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The NDST gene family in zebrafish: role of NDST1B in pharyngeal arch formation.

Filipek-Górniok B, Carlsson P, Haitina T, Habicher J, Ledin J, Kjellén L - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Husargatan 3, PO Box 582, SE-751 23, Uppsala, Sweden.

ABSTRACT
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.

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Morpholino knockdown of zebrafish ndst1b results in craniofacial cartilage defects at the early stages of development (3–7 dpf).Lateral and ventral view of a zebrafish larvae at 3 dpf injected with either control MO mix (A-B), MO1 (D-E) or MO2 (G-H) in combination with p53 MO (see Experimental Procedures). The black arrows point at the underdeveloped craniofacial cartilage of ndst1b morphants, while the red arrow heads point at the pectoral fins of the morphants, shortened in comparison to fins of the control larvae. At 5 dpf, the effects of the ndst1b MOs on zebrafish larvae were still visible. ndst1b MO treated larvae had shortened pectoral fins and body axis, smaller head and eyes, underdeveloped craniofacial cartilage and no swimming bladder (C,F,I). Confocal microscope images present a ventral view of Tg(fli1:EGFP) zebrafish larvae. GFP is expressed in endothelial and neural crest derived cells. Morphants displayed severe malformations in the pharyngeal cartilage structures (K, L) in comparison to control larvae (J). In contrast, chondrocyte stacking and flattening does not appear to be strongly affected, as compared to extl3-/- and ext2-/- mutant embryos (M-N) with clear chondrocyte stacking defects. The second pharyngeal arch (the ceratohyal), incorrectly localized in the ndst1b morphants, is indicated with a white arrow head (J-L). The incorrect localization is also seen after alcian blue staining of 7 dpf larvae injected with MO1, MO2 and half working dosage of these two combined (1/2 X (MO1 + MO2); O-R). Panel S of the figure represents quantification in percentage of the ceratohyal phenotype at day 6 of the development in phenotypic classes observed (chi-square test: *** p<0.001 comparing affected and nonaffected embryos). Confocal microscope images of the Tg(1.7col2a1a:mEGFP) fish (1–3) show the difference between reverted (1), perpendicular (2) and non-affected ceratohyals.
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pone.0119040.g006: Morpholino knockdown of zebrafish ndst1b results in craniofacial cartilage defects at the early stages of development (3–7 dpf).Lateral and ventral view of a zebrafish larvae at 3 dpf injected with either control MO mix (A-B), MO1 (D-E) or MO2 (G-H) in combination with p53 MO (see Experimental Procedures). The black arrows point at the underdeveloped craniofacial cartilage of ndst1b morphants, while the red arrow heads point at the pectoral fins of the morphants, shortened in comparison to fins of the control larvae. At 5 dpf, the effects of the ndst1b MOs on zebrafish larvae were still visible. ndst1b MO treated larvae had shortened pectoral fins and body axis, smaller head and eyes, underdeveloped craniofacial cartilage and no swimming bladder (C,F,I). Confocal microscope images present a ventral view of Tg(fli1:EGFP) zebrafish larvae. GFP is expressed in endothelial and neural crest derived cells. Morphants displayed severe malformations in the pharyngeal cartilage structures (K, L) in comparison to control larvae (J). In contrast, chondrocyte stacking and flattening does not appear to be strongly affected, as compared to extl3-/- and ext2-/- mutant embryos (M-N) with clear chondrocyte stacking defects. The second pharyngeal arch (the ceratohyal), incorrectly localized in the ndst1b morphants, is indicated with a white arrow head (J-L). The incorrect localization is also seen after alcian blue staining of 7 dpf larvae injected with MO1, MO2 and half working dosage of these two combined (1/2 X (MO1 + MO2); O-R). Panel S of the figure represents quantification in percentage of the ceratohyal phenotype at day 6 of the development in phenotypic classes observed (chi-square test: *** p<0.001 comparing affected and nonaffected embryos). Confocal microscope images of the Tg(1.7col2a1a:mEGFP) fish (1–3) show the difference between reverted (1), perpendicular (2) and non-affected ceratohyals.

Mentions: At 6 dpf, approximately 80% of the ndst1b morphant larvae injected with either translational blocking (MO1), or 55% of the ones injected with splice blocking mix (MO2) exhibited a distinct, uniform phenotype (Fig. 6S). Exon 6 was chosen as a splice MO target (MO2) since it contains the cysteine residue shown to be critical for N-deacetylase activity (S3 Fig.) [31, 32]. In addition control co-injection of a half working-concentration of MO1 and MO2 resulted in 50% penetrance and a similar phenotype as injection of MO1 or MO2 alone (Fig. 6R-S). For each of the performed morpholino injections, MO targeting p53 was included to avoid the off-target p53 up-regulation effect [24]. To confirm the specificity of the splice MO knockdown (MO2), RT-PCR was performed with cDNA synthesized from mRNA isolated from 24 hpf embryos. The obtained products matched the predicted lengths of the PCR products with and without exon 6 in the template mRNA. Their identities were also confirmed by sequencing of the PCR product (S3C Fig.).


The NDST gene family in zebrafish: role of NDST1B in pharyngeal arch formation.

Filipek-Górniok B, Carlsson P, Haitina T, Habicher J, Ledin J, Kjellén L - PLoS ONE (2015)

Morpholino knockdown of zebrafish ndst1b results in craniofacial cartilage defects at the early stages of development (3–7 dpf).Lateral and ventral view of a zebrafish larvae at 3 dpf injected with either control MO mix (A-B), MO1 (D-E) or MO2 (G-H) in combination with p53 MO (see Experimental Procedures). The black arrows point at the underdeveloped craniofacial cartilage of ndst1b morphants, while the red arrow heads point at the pectoral fins of the morphants, shortened in comparison to fins of the control larvae. At 5 dpf, the effects of the ndst1b MOs on zebrafish larvae were still visible. ndst1b MO treated larvae had shortened pectoral fins and body axis, smaller head and eyes, underdeveloped craniofacial cartilage and no swimming bladder (C,F,I). Confocal microscope images present a ventral view of Tg(fli1:EGFP) zebrafish larvae. GFP is expressed in endothelial and neural crest derived cells. Morphants displayed severe malformations in the pharyngeal cartilage structures (K, L) in comparison to control larvae (J). In contrast, chondrocyte stacking and flattening does not appear to be strongly affected, as compared to extl3-/- and ext2-/- mutant embryos (M-N) with clear chondrocyte stacking defects. The second pharyngeal arch (the ceratohyal), incorrectly localized in the ndst1b morphants, is indicated with a white arrow head (J-L). The incorrect localization is also seen after alcian blue staining of 7 dpf larvae injected with MO1, MO2 and half working dosage of these two combined (1/2 X (MO1 + MO2); O-R). Panel S of the figure represents quantification in percentage of the ceratohyal phenotype at day 6 of the development in phenotypic classes observed (chi-square test: *** p<0.001 comparing affected and nonaffected embryos). Confocal microscope images of the Tg(1.7col2a1a:mEGFP) fish (1–3) show the difference between reverted (1), perpendicular (2) and non-affected ceratohyals.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4359090&req=5

pone.0119040.g006: Morpholino knockdown of zebrafish ndst1b results in craniofacial cartilage defects at the early stages of development (3–7 dpf).Lateral and ventral view of a zebrafish larvae at 3 dpf injected with either control MO mix (A-B), MO1 (D-E) or MO2 (G-H) in combination with p53 MO (see Experimental Procedures). The black arrows point at the underdeveloped craniofacial cartilage of ndst1b morphants, while the red arrow heads point at the pectoral fins of the morphants, shortened in comparison to fins of the control larvae. At 5 dpf, the effects of the ndst1b MOs on zebrafish larvae were still visible. ndst1b MO treated larvae had shortened pectoral fins and body axis, smaller head and eyes, underdeveloped craniofacial cartilage and no swimming bladder (C,F,I). Confocal microscope images present a ventral view of Tg(fli1:EGFP) zebrafish larvae. GFP is expressed in endothelial and neural crest derived cells. Morphants displayed severe malformations in the pharyngeal cartilage structures (K, L) in comparison to control larvae (J). In contrast, chondrocyte stacking and flattening does not appear to be strongly affected, as compared to extl3-/- and ext2-/- mutant embryos (M-N) with clear chondrocyte stacking defects. The second pharyngeal arch (the ceratohyal), incorrectly localized in the ndst1b morphants, is indicated with a white arrow head (J-L). The incorrect localization is also seen after alcian blue staining of 7 dpf larvae injected with MO1, MO2 and half working dosage of these two combined (1/2 X (MO1 + MO2); O-R). Panel S of the figure represents quantification in percentage of the ceratohyal phenotype at day 6 of the development in phenotypic classes observed (chi-square test: *** p<0.001 comparing affected and nonaffected embryos). Confocal microscope images of the Tg(1.7col2a1a:mEGFP) fish (1–3) show the difference between reverted (1), perpendicular (2) and non-affected ceratohyals.
Mentions: At 6 dpf, approximately 80% of the ndst1b morphant larvae injected with either translational blocking (MO1), or 55% of the ones injected with splice blocking mix (MO2) exhibited a distinct, uniform phenotype (Fig. 6S). Exon 6 was chosen as a splice MO target (MO2) since it contains the cysteine residue shown to be critical for N-deacetylase activity (S3 Fig.) [31, 32]. In addition control co-injection of a half working-concentration of MO1 and MO2 resulted in 50% penetrance and a similar phenotype as injection of MO1 or MO2 alone (Fig. 6R-S). For each of the performed morpholino injections, MO targeting p53 was included to avoid the off-target p53 up-regulation effect [24]. To confirm the specificity of the splice MO knockdown (MO2), RT-PCR was performed with cDNA synthesized from mRNA isolated from 24 hpf embryos. The obtained products matched the predicted lengths of the PCR products with and without exon 6 in the template mRNA. Their identities were also confirmed by sequencing of the PCR product (S3C Fig.).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Husargatan 3, PO Box 582, SE-751 23, Uppsala, Sweden.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus