Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Podsumowanie

Herein we describe a procedure to capture live images of Drosophila gastrulation. This has enabled us to better understand the apical constriction involved in early development and further analyze mechanisms governing cellular movements during tissue structure modification.

Streszczenie

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. This morphological alteration is also recognized as epithelial to mesenchymal transition (EMT). Dysregulation of EMT is associated with fibrosis and cancer metastasis. There is emerging evidence that EMT is controlled by a number of molecular mechanisms. As such, many key genes that control apical constriction are also known to be important factors in the EMT observed in cancer metastasis. Like EMT during Drosophila gastrulation, epithelial cells can be induced to change their shape and be reprogrammed to redirect cell fate towards various other cell types. Here we provide a robust imaging method of Drosophila gastrulation to assay the initiation of morphogenetic cellular movements and cell fate identification during this stage of embryonic development. Using this method, we identify cell rearrangement at the time of gastrulation and demonstrate the importance of apical constriction during gastrulation using GFP labeled DE-cadherin.

Wprowadzenie

Gastrulation is the first set of morphologically dynamic events that occur during embryonic development of multicellular animals such as Drosophila^1,2. Interestingly, emerging evidence suggests that this process is regulated through the interplay between mechanical and molecular mechanisms³. Moreover, the epithelial to mesenchymal transition (EMT), which is a crucial process in gastrulation, is also implicated in human disease processes such as cancer metastasis^4-8. As such, many genes that control apical constriction are also known to be key factors in the EMT observed in cancer metastasis⁹. Thus, apical constriction at the time of gastrulation is an excellent model to investigate the aforementioned regulatory mechanisms and to enhance our understanding of cancer metastasis. The advantage of this technique is that we can observe cell movement at the time of gastrulation in real-time and therefore, we will be able to screen genes involved in gastrulation as well as cancer metastasis.

Although relatively unknown, cell-to-cell adhesion is thought to play a central role in apical constriction¹. Drosophila genetics is well suited for single cell level investigations exploring regulatory molecular mechanisms. This model will enable us to uncover the importance of apical constriction during gastrulation. Moreover, this method can be used to screen genes involved in cancer metastasis. Capturing live images of Drosophila gastrulation has further enabled us to understand in greater detail the molecular mechanisms governing tissue rearrangement. Herein, we provide a comprehensive description of a simple method to achieve this.

Protokół

NOTE: The transgenic flies used in this study include the following: DE-cad::GFP ¹⁰.

1. Preparation of Apple Plate

1. Prepare a mixture of 12.5 g agar, 125 mL 100% commercially available apple juice, 12.5 g glucose, and 375 mL H₂O. Heat the mixture in a microwave and pour it into a 3 cm cell culture dish. Store the mixture at 4 °C for future use.
2. After preparing the apple plate, add a thin layer of kneaded yeast paste on top of it to allow the flies to lay egg.

2. Embryo Preparation and Live Imaging Protocol

1. Place approximately 50 flies (25 males and 25 females) in a bottle to mate and subsequently lay eggs overnight on an apple plate covered in kneaded yeast. Set the apple juice plate at the mouth of the bottle. Keep the bottle upside down in an incubator. Wrap the plastic Drosophila stock bottles with aluminum foil to prompt the flies to lay more eggs.
2. The next morning, replace the old apple juice plate with a new one.
3. Let the flies lay eggs for 3 to 4 h by wrapping the bottle with aluminum foil.
4. Three to four hours later, collect the embryos in an embryo strainer from the apple juice plate using a brush or cotton bad and wash them with PBS. Wash the embryos 2 to 3 more times with PBS in 12-well culture dishes.
5. Dechorionate the embryos with 50% bleach for 5 min in 12-well culture dishes.
6. Following the dechorionation process, wash the embryos 2 to 3 more times with PBS.
7. Transfer the embryos to a 3 cm cell culture dish containing PBS and select stage 5 embryos under the microscope based on the level of transparency at their borders. Pipette the staged embryos using a 200 µL pipette tip.
8. Next, place two selected embryos on a glass coverslip, and remove excess PBS with finely twisted tissue paper. Orient the embryos dorsal side up on the coverslip using a finely twisted tissue paper. After that, attach the embryos to the glass coverslip with silicon grease using a fine needle and twisted tissue paper.
9. Add a small drop of halocarbon oil 700 on the embryos and place the coverslip containing the embryos upside down on the indented slide, leaving some space at the bottom of the slide.
10. Examine the embryos using a confocal imaging system, an Argon/488 laser, and an oil-immersion objective (63X).
    NOTE: Identifying the embryo at the appropriate developmental stage is important to capture images of the gastrulation event. Under these conditions, observe almost all embryos develop normally up to at least stage 14. The analyzed embryo can be cultured further to develop as an adult.

Wyniki

Here, we show the gastrulation events of the Drosophila embryo and a general overview of the embryo preparation procedure (Figure 1). Cell membranes are labeled using DE-cadherin::GFP and live imaging of cell movements is performed at the time of gastrulation in Drosophila (Figure 2). Since DE-cadherin GFP flies allow us to visualize cell adherence junctions, we are able to trace apical cell shape and movements using this system. More importantly...

Dyskusje

Although we have previously reported a similar procedure to capture live images of the gastrulation process in Drosophilla¹, the method we describe here is detailed and easy to trace endogenous cadherin expression and thus is quite useful for genetic screening of key factors involved in gastrulation. To maximize success with this imaging procedure, it is essential to use an indented slide. Mechanical pressure sometimes causes embryonic death. Therefore, it is also important to handle the embryos as ge...

Materiały

Name	Company	Catalog Number	Comments
Halocarbon oil 700	Sigma	MKBH 5726
Vacuum grease Silicone	Beckman	335148
Glass coverslip	Matsunami glass	Thickness No1	24 - 36 mm
Embryo stariner	Corning	Corning3477
Plastic Drosophilla Stock Bottles	Hitec	MKC-100
DE-Cadherin knock-in flies			REF (10)