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Spatiotemporal characteristics of eustachian tube development in C57BL/6 mice: Correlation between morphological and functional maturation

Developmental Dynamics

October 2024

DOI:10.1002/dvdy.753

Authors:

Hejie Li

Huazhong University of Science and Technology

Show all 9 authorsHide

Background The eustachian tube (ET), a critical conduit connecting the middle ear and nasopharynx, is essential for normal middle ear function. However, it remains one of the least understood anatomical structures due to its complexity and the challenges of in vitro manipulation. Historically, these challenges have hindered research into the morphology and function development of the ET. This study elucidates the spatiotemporal relationship of ET morpho‐functional maturation in mice, identifying key periods and factors that lay the theoretical foundation for exploring the molecular mechanisms of ET‐related diseases. Results We comprehensively characterized the ET development in C57BL/6 mice from embryonic day (E) 12.5 to postnatal day (P) 30, focusing on the development of cilia, secretory cells, surrounding glands, and macrophages. Immunostaining identified the localization and secretion patterns of the mucins Muc5b and Muc5ac within the ET. Additionally, using improved ET function assessment tools, we evaluated the developmental features of ET mucociliary clearance and ventilation functions. Conclusions In C57BL/6 mice, E16.5 marks a critical period for middle ear cavity and ET formation. Muc5b plays a foundational role during early stages, while Muc5ac enhances function in later stages. During P7‐11, despite morphological maturity, ET function remains underdeveloped but continues to improve with growth.

Schematic diagram of mouse ET tissue embedding and sectioning. (A) The mouse head was dissected along the sagittal line on the top of its skull, with the area near the nasal cavity being the lateral side of the ET, and the area near the laryngopharynx being the medial side of the ET. (B) Longitudinal section of the ET: By bisecting the mouse head, a longitudinal section is made, and the slices are continuously cut in the direction from the medial side toward the lateral side of the ET, as indicated by the arrow. (C) Arrows indicate spatial orientation of (B, D). (D) Cross section of the ET: Create a cross‐sectional view, with the direction of the slices continuously cutting from the area near the ET pharyngeal orifice toward the tympanic orifice, as indicated by the arrow. (E) The ET cross‐sectional specimens were sliced according to these three parts, and representative continuous sections were selected for each part. The dotted box was the spatial orientation map of the cross‐sectional specimens.

…

Histological examination of mouse ET development using H&E staining. (A–F) The ET longitudinal section of E12.5–16.5 and E19.5 in mice. A′–F′ show magnified views of the TTR and the junction between the ET and the MEC. (G–N) The morphological changes of the ET in prenatal and postnatal stages in cross‐sectional slices. Scale bars: 200 μm (A–F) and 100 μm (A′–F′, G–N). TTR, the tubotympanic recess; N, nasopharynx; OC, otic capsule; B, cranial base; MEC, middle ear cavity; TO, tympanic orifice; PO, pharyngeal orifice.

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The formation process of the cartilaginous and membranous parts of the mouse ET. H&E staining of representative sections of three parts of the ET (near the PO, middle segment, and near the TO) at each time point from P3 to P11. (A–E) Representative sections near PO; (A′–E′) representative sections in the middle segment; (A″–E″) representative sections near TO. The triangle indicates the cartilaginous structures of the ET. Yellow arrows show acinar cells exhibiting secretory activity. Scale bar: 100 μm. (G) The ET glands; m, mucous acinus; s, serous acinus; ma, mixed acinus.

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Observation of the developmental characteristics of mouse embryonic ET epithelium by immunofluorescence staining and SEM. H&E staining (A–F) and immunofluorescence (A′–F′, A–F″) of ciliated cells in the ET epithelium at E12.5–E16.5 and E19.5. Ciliated cells were labeled with anti‐acetylated alpha‐tubulin protein‐specific markers (blue: DAPI, green: α‐tubulin). Scale bar = 100 μm. (G–I) SEM images of embryonic mouse ET at E15.5, E16.5, and E18.5. The left column (G′–I′) corresponds to the PO of the ET, while the right column (G″–I″) corresponds to the part of the ET lumen, indicated by the yellow boxes in the main images. Original low‐magnification images (scale bar: 1 mm) and high‐magnification (scale bar: 100 μm). Areas magnified 10 times are indicated by black boxes. SP, soft palate; PO, pharyngeal orifice.

