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Genetic determinants of hyaloid and retinal vasculature in zebrafish.

Alvarez Y, Cederlund ML, Cottell DC, Bill BR, Ekker SC, Torres-Vazquez J, Weinstein BM, Hyde DR, Vihtelic TS, Kennedy BN - BMC Dev. Biol. (2007)

Bottom Line: Similar to the transient hyaloid vasculature in mammalian embryos, vessels are first found attached to the zebrafish lens at 2.5 days post fertilisation.Finally, we identify 9 genes with cell membrane, extracellular matrix and unknown identity that are necessary for zebrafish hyaloid and retinal vasculature development.Zebrafish have a retinal blood supply with a characteristic developmental and adult morphology.

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Affiliation: UCD School of Biomolecular, and Biomedical Sciences, University College Dublin, Dublin 4, Ireland. yolanda.alvarez@ucd.ie

ABSTRACT

Background: The retinal vasculature is a capillary network of blood vessels that nourishes the inner retina of most mammals. Developmental abnormalities or microvascular complications in the retinal vasculature result in severe human eye diseases that lead to blindness. To exploit the advantages of zebrafish for genetic, developmental and pharmacological studies of retinal vasculature, we characterised the intraocular vasculature in zebrafish.

Results: We show a detailed morphological and developmental analysis of the retinal blood supply in zebrafish. Similar to the transient hyaloid vasculature in mammalian embryos, vessels are first found attached to the zebrafish lens at 2.5 days post fertilisation. These vessels progressively lose contact with the lens and by 30 days post fertilisation adhere to the inner limiting membrane of the juvenile retina. Ultrastructure analysis shows these vessels to exhibit distinctive hallmarks of mammalian retinal vasculature. For example, smooth muscle actin-expressing pericytes are ensheathed by the basal lamina of the blood vessel, and vesicle vacuolar organelles (VVO), subcellular mediators of vessel-retinal nourishment, are present. Finally, we identify 9 genes with cell membrane, extracellular matrix and unknown identity that are necessary for zebrafish hyaloid and retinal vasculature development.

Conclusion: Zebrafish have a retinal blood supply with a characteristic developmental and adult morphology. Abnormalities of these intraocular vessels are easily observed, enabling application of genetic and chemical approaches in zebrafish to identify molecular regulators of hyaloid and retinal vasculature in development and disease.

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Adult zebrafish have a complex system of retinal blood vessels. A: Wholemount retina showing central major vessels that radiate into thinner vessels covering the entire inner surface of the retina. B: Example of disparity in branch number between left (5) and right (8) eyes. C: Transverse view of the blood vessels (green: Fli1-EGFP) overlaying the inner limiting membrane of the adult retina (blue: DAPI nuclear staining). Inset shows the same vessels overlying GCL nuclei in a flatmount preparation. D: At peripheral retinal regions, neighbouring vessels anastomose (inset), and elongated filopodia sprout from the capillaries suggesting active angiogenic remodelling. E-G: The diameter of vessels is thicker proximal to the optic disc and thinner peripherally. Numbers refer to the thickness of the vessel and the angle of measurement in reference to horizontal plane. H-K: Flatmount adult retinas immuno-labelled with retinal vasculature markers. H: collagen IV (red) stains the basal membrane of blood vessels. I: smooth muscle actin (SMA) stains vascular pericytes (green). J: Factor VIII labels endothelial cells (red). Cone photoreceptors label green as analysis was performed in Tg(3.2TαCP:EGFP) transgenic line [67]. K-L: Glial fibrillary acidic protein (GFAP) stains retinal vessels in adult zebrafish and a population of cells throughout the retina. GFAP (red), DAPI (blue) and fli1-EGFP (green). K: Flat-mounted retina, and L: Transverse view of the peripheral retina. Müller endfeet (asterisks) directly contact the endothelial cells (yellow co-staining). Inset in K: FITC channel turned off to highlight the GFAP reactivity of retinal vessels. ILM: inner limiting membrane; GCL: ganglion cell layer; IPL: inner plexiform layer; OPL: outer plexiform layer; INL: inner nuclear layer; ONL: outer nuclear layer; IOC: inner optic circle.
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Figure 1: Adult zebrafish have a complex system of retinal blood vessels. A: Wholemount retina showing central major vessels that radiate into thinner vessels covering the entire inner surface of the retina. B: Example of disparity in branch number between left (5) and right (8) eyes. C: Transverse view of the blood vessels (green: Fli1-EGFP) overlaying the inner limiting membrane of the adult retina (blue: DAPI nuclear staining). Inset shows the same vessels overlying GCL nuclei in a flatmount preparation. D: At peripheral retinal regions, neighbouring vessels anastomose (inset), and elongated filopodia sprout from the capillaries suggesting active angiogenic remodelling. E-G: The diameter of vessels is thicker proximal to the optic disc and thinner peripherally. Numbers refer to the thickness of the vessel and the angle of measurement in reference to horizontal plane. H-K: Flatmount adult retinas immuno-labelled with retinal vasculature markers. H: collagen IV (red) stains the basal membrane of blood vessels. I: smooth muscle actin (SMA) stains vascular pericytes (green). J: Factor VIII labels endothelial cells (red). Cone photoreceptors label green as analysis was performed in Tg(3.2TαCP:EGFP) transgenic line [67]. K-L: Glial fibrillary acidic protein (GFAP) stains retinal vessels in adult zebrafish and a population of cells throughout the retina. GFAP (red), DAPI (blue) and fli1-EGFP (green). K: Flat-mounted retina, and L: Transverse view of the peripheral retina. Müller endfeet (asterisks) directly contact the endothelial cells (yellow co-staining). Inset in K: FITC channel turned off to highlight the GFAP reactivity of retinal vessels. ILM: inner limiting membrane; GCL: ganglion cell layer; IPL: inner plexiform layer; OPL: outer plexiform layer; INL: inner nuclear layer; ONL: outer nuclear layer; IOC: inner optic circle.

Mentions: The morphology of the retinal vasculature in adult Tg(fli1:EGFP) zebrafish was characterised using fluorescent microscopy to directly visualise the vessels (Fig 1A–G and 1K–L). The retinal vasculature in wild type and albino animals was determined by immunohistochemistry and nuclear staining (Fig 1H–L and data not shown).


Genetic determinants of hyaloid and retinal vasculature in zebrafish.

Alvarez Y, Cederlund ML, Cottell DC, Bill BR, Ekker SC, Torres-Vazquez J, Weinstein BM, Hyde DR, Vihtelic TS, Kennedy BN - BMC Dev. Biol. (2007)

Adult zebrafish have a complex system of retinal blood vessels. A: Wholemount retina showing central major vessels that radiate into thinner vessels covering the entire inner surface of the retina. B: Example of disparity in branch number between left (5) and right (8) eyes. C: Transverse view of the blood vessels (green: Fli1-EGFP) overlaying the inner limiting membrane of the adult retina (blue: DAPI nuclear staining). Inset shows the same vessels overlying GCL nuclei in a flatmount preparation. D: At peripheral retinal regions, neighbouring vessels anastomose (inset), and elongated filopodia sprout from the capillaries suggesting active angiogenic remodelling. E-G: The diameter of vessels is thicker proximal to the optic disc and thinner peripherally. Numbers refer to the thickness of the vessel and the angle of measurement in reference to horizontal plane. H-K: Flatmount adult retinas immuno-labelled with retinal vasculature markers. H: collagen IV (red) stains the basal membrane of blood vessels. I: smooth muscle actin (SMA) stains vascular pericytes (green). J: Factor VIII labels endothelial cells (red). Cone photoreceptors label green as analysis was performed in Tg(3.2TαCP:EGFP) transgenic line [67]. K-L: Glial fibrillary acidic protein (GFAP) stains retinal vessels in adult zebrafish and a population of cells throughout the retina. GFAP (red), DAPI (blue) and fli1-EGFP (green). K: Flat-mounted retina, and L: Transverse view of the peripheral retina. Müller endfeet (asterisks) directly contact the endothelial cells (yellow co-staining). Inset in K: FITC channel turned off to highlight the GFAP reactivity of retinal vessels. ILM: inner limiting membrane; GCL: ganglion cell layer; IPL: inner plexiform layer; OPL: outer plexiform layer; INL: inner nuclear layer; ONL: outer nuclear layer; IOC: inner optic circle.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Adult zebrafish have a complex system of retinal blood vessels. A: Wholemount retina showing central major vessels that radiate into thinner vessels covering the entire inner surface of the retina. B: Example of disparity in branch number between left (5) and right (8) eyes. C: Transverse view of the blood vessels (green: Fli1-EGFP) overlaying the inner limiting membrane of the adult retina (blue: DAPI nuclear staining). Inset shows the same vessels overlying GCL nuclei in a flatmount preparation. D: At peripheral retinal regions, neighbouring vessels anastomose (inset), and elongated filopodia sprout from the capillaries suggesting active angiogenic remodelling. E-G: The diameter of vessels is thicker proximal to the optic disc and thinner peripherally. Numbers refer to the thickness of the vessel and the angle of measurement in reference to horizontal plane. H-K: Flatmount adult retinas immuno-labelled with retinal vasculature markers. H: collagen IV (red) stains the basal membrane of blood vessels. I: smooth muscle actin (SMA) stains vascular pericytes (green). J: Factor VIII labels endothelial cells (red). Cone photoreceptors label green as analysis was performed in Tg(3.2TαCP:EGFP) transgenic line [67]. K-L: Glial fibrillary acidic protein (GFAP) stains retinal vessels in adult zebrafish and a population of cells throughout the retina. GFAP (red), DAPI (blue) and fli1-EGFP (green). K: Flat-mounted retina, and L: Transverse view of the peripheral retina. Müller endfeet (asterisks) directly contact the endothelial cells (yellow co-staining). Inset in K: FITC channel turned off to highlight the GFAP reactivity of retinal vessels. ILM: inner limiting membrane; GCL: ganglion cell layer; IPL: inner plexiform layer; OPL: outer plexiform layer; INL: inner nuclear layer; ONL: outer nuclear layer; IOC: inner optic circle.
Mentions: The morphology of the retinal vasculature in adult Tg(fli1:EGFP) zebrafish was characterised using fluorescent microscopy to directly visualise the vessels (Fig 1A–G and 1K–L). The retinal vasculature in wild type and albino animals was determined by immunohistochemistry and nuclear staining (Fig 1H–L and data not shown).

Bottom Line: Similar to the transient hyaloid vasculature in mammalian embryos, vessels are first found attached to the zebrafish lens at 2.5 days post fertilisation.Finally, we identify 9 genes with cell membrane, extracellular matrix and unknown identity that are necessary for zebrafish hyaloid and retinal vasculature development.Zebrafish have a retinal blood supply with a characteristic developmental and adult morphology.

View Article: PubMed Central - HTML - PubMed

Affiliation: UCD School of Biomolecular, and Biomedical Sciences, University College Dublin, Dublin 4, Ireland. yolanda.alvarez@ucd.ie

ABSTRACT

Background: The retinal vasculature is a capillary network of blood vessels that nourishes the inner retina of most mammals. Developmental abnormalities or microvascular complications in the retinal vasculature result in severe human eye diseases that lead to blindness. To exploit the advantages of zebrafish for genetic, developmental and pharmacological studies of retinal vasculature, we characterised the intraocular vasculature in zebrafish.

Results: We show a detailed morphological and developmental analysis of the retinal blood supply in zebrafish. Similar to the transient hyaloid vasculature in mammalian embryos, vessels are first found attached to the zebrafish lens at 2.5 days post fertilisation. These vessels progressively lose contact with the lens and by 30 days post fertilisation adhere to the inner limiting membrane of the juvenile retina. Ultrastructure analysis shows these vessels to exhibit distinctive hallmarks of mammalian retinal vasculature. For example, smooth muscle actin-expressing pericytes are ensheathed by the basal lamina of the blood vessel, and vesicle vacuolar organelles (VVO), subcellular mediators of vessel-retinal nourishment, are present. Finally, we identify 9 genes with cell membrane, extracellular matrix and unknown identity that are necessary for zebrafish hyaloid and retinal vasculature development.

Conclusion: Zebrafish have a retinal blood supply with a characteristic developmental and adult morphology. Abnormalities of these intraocular vessels are easily observed, enabling application of genetic and chemical approaches in zebrafish to identify molecular regulators of hyaloid and retinal vasculature in development and disease.

Show MeSH
Related in: MedlinePlus