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Ledin, Johan
Publications (10 of 12) Show all publications
Haitina, T. (2018). Utvecklingsbiologi-: komplexa processer när en ny individ blir till. In: Reimegård, Lisa (Ed.), Fascinerande forskning: (pp. 38-42). Sweden: Nationellt resurscentrum för biologi och bioteknik, 1(1)
Open this publication in new window or tab >>Utvecklingsbiologi-: komplexa processer när en ny individ blir till
2018 (Swedish)In: Fascinerande forskning / [ed] Reimegård, Lisa, Sweden: Nationellt resurscentrum för biologi och bioteknik , 2018, Vol. 1, no 1, p. 38-42Chapter in book (Other (popular science, discussion, etc.))
Place, publisher, year, edition, pages
Sweden: Nationellt resurscentrum för biologi och bioteknik, 2018
National Category
Developmental Biology
Identifiers
urn:nbn:se:uu:diva-377775 (URN)9789197664790 (ISBN)
Available from: 2019-02-25 Created: 2019-02-25 Last updated: 2019-07-16Bibliographically approved
Varshney, G. K., Carrington, B., Pei, W., Bishop, K., Chen, Z., Fan, C., . . . Burgess, S. M. (2016). A high-throughput functional genomics workflow based on CRISPR/Cas9-mediated targeted mutagenesis in zebrafish. Nature Protocols, 11(12), 2357-2375
Open this publication in new window or tab >>A high-throughput functional genomics workflow based on CRISPR/Cas9-mediated targeted mutagenesis in zebrafish
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2016 (English)In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 11, no 12, p. 2357-2375Article in journal (Refereed) Published
Abstract [en]

The zebrafish is a popular model organism for studying development and disease, and genetically modified zebrafish provide an essential tool for functional genomic studies. Numerous publications have demonstrated the efficacy of gene targeting in zebrafish using CRISPR/Cas9, and they have included descriptions of a variety of tools and methods for guide RNA synthesis and mutant identification. However, most of the published techniques are not readily scalable to increase throughput. We recently described a CRISPR/Cas9-based high-throughput mutagenesis and phenotyping pipeline in zebrafish. Here, we present a complete workflow for this pipeline, including target selection; cloning-free single-guide RNA (sgRNA) synthesis; microinjection; validation of the target-specific activity of the sgRNAs; founder screening to identify germline-transmitting mutations by fluorescence PCR; determination of the exact lesion by Sanger or next-generation sequencing (including software for analysis); and genotyping in the F-1 or subsequent generations. Using these methods, sgRNAs can be evaluated in 3 d, zebrafish germline-transmitting mutations can be identified within 3 months and stable lines can be established within 6 months. Realistically, two researchers can target tens to hundreds of genes per year using this protocol.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-308888 (URN)10.1038/nprot.2016.141 (DOI)000386976800004 ()27809318 (PubMedID)
Funder
NIH (National Institute of Health)
Available from: 2016-12-01 Created: 2016-12-01 Last updated: 2018-01-13Bibliographically approved
Varshney, G. K., Zhang, S., Pei, W., Adomako-Ankomah, A., Fohtung, J., Schaffer, K., . . . Burgess, S. M. (2016). CRISPRz: a database of zebrafish validated sgRNAs. Nucleic Acids Research, 44(D1), D822-D826
Open this publication in new window or tab >>CRISPRz: a database of zebrafish validated sgRNAs
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2016 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no D1, p. D822-D826Article in journal (Refereed) Published
Abstract [en]

CRISPRz (http://research.nhgri.nih.gov/CRISPRz/) is a database of CRISPR/Cas9 target sequences that have been experimentally validated in zebrafish. Programmable RNA-guided CRISPR/Cas9 has recently emerged as a simple and efficient genome editing method in various cell types and organisms, including zebrafish. Because the technique is so easy and efficient in zebrafish, the most valuable asset is no longer a mutated fish (which has distribution challenges), but rather a CRISPR/Cas9 target sequence to the gene confirmed to have high mutagenic efficiency. With a highly active CRISPR target, a mutant fish can be quickly replicated in any genetic background anywhere in the world. However, sgRNA's vary widely in their activity and models for predicting target activity are imperfect. Thus, it is very useful to collect in one place validated CRISPR target sequences with their relative mutagenic activities. A researcher could then select a target of interest in the database with an expected activity. Here, we report the development of CRISPRz, a database of validated zebrafish CRISPR target sites collected from published sources, as well as from our own in-house large-scale mutagenesis project. CRISPRz can be searched using multiple inputs such as ZFIN IDs, accession number, UniGene ID, or gene symbols from zebrafish, human and mouse.

National Category
Biochemistry and Molecular Biology Evolutionary Biology
Identifiers
urn:nbn:se:uu:diva-282501 (URN)10.1093/nar/gkv998 (DOI)000371261700116 ()26438539 (PubMedID)
Funder
NIH (National Institute of Health), 1ZIAHG000183-14
Available from: 2016-04-05 Created: 2016-04-05 Last updated: 2017-11-30Bibliographically approved
Griffiths, G. W., Mueller, F., Ledin, J., Patton, E. E., Gjoen, T., Lobert, V. H., . . . Alestrom, P. (2016). Fish from Head to Tail: The 9th European Zebrafish Meeting in Oslo. Zebrafish, 13(2), 132-137
Open this publication in new window or tab >>Fish from Head to Tail: The 9th European Zebrafish Meeting in Oslo
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2016 (English)In: Zebrafish, ISSN 1545-8547, E-ISSN 1557-8542, Vol. 13, no 2, p. 132-137Article in journal (Refereed) Published
Abstract [en]

The 9th European Zebrafish Meeting took place recently in Oslo (June 28-July 2, 2015). A total of 650 participants came to hear the latest research news focused on the zebrafish, Danio rerio, and to its distant evolutionary relative medaka, Oryzias latipes. The packed program included keynote and plenary talks, short oral presentations and poster sessions, workshops, and strategic discussions. The meeting was a great success and revealed dramatically how important the zebrafish in particular has become as a model system for topics, such as developmental biology, functional genomics, biomedicine, toxicology, and drug development. A new emphasis was given to its potential as a model for aquaculture, a topic of great economic interest to the host country Norway and for the future global food supply in general. Zebrafish husbandry as well as its use in teaching were also covered in separate workshops. As has become a tradition in these meetings, there was a well-attended Wellcome Trust Sanger Institute and ZFIN workshop focused on Zebrafish Genome Resources on the first day. The full EZM 2015 program with abstracts can be read and downloaded from the EZM 2015 Web site zebrafish2015.org.

National Category
Developmental Biology Zoology
Identifiers
urn:nbn:se:uu:diva-283762 (URN)10.1089/zeb.2015.1212 (DOI)000372186600008 ()26859625 (PubMedID)
Available from: 2016-04-14 Created: 2016-04-14 Last updated: 2017-11-30Bibliographically approved
Habicher, J., Haitina, T., Eriksson, I., Holmborn, K., Dierker, T., Ahlberg, P. E. & Ledin, J. (2015). Chondroitin / Dermatan Sulfate Modification Enzymes in Zebrafish Development. PLoS ONE, 10(3), Article ID e0121957.
Open this publication in new window or tab >>Chondroitin / Dermatan Sulfate Modification Enzymes in Zebrafish Development
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 3, article id e0121957Article in journal (Refereed) Published
Abstract [en]

Chondroitin/dermatan sulfate (CS/DS) proteoglycans consist of unbranched sulfated polysaccharide chains of repeating GalNAc-GlcA/IdoA disaccharide units, attached to serine residues on specific proteins. The CS/DS proteoglycans are abundant in the extracellular matrix where they have essential functions in tissue development and homeostasis. In this report a phylogenetic analysis of vertebrate genes coding for the enzymes that modify CS/DS is presented. We identify single orthologous genes in the zebrafish genome for the sulfotransferases chst7, chst11, chst13, chst14, chst15 and ust and the epimerase dse. In contrast, two copies were found for mammalian sulfotransferases CHST3 and CHST12 and the epimerase DSEL, named chst3a and chst3b, chst12a and chst12b, dsela and dselb, respectively. Expression of CS/DS modification enzymes is spatially and temporally regulated with a large variation between different genes. We found that CS/DS 4-O-sulfotransferases and 6-O-sulfotransferases as well as CS/DS epimerases show a strong and partly overlapping expression, whereas the expression is restricted for enzymes with ability to synthesize di-sulfated disaccharides. A structural analysis further showed that CS/DS sulfation increases during embryonic development mainly due to synthesis of 4-O-sulfated GalNAc while the proportion of 6-O-sulfated GalNAc increases in later developmental stages. Di-sulfated GalNAc synthesized by Chst15 and 2-O-sulfated GlcA/IdoA synthesized by Ust are rare, in accordance with the restricted expression of these enzymes. We also compared CS/DS composition with that of heparan sulfate (HS). Notably, CS/DS biosynthesis in early zebrafish development is more dynamic than HS biosynthesis. Furthermore, HS contains disaccharides with more than one sulfate group, which are virtually absent in CS/DS.

National Category
Evolutionary Biology
Identifiers
urn:nbn:se:uu:diva-252231 (URN)10.1371/journal.pone.0121957 (DOI)000352083900161 ()25793894 (PubMedID)
Available from: 2015-05-05 Created: 2015-05-04 Last updated: 2017-12-04Bibliographically approved
Varshney, G. K., Pei, W., LaFave, M. C., Idol, J., Xu, L., Gallardo, V., . . . Burgess, S. M. (2015). High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9. Genome Research, 25(7), 1030-1042
Open this publication in new window or tab >>High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9
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2015 (English)In: Genome Research, ISSN 1088-9051, E-ISSN 1549-5469, Vol. 25, no 7, p. 1030-1042Article in journal (Refereed) Published
Abstract [en]

The use of CRISPR/Cas9 as a genome-editing tool in various model organisms has radically changed targeted mutagenesis. Here, we present a high-throughput targeted mutagenesis pipeline using CRISPR/Cas9 technology in zebrafish that will make possible both saturation mutagenesis of the genome and large-scale phenotyping efforts. We describe a cloning-free single-guide RNA (sgRNA) synthesis, coupled with streamlined mutant identification methods utilizing fluorescent PCR and multiplexed, high-throughput sequencing. We report germline transmission data from 162 loci targeting 83 genes in the zebrafish genome, in which we obtained a 99% success rate for generating mutations and an average germline transmission rate of 28%. We verified 678 unique alleles from 58 genes by high-throughput sequencing. We demonstrate that our method can be used for efficient multiplexed gene targeting. We also demonstrate that phenotyping can be done in the F-1 generation by inbreeding two injected founder fish, significantly reducing animal husbandry and time. This study compares germline transmission data from CRISPR/Cas9 with those of TALENs and ZFNs and shows that efficiency of CRISPR/Cas9 is sixfold more efficient than other techniques. We show that the majority of published "rules" for efficient sgRNA design do not effectively predict germline transmission rates in zebrafish, with the exception of a GG or GA dinucleotide genomic match at the 5' end of the sgRNA. Finally, we show that predicted off-target mutagenesis is of low concern for in vivo genetic studies.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-259105 (URN)10.1101/gr.186379.114 (DOI)000357356900010 ()26048245 (PubMedID)
Available from: 2015-07-28 Created: 2015-07-27 Last updated: 2017-12-04Bibliographically approved
Filipek-Gorniok, B., Carlsson, P., Haitina, T., Habicher, J., Ledin, J. & Kjellén, L. (2015). The Ndst Gene Family in Zebrafish: Role of Ndst1b in Pharyngeal Arch Formation. PLoS ONE, 10(3)
Open this publication in new window or tab >>The Ndst Gene Family in Zebrafish: Role of Ndst1b in Pharyngeal Arch Formation
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 3Article in journal (Refereed) Published
Abstract [en]

Heparan sulfate (HS) proteoglycans are ubiquitous components of the extracellular matrix and plasma membrane of metazoans. The sulfation pattern of the HS glycosaminoglycan chain is characteristic for each tissue and changes during development. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes catalyze N-deacetylation and N-sulfation during HS biosynthesis and have a key role in designing the sulfation pattern. We here report on the presence of five NDST genes in zebrafish. Zebrafish ndst1a, ndst1b, ndst2a and ndst2b represent duplicated mammalian orthologues of NDST1 and NDST2 that arose through teleost specific genome duplication. Interestingly, the single zebrafish orthologue ndst3, is equally similar to tetrapod Ndst3 and Ndst4. It is likely that a local duplication in the common ancestor of lobe-finned fish and tetrapods gave rise to these two genes. All zebrafish Ndst genes showed distinct but partially overlapping expression patterns during embryonic development. Morpholino knockdown of ndst1b resulted in delayed development, craniofacial cartilage abnormalities, shortened body and pectoral fin length, resembling some of the features of the Ndst1 mouse knockout.

National Category
Evolutionary Biology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-251802 (URN)10.1371/journal.pone.0119040 (DOI)000351277500060 ()25767878 (PubMedID)
Available from: 2015-04-28 Created: 2015-04-24 Last updated: 2017-12-04Bibliographically approved
Filipek-Gorniok, B., Holmborn, K., Haitina, T., Habicher, J., Oliveira, M. B., Hellgren, C., . . . Ledin, J. (2013). Expression of chondroitin/dermatan sulfate glycosyltransferases during early zebrafish development. Developmental Dynamics, 242(8), 964-975
Open this publication in new window or tab >>Expression of chondroitin/dermatan sulfate glycosyltransferases during early zebrafish development
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2013 (English)In: Developmental Dynamics, ISSN 1058-8388, E-ISSN 1097-0177, Vol. 242, no 8, p. 964-975Article in journal (Refereed) Published
Abstract [en]

Background: Chondroitin/dermatan sulfate (CS/DS) proteoglycans present in the extracellular matrix have important structural and regulatory functions. Results: Six human genes have previously been shown to catalyze CS/DS polymerization. Here we show that one of these genes, chpf, is represented by two copies in the zebrafish genome, chpfa and chpfb, while the other five human CS/DS glycosyltransferases csgalnact1, csgalnact2, chpf2, chsy1, and chsy3 all have single zebrafish orthologues. The putative zebrafish CS/DS glycosyltransferases are spatially and temporally expressed. Interestingly, overlapping expression of multiple glycosyltransferases coincides with high CS/DS deposition. Finally, whereas the relative levels of the related polysaccharide HS reach steady-state at around 2 days post fertilization, there is a continued relative increase of the CS amounts per larvae during the first 6 days of development, matching the increased cartilage formation. Conclusions: There are 7 CS/DS glycosyltransferases in zebrafish, which, based on homology, can be divided into the CSGALNACT, CHSY, and CHPF families. The overlap between intense CS/DS production and the expression of multiple CS/DS glycosyltransferases suggests that efficient CS/DS biosynthesis requires a combination of several glycosyltransferases.

Keywords
chondroitin sulfate, polymerase, CSGALNACT, CHSY, CHPF, zebrafish
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-204834 (URN)10.1002/dvdy.23981 (DOI)000321843400008 ()
Note

De två (2) första författarna delar förstaförfattarskapet.

Available from: 2013-08-13 Created: 2013-08-12 Last updated: 2017-12-06Bibliographically approved
Hayes, A. J., Reynolds, S., Nowell, M. A., Meakin, L. B., Habicher, J., Ledin, J., . . . Hammond, C. L. (2013). Spinal Deformity in Aged Zebrafish Is Accompanied by Degenerative Changes to Their Vertebrae that Resemble Osteoarthritis. PLoS ONE, 8(9), e75787
Open this publication in new window or tab >>Spinal Deformity in Aged Zebrafish Is Accompanied by Degenerative Changes to Their Vertebrae that Resemble Osteoarthritis
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 9, p. e75787-Article in journal (Refereed) Published
Abstract [en]

Age-related degenerative changes within the vertebral column are a significant cause of morbidity with considerable socio-economic impact worldwide. An improved understanding of these changes through the development of experimental models may lead to improvements in existing clinical treatment options. The zebrafish is a well-established model for the study of skeletogenesis with significant potential in gerontological research. With advancing age, zebrafish frequently develop gross deformities of their vertebral column, previously ascribed to reduced trunk muscle tone. In this study, we assess degenerative changes specifically within the bone and cartilage of the vertebral column of zebrafish at 1, 2 and 3-years of age. We show increased frequency and severity of spinal deformities/curvatures with age. Underlying the most severe phenotypes are partial or complete vertebral dislocations and focal thickening of the vertebral bone at the joint margins. MicroCT examination demonstrates small defects, fractures and morphological evidence suggestive of bone erosion and remodeling (i.e. osteophytes) within the vertebrae during aging, but no significant change in bone density. Light and electron microscopic examination reveal striking agerelated changes in cell morphology, suggestive of chondroptosis, and tissue remodelling of the vertebral cartilage, particularly within the pericellular micro-environment. Glycosaminoglycan analysis of the vertebral column by HPLC demonstrates a consistent, age-related increase in the yield of total chondroitin sulfate disaccharide, but no change in sulfation pattern, supported by immunohistochemical analysis. Immunohistochemistry strongly identifies all three chondroitin/dermatan sulphate isoforms (C-0-S, C-4-S/DS and C-6-S) within the vertebral cartilage, particularly within the pericellular micro-environment. In contrast, keratan sulfate immunolocalises specifically with the notochordal tissue of the intervertebral disc, and its labelling diminishes with age. In summary, these observations raise the prospect that zebrafish, in addition to modelling skeletal development, may have utility in modelling age-related degenerative changes that affect the skeleton during senescence.

National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-210236 (URN)10.1371/journal.pone.0075787 (DOI)000325025200062 ()
Available from: 2013-11-05 Created: 2013-11-04 Last updated: 2017-12-06Bibliographically approved
Holmborn, K., Habicher, J., Kasza, Z., Eriksson, A. S., Gorniok, B. F., Gopal, S., . . . Ledin, J. (2012). On the Roles and Regulation of Chondroitin Sulfate and Heparan Sulfate in Zebrafish Pharyngeal Cartilage Morphogenesis. Journal of Biological Chemistry, 287(40), 33905-33916
Open this publication in new window or tab >>On the Roles and Regulation of Chondroitin Sulfate and Heparan Sulfate in Zebrafish Pharyngeal Cartilage Morphogenesis
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2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 40, p. 33905-33916Article in journal (Refereed) Published
Abstract [en]

The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation of the common proteoglycan linkage tetrasaccharide were analyzed along with ext2 and extl3 mutants, predicted to have defective HS polymerization. Notably, the effects on HS and CS biosynthesis in the respective mutant strains were shown to differ from what had been hypothesized. In uxs1 and b3gat3 mutant larvae, biosynthesis of CS was shown to be virtually abolished, whereas these mutants still were capable of synthesizing 50% of the HS produced in control larvae. extl3 and ext2 mutants on the other hand were shown to synthesize reduced amounts of hypersulfated HS. Further, extl3 mutants produced higher levels of CS than control larvae, whereas morpholino-mediated suppression of csgalnact1/csgalnact2 resulted in increased HS biosynthesis. Thus, the balance of the Extl3 and Csgalnact1/Csgalnact2 proteins influences the HS/CS ratio. A characterization of the pharyngeal cartilage element morphologies in the single mutant strains, as well as in ext2;uxs1 double mutants, was conducted. A correlation between HS and CS production and phenotypes was found, such that impaired HS biosynthesis was shown to affect chondrocyte intercalation, whereas impaired CS biosynthesis inhibited formation of the extracellular matrix surrounding chondrocytes.

Keywords
Protein Linkage Region, Molecular-Cloning, Hspg Synthesis, Cell Polarity, Growth-Plate, Expression, Ext2, Proteoglycans, Glypican, Xylose
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-185209 (URN)10.1074/jbc.M112.401646 (DOI)000309602100071 ()
Note

De två första författarna delar förstaförfattarskapet.

De två sista författarna delar sistaförfattarskapet.

Available from: 2012-11-22 Created: 2012-11-21 Last updated: 2017-12-07Bibliographically approved
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