QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 20 April 2005
doi: 10.1242/dev.01834

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mantela, J. Articles by Pirvola, U. Search for Related Content PubMed PubMed Citation Articles by Mantela, J. Articles by Pirvola, U. Social Bookmarking                         What's this?

The retinoblastoma gene pathway regulates the postmitotic state of hair cells of the mouse inner ear

¹ Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland
² Departments of Medicine, Laboratory Medicine and Pathobiology and Medical Biophysics, Division of Cell and Molecular Biology, Toronto General Research Institute, University Health Network, University of Toronto, Toronto, Ontario M5G-2M1, Canada
³ Creighton University, Department of Biomedical Sciences, Omaha, NE 68178, USA

View larger version (103K): [in a new window]   Fig. 1. pRb expression in hair cells of the inner ear. pRb immunostaining; Rb in situ hybridization. (A,B) pRb staining in the early- and later-differentiating HCs of the embryonic saccule. (C,D) pRb and Rb expressions in HCs of the late-embryonic utricle. (E) A subpopulation of HCs is pRb-positive in the adult utricle. (F,G) pRb expression in inner and outer HCs of the cochlea at birth and early postnatal life. In addition to HCs, the greater epithelial ridge shows weak expression at birth. (H) In the mature organ of Corti, pRb expression is detected only in inner HCs. Supporting cells are negative at all stages. (I,J) pRb is not expressed in the hyperplastic sensory epithelium of utricle and cochlea of mgRb:Rb-/- mutants at E18.5. Abbreviations: wt, wild type; sac, saccule; ut, utricle; co, cochlea; IHC, inner hair cell; OHCs, outer hair cells; GER, greater epithelial ridge; HCs, hair cells; SCs, supporting cells. Arrows in G,H,J indicate HCs. Scale bar: 70 µm.

View larger version (126K): [in a new window]   Fig. 2. Proliferation and differentiation in the cochleas of mgRb:Rb-/- embryos. Myosin VI- and phospho-histone H3 immunostaining; Math1 and Fgf10 in situ hybridization. (A,A') At birth, myosin VI is expressed in inner and outer HCs of control cochleas and also in supernumerary HCs of mutants. (B-C') At late-embryogenesis, p27- and p75NTR-positive supporting cell populations (arrows) are seen both in controls and mutants. (D) A myosin VI-stained mitotic HC (arrow) in a mutant cochlea at birth. (E,E') At birth, phospho-histone H3 staining shows ectopic mitoses in the greater epithelial ridge and the region of inner and outer HCs in mutant cochleas. These are not present in controls. (E'') Most of these mitotic HCs with strongly stained condensed chromatin are located near the lumen. (F,F') At E14.5, phospho-histone H3-stained cells are seen in the caudal wall of the cochlea of both mutants and controls. (G,G') At birth, Math1 is expressed in cochlear HCs of controls and also in supernumerary HCs of mutants. (H,H') At birth, Fgf10 is expressed in the greater epithelial ridge of control cochleas and in the thickened ridge of mutants. Abbreviations: wt, wild type; ph3, phospho-histone H3; myo, myosin VI; GER, greater epithelial ridge; IHC, inner hair cell; OHCs, outer hair cells; CN, cochlear nerve. Scale bar: 70 µm for A-C',E-F'); 30 µm for D; 80 µm for G-H').

View larger version (139K): [in a new window]   Fig. 3. Mitosis and differentiation of vestibular hair cells of mgRb:Rb-/- embryos. Myosin VI- and phospho-histone H3-immunostaining. (A,A') When compared with controls at birth, utricular sensory epithelia of mutants are hyperplastic because of an excess of myosinVI-positive HCs, some of which have penetrated into the mesenchyme (arrows). (B,B') In contrast to the saccular sensory epithelium of controls in which HCs occupy the lumenal layer, HCs are distributed throughout the epithelium in mutants. (C,C') In contrast to controls, ampullary sensory epithelia of mutants are hyperplastic because of overproduction of myosin VI-positive HCs, some of which invade the mesenchyme (arrow). (D) An oblique section through the utricular sensory epithelium of a mutant shows mitotic figures (arrows) in myosin VI-stained HCs. (E) A cross-section through the utricle shows that mitotic HCs (arrows) have rounded shape and occupy both lumenal and deeper epithelial layers. (F-H') Phospho-histone H3-stained utricular (F,F'), saccular (G,G') and ampullary (H,H') sensory epithelia at birth show higher numbers of mitotic cells in mutants when compared with controls. (I,I') At E14.5, early-differentiating saccular HCs of mutants undergo mitoses, in contrast to controls. Differentiating HCs occupy the lumenal layer and have a large nucleus. Abbreviations: wt, wild type; myo, myosin VI; ph3, phospho-histone H3. Scale bar: 120 µm for A,A',F,F'); 70 µm for B-C',G-I'; 30 µm for D,E.

View larger version (144K): [in a new window]   Fig. 4. Stereociliary bundles of vestibular hair cells of mgRb:Rb-/- mutants at birth, as shown by espin-immunostaining. (A) Espin is localized to HC stereocilia of control utricules. (B) In mutants, espin-positive HCs are situated throughout the thickened utricular sensory epithelium. Several HCs at the surface show abnormal bundles. (C,D) High magnification views show HCs with aberrant stereocilia in the utricle and in the adjacent mesenchyme (arrow) of mutants. (E,F) In contrast to controls, mutants show espin-stained HCs at different levels of the ampullary sensory epithelium. HCs in the mesenchyme are also espin positive (arrow). (G,H) In addition to the distinct bundle abnormalities, stereociliary bundle morphologies comparable with controls are seen in some HCs of mutants, especially in ampullae. Abbreviations: wt, wild type; ut; utricle; amp, ampulla; SE, sensory epithelium; ME, mesenchyme; LU, lumen. Scale bar: 70 µm for A-F; 60 µm for G,H; 30 µm for C,D.

View larger version (72K): [in a new window]   Fig. 5. Apoptosis in the inner ear of mgRb:Rb-/- mutants at E18.5. (A-C) In a mutant cochlea, the myosin VI-stained region of supernumerary HCs, which comprises mitotic figures (A, short arrows), shows TUNEL-(B) and cleaved caspase 3-(C) positive cells. Views are from adjacent sections. Compare with normal morphology in Fig. 2A. (D-F) TUNEL- and caspase 3-positive cells are present in the myosin VI-stained region of a hyperplastic utricular sensory epithelium. Compare with normal morphology in Fig. 3A. Long arrows indicate apoptotic profiles. Abbreviations: co, cochlea; ut, utricle; myo, myosin VI; casp, cleaved caspase 3. Scale bar: 60 µm.

View larger version (135K): [in a new window]   Fig. 6. Mitosis, apoptosis and multinucleation of hair cells of mgRb:Rb-/- mutants at E18.5, as shown in semi-thin sections. (A) Normal utricular sensory epithelium shows HCs at the lumenal surface and underlying supporting cells. (B) A mutant utricle shows disorganized cell layering and HCs with an atypical nucleus or with two or more nuclei. Some of these HCs show near-normal stereociliary bundles. Some HCs seem to extend from the surface deep into the epithelium. (C) Mitotic cells with rounded morphology and nascent stereocilia (arrow) at the lumenal surface are intermixed with HCs with aberrant nuclear morphologies. A putative supporting cell that has translocated to the surface for mitosis is marked by an arrowhead. (D) Mitotic HCs (short arrows) are found mostly at the lumenal surface, but also in deeper layers, such as in this view where a cell undergoing mitosis is penetrating through the basal lamina. Scattered apoptotic profiles are also found (long arrows). (E,F) A mitotic cell (short arrow) showing signs of degeneration in the cytoplasm. Mitotic cells occasionally showed apoptotic figures (long arrow). (G,H) In contrast to controls, the ampullary sensory epithelia of mutants contain mitotic cells (arrows). In addition, several HCs with abnormal nuclei are found and many of them have stereociliary bundles with a morphology comparable with controls. (I,J) A transverse view through the organ of Corti of a normal cochlea shows four HCs and the underlying supporting cells. In the region of organ of Corti of mutants, high numbers of nuclei are seen, some of them having mitotic figures (short arrows) and a few apoptotic profiles (long arrow). Abbreviations: wt, wild type; ut, utricle; amp, ampulla; co, cochlea; LU, lumen, BL, basal lamina; ME, mesenchyme; HCs, hair cells; SCs, supporting cells; IHC, inner hair cell; OHCs, outer hair cells; P, pillar cell; Ds, Deiters' cell, H, Hensen's cell. Scale bar: 15 µm for A-J.

View larger version (127K): [in a new window]   Fig. 7. p21 expression in the developing inner ear and the phenotype of p21 knockout mice. p21 in situ hybridization; myosin VI and phospho-histone H3 immunostaining; Haematoxylin staining. (A,B) As shown in adjacent sections, p21 and myosin VI are expressed in utricular HCs (arrows) at E16.5. p21 is also found in the utricular non-sensory epithelium. (C) In the cochlea at E16.5, p21 is expressed in HCs and in stria vascularis, spiral limbus and cochlear ganglion neurons. (D,E) Histology of the utricular sensory epithelium of adult p21-/- mice is indistinguishable from controls, as shown by Haematoxylin staining. (F,G) Histology of the organ of Corti of adult p21 knockouts is comparable with controls. (H) Mitoses are not induced in the cochleas of p21-null mutants, as shown at P2 by phospho-histone H3-staining (compare with control in Fig. 2E). Arrows in G,H indicate HCs. Abbreviations: wt, wild type; ut, utricle; co, cochlea; HE, Haematoxylin; SL, spiral limbus; CG, cochlear ganglion; SV, stria vascularis; IHC, inner hair cell; OHCs, outer hair cells; NSE, non-sensory epithelium; myo, myosin VI; ph3, phospho-histone H3. Scale bar: 70 µm for A-C,H; 80 µm for D-G.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter