 |
 Previous Article | Next Article 
The Journal of Neuroscience, March 15, 1998, 18(6):2084-2096
Control of Neural Development and Function in a Thermoregulatory
Network by the LIM Homeobox Gene lin-11
Oliver
Hobert,
Tania
D'Alberti,
Yanxia
Liu, and
Gary
Ruvkun
Massachusetts General Hospital, Department of Molecular Biology,
Harvard Medical School, Department of Genetics, Boston, Massachusetts
02114
 |
ABSTRACT |
We show here that the lin-11 LIM homeobox gene is
expressed in nine classes of head, ventral cord, and tail neurons and
functions at a late step in the development of a subset of these
neurons. In a lin-11 null mutant, all
lin-11-expressing neurons are generated. Several of
these neurons, however, exhibit neuroanatomical as well as functional
defects. In the lateral head ganglion, lin-11 functions
in a neural network that regulates thermosensory behavior. It is
expressed in the AIZ interneuron that processes high temperature input
and is required for the function of AIZ in the thermoregulatory neural
network. Another LIM homeobox gene, ttx-3, functions in the antagonistic thermoregulatory interneuron AIY (Hobert et al., 1997 ). Thus, distinct LIM genes specify the functions of functionally related antagonistic interneurons within a neural network dedicated for
thermoregulatory processes. Both ttx-3 and
lin-11 expression are maintained throughout adulthood,
suggesting that these LIM homeobox genes play a role in the functional
maintenance of this neural circuit. We propose that particular LIM
homeobox genes specify the distinct features of functionally related
neurons that generate patterned behaviors.
Key words:
LIM homeobox gene; neurogenesis; thermotaxis; neural
function; axon pathfinding; axon fasciculation
| |
INTRODUCTION |
Genetic analysis of animals showing
behavioral or neuroanatomical defects has identified pathways that
control the generation and functional specification of neurons (Desai
et al., 1988; Ruvkun, 1997). Such studies have revealed sequential
steps to neurogenesis that are mediated by conserved regulatory
factors. Homeobox genes represent a class of regulatory factors that
control various stages of neurogenesis in Caenorhabditis
elegans and other organisms (Chalfie, 1993; Manak and Scott,
1994). For example, the unc-86 POU homeobox gene acts early
in neural development to couple cell lineage to neuroblast identity
(Finney and Ruvkun, 1990). After determination of neuroblast fate in
C. elegans, certain neuroblast migrations are regulated by a
homeobox gene (Salser and Kenyon, 1992). The determination of the
pattern of presynaptic input or neurotransmitter identity is also under
control of homeobox genes (Miller et al., 1992; White et al., 1992; Jin
et al., 1994).
Dedicated neural networks that subserve specific behaviors have been
identified in C. elegans. The cellular components of the
neuronal circuitry evoking the touch and tap responses and the neuronal
circuitry that regulates the response to temperature have been
characterized in detail, revealing the existence of dedicated sensory
neurons, interneurons, and motor neurons (Chalfie et al., 1985; Mori
and Ohshima, 1995; Wicks and Rankin, 1995). A genetic analysis of these
behaviors has identified several regulatory factors that are required
to specify functionally these neural circuits. For example, the
mec-3 LIM homeobox gene is required for the specification of
touch neurons within the touch circuit (Way and Chalfie, 1988), whereas
the ttx-3 LIM homeobox gene acts to control the function of
an interneuron in the thermoregulatory circuit (Hobert et al.,
1997).
LIM homeobox genes represent a particular subclass of homeobox genes
that encode proteins characterized by the presence of a DNA-binding
homeodomain and two LIM domains, each of which bears two Zn-finger-like
motifs (for review, see Dawid et al., 1995). LIM homeobox genes have
been identified across phylogeny and can be grouped into defined
subclasses (Fig. 1). The analysis of LIM homeobox genes in C. elegans and Drosophila has
suggested a critical role for this class of genes in neurogenesis. The
Drosophila apterous and islet genes control axon
pathfinding of particular neurons in the CNS (Lundgren et al., 1995;
Thor and Thomas, 1997). The C. elegans LIM homeobox genes
mec-3 and ttx-3 are each necessary for the
differentiation of particular neuronal cell types; the continuous
expression of these genes throughout adulthood suggests that they are
also required for neural maintenance (Way and Chalfie, 1991; Hobert et
al., 1997). Similarly, two other C. elegans LIM homeobox
genes identified from the genome sequence display a neural restricted
expression pattern that is maintained throughout adulthood (O. Hobert
and G. Ruvkun, unpublished observations).
View larger version (60K):
[in this window]
[in a new window]
|
Figure 1.
Subclasses of LIM homeobox genes. The nomenclature
used for vertebrate LIM homeobox genes has been proposed by Dawid et
al. (1995). Subclasses for which only one member has been identified to
date (e.g., C. elegans MEC-3) are not shown. The
dendrogram was constructed with the pileup algorithm in the Genetics
Computer Group software package using only the homeodomain sequences of the LIM homeodomain proteins because the evolutionary constraints on
variations within this functional domain are most pronounced and thus
produce the most significant relationship among various homeodomain
family members (Bürglin, 1995). CeISL-1 represents an ISLET
homolog recently identified in C. elegans (Hobert and Ruvkun, unpublished observations). BK64 and BK87 represent only partially characterized Drosophila LIM homeobox gene
sequences retrieved from a DNA-binding screen (Kalionis and O'Farrell,
1993). Because most of the C. elegans genome has been
sequenced, it is worthwhile to note that no LIM homeobox gene subclass
has more than one C. elegans member to date. Not all
vertebrate orthologs are shown. AP, Apterous;
C.e., C. elegans; D.m.,
fruit fly; G.g., chicken; H.r., ascidian;
H.s., human; M.m., mouse; and
X.l., frog.
|
|
The lin-11 LIM homeobox gene was first identified by the
defects in ectodermal cell lineage asymmetry caused by
lin-11 loss-of-function mutations (Ferguson and Horvitz,
1985; Freyd et al., 1990). Given the involvement of other LIM homeobox
genes in neurogenesis, we sought to determine whether lin-11
also plays a role in neural development or neural function. We find
that lin-11 is expressed in a specific set of postmitotic
neurons. lin-11 is not required for the generation of these
neurons but has a role in their functional specification. One of these
neurons has a function that is related to the function of a neuron
specified by the LIM homeobox gene ttx-3. The interneurons
AIZ and AIY act antagonistically in the neural network that subserves
thermoregulatory behavior, and the LIM genes lin-11 and
ttx-3, respectively, are necessary for events late in the
development of each neuron. These findings indicate that LIM homeobox
gene paralogs may serve homologous regulatory functions in the
development of related neurons.
| |
MATERIALS AND METHODS |
Strains and genetic procedures. The strains used in
this study are wild-type C. elegans Bristol strain (N2),
CB1467 [him-5(e1467)], MT633 [lin-11(n389);
him-5(e1467)], GR1052 [unc-86(n846)], and CB1372
[daf-7(e1372)]. lin-11(n389) represents a null
mutation in which the entire coding region of lin-11 is
deleted (Freyd et al., 1990). Double mutant strains were constructed as
follows: the daf-7(e1372); lin-11(n389) double mutant strain
was constructed by mating lin-11(n389); him-5(e1467) males
with daf-7(e1372) hermaphrodites. Nondauer lin-11,
daf-7 heterozygous cross-progeny was identified by raising animals
at 25°C. F2 animals homozygous for daf-7 were identified
at 25°C and recovered at 15°C, and homozygous lin-11 adults were scored for the lin-11-associated egg-laying
defect.
Expression constructs and generation of transgenic animals.
The plin-11-ABCDE-GFP reporter gene was constructed by
subcloning a genomic 10 kb fragment into the pPD95.75 vector, creating
a translational fusion of a BamHI site within the fifth exon
of lin-11 just upstream of the homeobox with the green
fluorescent protein (GFP) coding region. The
plin-11-CDE-GFP, plin-11-DE-GFP, and
plin-11-E-GFP constructs were injected as purified
restriction fragments obtained from the original
plin11-ABCDE-GFP construct using the ApaLI site
at nucleotide position  212 of the ATG start codon (for
plin-11-CDE-GFP), the BstEII site at position
1466 (for plin-11-DE-GFP), or the BglII site at
position 2564 (for plin-11-E-GFP) plus the EagI
site downstream of the GFP 3'-untranslated region. The plasmid or
restriction fragment DNA was injected at 50 ng/µl into N2 wild type
using pRF-4 (harboring a dominant rol-6 mutation) as an
injection marker (100 ng/µl). Multiple independent transgenic lines
were examined for expression. The plin-11-ABCDE-GFP and
plin-11-DE-GFP extrachromosomal arrays were integrated using a Stratalinker 1800 UV light source at 300 J/m2 and
were back-crossed several times. Neuroanatomical defects (see Fig. 4)
were identified by crossing the integrated plin-11-DE-GFP reporter array from wild-type into lin-11(n389) null mutant
animals. Because this allowed the comparison of reporter strains that
only differed in lin-11 gene activity and otherwise were
completely isogenic, artifacts induced by the reporter array could be
excluded.
