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
2. Preparation of Palate Explant Culture for Live Imaging
3. Confocal Time-lapse Imaging of Palate Explant
4. Data Image Analysis
NOTE: Here, Imaris was used. Similar data analyses may also be performed with ImageJ or Volocity software packages.
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 and Figure 2K-2O). Epithelial cells in deeper Z levels are also progressively displaced to the oral side of the MES contributing to the epithelial convergence process (Figure 2K). Posterior-directed cell migration was observed in middle palate MES (Figure 2F-2J). In addition, we found epithelial cell extrusion events during palate fusion suggesting that epithelial cells in the MES can be removed by this active process (Figure 2Q, 2T). The integrated epithelial seam will ultimately break down to achieve mesenchymal confluency (Figure 2S, 2T).
Lifeact-mRFPruby palate showed dynamic actin cytoskeletal rearrangements during palate fusion6. Filamentous actin became enriched at the border between epithelial and mesenchymal areas forming a cable-like structure along the anterior-posterior axis (Figure 2K-2T). Actomyosin contractility drives epithelial convergence and MES breakage because chemical inhibition of either myosin activity or actin polymerization blocked these dynamic processes6.
Software was used to trace cellular behaviors during palate fusion. The best method for cell tracking depends on the reporter signal. If a nuclear reporter mouse is used, the program can trace the center of fluorescent signals as cellular centers21. Nuclear GFP reporter mouse such as ROSA26GFP-NLS-lacZ (GNZ)22 can therefore be used to label epithelial cells using tissue-specific Cre lines; in our hands, this specific nuclear reporter exhibited faster bleaching over the long time-course used in live imaging. Therefore, we utilized the membrane-GFP reporter (ROSA26-mTmGflox) to follow epithelial cell movements in our studies. This required a method for identifying and tracing individual cells by a membrane signal. To solve this problem, we utilized a modified cell tracking method (Figure 3 and Protocol section 4).
Figure 1: Schematic view of mouse secondary palate dissection. (A) Side view of E14.5 mouse embryo. To dissect palatal shelves, the head was cut along the first white dotted line, #1. (B, C) The upper brain (above the second white line, #2) was removed. (D, E) Oral view of dissected head. Lower mandible (#3) was dissected and tongue (#4) was removed. (F) Posterior part of the head along the line #5 was cut out. (G-I) Both maxillae (#6, #7) and anterior part of upper jaw (#8) were removed. (J-L) Nasal septum (#9) was removed (M) GFP signals in the MES of dissected palate from a K14-cre; ROSA26mTmG mouse embryo. (N) Live imaging setup. The oral side of the palate explant was facing down to be imaged with inverted confocal microscope. MES is indicated with white arrowheads (F-I). The imaged area is indicated by white dotted boxes in L and M. Scale bars = 2 cm in A, 1 cm in B-I, 500 µm in J-M. Please click here to view a larger version of this figure.
Figure 2: Live imaging of palate explant cultures. (A-E) Live imaging of the anterior secondary palate from a K14-cre; ROSA26mTmG explant (5 µm depth). The white arrowhead shows a membrane protrusion from an epithelial cell (F-J) Live imaging of the middle palate from K14-cre; ROSA26mTmG explant (5 µm depth). White arrows indicate the direction of cell migrations from the anterior to posterior palate. (K-O) Live imaging of the anterior palate from Lifeact-mRFPruby explant (5 µm depth). Epithelial cell displacement (yellow arrow) to the oral surface and convergence to form an integrated MES with cable-like actin structures in the midline. (P-T) Live imaging of the anterior palate from Lifeact-mRFPruby explant (25 µm depth). The red arrow indicates a cell extrusion event (Q, T). Future breakage points of the seam are indicated by two vertical white arrows in S and T. Scale bars = 20 µm. All live imaging data in this figure is derived from experiments previously published in6. Please click here to view a larger version of this figure.
Figure 3: Data analysis. (A-D) To trace epithelial cells with membrane GFP signals from K14-cre; ROSA26mTmG palate explant (A), A surface was generated by volume rendering of the membrane GFP signal (B). Inverted images were generated after masking the created surface (C). Cellular centers were identified from the inverted images using a spots detection function in Imaris (D). (E) 3D reconstructions allow tracking cell movements in multiple directions. A: anterior, P: posterior, N: nasal, O: oral (F) 2D analysis generates a vantage plot showing cells migrating from anterior to posterior direction in middle palate MES. Scale bars = 20 µm in A-D, 10 µm in E-F. Please click here to view a larger version of this figure.
Video Figure 1: Live imaging of K14-cre; ROSA26-mTmGflox anterior palate (5 µm depth). Images were captured every 10 min (10 min/frame) for 16 h 20 min, Scale bar = 25 µm. This video is a different Z-position of a movie previously published in reference6. Please click here to view this video. (Right-click to download.)
Video Figure 2: Live imaging of K14-cre; ROSA26-mTmGflox middle palate (5 µm depth). Images were captured every 15 min (15 min/frame) for 13 h 30 min, Scale bar = 25 µm. This video has been previously published in reference6. Please click here to view this video. (Right-click to download.)
Video Figure 3: Live imaging of Lifeact-mRFPruby anterior palate (5 µm depth).
Images were taken every 10 min (10 min/frame) for 7 h 50 min. Scale bar = 20 µm. This video has been previously published in reference6. Please click here to view this video. (Right-click to download.)
Video Figure 4: Live imaging of Lifeact-mRFPruby anterior palate (25 µm depth).
Images were taken every 10 min (10 min/frame) for 7 h 50 min. Scale bar = 20 µm. This video is a different Z-position of a movie previously published in reference6. Please click here to view this video. (Right-click to download.)
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 | |
35mm 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 |
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-mTmGflox 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.
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-mTmGflox 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.
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-mTmGflox 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.