Opposing Activities of LIT-1/NLK and DAF-6/Patched-
Related Direct Sensory Compartment Morphogenesis in
Grigorios Oikonomou1, Elliot A. Perens1, Yun Lu1, Shigeki Watanabe2, Erik M. Jorgensen2, Shai
1Laboratory of Developmental Genetics, The Rockefeller University, New York, New York, United States of America, 2Howard Hughes Medical Institute, Department of
Biology, University of Utah, Salt Lake City, Utah, United States of America
Glial cells surround neuronal endings to create enclosed compartments required for neuronal function. This architecture is
seen at excitatory synapses and at sensory neuron receptive endings. Despite the prevalence and importance of these
compartments, how they form is not known. We used the main sensory organ of C. elegans, the amphid, to investigate this
issue. daf-6/Patched-related is a glia-expressed gene previously implicated in amphid sensory compartment morphogenesis.
By comparing time series of electron-microscopy (EM) reconstructions of wild-type and daf-6 mutant embryos, we show
that daf-6 acts to restrict compartment size. From a genetic screen, we found that mutations in the gene lit-1/Nemo-like
kinase (NLK) suppress daf-6. EM and genetic studies demonstrate that lit-1 acts within glia, in counterbalance to daf-6, to
promote sensory compartment expansion. Although LIT-1 has been shown to regulate Wnt signaling, our genetic studies
demonstrate a novel, Wnt-independent role for LIT-1 in sensory compartment size control. The LIT-1 activator MOM-4/TAK1
is also important for compartment morphogenesis and both proteins line the glial sensory compartment. LIT-1
compartment localization is important for its function and requires neuronal signals. Furthermore, the conserved LIT-1 C-
terminus is necessary and sufficient for this localization. Two-hybrid and co-immunoprecipitation studies demonstrate that
the LIT-1 C-terminus binds both actin and the Wiskott-Aldrich syndrome protein (WASP), an actin regulator. We use
fluorescence light microscopy and fluorescence EM methodology to show that actin is highly enriched around the amphid
sensory compartment. Finally, our genetic studies demonstrate that WASP is important for compartment expansion and
functions in the same pathway as LIT-1. The studies presented here uncover a novel, Wnt-independent role for the
conserved Nemo-like kinase LIT-1 in controlling cell morphogenesis in conjunction with the actin cytoskeleton. Our results
suggest that the opposing daf-6 and lit-1 glial pathways act together to control sensory compartment size.
Citation: Oikonomou G, Perens EA, Lu Y, Watanabe S, Jorgensen EM, et al. (2011) Opposing Activities of LIT-1/NLK and DAF-6/Patched-Related Direct Sensory
Compartment Morphogenesis in C. elegans. PLoS Biol 9(8): e1001121. doi:10.1371/journal.pbio.1001121
Academic Editor: Gian Garriga, UC Berkeley, United States of America
Received January 14, 2011; Accepted June 28, 2011; Published August 9, 2011
Copyright: ? 2011 Oikonomou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by NIH grants 1R01HD052677, 1R01NS073121, and 5R01NS064273 to SS, and NIH (NS034307) and NSF (0920069) to EMJ. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: Dyf, dye-filling defective; EM, electron microscopy; EMS, ethyl methanesulfonate; fEM, Fluorescence Electron Microscopy; GFAP, glial fibrillary
acidic protein; NLK, Nemo-like kinase; NLS-RFP, nuclearly localized dsRed fluorescent protein; PALM, photo-activated localization microscopy; SDS, sodium-
dodecylsulfate; SNP, single nucleotide polymorphism; WASP, Wiskott-Aldrich syndrome protein; WIP, WASP interacting protein.
* E-mail: firstname.lastname@example.org
Sensory organs are the gates through which information flows
into the nervous system. In many sensory organs, specialized glial
cells form a chemically isolated compartment around neuronal
receptive endings [1,2]. For example, in the skin, the mechano-
sensory Pacinian corpuscles consist of an unmyelinated nerve
ending that is surrounded by lamellae formed by a modified
Schwann glial cell . In the olfactory epithelium, sensory neurons
are ensheathed by glia-like sustentacular cells [4,5]. In the inner
ear, hair cells are surrounded by Deiter’s cells, which express the
glial marker glial fibrillary acidic protein (GFAP) ; and in the
vertebrate eye, retinal pigmented epithelial cells contact photore-
ceptor cell cilia . At least in some cases, the integrity of the glial
compartment is essential for proper sensory neuron function .
Glial compartments also enclose excitatory neuronal synapses in
the cerebellum and hippocampus [9,10], and are thought to be
important for synaptic function through limiting neurotransmitter
diffusion, and regulating levels of synaptic effectors. Despite the
prevalence of such glial compartments, little is known about their
To determine how such compartments form, we turned to the
major sense organ of the nematode Caenorhabditis elegans, the
amphid. C. elegans has two bilaterally symmetric amphids located
in the head . Each amphid consists of 12 sensory neurons,
which mediate many of the behavioral responses of the animal,
and two glial cells, the sheath and socket glia (Figure 1A, top).
Amphid neurons are bipolar, projecting an axon into the nerve
ring (the main neuropil of the animal) and extending a dendrite
anteriorly to the tip of the nose. The two amphid glia also extend
anterior processes collateral to the dendrites. At the nose tip,
sheath and socket glia form discrete single-cell tubular channels
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joined by adherens junctions (Figure 1A bottom). The resulting
two-cell channel compartment is open to the environment
anteriorly and surrounds and isolates the ciliated endings of
specific amphid sensory neurons. The socket portion of the
channel is lined with cuticle and serves as a conduit for cilia to
sample the animal’s environment . The sheath glial cell,
however, is an active secretory cell , releasing extracellular
matrix proteins, required for sensory neuron function, into the
sheath glia channel .
Previous studies demonstrated that the morphogenesis of this
compartment depends on the Patched-related gene daf-6 [12–14],
which acts within glia [14,15]. Although the primary defects in daf-
6 mutants were not characterized, these studies demonstrated that
glial compartment formation employs mechanisms shared with the
genesis of other tubular structures in the animal, including the
vulva and excretory system . Similarly, the C. elegans
Dispatched-related protein CHE-14 seems to play important roles
in the formation of the amphid sensory compartment and other
tubular organs [14,16].
Here we demonstrate a primary function for daf-6 in restricting
sensory compartment size and show that the conserved MAP kinase
LIT-1/NLK acts in counterbalance to DAF-6 to promote compart-
ment expansion. Although LIT-1 is an important component of the
Wnt signaling pathway in C. elegans , our studies argue against a
role for Wnt in compartment size control. However, the previously
characterized LIT-1 activator MOM-4/TAK1 is important for
amphid sensory compartment morphogenesis. LIT-1 and MOM-4
co-localize to the amphid sensory compartment, and LIT-1
localization requires its highly conserved carboxy-terminal region.
actin and with the Wiskott-Aldrich syndrome protein (WASP), a
regulator of actin polymerization . Actin is highly enriched
around the amphid pocket, and WASP appears to act in the same
pathway as LIT-1 to influence compartment morphogenesis.
Our studies reveal two opposing activities, one mediated by
DAF-6, the other by LIT-1, which, together with glial cytoskeletal
proteins, drive sensory compartment morphogenesis.
daf-6/Patched-Related Inhibits Amphid Sensory Channel
The amphid sheath glial cell forms a compartment that
surrounds the ciliated endings of amphid sensory neurons,
constraining them into a tight bundle (Figure 1A–C). Within this
bundle, 10 sensory cilia are stereotypically arranged in three
successive columns containing 3, 4, and 3 cilia, respectively
(Figure 1C; ). We previously reported the cloning and
characterization of daf-6, a Patched-related gene required for
amphid channel morphogenesis . In daf-6 mutant adults, the
amphid channel is grossly enlarged, the socket and sheath glia
channels are not continuous, and distal portions of sensory cilia are
neither bundled nor exposed to the environment (Figure 1D and
At least two interpretations of this phenotype are possible: First,
daf-6 might act to open the sheath glia channel at its anterior end.
Thus in daf-6 mutants, the channel pocket would form but would
remain sealed, and would continuously enlarge as matrix material
is deposited. Second, daf-6 might act to constrain the luminal
diameter of the sheath glia channel. Thus, in daf-6 mutants, the
sheath and socket glia would properly align and form an open
compartment, yet without lateral constraints on its size, the sheath
channel would expand circumferentially. In this latter model, loss
of the sheath-socket junction would be a later secondary defect.
To discriminate between these possibilities, we used electron
microscopy (EM) to follow the development of amphid sensory
compartments in wild-type and daf-6(e1377) mutant embryos. We
used high-pressure freezing to fix embryos at several time points
between 300 and 450 min post-fertilization, the time period
during which the amphid is generated , collected serial
sections, and assessed channel morphology.
By 380 min, sensory dendrites that have not yet formed cilia are
evident in wild-type embryos. The tips of these dendrites are
laterally ensheathed by the sheath glial cell, but the sheath cell also
forms a cap blocking the anterior portion of the compartment and
preventing access of neuronal processes to the socket (Figure S1).
By 400 min, a well-defined amphid primordium is formed in
wild-type embryos (Figures 1F and S1). The sheath glia cap is gone
and the open channel is continuous with the socket glia channel.
At this stage, the socket channel is devoid of neuronal processes as
dendritic tips have yet to extend cilia. Instead, a dense
arrangement of filaments traverses the socket channel and forms
a link between the tips of the sensory dendrites and the outside of
the embryo (asterisk in Figure 1F). These filaments are consistent
with an extracellular matrix proposed to anchor dendrites during
retrograde extension . Although cilia have not yet formed,
structures resembling basal bodies (the initial sites of cilia
construction) are visible at dendrite endings (arrow in Figure 1F).
In daf-6 mutant embryos, the initial stages of amphid
development are unperturbed (n=3). By 400 min, the sheath
and socket channels are aligned and open. Dendrites lacking cilia,
but containing basal body-like structures, reside within the sheath
channel, while filaments emanating from the dendrite tips and
traversing the sheath and socket channels are seen (Figure 1H).
However, only slightly later, at 420 min and before cilia have
formed, bloating of the amphid sheath channel is apparent, and
dendrites begin to unbundle (Figure 1I, compare to Figure 1G).
These studies indicate that daf-6 is not required for aligning the
sheath and socket channels or for opening the amphid sensory
compartment. Rather, daf-6 seems to function in restricting
The nervous system of most animals consists of two
related cell types, neurons and glia. A striking property of
glia is their ability to ensheath neuronal cells, which can
help increase the efficiency of synaptic communication
between neurons. Sensory neuron receptive endings in
the periphery, as well as excitatory synapses in the central
nervous system, often lie within specialized compartments
formed by glial processes. Despite the prevalence of these
compartments, and their importance for neuronal function
and signal transmission, little is known about how they
form. We have used the amphid, the main sensory organ
of the worm Caenorhabditis elegans, to investigate glial
sensory compartment morphogenesis. We demonstrate
that the glia-expressed gene daf-6/Patched-related acts to
restrict the size of the sensory compartment, while the
Nemo-like kinase lit-1 acts within glia in the opposite
direction, to promote sensory compartment expansion.
We show that LIT-1 localizes to the sensory compartment
through a highly conserved domain. This domain can
interact both with actin, which outlines the compartment,
and with the regulator of actin polymerization WASP,
which acts in the same pathway as lit-1. We postulate that
Nemo-like kinases could have broader roles as regulators
of cellular morphogenesis, in addition to their traditional
role in regulating the Wnt signaling pathway.
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Figure 1. daf-6 restricts amphid sensory compartment size. In longitudinal sections and diagrams (A, B, D, F, and H) anterior is left. White scale
bars, 10 mm. Black scale bars, 1 mm. (A) Schematic of the C. elegans amphid. Top: Each amphid consists of 12 neurons (only one is depicted here) and
two glial cells, the sheath and the socket. Bottom: Detail of the anterior tip of the amphid. Matrix is secreted by the Golgi apparatus. tj, tight junction.
Adapted from . (B, D) The ASER neuron and the amphid sheath glia visualized, respectively, with mCherry (red; driven by the gcy-5 promoter) and
GFP (green; driven by the T02B11.3 amphid sheath promoter  in a wild-type (B) or daf-6(e1377) (D) animal (transgenes nsEx2766 and nsEx2752,
respectively)). The ASER neuron extends a single cilium through the length of the amphid channel in the wild type (arrow). In the mutant, the cilium is
bent and not exposed to the environment, and the amphid pocket is bloated (asterisk). (C, E) Electron micrograph of a cross-section through the
anterior portion of the amphid sheath glia channel in an adult wild-type animal (C) or a daf-6(e1377) adult mutant (E). Arrow in (C), sensory cilium. Red
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Loss of lit-1/NLK Restores Amphid Sensory Compartment
Morphology and Function to daf-6 Mutants
The abnormal expansion of the amphid sensory compartment
in daf-6 mutants suggests that active processes promote compart-
ment expansion and that these processes are balanced by daf-6
activity during development. We surmised that mutations in genes
promoting compartment expansion might, therefore, counteract
the loss of daf-6 and restore compartment size and function.
To identify such genes, we screened for mutants able to
generate a normal compartment in the absence of daf-6 function,
taking advantage of an easily scored daf-6 mutant defect: the
inability to form dauer larvae. Dauer is an alternative develop-
mental state induced by starvation and perception of high
concentration of dauer pheromone. Dauer animals are highly
resistant to environmental insults and can survive in the presence
of 1% sodium-dodecylsulfate (SDS) . daf-6 mutants fail to
become dauer larvae, presumably due to their sensory deficits
, and are thus killed by exposure to SDS. We therefore
randomly mutagenized animals homozygous for the strong loss-of-
function daf-6(e1377) allele  using ethyl methanesulfonate
(EMS), allowed F2 animals to starve, and treated them with SDS.
