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 ...

Research

JoVE Journal

Developmental Biology

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The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended ...

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Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

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Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Assessing Lysosomal Alkalinization in the Intestine of Live Caenorhabditis elegans

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The nematode Caenorhabditis elegans (C. elegans) is a model system that is widely used to study longevity and developmental pathways. Such studies are ...

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

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Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases ...