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doi: 10.1242/10.1242/dev.00481

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Laminin subunits and their role in C. elegans development

Cheng-chen Huang^1,*, David H. Hall^2,*, Edward M. Hedgecock³, Gautam Kao¹, Vassiliki Karantza¹, Bruce E. Vogel⁴, Harald Hutter⁵, Andrew D. Chisholm⁶, Peter D. Yurchenco¹ and William G. Wadsworth^1,

¹ Department of Pathology, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
² Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, NY 104661, USA
³ Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA
⁴ Medical Biotechnology Center, University of Maryland Biotechnology Institute, 725 West Lombard Street, Baltimore, MD 21201, USA
⁵ Max-Planck-Institut Für Medizinische Forchung, Heidelberg, 69120 Germany
⁶ Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, CA 95064, USA
^* These authors contributed equally to the paper

View larger version (23K): [in a new window]   Fig. 1. Modular organization of C. elegans laminin A and laminin B. The N-terminal (LN, domain VI) and the internal globular (L4, domain IV) modules are represented by filled ovals. The rod-like epidermal growth factor-like (LE) repeats are indicated by rectangles. The coiled-coil forming domains are shown as open ovals. The C-terminal G-repeat (G) modules are indicated by half circles. The approximate locations of different epi-1mutations are shown (arrows). Laminin B domains and the alterations of rh27, rh92, rh199 and rh200 have been previously presented (Zhu et al., 2000).

View larger version (83K): [in a new window]   Fig. 2. Localization of laminin A. Wild-type animals were stained using laminin A antibodies. (A) The laminin A protein is detected in the late gastrula between the rows of intestinal (i) and pharyngeal (p) precursor cells, the flanking myoblast cells (m), and the epidermoblast cells (e). Mid-plane optical section. (B) During embryo elongation, laminin A is strongly detected between muscle and epidermal cells. Shown is staining associated with the two dorsal muscle quadrants. Dorsal lateral optical section. (C) In two- and threefold embryos, intense staining for laminin A is observed at the basement membranes associated with the pharynx and intestine. Staining is also detected at the nerve ring (nr). (D) In larvae and adults, staining is detected at the spermatheca (s) where the protein localizes between individual endothelial cells. (E) In larvae and adults, staining is weak at pharyngeal and intestinal basement membranes but is stronger at the nerve ring. (F) Laminin A is also detected in the basement membranes associated with the excretory canal (c). Anterior is towards the left, dorsal towards the top. Scale bars: 10 µm.

View larger version (68K): [in a new window]   Fig. 3. Laminin A is associated with neurons. Immunofluorescence micrograph of nid-1(+) (A,B) and nid-1(ur41) (C,D) animals that express GFP in right fascicle axons of the ventral nerve cord were co-stained with anti-GFP (A,C) and anti-laminin A (B,D) antiserum. In wild type, there is an average of 54 axons in the right fascicle and six in the left (Hedgecock et al., 1990). (C) In nid-1(ur41) mutants, several axons from the right fascicle are mispositioned to the left fascicle (Kim and Wadsworth, 2000). (D) Laminin A associates with the mispositioned axons in the left fascicle (arrows). Ventral aspect. The midline is defined by the vulva (*) and midline motor neuron cell bodies (arrowheads in C). Anterior is towards the left, dorsal towards the top.

View larger version (153K): [in a new window]   Fig. 4. Localization of Laminin B. Wild-type animals were stained using Laminin B antibodies. (A) The Laminin B protein is detected in the late gastrula between the rows of intestinal (i) and pharyngeal (p) precursor cells, the flanking myoblast cells (m), and the epidermal cells (e). Mid-plane optical section. (B) Laminin B is localized to the muscle and epidermis basement membranes in late stage embryos, larvae and adults. The protein has different distribution patterns in the pseudocoelom/epidermis (p/e) and muscle/epidermis (m/e) basement membranes. Lateral view showing two muscle quadrants, each two cells wide. The muscle cells are anchored to the epidermis through connections involving the muscle/epidermis basement membrane. The lateral epidermal ridge separates the quadrants. (C) In L3 and L4 larvae, Laminin B is localized to the gonad basement membrane and is associated with the distal tip cell (dtc), a migratory cell that helps form the gonad (g). (D) In late stage embryos, larvae and adults, Laminin B is localized to the basement membranes that separate the intestine (i), rectal epithelium (a; anus), epidermis and pseudocoelom. (E) Laminin B is localized to the basement membrane that encloses the gonad (g) primordium cells in the L1 and L2 larval stages. (F) Laminin B is detected in the basement membranes associated with intestinal muscle (im) and anal depressor muscle (am). (G) Laminin B is detected in vulval muscle (vm) basement membrane and somatic gonad (g; uterine epithelium and/or uterine muscle). Staining of all specialized muscles is asymmetric: stronger near their anchorage to bodywall or other epithelia. (H) Laminin B is associated with coelomocytes (cc). Scale bars: 20 µm for A-E.

View larger version (110K): [in a new window]   Fig. 5. Laminin B forms a repeating network in the muscle/epidermis basement membrane. Immunofluorescence micrograph of the same wild-type animal co-stained with Laminin B (green) and myosin heavy chain B (red) antibodies. (A) Laminin B is distributed in a grid-like pattern on the muscle surface. The signal appears as periodic dots (arrowheads) and as regularly spaced bands running circumferentially and longitudinally between the dots. The signal is also enhanced at muscle-muscle cell boundaries (arrow). (B) Staining showing myosin thick filament structure in the muscle cell. Thick filaments emanate from the M-line (small arrow) and interact with thin filaments that are in turn anchored to dense bodies, which are functionally analogous to vertebrate Z-lines. The arrowheads indicate the region where dense bodies are located; large arrow indicates muscle-muscle cell boundaries. (C) Superimposition of the two images above showing the relationship of the extracellular laminin network to the myofilament lattice. The longitudinal laminin band and dots corresponds to the thin-filament-containing regions of the muscle.

View larger version (38K): [in a new window]   Fig. 6. Laminin A and Laminin B segregate to different basement membranes. Immunofluorescence micrograph of embryos co-stained with Laminin A (red) and Laminin B (green) antibodies. (A) By late gastrulation, both subunits are deposited between the germ layers. While staining for Laminin A is intense around the pharyngeal precursors (p), both subunits are detected between the muscle (m), intestinal (i) and epidermal (e) precursor cells. (B) As the embryo elongates, areas of distinct Laminin A and Laminin B composition can be distinguished between the developing pharynx/intestine (arrowheads) and body wall muscle (arrows). (C) Co-staining of a late stage mnDf90 embryo, in which the pharynx and intestine has detached from the body wall muscle. Laminin A associates with the pharynx and intestine (large arrows) and Laminin B associates with the body wall muscles (arrowheads), suggesting that these basement membranes, which are juxtaposed in wild-type animals, each retain a unique subunit composition. Juxtaposed muscle/epidermis associated membranes appear yellow (small arrows). Anterior is towards the left, dorsal towards the top. Scale bars: 10 µm.

View larger version (118K): [in a new window]   Fig. 7. Expression of the Laminin genes, lam-3 and epi-1. To observe epi-1 RNA expression, embryos were hybridized with antisense epi-1 probes (left panels in A-C) and stained for DNA with DAPI (right panels in A-C). To observe the expression pattern of lam-3, the lam-3 promoter was used to drive GFP expression (D-F). Anterior is leftwards. (A) The endodermal and mesodermal precursor cells sink inwards from the ventral surface as gastrulation begins. epi-1 RNA is detected in the nuclei of the ingressing cells (arrows). (B) At midgastrulation, epi-1 RNA is detected in the cytoplasm of intestinal, pharyngeal and myoblast cells, but not epidermal (e) precursor cells. (C) At the beginning of elongation, expression is detected primarily in the body wall myoblast cells (m). Expression is weak or is not detected in intestinal (i), pharyngeal (p) and epidermal cells (e). (D) By the onset of elongation, lam-3::gfp is expressed in pharyngeal (p), intestinal (i) and epidermal cells (e), but not in myoblast cells. As the embryo elongates, expression in all these cells declines. (E) In larvae and adults, lam-3::gfp expression is detected in spermatheca (sp) cells. The fluorescence image is superimposed on top of the DIC image of the same animal to show the uterus (u). (F) In larvae and adults, lam-3::gfp expression is also detected in pharyngeal muscle cells (p). Scale bars: 10 µm in D-F.

