3915
Development 125, 3915-3923 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
DEV8544
Function and specificity of LIM domains in Drosophila nervous system and
wing development
David D. O’Keefe1, Stefan Thor2 and John B. Thomas2,*
1Department of Neurosciences, School of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California
92093, USA
2The Salk Institute for Biological Studies, PO Box 85800, San Diego, CA 92186, USA
*Author for correspondence (e-mail: [email protected])
Accepted 13 July; published on WWW 7 September 1998
SUMMARY
LIM domains are found in a variety of proteins, including
cytoplasmic and nuclear LIM-only proteins, LIMhomeodomain (LIM-HD) transcription factors and LIMkinases. Although the ability of LIM domains to interact
with other proteins has been clearly established in vitro and
in cultured cells, their in vivo function is unknown. Here
we use Drosophila to test the roles of the LIM domains of
the LIM-HD family member Apterous (Ap) in wing and
nervous system development. Using a rescuing assay of the
ap mutant phenotype, we have found that the LIM domains
are essential for Ap function. Furthermore, expression of
LIM domains alone can act in a dominant-negative fashion
to disrupt Ap function. The Ap LIM domains can be
replaced by those of another family member to generate
normal wing structure, but LIM domains are not
interchangeable during axon pathfinding of the Ap
neurons. This suggests that the Ap LIM domains mediate
different protein interactions in different developmental
processes, and that LIM domains can participate in
conferring specificity of target gene selection.
INTRODUCTION
O’Farrell, 1993). Therefore, specificity of target gene selection
is not likely to be controlled entirely by the DNA-binding
properties of the homeodomain.
In addition to the DNA-binding homeodomain, members of
the LIM-HD family of transcription factors have two highly
conserved domains in the N-terminal portion of the protein,
called the LIM domains (Freyd et al., 1990; Karlsson et al.,
1990). LIM domains received their name from the first three
founding members of the family: C. elegans Lin-11 and Mec3, plus mammalian Islet-1. LIM domains are cysteine-rich,
zinc-finger like domains (Michelsen et al., 1993; Archer et al.,
1994) which do not bind DNA, but instead have been shown
to mediate protein-protein interactions (Schmeichel and
Beckerle, 1994; Arber and Caroni, 1996, for recent review see
Jurata and Gill, 1998).
Interactions between LIM domains and a number of different
proteins have been well characterized. One family of interacting
proteins binds the LIM domains of LIM-HD and nuclear LIMonly proteins with high affinity. This family includes mouse
NLI/Ldb1/Clim-2, Xenopus Xlbd1, Zebrafish Ldb1-4, and
Drosophila Chip (Agulnick et al., 1996; Jurata et al., 1996;
Bach et al., 1997; Morcillo et al., 1997; Toyama et al., 1998).
NLI binds LIM domains via a specific LIM interacting domain,
and is also capable of self-dimerization (Jurata and Gill, 1997).
In this manner, NLI forms a bridge between two LIM-HD
proteins, building homo- or heterodimeric complexes of LIM-
The LIM-homeodomain (LIM-HD) gene family encodes a large
group of transcriptional regulators, whose members have been
implicated in a variety of developmental processes. Early
embryonic patterning events (Shawlot and Behringer, 1995; Taira
et al., 1997), limb and eye formation (Vogel et al., 1995; Porter
et al., 1997), and the development of imaginal discs in Drosophila
(Cohen et al., 1992; Curtiss and Heilig, 1997) all depend on the
function of LIM-HD family members. LIM-HD proteins are also
expressed in discrete subpopulations of developing neurons (Way
and Chalfie, 1989; Ericson et al., 1992; Tsuchida et al., 1994;
Appel et al., 1995; Matsumoto et al., 1996; Varela-Echavarria et
al., 1996) and play key roles in their differentiation (Way and
Chalfie, 1988; Lundgren et al., 1995; Pfaff et al., 1996; Sheng et
al., 1996; Hobert et al., 1997; Porter et al., 1997; Sheng et al.,
1997; Thor and Thomas, 1997; Hobert et al., 1998).
Although many of the developmental processes that the
LIM-HD transcription factors control have been elucidated, the
mechanisms by which they regulate target genes remains
unknown. It is generally thought that these factors become
localized to the regulatory regions of appropriate target genes
through recognition of specific genomic sequences by their
homeodomains. However, many homeodomains, including
those of the LIM-HD class, appear to bind similar target
sequences in vitro (Hoey and Levine, 1988; Kalionis and
Key words: Drosophila melanogaster, Nervous system,
Development, LIM domain, Homeodomain
3916 D. D. O’Keefe, S. Thor and J. B. Thomas
HD transcription factors (Jurata et al., 1998). In addition to
binding the LIM domain of the LIM-HD family member
Apterous (Ap), the Drosophila NLI homolog, Chip, genetically
interacts with ap in the developing wing, evidence that the ApChip interaction is functional (Morcillo et al., 1997). LIM
domains can also bind directly to other transcription factors,
resulting in synergistic activation of transcription in cultured
cells. For example E47, a bHLH transcription factor, binds the
LIM domains of Lmx-1 (Johnson et al., 1997), and the POU
domain of Pit-1 binds the LIM domains of Lhx-3 (Bach et al.,
1995). In contrast to NLI-LIM interactions, which appear to be
global since NLI binds all nuclear LIM domains tested,
interactions between LIM domains and other transcription
factors are highly specific. For example, E47 binds Lmx-1, but
does not interact with Islet-1 (Johnson et al., 1997). Collectively
these studies suggest that LIM domains play a critical role in
the assembly of protein complexes necessary for target gene
regulation and indicate that LIM domains mediate a variety of
specific protein interactions.
While the nature of protein interactions mediated by LIM
domains are beginning to be elucidated in vitro, the functional
contribution of the LIM domains in vivo is virtually unknown.
Here we use Drosophila to investigate the role of the LIM
domains of the LIM-HD member Apterous (Ap). ap plays a
key role in a number of developmental processes, including
wing and haltere development (Butterworth and King, 1965;
Cohen et al., 1992; Diaz-Benjumea and Cohen, 1993; Blair et
al., 1994), muscle differentiation (Bourgouin et al., 1992), axon
guidance within the embryonic nervous system (Lundgren et
al., 1995) and juvenile hormone production (Altartz et al.,
1991). Adults mutant for ap eclose at a low frequency and are
wingless, highly uncoordinated, sterile and short-lived, dying
within 1-2 days after eclosion. We have chosen to examine two
aspects of the ap phenotype in detail: wing development and
embryonic axon pathfinding.
Using the GAL4 system (Brand and Perrimon, 1993) to
direct expression of transgenes specifically in ap-expressing
cells, we have found that the LIM domains, as well as the
homeodomain, are essential for Ap function. Furthermore,
expression of LIM domains alone can act in a dominantnegative fashion to disrupt Ap function, causing ap mutant
phenotypes. Analysis of chimeric LIM-HD proteins establishes
that LIM domains are interchangeable between family
members in the generation of wing structure. In contrast, for
axon pathfinding of the Ap neurons, the LIM domains confer
functional specificity and cannot be replaced by those of
another family member. This suggests that the Ap LIM
domains mediate qualitatively different protein interactions in
these two developmental processes.
MATERIALS AND METHODS
DNA constructs
All manipulations of the ap cDNA are based on the published full length
cDNA clone (Bourgouin et al., 1992) in pKS Bluescript vector
(Stratagene). UAS-ap was generated by XbaI excision of the entire ap
open-reading frame from pKS16-5 and insertion into the pUAS vector
(Brand and Perrimon, 1993). To generate UAS-ap∆HD, sequences
encoding aa 1-338 were amplified from pKS16-4 by polymerase chain
reaction (PCR), ligated into pKS by TA cloning (Marchuk et al., 1990),
then excised with XbaI and cloned into pUAS. For UAS-ap∆LIM, PCR-
based mutagenesis (Ho et al., 1989) was used to eliminate sequences
encoding aa 148-260 (the two LIM domains). The resulting PCR
fragment was inserted into the pUAS XbaI site as above. The UAS-lim3
construct comprised a HindIII/NotI digested lim3 cDNA fragment from
pNB40-lim3 (S. T., S. G. E. Andersson, A. Tomlinson and J. B. T.,
unpublished) to which XbaI linkers were ligated for subsequent cloning
into pUAS. For UAS-lim3:ap, sequences encoding aa 1-167 of Lim3
(including the LIM domains) were PCR amplified from pNB40-lim3
adding a 3′ EcoRV restriction site. This PCR fragment was TA cloned
into pKS to create pKS-lim3LIM. Concurrently, sequences encoding aa
339-470 of ap (including the homeodomain) were PCR amplified from
pKS16-5 adding a 5′ EcoRV restriction site and TA cloned into pKS to
create pKS-apHD. Sequences encoding the Lim3 LIM domain were
excised from pKS-lim3LIM using EcoRV and XbaI and inserted into the
EcoRV site of pKS-apHD. The entire lim3:ap chimera was then cloned
into the XbaI site of pUAS. For UAS-islet∆HD, sequences encoding aa
1-226 were PCR amplified and inserted into the pUAS XhoI site as
above. UAS-tau-lacZ was made by EcoRI excision of tau-lacZ (Callahan
and Thomas, 1994) from pKS and transferred into pUAS.
Fly strains and genetics
P-element transformation was carried out as described previously
(Rubin and Spradling, 1982). For each UAS transgene, multiple lines
were generated and checked for expression using the anti-Ap
antibody. Insertions for each UAS element were recombined onto a
UAS-tau-lacZ chromosome to label expressing cells. These
chromosomes were then crossed into an apP44 mutant background.
Each line was then crossed to apGAL4 to analyze the rescuing ability
of the transgene (apGAL4/Cyo,wg-lacZ x apP44/Cyo,wg-lacZ; UAStau-lacZ, UAS-transgene). Mutants were identified by using a CyO
balancer chromosome marked with wg-lacZ. Since UAS-tau-lacZ had
a dominant effect when expressed in the wing disc, this transgene was
omitted for wing analysis. All fly crosses and embryo collections were
carried out at 25°C unless otherwise indicated.
Immunohistochemistry
Embryo dissections and HRP immunostainings were performed as
described previously (Callahan and Thomas, 1994). For fluorescence
immunostaining to detect β-Gal and Ap simultaneously, dissected
embryos were first incubated with rabbit anti-β-Gal antibody (Cappel;
diluted 1:10,000) at 4°C overnight, followed by a biotin-conjugated
secondary antibody (Vector; diluted 1:500) and finally Cy-3conjugated streptavidin (Jackson Immunoresearch Labs; diluted
1:400). Following a twenty minute re-fixation, embryos were
incubated with rat anti-Ap (Lundgren et al., 1995, diluted 1:1000) at
4°C overnight, and then an FITC-conjugated secondary antibody
(Vector; diluted 1:200). X-Gal staining was performed according to
Klämbt et al. (1991) on dissected wing discs fixed for 10 minutes in
4% paraformaldehyde. Confocal microscopy was performed as
described previously (Callahan et al., 1996).
RESULTS
The ap mutant phenotype
During larval development ap is expressed in the dorsal
compartment of the wing disc, the region giving rise to the
notum, scutelum, wing hinge and dorsal surface of the wing
blade (Cohen et al., 1992). ap functions to restrict these cells to
a dorsal identity, and is necessary for normal wing margin
formation (Diaz-Benjumea and Cohen, 1993; Blair et al., 1994).
In flies homozygous for apP44, a null allele of ap (Bourgouin et
al., 1992), the wing is completely eliminated. Within the
embryonic ventral nerve cord (VNC), ap is expressed by three
of the approximately 200 neurons in each abdominal
LIM domain function 3917
hemisegment (Bourgouin et al., 1992). Using promoter fusions
to the axon-targeting tau-lacZ reporter (Callahan and Thomas,
1994), we previously showed that the ap-expressing neurons are
interneurons that extend axons ipsilaterally and anteriorly along
a single pathway within each longitudinal connective (Lundgren
et al., 1995). Upon reaching the adjacent anterior segment the
Ap neurons tightly fasciculate with their homologues, forming
a discrete axon bundle running the length of the VNC (Fig. 1F).
In apP44 mutant embryos the ap neurons fail to
recognize their appropriate pathway and instead
wander within the connective, failing to fasciculate
with one another.
A full-length ap transgene rescues the ap mutant
phenotype
We first tested whether rescue of the ap mutant phenotype was
possible by re-supplying wild-type function using the apGAL4
allele to drive expression of an ap cDNA (Fig. 2). A single copy
of a UAS-ap transgene almost completely rescues the wing
defects of apGAL4/apP44 individuals (Fig. 3A). The wing blade
is of appropriate size with a normal margin and vein pattern,
apGAL4 directs transgene expression
specifically in ap cells
To express the Ap variants in this study we made use
of the GAL4/UAS system (Brand and Perrimon,
1993). We used a P[GAL4] enhancer trap insertion in
the ap locus, isolated by Calleja et al. (1996). We found
that this P[GAL4] line, which we have named apGAL4,
is capable of driving reproducibly high levels of UAS
transgene expression in the ap cells within the wing
disc and the central nervous system (CNS). In
apGAL4/+ individuals carrying a UAS-lacZ transgene,
β-galactosidase (β-gal) activity is specifically localized
to the cells of the dorsal compartment of the wing disc
where ap is normally expressed (Fig. 1A). We
determined the identity and behavior of the GAL4expressing cells in the CNS using a UAS-tau-lacZ
transgene to label axon processes. Double labeling of
apGAL4/+; UAS-tau-lacZ/+ embryos for Ap and β-gal
revealed that apGAL4 drives high levels of Tau-β-gal
specifically in ap neurons, and that the axon processes
of these neurons project normally within the
connectives (Fig. 1B).
apGAL4 is a strong mutant allele of ap
By several criteria, apGAL4 acts as a strong
hypomorphic allele of ap. apGAL4/apP44 individuals
show only a slightly more extreme wing phenotype
than apGAL4 homozygotes. Like apP44 homozygotes,
apGAL4/apP44 individuals have no wings, but often
have a ribbon-like outgrowth in the wing region that
lacks any recognizable structures (Fig. 1G). This
ribbon-like outgrowth is similar to that observed when
apP44 is placed in trans to the temperature-sensitive
allele apts78j (Wilson, 1981) and the flies are raised at
non-permissive temperatures (data not shown). Such
an outgrowth is rarely seen in apP44 homozygotes,
suggesting that apGAL4 retains a low level of ap
function. Like null mutants, apGAL4 mutant
individuals are sterile, uncoordinated and exhibit
precocious adult death.
Within the CNS of apGAL4/apP44 embryos, Ap
protein levels are undetectable and axon pathfinding
errors, as assayed with a UAS-tau-lacZ reporter
transgene, are indistinguishable from those of apP44
homozygotes (Fig. 1C,H). As in apP44 homozygotes,
the Ap neurons still project anteriorly but fail to
choose a common pathway, wandering within the
connectives and remaining highly defasciculated.
Fig. 1. apGAL4 directs transgene expression in ap cells and is a mutant allele of
ap. (A) An apGAL4/+;UAS-lacZ/+ third instar larval wing disc stained with XGal. β-gal activity is confined to the dorsal compartment of the wing disc
where ap is normally expressed. (B-D) Double immunofluorescence labeling
for Apterous (green) and Tau-β-gal (red) in dissected embryonic ventral nerve
cords (stage 16-17). The left hemisegments of two adjacent abdominal
segments are shown. In apGAL4/+; UAS-tau-lacZ embryos (B), Tau-β-gal
immunoreactivity is restricted to the three neurons per hemisegment that
express Ap. Arrow points to the three Ap neurons in the top hemisegment.
Slight variations in Tau-β-gal levels may reflect variability in the strength of
GAL4 transactivation. In apGAL4/apP44;UAS-tau-lacZ/+ mutant embryos (C),
there is no detectable Ap immunoreactivity in the Tau-β-gal-expressing Ap
neurons. (D) Introduction of a single UAS-ap transgene into an apGAL4/apP44
mutant background (genotype = apGAL4/apP44;UAS-ap,UAS-tau-lacZ/+)
restores Ap immunoreactivity to the Tau-β-gal-expressing Ap neurons.
(E,G) Adult apGAL4/+ heterozygotes (E) have wild-type wings, whereas
apGAL4/apP44 mutants (G) show a strong hypomorphic ap mutant phenotype in
which the wings are reduced to unstructured ribbon-like protrusions (arrow).
(F,H) Dissected embryonic VNCs from individuals carrying a UAS-tau-lacZ
transgene to visualize the Ap neurons. Embryos were stained for Tau-β-gal
using HRP immunohistochemistry. In apGAL4/+;UAS-tau-lacZ/+ embryos (F),
the Ap neurons are tightly fasciculated, forming a discrete axon bundle within
the connectives (arrowhead). Arrow points to the cell body of the dorsal Ap
neuron; the two ventral cell bodies are out of the focal plane. In
apGAL4/apP44;UAS-tau-lacZ/+ mutant embryos (H), the Ap neurons exhibit
pathfinding errors, wandering within the connectives and failing to fasciculate
with one another (arrowhead).
3918 D. D. O’Keefe, S. Thor and J. B. Thomas
LIM
apGAL4
Ap
HD
Ap LIM
Ap HD
Lim3
Lim3:Ap
Fig. 2. Schematic representation of transgenes tested for their ability
to rescue the ap phenotype. Full-length, truncated and chimeric
versions of the ap and lim3 cDNAs were cloned downstream of the
GAL4 Upstream Activating Sequences (UAS), enabling apGAL4 to
express these transgenes specifically in ap cells. The Ap protein is
470 amino acids (aa) in length. Ap∆LIM eliminates aa 148-260
comprising the two LIM domains. Ap∆HD truncates the protein
between the LIM and homeodomain at aa 338 (the homeodomain is
aa 367-426). Lim3 is 442 aa in length. The Lim3:Ap chimera fuses
aa 1-169 of Lim3 (containing the LIM domains) to aa 339-470 of Ap
(containing the homeodomain).
but is held at right angles away from the body, resembling the
phenotype of mild ap hypomorphic mutations in which only the
hinge region is affected (Wilson, 1981; Bourgouin et al., 1992).
This mild wing defect may result from failure of apGAL4 to drive
completely wild-type expression in the developing wing disc,
or from the inherent delay in the timing of expression using
GAL4/UAS-mediated transactivation (Brand and Perrimon,
1993; Lin et al., 1995). A single copy of UAS-ap is also capable
of rescuing both the sterility and uncoordinated behavior of
apGAL4/apP44 individuals.
