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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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Three-dimensional reconstructions of adult rescued swr inner ears.(A–F) Three-dimensional reconstructions of the inner ears of two adult rescued swr fish (swirl 1 (A, D) and swirl 2 (B, E)) and an age-matched wild-type fish (C, F). Semicircular canals are absent in the rescued swr fish ears, while the ampullae, crus commune, utricle, saccule and lagena are present and appear normal. Note that the pars superior (ampullae, crus commune and utricle) of swirl 1 is twisted relative to the pars inferior (saccule and lagena), about the point marked with the arrow (we are unable to tell whether or not this is an experimental artefact). The kink in the lateral canal of the wild-type ear (C) is an artefact. The asterisk marks the transverse canal that links the two saccules across the midline. (G, H) Sketch of the inner ears of adult rescued swr and wild-type fish. Shading in the wild-type ear indicates the regions missing from rescued swr ears. Abbrevations: a-amp, anterior ampulla; l-amp, lateral ampulla; p-amp, posterior ampulla; a-canal, anterior semicircular canal; l-canal, lateral semicircular canal; p-canal, posterior semicircular canal; cc, crus commune. swirl 1 is a smad5-rescued swrdc24 fish; swirl 2 is a bmp2b-rescued swrta72 fish. A–C, G and H are lateral views of left hand ears, with anterior to the left; D–F are dorsal views. Scale bar, 500 µm.
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pone-0004368-g002: Three-dimensional reconstructions of adult rescued swr inner ears.(A–F) Three-dimensional reconstructions of the inner ears of two adult rescued swr fish (swirl 1 (A, D) and swirl 2 (B, E)) and an age-matched wild-type fish (C, F). Semicircular canals are absent in the rescued swr fish ears, while the ampullae, crus commune, utricle, saccule and lagena are present and appear normal. Note that the pars superior (ampullae, crus commune and utricle) of swirl 1 is twisted relative to the pars inferior (saccule and lagena), about the point marked with the arrow (we are unable to tell whether or not this is an experimental artefact). The kink in the lateral canal of the wild-type ear (C) is an artefact. The asterisk marks the transverse canal that links the two saccules across the midline. (G, H) Sketch of the inner ears of adult rescued swr and wild-type fish. Shading in the wild-type ear indicates the regions missing from rescued swr ears. Abbrevations: a-amp, anterior ampulla; l-amp, lateral ampulla; p-amp, posterior ampulla; a-canal, anterior semicircular canal; l-canal, lateral semicircular canal; p-canal, posterior semicircular canal; cc, crus commune. swirl 1 is a smad5-rescued swrdc24 fish; swirl 2 is a bmp2b-rescued swrta72 fish. A–C, G and H are lateral views of left hand ears, with anterior to the left; D–F are dorsal views. Scale bar, 500 µm.

Mentions: To obtain a clearer picture of the morphological defects, we used the sections to generate three-dimensional reconstructions of the inner ears of three rescued swr fish (two bmp2b-rescued swrta72 and one smad5-rescued swrtdc24) and two wild-type fish. We obtained full reconstructions of three rescued swr ears from two fish and two partial reconstructions from a single fish. We also obtained three fully reconstructed ears from the two wild-type fish (Fig. 2 and Supplementary Videos S1, S2). The reconstructions confirmed that all three rescued swr specimens (five ears examined) lacked semicircular canal ducts. The ampullae, crus commune and the posterior end of the lateral canal were present in all cases, but ended blindly. In the three rescued swr ears in which the vestibular chambers were reconstructed, the utricule, saccule and lagena were all present and grossly normal in size and morphology (Fig. 2 and Supplementary Videos S1, S2).


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)

Three-dimensional reconstructions of adult rescued swr inner ears.(A–F) Three-dimensional reconstructions of the inner ears of two adult rescued swr fish (swirl 1 (A, D) and swirl 2 (B, E)) and an age-matched wild-type fish (C, F). Semicircular canals are absent in the rescued swr fish ears, while the ampullae, crus commune, utricle, saccule and lagena are present and appear normal. Note that the pars superior (ampullae, crus commune and utricle) of swirl 1 is twisted relative to the pars inferior (saccule and lagena), about the point marked with the arrow (we are unable to tell whether or not this is an experimental artefact). The kink in the lateral canal of the wild-type ear (C) is an artefact. The asterisk marks the transverse canal that links the two saccules across the midline. (G, H) Sketch of the inner ears of adult rescued swr and wild-type fish. Shading in the wild-type ear indicates the regions missing from rescued swr ears. Abbrevations: a-amp, anterior ampulla; l-amp, lateral ampulla; p-amp, posterior ampulla; a-canal, anterior semicircular canal; l-canal, lateral semicircular canal; p-canal, posterior semicircular canal; cc, crus commune. swirl 1 is a smad5-rescued swrdc24 fish; swirl 2 is a bmp2b-rescued swrta72 fish. A–C, G and H are lateral views of left hand ears, with anterior to the left; D–F are dorsal views. Scale bar, 500 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2629815&req=5

pone-0004368-g002: Three-dimensional reconstructions of adult rescued swr inner ears.(A–F) Three-dimensional reconstructions of the inner ears of two adult rescued swr fish (swirl 1 (A, D) and swirl 2 (B, E)) and an age-matched wild-type fish (C, F). Semicircular canals are absent in the rescued swr fish ears, while the ampullae, crus commune, utricle, saccule and lagena are present and appear normal. Note that the pars superior (ampullae, crus commune and utricle) of swirl 1 is twisted relative to the pars inferior (saccule and lagena), about the point marked with the arrow (we are unable to tell whether or not this is an experimental artefact). The kink in the lateral canal of the wild-type ear (C) is an artefact. The asterisk marks the transverse canal that links the two saccules across the midline. (G, H) Sketch of the inner ears of adult rescued swr and wild-type fish. Shading in the wild-type ear indicates the regions missing from rescued swr ears. Abbrevations: a-amp, anterior ampulla; l-amp, lateral ampulla; p-amp, posterior ampulla; a-canal, anterior semicircular canal; l-canal, lateral semicircular canal; p-canal, posterior semicircular canal; cc, crus commune. swirl 1 is a smad5-rescued swrdc24 fish; swirl 2 is a bmp2b-rescued swrta72 fish. A–C, G and H are lateral views of left hand ears, with anterior to the left; D–F are dorsal views. Scale bar, 500 µm.
Mentions: To obtain a clearer picture of the morphological defects, we used the sections to generate three-dimensional reconstructions of the inner ears of three rescued swr fish (two bmp2b-rescued swrta72 and one smad5-rescued swrtdc24) and two wild-type fish. We obtained full reconstructions of three rescued swr ears from two fish and two partial reconstructions from a single fish. We also obtained three fully reconstructed ears from the two wild-type fish (Fig. 2 and Supplementary Videos S1, S2). The reconstructions confirmed that all three rescued swr specimens (five ears examined) lacked semicircular canal ducts. The ampullae, crus commune and the posterior end of the lateral canal were present in all cases, but ended blindly. In the three rescued swr ears in which the vestibular chambers were reconstructed, the utricule, saccule and lagena were all present and grossly normal in size and morphology (Fig. 2 and Supplementary Videos S1, S2).

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