The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging

Summary

The transparent C. elegans intestine can serve as an "in vivo tissue chamber" for studying apicobasal membrane and lumen biogenesis at the single-cell and subcellular level during multicellular tubulogenesis. This protocol describes how to combine standard labeling, loss-of-function genetic/RNAi and microscopic approaches to dissect these processes on a molecular level.

Abstract

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism's genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

Introduction

The generation of cellular and subcellular asymmetries, such as the formation of polarized membrane domains, is crucial for the morphogenesis and function of cells, tissues and organs¹. Studies on polarized membrane biogenesis in epithelia remain a technical challenge, since directional changes in the allocation of subcellular components depend upon multiple consecutive and coincident extracellular and intracellular signals that are difficult to separate in most models and strongly depend on the model system. The model presented here - the single-layered Caenorhabditis elegans intestine - is a tissue of exquisite simplicity. Together w....

Protocol

1 . Labeling the C. elegans intestine

Note: See the accompanying paper by the authors on the analysis of excretory canal tubulogenesis² for the construction of tissue specific fluorescent marker plasmids and the generation of transgenic animals, including discussions on transcriptional and translational fusion proteins (the latter required for the subcellular localization of a molecule of interest). These procedures can be adapted by using specific promoters to drive the molecule of interest to the intestine. See Table 1 for examples of molecules proven useful for visualizing C. elegans

Results

This protocol describes how to molecularly analyze and visualize polarized membrane biogenesis and lumen morphogenesis in the C. elegans intestine, at the single cell and subcellular level. The twenty-cell single-layered C. elegans intestine is formed by directed cell division and migration during mid embryogenesis. At this time, polarized membrane domains become established, yet de novo polarized membrane biogenesis continues in the mature but expanding epithel.......

Discussion

This protocol describes how to combine standard loss-of-function genetic/RNAi and imaging (labeling and microscopic) approaches to take advantage of the C. elegans intestinal epithelium as a model for the visual and molecular dissection of in vivo polarized membrane and lumen biogenesis.

Labeling

This protocol focuses on antibody staining. In situ labeling by antibodies is a highly specific alternative approach to labeling.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Antibody staining
poly-L-lysine	Sigma	P5899
Methanol	Fisher Scientific	A452-4
Acetone	Fisher Scientific	A949SK-4
Tween	Fisher Scientific	50-213-612
Permount	Fisher Scientific	SP15-100
Powdered milk	Sigma	MT409-1BTL
Primary antibodies
MH27 (mouse)			Concentration: 1:20 Resources: Developmental Studies Hybridoma Bank.
MH33 (mouse)			Concentration: 1:10 Resources: Developmental Studies Hybridoma Bank.
anti-ICB4 (rabbit)			Concentration: 1:5 Resources: A gift from Mario de Bono (Medical Research Council, England)
anti-PAR-3 (rabbit)			Concentration: 1:50 Resources: A gift from Kenneth J. Kemphues (Cornell University)
Secondary antibodies
Alexa Floor 568 (anti-rabbit)	ABCam	AB175471	Concentration: 1:200
Cy5 (anti-mouse)	Life technologies	A10524	Concentration: 1:200
TRITC (anti-rabbit)	Invitrogen	T2769	Concentration: 1:200
FITC (anti-mouse)	Sigma	F9006	Concentration: 1:100
Labeled chemicals
Texas Red-Phalloidin			Concentration: 1:100 Resources: Molecular Probes-T7471
Materials
Vacuum Grease Silicone	Beckman	335148
Microscope slides	Fisher Scientific	4448
Microscope coverslips (22x22-1)	Fisher Scientific	12-542-B
C. elegans related			see reference29 for standard C. elegans culture and maintenance procedures.
LB Medium and plates			see reference29 for protocols.
Tryptone	Acros Organics	611845000
Yeast Extract	BD Biosciences	212750
NaCl	Sigma	S7653
Bacto Agar	BD Biosciences	214040
Ampicillin	Sigma	A0116
Tetracycline	Fisher Scientific	BP912
M9 Medium			see reference29 for protocols.
NaCl	Sigma	S7653
KH2PO4	Sigma	P0662
Na2HPO4	Sigma	S7907
MgSO4	Sigma	M2773
NGM plates			see reference29 for protocols.
NaCl	Sigma	S7653
Peptone	BD Biosciences	211677
Tryptone	Acros Organics	611845000
Bacto Agar	BD Biosciences	214040
MgSO4	Sigma	M2773
CaCl2	Sigma	C3881
Cholesterol	Sigma	C8667
K2HPO4	Sigma	P3786
KH2PO4	Sigma	P0662
RNAi plates			see reference30 for protocols.
NaCl	Sigma	S7653
Peptone	BD Biosciences	211677
Tryptone	Acros Organics	611845000
Bacto Agar	BD Biosciences	214040
MgSO4	Sigma	M2773
CaCl2	Sigma	C3881
Cholesterol	Sigma	C8667
K2HPO4	Sigma	P3786
KH2PO4	Sigma	P0662
IPTG	US Biological	I8500
Carbenicillin	Fisher Scientific	BP2648
NaOH	Fisher Scientific	SS266-1
Sodium hypochlorite	Fisher Scientific	50371500
Bacteria
OP50 bacteria	CGC
HT115 bacteria	CGC
Genome-wide RNAi libraries Ahringer genome-wide RNAi feeding library (ref 30,49,50)	Source BioScience
C. elegans ORF-RNAi feeding library (ref51)	Source BioScience
Imaging related
Sodium azide	Fisher Scientific	BP9221-500
Equipment
dissecting microscope	Nikon	SMZ-U
dissecting microscope equipped with a high-power stereo fluorescence attachment (Kramer Scientific), CCD camera with Q capture software and X-Cite fluorescent lamp (Photonic Solutions)	Olympus	SZX12
Laser-scanning confocal microscope	Leica Microsystem	TCS SL
laser-scanning confocal mounted on an ECLIPSE Ti-E inverted microscope	Nikon	C2