Cell identifications and scoring of neuroanatomical defects.
Identification of neurons that express lin-11-GFP was done
by monitoring cell body position and axon morphology in larval stage L1
and adult animals using light microscopy. Additionally, staining with
the fluorescent dye DiI, which fills defined subsets of head and tail
neurons, was undertaken (as described by Hedgecock et al., 1985) on
transgenic GFP reporter gene-expressing animals to compare green GFP
fluorescence with red dye fluorescence using different microscope
filter sets. The amphid neuron ADL and the phasmid neuron PHA showed
overlapping GFP and dye staining; the relative positions of adjacent
GFP reporter gene-expressing neurons as well as Nomarski optics were
used to identify neighboring cells in the head and tail ganglia. The
wild-type position of cells and the axon morphology of neurons have
been described by Sulston et al. (1983) and White et al. (1986).
Neuroanatomical defects (see Fig. 4) were identified by crossing the
integrated plin-11-DE-GFP reporter array from wild-type into
lin-11(n389) null mutant animals. VC neuroanatomical defects
(see Fig. 6) were identified with a plin-11-B-GFP; rol-6
extrachromosomal array; four independent transgenic lines were examined
and showed similar defects. Neuroanatomical defects were quantified by
picking a random amount of transgenic, reporter gene-bearing animals
(numbers given in the text) and were scored according to the categories
described in the text.
Behavioral assays. Single worm thermotaxis assays were
performed as described previously (Mori and Ohshima, 1995). Briefly, single animals were placed on 8 cm agar plates, which were then inverted, and a vial of frozen acetic acid (2.7 cm in diameter) was
placed in the center on top of the agar plate to create a radial
temperature gradient from 16 to 25°C. Animals were allowed to migrate
in this radial temperature gradient for 1.5 hr, and their position and
tracks on the agar plate were scored. For dauer formation assays, the
synchronized brood of the respective genotypes was raised at different
temperatures and scored for characteristic dauer features (Riddle and
Albert, 1997). Because of the variability of dauer formation caused by
subtle changes in the environment, comparisons of different genotypes
were only made with assays that were done in parallel. Each assay was
performed in triplicate.
| |
RESULTS |
Expression pattern of a lin-11-GFP reporter gene
The neural specific expression of many LIM homeobox genes in
C. elegans (Freyd, 1991; Way and Chalfie, 1991; Hobert et
al., 1997) (Hobert and Ruvkun, unpublished observations) prompted us to
investigate the temporal and spatial control of expression of a
lin-11-GFP reporter gene construct in the nervous system of
C. elegans. A genomic DNA fragment bearing 6.5 kb of
5'-upstream sequences as well as the first five exons and four introns
of lin-11 was fused to GFP just upstream of the homeobox,
transformed into C. elegans, and integrated into chromosomes
to yield stable reporter gene expression. lin-11-GFP
expression can be observed from late embryonic stages throughout larval
and adult stages. Because of their characteristic position (Sulston et
al., 1983), the lin-11-GFP-expressing cells were identified
in early larval stages. At the L1 stage, lin-11-GFP
expression is exclusively confined to neurons in the head ganglia and
the lumbar ganglion in the tail (Fig. 2).
The neurons that express lin-11-GFP in the head ganglion are
the sensory neurons ADF and ADL and the interneurons AIZ and RIC (Fig.
3). The expression of lin-11
in head sensory neurons correlates with the expression of the
vertebrate lin-11 homolog Lhx1 in head sensory
structures (Barnes et al., 1994). Weak lin-11-GFP expression
can be observed in the interneuron AVG that sends a process along the
ventral cord. A role for lin-11 in AVG is suggested by the
observation of Baumeister et al. (1996) that in lin-11
mutant animals unc-86 expression is aberrantly activated in
AVG, suggesting that lin-11 normally represses
unc-86 expression in AVG. The identity of another head
neuron pair that exhibits lin-11-GFP expression could not be
unambiguously determined, but because of its characteristic axonal
morphology in the ventral cord (White et al., 1986), we tentatively
assigned this pair of neurons as either the AVH or AVJ interneuron. The
expression pattern of lin-11-GFP in the head ganglia is
summarized in Figure 3.
View larger version (74K):
[in this window]
[in a new window]
|
Figure 2.
Expression pattern analysis of the
lin-11 gene. GFP fluorescence of different-staged
animals carrying integrated lin-11-GFP reporter gene
constructs are shown. The larval stages are indicated. The white
arrow in the embryonic stages depicts an outgrowing axon from a
neuronal cell body. Note that the GFP reporter construct does not
contain the nuclear localization sequence of LIN-11; thus the axon and
cell bodies fluoresce uniformly. Also note that because of differential
planes of focus, not all neurons can be seen in every
panel (e.g., in A, not all neurons in the
lumbar ganglion are in the same plane of focus). A,
C, F, Lateral view. B,
D, E, Ventral and dorsal views. All
animals shown (except those in E and
F) carry an integrated
plin-11-ABCDE-GFP reporter gene construct (see Fig. 4).
Note that in the early L2 animal in D, the VC motor neurons are not matured (they have not sent
out their axonal projections) (Li and Chalfie, 1990), so that in the
ventral cord, only the AVG, AVH/AVJ, and
PVQ interneurons stain. The white triangles in D point to the left
(PVQL) and right (AVG,
AVH/AVJ, and PVQR) ventral cord tracts.
E, F, Transgenic
plin-11-B-GFP animals, which display staining of the
VC motor neurons and the vulva, are
shown. The expression in the VC motor neurons begins at
late larval stages after the postembryonic birth and axonal outgrowth
of the VC neurons. Staining of tail neurons decreases in
adulthood, whereas expression in the head neurons perdures. Expression
in the vulval precursors cells is confined to the generation of the
vulva during middle to late larval stages. Because the numbers of cells staining in these early larval stages does not appear
to differ from the number of embryonically staining cells and because
the onset of embryonic GFP staining correlates with the birth of these
neurons, we conclude that the embryonic cells expressing
lin-11-GFP are exclusively neurons. An essentially similar expression pattern can be observed using a lacZ-reporter gene
fusion (Freyd, 1991). The expression of lin-11 in the
AVG interneuron is considerably weaker than that in the
other head neurons.
|
|
View larger version (30K):
[in this window]
[in a new window]
|
Figure 3.
lin-11-expressing neurons in the head
ganglion. The positions of the neurons in the anterior part of
C. elegans are schematically shown. The drawing is
adapted from Sulston et al. (1983) and White et al. (1986). The
lin-11-expressing neurons are shaded, and
their axonal projections are shown. Functions have been assigned to some of the lin-11-expressing neurons by laser ablation
and subsequent behavioral assays. ADF, See Bargmann and
Horvitz (1991a,b) and Schackwitz et al. (1996); ADL, see
Troemel et al. (1995); AIZ, see Mori and Ohshima (1995)
and Sze and Ruvkun (unpublished observations); and AVG,
see Durbin (1987).
|
|
Motor neurons within the vertebrate spinal cord express a specific set
of LIM homeobox genes (Tsuchida et al., 1994). We find that
lin-11-GFP is also expressed in motor neurons of the
C. elegans ventral cord (Fig. 2). lin-11-GFP is
activated in the six VC ventral cord motor neurons after they are
generated during the L1 stage. Although their pattern of connectivity
suggests a function in egg laying (White et al., 1986; Li and Chalfie,
1990), it has not been reported what kind of defects are caused by
microsurgical removal of the VC ventral cord motor neurons. Adult
lin-11 null mutant animals display uncoordinated backward
locomotion (Freyd, 1991). It is unclear, however, whether this defect
can be related to a function of lin-11 in the VC motor
neurons.
The lin-11-GFP-expressing tail neurons were identified as
the PHA phasmid sensory neurons and the PVQ ventral cord interneurons, one of which (PVQL) extends a process on the left side of the ventral
cord (Fig. 2). PVQ is a pioneer neuron of the ventral cord (Durbin,
1987) and required for correct axon fasciculation of follower neurons
such as the HSN motor neuron (Garriga et al., 1993). HSN fasciculation
also requires lin-11 gene activity (Garriga et al., 1993).
The expression of lin-11 in PVQ but not HSN suggests that
PVQ requires lin-11 to provide fasciculation cues for HSN. It is interesting to note that all the neurons that express
lin-11 in the ventral cord are directly connected to other
lin-11-expressing neurons; PHA forms synapses with AVG, PVQ,
and AVH, and the VC neurons synapse onto PVQ (White et al., 1986). The
functional significance of this observation is unclear, however (see
Discussion).
Outside the nervous system, lin-11-GFP is expressed in cells
of the developing vulva (Fig. 2), which correlates with the function of
lin-11 in these cells (Freyd et al., 1990).
lin-11 expression in the vulval precursor cells is dynamic
and disappears once the vulva is formed. lin-11-GFP
expression is also observed in the spermatheca (data not shown). A
lin-11-LacZ-reporter gene fusion reveals a similar
expression pattern (Freyd, 1991).
Temporal control of lin-11 expression
The vertebrate lin-11 homolog Lhx1 has been
shown to regulate embryonic inductions in pregastrulation stages (Taira
et al., 1994; Shawlot and Behringer, 1995). In this regard, it is
remarkable that no embryonic defects can be observed in
lin-11 mutants (Ferguson and Horvitz, 1985; Freyd et al.,
1990). We corroborated the apparent absence of a role for
lin-11 in early embryogenesis by determining at which
embryonic stage lin-11-GFP expression is activated. We found
no detectable expression of lin-11-GFP in embryonic blastula and gastrula stages (Fig. 2; data not shown). lin-11-GFP
expression is first observed in embryonic stages (~400 min) long
after the beginning of gastrulation (at 100 min) (Sulston et al.,
1983). The observed ~30 min delay between the onset of GFP protein
expression and GFP fluorescence observed in C. elegans (G. Seydoux, personal communication) does not seem to be sufficient to
account for the delay. To exclude any GFP maturation artifacts of our
GFP reporter construct, we performed anti-GFP antibody staining on
lin-11-GFP-expressing embryos and confirmed the
postgastrulation embryonic expression of the lin-11-GFP
reporter (data not shown). Moreover, a lin-11-lacZ fusion,
the enzymatic activity of which is not subject to maturation, shows a
similar onset of expression (Freyd, 1991). Thus, lin-11 expression is activated significantly later in development than is that
of its vertebrate homologs. The lack of early embryonic defects in a
lin-11 null mutant (Ferguson and Horvitz, 1985; Freyd et
al., 1990) is consistent with this observation.
lin-11 expression is activated either significantly after
the birth of the respective neuron (ADF, ADL, AIY, AVG, AVH/AVJ, or VC)
or at approximately the time the respective neuron is born (RIC). In
the case of the VC motor neurons, this temporal control is particularly
obvious; the VC motor neurons are formed in the L1 larval stage
(Sulston et al., 1983) and extend their axonal projections along the
ventral cord in L3 larval stages (Li and Chalfie, 1990).
lin-11-GFP expression can first be observed in late larval
stages once the VC motor neurons have sent out their axonal projections
(Fig. 2). These observations demonstrate that activation of
lin-11 is a postmitotic event.
The continuous expression of a transcription factor gene throughout the
life of an animal suggests a role in maintenance of cellular function.
In adult C. elegans, we find that lin-11
expression is maintained at similar levels in all classes of head
neurons throughout adulthood (Fig. 2). In contrast, expression in the tail neurons fades at late larval stages and cannot be observed in
adult animals. These observations suggest a role for lin-11 in the maintenance of neural function of head neurons. The
perdurability of lin-11-GFP is not attributable to an
artifactual stability of the reporter gene construct for several
reasons. First, lin-11-GFP expression fades in tail neurons
in postlarval stages, whereas it perdures in head neurons. Second,
lin-11-GFP expression is rapidly turned off when
unc-86, an upstream transcriptional regulator of
lin-11, is conditionally turned off (J. Sze and G. Ruvkun, unpublished observations). Third, a ttx-3-GFP fusion gene is
similarly expressed throughout the life of the animal, and in this case the expression is dependent on ttx-3(+) activity, showing
that the maintenance of GFP expression is an active process in this neuron (Hobert et al., 1997).
Defined cis-regulatory elements are responsible for
lin-11 gene expression
Deletion mutants of the original lin-11-GFP reporter
gene define the control elements for lin-11 expression (Fig.
4). A 3.5 kb region upstream of the
putative ATG start codon (B element) mediates expression of
lin-11 in the developing vulva, the spermatheca, and the
ventral cord motor neurons (Fig. 4). The cis-regulatory elements that mediate lin-11 expression in neurons of the
head and tail ganglion (DE elements) are localized to a region
downstream to the putative translational start site (Fig. 4).
Transgenes bearing an even shorter cis-regulatory element (E
element) express only weakly in head neurons (Fig. 4). Interestingly,
although the DE elements mediate initiation of lin-11
expression in head neurons, they do not maintain lin-11
expression throughout adulthood in all neurons (data not shown),
suggesting that initiation and maintenance regulatory elements are
separable.
View larger version (24K):
[in this window]
[in a new window]
|
Figure 4.
Definition of regulatory elements driving
lin-11 expression. The expression of various deletion
derivatives of the original plin-11-ABCDE-GFP reporter
construct was monitored in different tissues. The genomic structure of
the lin-11 gene plus the preceding gene
(ZC247.4) is shown. The expression of the
plin-11-CDE-GFP and plin-11-DE-GFP
reporter constructs entirely recapitulates the neural expression of
plin-11-ABCDE-GFP. Notably, some additional head neurons
plus (in a few animals) some ventral and dorsal cord motor neurons
weakly express plin-11-DE-GFP, arguing that the ABC
elements contain negative regulatory elements. Note that the deletion
derivatives plin-11-CDE-GFP,
plin-11-DE-GFP, and plin-11-E-GFP do not
contain the original translational start site
(right-angle arrow) of the
lin-11 gene; these constructs presumably use either a
potential downstream translational start site (stippled
arrow) or the translational start site of the
green fluorescent protein. Vulva, Cells
of the developing vulva; VC, ventral cord motor neurons; and Sper, spermatheca.
|
|
It is remarkable that the regulatory elements responsible for driving
lin-11 expression in different classes of head neurons that
are not closely related by lineage (Sulston et al., 1983) are confined
to a small genomic 1.1 kb region downstream of the putative
lin-11 translational start site. The location of
transcriptional regulatory elements within introns has been observed in
various neuronally expressed genes, such as nestin in vertebrates
(Zimmerman et al., 1994), eyeless in Drosophila
(Quiring et al., 1994), or unc-86 (Baumeister et al., 1996)
and ttx-3 (Hobert and Ruvkun, unpublished
observations) in C. elegans. This observation underscores the compactness of the C. elegans genome, both in terms of
gene structure and the structure of gene regulatory
elements.
The paired-homeobox gene Pax-6 is involved in neuronal
patterning (Stoykova et al., 1996) and influences the combinatorial code of LIM homeobox genes that is thought to determine neuronal identity in the vertebrate brain and spinal cord (Tsuchida et al.,
1994; Ericson et al., 1997; Osumi et al., 1997). Because the C. elegans Pax-6 ortholog vab-3 is also involved in head
patterning (Chisholm and Horvitz, 1995), we determined whether
expression of the Lhx1/5 ortholog lin-11 in
either the head ganglion or in the ventral cord is under control of
vab-3. However, the neuronally expressed
plin-11-DE-GFP construct shows normal expression in animals
with a loss-of-function vab-3(e648) mutation (data not shown).
LIN-11 does not autoregulate its expression
The only two other LIM homeobox genes in C. elegans for which loss-of-function phenotypes have been described
are mec-3 (Way and Chalfie, 1988) and ttx-3
(Hobert et al., 1997). In both cases, the respective LIM homeobox gene
is expressed throughout adulthood and is required for the maintenance
of its own expression (Way and Chalfie, 1991; Hobert et al., 1997). We
similarly observe continuous lin-11-GFP expression
throughout adulthood, but we find that lin-11 gene activity
is not required for maintenance of its expression; no changes in the
intensity of lin-11-GFP reporter gene expression were
observed when comparing wild-type with lin-11 null mutant
animals (compare Figs. 2 and 5). This
comparison was performed with an integrated transgenic array that was
crossed from wild-type to mutant animals, excluding artifacts
attributable to different transgenes. Factors other than
lin-11 must be required for maintenance of lin-11
expression. In fact, J. Sze and G. Ruvkun have recently shown that
continuous unc-86 gene activity is required for maintenance
of lin-11 expression in the AIZ interneuron, the only
overlap between the unc-86 and lin-11 expression
patterns (Sze and Ruvkun, unpublished observations).
View larger version (77K):
[in this window]
[in a new window]
|
Figure 5.
lin-11-expressing neurons are formed but
display neuroanatomical defects in lin-11 null mutants.
To visualize the lin-11-expressing neurons in
lin-11(n389) null mutants, we crossed an integrated plin-11-DE-GFP reporter gene into
lin-11(n389) null mutant animals. Because of its
structure (see Fig. 4), this reporter gene is not expected to have any
lin-11 gene activity. Young adult animals (aligned with
anterior to the left and posterior to the
right) with characteristic neuronal defects are shown.
The reporter gene-expressing neurons in the head ganglion (most
anterior-located cell bodies) are approximately similar in position and
main axonal morphology to those in wild-type animals. Also, no obvious
differences in fluorescence intensity can be observed (compare with
Fig. 2). The transgenic animals carry the rol-6 reporter
gene as a marker, resulting in twisted worms. The white
triangles (II, IV,
V) point to the ventral cord. Note that a single
animal can display several neuroanatomical defects at once or,
alternatively, only a subset of them. There seems to be no linkage of
the four classes of neuroanatomical defects shown in the
table. I, V, The
lower arrow (a) points to a
process from the posteriorly displaced neuron AIZ that joins the
ventral cord in an aberrant path toward the ventral cord. Normally, AIZ
follows the amphid commissure to join the ventral cord (Fig. 3).
I, II, The uppermost arrow
(d) points to a posteriorly displaced neuron,
most likely the AIZ interneuron. We define posterior displacement as
the placement of the AIZ/RIC interneurons at locations significantly
posterior to the posterior bulb of the pharynx. In wild type, the
positions of AIZ, RIC, and other cells in the posterior lateral ganglia
display some natural variability in their relative positions with
respect to the posterior bulb of the pharynx; however, they always
remain in close proximity to the pharyngeal bulb. In
lin-11 mutants, however, AIZ and RIC are displaced to a
position significantly posterior to the posterior bulb of the pharynx.
II, The posterior arrow marks a
prematurely terminated process in the ventral cord. Note the
fasciculation defects of processes in the ventral cord.
III-V, Axons projecting posteriorly
through a random path are shown (p).
|
|
LIN-11-expressing neurons are generated but defective in
lin-11 null mutants
The lin-11 gene was identified based on defects
in vulval cell lineages (Ferguson and Horvitz, 1985). The neuronal
expression pattern of lin-11, which we have described above,
led us to investigate further what role lin-11 might play in
neurogenesis or neural function. We first wanted to determine whether
lin-11 gene activity is required for the generation of the
lin-11-expressing neurons or whether lin-11
affects neuronal development after the initial generation of neurons,
as suggested by the temporal regulation of lin-11
expression. We visualized the lin-11-expressing neurons in
lin-11(n389) null mutant animals using a
lin-11-GFP reporter gene construct. lin-11(n389)
represents a null mutation in which the entire coding region of
lin-11 is deleted (Freyd et al., 1990). Because the
plin-11-ABCDE-GFP reporter gene construct (Fig. 4) exhibits
partial rescuing activity (data not shown), we used the plin-11-DE-GFP intronic promoter construct that has no
lin-11 gene activity and that entirely recapitulates the
head neuronal expression pattern of lin-11. We crossed the
chromosomally integrated plin-11-DE-GFP reporter gene array
into lin-11 null mutant animals. As judged by cell number
and characteristic axon morphologies, we observed that all
lin-11-expressing neurons are present in lin-11
null mutant animals (Fig. 5), arguing that lin-11 plays no
major role in the generation of the lin-11-expressing
neurons.
We do, however, observe subtle but readily detectable neuroanatomical
defects in >60% of lin-11 mutant animals. Representative examples are shown in Figure 5. Because the main axonal trajectories and the approximate cellular position are not severely disturbed, these
types of defects are indicative of neural specification defects rather
than lineage defects. The most obvious defects are (1) the presence of
additional posteriorly directed processes that do not follow any
specific path in the ventral or in the dorsal nerve cord and that
terminate at random positions and (2) posteriorly displaced cell
positions of some neurons, the axonal projections of which take unusual
paths (Fig. 5). The identities of the posteriorly displaced neuronal
cell bodies are most likely AIZ and RIC. It is possible that the
posterior displacement of AIZ and RIC represents a migration defect of
these neurons. The axons of the posteriorly displaced AIZ and RIC
neurons often follow an aberrant path into the ventral cord (Fig.
5I,V, arrow a) that is distinct from the
amphid commissure along which AIZ and RIC normally send their axonal
projections into the ventral cord (see amphid commissure in Fig. 3).
Examples of posteriorly directed processes are shown in Figure 5
(III-V, arrow p). Their origins are often hard to trace, but we observed cases in which they originate either from the posteriorly displaced neurons or, alternatively, from
neurons in the more anterior head ganglion. These abnormalities are not
observed in wild-type animals expressing the same, integrated plin-11-DE-GFP reporter gene construct. Occasionally, one of
the processes in the ventral cord (AVG or AVH/AVJ) terminates
prematurely in the lin-11 mutant (Fig.
5II). Aberrant branching of posteriorly directed
processes can also be observed (Fig. 5III,IV).
Neuroanatomical defects of the ADF and ADL sensory neurons could not be
observed; however, the axonal processes of ADF and ADL are difficult to follow because they fasciculate and run along with most other lin-11-expressing neurons in the amphid commissure and the
nerve ring (Fig. 3).
Because lin-11 is also expressed in some neurons of the
ventral cord, we examined their fate in lin-11 null mutant
animals. The plin-11-B-GFP reporter gene construct (Fig. 4)
was used to monitor VC motor neuron anatomy in lin-11 mutant
animals. This reporter gene construct allows visualization of the VC
motor neuron axonal projections without the confounding axonal
processes that emanate from the lin-11-expressing head or
tail neurons. We found that lin-11 gene activity is not
required for the VC motor neurons to be generated or to extend their
processes along the ventral cord (Fig.
6). However, in >50% of the animals
examined, the VC motor neurons displayed a defasciculated phenotype
(Fig. 6). Normally, the six VC motor axons run parallel to one another
and form multiple connections to each other as well as to other neurons
in the ventral cord (White et al., 1986). Note that the head neurons
that send processes along the ventral cord also show fasciculation
defects (Fig. 5). The fasciculation defects we observe in the VC
ventral cord motor neurons might be caused by a cell-autonomous
requirement for lin-11 in the VCs. Alternatively, these
defects could be caused by defects in the lin-11-expressing
AVG motor neuron, which is a pioneer neuron in the ventral cord
(Durbin, 1987) and the laser ablation of which affects the bundling of
neurons in the ventral cord, causing aberrant transfer and crossing of
bundles (Durbin, 1987). In particular, several V- and D-type motor
neurons display axonal defects if AVG is laser-ablated (Durbin, 1987).
We addressed whether lin-11 might have a role in AVG in
guiding follower neurons by visualizing in lin-11 null
mutant animals several GABAergic V- and D-motor neurons in the ventral
cord with an anti-GABA antibody (kindly provided by H. R. Horvitz;
McIntire et al., 1993). No obvious axonal defects could be detected
(data not shown). The expression of lin-11 in the PHA
phasmid neurons also prompted us to examine their neuroanatomy in
lin-11 mutant animals. Labeling the PHA sensory neuron with
the fluorescent dye DiI, which fills amphid and phasmid sensory neurons
(Hedgecock et al., 1985), we observed no obvious neuroanatomical
defects of this pair of neurons in lin-11 null mutant
animals (data not shown).
View larger version (69K):
[in this window]
[in a new window]
|
Figure 6.
Motor neuron defects in lin-11
null-mutant animals. The VC motor neurons were visualized using the
plin-11-B-GFP reporter gene described in Figure 4. The
VC motor neurons are the only neurons expressing this reporter, which
allows us to visualize their anatomy independent of the ventral cord
axonal processes of the lin-11-expressing head and tail
neurons AVG, AVH/AVJ, and PVQ. The large white arrow
points to one of the three VC motor neuron cell bodies shown; the
small white arrows in the upper panel
point to the fasciculated VC processes (up to six, depending on the
position in the ventral cord) (White et al., 1986) in wild-type animals, whereas in the lower panels, the
arrows point to the defasciculated VC processes in
lin-11 null mutants. Note that the animals are slightly
twisted and lie upside down because of the rol-6 marker,
which allows us to obtain ventral views of the animals. The penetrance
of the defect was determined by picking randomly >20 animals of each
genotype that carried the transgenic marker rol-6 and
the GFP reporter construct as an extrachromosomal array and by scoring
their defects. Similar defects were observed in four independent
transgenic lin-11 mutant lines.
|
|
lin-11 mutants display thermotactic defects
Some of the head neurons that express
lin-11 have been assigned functions by laser ablation and
behavioral assays (Fig. 3). This type of analysis has revealed that the
head interneuron AIZ plays a pivotal role in thermotaxis (Mori and
Ohshima, 1995). Thermotaxis represents a learning paradigm in which the
animal displays the capacity to sense and memorize its cultivation
temperature so that it will migrate to its cultivation temperature when
placed on a thermal gradient (Hedgecock and Russell, 1975). The
thermotactic response relies on the output of two balanced and opposing
temperature-processing pathways, one governed by the AIY and the other
by the AIZ interneuron (Fig.
7A). Laser ablation of the AIY
interneuron causes a cryophilic phenotype because of the unregulated
activity of the other unaffected pathway, whereas laser ablation of the
AIZ interneuron causes a thermophilic phenotype (Mori and Ohshima,
1995). Because lin-11 is expressed in AIZ and because
lin-11 mutations cause AIZ neuroanatomical defects, we
tested whether the absence of lin-11 gene activity affects
thermotaxis. Thermotaxis assays on lin-11 null mutant animals revealed a thermophilic phenotype (Fig. 7B), which
is similar to laser ablation of AIZ. The assignment of thermotactic function with AIZ is underscored by the similar thermophilic phenotype of unc-86 mutant animals (Fig. 7B) (Mori and
Ohshima, 1995), which do not generate the AIZ interneuron because of a
cell lineage defect (Finney and Ruvkun, 1990). Because AIZ is formed
but apparently not functional in lin-11 mutant animals (Fig.
5), we conclude that lin-11 is required for the functional
specification of AIZ.
View larger version (30K):
[in this window]
[in a new window]
|
Figure 7.
lin-11 null mutants display defects in
thermotaxis. A, Schematic representation of the neural
pathway subserving thermotaxis as revealed by laser ablation (Mori and
Ohshima, 1995). Laser ablation of a neuron from one of the two pathways
leads to uncontrolled activity of the other pathway, thus causing a
thermophilic (laser ablation of AIZ) or cryophilic (laser ablation of
AIY) phenotype. Sensory neurons are depicted by
triangles; interneurons are indicated by
hexagons. The connectivity of the neurons was delineated
by White et al. (1986). B, lin-11(n389)
null mutants exhibit a thermophilic phenotype. Animals were raised at
15°C and then tested on a thermal gradient. The mean values of three
(Figure legend continues) representative assays are shown; in each assay, single
animals (using 10-15 lin-11, 9-15 control, and 5-15
unc-86 animals) from each different genotype were tested
in parallel. The tracking of animals in the thermal gradient was
classified into three categories: animals that have learned to remember
the 15°C cultivation temperature and move to and track within the
16°C region of the thermal gradient (gray bars), animals that move irrespective of their cultivation
temperature to higher temperatures (red bars;
thermophilic), and animals that track within the 16°C region of the
gradient but occasionally also outside this region (hatched red
bars). lin-11(n389) hermaphrodites display
slightly uncoordinated movement, which hampers the thermotaxis assay.
Because lin-11(n389) males do not exhibit this
uncoordinated phenotype, lin-11(n389), him-5(e1467)
males were assayed for thermotactic behavior. Consequently,
him-5(e1467) males were used as control animals.
unc-86(n846) animals lack AIZ because of a cell lineage defect (Finney and Ruvkun, 1990) and thus display a strong thermophilic phenotype (Mori and Ohshima, 1995).
|
|
Another temperature-controlled behavior is the execution of the
dauer developmental program (Golden and Riddle, 1984). Specific sensory
inputs that are indicative of harsh environmental conditions, such as
low amounts of food, crowding, and high temperature, are processed by
the C. elegans nervous system to cause arrest of C. elegans larvae at the dauer stage (Riddle and Albert, 1997). Considering the thermotactic defect of lin-11 (Fig.
7B), we asked whether lin-11 loss of function
also affects temperature modulation of dauer arrest. We observed a
small but significant suppression of daf-7-induced dauer
formation at 25°C (for daf-7(e1372); lin-11(n389), 15 ± 4% suppression; n = 928; for
daf-7(e1372), 0% suppression; n > 100).
However, because a variety of unrelated genes also display weak
suppression of dauer formation, we cannot conclude that
lin-11 affects dauer formation specifically through the
thermosensory neural circuit.
| |
DISCUSSION |
Specification of a neural network by two LIM homeobox genes
C. elegans thermotactic behavior represents one of the
few animal behaviors the neuronal components of which have been well defined, revealing the existence of a specific neural regulatory network that subserves this behavior (Mori and
Ohshima, 1995). The organization of this network into two parallel,
warm- and cold-processing thermoregulatory pathways is remarkably
similar to that of thermocontrol in vertebrates (Boulant and Dean,
1986). The molecular basis for the generation and function of this
C. elegans pathway may therefore be related to the mechanism
by which vertebrate thermoregulation is achieved.
We have shown previously that the AIY interneuron that is necessary for
thermophilic responses requires a LIM homeobox gene, ttx-3,
for its functional specification (Hobert et al., 1997). In the case of
ttx-3, it is expressed only in AIY, and the lack of
ttx-3 gene activity affects AIY structure and the
thermoregulatory function assigned to AIY by laser ablation. Our
assignment of lin-11 function in thermoregulation to the AIZ
interneuron involves a similar argument. (1) A lin-11-GFP
fusion gene is expressed in AIZ, (2) the neuroanatomy of AIZ is
affected in a lin-11 null mutant, and (3) the
thermoregulatory function assigned to AIZ by laser ablation is affected
similarly in a lin-11 null mutant. Although we cannot
exclude the possibility that the thermotactic defect of
lin-11 mutants is because of defects in other
lin-11-expressing neurons, we consider this unlikely for the
following reasons. First, none of the other
lin-11-expressing neurons has been implicated in thermotaxis
(Mori and Ohshima, 1995); second, unc-86 causes similar
thermotactic defects (Fig. 7B), and the only overlap between unc-86 and lin-11 expression in postmitotic head
neurons is in AIZ. Thus we suggest that the lin-11 LIM
homeobox gene acts in the AIZ interneuron that is functionally
antagonistic to the AIY ttx-3-expressing interneuron (Fig.
7A).
The entry of C. elegans into the developmentally arrested
dauer stage is also under thermosensory control (Golden and Riddle, 1984; Hobert et al., 1997). Our finding that lin-11 affects
dauer formation could be explained with a defect in thermoregulation of
dauer formation. This hypothesis is consistent with the expression of
lin-11-GFP in the thermoregulatory AIZ interneuron. However, the effect of lin-11 on dauer formation is considerably
weaker than the effect of ttx-3 on dauer formation (Hobert
et al., 1997) and could be an effect of lin-11 unrelated to
thermocontrol of dauer formation [e.g., lin-11 could affect
sensory function of the ADF neuron, which controls the dauer program
(Bargmann and Horvitz, 1991a)]. Thus, although ttx-3 and
lin-11 are clearly both required for thermotactic behavior,
it remains an open issue as to how much the neural and genetic
components that couple thermosensory information to a motor output are
the same as the neural components that couple to dauer development.
Perhaps the major output to neuroendocrine control is via AIY and
not AIZ.
The major thermoregulatory organ of vertebrates, the hypothalamus,
contains distinguishable warm- and cold-sensing temperature-processing units (Boulant and Dean, 1986) that may be homologous to the
antagonistic high and low temperature-sensing pathways of the C. elegans thermotactic response pathway (Fig. 7A).
lin-11 and ttx-3 in C. elegans, and their homologs in mammals, may thus mediate the development of two
components of this phyletically conserved thermal-processing network.
In support of this hypothesis, the vertebrate ttx-3 homolog Lhx2 and the lin-11 homologs Lhx1 and
Lhx5 are expressed in the diencephalon, which gives rise to
the thermoregulatory hypothalamus (Fuji et al., 1994; Porter et al.,
1997; Sheng et al., 1997).
The AIY and AIZ neurons are formed in animals bearing null mutations in
ttx-3 and lin-11, respectively, but these neurons show subtle anatomical defects suggesting that they fail to execute late events in neurogenesis. For example, TTX-3 and LIN-11 may regulate
genes that are directly involved in axonal pathfinding, so that the
patterns of neuroanatomical defects observed in both ttx-3
and lin-11 mutant animals can be explained on the basis of
direct disturbances in axonal pathfinding. Alternatively, these genes
may regulate the expression of genes that mediate synaptic connectivity
or synaptic activity, and these neural defects may be secondary
consequences of the absence of synaptic signaling. Synaptic
connectivity-dependent sprouting of additional processes has been
observed in other systems (e.g., Henderson et al., 1983).
The expression and function of two genes from the same gene family in
two parallel neural pathways provokes the question as to whether
ttx-3 and lin-11 are differentially instructive
to determine specific AIY and AIZ features, respectively.
Alternatively, they could act permissively to regulate the same set of
downstream target genes in order to determine features that are common
to AIY and AIZ [e.g., their connection to the thermotactic RIA
interneuron, which is a postsynaptic target of both AIY and AIZ and
required for thermotactic behavior (Mori and Ohshima, 1995)]. In
Drosophila, the paired-class homeobox genes prd,
gsb, and gsbn are differentially expressed but
can functionally substitute for one another (Li and Noll, 1994). Their
divergent roles may have simply evolved by changes in deployment. The
compactness and modularity of the cis-regulatory elements
for lin-11 (Fig. 4) and ttx-3 (Hobert and Ruvkun,
unpublished observations) would facilitate the evolution of distinct
developmental function by acquisition of distinct regulatory elements.
In summary, our data suggest that distinct LIM homeobox genes may
determine the features of functionally related neurons of neural
circuits that generate specific behaviors.
LIN-11 function in the ventral cord
We find that lin-11-expressing motor neurons in the
ventral cord are formed in lin-11 null mutant animals but
display neuroanatomical defects. There are six VC motor neurons in the
ventral cord, the axonal projections of which fasciculate and run in
parallel in the ventral cord to form chemical and electrical synapses
with each other and with other neurons and target muscles. We find that
lin-11 gene activity is not required for the generation or the axonal outgrowth of these neurons but required for them to fasciculate correctly. It is possible that LIN-11 regulates downstream target genes that are required for homophilic interaction of the VC
motor neurons; alternatively, these downstream target genes might be
required for correct fasciculation with other neurons in the ventral
cord. It is also possible that the VC motor neuron fasciculation
defects are caused by absence of lin-11 function in the AVG
neuron, which is a pioneer in the ventral cord (Durbin, 1987); however,
because we do not observe defects in other ventral cord motor neurons
that require AVG to fasciculate, such as the V- and D-type motor
neurons (Durbin, 1987), we consider this possibility less likely.
The HSN motor neuron requires the PVQ ventral cord pioneer to
fasciculate correctly (Garriga et al., 1993). Moreover, Garriga et al.
(1993) have shown that lin-11 gene activity is required for
proper HSN fasciculation. Our identification of PVQ, but not HSN, as
one of the lin-11-expressing neurons is consistent with a
hypothesis in which lin-11 acts in PVQ to affect HSN
morphology. The function of lin-11 in the VC motor neurons
and PVQ neurons might thus be similar in that LIN-11 could to be
required to provide cues for selective fasciculation of neurons in the
ventral cord. For example, LIN-11 could regulate the expression of
adhesive cell surface receptors that mediate fasciculation between VC
neurons and between PVQ pioneer and HSN follower axons. It is also
intriguing to note that all the lin-11-expressing neurons in
the ventral cord have at least one synaptic partner that also expresses
lin-11. VC synapses with PVQ and HSN; PHA synapses with AVG,
AVH, and PVQ; and PVQ synapses with VC and HSN. Thus, another
hypothesis posits that lin-11 is required to determine
synaptic connectivity and that the fasciculation defects of the VC and
HSN neurons are a secondary consequence of missing synaptic inputs.
Although we do not observe fasciculation defects of the
lin-11-expressing head neurons in lin-11 mutants,
the process outgrowth and migration defects of the head neurons could
also be explained by misregulation of cell surface molecules required
for correct axonal guidance or synaptic activity.
The vertebrate lin-11 ortholog Lhx1 is also
expressed in motor neurons of the spinal cord. Lhx1 null
mutant mice die because of early embryonic defects (Shawlot and
Behringer, 1995); thus the importance of Lhx1 in motor
neurons could not be addressed to date. Regarding the function of
lin-11, it is conceivable that Lhx1 might act in
a similar manner in determining axonal morphology of spinal cord motor
neurons.
A conserved neural function for LIM homeobox genes
Loss-of-function studies in C. elegans and
Drosophila have shown that LIM homeobox genes display a
postmitotic function late in neural development. C. elegans
mec-3 acts in touch cell receptor specification (Way and Chalfie,
1988, 1991); ttx-3 (Hobert et al., 1997) and
lin-11 as shown here act in thermotactic interneuron specification. All three genes continue to be expressed at high levels
throughout adulthood. Two recently identified C. elegans LIM
homeobox genes are also expressed in a very restricted subset of
postmitotic neurons, and their expression is also maintained throughout
adulthood (Hobert and Ruvkun, unpublished observations). Axonal
pathfinding defects have been described in Drosophila
mutants of the LIM homeobox genes apterous and
islet (Lundgren et al., 1995; Thor and Thomas, 1997);
Drosophila islet also controls neurotransmitter identity
(Thor and Thomas, 1997). Also, vertebrate LIM homeobox genes from all
LIM homeodomain subclasses (Fig. 1) have been shown to be expressed
continuously throughout adulthood, mostly in the brain (Thor et al.,
1991; Taira et al., 1992; Xu et al., 1993; Barnes et al., 1994; Fuji et
al., 1994; Gong et al., 1995; Sheng et al., 1997). Taken together,
these observations suggest a common theme for the function of LIM
homeobox genes late in neural differentiation, potentially in the
establishment of features required for synaptic signaling as well as
for the maintenance of the differentiated state of neurons. Additional
and relatively specialized functions of LIM homeobox genes, such as
lin-11 function in vulva formation (Ferguson and Horvitz,
1985; Freyd et al., 1990), apterous function in wing
development (Cohen et al., 1992), Lhx3 function in pituitary development (Sheng et al., 1996), or Lim1 function in
organizer activity (Taira et al., 1994; Shawlot and Behringer, 1995),
might have been co-opted by specific phyla at different stages of
evolution.
| |
FOOTNOTES |
Received Sept. 26, 1997; revised Dec. 17, 1997; accepted Jan. 6, 1998.
This work is supported in part by Hoechst AG to G.R. O.H. is
supported by a postdoctoral fellowship from the Human Frontiers Science
Program. We thank Cori Bargmann, Gwen Acton, members of the Ruvkun
laboratory, and anonymous reviewers for helpful suggestions on this
manuscript, G. Seydoux for comments and communicating unpublished
observations, the Caenorhabditis Genetics Center (funded by the National Institutes of Health Center for Research Resources) for
supplying strains, A. Fire for the GFP vector pPD95.75, Bob Horvitz for
anti-GABA-antibody, and A. Coulson and the C. elegans sequencing centers for their updates on the status of the genome sequencing project. We are particularly grateful to Cori Bargmann for
the initial identification of lin-11-lacZ-expressing
neurons and to Hitoshi Sawa and Bob Horvitz for providing the
plin-11-B-GFP construct shown in Figure 4.
Correspondence should be addressed to Dr. Gary Ruvkun, Massachusetts
General Hospital, Department of Molecular Biology, Wellman 8, Boston,
MA 02114.
| |
REFERENCES |
-
Bargmann CI,
Horvitz HR
(1991a)
Control of larval development by chemosensory neurons in Caenorhabditis elegans.
Science
251:1243-1246[Abstract/Free Full Text].
-
Bargmann CI,
Horvitz HR
(1991b)
Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans.
Neuron
7:729-742[Web of Science][Medline].
-
Barnes JD,
Crosby JL,
Jones CM,
Wright CVE,
Hogan BLM
(1994)
Embryonic expression of Lim-1, the mouse homolog of Xenopus Xlim-1, suggests a role in lateral mesoderm differentiation and neurogenesis.
Dev Biol
161:168-178[Web of Science][Medline].
-
Baumeister R,
Liu Y,
Ruvkun G
(1996)
Lineage-specific regulators couple cell lineage asymmetry to the transcription of the Caenorhabditis elegans POU gene unc-86 during neurogenesis.
Genes Dev
10:1395-1410[Abstract/Free Full Text].
-
Boulant JA,
Dean JB
(1986)
Temperature receptors in the central nervous system.
Annu Rev Physiol
48:639-654[Web of Science][Medline].
-
Bürglin TR
(1995)
The evolution of homeobox genes.
In: Biodiversity and evolution (Arai R,
Kato M,
Doi Y,
eds), pp 291-336. Tokyo: The National Science Museum Foundation.
-
Chalfie M
(1993)
Homeobox genes in Caenorhabditis elegans.
Curr Opin Genet Dev
3:275-277[Medline].
-
Chalfie M,
Sulston JE,
White JG,
Southgate E,
Thomson JN,
Brenner S
(1985)
The neural circuit for touch sensitivity in C. elegans.
J Neurosci
5:956-964[Abstract].
-
Chisholm AD,
Horvitz HR
(1995)
Patterning of the Caenorhabditis elegans head regions by the Pax-6 family member vab-3.
Nature
377:52-55[Medline].
-
Cohen B,
McGuffin ME,
Pfeifle C,
Segal D,
Cohen SM
(1992)
Apterous, a gene required for imaginal disc development in Drosophila encodes a member of the LIM family of developmental regulatory proteins.
Genes Dev
6:715-729[Abstract/Free Full Text].
-
Dawid IB,
Toyama R,
Taira M
(1995)
LIM domain proteins.
CR Acad Sci Paris/Life Sci
318:295-306.
-
Desai C,
Garriga G,
McIntire SL,
Horvitz HR
(1988)
A genetic pathway for the development of the Caenorhabditis elegans HSN motor neurons.
Nature
336:638-646[Medline].
-
Durbin RM
(1987)
In: Studies on the development of organisation of the nervous system of Caenorhabditis elegans. PhD thesis University of Cambridge, Cambridge, UK.
-
Ericson J,
Rashbass P,
Schedl A,
Brenner-Morton S,
Kawakami A,
van Heyningen V,
Jessell TM,
Briscoe J
(1997)
Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling.
Cell
90:169-180[Web of Science][Medline].
-
Ferguson EL,
Horvitz HR
(1985)
Identification and characterization of 22 genes that affect the vulval cell lineages of the nematode C. elegans.
Genetics
110:17-72[Abstract/Free Full Text].
-
Finney M,
Ruvkun G
(1990)
The unc-86 gene product couples cell lineage and cell identity in C. elegans.
Cell
63:895-905[Web of Science][Medline].
-
Freyd G
(1991)
In: Molecular analysis of the Caenorhabditis elegans cell lineage gene lin-11. PhD thesis Massachusetts Institute of Technology.
-
Freyd G,
Kim SK,
Horvitz HR
(1990)
Novel cysteine-rich motif and homeodomain in the product of the Caenorhabditis elegans cell lineage gene lin-11.
Nature
344:876-879[Medline].
-
Fuji T,
Pichel JG,
Taira M,
Toyama R,
Dawid IB,
Westphal H
(1994)
Expressions patterns of the murine LIM class homeobox gene lim1 in the developing brain and excretory system.
Dev Dyn
199:73-83[Web of Science][Medline].
-
Garriga G,
Desai C,
Horvitz HR
(1993)
Cell interactions control the direction of outgrowth, branching and fasciculation of the HSN axons in Caenorhabditis elegans.
Development
117:1071-1087[Abstract].
-
Golden JW,
Riddle DL
(1984)
A pheromone-induced developmental switch in C. elegans: temperature-sensitive mutants reveal a wild-type temperature-dependent process.
Proc Natl Acad Sci USA
81:819-823[Abstract/Free Full Text].
-
Gong Z,
Hui CC,
Hew CL
(1995)
Presence of isl-1-related LIM domain homeobox genes in teleost and their similar patterns of expression in brain and spinal cord.
J Biol Chem
270:3335-3345[Abstract/Free Full Text].
-
Hedgecock EM,
Russell RL
(1975)
Normal and mutant thermotaxis in the nematode Caenorhabditis elegans.
Proc Natl Acad Sci USA
72:4061-4065[Abstract/Free Full Text].
-
Hedgecock EM,
Culotti JG,
Thomson JN,
Perkins LA
(1985)
Axonal guidance mutants of Caenorhabditis elegans identified by filling sensory neurons with fluorescin dyes.
Dev Biol
111:158-170[Web of Science][Medline].
-
Henderson CE,
Huchet M,
Changeux JP
(1983)
Denervation increases a neurite-promoting activity in extracts of skeletal muscle.
Nature
302:609-611[Medline].
-
Hobert O,
Mori I,
Yamashita Y,
Honda H,
Ohshima Y,
Liu Y,
Ruvkun G
(1997)
Regulation of interneuron function in the C. elegans thermoregulatory pathway by the ttx-3 LIM homeobox gene.
Neuron
19:345-357[Web of Science][Medline].
-
Jin Y,
Hoskins R,
Horvitz HR
(1994)
Control of type-D GABAergic neuron differentiation by C. elegans UNC-30 homeodomain protein.
Nature
372:780-782[Medline].
-
Kalionis B,
O'Farrell PH
(1993)
A universal target sequence is bound in vitro by diverse homeodomains.
Mech Dev
43:57-70[Web of Science][Medline].
-
Li C,
Chalfie M
(1990)
Organogenesis in C. elegans: positioning of neurons and muscles in the egg-laying system.
Neuron
4:681-695[Web of Science][Medline].
-
Li X,
Noll M
(1994)
Evolution of distinct developmental functions of three Drosophila genes by acquisition of different cis-regulatory regions.
Nature
367:83-87[Medline].
-
Lundgren SE,
Callahan CA,
Thor S,
Thomas JB
(1995)
Control of neuronal pathway selection by the Drosophila LIM homeodomain gene apterous.
Development
121:1769-1773[Abstract].
-
Manak JR,
Scott MP
(1994)
A class act: conservation of homeodomain protein function.
Development [Suppl]
120:61-71.
-
McIntire SL,
Jorgensen E,
Kaplan J,
Horvitz HR
(1993)
The GABAergic nervous system of Caenorhabditis elegans.
Nature
364:337-341[Medline].
-
Miller DM,
Shen MM,
Shamu CE,
Bürglin TR,
Ruvkun G,
Dubois ML,
Ghee M,
Wilson L
(1992)
C. elegans unc-4 gene encodes a homeodomain protein that determines the pattern of synaptic input to specific motor neurons.
Nature
355:841-845[Medline].
-
Mori I,
Ohshima Y
(1995)
Neural regulation of thermotaxis in Caenorhabditis elegans.
Nature
376:344-348[Medline].
-
Osumi N,
Hirota A,
Ohuchi H,
Nakafuku M,
Limura T,
Kuratani S,
Fujiwara M,
Noji S,
Eto K
(1997)
Pax6 is involved in the specification of hindbrain motor neuron subtype.
Development
124:2961-2972[Abstract].
-
Porter FD,
Drago J,
Xu Y,
Cheema S,
Huang SP,
Lee E,
Grinberg A,
Massalas JS,
Bodine D,
Alt FW,
Westphal H
(1997)
Lhx2, a LIM homeobox gene, is required for eye, forebrain and definitive erythrocyte development.
Development
124:2935-2944[Abstract].
-
Quiring R,
Walldorf W,
Kloter U,
Gehring WJ
(1994)
Homology of the eyeless gene of Drosophila to the small eye gene in mice and aniridia in humans.
Science
265:785-789[Abstract/Free Full Text].
-
Riddle DL,
Albert PS
(1997)
Genetic and environmental regulation of dauer larva development.
In: C. elegans II (Riddle DL,
Blumenthal T,
Meyer BJ,
Priess JR,
eds), pp 739-768. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
-
Ruvkun G
(1997)
Patterning the nervous system.
In: C. elegans II (Riddle DL,
Blumenthal T,
Meyer BJ,
Priess JR,
eds), pp 543-582. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
-
Salser SJ,
Kenyon C
(1992)
Activation of a C. elegans Antennapedia homologue in migrating cells controls their direction of migration.
Nature
355:255-258[Medline].
-
Schackwitz WS,
Inoue T,
Thomas JH
(1996)
Chemosensory neurons function in parallel to mediate a pheromone response in C. elegans.
Neuron
17:719-728[Web of Science][Medline].
-
Shawlot W,
Behringer RR
(1995)
Requirement for Lim1 in head-organizer function.
Nature
374:425-430[Medline].
-
Sheng HZ,
Zhadanov AB,
Mosinger Jr B,
Fujii T,
Bertuzzi S,
Grinberg A,
Lee EJ,
Huang SP,
Mahon KA,
Westphal H
(1996)
Specification of pituitary cell lineages by the LIM homeobox gene Lhx3.
Science
272:1004-1007[Abstract].
-
Sheng HZ,
Bertuzzi S,
Chiang C,
Shawlot W,
Taira M,
Dawid IB,
Westphal H
(1997)
Expression of murine Lhx5 suggests a role in specifying the forebrain.
Dev Dyn
208:266-277[Web of Science][Medline].
-
Stoykova A,
Walther C,
Gruss P
(1996)
Forebrain patterning defects in Small eye mutant mice.
Development
122:3453-3465[Abstract].
-
Sulston JE,
Schierenberg E,
White JG,
Thomson JN
(1983)
The embryonic cell lineage of the nematode Caenorhabditis elegans.
Dev Biol
100:64-119[Web of Science][Medline].
-
Taira M,
Jamrich M,
Good PJ,
Dawid IB
(1992)
The LIM domain-containing homeobox gene Xlim-1 is expressed specifically in the organizer regions of Xenopus gastrula embryos.
Genes Dev
6:356-366[Abstract/Free Full Text].
-
Taira M,
Otani H,
Saint-Jeannet JP,
Dawid IB
(1994)
Role of the LIM class homeodomain protein Xlim-1 in neural and muscle induction by the Spemann organizer in Xenopus.
Nature
372:677-679[Medline].
-
Thor S,
Thomas JB
(1997)
The Drosophila islet gene governs axon pathfinding and neurotransmitter identity.
Neuron
18:397-409[Web of Science][Medline].
-
Thor S,
Ericson J,
Brännström T,
Edlund T
(1991)
The homeodomain LIM protein Isl-1 is expressed in subsets of neurons and endocrine cells in the adult rat.
Neuron
7:881-889[Web of Science][Medline].
-
Troemel E,
Chou JH,
Dwyer ND,
Colbert HA,
Bargmann CI
(1995)
Divergent seven transmembrane receptors are candidate chemosensory receptors in C. elegans.
Cell
83:207-218[Web of Science][Medline].
-
Tsuchida T,
Ensini M,
Morton SB,
Baldassare M,
Edlund T,
Jessell TM,
Pfaff SL
(1994)
Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes.
Cell
79:957-970[Web of Science][Medline].
-
Way JC,
Chalfie M
(1988)
Mec-3, a homeobox-containing gene that specifies differentiation of the touch receptor neurons in C. elegans.
Cell
54:5-16[Web of Science][Medline].
-
Way JC,
Chalfie M
(1991)
The mec-3 gene of Caenorhabditis elegans requires its own product for maintained expression and is expressed in three neuronal types.
Genes Dev
3:1823-1833[Abstract/Free Full Text].
