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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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Semicircular canal ducts are absent, but sensory patches are present, in the inner ears of adult rescued swr zebrafish.(A–F) 10 µm resin parasagittal sections through the inner ears of adult rescued swr zebrafish and age-matched wild-types (anterior to the left, dorsal to the top). Note the absence of semicircular canal ducts in the rescued swr ear (A), which are clearly present in the wild-type ear in an equivalent section (B, showing lumens of anterior and posterior canal ducts). All sensory patches detected in the wild-type ears are also present in the rescued swr ears. Differences in the orientation of the lagenar macula (E, F) reflect a slight difference in the position of the wild-type and swr sections for this pair of panels. (G–J) Higher magnification views of the posterior crista (G, H) and utricular macula (I, J). The structure of the sensory patches is similar in both rescued swr and wild-type ears. Abbreviations: ac, anterior crista; lc, lateral crista; pc, posterior crista; ascc, anterior semicircular canal duct; lscc, lateral semicircular canal duct; cc, crus commune; ut, utricular macula; s, saccular macula; lg, lagenar macula; br, brain; s, somite; gl, gill. Scale bar, (A–F) 400 µm; (G–J) 50 µm.
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pone-0004368-g001: Semicircular canal ducts are absent, but sensory patches are present, in the inner ears of adult rescued swr zebrafish.(A–F) 10 µm resin parasagittal sections through the inner ears of adult rescued swr zebrafish and age-matched wild-types (anterior to the left, dorsal to the top). Note the absence of semicircular canal ducts in the rescued swr ear (A), which are clearly present in the wild-type ear in an equivalent section (B, showing lumens of anterior and posterior canal ducts). All sensory patches detected in the wild-type ears are also present in the rescued swr ears. Differences in the orientation of the lagenar macula (E, F) reflect a slight difference in the position of the wild-type and swr sections for this pair of panels. (G–J) Higher magnification views of the posterior crista (G, H) and utricular macula (I, J). The structure of the sensory patches is similar in both rescued swr and wild-type ears. Abbreviations: ac, anterior crista; lc, lateral crista; pc, posterior crista; ascc, anterior semicircular canal duct; lscc, lateral semicircular canal duct; cc, crus commune; ut, utricular macula; s, saccular macula; lg, lagenar macula; br, brain; s, somite; gl, gill. Scale bar, (A–F) 400 µm; (G–J) 50 µm.

Mentions: To investigate the causes of the behavioural defect further, we analysed inner ear morphology in the rescued swr adults by histological sectioning. We cut resin sections at 10 µm through the heads of six rescued homozygous adult swr fish (three bmp2b-rescued swrta72, two smad5-rescued swrtdc24 and one smad5-rescued swrtc300) and three age-matched wild-type fish. Examination of these sections indicated that the semicircular canal ducts were absent in all six rescued swr mutant specimens (Fig. 1). The ampullae, utricule, saccule and lagena were all present, however, and contained sensory patches (cristae, utricular macula, saccular macula and lagenar macula, respectively) of apparently normal morphology (Fig. 1).


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)

Semicircular canal ducts are absent, but sensory patches are present, in the inner ears of adult rescued swr zebrafish.(A–F) 10 µm resin parasagittal sections through the inner ears of adult rescued swr zebrafish and age-matched wild-types (anterior to the left, dorsal to the top). Note the absence of semicircular canal ducts in the rescued swr ear (A), which are clearly present in the wild-type ear in an equivalent section (B, showing lumens of anterior and posterior canal ducts). All sensory patches detected in the wild-type ears are also present in the rescued swr ears. Differences in the orientation of the lagenar macula (E, F) reflect a slight difference in the position of the wild-type and swr sections for this pair of panels. (G–J) Higher magnification views of the posterior crista (G, H) and utricular macula (I, J). The structure of the sensory patches is similar in both rescued swr and wild-type ears. Abbreviations: ac, anterior crista; lc, lateral crista; pc, posterior crista; ascc, anterior semicircular canal duct; lscc, lateral semicircular canal duct; cc, crus commune; ut, utricular macula; s, saccular macula; lg, lagenar macula; br, brain; s, somite; gl, gill. Scale bar, (A–F) 400 µm; (G–J) 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2629815&req=5

pone-0004368-g001: Semicircular canal ducts are absent, but sensory patches are present, in the inner ears of adult rescued swr zebrafish.(A–F) 10 µm resin parasagittal sections through the inner ears of adult rescued swr zebrafish and age-matched wild-types (anterior to the left, dorsal to the top). Note the absence of semicircular canal ducts in the rescued swr ear (A), which are clearly present in the wild-type ear in an equivalent section (B, showing lumens of anterior and posterior canal ducts). All sensory patches detected in the wild-type ears are also present in the rescued swr ears. Differences in the orientation of the lagenar macula (E, F) reflect a slight difference in the position of the wild-type and swr sections for this pair of panels. (G–J) Higher magnification views of the posterior crista (G, H) and utricular macula (I, J). The structure of the sensory patches is similar in both rescued swr and wild-type ears. Abbreviations: ac, anterior crista; lc, lateral crista; pc, posterior crista; ascc, anterior semicircular canal duct; lscc, lateral semicircular canal duct; cc, crus commune; ut, utricular macula; s, saccular macula; lg, lagenar macula; br, brain; s, somite; gl, gill. Scale bar, (A–F) 400 µm; (G–J) 50 µm.
Mentions: To investigate the causes of the behavioural defect further, we analysed inner ear morphology in the rescued swr adults by histological sectioning. We cut resin sections at 10 µm through the heads of six rescued homozygous adult swr fish (three bmp2b-rescued swrta72, two smad5-rescued swrtdc24 and one smad5-rescued swrtc300) and three age-matched wild-type fish. Examination of these sections indicated that the semicircular canal ducts were absent in all six rescued swr mutant specimens (Fig. 1). The ampullae, utricule, saccule and lagena were all present, however, and contained sensory patches (cristae, utricular macula, saccular macula and lagenar macula, respectively) of apparently normal morphology (Fig. 1).

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