Anat Cell Biol.  2011 Dec;44(4):274-283. 10.5115/acb.2011.44.4.274.

Morphological evidences in circumvallate papilla and von Ebners' gland development in mice

• Sohn WJ
• Gwon GJ
• An CH
• Moon C
• Bae YC
• Yamamoto H
• Lee S
• Kim JY

• KMID: 1447441
• DOI: http://doi.org/10.5115/acb.2011.44.4.274

Abstract

In rodents, the circumvallate papilla (CVP), with its underlying minor salivary gland, the von Ebners' gland (VEG), is located on the dorsal surface of the posterior tongue. Detailed morphological processes to form the proper structure of CVP and VEG have not been properly elucidated. In particular, the specific localization patterns of taste buds in CVP and the branching formation of VEG have not yet been elucidated. To understand the developmental mechanisms underlying CVP and VEG formation, detailed histological observations of CVP and VEG were examined using a three-dimensional computer-aided reconstruction method with serial histological sections and pan-Cytokeratins immunostainings. In addition, to define the developmental processes in CVP and VEG formation, we examined nerve innervations and cell proliferation using microinjections of AM1-43 and immunostainings with various markers, including phosphoinositide 3-kinase, Ki-67, PGP9.5, and Ulex europaeus agglutinin 1 (UEA1). Results revealed specific morphogenesis of CVP and VEG with nerve innervations patterns, evaluated by the coincided localization patterns of AM1-43 and UEA1. Based on these morphological and immunohistochemical results, we suggest that nerve innervations and cell proliferations play important roles in the positioning of taste buds in CVP and branching morphogenesis of VEG in tongue development.

Keyword

Circumvallate papilla; von Ebners glands; Epithelial differentiation; AM1-43 microinjections; 3D computer-aided reconstruction

MeSH Terms

Animals
Cell Proliferation
Mice
Microinjections
Morphogenesis
Rodentia
Salivary Glands, Minor
Taste Buds
Tongue
Ulex
von Ebner Glands

Figure

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  Fig. 1 Three-dimensional (3D) computer-aided reconstruction after pan-Cytokeratins (pan-Cks) immunostaining of circumvallate papilla (CVP) at embryonic day 16 (E16). (A) Serial frontal sections after pan-Cks immunohistochemistry. The number on each slide indicates the the order of slide from anterior to posterior. Gray colors demarcate the pan-Cks positive epithelial tissues. (B1-5) 3D computer-aided reconstruction of CVP epithelium at E16. Gray colors indicate the positive cells of pan-Cks. (B6) Scanning electron microscopic (SEM) observations of E16 CVP. ANT, anterior; POST, posterior.

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  Fig. 2 Three-dimensional (3D) computer-aided reconstruction after pan-Cytokeratins (pan-Cks) immunostainings of circumvallate papilla (CVP) at postnatal day 3 (PN3). (A) Serial frontal sections after the pan-Cks immunohistochemistry. The number on each slide indicates the order of slide from anterior to posterior. Gray colors demarcate the pan-Cks positive epithelial tissues. (B) 3D computer-aided reconstruction of CVP epithelium at PN3 is presented, with the gray color indicating the positive cells of pan-Cks. ANT, anterior; POST, posterior.

• 

  Fig. 3 Immunostaining of circumvallate papilla (CVP) and von Ebners' gland (VEG) using various markers for taste buds, nerves, and cell proliferation. (A, B) Specific localization patterns of Ulex europaeus agglutinin 1 (UEA1) in the taste bud regions of CVP epithelium in adult mice. (C, D) PGP9.5 localization patterns in the mesenchymal core regions of apex part of CVP in adult mice. (E, F) Cell proliferations occur mainly in the VEG forming regions of CVP epithelium at embryonic day 16 (E16) and postnatal day 3 (PN3). (G, H) Phosphoinositide 3-kinase (PI3K), a well-known mediator in the receptor tyrosine kinase pathway, localizes in the VEG forming epithelium at E16 and PN3. Scale bars=500 µm (A, C), 20 µm (B, D), 50 µm (E-H).

• 

  Fig. 4 Whole mount observations of AM1-43 microinjected tongues. (A-D) Twenty-four hour after the in utero injections of AM1-43, positive localizations are detected in the circumvallate papilla (CVP) and fungiform papillae regions. (E-H) After intra-peritoneal injections of AM1-43 for 24 h, fungiform papillae and CVP show much stronger positive reactions of AM1-43 than those of embryonic day 16 (E16). PN3, postnatal day 3; BF, bight field; FL, fluorescent. Scale bars=1 mm (A, B, E, F), 250 µm (C, D), 500 µm (G, H).

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  Fig. 5 Analysis of co-localization patterns of AM1-43 with Ulex europaeus agglutinin 1 (UEA1) and PGP9.5. After AM1-43 observations using frontal frozen sections, immunostaining was examined using UEA1 and PGP9.5, respectively. (A1-3) At embryonic day 16 (E16), (B1-3) at postnatal day 3 (PN3), co-localization patterns are examined between AM1-43 and UEA1. (C1-3) At E16, (D1-3) at PN3, co-localization patterns are examined between AM1-43 and PGP9.5. (A1, B1, C1, D1) Immunostaining results, (A2, B2, C2, D2) fluorescent observations of AM1-43, (A3, B3, C3, D3) merged figures. Arrows indicate positive reactions of markers and arrow heads indicate positive localization patterns of AM1-43. Scale bars=50 µm.

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