Resistant animals could have suppressed the daf-6 amphid sensory
compartment defects or could have constitutively activated a more
downstream step in dauer formation. To distinguish between these
mutant classes, we examined the ability of amphid sensory neurons
to fill with dye provided in the medium. When exposed to a
solution of the lipophilic dye DiI, wild-type animals readily take up
the dye into exposed amphid neurons. daf-6 animals fail to do so,
presumably due to their defective amphid sensory compartments
(Figure S2A–C) [13,23].
From a screen of 60,000 mutagenized genomes we identified
seven mutants that survived SDS treatment and that dye filled
properly. We further characterized one of these daf-6 suppressors,
given the allele designation ns132. As shown in Figure 2A,
approximately 40% of ns132; daf-6(e1377) animals are able to take
up dye in at least one amphid. Likewise, the ns132 allele was able
to suppress amphid channel defects in another daf-6 mutant,
n1543, supporting the notion that ns132 is a bypass suppressor
To further confirm the rescue of the daf-6 amphid defects in
ns132; daf-6(e1377) animals, we examined amphid sensory
compartments using fluorescence microscopy. We found that cilia
in these double mutants projected through a compartment of
normal appearance (Figure 2B, compare to Figure 1D). In
addition, ns132; daf-6(e1377) individuals that displayed normal
dye filling in one of the two amphids had one amphid channel that
resembled a wild-type channel by EM serial reconstruction
(Figure 2C; n=3). Interestingly, even in rescued amphids, cilia
packing was more variable compared to the regular 3:4:3 packing
observed in wild-type animals, and the amphid sensory compart-
ment was somewhat wider than normal (Figure 2C, compare to
Figure 1C), perhaps reflecting a partial suppression of the daf-6
We used single nucleotide polymorphism (SNP) mapping and
transgenic rescue methods (Figure S2D) to identify the gene
defective in ns132 animals as lit-1. lit-1 encodes a Ser/Thr MAP
kinase that is highly conserved from C. elegans to mammals.
Supporting this assignment, a genomic region containing lit-1
(Figures 2A and S2E), as did a transgene in which the lit-1
promoter region (2.5 kb upstream of the lit-1 start codon) drives
the lit-1 cDNA (Figure 2A). Furthermore, a temperature-sensitive
mutation in lit-1, t1512, also suppressed the dye-filling defects of
daf-6(n1543) mutants (Figure 2A). Finally, we found that animals
containing the ns132 allele have a C-to-T mutation in the coding
region of lit-1, converting codon 437, encoding glutamine, to a
stop codon. This mutation is predicted to result in a truncated
LIT-1 protein (Figure 2D) lacking the last 26 amino acids of the
highly conserved carboxy-terminal (C-terminal) domain.
LIT-1 Functions in Amphid Glia During Compartment
To determine in which cells lit-1 functions to regulate
compartment development, we first examined its expression
pattern by generating animals harboring a transgene in which
the lit-1 promoter drives expression of a nuclearly localized dsRed
fluorescent protein (NLS-RFP). We found that lit-1 is expressed in
amphid sheath glia (Figure 3A), among other cells. In addition, the
expression pattern of this reporter partially overlaps with that of
ptr-10 (Figure 3B), a gene expressed in ensheathing glia of other
sensory organs , suggesting that lit-1 could act in compartment
formation in other C. elegans sensory structures as well.
Next, we pursued cell-specific rescue experiments to determine
in which cells lit-1 can act to regulate compartment morphogen-
esis. We generated lit-1(ns132); daf-6(e1377) animals containing a
transgene in which a lin-26 promoter fragment drives expression of
the lit-1 cDNA in glia, but not neurons, of embryos at the time of
amphid sensory compartment formation . We found that
transgenic animals were rescued (Figure 3C), supporting the
notion that lit-1 can act in glia to regulate compartment
morphology. Importantly, expression of the lit-1 cDNA in amphid
sensory neurons during the time of amphid morphogenesis (using
the dyf-7 promoter; ) failed to rescue lit-1(ns132); daf-6(e1377)
animals (Figure 3C).
To determine whether lit-1 can control amphid sensory
compartment structure after compartment formation is complete,
we examined lit-1(ns132); daf-6(e1377) animals expressing the lit-1
cDNA under the control of the sheath glia-specific vap-1 promoter.
vap-1 expression begins in late embryos , after the compart-
ment has formed. We found that these transgenic animals were not
rescued (Figure 3C), supporting the conclusion that lit-1 is required
within amphid sheath glia at the time of amphid morphogenesis to
influence compartment formation.
Finally, to ascertain whether the kinase activity of LIT-1 is
required, we generated a mutant lit-1 cDNA that disrupts the ATP
binding domain (VALKK to VALGK) and which has been shown
to eliminate LIT-1 kinase activity in vitro . lit-1(ns132); daf-
6(e1377) animals carrying a lin-26 promoter::LIT-1(K97G) cDNA
transgene still displayed 30% dye filling, similar to controls,
suggesting that LIT-1 kinase activity is indeed required for glial
compartment morphogenesis (Figure 3C). None of the transgenes
used in Figure 3C had an effect on the dye filling of wild-type
arrowheads indicate subcortical electron dense material. Arrow in (E), bent cilium. Asterisk, bloated sheath glia channel. Note difference in
magnification between (C) and (E). (F, H) Longitudinal section through the amphid primordium of a wild-type (F) or daf-6(e1377) (H) embryo at
approximately 400 min of development. Asterisk, filaments. Arrow, basal body. (G, I) Cross-section through the amphid primordium of a wild-type (G)
or daf-6(e1377) (I) embryo at approximately 420 min of development. Arrow, basal body. Asterisk, bloated channel. Note difference in magnification
between (G) and (I). See also Figure S1.
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lit-1 Promotes Amphid Sensory Compartment Expansion
Since daf-6 normally acts to restrict amphid sensory compart-
ment expansion, the observation that lit-1 mutations suppress daf-6
suggests that lit-1 may normally promote compartment growth.
Consistent with this idea, the lit-1(ns132) allele enhances the dye-
filling defects of che-14(ok193) mutants (Figure 4A). CHE-14
protein is similar to the Drosophila and mammalian protein
Dispatched, and is important for apical secretion and amphid
sensory compartment morphogenesis , suggesting a role in
lumen expansion. The enhancement of che-14 defects by lit-
1(ns132) suggests that both genes may be involved in this process.
To further test the idea that lit-1 promotes compartment
expansion, we examined lit-1(ns132) single mutants for dye-filling
abnormalities; however, no defects were observed (Figure 4B),
suggesting that amphid morphology in these animals may be
normal. However, two observations suggest that ns132 is a weak
Figure 2. Loss of lit-1 suppresses the loss of daf-6. (A) Dye-filling assay for indicated genotypes (n$90). The lit-1(t1512) strain also contained the
unc-32(e189) mutation. unc-32(e189) does not affect dye filling (unpublished data). Error bars, standard error of the mean (SEM). (B) The ASER neuron
and the amphid sheath glia, visualized with mCherry (red) and GFP (green), respectively, in a lit-1(ns132); daf-6(e1377) animal (transgene nsEx2761).
Arrow, ASER cilium. Left is anterior. Scale bar, 10 mm. (C) Electron micrograph of a cross-section through the amphid sheath channel of a lit-1(ns132);
daf-6(e1377) adult animal. Arrow, cilium. Scale bar, 1 mm. (D) Top: Schematic of the LIT-1 protein. Light blue, non-conserved N-terminal domain. Red,
conserved kinase domain. Dark blue, conserved C-terminal domain. Bottom: Alignment of the region truncated in lit-1(ns132) from different species.
See also Figure S2.
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allele of lit-1. First, the ns132 lesion truncates only 26 amino acids
from the C-terminus of the LIT-1 protein and leaves the kinase
domain intact (Figure 2D). Second, null alleles of lit-1 are
embryonic lethal [17,26], whereas ns132 mutants are fully viable.
To examine the consequences of more severe defects in lit-1
function, we turned to animals homozygous for the lit-1(t1512)
temperature-sensitive allele. lit-1(t1512ts) animals grow nearly
normally at 15uC, but exhibit early embryonic lethality at 25uC
. At 20uC, some lit-1(t1512ts) embryos escape lethality and
grow to adulthood. We reasoned that in some of these escapers,
LIT-1 activity could be low enough to allow us to discern defects in
amphid morphogenesis. Indeed, as shown in Figure 4B, nearly
50% of lit-1(t1512ts) adults grown at 20uC exhibit defects in a
sensitized amphid dye-filling assay (this assay was developed to
detect weak defects in dye filling; see Experimental Procedures).
These results suggest that amphid structure, and perhaps
compartment morphogenesis, has been perturbed in these
To assess whether compartment morphology is indeed per-
turbed, we performed serial-section EM on dye-filling defective
adult lit-1(t1512ts) animals raised at 20uC (n=3). Whereas in wild-
type animals a cross-section through the sheath channel
immediately posterior to the socket-sheath boundary (yellow line
in Figure 4C) reveals the stereotypical 3:4:3 arrangement of the 10
channel cilia, in lit-1(t1512ts) mutants (Figure 4D), the amphid
sensory compartment has a smaller diameter and contains fewer
cilia. Fewer cilia are also found in the socket channel in lit-
1(t1512ts) animals (unpublished data). Furthermore, in wild-type
animals, cross-sections roughly 1 mm posterior to the sheath-socket
junction (blue line in Figure 4C) reveal a less packed arrangement
of cilia that are loosely surrounded by the sheath glia membrane;
by contrast, in lit-1(t1512ts) animals the sheath glia is tightly
wrapped around individual cilia (arrowheads in Figure 4D),
consistent with the idea that compartment diameter is reduced.
Importantly, despite the posterior displacement of some cilia in lit-
1(t1512ts) animals, the total number of cilia is normal (blue section
in Figure 4D).
Taken together, the che-14, dye-filling, and EM studies suggest
that lit-1 opposes daf-6 by promoting channel expansion during
Mutation of the MAP Kinase Kinase Kinase mom-4/TAK1
Also Suppresses the Compartment Defects of daf-6
The kinase activity of LIT-1 was previously shown to depend on
MOM-4/TAK1, a MAP kinase kinase kinase. MOM-4 increases
LIT-1 kinase activity in vitro and mutations in mom-4 interact
genetically with mutations in lit-1 during anterior/posterior
polarity establishment in early embryos . We therefore tested
whether mutations in mom-4 could also suppress the dye-filling
defects of daf-6 mutants. While complete loss of mom-4, like loss of
lit-1, leads to early embryonic lethality, some animals homozygous
for a temperature-sensitive allele of mom-4, ne1539ts, can escape
lethality. We found that whereas only 1% of mom-4(ne1539ts); daf-
6(e1377) double-mutant escapers grown at 15uC exhibit suppres-
sion of the daf-6 dye-filling defect, 18% of surviving animals grown
at 20uC can take up dye (p,1026, Chi-squared test; Figure 5A).
This observation suggests that mom-4 acts similarly to lit-1 in
Figure 3. Suppression of daf-6 mutations requires loss of lit-1 in glia. (A) Image of an amphid sheath glial cell body expressing lit-1p::NLS-RFP
(red; in nucleus) and vap-1p::GFP (green) (transgene nsEx2308). Yellow, overlapping expression. Left is anterior. Scale bar, 10 mm. (B) Image of an adult
(head) expressing lit-1p::GFP (green) and ptr-10p::NLS-RFP (red) (transgene nsEx2159). Arrows, cells with overlapping expression. Left is anterior. Scale
bar, 10 mm. (C) Dye-filling assay for indicated genotypes (n$90). None of the transgenes had an effect on the dye filling of wild type animals (n.100,
unpublished data). Error bars, SEM. p value calculated using Chi-squared test.
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Figure 4. LIT-1 is required for amphid sensory compartment morphogenesis. (A, B) Dye filling in animals carrying the indicated mutations
(n$100). Error bars, SEM. In (B) a sensitized dye-filling assay was used (see Experimental Procedures). (C) Left: Schematic of the arrangement of the
cilia (red) and the sheath glial channel (green) in a wild-type adult animal. Not all cilia are depicted. Right: electron micrograph of cross-sections of the
amphid channel. Section outlined in yellow is just below the socket-sheath junction; blue outlined section is approximately one micron posterior.
Scale bars, 1 mm. (D) Same as in (C), but for a dye-filling defective lit-1(t1512) adult animal. The panel arrangement is a reflection of the one in (C).
Arrowheads, tight ensheathment of individual cilia by the sheath glia. Scale bars, 1 mm.
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To test whether mom-4, like lit-1, acts within glia to regulate
amphid morphogenesis, we constructed mom-4(ne1539ts); daf-
6(e1377) double mutants expressing a lin-26 promoter::GFP::-
mom-4 cDNA transgene. When these animals were grown at 20uC,
only 7% filled with dye (Figure 5A), consistent with the hypothesis
that mom-4 acts within glia during early amphid morphogenesis,
similar to lit-1.
To assess whether mom-4 and lit-1 function in the same pathway
to promote channel expansion, we examined dye filling in daf-6
mutants that were also homozygous for both lit-1(ns132) and mom-
4(ne1539ts) alleles. We found that the mom-4; lit-1; daf-6 triple
mutant is viable at both 15uC and 20uC and is not suppressed to a
greater extent than lit-1; daf-6 double mutants at either
temperature (Figure 5A). This result is consistent with the idea
that lit-1 and mom-4 function in the same pathway to control
channel expansion, similar to their established roles in embryonic
The roles of lit-1 and mom-4 in Wnt signaling in C. elegans have
been extensively studied [28,29]. In this context, MOM-4 activates
LIT-1, which then forms a complex with the b-catenin WRM-1.
The LIT-1/WRM-1 complex phosphorylates the C. elegans TCF/
LEF transcription factor POP-1, resulting in reduction (but not
elimination) of POP-1 nuclear levels and activation of transcrip-
tion (Figure 5B) [17,27,30,31]. We therefore examined animals
containing mutations in Wnt signaling components for defects in
dye filling, or for suppression of the daf-6 dye-filling defects.
Surprisingly, mutations in Wnt-encoding genes, the C. elegans
Wntless homolog mig-14, required for Wnt protein secretion, Wnt
receptors, b-catenins, or pop-1/TCF/LEF, the main LIT-1 target
in the Wnt signaling pathway, have no effect on dye filling and
show no, or minimal, suppression of daf-6 (Table S1).
Although we cannot eliminate the possibility that multiple
redundant Wnt pathways contribute to channel formation and
that these operate through LIT-1 targets other than POP-1, the
most parsimonious interpretation of our data is that the MOM-4/
LIT-1 kinase module operates independently of Wnt signaling to
promote expansion of the amphid glial compartment.
LIT-1 and MOM-4 Proteins Localize to the Amphid
To determine where within the amphid sheath glia LIT-1 and
MOM-4 are localized, we generated animals expressing either a
rescuing GFP::MOM-4 or a rescuing GFP::LIT-1 fusion protein
within amphid sheath glia using the T02B11.3 amphid sheath
promoter . Strikingly, we found that both fusion proteins were
tightly associatedwith theamphid
(Figure 6A and 6B).
To determine whether LIT-1 localization requires functional
mom-4, we examined localization of the GFP::LIT-1 fusion protein
in mom-4(ne1539ts) single mutants at 20uC. GFP::LIT-1 was
properly localized in all animals we observed (n=44), suggesting
that LIT-1 localizes to the sheath channel independently of its
The DAF-6 protein is mislocalized in animals lacking
neuronal cilia, accumulating only at the sheath-socket junction
rather than along the length of the sheath glia channel . To
examine whether LIT-1 also requires cilia to properly localize,
we examined animals harboring a loss-of-function mutation in
daf-19, which encodes a transcription factor required for
ciliogenesis. Our previous EM studies demonstrated that, despite
minor defects, a channel of normal length is generated in these
mutants . As shown in Figure 6C, in daf-19 mutants, LIT-1
no longer lines the entire channel, but is restricted to its anterior
aspect. Thus, neuronal signals are required for LIT-1 glial
Figure 5. mom-4/TAK1 mutations suppress the loss of daf-6. (A) Dye filling in animals of the indicated genotypes (n$90). The alleles used are:
daf-6(n1543), lit-1(ns132), mom-4(ne1539). daf-6 is marked with unc-3(e151) in all strains except for mom-4; daf-6. unc-3(e151) does not affect dye filling
(unpublished data). Error bars, SEM. p value calculated using Chi-squared test. (B) Schematic of Wnt signaling during endoderm specification in C.
elegans. In contrast to the LIT-1 MAPK module (red), Wnt signaling does not appear to be involved in amphid sheath channel formation (see text and
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The C-Terminus of LIT-1 Is Necessary and Sufficient for
Amphid Sensory Compartment Localization
The channel localization of LIT-1 raised the possibility that in
lit-1(ns132) mutants, LIT-1 localization might be disrupted. To test
this, we expressed GFP-tagged LIT-1(Q437Stop) (the mutation
corresponding to ns132) in wild-type animals and examined its
localization. While GFP::LIT-1 reproducibly lines the amphid
sensory compartment, GFP::LIT-1(Q437Stop) fails to localize in
about one-third of animals and is instead diffusely distributed
throughout the cell (Figure 6D and 6G). This result suggests that
the highly conserved C-terminal region of LIT-1 may be required
for compartment localization. In addition, the fraction of animals
in which GFP::LIT-1(Q437Stop) is mislocalized (31%, Figure 6G)
mirrors the fraction of daf-6 mutants suppressed by the lit-1(ns132)
allele (Figure 2A), raising the possibility that mislocalization may
account for the suppression we observed.
The observation that GFP::LIT-1(Q437Stop) still localizes to
the amphid channel in some animals raised the possibility that the
C-terminal 26 amino acids may represent only a portion of the full
targeting domain. To test this idea, we generated animals
expressing a GFP::LIT-1DCt fusion protein in which all sequences
downstream of the kinase domain are deleted. We found that in
these animals LIT-1 never accumulated at the amphid sensory
compartment, and was diffusely distributed throughout the cell
(Figure 6E and 6G), demonstrating that the C-terminal domain is
necessary for LIT-1 compartment localization.
To determine whether the C-terminal domain of LIT-1 is
sufficient for channel localization, we generated animals express-
Figure 6. LIT-1 and MOM-4 localize to the amphid sensory compartment. (A–F) Images of adult animals expressing the indicated GFP fusion
proteins. Animals are otherwise wild-type except in (C). daf-19(m86) animals also carried the daf-16(mu86) allele to prevent dauer entry. The T02B11.3
amphid sheath promoter  was used to drive all constructs. Transgenes depicted: nsEx2606 (A), nsEx2840 (B), nsEx2829 (C), nsEx2609 (D), nsEx2747
(E), and nsEx2626 (F). Anterior is to the left. Scale bars, 10 mm. (G) Quantification of channel localization of indicated LIT-1 protein fusions (n$100). See
also Figure S3.
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ing a GFP::LIT-1 C-terminal domain fusion protein. Remarkably,
we found that this fusion protein accumulated at the amphid
sensory compartment in a pattern identical to that of full-length
LIT-1 (Figure 6F and 6G).
Previous work showed that LIT-1 also localizes to the cell
nucleus [30,33,34], and we found this to be the case for amphid
sheath glia as well (Figure S3). However, disruption of the C-
terminal domain of LIT-1 does not result in its exclusion from the
nucleus (Figure S3), suggesting that nuclear functions of LIT-1
may not be abrogated in lit-1(ns132) mutants.
Although the C-terminal domain of LIT-1 is highly conserved
from C. elegans to mammals, its function is not well studied. Our
studies demonstrate that this domain is both necessary and
sufficient for LIT-1 localization to the amphid sensory compart-
ment, and suggest that proper localization is important for LIT-1
function in compartment formation.
ACT-4 Interacts with the C-terminal Domain of LIT-1 and
Is Enriched around the Amphid Sensory Compartment
Because of the importance of the LIT-1 C-terminal domain in
compartment localization, we used this domain as bait in a yeast
two-hybrid screen with the aim of identifying proteins that interact
From a screen of approximately 106clones, we identified 26
positive clones (Table S2, Figure 7A). While some clones were
isolated multiple times, others were found only once, suggesting
that our screen was not saturated. We were intrigued that 4 of the
26 interacting clones identified encoded the C. elegans actin protein
ACT-4. EM studies of the amphid sheath glia channel had
previously shown that the channel is lined by an electron dense
subcortical layer (red arrowheads in Figure 1C) . A similar
layer can be seen in other highly secreting cells such as pancreatic
acinar cells and adrenal chromaffin cells. In these cells, this
electron dense layer has been demonstrated to be enriched in actin
To determine whether ACT-4 might be part of the electron-
dense subcortical layer near the amphid sensory compartment, we
examined animals expressing a GFP::ACT-4 fusion protein in
GFP::ACT-4 was seen throughout the cell, it was highly enriched
at the amphid sensory compartment (Figure 7B). We wondered
whether other actin proteins also accumulate at the channel and,
therefore, generated animals expressing a protein fusion of GFP to
ACT-1. Again, we found increased channel localization (unpub-
lished data), suggesting that actin filaments may be components of
the subcortical density.
To examine the localization pattern of ACT-4 at higher
resolution, we used scanning EM coupled with photo-activated
localization microscopy (PALM). In this method, serial sections are
imaged by scanning EM and using single-molecule fluorescence of
mEos::ACT-4 . Images are then superimposed, using fiduciary
markers (fluorescent gold beads), to reveal the subcellular
localization of fluorescent proteins. As shown in Figure 7C, at
the anterior portion of the amphid channel, where an electron
dense subcortical region has been described, mEos::ACT-4 is
localized near the sensory compartment membrane (blue trace).
mEos::ACT-4 does not localize to the sensory compartment in
more posterior sections (Figure 7D, 2 mm posterior to 7C), which
should lack the subcortical electron density. These observations
support the notion that actin is intimately associated with the glial
sensory compartment and that the subcortical density may be
composed at least in part of actin.
We also found that GFP::ACT-4 was properly localized in lit-
1(ns132) mutants (n=50), suggesting that actin accumulates
around the sensory compartment independently of lit-1, and
consistent with the possibility that actin may recruit LIT-1. To test
this possibility we tried to disturb GFP::ACT-4 localization by
treating the animals with an inhibitor of actin polymerization,
cytochalasin D. After a 2 h incubation with 1 mM of the drug, the
cell bodies of the sheath glia assumed a rounded morphology,
indicative of breakdown of the actin cytoskeleton. However, the
sensory compartment localization of neither GFP::ACT-4 nor
GFP::LIT-1 was disturbed (unpublished data). This result suggests
that the subcortical actin around the amphid channel could be
part of a stable structure with a lower turnover rate than the rest of
the actin cytoskeleton.
Similarly, LIT-1, MOM-4, and ACT-4 all localized to the
sensory compartment in daf-6(n1543) mutants (Figure S4),
suggesting that DAF-6 is not involved in recruiting these proteins.
The Actin Regulator WASP Binds LIT-1 and Is Required for
Sensory Compartment Expansion in daf-6 Mutants
In addition to actin, our two-hybrid studies suggested that the
LIT-1 C-terminal domain can also bind to the proline-rich region
of WSP-1, the C. elegans homolog of the Wiskott-Aldrich Syndrome
Protein (WASP) (Table S2, Figure 7A). Furthermore, we could
immunoprecipitate the LIT-1 C-terminal domain using WSP-1
from cultured Drosophila S2 cells co-expressing both proteins
(Figure 7H), suggesting that LIT-1 and WSP-1 can interact.
Although GFP::WSP-1 expressed in amphid sheath glia is diffusely
localized (unpublished data), co-expression with mCherry::LIT-1
revealed partial co-localization (Figure 7E–G), supporting the
notion that LIT-1 and WSP-1 may interact in vivo.
To determine whether wsp-1 plays a role in amphid morpho-
genesis, we examined wsp-1(gm324) mutants, which, unlike actin
mutants, are viable . We did not find any defects in dye filling
in the single mutant. However, wsp-1(gm324) suppresses the daf-
6(n1543) dye-filling defects (Figure 7I). Furthermore, daf-6 mutants
homozygous for both lit-1(ns132) and wsp-1(gm324) were as dye-
filling defective as lit-1(ns132); daf-6(n1543) mutants alone,
consistent with the hypothesis that LIT-1 and WSP-1 act in the
Interestingly, we found that overexpression of a GFP::LIT-1
fusion protein results in abnormal glial morphology (Figure S5B,
compare to Figure S5A) and distorted sensory compartment
morphology (Figure S5C, compare to Figure 6A). This result,
together with the genetic and physical interactions between LIT-1
and actin and LIT-1 and WASP, are consistent with the possibility
that LIT-1 facilitates glial morphogenesis by regulating actin
lit-1 Regulates the Morphogenesis of a Subcellular
LIT-1 is the C. elegans homolog of Nemo-like kinase (NLK) ,
a Serine/Threonine kinase originally described in Drosophila .
In C. elegans, lit-1 (loss of intestine) was first identified for its role in
endoderm specification during early embryogenesis . Subse-
quent work established lit-1 as a component of the Wnt/b-catenin
asymmetry pathway that directs many cell fate decisions in C.
elegans [28,29]. NLK also plays roles in control of the Wnt [41,42],
TGFb , and Notch  signaling pathways in vertebrates.
Although LIT-1/NLK has been implicated in cell fate
determination, we identified lit-1 mutations as suppressors of
lesions in daf-6, a gene that affects morphogenesis of the amphid
glial sensory compartment, but not glial cell fate. Indeed, lit-1
single mutants seem to have well-specified amphid components.
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Furthermore, despite an established connection between lit-1 and
the Wnt/b-catenin asymmetry pathway (a major regulator of cell
fate decisions in C. elegans), we found no evidence linking Wnt
signaling to amphid morphogenesis (Table S1). These observations
are consistent with the idea that the role of lit-1 in sensory organ
morphogenesis does not involve cell fate decisions, but instead
reflects a novel function in cellular morphogenesis.
Within the context of cell fate decisions, LIT-1/NLK often acts
by impinging upon the activity of nuclear transcription factors
[30,43,44]. It is unclear whether the role of lit-1 in sensory organ
morphogenesis might also involve transcriptional regulation. The
C-terminal domain of LIT-1 is required for its role in amphid
morphogenesis and for its amphid channel localization, but it is
not essential for the ability of LIT-1 to enter the nucleus. This
suggests that LIT-1 may exert its primary influence on channel
morphogenesis at the channel itself. However, LIT-1 C-terminus
can interact not only with cytoskeletal proteins (actin and WASP)
but also with the transcription factors ZTF-16 and MEP-1 (Table
S2). Thus, while it is likely that sensory compartment localization
is important for LIT-1 function, we cannot rule out the possibility
that LIT-1 has independent relevant functions in the nucleus.
Opposing Activities of lit-1 and daf-6 Direct Sensory
Our results suggest that daf-6 and lit-1 direct the morphogenesis
of the sheath glia sensory compartment by exerting opposing
influences. In daf-6 mutants, neurons and glia form an amphid
primordium in which all components are initially linked and
aligned; however, the sensory compartment expands abnormally.
