led to an expansion of spi1+ cell numbers in the posterior intermediate cell mass ( Figure 2D,N).…”

Section: Molecular Analysis Of Hematologic Compartmentsmentioning

confidence: 95%

Alternative TEL-JAK2 fusions associated with T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia dissected in zebrafish

et al. 2012

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The online version of this article has a Supplementary Appendix. BackgroundChromosomal translocations resulting in alternative fusions of the human TEL (ETV6) and JAK2 genes have been observed in cases of acute lymphoblastic leukemia and chronic myelogenous leukemia, but a full understanding of their role in disease etiology has remained elusive. In this study potential differences between these alternative TEL-JAK2 fusions, including their lineage specificity, were investigated. Design and MethodsTEL-JAK2 fusion types derived from both T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia were generated using the corresponding zebrafish tel and jak2a genes and placed under the control of either the white blood cell-specific spi1 promoter or the ubiquitously-expressed cytomegalovirus promoter. These constructs were injected into zebrafish embryos and their effects on hematopoiesis examined using a range of molecular approaches. In addition, the functional properties of the alternative fusions were investigated in vitro. ResultsInjection of the T-cell acute lymphoblastic leukemia-derived tel-jak2a significantly perturbed lymphopoiesis with a lesser effect on myelopoiesis in zebrafish embryos. In contrast, injection of the atypical chronic myelogenous leukemia-derived tel-jak2a resulted in significant perturbation of the myeloid compartment. These phenotypes were observed regardless of whether expressed in a white blood cell-specific or ubiquitous manner, with no overt cellular proliferation outside of the hematopoietic cells. Functional studies revealed subtle differences between the alternative forms, with the acute lymphoblastic leukemia variant showing higher activity, but reduced downstream signal transducer and activator of transcription activation and decreased sensitivity to JAK2 inhibition. JAK2 activity was required to mediate the effects of both variants on zebrafish hematopoiesis. ConclusionsThis study indicates that the molecular structure of alternative TEL-JAK2 fusions likely contributes to the etiology of disease. The data further suggest that this class of oncogene exerts its effects in a cell lineage-specific manner, which may be due to differences in downstream signaling.Key words: oncogenesis, TEL-JAK2, leukemia, zebrafish.Citation: Onnebo SMN, Rasighaemi P, Kumar J, Liongue C, and Ward AC. Alternative TEL-JAK2 fusions associated with T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia dissected in zebrafish. Haematologica 2012;97(12):1895-1903. doi:10.3324/haematol.2012 This is an open-access paper.Alternative TEL-JAK2 fusions associated with T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia dissected in zebrafish ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n

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“…In some cases, these screens were designed to focus on particular aspects (e.g. secreted proteins) (Christiansen et al, 2001;Crosier et al, 2001;Nguyen et al, 2001;Thut et al, 2001;Yoda et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Large-scale expression screening by automated whole-mount in situ hybridization

Quiring

Wittbrodt

Henrich

et al. 2004

Mechanisms of Development

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Gene expression profiling is an important component of functional genomics. We present a time and cost efficient high-throughput whole-mount in situ technique to perform a large-scale gene expression analysis in medaka fish (Oryzias latipes) embryos. Medaka is a model system ideally suited for the study of molecular genetics of vertebrate development. Random cDNA clones from an arrayed stage 20 medaka plasmid library were analyzed by whole-mount in situ hybridization on embryos of three representative stages of medaka development. cDNA inserts were colony PCR amplified in a 384-format. The PCR products were used to generate over 2000 antisense RNA digoxigenin probes in a high-throughput process. Whole-mount in situ hybridization was carried out in a robot and a broad range of expression patterns was observed. Partial cDNA sequences and expression patterns were documented with BLAST results, cluster analysis, images and descriptions, respectively; collectively this information was entered into a web-based database, "MEPD" (http://www.embl-heidelberg.de/mepd/), that is publicly accessible.

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In situ hybridization screen in zebrafish for the selection of genes encoding secreted proteins

Cited by 19 publications

References 53 publications

Regulation of Zebrafish Hatching by Tetraspanin cd63

Regulation of Zebrafish Hatching by Tetraspanin cd63

Alternative TEL-JAK2 fusions associated with T-cell acute lymphoblastic leukemia and atypical chronic myelogenous leukemia dissected in zebrafish

Large-scale expression screening by automated whole-mount in situ hybridization

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