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Developmental characteristics of postnatal ET epithelium in mice. (A–I) Representative H&E staining of longitudinal sections of ET at various time points from P3 to P30. (A′–I′) and (A″–I″) depict magnified views of the ET tympanic and pharyngeal orifice, respectively. (J′–O′) Immunofluorescence staining of ciliated cells in the cross‐sectional sections of ET from P9 onward. Blue (DAPI) and green (α‐tubulin). Red arrows indicate the regions devoid of ciliated cell expression in the ET cartilaginous part. (J–O) H&E staining of the cross‐sectional sections of ET adjacent to immunofluorescence staining. scale bar = 200 μm (A–I, J–O″); scale bar = 100 μm (A′–I′, A″–I″). G, glands; GC, goblet cells; Cp, the cartilaginous part; Mp, the membranous part.

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RESEARCH ARTICLE

Spatiotemporal characteristics of eustachian tube

development in C57BL/6 mice: Correlation between

morphological and functional maturation

Xuan Yu

1,2

| Huimin Zhang

1,2

| Hejie Li

1,2

| Xingqian Shen

1,2

Wenting Yu

1,2

| Ting Li

1,2

| Xiaoye Chen

1,2

| Shimin Zong

1,2

Hongjun Xiao

1,2,3

Department of Otorhinolaryngology-

Head and Neck Surgery, Union Hospital,

Tongji Medical College, Huazhong

University of Science and Technology,

Wuhan, China

Institute of Otorhinolaryngology-Head

and Neck Surgery, Tongji Medical

College, Huazhong University of Science

and Technology, Wuhan, China

Hubei Province Clinical Research Center

for Deafness and Vertigo, Wuhan, China

Correspondence

Hongjun Xiao and Shimin Zong,

Department of Otorhinolaryngology-Head

and Neck Surgery, Union Hospital, Tongji

Medical College, Huazhong University of

Science and Technology, Wuhan 430022,

China.

Email: xhjent_whxh@hust.edu.cn and

Email: 2018xh0090@hust.edu.cn

Funding information

Key Research and Development Program

of Hubei Province Project, Grant/Award

Number: 2021BCA144; National Natural

Science Foundation of China,

Grant/Award Numbers: 82071057,

82101229

Abstract

Background: The eustachian tube (ET), a critical conduit connecting the mid-

dle ear and nasopharynx, is essential for normal middle ear function. However, it

remains one of the least understood anatomical structures due to its complexity

and the challenges of in vitro manipulation. Historically, these challenges have

hindered research into the morphology and function development of the ET. This

study elucidates the spatiotemporal relationship of ET morpho-functional matura-

tion in mice, identifying key periods and factors that lay the theoretical foundation

for exploring the molecular mechanisms of ET-related diseases.

Results: We comprehensively characterized the ET development in C57BL/6

mice from embryonic day (E) 12.5 to postnatal day (P) 30, focusing on the

development of cilia, secretory cells, surrounding glands, and macrophages.

Immunostaining identified the localization and secretion patterns of the

mucins Muc5b and Muc5ac within the ET. Additionally, using improved ET

function assessment tools, we evaluated the developmental features of ET

mucociliary clearance and ventilation functions.

Conclusions: In C57BL/6 mice, E16.5 marks a critical period for middle ear

cavity and ET formation. Muc5b plays a foundational role during early stages,

while Muc5ac enhances function in later stages. During P7-11, despite mor-

phological maturity, ET function remains underdeveloped but continues to

improve with growth.

KEYWORDS

eustachian tube, eustachian tube function tests, glands, macrophage, morphogenesis, mucins

1|INTRODUCTION

The eustachian tube (ET) is the only channel connecting

the middle ear and the nasopharynx. Its main functions

include equalizing the pressure inside and outside the mid-

dle ear, clearing middle ear mucosal secretions, and protect-

ing the middle ear from sound and pathogens and

secretions from the nasopharynx. During embryonic devel-

opment, the ET originates from the proximal part of the

first pharyngeal pouch (tubotympanic recess).

1,2

It is linedXuan Yu, Huimin Zhang, and Hejie Li contributed equally to this work.

Received: 27 May 2024 Revised: 25 August 2024 Accepted: 24 September 2024

DOI: 10.1002/dvdy.753

ResearchGate has not been able to resolve any citations for this publication.

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Salivary glands at the pharyngeal ostium of the Eustachian tube are already described in histological literature

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