Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Summary

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

Abstract

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Introduction

For nearly 50 years the nematode Caenorhabditis elegans established itself as a powerful model to study important questions in development, neurobiology, evolution, host-pathogen interactions, etc.¹ The strength of this model in the study of development lies in: its short life cycle of 3 days; the ease with which these animals can be genetically altered; its transparency that enables the observation of cell displacement and morphology in living animals and its development that is mostly extra-uterine. The developmental stages of the nematode involve embryogenesis and four larval stages (L1 to L4), followed by adulthood. During embryonic de....

Protocol

1. Characterization of Early Elongation Defects in WT and Mutant Animals

1. Mounting Embryos for Normarski DIC Microscopy
   1. Prepare the following culture media and material:
      1. M9 Buffer, dissolve 12.8 g/L Na₂HPO₄•7H₂O, 3 g/L KH₂PO₄, 5 g/L NaCl, 0.25 g/L MgSO₄•7H₂O in distilled water and autoclave to sterilize.
      2. NGM plates, dissolve 3 g/L NaCl, 16 g/L agar, 2.5 g/L bactopeptone in ddH₂O. Autoclave and add 1 mM MgSO₄, 1 mM CaCl₂, 1 mM phosphate buffer and 5 µg/ml cholesterol. Po....

Results

Head-, Tail- and Head/Tail-width Ratio are Robust Metrics.

The protocols described here have been successfully used to characterize the function of regulators and effectors of Rho GTPases pix-1, pak-1 and let-502 during early elongation⁷. pix-1 and .......

Discussion

This protocol describes novel metrics to characterize early and late phases of embryonic elongation.

In section 1, the critical step is the potential presence of bacteria on the pad. The embryos are hermetically enclosed between the pad and the coverslip during image acquisition. Sealing the slide is required to avoid desiccation of the animals during acquisition that lasts more than two hours. To our knowledge, none of the sealers used to mount agarose pads between slide and coverslip are air.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agar	BioShop	AGR001.500
Agarose	Bioshop	AGA001.500
CaCl2 (Calcium Chloride)	Bio Basic Inc.	CT1330
Cholesterol	Sigma-aldrich	C8667
Cleaning solution	union Biometrica	300-5072-000
Glass coverslips	Fisherbrand	12-542B
Glass slides	Fisherbrand	12-552-3
High fluorescent control particles	union Biometrica	310-5071-001
K2HPO4 (potassium phosphate, dibasic)	Bio Basic Inc.	PB0447
K2HPO4 (potassium phosphate, monobasic)	Bio Basic Inc.	PB0445
MgSO4 (magnesium sulfate)	Sigma-aldrich	230391
Na2HPO4( sodium phosphate, dibasic)	Bio Basic Inc.	SDB0487
NaCl (sodium chloride)	Bio Basic Inc.	DB0483
Pebeo Drawing Gum 45 ml	pébéo	PDG033000	any art/craft store
Peptone	BioShop	PEP403.500
Sheath buffer	union Biometrica	300-5070-100
COPAS Biosort	union Biometrica