Conversely, in lit-1 mutants, the sensory compartment is too
narrow. Mutations in lit-1 can correct for the loss of daf-6; thus, lit-
1; daf-6 double mutants have relatively normal glial channels. A
situation that mimics lit-1; daf-6 double mutants arises in animals
with mutations in genes controlling neuronal cilia development. In
these animals, channel localization of LIT-1, as well as DAF-6, is
perturbed. Consistent with the lit-1; daf-6 phenotype, channel
formation is only mildly defective in these mutants .
The observation that lit-1 loss-of-function mutations suppress
daf-6 null alleles argues that lit-1 cannot function solely upstream
of daf-6 in a linear pathway leading to channel formation. Our
data, however, are consistent with the possibility that daf-6
functions upstream of lit-1 to inhibit lit-1 activity. Alternatively,
lit-1 and daf-6 may act in parallel. Our studies do not currently
allow us to distinguish between these models.
Vesicles, the Actin Cytoskeleton, and Sensory
How might DAF-6 restrict the size of glial sensory compart-
ments? Electron micrographs of the C. elegans amphid reveal the
presence of highly organized Golgi stacks near the amphid
channel. These images also show vesicles, containing extracellular
matrix, that appear to be released by the sheath glia into the
channel (Figure 1A) . These studies suggest that vesicular
secretion may play a role in channel morphogenesis. Interestingly,
DAF-6 is related to Patched, a protein implicated in endocytosis of
the Hedgehog ligand, and the C. elegans Patched gene ptc-1 is
proposed to regulate vesicle dynamics during germ-cell cytokinesis
. Furthermore, DAF-6 can be seen in punctate structures,
which may be vesicles , and DAF-6 and CHE-14/Dispatched
function together in tubulogenesis [14,16], a process hypothesized
to require specialized vesicular transport. Together these obser-
vations raise the possibility that DAF-6 may restrict amphid
sensory compartment expansion by regulating vesicle dynamics in
the sheath glia .
If indeed DAF-6 controls membrane dynamics, it is possible that
LIT-1, which localizes to and functions at the sheath glia channel,
also interfaces with such processes. How might LIT-1 localize to
the glial sensory compartment and control vesicle dynamics?
Previous studies suggest that cortical localization of LIT-1 requires
it to stably interact with WRM-1/b-catenin [33,34]. In the sheath
glia, however, we found that wrm-1 is not required for sensory
compartment morphogenesis or for LIT-1 localization and that
LIT-1 and WRM-1 do not co-localize to the amphid sensory
compartment (unpublished data). Instead, we found that LIT-1
physically interacts with actin and that actin is highly enriched
around the amphid sensory compartment. Thus, actin might serve
as a docking site for LIT-1. The interaction between LIT-1 and
actin may not be passive. Indeed, we showed that LIT-1 also binds
to WASP, and mutations in wsp-1/WASP suppress daf-6 similarly
to mutations in lit-1. Furthermore, WASP activity is stimulated by
phosphorylation of Serines 483 and 484 , suggesting that LIT-
1, a Ser/Thr kinase, could activate WASP to promote actin
Remodeling of the cortical actin cytoskeleton plays important
roles in several aspects of membrane dynamics . For example,
WASP-dependent actin polymerization has a well-established role
in promoting vesicle assembly during clathrin-mediated endocy-
tosis . Recent work has demonstrated positive roles for actin
polymerization in exocytosis as well [49,50]. In pancreatic acinar
cells, secretory granules become coated with actin prior to
membrane fusion , and in neuroendocrine cells, actin
polymerization driven by WASP stimulates secretion . During
Drosophila myoblast fusion, actin polymerization, dependent on
WASP and WASP interacting protein (WIP), is required for
targeted exocytosis of prefusion vesicles , and antibodies
against WASP inhibit fusion of purified yeast vacuoles . An
attractive possibility, therefore, is that LIT-1 might regulate
sensory compartment morphogenesis by altering vesicle trafficking
through WASP-dependent actin polymerization.
Glial ensheathment is a feature of many animal sensory organs
and synapses, and LIT-1 and WASP are highly conserved,
suggesting that our studies may be broadly relevant. Interestingly,
Figure 7. The actin cytoskeleton is involved in amphid sensory compartment morphogenesis. (A) Growth assay (left) and quantitative b-
galactosidase enzymatic activity assay (right) demonstrating the interaction between LexA fused to the LIT-1 carboxy-terminal domain and GAD
fused to fragments of ACT-4 or WSP-1. Error bars, standard deviation. f, fragment. 2WL, medium without Tryptophan and Leucine. –WLH, medium
without Tryptophan, Leucine, and Histidine. 3AT, 3-amino-1,2,4-triazole. A.U., arbitrary units. (B) Amphid channel localization of GFP::ACT-4 (transgene
nsEx2876). Anterior is to the left. Scale bar, 10 mm. (C, D) fEM (see Experimental Procedures) of a cross-section through the amphid channel (blue
trace) just below the socket-sheath junction (C) or 2 mm posterior (D). White puncta indicate mEos::ACT-4 localization. Transgene used nsEx2970.
Asterisks, cilia. Scale bars, 1 mm. (E–G) Co-localization of GFP::WSP-1 and mCherry::LIT-1 at the amphid sensory compartment (transgene nsEx3245).
The T02B11.3 amphid sheath promoter  was used to drive all constructs. Anterior is to the left. Scale bars, 10 mm. (H) The carboxy-terminal domain
of LIT-1 co-immunoprecipitates with WSP-1. Drosophila S2 cells were transfected with HA::eGFP::LIT-1Ct and with or without MYC::WSP-1. Cell lysates
were immunoprecipitated using anti-MYC-conjugated agarose beads and analyzed by anti-HA immunoblot. (I) Dye filling in animals of the indicated
genotypes (n$90). The alleles used are: daf-6(n1543), lit-1(ns132), wsp-1(gm324). daf-6 is marked with unc-3(e151) in all strains. unc-3(e151) does not
affect dye filling (unpublished data). Error bars, SEM. See also Table S2.
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LIT-1 was recently shown to be required for cell invasion through
basement membranes in C. elegans and in metastatic carcinoma
cells , processes that require extensive remodeling of the actin
cytoskeleton. Our results may, thus, represent a general mecha-
nism for regulating cell shape changes using localized interactions
of LIT-1/NLK with cytoskeletal proteins.
Materials and Methods
Strains, Plasmid Construction, and lit-1 Mapping and
See Supporting Information.
Animals were washed off NGM plates with M9 buffer,
resuspended in a solution of 10 mg/mL of DiI (1,19-dioctadecyl-
D282), and rotated in the dark for 1.5 h at room temperature.
Animals were then transferred to a fresh NGM plate, anaesthe-
tized with 20 mM sodium azide, and observed using a dissecting
microscope equipped with epifluorescence. Animals in which none
of the amphid neurons filled with dye were scored as dye-filling
defective (Dyf). For the sensitized dye-filling assay, 1 mg/mL of DiI
was used, and the incubation time was 15 min. Animals were
scored as dye filling defective (Dyf) if either one or two amphids
failed to fill.
Transmission Electron Microscopy and Fluorescence
Electron Microscopy (fEM)
See Supporting Information and .
Fluorescence Microscopy and Image Analysis
Images were acquired using a DeltaVision Image Restoration
Microscope (Applied Precision) equipped with a 606/NA 1.42
Plan Apo N oil immersion objective (Olympus) and a Photometrics
CoolSnap camera (Roper Scientific), or an Upright Axioplan LSM
510 laser scanning confocal microscope (Zeiss) equipped with a C-
Apochromat 406/NA 1.2 objective. Acquisition, deconvolution,
and analysis of images from the DeltaVision system were
performed with Softworx (Applied Precision); images from the
confocal microscope were acquired and analyzed using LSM 510
Yeast Two-Hybrid Screen
LexA::LIT-1Ct was used as bait in a Y2H screen using the
DUALHhybrid kit (Dualsystems Biotech) in conjunction with the
C. elegans Y2H cDNA library (Dualsystems Biotech), as described
by the manufacturer. For the growth assay, cultures growing on
Synthetic Complete Dextrose –Tryptophan, –Leucine (SCD –WL)
plates were resuspended in water to OD660=0.1. 5 mL of each
culture were seeded on SCD –WL plates and SCD –WL, –
Histidine (H) plates + 1 mM 3AT (3-amino-1,2,4-triazole) to select
for HIS3 expression. b-galactosidase assay was performed using
the yeast b-galactosidase assay kit (Thermo Scientific).
Protein Interaction Studies
Drosophila S2 cells (Invitrogen) cultured at 25uC were transfected
with FuGene HD (Roche), incubated for 3 d, and lysed in 1 mL of
IP buffer (60 mM Tris HCl, pH 8.0, 1% Tergitol type NP-40
(Sigma), 10% glycerol, 16Complete protease inhibitor cocktail
(Roche), 16PhoStop phosphatase inhibitor cocktail (Roche)).
100 mL of lysate was stored on ice as input. Immunoprecipitation
was performed with the remaining lysate for 2 h at 4uC, using goat
anti-myc-conjugated agarose beads (Genetex). Immunoprecipitat-
ed complexes were released from the beads with 100 mL of sample
buffer (same as IP buffer with the addition of 2% sodium
dodecylsulfate (SDS), 0.1 M Dithiothreitol (DTT), and 0.01%
bromophenol blue). Samples were analyzed on NuPage 4%–12%
Bis-Tris gels (Invitrogen). Immunoblotting was performed using
rat monoclonal anti-HA 3F10 coupled to horseradish peroxidase
(HRP) (Roche), 1:2,000; rabbit polyclonal anti-myc (AbCam),
1:5,000; And goat polyclonal anti-rabbit (Pierce) coupled to HRP,
wild-type embryos. Electron micrographs of cross-sections through
the amphid primordium in wild-type animals. Top: At approxi-
mately 380 min after fertilization, the amphid pocket is blocked
anteriorly by a cap formed by the sheath glia (left). More
posteriorly (middle and right), the sheath wraps around the
dendrites of the amphid neurons. Bottom: At approximately
400 min after fertilization, the amphid channel is open, with
filaments (asterisk) visible at the level of the socket (left; arrow
indicates socket self junction). More posteriorly (middle and right),
the sheath glia wraps around the dendrites of the amphid neurons.
Filaments (asterisk) can be seen in the middle section.
Amphid sensory compartment morphogenesis in
cloning. (A–C) Fluorescence images of (A) wt, (B) daf-6(e1377),
and (C) lit-1(ns132); daf-6(e1377) animals after incubation for 1.5 h
in 10 mg/mL of DiI (red). Scale bars, 50 mm. (D) Using SNP
mapping (see Supplemental Materials and Methods, Text S1),
ns132 was mapped to the right end of chromosome III, distal to
the SNP F54F12:17329 at genetic position +20.72. The cosmids
ZK520, ZK525, W96F12, and K08E3 were used for the
construction of transgenic strains (see panel E). (E) Dye-filling in
animals of the indicated genotypes (n$90). The alleles used were
daf-6(e1377) and lit-1(ns132). lit-1 genomic and lit-1(ns132) genomic
correspond to constructs pGO1 and pGO2, respectively (see
Supplemental Materials and Methods, Text S1).
Dye-filling assay and lit-1(ns132) mapping and
disruption of the LIT-1 carboxy-terminal domain. (A–C) Fluores-
cence images of sheath glia cell body and nucleus in animals
transgenic for the indicated GFP::LIT-1 fusion protein. Trans-
genes depicted: nsEx2606 (A), nsEx2609 (B), nsEx2747 (C). Arrow,
cell nucleus. Scale bar, 10 mm. The T02B11.3 promoter was used
to drive all constructs.
Nuclear localization of LIT-1 is not abrogated by
4, and ACT-4 are independent of daf-6. (A–C) Fluorescence
images of adult daf-6(n1543) animals expressing the indicated GFP
fusion proteins. The T02B11.3 amphid sheath promoter was used
to drive all constructs. Trangenes depicted: nsEx2606 (A),
nsEx2840 (B), nsEx2876 (C). Anterior is to the left. Scale bars,
Sensory compartment localization of LIT-1, MOM-
disrupts cellular morphology. (A) Fluorescence projection image of
the sheath glia promoter F16F9.3 driving dsRed (transgene
nsEx3272). (B) Fluorescence projection image of a transgenic
animal carrying a high copy number of the T02B11.3 amphid
sheath promoter driving GFP::LIT-1 (transgene nsEx2619).
Compare the extensive branching of the sheath glia process with
Overexpression of LIT-1 within the sheath glia
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PLoS Biology | www.plosbiology.org 13 August 2011 | Volume 9 | Issue 8 | e1001121
(B). (C) Fluorescence image of the sensory compartment of an Download full-text
animal with the same genotype as the one in (B). Compare with
Figure 6A. Anterior is to the left. Scale bars, 10 mm.
affect amphid morphogenesis.
Components of the Wnt signaling pathway do not
proteins that interact with the carboxy-terminal domain of LIT-1.
Clones identified from a yeast-two-hybrid screen for
Supplemental Materials and Methods.
We thank Craig Mello and Cori Bargmann for strains, Yuji Kohara for
cDNA clones, the Sanger Center for cosmids, Maya Tevlin for sharing
microarray data, Max Heiman for advice on imaging, Catherine
Oikonomou and Fred Cross for help with yeast techniques, and members
of the Shaham lab for comments on the manuscript. Some nematode
strains used in this work were provided by the Caenorhabditis Genetics
Center, which is funded by the NIH National Center for Research
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: GO EAP EMJ
SS. Performed the experiments: GO EAP SW YL. Analyzed the data: GO
EAP SW EMJ SS.
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Shaping Glial Tubes
PLoS Biology | www.plosbiology.org14 August 2011 | Volume 9 | Issue 8 | e1001121