Name: 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
Uploaded: 2017-10-03T13:00:00
Description: Watch this Scientific Journal Video about The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Anti

We thank Mario de Bono (MRC Laboratory of Molecular Biology, Cambridge, UK), Kenneth J. Kemphues (Cornell University, Ithaca, USA), Michel Labouesse (Institut de Biologie Paris Seine, Universite&#769; Pierre et Marie Curie, Paris, France), Gr&#233;goire Michaux (Universit&#233; deRennes 1, Rennes, France) and the CGC, funded by NIH Office of Research Infrastructure Programs (P40 OD010440), for strains and antibodies. This work was supported by grants NIH GM078653, MGH IS 224570 and SAA 223809 to V.G.

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 d...

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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...

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 with the single-cell C. elegans excretory canal (see accompanying paper on polarized membrane biogenesis in the C. elegans excretory canal)2, it provides several unique advantages for the identification and characterization of molecules required for polarized membrane biogenesis. The conservation of molecular polarity cues from yeast to man make this simple invertebrate organ an excellent &#34;in vivo tissue chamber&#34; to address questions on epithelial polarity that are of direct relevance to the human system, which is still far too complex to allow the visual dissection of these events at the single cell level in vivo.
Although multiple conserved polarity cues from (1) the extracelluar matrix, (2) the plasma membrane and its junctions, and (3) intracellular vesicular trafficking have been identified3, the underlying principles of their integration in the process of polarized epithelial membrane and tissue biogenesis is poorly understood4. The classical single-cell in vivo models (e.g.S. cerevisiae and the C. elegans zygote) have been instrumental in defining the principles of polarized cell division and anterior-posterior polarity and have identified critical membrane-associated polarity determinants (the small GTPases/CDC-42, the partitioning-defective PARs)5,6, but they depend upon unique symmetry breaking cues (bud scar, sperm entry) and lack junction-secured apicobasal membrane domains and, presumably, the corresponding intracellular apicobasal sorting machinery. Our current knowledge about the organization of polarized trafficking in epithelia, however, primarily relies on mammalian 2D monocultures7, which lack physiological extracellular and developmental cues that can change positions of membrane domains and directions of trafficking trajectories (a switch from 2D to 3D in vitro culture systems alone suffices to invert membrane polarity in MDCK (Madin-Darby canine kidney) cells)8. In vivo developmental studies on epithelial polarity in invertebrate model organisms were initially conducted in flat epithelia, for instance in the Drosophila melanogaster epidermis, where they identified the critical contribution of junction dynamics for polarized cell migration and cell sheet movement9, and of endocytic trafficking for polarity maintenance10. The 3D in vitro and in vivo analysis of lumen morphogenesis in tubular epithelia in MDCK cells and in the C. elegans intestine, respectively, have recently identified the requirement of intracellular trafficking for de novo (apical) domain and lumen biogenesis and positioning11,12,13. The thickness of tubular (versus flat) epithelial cells is an advantage for the 3D analysis of subcellular asymmetries since it permits a superior visual distinction of the apical-lumenal membrane, apico-lateral junctions, the lateral membrane, and the positions of intracellular organelles. To these visual advantages, the C. elegans model adds the in vivo setting, developmental axis, transparency, simplicity of body plan, invariant and defined cell lineage, analytical (genetic) and additional advantages described below.
C. elegans itself is a roundworm of tubular structure whose transparency and simple architecture make its likewise tubular internal organs directly accessible to the visual analysis of tube and lumen morphogenesis. The twenty cells of its intestine (21 or 22 cells on occasion)14 are derived from a single progenitor cell (E) and develop from a double-layered epithelium by one intercalation step into a bilaterally symmetrical tube of nine INT rings (four cells in the first ring; Figure 1 schematic)14,15,16. The intestine&#39;s lineage and tissue analysis, initially determined by Nomarski optics via nuclear identities17and subsequently by fluorescence microscopy via labeled membranes, has provided critical insights into its morphogenesis, in particular the cell-autonomous and cell-non-autonomous requirements for its directional cell divisions and movements (e.g., intercalation, right-left asymmetries, anterior and posterior tube rotation)14,18. Early endodermal cell specification and the gene regulatory network controlling the development of this clonal model organ are well characterized19,20. The focus here, however, is on the analysis of polarized membrane and lumen biogenesis in single tubular cells, and of the intracellular asymmetries of endomembranes, cytoskeletal structures and organelles that accompany this process. The analysis is facilitated by the simplicity of this tube, where all apical membranes (on the ultrastructural level distinguished by microvilli) face the lumen and all basal membranes face the outer tube surface, with lateral membranes contacting each other, separated from the apical membrane by junctions (Figure 1 schematic; see references (16,21) for the C. elegans-specific organization of tight and adherens junction components). Apical membrane biogenesis is thus coincident with lumen morphogenesis. Furthermore, the size of adult intestinal cells - the largest cells of this small animal (with exception of the excretory cell) - approximate the size of a mammalian cell, permitting the in vivo visual tracking of subcellular elements, e.g. vesicle trajectories, that is typically attempted in vitro in a culture dish.
