Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

Summary

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

Abstract

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Introduction

While there are protocols describing how to image cell division in the early C. elegans embryo, this protocol describes how to image cell division within a tissue during mid embryogenesis. One of the major challenges in imaging organisms during development has been their sensitivity to phototoxicity. However, increased accessibility to spinning disk confocal microscopes or swept field microscopes has permitted more widespread imaging applications. Both systems use solid state lasers and scattered light, limiting the levels of UV that the organisms are exposed to. However, widefield stands can still be used for imaging in vivo, particularly if they ar....

Protocol

1. Preparation of Plates for Maintaining Worm Strains and Performing RNAi

1. Nematode Growth Media Plates
   1. Plates
      1. Prepare Nematode Growth Media (NGM) plates to maintain worm strains and to perform genetic crosses. Combine 3 g NaCl, 17 g Agar and 2.5 g BactoPeptone with 1 L of distilled water in a 2 L flask and add a metal stirring bar.
      2. Autoclave the flask to dissolve the agar and to sterilize the media. Then place the flask on a stir plate and allow the media to cool while stirring.
      3. Once the media has cooled down and is still somewhat warm to touch (45-50 °C), add 1 ml 1 M CaCl₂, 1 ml 1 M M....

Results

This experimental protocol describes how to image cell division in C. elegans embryos during mid embryogenesis. In particular, it describes how to image neuroblasts, which may facilitate epidermal morphogenesis. Epidermal morphogenesis occurs due to a combination of epidermal cell shape changes, migration and adhesion, but also relies on chemical or mechanical cues from the underlying neuroblasts (Figure 1B). The neuroblasts secrete guidance cues that are received by receptors on the surface of .......

Discussion

This protocol describes the use of various types of microscopy to image cell divisions during mid embryogenesis. In particular, this protocol highlights how to image the division of neuroblasts, cells that may facilitate epidermal morphogenesis. Cell-cell communication is important for tissue formation during metazoan development and C. elegans is an excellent model to study tissue formation in vivo. One event that nicely portrays the interplay of tissues is epidermal morphogenesis, which covers the emb.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	BioShop Canada Inc.	#AGR001.1	For making C. elegans NGM and RNAi plates
Agar	Bio Basic Inc.	#9002-18-0	For making bacteria LB agar plates
Agarose	BioShop Canada Inc.	#AGA001.500
Anti-mouse Alexa Fluor 488 antibody	Life Technologies Corporation (Invitrogen)	#A11029
Anti-mouse anti-GFP antibody	Roche Applied Science	#11814460001
Anti-rabbit Alexa Fluor 568 antibody	Life Technologies Corporation (Invitrogen)	#A11011
Ampicillin	BioShop Canada Inc.	#AMP201.5	Store powder at 4 °C and dissolved ampicillin at -20 °C
Bactopetone  (peptone-A)	Bio Basic Inc.	#G213
CaCl2 (calcium chloride)	BioShop Canada Inc.	#C302.1
Cholesterol	BioShop Canada Inc.	#CHL380.25	Dissolve in ethanol
DAPI	Sigma-Aldrich	#D9542	Use to stain nucleic acids (DNA)
Glycerol	BioShop Canada Inc.	#GLY001.1
IPTG (isopropylthio-β-galactoside)	Bio Basic Inc.	#367-93-1	Store powder and dissolved IPTG at -20 °C
KH2PO4 (potassium phosphate, monobasic)	BioShop Canada Inc.	#PPM666.1
K2HPO4 (potassium phosphate, dibasic)	BioShop Canada Inc.	#PPD303.1
L4440  (feeding vector)	Addgene	#1654	Keep as glycerol stock at -80 °C
MgSO4   (magnesium sulfate)	BioShop Canada Inc.	#MAG511.500
NaCl (sodium chloride)	Bio Basic Inc.	#7647-14-5
Na2HPO4 (sodium phosphate, dibasic)	Bio Basic Inc.	#7558-79-4
Normal Donkey Serum (NDS)	Wisent Bioproducts	#035-110
n-Propyl-3,4,5-trihydroxybenzoate (propyl gallate)	Alfa Aesar	#A10877
Poly-L-lysine	Sigma-Aldrich	#P8920	For optimal results coat microscope slides three times
Streptomycin	BioShop Canada Inc.	#STP101.50	Store powder at 4 °C and dissolved streptomycin at -20 °C
Tetracyclin	BioShop Canada Inc.	#TET701.10	Store powder at 4 °C and dissolved tetracycline at -20 °C
Tween-20	Bio Basic Inc.	CAS#9005-64-5
Tryptone	BioShop Canada Inc.	#TRP402.500
Yeast Extract	Bio Basic Inc.	#8013-01-2