探寻调控小鼠肺部发育的信号转导途径的体外外植体培养

Changfu Yao; Gianni Carraro; Barry R. Stripp

doi:10.21769/BioProtoc.2852

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Peer-reviewed

In vitro Explant Cultures to Interrogate Signaling Pathways that Regulate Mouse Lung Development

探寻调控小鼠肺部发育的信号转导途径的体外外植体培养

CY Changfu Yao ^*

GC Gianni Carraro ^* email

BS Barry R. Stripp email

(*contributed equally to this work) 发布: 2018年05月20日第8卷第10期 DOI: 10.21769/BioProtoc.2852 浏览次数: 6508

评审: Giusy TornilloZhenguang ZhangGunjan Mehta

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Abstract

Early mouse lung development, including specification of primordia, patterning of early endoderm and determination of regional progenitor cell fates, is tightly regulated. The ability to culture explanted embryonic lung tissue provides a tractable model to study cellular interactions and paracrine factors that regulate these processes. We provide up-to-date protocols for the establishment of this culture model and its application to investigate hedgehog signaling in the developing lung.

Keywords: Mouse embryonic lung (小鼠胚胎肺)

In vitro (体外)

Explant culture (外植体培养)

Background

Mouse lung development initiates as an endodermal diverticulum of the anterior foregut endoderm at day 9.5 postcoitum (E9.5), with subsequent closure of a proximal tracheoesophageal septum for the formation of distinct tracheal and esophageal tubes (Minoo and King, 1994). Subsequent branching of primitive endodermal tubes yields a planar lung structure by E12.5, with subsequent orthogonal branches yielding three-dimensional structure characteristic of the mature lung (Metzger et al., 2008). The planar structure of lung rudiments isolated prior to E12.5 is suitable for in vitro culture at an air liquid interface (Carraro et al., 2010; Del Moral and Warburton, 2010). Embryonic lung is isolated by dissection using a stereo microscope either under bright field illumination or by fluorescence illumination when coupled with lineage tracing and fluorescent reporters. Herein we describe the use of a Shh^Cre/Rosa^mTmG reporter mouse allowing Cre-mediated activation of membrane-localized GFP within anterior foregut endoderm from approximately E8.75 (Montgomery et al., 2007; Goss et al., 2009; Yao et al., 2017). Accordingly lung endoderm is visualized by green fluorescence and surrounding tissue by red fluorescence, allowing clear identification and microdissection of developing endodermal structures, including the lung, and imaging during in vitro culture.

Materials and Reagents

Whole embryonic lung isolation
1. BD 1 ml TB syringe 26 G (BD, catalog number: 309625 )
2. 50 ml conical tube (Denville Scientific, catalog number: C1062-P (1000799))
3. Petri dish (Greiner Bio One International, catalog number: 663161 )
4. Shh^Cre mice (THE JACKSON LABORATORY, catalog number: 005622 )
5. Rosa^mTmG/+ mice (THE JACKSON LABORATORY, catalog number: 007576 )
6. Mouse embryonic lungs from Shh^Cre/+, Rosa^mTmG/+ mice
  Note: Mouse embryonic lungs from Shh^Cre/+; Rosa^mTmG/+ mice were harvested between E10.5 and E12.5. The day of vaginal plug detection was considered to be E0.5
7. General anesthesia: Ketamine (VET one, NDC 13985-702-10) and xylazine (AnaSed Injection, NDC 59339-110-20)
8. 70% ethanol (Fisher Scientific, catalog number: BP8201500 )
9. Phosphate buffered saline (PBS) (1x), liquid, without calcium and magnesium (Corning, catalog number: 21-040-CV )
10. Penicillin-streptomycin (Thermo Fisher Scientific, Gibco^TM, catalog number: 15070063 )
11. PBS with P/S (see Recipes)
Whole embryonic lung culture
1. 12 wells plate (Denville Scientific, catalog number: T1012 )
2. Disposable transfer pipettes, sterile (VWR, catalog number: 414004-036 )
3. Razor blade (VWR, catalog number: 55411-050 )
4. Nuclepore Polycarbonate Track-Etch membrane (13 mm, 8 μm) (GE Healthcare, catalog number: 150446 )
5. Dulbecco’s modified Eagle medium: Nutrient Mix F-12 (DMEM/F12) (1x), liquid, 1:1 Contains GlutaMAX, but no HEPES buffer (Thermo Fisher Scientific, Gibco^TM, catalog number: 10565042 )
6. Penicillin-streptomycin (P/S) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15070063 )
7. BenchMark fetal bovine serum (Gemini Bio-Products, catalog number: 100-106 )
8. Embryonic lung culture medium (see Recipes)

Equipment

Surgical instruments including:
1. 2 Dumont #5 forceps (Fine Science Tools, catalog number: 11295-10 )
2. Moria^® spoon (perforated) (Fine Science Tools, catalog number: 10370-17 )
Mouse necropsy instrument set including:
1. Metzenbaum (Lahey) Scissors (Roboz Surgical Instrument, catalog number: RS-6950 )
2. Micro Dissecting Scissors 4" Blunt (Roboz Surgical Instrument, catalog number: RS-5980 )
3. Graefe Forceps Straight (Roboz Surgical Instrument, catalog number: RS-5130 )
4. Graefe Forceps Curved (Roboz Surgical Instrument, catalog number: RS- 5135 )
CO₂ Incubator (Thermo Fisher Scientific, model: Heracell^TM 150i )
Fluorescent Stereo Microscope (Carl Zeiss, model: Zeiss SteREO Discovery.V8 , with 8x magnification) equipped with 5 MP, 36 bit, Peltier cooled Zeiss Axiocam MRc5 camera (Carl Zeiss, model: AxioCam MRc 5 )

Software

Zen blue software (Carl Zeiss)
PRISM software version 7

Procedure

English

中文翻译

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如何引用

Yao, C., Carraro, G. and Stripp, B. R. (2018). In vitro Explant Cultures to Interrogate Signaling Pathways that Regulate Mouse Lung Development. Bio-protocol 8(10): e2852. DOI: 10.21769/BioProtoc.2852.