For the purpose of this cellular and subcellular analysis, appropriate labeling is critical. Intestinal endo- or plasma-membrane domains, junctions, cytoskeletal structures, nuclei and other subcellular organelles can be visualized by labeling their specific molecular components. Many such components have been characterized and continue to be discovered (Table 1 gives a few examples and refers to resources). For instance, various molecules distinguishing the tubular and/or vesicular compartments of the intestinal endomembrane system, from the ER to the Golgi via post-Golgi vesicles to the plasma membrane, have been identified22. The specific proteins (as well as lipids and sugars) can either be labeled directly, or indirectly via binding proteins. This protocol focuses on in situ antibody staining of fixed specimens, one of two standard labeling techniques (see the accompanying paper on excretory canal tubulogenesis for a description of the other technique2 - in vivo labeling via fluorescent protein fusions - which is directly applicable to the intestine; Table 2 provides examples of intestine-specific promoters that can be used to drive expression of such fusion proteins to the intestine). Double- or multiple labeling with either approach, or with a combination of both plus additional chemical staining, allows greater in-depth visual resolution and the examination of spatial and temporal changes in co-localization and recruitment of specific molecules or of subcellular components (Figure 2). The fixation and staining procedures described in this protocol support preservation of green fluorescent protein (GFP) labeling during immunostaining procedures. For imaging, key points of the detection and characterization of tubulogenesis phenotypes via standard microscopic procedures (fluorescence dissecting and confocal microscopy) are described (Figure 3, 4). These can be extended to higher resolution imaging approaches, for instance superresolution microscopy and transmission electron microscopy (not described here).
A key strength of this system is the ability to analyze polarity in individual cells at different developmental stages, from embryogenesis through adulthood. For instance, apical membrane domain and lumen biogenesis can be tracked throughout development at the single-cell level via labeling with ERM-1, a highly conserved membrane-actin linker of the Ezrin-Radixin-Moesin family23,24. ERM-1 visualizes apical membrane biogenesis (1) during embryonic tube morphogenesis, when it occurs concomitantly with polarized cell division and migration (cells move apically around the lumen during intercalation)15; (2) during late embryonic and larval tube extension that proceeds in the absence of cell division or migration; and (3) in the adult intestine, where polarized membrane domains are maintained (Figure 1). In the expanding post-mitotic larval epithelium, de novo polarized membrane biogenesis can thus be separated from polarized tissue morphogenesis, which is not possible in most in vivo and in vitro epithelial polarity models, including those with single-cell resolution (e.g. the 3D MDCK cyst model8). With labeling for other components, this setting provides the opportunity (particularly at the L1 larval stage when cells have a higher cytoplasm/nucleus ratio) to distinguish those intracellular changes that are specific to polarized membrane biogenesis (e.g. the reorientation of trafficking trajectories) from those concomitantly required for polarized cell division and migration.
The genetic versatility of C. elegans is well known25and makes it a powerful model system for the molecular analysis of any biological question. A study on morphogenesis, for instance, can start with a wild-type strain, a transgenic strain where the structure of interest (e.g. a membrane) is labeled with a fluorescent marker, or with a loss- or gain-of-function mutant with a defect in this structure. A typical reverse genetic study may generate a mutant where the gene of interest is deleted in the germline (e.g. by a targeted deletion), modified by mutagenesis (typically producing point mutations with consequent loss, reduction, or gain in function of the gene), or where its transcript is reduced by RNAi. The ease of RNAi by feeding in C. elegans26 also lends itself to the design of targeted screens that examine a larger group of genes of interest. A genetic model organism&#39;s arguably greatest strength is the ability to conduct in vivo forward screens (e.g. mutagenesis, systematic or genome-wide RNAi screens) that permit an unbiased inquiry into the molecular cause for a phenotype of interest. For instance, an unbiased visual C. elegans RNAi tubulogenesis screen, starting with a transgenic animal with ERM-1-labeled apical membranes, discovered an intriguing reversible intestinal polarity conversion and ectopic lumen phenotype, used here as an example for this type of analysis. This screen identified the depletion of glycosphingolipids (GSLs; obligate membrane lipids, identified via their GLS-biosynthetic enzymes) and components of the vesicle coat clathrin and its AP-1 adaptor as the specific molecular defects causing this polarity conversion phenotype, thereby characterizing these trafficking molecules as in vivo cues for apical membrane polarity and lumen positioning12,13. When starting with a specific genetic mutation/morphogenesis phenotype, such screens (or single genetic/RNAi interaction experiments) can also examine functional interactions between two or multiple genes of interest (see accompanying paper on the excretory canal for an example of such an analysis)2. This protocol focuses on RNAi which, in addition to its ability to directly identify the gene whose loss causes the phenotype in forward screens, provides specific advantages for the analysis of morphogenesis. Since gene products directing morphogenesis often work in a dose-dependent fashion, RNAi is usually successful in generating a spectrum of phenotypes. The ability to generate informative partial-loss-of-function phenotypes also helps to address the problem that the majority of important tubulogenesis genes are essential and that their losses cause sterility and early embryonic lethality. This protocol includes conditional RNAi strategies to overcome this difficulty and suggests ways to optimize the generation of a broader spectrum of phenotypes, such as an allelic series produced by mutagenesis.

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

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

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&#160;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&#39;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.

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...

Watch this Scientific Journal Video about The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Anti

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 Analy

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.

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

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the ...

The overall goal of this combination of standard methods is to demonstrate how the C.elegans intestine can be used for the in vivo analysis of polarized membrane biogenesis and lumen morphogenesis at the single-cell and sub-cellular level. This method can help answer key questions in the polarity and biogenesis fields such as how apical versus basolateral membrane domains and other sub-cellular asymmetries are established and maintained during and after morphogenesis. The main advantage of this model system is that this transparent single layer of only 20 cells can survive in vivo tissue chamber to dissect single cell polarized membrane and lumen biogenesis.The techniques can be extended to the analysis of other C.Elegans morphogenesis phenotypes the focus here is on the analysis of membrane biogenesis, specifically on apical membrane and lumen biogenesis. The procedures include refined imaging techniques in fixed and alive animals to distinguish membrane domains and sub-cellular components and to identify lumen morphogenesis phenotypes in a C.elegans intestine. This video on intestinal tubulogenesis is accompanied by a video on excretory canal tubulogenesis mutually informative models where the in vivo analysis of polarized membrane biogenesis.With regard to labeling the generation of transgenic animals with fluorescently labeled fusion proteins for live imaging is described in the accompanying video on the analysis of excretory canal tubulogenesis. Such animals can be directly used in RNAi experiments. Third generation is typically the first step in this combination of procedures.This video starts with the alternative labeling method antibody staining for which animals must be fixed. The different labeling procedures can be combined with immunohistochemistry typically being used for the final imaging analysis. This protocol also has examples of intestine specific membrane markers promoters and other resources useful for both labeling procedures.After demonstrating immunohistochemistry this video uses a transgenic strain that labels the intestinal apical membrane with ERM-1 GFP to search for polarity and lumen morphogenesis phenotypes using RNAi. First prepare a slide to fix the worms. Pipette 30 microliters of poly-L-lysine onto a slide and then set a second slide onto the other and drop them together to spread the solution evenly onto both.Now peel apart the two slides and let them air dry for 30 minutes. Next place a flat metal block into a container filled with liquid nitrogen. Wash the plates with M9 solution and transfer about 10 microliters of M9 containing the worms.Alternatively pick the worms and add M9 to the slide. Once the worms are transferred gently drop a cover slip cross ways onto them such that the cover slip over hangs one edge. Then gently press the cover slip without making any lateral movements.Now immediately transfer the slide to the metal block in liquid nitrogen and let it freeze. After five minutes flick off the cover slip using the overhanging edge. Do this swiftly to get the proper crack of the cuticle.Next immerse the slide in a methanol filled glass Coplin jar at minus 20 degrees Celsius. After five minutes transfer the slide to an acetone filled glass Coplin jar for another five minutes at the same tempature. The slides can be stained directly or stored at minus 20 degrees Celsius.Remove the slide from acetone and let it air dry at room temperature. Next mark the location for a circular wall of petroleum jelly on the underside of the slide then build up the jelly wall. Do not rub off the jelly during this procedure.Next prepare a wet chamber in a bin with wet paper towels and place the slide onto a rack within. There should be no contact between the towels and slide or between adjacent slides. Now pipette enough PBS into the jelly circle to cover the area.Gently place a pipette tip at the edge of the circle and allow fluid to smoothly disperse over the worms. Then close the lid and wait five minutes. After five minutes tilt the slide and slowly aspirate the PBS taking care not to lose worms from the slide.Next carefully fill the jelly circle with freshly prepared blocking solution. And let the slide incubate for 15 minutes at room tempature. After 15 minutes replace the blocking solution with blocking solution containing the primary antibody.And incubate the slide at four degrees Celsius overnight. The next day remove the primary antibody and wash the slide by applying and removing blocking solution three times. Allow each block application to incubate for 10 minutes.Next add the secondary antibody diluted in blocking solution and incubate the slide for an hour at room temperature. Thereafter wash the slide two times with blocking solution and then once with PBS. Then remove as much of the PBS as possible without permitting the specimen to dry out and carefully remove the jelly around the specimen.Apply a drop of mounting medium onto the specimen followed by a cover slip and a nail polish seal. Then store the slides in the dark at four degrees Celsius. Transfer the RNAi library plate from minus 80 degrees Celsius to dry ice.Remove the sealing tape. And transfer the clone of interest onto selective auger. Then streak out the clone and grow the bacteria over night.Before returning the library plate to the minus 80 degrees Celsius freezer apply new sealing tape. The next day inoculate colonies from the plate into one milliliter aliquots of liquid medium containing ampicillin. Then shake them for about 14 hours at 37 degrees Celsius.The following day seed 200 microliters per clone onto separate RNAi plates. Let the plates dry and induce the RNAi production with an overnight incubation at room tempature. A day later transfer five L4 stage worms to each RNAi plate labeled with the specific gene that is targeted.Then transfer the plates to an incubator typically at 20 degrees Celsius. First examine control versus RNAi animals under bright light at low and high magnification. Next examine control versus RNAi animals under fluorescent light under the appropriate filters.Systematically scan a plate with RNAi animals from the upper left to the lower right of the entire plate searching for phenotypes. Look for animals of interest. For a closer examination of the selected RNAi phenotypes focus on the intestine and use the the zoom to assess tubulogenesis and lumen morphogenesis phenotypes.To examine the worms of interest under confocal microscopy first mount and immobilize them by hand make a thin circle of vacuum grease or petroleum jelly on a glass slide. The grease layer should be thin not thicker than 0.1 millimeter. Next add about 3.5 microliters of 10 millimolar sodium azide into the middle of the circle.Then swiftly pick worms of interest into the solution under a dissecting microscope before the solution dries. Then gently apply a cover slip and image the animals within 30 minutes. Use mild pressure and under the microscope verify that the worms are not floating in solution.Under the confocal microscope find the worms under low power and focus. Then use a 60 or 100X oil immersion objective to view and image the intestine. Next examine the animal under fluorescent light with the appropriate channel to select areas that display the cellular defect of interest.Move swiftly to avoid photo bleaching. Then switch to laser scanning and restrict the image to the intestine by setting scanning boundaries at its dorsal and ventral side. Capture an image series and later merge the images into a single projection image.This protocol describes how to molecularly analyze and visually dissect polarized membrane biogenesis in the developing C.elegans intestine where apical membrane and lumen morphogenesis coincide. Apical and basolateral membrane domains, membrane associated junctions and the intestinal lumen could be resolved and their relation to each other could be examined throughout development. These sub-cellular components could be labeled by fluorescently tagged antibodies or fluorescent fusion proteins for double or triple labeling as described in the accompanying paper.Gross lumen defects could be quickly scanned by using a dissecting fluorescence microscope. The apical membranes cytoskeleton linker ERM-1 coupled to GFP is a robustly expressed marker useful for a RNAi screen. An array of novel intestinal lumen morphogenesis and polarity phenotypes along with their underlying gene defects were uncovered when using this marker.Then higher magnification images acquired by a confocal laser scanning were used to more carefully examine the phenotypes to better understand the underlying defects. Some phenotypes could be quantified. Quantification was used to compare such defects and track their progression through development.After watching this video you should have a good understanding of how to label intestinal membrane and endo membrane compartments by immuno staining and how to analyze function RNAi phenotypes of polarized membrane and lumen biogenesis.

Research

JoVE Journal

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