Live Imaging of Mouse Secondary Palate Fusion

Summary

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Abstract

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmG^flox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Introduction

Tissue fusion is an important step in the development of multiple organs. Major human birth defects such as cleft lip and palate, spina bifida and malformations of the heart can result from defects in tissue fusion¹. Mouse secondary palate fusion has been extensively studied to identify the cellular and molecular mechanisms controlling tissue fusion in development^2,3,4. In the mouse, secondary palate development starts at around E11.5 with the outgrowth of a secondary palatal shelf from each of the bilateral maxillary processes. Initial growth of the p....

Protocol

All animal experiments were performed in accordance with the protocols of the University of California at San Francisco Institutional Animal Care and Use Committee.

1. Preparation of Live Imaging Media

1. Preparation of liquid culture media
   1. Add 20% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 200 µg/mL L-ascorbic acid, 15 mM HEPES to Dulbecco's Modified Eagle Medium (DMEM)/F12 media.
      NOTE: Live imaging media was freshly made right before the imaging. This media contains phenol-red which may lead to phototoxicity over long time courses of ....

Results

Live imaging of palate explant culture revealed multiple cellular processes mediating palate fusion⁶. Initial contacts between two epithelial cells are made by membrane protrusions (Figure 2B-2E). When two epithelial layers meet, they form a multi-cell layered MES followed by intercalation to make an integrated single cell-layer MES through epithelial convergence(Figure 2A-2E

Discussion

Live imaging of tissue morphogenesis with 3D organ explant culture can provide detailed information regarding cellular processes that cannot be shown in conventional staining analysis of fixed tissue sections. Using in vitro explant culture of mouse embryonic secondary palate, we observed several interesting cellular behaviors leading us to propose a novel mechanism of palate fusion that involves epithelial convergence and cell extrusion.

One common challenge in studies of this type i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
DMEM/F12	Life technology	11330-032
Fetal Bovine Serum	Life technology	16000-044
L-glutamine	Life technology	25030-081
L-Ascorbic acid	Sigma	A4544-100G
Pennicillin/Streptomycin	Life technology	15140-122
Low melting agarose	BioExpress	E-3111-125
35 mm glass bottom dish	MatTek	P35G-1.5-10-C
Petrolieum Jelly (Vaseline)	Sigma	16415-1Kg
Mice
Keratin14-cre		MGI: J:65294	Allele = Tg(KRT14-cre)1Amc
ROSA26mTmG		MGI: J:124702	Allele = Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo
Lifeact-mRFPruby		MGI: J:164274	Allele = Tg(CAG-mRuby)#Rows
Microscope
White Light SP5 confocal microscope	Leica Microsystems
Cell Observer spinning disk confocal microscope	Zeiss Microscopy