Studying Wnt Signaling During Patterning of Conducting Airways

Summary

The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development.

Abstract

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

Introduction

Respiratory tract development is initiated by embryonic day 9 (E9) with the appearance of Nkx2.1 positive cells in the ventral endodermal foregut^1,2. Esophageal-tracheal tube separation will resolve by E11.5 when the tubes can be distinguished as distinct entities, each surrounded by mesenchymal tissue³. Wnt signaling plays a key role in the specification of the respiratory tract as deletion of Wnt2 and Wnt2b, expressed by the splanchnic mesenchyme and deletion of β-catenin from the endodermal respiratory epithelium will result in lung agenesis^4,5. Our previous studies determined that deletion of Wls....

Protocol

Animals were housed in pathogen-free conditions. Mice were handled according to protocols approved by CCHMC Institutional Animal Care and Use Committee (Cincinnati, OH USA). Mice utilized throughout these studies were maintained in a mixed background.

1. Whole Mount X-galactosidase Staining

1. Euthanize pregnant female at E11.5 to E14.5, by CO₂ inhalation. Place animals in CO₂ chamber, charge the chamber with CO₂. Maintain animals in chamber for minimum of 5 min. Perform secondary method of euthanasia by cervical dislocation.
2. Clean abdominal area with ethanol (EtOH) 70% and perform laparot....

Results

Wnt/β-catenin activity

Whole mount Lac-Z staining was detected in tracheal-lung tissue of embryos isolated from reporter Axin2^Lac-Z mice¹¹. Sites of staining indicate Wnt/β-catenin activity. Analysis of sections of whole mount staining determined that Wnt/β-catenin activity was present in the mesenchyme of the trachea and in mesenchyme of peripheral regions of developing lungs. In Wl.......

Discussion

Events underlying the morphogenesis of the respiratory tract are not completely understood, particularly the processes required for the patterning of the conducting airways. Previous studies have utilized ex vivo techniques wherein developing explants are cultured at the air-liquid interphase or embedded in matrigel^21,22. These studies have shown how growth factors influence the patterning of the developing trachea and the formation of tracheal cartilage. A limitation to these studies is that the arch.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti Sox9 ab.	Millipore	AB5535	1:400 , rabbit
Anti Sox9 ab.	Santa Cruz	Sc-20095	1:50, rabbit
Anti Smooth Muscle Actin ab.	Sigma	A5228	1:2k, mouse
Anti NKX2.1 ab.	Seven Hills	n/a	1:100, guinea pig
Anti NKX2.1 ab.	Seven Hills	n/a	1:400, mouse
Anti Brdu ab.	Abcam	AB1893	1:200, sheep
Anti Brdu ab.	Santa Cruz	Sc-32323	1:4k, mouse
PNA Lectin	Sigma	L 7381
Secondary antibodies	Life technologies		Alexa fluor Molecular probes
K3Fe(CN)6	Sigma	P8131
K4Fe(CN)6	Sigma-Aldrich	P3289
MgCl2	Sigma-Aldrich	M9272
NaDOC	Life Technologies	89905
NP4O	Life Technologies	85124
Alcian Blue 8GX	Sigma	A-3157
Fisher brand super-frost plus	Fisher	12-550-15
PFA (16%)	EMS	15710
PBS	Gibco	70011-044
Fetal Calf Serum	Sigma	11K413
Blocking reagent	Invitrogen		Component of TSA kit #2    ( T20932)
BrDu	Sigma	B5002-5g
Vectashield mounting medium	Vector labs	H-1000
Permount	Fisher	SP15-500
Tissue-loc cassettes Histoscreen	Fisher	C-0250-GR
Biopsy cassettes	Premiere	BC0109	Available in different colors
Nuclear fast red  Kernechtrot 0.1%	Sigma	N3020
Citric acid	Sigma	C1909-500G
Sodium citrate tribasic dihydrate	Sigma	S4641-1Kg
Trizma hydrochloride	Sigma	T5941-500G
Xylene	Pharmco-AAPER	399000000
Ethanol	Pharmco-AAPER	111000200
Micro knives	FST	10318-14
Dumont #5 ceramic coated	FST	11252-50
Dumont #5CO	FST	11295-20
Dumont # 5	FST	91150-20
Thermo/Shandon Excelsior ES	Thermo Fisher
Microtome	Leica	RM2135
Nikon i90	Nikon		Wide field microscope
NikonA1Rsi	Nikon		Confocal microscopy. Settings:NikonA1 plus camera, scanner: Galvano, detector:DU4. Optics Plan Apo lambda 10X. Modality: Widefield fluorescence laser confocal.
Leica MS 16 FA	Leica		Fluorescence Dissecting microscope
Zeiss	Zeiss		Automated fluorescence microscope
Leica Application suite	Leica		Leica imaging software
NIS	Nikon		Nikon imaging software
IMARIS	Bitplane		Imaging processing software