Haddon CM and Lewis JH (1991), Hyaluronan as a propellant for epithelial movem...

XB-ART-24797

Development 1991 Jun 01;1122:541-50.

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Hyaluronan as a propellant for epithelial movement: the development of semicircular canals in the inner ear of Xenopus.

Haddon CM , Lewis JH .

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The membranous labyrinth of the inner ear, with its three semicircular canals, originates from a simple spheroidal otic vesicle. The process is easily observed in Xenopus. The vesicle develops three dorsal outpocketings; from the two opposite faces of each outpocketing pillars of tissue are protruded into the lumen; and these paired 'axial protrusions' eventually meet and fuse, to form a column of tissue spanning the lumen of the outpocketing like the hub of a wheel, with a tube of epithelium forming the semicircular canal around the periphery. Each axial protrusion consists of epithelium encasing a core of largely cell-free extracellular matrix that stains strongly with alcian blue. In sections, at least 60% of the stainable material is removed by treatment with Streptomyces hyaluronidase. When Streptomyces hyaluronidase is microinjected into the core of a protrusion in vivo, the protrusion collapses and the corresponding semicircular canal fails to form. Hyaluronan (hyaluronic acid) in the core of the protrusion therefore seems to be essential in driving the extension of the protrusion. Autoradiography with tritiated glucosamine indicates that the hyaluronan-rich matrix is synthesised by the epithelium covering the tip of the protrusion; the basal lamina here appears to be discontinuous. These findings indicate that the epithelium of the axial protrusion propels itself into the lumen of the otocyst by localised synthesis of hyaluronan. Hyaluronan may be used in a similar way in the development of other organs, such as the heart and the secondary palate.

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Species referenced: Xenopus laevis
Genes referenced: cspg4b elavl2 fubp1
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	Fig. 1. Clay models of a right inner ear of Xenopus at three successive stages in the formation of the semicircular canals. Lateral views above, medial views below. (A) Stage 44: slight indentations represent the axial protrusions beginning to extend into the ear lumen. (B) Stage 47: three stubby semicircular canals have formed through the fusion of the axial protrusions. The dorsal structure is the endolymphatic sac at the end of the endolymphatic duct. (C) Stage 48+: The semicircular canals have extended and become thinner. (Endolymphatic duct and sac not shown).
	Fig. 2. Schematic cut-away views of three stages in the formation of a semicircular canal. (A) Initial outpocketing of otocyst epithelium. (B) Axial protrusions extend into lumen. (C) Axial protrusions meet and fuse, creating a semicircular canal.
	Fig. 3. Dorsal view of an intact tadpole at stage 46, showing the location of the ears adjacent to the hindbrain. A blue pigment (monastral blue) has been injected into the lumen of the ear to make its outline clear. Scale bar=100um.
	Fig. 4. Araldite sections showing the formation of a semicircular canal. (A) A pair of axial protrusions (arrows) filled with extracellular matrix grow into the ear lumen. Note the thickened appearance of the epithelium covering the rounded tips of the protrusions. (B) The protrusions extend and meet, fusing at their tips. Cell death occurs at the fusion plate (arrow). (C) Epithelial remodelling occurs creating a smooth cylinder of epithelium covering a column whose core, initially acellular, later becomes seeded with mesenchymal cells. The completed canal lies perpendicular to the plane of the page. Scale bar=80um.
	Fig. 5. S.E.M. of an axial protrusion with its apical epithelium removed, viewed end-on from the luminal side. Note the finely fibrous matrix in the core. Scale bar=10um.
	Fig. 6. Fibrowax sections stained with alcian blue at pH 2.5 and counterstained with neutral red. (A) Control section. (B) Adjacent section treated with Streptomyces hyaluronidase (lmgml"1) for 4h at 37Â°C before alcian blue staining. The enzyme has removed most of the alcian-blue-stainable material from the core of the axial protrusion (arrow). Scale bar = 75um
	Fig. 7. Graph showing the amount of alcian-blue stainable material remaining in the core of an axial protrusion following a 4-h digestion with Streptomyces hyaluronidase at various concentrations. The intensity of stain was measured photometrically as described in 'Materials and methods'; the 'relative stain intensity' plotted along the abscissa is the stain intensity for the given enzyme concentration divided by the intensity for an adjacent control section exposed to zero enzyme concentration. (Thus the relative stain intensity for zero enzyme concentration is exactly 1.0 by definition). The solid circles represent a series of measurements made on multiple sections of a single tadpole. The hollow square at l.Omgml"1 represents a set of measurements on sections of 10 different tadpoles. The error bars represent the standard error of the mean. The curve is drawn by eye.
	Fig. 8. Transverse section of a tadpole 2 days after an injection of Streptomyces hyaluronidase (â€”0.01 nl, lmgml"1) into the lateral axial protrusion in the right ear. On the control (left) side, the axial protrusions have met and fused to form the hub (arrow) of a semicircular canal (asterisk). By looking at serial sections, we confirmed that nothing remained of the injected protrusion and its partner except the two low pimples visible in this section. On the injected side, the protrusions have regressed and no canal has been formed. Scale bar=160um.
	Fig. 9. Collapse of an axial protrusion after Streptomyces hyaluronidase (0.01 nl, lmgml ') is injected into its core, as seen in dorsal views of the ear of a live, anaesthetised stage 46.5 tadpole. (A) Just before injection of enzyme into the lateral posterior axial protrusion (arrow). (B) The same specimen Imin after injection: the protrusion is already beginning to deflate. (C) After 10min the protrusion has collapsed to less than half its former size. Scale bar=70um.
	Fig. 10. (A) The chondroitin-sulphate distribution in the inner ear at stage 46.5 as revealed by immunofluorescence with the CS56 antibody. (B) The pattern of cell nuclei revealed by DAPI staining. Note that there is CS56 immunoreactivity in the epithelium of an axial protrusion (arrow) and as expected in the cartilage around the ear but not in the core of the protrusion. Scale bar=50um.
	Fig. 11. T.E.M. sections showing (A) a portion of the squamous epithelium near the base of an axial protrusion, and (B) a portion of the rather thicker epithelium covering the tip of the same protrusion. In (A) one can see a well-defined basal lamina; in (B) only an amorphous basal fuzz is present. Scale bar=um.
	Fig. 12. Immunofluorescence with an anti-laminin antiserum on fibrowax sections, viewed with a confocal scanning microscope. (A) An axial protrusion just beginning to form: there appears to be a continuous layer of laminin beneath the epithelium. (B) A well-developed axial protrusion: the layer of laminin appears discontinuous beneath the epithelium covering the distal part of the protrusion. Scale bar=45um.
	Fig. 13. Autoradiographs showing the pattern of [3H]glucosamine incorporation in extending axial protrusions and adjacent tissues. Bright-field views on the left, dark-field views on the right. (A) Explant pulsed with [3H]glucosamine at stage 46 and fixed after lh. The epithelium of two rudimentary axial protrusions is heavily labelled; neighbouring regions of otic epithelium are not. Scale bar=50,um. (B) Explant pulsed for lh with [3H]glucosamine at stage 46 and then cultured for 20 h in nonradioactive medium before fixation. Most of the label has cleared from the epithelium and is seen instead in the extracellular matrix, both in the acellular core of the protrusion and in other tissues such as cartilage, which is also rich in GAGS. In this specimen, the starred region (less heavily labelled) is not a growing axial protrusion but an already formed axis of a semicircular canal, with flattened, inactive epithelium and mesenchymal cells already populating its core. Scale bar=50um.