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 are outfitted with cameras with high sensitivity (e.g. EMCCD), aperture control and light control (e.g. LEDs or adjustable mercury bulbs). This protocol describes how to use either a confocal based system or a widefield system to image cell division within developing C. elegans embryos. As an example, we describe how to image neuroblast cell division. Neuroblasts may facilitate epidermal morphogenesis by providing chemical or mechanical cues to the overlying epidermal cells, and provide an excellent example of the importance of intercellular communication in the formation of tissues.

Caenorhabditis elegans is an ideal model organism for microscopy based studies due to its transparency and simple tissue organization¹. Furthermore, C. elegans is amenable to genetic methods and RNAi, and since many of its genes have human homologues, it can be used to identify conserved mechanisms for tissue formation^2-5. In C. elegans, formation of the epidermis occurs during mid embryogenesis, when the embryo has >300 cells. Epidermal morphogenesis consists of several major phases, during which the embryo is enclosed in a layer of epidermal cells that constrict and extend to transform the embryo from an ovoid form into the elongated shape of a worm⁶. Ventral enclosure describes one of these morphogenetic events, when the ventral epidermal cells migrate towards the ventral midline to cover the ventral surface of the embryo (Figure 1). First, two pairs of anterior located leading edge cells migrate towards the ventral midline, where they adhere and fuse with their contralateral neighbors⁶. This is followed by migration of the posterior located pocket cells, which form wedge like shapes creating a ventral pocket^6-7. The mechanism that closes the pocket is not well understood. One possibility is that a supracellular actin myosin contractile structure ties the pocket cells together in a purse string like fashion, similar to wound healing⁸. Interestingly, migration of some of the pocket cells is mediated by specific subsets of underlying neuroblasts⁹ (neuronal precursors that are found underneath the epidermis; Figure 1B).

Previous studies showed that the neuroblasts regulate ventral epidermal cell migration and ventral enclosure. VAB-1 (Ephrin receptor) and VAB-2 (Ephrin ligand) are highly expressed in the neuroblasts and facilitate the sorting of anterior and posterior neuroblasts from one another, and mutations in vab-1 or vab-2 cause ventral enclosure phenotypes^10-13. However, promoter rescue experiments showed that vab-1 is also required in the overlying ventral epidermal cells and receptors for other guidance cues secreted by the neuroblasts are expressed in the ventral epidermal cells⁹. Although mutations in any of these receptors cause ventral enclosure phenotypes, it is not clear if defects arise due to problems in neuroblast positioning or due to failure of the ventral epidermal cells to respond to guidance cues¹⁴. Altering the division of neuroblasts without affecting their ability to secrete guidance cues could shed light on the role of neuroblasts and their ability to provide mechanical input during epidermal morphogenesis. Recently, it was found that a cell division gene, ani-1 (anillin) is highly expressed in neuroblasts (Figure 2A) and its depletion causes neuroblast division defects. Interestingly, these embryos display ventral enclosure phenotypes (Fotopoulos, Wernike and Piekny, unpublished observations).

Anillin is required for cell division, and particularly for cytokinesis, which describes the process where a mother cell physically divides into two daughter cells. Cytokinesis is driven by the formation of an actomyosin contractile ring, which needs to be tightly controlled in space and time to ensure that it is properly coupled with sister chromatid segregation. The master regulator of metazoan cytokinesis is RhoA (RHO-1 in C. elegans), a small GTPase that is active in its GTP-bound form. The GEF Ect2/ECT-2 activates RhoA, after which RhoA-GTP interacts with downstream effectors that form the contractile ring and mediate its ingression¹⁵. Anillin is a multi domain protein that binds to RhoA via its C-terminus and to actin and myosin via its N-terminus. Anillin is required to stabilize the position of the contractile ring in mammalian or Drosophila S2 cells¹⁶. Anillin depletion causes contractile rings to undergo lateral oscillations, and cytokinesis eventually fails forming multinucleate cells^17-19. Interestingly, although C. elegans ani-1 coordinates actomyosin contractility in the early embryo, it is not essential for cytokinesis. However, as described above, ani-1 is required for neuroblast cytokinesis during mid-embryogenesis (Fotopoulos, Wernike and Piekny, unpublished observations). Cytokinesis failure would alter the number and position of neuroblasts and could affect the location of chemical guidance cues, or it could alter the mechanical properties of the tissue. Both models highlight the nonautonomous role of neuroblasts for ventral enclosure, and the importance of tissue-tissue communication during embryonic development.

This experimental protocol describes how to image cell division during C. elegans mid embryogenesis using fluorescence microscopy. The majority of experiments studying the mechanisms of cell division were performed in single cells within culture dishes (e.g. HeLa or S2 cells) or in early embryos with a limited number of cells (e.g. C. elegans one-cell embryo, Xenopus, or echinoderm embryos). However, it is important to also study cell division within tissues, as there are external cues that can influence the timing and placement of the division plane. Furthermore, cells may provide chemical or mechanical cues to influence the development of neighboring tissues, and it is important to understand how intercellular communication helps tissues to form during development.

Protocol

1. Preparation of Plates for Maintaining Worm Strains and Performing RNAi Nematode Growth Media Plates Plates 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. 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 whi…

Representative 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

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 488 antibody	Life Technologies Corporation (Invitrogen)	#A11029
Anti-mouse anti-GFP antibody	Roche Applied Science	#11814460001
Anti-rabbit Alexa 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