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A late role for bmp2b in the morphogenesis of semicircular canal ducts in the zebrafish inner ear.

Hammond KL, Loynes HE, Mowbray C, Runke G, Hammerschmidt M, Mullins MC, Hildreth V, Chaudhry B, Whitfield TT - PLoS ONE (2009)

Bottom Line: Our findings demonstrate a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear.This is the first demonstration of a developmental role for any gene during post-embryonic stages of otic morphogenesis in the zebrafish.Despite differences in the early stages of semicircular canal formation between zebrafish and amniotes, the role of Bmp2 in semicircular canal duct outgrowth is likely to be conserved between different vertebrate species.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Developmental and Biomedical Genetics and Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom.

ABSTRACT

Background: The Bone Morphogenetic Protein (BMP) genes bmp2 and bmp4 are expressed in highly conserved patterns in the developing vertebrate inner ear. It has, however, proved difficult to elucidate the function of BMPs during ear development as mutations in these genes cause early embryonic lethality. Previous studies using conditional approaches in mouse and chicken have shown that Bmp4 has a role in semicircular canal and crista development, but there is currently no direct evidence for the role of Bmp2 in the developing inner ear.

Methodology/principal findings: We have used an RNA rescue strategy to test the role of bmp2b in the zebrafish inner ear directly. Injection of bmp2b or smad5 mRNA into homozygous mutant swirl (bmp2b(-/-)) embryos rescues the early patterning defects in these mutants and the fish survive to adulthood. As injected RNA will only last, at most, for the first few days of embryogenesis, all later development occurs in the absence of bmp2b function. Although rescued swirl adult fish are viable, they have balance defects suggestive of vestibular dysfunction. Analysis of the inner ears of these fish reveals a total absence of semicircular canal ducts, structures involved in the detection of angular motion. All other regions of the ear, including the ampullae and cristae, are present and appear normal. Early stages of otic development in rescued swirl embryos are also normal.

Conclusions/significance: Our findings demonstrate a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear. This is the first demonstration of a developmental role for any gene during post-embryonic stages of otic morphogenesis in the zebrafish. Despite differences in the early stages of semicircular canal formation between zebrafish and amniotes, the role of Bmp2 in semicircular canal duct outgrowth is likely to be conserved between different vertebrate species.

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Sensory patches and fusion plates are present in the inner ears of 5 dpf rescued swr embryos.Projected confocal z-stacks through inner ears of 5 dpf embryos stained with FITC-conjugated phalloidin, revealing the actin-rich stereociliary bundles of the sensory hair cells and the cortical actin in every cell. (A–D) Lateral views; anterior to left, dorsal to top. (A, B) Lateral plane of focus, showing the cristae, utricular macula and the fusion plates (arrowheads) between the semicircular canal projections. (C, D) More medial plane of focus showing the saccular macula. (E, F) Dorsal views; anterior to left, medial to top. Images shown are composites of two sets of projected z-stacks as the anterior crista is in a more dorsal plane of focus than the remaining sensory patches. Abbreviations: sm, saccular macula; um, utricular macula. Cristae are indicated with an asterisk. Scale bar, 50 µm.
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pone-0004368-g003: Sensory patches and fusion plates are present in the inner ears of 5 dpf rescued swr embryos.Projected confocal z-stacks through inner ears of 5 dpf embryos stained with FITC-conjugated phalloidin, revealing the actin-rich stereociliary bundles of the sensory hair cells and the cortical actin in every cell. (A–D) Lateral views; anterior to left, dorsal to top. (A, B) Lateral plane of focus, showing the cristae, utricular macula and the fusion plates (arrowheads) between the semicircular canal projections. (C, D) More medial plane of focus showing the saccular macula. (E, F) Dorsal views; anterior to left, medial to top. Images shown are composites of two sets of projected z-stacks as the anterior crista is in a more dorsal plane of focus than the remaining sensory patches. Abbreviations: sm, saccular macula; um, utricular macula. Cristae are indicated with an asterisk. Scale bar, 50 µm.

