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A central function for perlecan in skeletal muscle and cardiovascular development.

Zoeller JJ, McQuillan A, Whitelock J, Ho SY, Iozzo RV - J. Cell Biol. (2008)

Bottom Line: In the perlecan morphants, primary intersegmental vessel sprouts, which develop through angiogenesis, fail to extend and show reduced protrusive activity.The phenotype is partially rescued by microinjection of human perlecan or endorepellin.These findings indicate that perlecan is essential for the integrity of somitic muscle and developmental angiogenesis and that endorepellin mediates most of these biological activities.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Anatomy, and Cell, Thomas Jefferson University, Philadelphia, PA 19107, USA.

ABSTRACT
Perlecan's developmental functions are difficult to dissect in placental animals because perlecan disruption is embryonic lethal. In contrast to mammals, cardiovascular function is not essential for early zebrafish development because the embryos obtain adequate oxygen by diffusion. In this study, we use targeted protein depletion coupled with protein-based rescue experiments to investigate the involvement of perlecan and its C-terminal domain V/endorepellin in zebrafish development. The perlecan morphants show a severe myopathy characterized by abnormal actin filament orientation and disorganized sarcomeres, suggesting an involvement of perlecan in myopathies. In the perlecan morphants, primary intersegmental vessel sprouts, which develop through angiogenesis, fail to extend and show reduced protrusive activity. Live videomicroscopy confirms the abnormal swimming pattern caused by the myopathy and anomalous head and trunk vessel circulation. The phenotype is partially rescued by microinjection of human perlecan or endorepellin. These findings indicate that perlecan is essential for the integrity of somitic muscle and developmental angiogenesis and that endorepellin mediates most of these biological activities.

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Classification and verification of the perlecan morphant phenotype. (A) All observed perlecan morphant phenotypes can be classified according to the degree of body twisting as presented for MO-DI. Categories include mild (least striking twisted body but noted tail phenotype), moderate (general twisted body usually accompanied by curly tail up), and severe (significant body plan shortening accompanied by twisting of the tail). (B) Mean observed frequencies of the twisted body classes from MO-DI perlecan morphant embryos (n = 232). Error bars represent ±SEM. (C) Phenotypic overview of perlecan splice junction–blocking MO-DIII. (D) RT-PCR verification of domain III splice junction–blocking morpholino effect. Note the band shift in morpholino-injected embryos (lane 5) versus uninjected embryos (lane 4), verifying the splice-blocking/intron retaining effect of MO-DIII. Lane 1, DNA ladder; lane 2, domain III PCR from zebrafish cDNA template; lane 3, domain III PCR from genomic DNA template; lane 4, domain III PCR from uninjected embryos' cDNA template; lane 5, domain III PCR from MO-DIII–injected embryos' cDNA template. The bottom bands in lanes 4 and 5 represent the β-actin control. Template from lanes 4 and 5 were derived from total RNA isolated from 23 embryos. See Fig. 1 A for additional details regarding the targeting positions of the morpholinos. (E–G) Whole mount immunohistochemistry for verification of domain III morpholino-based knockdown of perlecan. (E) Control uninjected embryo (2 dpf) shows perlecan expression throughout the trunk musculature and vasculature. (F and G) MO-DIII embryos (2 dpf) show significantly reduced perlecan protein levels in the trunk, with only minimal staining detected in the head and tail. A and E–G are left-side views with dorsal up and anterior to the left. Bars, 500 μm.
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fig3: Classification and verification of the perlecan morphant phenotype. (A) All observed perlecan morphant phenotypes can be classified according to the degree of body twisting as presented for MO-DI. Categories include mild (least striking twisted body but noted tail phenotype), moderate (general twisted body usually accompanied by curly tail up), and severe (significant body plan shortening accompanied by twisting of the tail). (B) Mean observed frequencies of the twisted body classes from MO-DI perlecan morphant embryos (n = 232). Error bars represent ±SEM. (C) Phenotypic overview of perlecan splice junction–blocking MO-DIII. (D) RT-PCR verification of domain III splice junction–blocking morpholino effect. Note the band shift in morpholino-injected embryos (lane 5) versus uninjected embryos (lane 4), verifying the splice-blocking/intron retaining effect of MO-DIII. Lane 1, DNA ladder; lane 2, domain III PCR from zebrafish cDNA template; lane 3, domain III PCR from genomic DNA template; lane 4, domain III PCR from uninjected embryos' cDNA template; lane 5, domain III PCR from MO-DIII–injected embryos' cDNA template. The bottom bands in lanes 4 and 5 represent the β-actin control. Template from lanes 4 and 5 were derived from total RNA isolated from 23 embryos. See Fig. 1 A for additional details regarding the targeting positions of the morpholinos. (E–G) Whole mount immunohistochemistry for verification of domain III morpholino-based knockdown of perlecan. (E) Control uninjected embryo (2 dpf) shows perlecan expression throughout the trunk musculature and vasculature. (F and G) MO-DIII embryos (2 dpf) show significantly reduced perlecan protein levels in the trunk, with only minimal staining detected in the head and tail. A and E–G are left-side views with dorsal up and anterior to the left. Bars, 500 μm.