-
White JG,
Southgate E,
Thomson JN,
Brenner S
(1986)
The structure of the nervous system of the nematode Caenorhabditis elegans.
Philos Trans R Soc Lond [Biol]
314:1-340.
-
White JG,
Southgate E,
Thomson JN
(1992)
Mutations in the Caenorhabditis elegans unc-4 gene alter the synaptic input to ventral cord motor neurons.
Nature
355:838-841[Medline].
-
Wicks SR,
Rankin CH
(1995)
Integration of mechanosensory stimuli in C. elegans.
J Neurosci
15:2434-2444[Abstract].
-
Xu Y,
Baldassare M,
Fisher P,
Rathbun G,
Oltz EM,
Yancopoulos GD,
Jessell TM,
Alt FW
(1993)
LH-2: a LIM/homeodomain gene expressed in developing lymphocytes and neural cells.
Proc Natl Acad Sci USA
90:227-231[Abstract/Free Full Text].
-
Zimmerman L,
Lendahl U,
Cunningham M,
McKay R,
Parr B,
Gavin B,
Mann J,
Vassileva G,
McMahon A
(1994)
Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors.
Neuron
12:11-24[Web of Science][Medline].
Copyright © 1998 Society for Neuroscience 0270-6474/98/1862084-13$05.00/0
This article has been cited by other articles:
|
|
|
|
 
R. Jovelin
Rapid Sequence Evolution of Transcription Factors Controlling Neuron Differentiation in Caenorhabditis
Mol. Biol. Evol.,
October 1, 2009;
26(10):
2373 - 2386.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Kuhara and I. Mori
Molecular Physiology of the Neural Circuit for Calcineurin-Dependent Associative Learning in Caenorhabditis elegans.
J. Neurosci.,
September 13, 2006;
26(37):
9355 - 9364.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Cordes, C. A. Frank, and G. Garriga
The C. elegans MELK ortholog PIG-1 regulates cell size asymmetry and daughter cell fate in asymmetric neuroblast divisions
Development,
July 15, 2006;
133(14):
2747 - 2756.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Broday, I. Kolotuev, C. Didier, A. Bhoumik, B. P. Gupta, P. W. Sternberg, B. Podbilewicz, and Z. Ronai
The small ubiquitin-like modifier (SUMO) is required for gonadal and uterine-vulval morphogenesis in Caenorhabditis elegans
Genes & Dev.,
October 1, 2004;
18(19):
2380 - 2391.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Zhang, I. Sokolchik, G. Blanco, and J. Y. Sze
Caenorhabditis elegans TRPV ion channel regulates 5HT biosynthesis in chemosensory neurons
Development,
April 1, 2004;
131(7):
1629 - 1638.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Hutter
Extracellular cues and pioneers act together to guide axons in the ventral cord of C. elegans
Development,
November 15, 2003;
130(22):
5307 - 5318.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. G. Clark and C. Chiu
C. elegans ZAG-1, a Zn-finger-homeodomain protein, regulates axonal development and neuronal differentiation
Development,
August 15, 2003;
130(16):
3781 - 3794.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Y. Sze and G. Ruvkun
Activity of the Caenorhabditis elegans UNC-86 POU transcription factor modulates olfactory sensitivity
PNAS,
August 5, 2003;
100(16):
9560 - 9565.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. P. Gupta, M. Wang, and P. W. Sternberg
The C. elegans LIM homeobox gene lin-11 specifies multiple cell fates during vulval development
Development,
June 15, 2003;
130(12):
2589 - 2601.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. Esmaeili, J. M. Ross, C. Neades, D. M. Miller III, and J. Ahringer
The C. elegans even-skipped homologue, vab-7, specifies DB motoneurone identity and axon trajectory
Development,
March 4, 2003;
129(4):
853 - 862.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. Aurelio, T. Boulin, and O. Hobert
Identification of spatial and temporal cues that regulate postembryonic expression of axon maintenance factors in the C. elegans ventral nerve cord
Development,
February 1, 2003;
130(3):
599 - 610.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. A. Scott, M. S. Avidan, and C. M. Crowder
Regulation of Hypoxic Death in C. elegans by the Insulin/IGF Receptor Homolog DAF-2
Science,
June 28, 2002;
296(5577):
2388 - 2391.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. S. Fay, S. Keenan, and M. Han
fzr-1 and lin-35/Rb function redundantly to control cell proliferation in C. elegans as revealed by a nonbiased synthetic screen
Genes & Dev.,
February 15, 2002;
16(4):
503 - 517.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. R. Sarafi-Reinach, T. Melkman, O. Hobert, and P. Sengupta
The lin-11 LIM homeobox gene specifies olfactory and chemosensory neuron fates in C. elegans
Development,
September 1, 2001;
128(17):
3269 - 3281.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Altun-Gultekin, Y. Andachi, E. L. Tsalik, D. Pilgrim, Y. Kohara, and O. Hobert
A regulatory cascade of three homeobox genes, ceh-10, ttx-3 and ceh-23, controls cell fate specification of a defined interneuron class in C. elegans
Development,
June 1, 2001;
128(11):
1951 - 1969.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Fay and M Han
Mutations in cye-1, a Caenorhabditis elegans cyclin E homolog, reveal coordination between cell-cycle control and vulval development
Development,
January 9, 2000;
127(18):
4049 - 4060.
[Abstract]
[PDF]
|
|
|
|
|
|
|
S. Certel, P. Clyne, J. Carlson, and W. Johnson
Regulation of central neuron synaptic targeting by the Drosophila POU protein, Acj6
Development,
January 6, 2000;
127(11):
2395 - 2405.
[Abstract]
[PDF]
|
|
|
|
|
|
|
H. A. Tissenbaum, J. Hawdon, M. Perregaux, P. Hotez, L. Guarente, and G. Ruvkun
A common muscarinic pathway for diapause recovery in the distantly related nematode species Caenorhabditis elegans and Ancylostoma caninum
PNAS,
January 4, 2000;
97(1):
460 - 465.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Much, D. Slade, K Klampert, G Garriga, and B Wightman
The fax-1 nuclear hormone receptor regulates axon pathfinding and neurotransmitter expression
Development,
January 2, 2000;
127(4):
703 - 712.
[Abstract]
[PDF]
|
|
|
|
|
|
|
Y. Zhao, Y.-J. Guo, A. C. Tomac, N. R. Taylor, A. Grinberg, E. J. Lee, S. Huang, and H. Westphal
Isolated cleft palate in mice with a targeted mutation of the LIM homeobox gene Lhx8
PNAS,
December 21, 1999;
96(26):
15002 - 15006.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. R. Winnier, J. Y.-J. Meir, J. M. Ross, N. Tavernarakis, M. Driscoll, T. Ishihara, I. Katsura, and D. M. Miller III
UNC-4/UNC-37-dependent repression of motor neuron-specific genes controls synaptic choice in Caenorhabditis elegans
Genes & Dev.,
November 1, 1999;
13(21):
2774 - 2786.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
N. Wittenburg and R. Baumeister
Thermal avoidance in Caenorhabditis elegans: An approach to the study of nociception
PNAS,
August 31, 1999;
96(18):
10477 - 10482.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Sagasti, O. Hobert, E. R. Troemel, G. Ruvkun, and C. I. Bargmann
Alternative olfactory neuron fates are specified by the LIM homeobox gene lim-4
Genes & Dev.,
July 15, 1999;
13(14):
1794 - 1806.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
Y. Zhao, H. Z. Sheng, R. Amini, A. Grinberg, E. Lee, S. Huang, M. Taira, and H. Westphal
Control of Hippocampal Morphogenesis and Neuronal Differentiation by the LIM Homeobox Gene Lhx5
Science,
May 14, 1999;
284(5417):
1155 - 1158.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
A. Newman, G. Acton, E Hartwieg, H. Horvitz, and P. Sternberg
The lin-11 LIM domain transcription factor is necessary for morphogenesis of C. elegans uterine cells
Development,
January 12, 1999;
126(23):
5319 - 5326.
[Abstract]
[PDF]
|
|
|
|
|
|
|
O Hobert, K Tessmar, and G Ruvkun
The Caenorhabditis elegans lim-6 LIM homeobox gene regulates neurite outgrowth and function of particular GABAergic neurons
Development,
January 4, 1999;
126(7):
1547 - 1562.
[Abstract]
[PDF]
|
|
|
|
|
|
|
J. S. Duerr, D. L. Frisby, J. Gaskin, A. Duke, K. Asermely, D. Huddleston, L. E. Eiden, and J. B. Rand
The cat-1 Gene of Caenorhabditis elegans Encodes a Vesicular Monoamine Transporter Required for Specific Monoamine-Dependent Behaviors
J. Neurosci.,
January 1, 1999;
19(1):
72 - 84.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. O'Keefe, S Thor, and J. Thomas
Function and specificity of LIM domains in Drosophila nervous system and wing development
Development,
January 10, 1998;
125(19):
3915 - 3923.
[Abstract]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction
