Molecular Biology of the Cell
Vol. 19, 3898–3908, September 2008
Caenorhabditis elegans Teneurin, ten-1, Is Required for
Gonadal and Pharyngeal Basement Membrane Integrity
and Acts Redundantly with Integrin ina-1
and Dystroglycan dgn-1
Agnieszka Trzebiatowska,* Ulrike Topf,* Ursula Sauder,†Krzysztof Drabikowski,*
and Ruth Chiquet-Ehrismann*
*Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, CH-4058 Basel,
Switzerland; and†Microscopy Center, Pharmazentrum, University of Basel, CH-4056 Basel, Switzerland
Submitted January 14, 2008; Revised June 27, 2008; Accepted July 1, 2008
Monitoring Editor: Jean E. Schwarzbauer
The Caenorhabditis elegans teneurin ortholog, ten-1, plays an important role in gonad and pharynx development. We
found that lack of TEN-1 does not affect germline proliferation but leads to local basement membrane deficiency and early
gonad disruption. Teneurin is expressed in the somatic precursor cells of the gonad that appear to be crucial for gonad
epithelialization and basement membrane integrity. Ten-1 null mutants also arrest as L1 larvae with malformed pharynges
and disorganized pharyngeal basement membranes. The pleiotropic phenotype of ten-1 mutant worms is similar to
defects found in basement membrane receptor mutants ina-1 and dgn-1 as well as in the mutants of the extracellular
matrix component laminin, epi-1. We show that the ten-1 mutation is synthetic lethal with mutations of genes encoding
basement membrane components and receptors due to pharyngeal or hypodermal defects. This indicates that TEN-1 could
act redundantly with integrin INA-1, dystroglycan DGN-1, and laminin EPI-1 in C. elegans development. Moreover, ten-1
deletion sensitizes worms to loss of nidogen nid-1 causing a pharynx unattached phenotype in ten-1;nid-1 double mutants.
We conclude that TEN-1 is important for basement membrane maintenance and/or adhesion in particular organs and
affects the function of somatic gonad precursor cells.
Teneurins are large transmembrane proteins that play im-
portant roles in cell signaling and cell adhesion (Tucker and
Chiquet-Ehrismann, 2006; Tucker et al., 2007). Teneurins are
phylogenetically conserved among metazoans and they
were described in several species, including ten-1 in Caeno-
rhabditis elegans (Drabikowski et al., 2005), ten-m/odz and
ten-a in Drosophila (Baumgartner et al., 1994; Levine et al.,
1994; Fascetti and Baumgartner, 2002; Rakovitsky et al.,
2007), zebrafish (Mieda et al., 1999), and in chicken (Minet et
al., 1999; Tucker et al., 2000; Tucker et al., 2001; Rubin et al.,
2002) and mouse (Oohashi et al., 1999; Ben-Zur et al., 2000;
Zhou et al., 2003). In vertebrates, the four teneurin paralogs
were named teneurin-1 to -4, ten-m1 to -m4, or odz-1 to -4.
The extracellular domain of all teneurins is composed of
eight tenascin-type EGF-like repeats, a region of conserved
cysteines, and YD repeats that are also found in a few
bacterial proteins (Minet and Chiquet-Ehrismann, 2000). The
intracellular domain contains proline-rich stretches and pu-
tative tyrosine phosphorylation sites but is less conserved
than the extracellular part and cannot be aligned in a linear
way between the phyla. Teneurins are thought to interact in
a homophilic manner (Oohashi et al., 1999; Rubin et al., 2002;
Bagutti et al., 2003; Leamey et al., 2008) and to date, no other
ligand has been identified.
The name “teneurins” refers to their high expression in
the developing and adult nervous system (Mieda et al.,
1999; Oohashi et al., 1999; Otaki and Firestein, 1999; Ben-
Zur et al., 2000; Tucker et al., 2000; Rubin et al., 2002; Zhou
et al., 2003). In the developing mouse cortex, all teneurins
are expressed in distinctive gradients and may be re-
quired for neocortical patterning (Li et al., 2006). Several
reports point out their role in the development of visual
pathways. Leamey et al. (2008) have found that teneurins
are up-regulated in visual versus somatosensory areas of
the neocortex. Moreover, expression of different teneurins
is largely nonoverlapping and can be found in intercon-
nected regions of the developing visual system (Rubin et
al., 1999, 2002; Kenzelmann et al., 2008; Leamey et al.,
2008). For instance, teneurin-1 staining is found in the
tectofugal pathway, and teneurin-2 is primarily expressed
in the thalamofugal pathway. In addition, teneurins were
shown to promote neurite outgrowth in vitro (Minet et al.,
1999; Rubin et al., 1999) and in vivo (Leamey et al., 2008),
suggesting an important function for teneurins in axon
guidance and target recognition. Recently, the first verte-
brate teneurin knockout was described (Leamey et al.,
2007). Teneurin-3 regulates eye-specific patterning in the
This article was published online ahead of print in MBC in Press
on July 16, 2008.
Address correspondence to: Ruth Chiquet-Ehrismann (Ruth.Chiquet@
Abbreviations used: BM, basement membrane; DIC, differential in-
terference contrast; L1, first larval stage; L2, second larval stage; L3,
third larval stage; L4, fourth larval stage; Pun, pharynx unattached;
SGP, somatic gonad precursor cells.
3898© 2008 by The American Society for Cell Biology
visual system, and the knockout mice show impaired
Beside prominent expression in the nervous system, te-
neurins are also found in nonneuronal tissues. They are
expressed in alternating parasegments in the fly embryo, as
well as in cardiac cells, muscle attachment sites, and the
tracheal system in Drosophila (Baumgartner and Chiquet-
Ehrismann, 1993; Baumgartner et al., 1994). In the chicken
teneurins are found in limb buds, branchial arches, and
somites (Tucker et al., 2000, 2001), and in C. elegans ten-1 is
expressed in gonadal somatic cells, pharynx, and muscles
(Drabikowski et al., 2005). Teneurin expression in each of
these tissues is often associated with pattern formation and
The in vivo function of teneurins is mainly inferred from
studies of C. elegans and Drosophila mutants. Mutation of the
fly ten-m gene causes embryonic lethality due to the fusion
of adjacent denticle belts (Baumgartner et al., 1994; Levine et
al., 1994). Moreover, defects in the ventral nerve cord, car-
diac cells and eye patterning are found in late ten-m mutant
embryos (Levine et al., 1994; Kinel-Tahan et al., 2007). Similar
defects in cuticle and eye development have been observed
for the second Drosophila teneurin gene, ten-a (Rakovitsky et
al., 2007). In C. elegans, deletion in the ten-1 gene causes a
pleiotropic phenotype, including gonad disorganization,
nerve cord defasciculation, and defects in distal tip cell
migration and axonal pathfinding (Drabikowski et al., 2005).
The single teneurin ortholog in C. elegans, ten-1, is under
control of alternative promoters giving rise to two protein
variants. The isoforms differ only in their intracellular do-
mains. Their expression patterns are complex but mostly
nonoverlapping: TEN-1 long (TEN-1L) is found mainly in
the mesoderm, including pharynx, somatic gonad, and var-
ious muscles and neurons, and TEN-1 short (TEN-1S) is
predominantly expressed in some hypodermal cells and in a
subset of neurons (Drabikowski et al., 2005).
We report here the role of TEN-1 in gonadal basement
membrane maintenance, as well as in epidermal and pha-
ryngeal development. Mutation of the ten-1 gene leads to
gonad rupture and sterility. Germ cell leakage from the
gonads has also been reported for basement membrane mu-
tants, e.g., integrin ? ina-1, dystroglycan dgn-1, and laminin
?B epi-1 (Baum and Garriga, 1997; Huang et al., 2003; John-
son et al., 2006). Furthermore, the genetic interactions be-
tween ten-1, ina-1, dgn-1, epi-1, and nid-1 suggest that te-
neurin, integrin, and dystroglycan have related and partly
redundant functions in C. elegans development.
MATERIALS AND METHODS
General Methods and C. elegans Strains
C. elegans strains were maintained at 20°C as described (Brenner, 1974). The
following strains were used in this study: wild-type N2, variety Bristol,
CH120: cle-1(cg120) I, CB444: unc-52(e444) II, VC518: ten-1(ok641) III; TM0651:
ten-1(tm651) III; NG39: ina-1(gm39) III; NG144: ina-1(gm144) III; CB189: unc-
32(e189) III; CX2914: nDf16/dpy-17(e164) unc-32(e189) III; CH119: nid-1(cg119)
V; CH121: dgn-1(cg121)/dpy-6(e14) unc-115(mn481) X. The tm651 deletion re-
moves nucleotides R13F6: 3661-4550 of the ten-1 coding sequence.
The following GFP marker strains were used: RU7: kdEx7 [ten-1a::gfp];
RU97: ten-1(ok641) kdEx45 [F36A3, III]; JK2049: qIs19 [lag-2::gfp]; SS0747: bnIs1
[pie-1::GFP::PGL-1] (gift of Susan Strome, University of California, Santa Cruz,
CA); IM253: urEx131 [lam-1::gfp] (gift of William Wadsworth, Robert Wood
Johnson Medical School, Piscataway, NJ), CH1878: dgn-2(ok209) dgn-3(tm1092)
dgn-1(cg121); cgEx308 [DGN-1::GFP] (gift of James Kramer, Northwestern
University Medical School, Chicago, IL).
Double mutant worms were maintained as [ten-1(ok641);ina-1(gm144);
kdEx45], [ten-1(ok641/?);nid-1(cg119)], [ten-1(ok641);dgn-1(cg121/?); kdEx45] or
[ten-1(ok641/?);dgn-1(cg121); cgEx308] strains and genotyped by PCR for the
Constructs and Plasmids
The translational Pten-1a::GFP::TEN-1L minigene reporter construct was gen-
erated by cloning SpeI-HindIII cDNA fragment and HindIII-XhoI genomic
fragment of TEN-1 long variant into p123T vector (Mo Bi Tec, Goettingen,
Germany). The following restriction sites were introduced into the primers:
SpeI and XhoI flanking the ten-1 coding sequence, SacII at the 5? end of the
ten-1a promoter, and ApaI downstream of the 3? UTR.
The long intracellular domain, transmembrane domain, and a short frag-
ment of the extracellular part were amplified using 5?-AACAGTCTAC-
CGAATCCCAACC-3? and 5?-ATAACTAGTATGTTCCAGCACAGGTAAA-
CTACCACG-3? primers and cDNA from mixed stage N2 worms as a tem-
plate. For the extracellular domain of ten-1 we used 5?-GCTGAAATAC-
CCACTCGCCAGC-3? and 5?-ATCTCGAGCTATTCAGATTTTCGGAACT-
TCC-3? primers and R06H12 cosmid as a template. The sequence encoding
green fluorescent protein (GFP) was amplified from pPD117.01 vector and
its NcoI site was mutated to CCTTGG. GFP was fused by PCR to the
N-terminus of the ten-1 cDNA fragment, which was cloned into SpeI-NcoI
sites of ten-1 minigene. Hemagglutinin (HA) tag was added at the C-
terminus of ten-1 coding sequence by PCR and cloned into HpaI-XhoI sites.
The Pten-1a::GFP::TEN-1L construct contained 4235 base pairs of the ten-1a
promoter and a 512-base pair sequence downstream of the stop codon. PCR
fragments were generated with Pfu Turbo DNA polymerase (Stratagene,
La Jolla, CA).
Transgenic lines were generated as previously described (Mello et al., 1991).
The Pten-1a::GFP::TEN-1L plasmid was injected into ten-1(ok641) mutant
worms. Injections of GFP::TEN-1 minigene at low concentration (5 ng/?l)
resulted in a very weak GFP fluorescence, mainly in the nervous system.
Therefore, we injected the worms with high concentrations of the transgene
(40 ng/?l) and obtained several lines giving stronger GFP fluorescence. We
used pRF4 [rol-6] as a coinjection marker. This resulted in the line RU152:
kdEx121 [Pten-1a::GFP::TEN-1L] used in this study.
RNA-mediated interference (RNAi) was performed as described (Kamath and
Ahringer, 2003). The K08C7.3 RNAi clone was obtained from the Ahringer
feeding library. Wild-type and ten-1(ok641) synchronized L4 hermaphrodites
were placed on RNAi plates and grown at 15°C for 72 h. Single adult worms
were placed on fresh RNAi plates and allowed to lay eggs for 24 h. These
plates were examined for 3 d to determine embryonic lethality and postem-
Immunostaining of C. elegans Larvae
C. elegans larvae were prepared as previously described (Finney and Ruvkun,
1990). Fixed animals were blocked overnight at 4°C in PBS containing 0.1%
Triton X-100 (Triton) and 10% goat serum. Samples were incubated with an
antibody against collagen IV LET-2 (NW68, kind gift of James Kramer)
overnight at 4°C, washed in PBS containing Triton, and incubated with
fluorescein conjugated goat anti-rabbit secondary antibody overnight at room
temperature. Finally, fixed larvae were washed in PBS containing Triton and
Hoechst, followed by PBS alone.
Worms were washed in M9 and anesthetized in 8% ethanol in M9 for 5 min.
They were placed in a fixative (2.5% glutaraldehyde, 1% paraformaldehyde in
0.1M sucrose, and 10 mM PBS, pH 7.4), cut open with a needle at both anterior
and posterior ends, and fixed for 2 h. Worms were embedded in 2% agarose,
cut into small blocks, and washed three times in PBS. Subsequently, pieces
were fixed with a second solution (1% osmium tetroxide, 1.5% potassium
ferrocyanide in PBS) for 2 h and washed three times in water. Worms were
stained with 1% uranyl acetate for 1 h. Samples were dehydrated in ethanol
(10 min in 50% ethanol, 10 min in 70% ethanol, 10 min in 90% ethanol, and 10
min in 100% ethanol) and acetone (10 min). Blocks with worms were embed-
ded in Epon resin (Fluka, Buchs, Switzerland): first in Epon-acetone (1:1) for
1–2 h and then in pure resin for 2–4 h. Samples polymerized for 24–48 h at
60°C and in 60-nm sections were prepared with Ultracut E. Sections were
stained in uranyl acetate for 60 min and then 2 min in Millonig’s lead acetate
stain. Pictures were taken on Philips Morgagni 80 KV microscope (Eindhoven,
Young adult hermaphrodites were placed on separate plates and allowed to
lay eggs for 24 h. The progeny were analyzed for embryonic and postembry-
onic phenotypes: lethality, larval arrest, sterility, and bursting at the vulva.
TEN-1 Function in Basement Membranes
Vol. 19, September 20083899
Drosophila ten(m) /odd Oz gene, in the central nervous system. Mech. Dev. 87,
Miner, J. H., and Yurchenco, P. D. (2004). Laminin functions in tissue mor-
phogenesis. Annu. Rev. Cell Dev. Biol. 20, 255–284.
Minet, A. D., and Chiquet-Ehrismann, R. (2000). Phylogenetic analysis of
teneurin genes and comparison to the rearrangement hot spot elements of E.
coli. Gene 257, 87–97.
Minet, A. D., Rubin, B. P., Tucker, R. P., Baumgartner, S., and Chiquet-
Ehrismann, R. (1999). Teneurin-1, a vertebrate homologue of the Drosophila
pair-rule gene ten-m, is a neuronal protein with a novel type of heparin-
binding domain. J. Cell Sci. 112, 2019–2032.
Muriel, J. M., Dong, C., Hutter, H., and Vogel, B. E. (2005). Fibulin-1C and
Fibulin-1D splice variants have distinct functions and assemble in a hemicen-
tin-dependent manner. Development 132, 4223–4234.
Oohashi, T., Zhou, X. H., Feng, K., Richter, B., Morgelin, M., Perez, M. T., Su,
W. D., Chiquet-Ehrismann, R., Rauch, U., and Fassler, R. (1999). Mouse
ten-m/Odz is a new family of dimeric type II transmembrane proteins ex-
pressed in many tissues. J. Cell Biol. 145, 563–577.
Otaki, J. M., and Firestein, S. (1999). Neurestin: putative transmembrane
molecule implicated in neuronal development. Dev. Biol. 212, 165–181.
Previtali, S. C., Dina, G., Nodari, A., Fasolini, M., Wrabetz, L., Mayer, U.,
Feltri, M. L., and Quattrini, A. (2003). Schwann cells synthesize alpha7beta1
integrin which is dispensable for peripheral nerve development and myeli-
nation. Mol. Cell. Neurosci. 23, 210–218.
Rakovitsky, N., Buganim, Y., Swissa, T., Kinel-Tahan, Y., Brenner, S., Cohen,
M. A., Levine, A., and Wides, R. (2007). Drosophila Ten-a is a maternal
pair-rule and patterning gene. Mech. Dev. 124, 911–924.
Rubin, B. P., Tucker, R. P., Brown-Luedi, M., Martin, D., and Chiquet-Ehris-
mann, R. (2002). Teneurin 2 is expressed by the neurons of the thalamofugal
visual system in situ and promotes homophilic cell-cell adhesion in vitro.
Development 129, 4697–4705.
Rubin, B. P., Tucker, R. P., Martin, D., and Chiquet-Ehrismann, R. (1999).
Teneurins: a novel family of neuronal cell surface proteins in vertebrates,
homologous to the Drosophila pair-rule gene product Ten-m. Dev. Biol. 216,
Tucker, R. P., and Chiquet-Ehrismann, R. (2006). Teneurins: a conserved
family of transmembrane proteins involved in intercellular signaling during
development. Dev. Biol. 290, 237–245.
Tucker, R. P., Chiquet-Ehrismann, R., Chevron, M. P., Martin, D., Hall, R. J.,
and Rubin, B. P. (2001). Teneurin-2 is expressed in tissues that regulate limb
and somite pattern formation and is induced in vitro and in situ by FGF8.
Dev. Dyn. 220, 27–39.
Tucker, R. P., Kenzelmann, D., Trzebiatowska, A., and Chiquet-Ehrismann, R.
(2007). Teneurins: transmembrane proteins with fundamental roles in devel-
opment. Int. J. Biochem. Cell Biol. 39, 292–297.
Tucker, R. P., Martin, D., Kos, R., and Chiquet-Ehrismann, R. (2000). The
expression of teneurin-4 in the avian embryo. Mech. Dev. 98, 187–191.
Zhou, X. H., Brandau, O., Feng, K., Oohashi, T., Ninomiya, Y., Rauch, U., and
Fassler, R. (2003). The murine Ten-m/Odz genes show distinct but overlap-
ping expression patterns during development and in adult brain. Gene Expr.
Patterns 3, 397–405.
A. Trzebiatowska et al.
Molecular Biology of the Cell3908