Mentions: To asses the developmental roles of perlecan, we selectively blocked the translation of perlecan mRNA using morpholino antisense oligonucleotides (MO), a specific translation inhibitor in zebrafish (Nasevicius and Ekker, 2000). We initially used two morpholinos of nonoverlapping sequence: one directed against the translation start site (MO-DI) of perlecan and the other encompassing the splice donor site of the initial exon of domain V/endorepellin (MO-DV; Fig. 1 A). Embryos at the one- to two-cell stage were injected with 2.5–10 ng morpholino, and embryos from the same matings were injected with an equal volume of phenol red/nuclease-free water mixture as a control. Higher morpholino concentrations (5–10 ng) caused significant lethality, especially for MO-DV; thus, we used 2.5 ng or less of the morpholinos. Both morpholinos produced an identical phenotype >90% (n = 358; P < 0.001; 10 independent experiments) with severe defects in the cardiovascular and musculoskeletal systems that became visible at 1 dpf (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200708022/DC1) and became progressively more apparent as development proceeded (Fig. S2, B–E). At 2 dpf, the morphants displayed a pronounced curvature of the tail and trunk, which could be classified as mild, moderate, or severe twisting of the body (Fig. 3, A and B). The morphants exhibited either no escape response or uncoordinated movements in response to tactile stimuli and often swam in a circular fashion (Video 1).


A central function for perlecan in skeletal muscle and cardiovascular development.

Zoeller JJ, McQuillan A, Whitelock J, Ho SY, Iozzo RV - J. Cell Biol. (2008)

Classification and verification of the perlecan morphant phenotype. (A) All observed perlecan morphant phenotypes can be classified according to the degree of body twisting as presented for MO-DI. Categories include mild (least striking twisted body but noted tail phenotype), moderate (general twisted body usually accompanied by curly tail up), and severe (significant body plan shortening accompanied by twisting of the tail). (B) Mean observed frequencies of the twisted body classes from MO-DI perlecan morphant embryos (n = 232). Error bars represent ±SEM. (C) Phenotypic overview of perlecan splice junction–blocking MO-DIII. (D) RT-PCR verification of domain III splice junction–blocking morpholino effect. Note the band shift in morpholino-injected embryos (lane 5) versus uninjected embryos (lane 4), verifying the splice-blocking/intron retaining effect of MO-DIII. Lane 1, DNA ladder; lane 2, domain III PCR from zebrafish cDNA template; lane 3, domain III PCR from genomic DNA template; lane 4, domain III PCR from uninjected embryos' cDNA template; lane 5, domain III PCR from MO-DIII–injected embryos' cDNA template. The bottom bands in lanes 4 and 5 represent the β-actin control. Template from lanes 4 and 5 were derived from total RNA isolated from 23 embryos. See Fig. 1 A for additional details regarding the targeting positions of the morpholinos. (E–G) Whole mount immunohistochemistry for verification of domain III morpholino-based knockdown of perlecan. (E) Control uninjected embryo (2 dpf) shows perlecan expression throughout the trunk musculature and vasculature. (F and G) MO-DIII embryos (2 dpf) show significantly reduced perlecan protein levels in the trunk, with only minimal staining detected in the head and tail. A and E–G are left-side views with dorsal up and anterior to the left. Bars, 500 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2315682&req=5

fig3: Classification and verification of the perlecan morphant phenotype. (A) All observed perlecan morphant phenotypes can be classified according to the degree of body twisting as presented for MO-DI. Categories include mild (least striking twisted body but noted tail phenotype), moderate (general twisted body usually accompanied by curly tail up), and severe (significant body plan shortening accompanied by twisting of the tail). (B) Mean observed frequencies of the twisted body classes from MO-DI perlecan morphant embryos (n = 232). Error bars represent ±SEM. (C) Phenotypic overview of perlecan splice junction–blocking MO-DIII. (D) RT-PCR verification of domain III splice junction–blocking morpholino effect. Note the band shift in morpholino-injected embryos (lane 5) versus uninjected embryos (lane 4), verifying the splice-blocking/intron retaining effect of MO-DIII. Lane 1, DNA ladder; lane 2, domain III PCR from zebrafish cDNA template; lane 3, domain III PCR from genomic DNA template; lane 4, domain III PCR from uninjected embryos' cDNA template; lane 5, domain III PCR from MO-DIII–injected embryos' cDNA template. The bottom bands in lanes 4 and 5 represent the β-actin control. Template from lanes 4 and 5 were derived from total RNA isolated from 23 embryos. See Fig. 1 A for additional details regarding the targeting positions of the morpholinos. (E–G) Whole mount immunohistochemistry for verification of domain III morpholino-based knockdown of perlecan. (E) Control uninjected embryo (2 dpf) shows perlecan expression throughout the trunk musculature and vasculature. (F and G) MO-DIII embryos (2 dpf) show significantly reduced perlecan protein levels in the trunk, with only minimal staining detected in the head and tail. A and E–G are left-side views with dorsal up and anterior to the left. Bars, 500 μm.
Mentions: To asses the developmental roles of perlecan, we selectively blocked the translation of perlecan mRNA using morpholino antisense oligonucleotides (MO), a specific translation inhibitor in zebrafish (Nasevicius and Ekker, 2000). We initially used two morpholinos of nonoverlapping sequence: one directed against the translation start site (MO-DI) of perlecan and the other encompassing the splice donor site of the initial exon of domain V/endorepellin (MO-DV; Fig. 1 A). Embryos at the one- to two-cell stage were injected with 2.5–10 ng morpholino, and embryos from the same matings were injected with an equal volume of phenol red/nuclease-free water mixture as a control. Higher morpholino concentrations (5–10 ng) caused significant lethality, especially for MO-DV; thus, we used 2.5 ng or less of the morpholinos. Both morpholinos produced an identical phenotype >90% (n = 358; P < 0.001; 10 independent experiments) with severe defects in the cardiovascular and musculoskeletal systems that became visible at 1 dpf (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200708022/DC1) and became progressively more apparent as development proceeded (Fig. S2, B–E). At 2 dpf, the morphants displayed a pronounced curvature of the tail and trunk, which could be classified as mild, moderate, or severe twisting of the body (Fig. 3, A and B). The morphants exhibited either no escape response or uncoordinated movements in response to tactile stimuli and often swam in a circular fashion (Video 1).

Bottom Line: In the perlecan morphants, primary intersegmental vessel sprouts, which develop through angiogenesis, fail to extend and show reduced protrusive activity.The phenotype is partially rescued by microinjection of human perlecan or endorepellin.These findings indicate that perlecan is essential for the integrity of somitic muscle and developmental angiogenesis and that endorepellin mediates most of these biological activities.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Anatomy, and Cell, Thomas Jefferson University, Philadelphia, PA 19107, USA.

ABSTRACT
Perlecan's developmental functions are difficult to dissect in placental animals because perlecan disruption is embryonic lethal. In contrast to mammals, cardiovascular function is not essential for early zebrafish development because the embryos obtain adequate oxygen by diffusion. In this study, we use targeted protein depletion coupled with protein-based rescue experiments to investigate the involvement of perlecan and its C-terminal domain V/endorepellin in zebrafish development. The perlecan morphants show a severe myopathy characterized by abnormal actin filament orientation and disorganized sarcomeres, suggesting an involvement of perlecan in myopathies. In the perlecan morphants, primary intersegmental vessel sprouts, which develop through angiogenesis, fail to extend and show reduced protrusive activity. Live videomicroscopy confirms the abnormal swimming pattern caused by the myopathy and anomalous head and trunk vessel circulation. The phenotype is partially rescued by microinjection of human perlecan or endorepellin. These findings indicate that perlecan is essential for the integrity of somitic muscle and developmental angiogenesis and that endorepellin mediates most of these biological activities.

Show MeSH
Related in: MedlinePlus