Mentions: The absence of canal duct tissue in adult fish led us to examine otic development in rescued swr mutants at embryonic and early larval stages. To analyse the development of sensory patches and semicircular canals, we stained smad5-RNA rescued swrta72 and swrtdc24 embryos and wild-type controls at 48 hpf to 7 dpf (days post fertilisation) with FITC-conjugated phalloidin. This marks both cortical actin and the actin-rich stereocilia of sensory hair cell bundles, allowing both general ear morphology and the sensory patches to be visualised (see, for example, [18]). In the wild-type ear at 48 hpf, hair cells have differentiated in two sensory patches, the utricular and saccular maculae. At the same stage, projections of epithelium that will form the hubs of the semicircular canals begin to ingress into the vesicle, meeting at a fusion plate to form pillars by approximately 72 hpf. Hair cells in the cristae are present by 60 hpf [19]. At all stages examined, both sensory patch morphology and semicircular canal development appeared normal in the rescued swr ears, including the formation of semicircular canal fusion plates (Fig. 3), although we cannot rule out the presence of any subtle defects. Of thirteen rescued swr embryos examined (four at 48 hpf, two at 72 hpf, five at 5 dpf and two at 7 dpf) only one of the 5 dpf fish did not show a wild-type phenotype. In the ears of this fish, all sensory patches and semicircular canal pillars were present, but the posterior macula was disorganised (data not shown). As the balance defect in rescued swr adults is fully penetrant, and all six sets of adult sections examined show a consistent phenotype, the posterior macula defect in this individual is likely to be non-specific rather than due to a specific lack of bmp2b. Overall, our data demonstrate a late requirement for bmp2b in the development of semicircular canals in the zebrafish that becomes manifest after 7 dpf.


A late role for bmp2b in the morphogenesis of semicircular canal ducts in the zebrafish inner ear.

Hammond KL, Loynes HE, Mowbray C, Runke G, Hammerschmidt M, Mullins MC, Hildreth V, Chaudhry B, Whitfield TT - PLoS ONE (2009)

Sensory patches and fusion plates are present in the inner ears of 5 dpf rescued swr embryos.Projected confocal z-stacks through inner ears of 5 dpf embryos stained with FITC-conjugated phalloidin, revealing the actin-rich stereociliary bundles of the sensory hair cells and the cortical actin in every cell. (A–D) Lateral views; anterior to left, dorsal to top. (A, B) Lateral plane of focus, showing the cristae, utricular macula and the fusion plates (arrowheads) between the semicircular canal projections. (C, D) More medial plane of focus showing the saccular macula. (E, F) Dorsal views; anterior to left, medial to top. Images shown are composites of two sets of projected z-stacks as the anterior crista is in a more dorsal plane of focus than the remaining sensory patches. Abbreviations: sm, saccular macula; um, utricular macula. Cristae are indicated with an asterisk. Scale bar, 50 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2629815&req=5

pone-0004368-g003: Sensory patches and fusion plates are present in the inner ears of 5 dpf rescued swr embryos.Projected confocal z-stacks through inner ears of 5 dpf embryos stained with FITC-conjugated phalloidin, revealing the actin-rich stereociliary bundles of the sensory hair cells and the cortical actin in every cell. (A–D) Lateral views; anterior to left, dorsal to top. (A, B) Lateral plane of focus, showing the cristae, utricular macula and the fusion plates (arrowheads) between the semicircular canal projections. (C, D) More medial plane of focus showing the saccular macula. (E, F) Dorsal views; anterior to left, medial to top. Images shown are composites of two sets of projected z-stacks as the anterior crista is in a more dorsal plane of focus than the remaining sensory patches. Abbreviations: sm, saccular macula; um, utricular macula. Cristae are indicated with an asterisk. Scale bar, 50 µm.
Mentions: The absence of canal duct tissue in adult fish led us to examine otic development in rescued swr mutants at embryonic and early larval stages. To analyse the development of sensory patches and semicircular canals, we stained smad5-RNA rescued swrta72 and swrtdc24 embryos and wild-type controls at 48 hpf to 7 dpf (days post fertilisation) with FITC-conjugated phalloidin. This marks both cortical actin and the actin-rich stereocilia of sensory hair cell bundles, allowing both general ear morphology and the sensory patches to be visualised (see, for example, [18]). In the wild-type ear at 48 hpf, hair cells have differentiated in two sensory patches, the utricular and saccular maculae. At the same stage, projections of epithelium that will form the hubs of the semicircular canals begin to ingress into the vesicle, meeting at a fusion plate to form pillars by approximately 72 hpf. Hair cells in the cristae are present by 60 hpf [19]. At all stages examined, both sensory patch morphology and semicircular canal development appeared normal in the rescued swr ears, including the formation of semicircular canal fusion plates (Fig. 3), although we cannot rule out the presence of any subtle defects. Of thirteen rescued swr embryos examined (four at 48 hpf, two at 72 hpf, five at 5 dpf and two at 7 dpf) only one of the 5 dpf fish did not show a wild-type phenotype. In the ears of this fish, all sensory patches and semicircular canal pillars were present, but the posterior macula was disorganised (data not shown). As the balance defect in rescued swr adults is fully penetrant, and all six sets of adult sections examined show a consistent phenotype, the posterior macula defect in this individual is likely to be non-specific rather than due to a specific lack of bmp2b. Overall, our data demonstrate a late requirement for bmp2b in the development of semicircular canals in the zebrafish that becomes manifest after 7 dpf.

Bottom Line: Our findings demonstrate a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear.This is the first demonstration of a developmental role for any gene during post-embryonic stages of otic morphogenesis in the zebrafish.Despite differences in the early stages of semicircular canal formation between zebrafish and amniotes, the role of Bmp2 in semicircular canal duct outgrowth is likely to be conserved between different vertebrate species.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Developmental and Biomedical Genetics and Department of Biomedical Science, University of Sheffield, Sheffield, United Kingdom.

ABSTRACT

Background: The Bone Morphogenetic Protein (BMP) genes bmp2 and bmp4 are expressed in highly conserved patterns in the developing vertebrate inner ear. It has, however, proved difficult to elucidate the function of BMPs during ear development as mutations in these genes cause early embryonic lethality. Previous studies using conditional approaches in mouse and chicken have shown that Bmp4 has a role in semicircular canal and crista development, but there is currently no direct evidence for the role of Bmp2 in the developing inner ear.

Methodology/principal findings: We have used an RNA rescue strategy to test the role of bmp2b in the zebrafish inner ear directly. Injection of bmp2b or smad5 mRNA into homozygous mutant swirl (bmp2b(-/-)) embryos rescues the early patterning defects in these mutants and the fish survive to adulthood. As injected RNA will only last, at most, for the first few days of embryogenesis, all later development occurs in the absence of bmp2b function. Although rescued swirl adult fish are viable, they have balance defects suggestive of vestibular dysfunction. Analysis of the inner ears of these fish reveals a total absence of semicircular canal ducts, structures involved in the detection of angular motion. All other regions of the ear, including the ampullae and cristae, are present and appear normal. Early stages of otic development in rescued swirl embryos are also normal.

Conclusions/significance: Our findings demonstrate a critical late role for bmp2b in the morphogenesis of semicircular canals in the zebrafish inner ear. This is the first demonstration of a developmental role for any gene during post-embryonic stages of otic morphogenesis in the zebrafish. Despite differences in the early stages of semicircular canal formation between zebrafish and amniotes, the role of Bmp2 in semicircular canal duct outgrowth is likely to be conserved between different vertebrate species.

Show MeSH
Related in: MedlinePlus