View larger version (207K): [in a new window]   Fig. 8. Basement membranes are disrupted in laminin B mutants. Mutations in the laminin epi-1 gene disrupt the integrity of basement membranes. In the body cavity, multiple layers, large whorls (wh) and clumping (cl) of material are common in adult animals. This electron micrograph shows a region between the gonad and intestine, a germ cell (g) and yolk (y) are indicated. Scale bar: 5 µm.

View larger version (185K): [in a new window]   Fig. 9. Laminin A-deficient animals. (A) The pharynx (p) of predicted lam-3 null mutants do not properly form; cell bodies (arrowheads) are mispositioned into the surrounding tissues. In this animal the pharyngeal muscle cells (p) were visualized by expression of a myo-2::gfp transgene. (B) Electron micrograph of a lam-3 mutant reveals that the pharynx, which is normally cylindrical, is distorted because of the displacement of pharyngeal muscle (pm) and marginal (pmc) cells. The pharyngeal basement membrane is discontinuous and pharyngeal cells directly adhere to the body wall muscle (m) and epidermis (e) cells of the surrounding tissues (arrowheads). Asterisk indicates the lumen. (C,D) In the pharyngeal cells of lam-3 mutants, the apical membrane domain appears to develop normally as judged by the adherens junctions (C, arrowheads) that form by the lumen (asterisk). In addition, ectopic adherens junctions (D, arrow) also form at what should be the basolateral side of cells. Myofilaments in muscles and intermediate filaments in marginal cells may not assume their normal radial orientation (C, double-headed arrows). (E) In some cases, the lateral cell membrane appears greatly reduced in lam-3 mutants. Increased space between cells with what appears to be excess basement membrane forms between adjacent pharyngeal cells (arrowheads). Scale bars: 2 µm in B-E.

View larger version (217K): [in a new window]   Fig. 10. Early embryonic lethality in epi-1 mutants. A homozygous epi-1(rh199) embryo inside of the heterozygous parent shows cells (asterisks) that are separated and detached from the tissues. The detachment of cells during embryonic development and the failure of organogenesis cause embryonic lethality in predicted epi-1 null mutants. The eggshell (arrows) is slightly shrunk owing to the fixation. Scale bar: 5 µm.

View larger version (66K): [in a new window]   Fig. 11. Cell polarization, cell proliferation, cell differentiation and migration defects in epi-1 mutants. (A) In this predicted epi-1 null mutant, epi-1(rh199), cell polarity is disrupted such that, in the muscles, additional dense bodies (arrows) form ectopically on the pseudocoelomic side of the cell, and sarcomeres (s') organize in an unusual position away from the epidermis. Normally positioned dense bodies (arrowheads) and sarcomeres (s) are observed on the epidermal (e) side of the muscle cells. Dense bodies are analogous to vertebrate Z-lines and function to maintain the alignment of the thin filaments. A displaced muscle cell distorts the shape of a nerve (n). (B) Strong epi-1 alleles cause sterility owing to failure of gonadogenesis during the third larval stage. The gonadal basement membrane is weakened or missing, and the gonadal sheath fails to enclose the germline, which permits germ cells to escape into the body cavity and to invade neighboring tissues. In this epi-1(rh165) mutant, germ cells (asterisks) have invaded the intestine (i). A basement membrane separates the intestine from the gonad arm below it, however no basement membrane separates the gonad (g) and the thin layer of epidermis (e) and here the tissues adhere to one another (arrows). (C) In epi-1 mutants, the development of the body wall muscles is compromised. In this epi-1(rh165) mutant, the muscle cells (m) of a quadrant show incomplete differentiation. The organization of the sarcomere is primitive, with poor segregation of thick and thin filaments and little evidence of dense bodies to anchor them. The entire muscle has failed to settle closely onto the bodywall and the intervening epidermis (e), normally a thin layer, is abnormally wide (double-headed arrows). (D) Axon migration and nerve positioning defects are often observed in epi-1 mutants. In this epi-1(rh191) mutant, the right bundle of the ventral cord (white arrow) is mispositioned to the dorsal side of the ventral epidermal ridge (e). At the normal position of the right bundle, four or five axons are seen (black arrow). In addition, two or three axons are mispositioned to the lateral side of the ridge (arrowheads). Interestingly, a basement membrane appears to be associated with each of these individual axons, a phenotype never observed in wild type. Scale bars: 5 µm in A; 10 µm in B; 2.5 µm in C; 1 µm in D.