To examine the ap neurons in these rescued individuals, we
recombined the UAS-tau-lacZ and UAS-ap transgenes onto the
same chromosome. Within the VNC of apGAL4/apP44 embryos
carrying one copy each of UAS-ap and UAS-tau-lacZ, Ap
immunoreactivity is clearly restored in the Ap neurons (Fig.
1D) and the behavior of these neurons is indistinguishable from
wild-type (Figs 3B, 6). The axons of the Ap neurons in these
rescued individuals fasciculate tightly with one another and
choose a single pathway as they project anteriorly within the
connectives. Thus, re-supplying ap function with UAS-ap fully
rescues the nervous system phenotype.
Both the LIM and homeodomain of Ap are
necessary for function
We capitalized on the apGAL4/UAS-ap-mediated phenotypic
rescue to ask whether the Ap LIM domains are required for
function. To generate Ap∆LIM, we specifically eliminated the
LIM domains, and left the rest of the protein intact (Fig. 2).
This Ap derivative is unable to rescue any element of the ap
phenotype when expressed using apGAL4 in ap mutant cells,
although Ap∆LIM protein is present at high levels and is
properly localized to the nucleus as assayed with the anti-Ap
antibody (data not shown). In apGAL4/apP44; UAS-ap∆LIM/+
adults, the wings remain ribbon-like outgrowths, devoid of any
identifiable structures (Fig. 3C). Using the UAS-tau-lacZ
transgene we found that the ap neurons remain defasciculated,
Fig. 3. The LIM domain and homeodomain of Ap are necessary for
function. Pairs of panels show the wing and embryonic CNS
phenotypes of apGAL4/apP44 mutant individuals carrying a single
copy of the UAS transgene denoted. To visualize the Ap neurons in
the CNS, a UAS-tau-lacZ reporter transgene was included.
Embryos were stained for Tau-β-gal using HRP
immunohistochemistry. (A,B) Full-length Ap rescues the wing and
embryonic CNS ap phenotypes. (A) The wings are of normal size,
shape and veination, although they are held away from the body.
(B) Within the CNS, the Ap neurons recognize their correct
pathway and are tightly fasciculated (arrowhead), indistinguishable
from wild-type. (C,D) Ap∆LIM, a LIM domain-deleted version of
Ap, is unable to rescue either the wing or CNS ap mutant
phenotypes. Wings remain ribbon-like protrusions (arrow) (C) and
the Ap neurons remain highly defasciculated, choosing multiple
pathways within the connectives (arrowhead) (D). These
individuals are indistinguishable from apGAL4/apP44 mutants.
(E,F) Ap∆HD, a homeodomain-deleted version of Ap, is unable to
rescue either the wing or CNS ap phenotypes. (E) The ribbon-like
outgrowth observed in apGAL4/apP44 mutants is entirely eliminated
(arrow), suggesting that Ap∆HD acts as a dominant-negative factor
in the wing to disrupt residual ap function. (F) In the CNS the Ap
neurons remain highly defasciculated (arrowhead),
indistinguishable from apGAL4/apP44 mutants. Quantitation of the
CNS phenotypes are shown in Fig. 6.
indistinguishable from those of apGAL4/apP44 mutant
individuals (Figs 3D, 6). Therefore, the LIM domains are
essential for ap function.
We also tested whether the Ap homeodomain is necessary for
function. To generate Ap∆HD, we truncated Apterous between
the LIM domains and the homeodomain, thereby eliminating
the homeodomain and the C-terminal end of the protein (Fig.
2). Like Ap∆LIM, this construct is unable to rescue either the
ap wing or CNS phenotypes when expressed in ap mutant cells
under the control of apGAL4, indicating that the homeodomain
is also required for ap function (Figs 3E,F, 6).
LIM domain function 3919
Fig. 4. Expression of LIM domains disrupts ap function in the
developing wing. (A) Expression of Ap∆HD in apGAL4/+;UASap∆HD/+ flies produces a dominant wing phenotype consisting of
blisters (arrow) and margin defects (arrowhead). (B) The dominant
phenotype of UAS-ap∆HD is suppressed completely with a single
UAS-ap transgene (genotype = apGAL4/+;UAS-ap∆HD/UAS-ap).
(C) apGAL4/apts78j flies have wings with blisters (arrow) and margin
defects (arrowhead) when raised at 25°C. (D) Introduction of a single
UAS-ap∆HD transgene into apGAL4/apts78j individuals raised at 25°C
(genotype = apGAL4/apts78j;UAS-ap∆HD/+) eliminates the wings
altogether (arrow), mimicking the ap null mutant phenotype. (E) A
single UAS-isl∆HD transgene also disrupts wing formation in
apGAL4/+ individuals, causing blistering and loss of margins (arrow).
(F) The UAS-isl∆HD dominant phenotype is suppressed completely
with a UAS-ap transgene (genotype = apGAL4/+;UAS-isl∆HD/UAS-ap).
ap∆HD disrupts ap function
We found that Ap∆HD, but not Ap∆LIM, is capable of modifying
the wing phenotype of apGAL4/apP44 flies. In apGAL4/apP44; UASap∆HD/+ flies the ribbon-like outgrowth commonly observed in
apGAL4/apP44 individuals is entirely eliminated, suggesting that
the Ap∆HD protein acts in a dominant-negative fashion to disrupt
residual ap function (Fig. 3E).
To further examine the potential dominant-negative activity
of Ap∆HD, we analyzed its effects in apGAL4/+ heterozygous
individuals, which have normal wings. The wings of apGAL4/+
flies carrying a single copy of UAS-ap∆HD are blistered and
exhibit numerous margin defects (Fig. 4A). The dorsal and
ventral wing surfaces often fail to fuse, resulting in a fluidfilled balloon-like structure. Supplying additional wild-type ap
function with a copy of UAS-ap (genotype = apGAL4/+; UASap∆HD/UAS-ap) fully restores wild-type wing structure,
indicating that Ap∆HD interferes with ap function (Fig. 4B).
We further tested whether Ap∆HD could modify the
phenotypes of an intermediate ap hypomorphic allelic
combination. The apts78j temperature-sensitive allele has nearly
normal wing morphology when raised at 15°C, but completely
Fig. 5. LIM domains are interchangeable for wing formation but not
axon pathfinding of the Ap neurons. Pairs of panels show the wing
and embryonic CNS phenotypes of apGAL4/apP44 mutant individuals
carrying a single copy of the UAS transgene denoted. To visualize the
Ap neurons in the CNS, a UAS-tau-lacZ reporter transgene was
included. (A,B) A UAS-lim3 transgene partially rescues both the wing
(genotype = UAS-lim3/+;apGAL4/apP44) and CNS ap phenotypes. The
wings are stunted and devoid of normal veination, but do have a
rudimentary margin marked by a bristle row (arrow) (A). Within the
CNS, the Ap neurons are more highly fasciculated than in
apGAL4/apP44 individuals, but still exhibit numerous pathfinding errors
(arrowheads, B). (C,D) Lim3:Ap, a fusion of the Lim3 LIM domain
to the Ap homeodomain, rescues the ap wing phenotype to the same
extent as full-length Ap (genotype = apGAL4/apP44;UAS-lim3:ap/+)
(C), but only partially rescues the CNS phenotype, to an extent
indistinguishable from UAS-lim3. Arrowheads point to
defasciculation (top) and midline crossing (bottom) pathfinding errors
(D). Quantitation of the CNS phenotypes are shown in Fig. 6.
lacks all wing structures at 29°C (Wilson, 1981). When raised
at 18°C, apGAL4/apts78j flies have defective wing margins and
unfused wing surfaces (Fig. 4C), a phenotype similar to the
ap∆HD dominant effect in an apGAL4/+ heterozygous
background. Introduction of the UAS-ap∆HD transgene into
this hypomorphic mutant background at 18°C (genotype =
apts78j/apGAL4; UAS-ap∆HD/+) eliminates the wing blade
altogether (Fig. 4D). Therefore, in a genetic background in
which ap functional levels are lowered sufficiently, expression
of the Ap∆HD variant mimics the null mutant phenotype.
The same dominant effect on wing development is also
observed using a HD-deleted version of Islet (Isl), a different
LIM-HD protein normally not expressed in the wing disc (Thor
and Thomas, 1997). In apGAL4/+ individuals carrying a UASisl∆HD transgene, wing structure is severely disrupted (Fig.
4E). As found for Ap∆HD, the Isl∆HD dominant effect is
suppressed by the addition of full-length Ap with a UAS-ap
transgene (Fig. 4F). Thus, the ability to act as a dominantnegative inhibitor of Ap function in the wing is not restricted to
the Ap LIM domains.
In contrast to the wing, neither Ap∆HD nor Isl∆HD has any
dominant effects on the development of the ap neurons within
the CNS. This suggests that for these two developmental
processes, generation of wing structures and axon pathfinding,
there are differences in the protein interactions involving the
Ap LIM domains.
3920 D. D. O’Keefe, S. Thor and J. B. Thomas
Fasciculated segments per embryo (%)
100
A
Wing
80
*
Ch
ip
60
Apterous
*
40
*
B
20
Interneurons
ap
G
A
L4
ap
/+
G
A
L4
/a
pP
44
+
U
A
Sap
+
U
A
Sap
LI
+
M
U
A
Sap
H
D
+
U
A
Slim
+
3
U
A
Slim
3:
ap
0
∆
∆
Apterous
X
apGAL4/apP44
Fig. 6. Quantitation of axon pathfinding data. Each embryonic
segment was scored either as fasciculated or defasciculated.
Fasciculated segments contained only two bundles of axons with no
axons crossing the midline or choosing a separate pathway. For each
embryo, all eight abdominal segments were scored and the
percentage of fasciculated segments was calculated. An average of
12 embryos were examined for each genotype (minimum = 9).
Asterisks denote a statistically significant improvement in axon
fasciculation compared to the apGAL4/apP44 genotype (P≤0.05,
Student’s t-test). Error bars indicate standard error of the mean.
Ap and Lim3 are not interchangeable
The above results establish that the LIM domains are essential
for Ap function. We next tested whether the LIM domains of
a different LIM-HD family member might be interchangeable
with those of Ap. For these studies we chose Drosophila Lim3,
which is normally expressed in subsets of post-mitotic neurons,
none of which co-express Ap (S. T., S. G. E. Andersson, A.
Tomlinson and J.B.T., unpublished). Conservation of LIM
domain sequence within the LIM-HD family ranges from 25%
to 86% aa identity. Ap and Lim3 are relatively divergent,
sharing only 37% identity within the LIM domains.
Expression of Lim3 in Ap cells results in lethality during
larval or early pupal stages. To circumvent these lethal effects
we raised individuals carrying both apGAL4 and UAS-lim3 at
18°C to reduce levels of GAL4-mediated transactivation
(Staehling-Hampton et al., 1994). At this temperature a small
number of apGAL4/apP44; UAS-lim3/+ individuals emerge and
these flies have small outgrowths of tissue in the hinge region
of the thorax (Fig. 5A). Although unstructured, the presence of
a row of bristles on this tissue suggests the development of a
rudimentary dorsal/ventral margin, and indicates that Lim3 can
partially rescue the ap wing phenotype.
Although non-viable at 25°C, apGAL4/apP44; UAS-lim3/+
embryos survive through embryonic stages, allowing us to
examine the behavior of the ap mutant interneurons ectopically
expressing Lim3. Using the UAS-tau-lacZ transgene, we found
that Lim3 partially rescues the ap neuronal pathfinding defects
(Figs 5B, 6). The axons are more highly fasciculated than those
Ch
ip
Apterous
Fig. 7. Model of protein interactions mediated by the Ap LIM
domains. (A) Our data suggests that during wing formation the
primary function of the Ap LIM domains is to interact with Chip.
This interaction is required since Ap∆LIM fails to rescue the ap wing
phenotype and it can be disrupted by the expression of LIM domains
alone. The Lim3:Ap chimera rescues the wing phenotype to the same
extent as full length Ap, likely because the Lim3 LIM domains also
bind Chip. Thus, interchanging the LIM domains has no effect on
formation of the Ap-Chip complex in the generation of wing
structure. (B) During pathfinding of the Ap neurons, the Ap LIM
domains are involved in additional protein interactions independent
of Chip. These interactions are specific to the Ap LIM domains and
cannot be mediated by the Lim3 LIM domains. Taken together, this
data suggests that LIM domains mediate different types of protein
interactions in different developmental processes.
in ap mutants, but 74% of the segments still display clear
pathfinding errors. Thus, although Lim3 promotes some degree
of axon fasciculation and the formation of a rudimentary wing
margin in ap mutants, it is not interchangeable with Ap.
LIM domains are interchangeable for wing formation
but not axon pathfinding
To determine whether LIM domains are interchangeable
between Lim3 and Ap, we created a fusion between the Nterminal half of Lim3, including the LIM domains, to the Cterminal half of Ap, containing the homeodomain (Fig. 2). This
Lim3:Ap chimera, rescues the ap wing phenotype to the same
extent as full-length Ap (Fig. 5C). apGAL4/apP44; UASlim3:ap/+ flies emerge at a high frequency, and their wings are
of appropriate size and shape, displaying wild-type vein
patterns and wing margins. The wings rescued with the
Lim3:Ap chimera are held away from the body, revealing the
same mild hinge defect seen with UAS-ap. Both the sterility
LIM domain function 3921
and uncoordinated behavior of apGAL4/apP44 individuals also
can be rescued with UAS-lim3:ap.
In contrast to its ability to rescue the wing phenotype, the
Lim3:Ap chimera only partially rescues the axon pathfinding
phenotype. The level of rescue seen with the Lim3:Ap chimera
is not significantly different from Lim3 itself (Figs 5D, 6).
Thus, despite their sequence divergence, the Ap and Lim3 LIM
domains are interchangeable in the generation of normal wing
structure, but not in the control of pathfinding of the Ap
neurons, revealing a functional difference between the Ap and
Lim3 LIM domains.
DISCUSSION
The Ap LIM domains are essential for function
Although the ability of LIM domains to interact with other
proteins has been clearly established in vitro and in cultured
cells, their in vivo role is unknown. Within the LIM-HD family,
it has been suggested that LIM domains function to negatively
regulate LIM-HD activity by interfering with the ability of the
homeodomain to bind DNA. This model is based primarily on
two observations. First, LIM-less versions of Islet-1 and Mec3 bind target DNA sequences more effectively in vitro than the
full length proteins (Sanchez-Garcia et al., 1993; Xue et al.,
1993). Second, site-directed mutagenesis of the LIM domains
of Xlim-1 potentiates its ability to form secondary axes when
misexpressed in Xenopus embryos (Taira et al., 1994).
However, our genetic data indicate that negative regulation of
LIM-HD function is not the primary role for the LIM domains.
LIM-less versions of Ap clearly do not act as activated forms
of the protein, as the Xlim-1 data might predict, but instead are
incapable of mediating any discernible ap function within the
wing disc or CNS.
LIM domains act as dominant negative factors in the
wing
Not only does expression of ap∆HD fail to rescue the ap
mutant phenotype, but it disrupts normal ap function in the
wing disc. This result supports previous studies in which
overexpression of Islet-3 LIM domains was found to disrupt
eye and tectal development in the zebrafish embryo,
presumably due to a dominant-negative effect of the LIM
domains on Islet-3 function (Kikuchi et al., 1997). Our finding
that Isl∆HD was just as effective in disrupting ap function as
Ap∆HD suggests that in some circumstances LIM domains
show little or no specificity when interfering with LIM-HD
function. The most likely explanation for this lack of specificity
is that the Ap LIM domains normally mediate a functional
complex with Chip (Morcillo et al., 1997). Extrapolating from
studies of NLI binding (Jurata et al., 1996), Chip is likely to
be capable of binding the LIM domains of most, if not all,
LIM-HD proteins. Thus we would predict that in addition to
Isl∆HD, a ∆HD variant of any family member would compete
with full length Ap for binding to Chip and prevent formation
of a functional complex.
Why do we not see a dominant effect of Ap∆HD and Isl∆HD
in the CNS? One possibility is that Chip or Ap (or both) are
expressed at sufficiently high levels in the Ap neurons that they
are refractory to the levels of Ap∆HD driven by apGAL4.
Alternatively, Ap function in axon pathfinding may require
other, non-Chip-dependent interactions that are unaltered by
Ap∆HD. The latter possibility is supported by the finding that
the LIM domain-dependent synergism exhibited by Lmx-1 and
E47 in transcriptional activation cannot be abolished by
overexpressing LIM domains (German et al., 1992).
Ap and Lim3 are not interchangeable
While there is a clear difference in the ability of Ap and Lim3
to rescue the ap mutant phenotype, Lim3 is capable of partial
rescue. In ap mutants, Lim3 can induce a wing-like structure
with an irregular margin and unfused dorsal and ventral
surfaces. In the CNS, Lim3 enables Ap mutant interneurons to
fasciculate more tightly. This result is surprising given the
divergence between the two family members (only 37% aa
identity within the LIM domains and 46% within the
homeodomain). Although the molecular basis for this partial
rescue remains unknown, there are two extremes among the
various possibilities. First, when expressed in Ap cells, Lim3
may regulate those genes normally regulated by Ap, albeit not
as effectively. However, it seems unlikely that Lim3 regulates
exactly the same set of genes as Ap since individuals that
misexpress high levels of Lim3 using apGAL4 (at 25oC) are
completely non-viable, while individuals expressing high levels
of full-length Ap under identical conditions are viable. The
other extreme predicts that Lim3 regulates an entirely different
set of effector target genes, but that these effectors serve similar
functions and are sufficient to give some degree of phenotypic
rescue. For example, within the CNS, Ap and Lim3 control
specific pathfinding behaviors of neurons that normally express
them, presumably by regulating the expression of distinct sets
of cell recognition molecules (Lundgren et al., 1995; S. T., S.
G. E. Andersson, A. Tomlinson and J. B. T., unpublished). Thus
it is conceivable that both LIM-HD members regulate similar
types of target genes involved in controlling pathway
recognition and axon fasciculation.
Specificity of LIM domains
Our results with the Lim3:Ap chimera demonstrate that in the
developing wing the LIM domains of Ap and Lim3 are
completely interchangeable. The simplest explanation for this
interchangeability is that the primary function of the Ap LIM
domains is to bind Chip. Thus, any nuclear LIM domain would
be able to carry out this function. Based on studies of Chip and
NLI (Morcillo et al., 1997; Jurata et al., 1998), we predict that
in the developing wing Ap forms Chip-mediated homodimers
or possibly heterodimers with another, as yet unidentified LIMHD family member (Fig. 7A).
In contrast, our analysis of the Lim3:Ap chimera in the CNS
indicates that the Ap and Lim3 LIM domains are not
interchangeable. While full-length Ap fully rescues the ap
pathfinding phenotype, Lim3:Ap shows only partial rescue, to
an extent no greater than Lim3 itself. This result suggests that
there are functional differences between LIM domains, a
notion supported by the finding that LIM domains of different
LIM-HD family members show specificity in their ability to
bind other proteins (e.g., Jurata et al., 1998). Whereas Ap-Chip
complexes may be sufficient to regulate target genes in the
wing disc, in the Ap neurons it is likely that additional
interactions with other factors are required for full Ap function,
and that these interactions are specific to the Ap LIM domains
(Fig. 7B).
3922 D. D. O’Keefe, S. Thor and J. B. Thomas
Our findings support the notion that interactions among
transcription factors play critical roles in the specificity of
target gene regulation (Xue et al., 1993; Copeland et al., 1996;
Guichet et al., 1997; Yu et al., 1997; Zelzer et al., 1997). For
example, within the bHLH/PAS family of transcription factors,
it is the protein-interacting PAS domain and not the DNA
binding domain which dictates target specificity (Zelzer et al.,
1997). It has also been shown that a homeodomain-deleted
version of the Fushi tarazu (Ftz) protein is capable of regulating
ftz-dependent segmentation, presumably through interactions
with other transcription factors such as the pair-rule protein
Paired (Copeland et al., 1996) or the nuclear hormone receptor
Ftz-F1 (Guichet et al., 1997; Yu et al., 1997). Our analysis of
the LIM-HD protein Apterous further underscores the
importance of protein-protein interactions in transcription
factor function, and suggests that LIM domains are involved in
different types of protein-protein interactions in different
developmental processes.
We thank Andrew Tomlinson for providing the lim-3 cDNA; Rusty
Gage and Dan Peterson for help with confocal imaging; Don Van
Meyel and Greg Lemke for critical reading of the manuscript; Linda
Jurata and Gordon Gill for insightful discussions; the members of the
Thomas lab for providing advice, technical support and good humor;
Karolyn Ronzano for her contributions beyond measure; and Blanca
in San Felipe for all the delicious fish tacos. This work was supported
by an NIH Training Grant to D. D. O’K., grants from the NIH to J.
B. T. and a Human Frontiers Science Program Long-term Fellowship
to S. T.
REFERENCES
Agulnick, A. D., Taira, M., Breen, J. J., Tanaka, T., Dawid, I. B. and
Westphal, H. (1996). Interactions of the LIM-domain-binding factor Lbd1
with LIM homeodomain proteins. Nature 384, 270-272.
Altartz, M., Applebaum, S. W., Richard, D. S., Gilbert, L. I. and Segal, D.
(1991). Regulation of juvenile hormone synthesis in wild-type and ap
mutant Drosophila. Mol. Cell. Endocrinol. 81, 205-216.
Appel, B., Korzh, V., Glasgow, E., Thor, S., Edlund, T., Dawid, I. B. and
Eisen, J. S. (1995). Motorneuron fate specification revealed by patterned
LIM homeobox gene expression in embryonic zebrafish. Development 121,
4117-4125.
Arber, S. and Caroni, P. (1996). Specificity of single LIM motifs in targeting
and LIM/LIM interactions in situ. Genes Dev. 10, 289-300.
Archer, V. E. V., Breton, J., Sanchez-Garcia, I., Osada, H., Forster, A.,
Thomson, A. J. and Rabbitts, T. H. (1994). Cysteine-rich LIM domains
of LIM-homeodomain and LIM-only proteins contain zinc but not iron.
Proc. Natl. Acad. Sci. USA 91, 316-320.
Bach, I., Carriere, C., Ostendorff, H. P., Andersen, B. and Rosenfeld, M.
G. (1997). A family of LIM domain-associated cofactors confer
transcriptional synergism betwwn LIM and Otx homeodomain proteins.
Genes Dev. 11, 1370-1380.
Bach, I., Rhodes, S. J., Pearse, R. V. I., Heinzel, T., Gloss, B., Scully, K.
M., Sawchenko, P. E. and Rosenfeld, M. G. (1995). P-Lim, a LIM
homeodomain factor, is expressed during pituitary organ and cell
commitment and synergizes with Pit-1. Proc. Natl. Acad. Sci. USA 92, 27202724.
Blair, S. S., Brower, D. L., Thomas, J. B. and Zavortink, M. (1994). The
role of apterous in the control of doroventral compartmentalization and PS
integrin gene expression in the developing wing of Drosophila.
Development 120, 1805-1815.
Bourgouin, C., Lundgren, S. E. and Thomas, J. B. (1992). apterous is a
Drosophila LIM domain gene required for the development of a subset of
embryonic muscles. Neuron 9, 549-561.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means
of altering cell fates and generating dominant phenotypes. Development 118,
401-415.
Butterworth, F. M. and King, R. C. (1965). The developmental genetics of
ap mutants of Drosophila melanogaster. Genetics 52, 1153-1174.
Callahan, C. A., Bonkovsky, J. L., Scully, A. L. and Thomas, J. B. (1996).
derailed is required for muscle attachment site selection in Drosophila.
Development 122, 2761-2767.
Callahan, C. A. and Thomas, J. B. (1994). Tau-β-galactosidase, an axontargeted fusion protein. Proc. Natl. Acad. Sci. USA 91, 5972-5976.
Calleja, M., Moreno, E., Pelaz, S. and Morata, G. (1996). Visualization
of Gene Expression in Living Adult Drosophila. Science 274, 252255.
Cohen, B., McGuffin, M. E., Pfeifle, C., Segal, D. and Cohen, S. M.
(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.
Copeland, J. W. R., Nasiadka, A., Dietrich, B. H. and Krause, H. M.
(1996). Patterning of the Drosophila embryo by a homeodomain-deleted Ftz
polypeptide. Nature 379, 162-165.
Curtiss, J. and Heilig, J. S. (1997). Arrowhead Encodes a LIM Homeodomain
Protein That Distinguishes Subsets of Drosophila Imaginal Cells. Dev. Biol.
190, 129-141.
Diaz-Benjumea, F. J. and Cohen, S. M. (1993). Interaction between dorsal
and ventral cells in the imaginal disc directs wing development in
Drosophila. Cell 75, 741-752.
Ericson, J., Thor, S., Edlund, T., Jessell, T. M. and Yamada, T. (1992). Early
stages of motor neuron differentiation revealed by expression of homeobox
gene Islet-1. Science 256, 1555-60.
Freyd, G., Kim, S. K. and Horvitz, H. R. (1990). Novel cysteine-rich motif
and homeodomain in the product of the Caenorhabditis elegans cell lineage
gene lin-11. Nature 344, 876-879.
German, M. S., Wang, J., Chadwick, R. B. and Rutter, W. J. (1992).
Synergistic activation of the insulin gene by a LIM-homeo protein and a
basic helix-loop-helix protein: building a functional insulin minienhancer
complex. Genes Dev. 6, 2165-2176.
Guichet, A., Copeland, J. W. R., Erdelyl, M., Hlousek, D., Zavorszky, P.,
Ho, J., Brown, S., Percival-Smith, A., Krause, H. M. and Ephrussi, A.
(1997). The nuclear receptor homologue Ftz-F1 and the homeodomain
protein Ftz are mutually dependent cofactors. Nature 385, 548-552.
Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K. and Pease, L. R. (1989).
Site-directed mutagenesis by overlap extension using the polymerase chain
reaction. Gene 77, 51-59.
Hobert, O., D’Alberti, T., Liu, Y. and Ruvkun, G. (1998). Control of neural
development and function in a thermoregulatory network by the LIM
homeobox gene lin-11. J. Neurosci 18, 2084-96.
Hobert, O., Mori, I., Yamashita, Y., Honda, H., Ohshima, Y., Lui, Y. and
Ruvkun, G. (1997). Regulation of Interneuron Function in the c. elegans
Thermoregulatory Pathway by the ttx-3 LIM Homeobox Gene. Neuron 19,
345-357.
Hoey, T. and Levine, M. (1988). Divergent homeo box proteins recognize
similar DNA sequences in Drosophila. Nature 332, 858-861.
Johnson, J. D., Zhang, W., Rudnick, A., Rutter, W. J. and German, M. S.
(1997). Transcriptional Synergy between LIM-Homeodomain Proteins and
Basic Helix-Loop-Helix Proteins: the LIM2 Domain Determines Specificity.
Mol. Cell. Biol. 17, 3488-3496.
Jurata, L. W. and Gill, G. N. (1997). Functional analysis of the nuclear LIM
domain interactor NLI. Mol. Cell. Biol. 17, 5688-5698.
Jurata, L. W. and Gill, G. N. (1998). Structure and Function of LIM
Domains. Curr. Top. Microbiol. Immunol. 228, 75-113.
Jurata, L. W., Kenny, D. A. and Gill, G. N. (1996). Nuclear LIM interactor,
a rhombotin and LIM homeodomain interacting protein, is expressed
early in neuronal development. Proc. Natl. Acad. Sci. USA 93, 1169311698.
Jurata, L. W., Pfaff, S. L. and Gill, G. N. (1998). The nuclear LIM domain
interactor NLI mediates homo- and heterodimerization of LIM domain
transcription factors. J. Biol. Chem. 273, 3152-7.
Kalionis, B. and O’Farrell, P. H. (1993). A universal target sequence is bound
in vitro by diverse homeodomains. Mech. Dev. 43, 57-70.
Karlsson, O., Thor, S., Norberg, T., Ohlsson, H. and Edlund, T. (1990).
Insulin gene binding protein Isl-1 is a member of a novel class of
proteins containing both a homeo- and a Cys-His domain. Nature 344,
879-882.
Kikuchi, Y., Segawa, H., Tokumoto, M., Tsubokawa, T., Hotta, Y.,
Uyemura, K. and Okamoto, H. (1997). Ocular and Cerebellar Defects in
Zebrafish Induced by Overexpression of the LIM Domains of the Islet-3
LIM/Homeodomain Protein. Neuron 18, 369-382.
LIM domain function 3923
Klämbt, C., Jacobs, J. R. and Goodman, C. S. (1991). The midline of the
Drosophila central nervous system: a model for the genetic analysis of cell
fate, cell migration, and growth cone guidance. Cell 64, 801-815.
Lin, D. M., Auld, V.J. and Goodman, C. S. (1995). Targeted neuronal
ablation in the Drosophila embryo: pathfinding by follower growth cones in
the absence of pioneers. Neuron 14, 707-715.
Lundgren, S. E., Callahan, C. A., Thor, S. and Thomas, J. B. (1995).
Control of neuronal pathway selection by the Drosophila LIM
homeodomain gene apterous. Development 121, 1769-1773.
Marchuk, D., Drumm, M., Saulino, A. and Collins, F. S. (1990).
Construction of T-vectors, a rapid and general system for direct cloning of
unmodified PCR products. Nuc. Acids Res. 19, 1154.
Matsumoto, K., Tanaka, T., Furuyama, T., Kashihara, Y., Ishii, N.,
Tohyama, M., Kitanaka, J., Takemura, M., Mori, T. and Wanaka, A.
(1996). Differential expression of LIM-homeodomain genes in the
embryonic murine brain. Neurosci. Lett. 211, 147-150.
Michelsen, J. W., Schmeichel, K. L., Beckerle, M. C. and Winge, D. R.
(1993). The LIM motif defines a specific zinc-binding protein domain. Proc.
Natl. Acad. Sci. USA 90, 4404-4408.
Morcillo, P., Rosen, C., Baylies, M. K. and Dorsett, D. (1997). Chip, a
widely expressed chromosomal protein required for segmentation and
activity of a remote wing margin enhancer in Drosophila. Genes Dev. 11,
2729-2740.
Pfaff, S. L., Mendelsohn, C. L., Stewart, C. L., Edlund, T. and Jessell, T.
M. (1996). Requirement for LIM Homeobox Gene Isl-1 in Motor Neuron
Generation Reveals a Motor Neuron-Dependent Step in Interneuron
Differentiation. Cell 84, 309-320.
Porter, F. D., Drago, J., Xu, Y., Cheema, S. S., Wassif, C., Huang, S., Lee,
E., Grinberg, A., Massalas, J. S., Bodine, D., Alt, F. and Westphal, H.
(1997). Lhx2, a LIM homeobox gene, is required for eye, forebrain, and
definitive erythrocyte development. Development 124, 2935-2944.
Rubin, G. M. and Spradling, A. C. (1982). Genetic transformation of
Drosophila with transposable element vectors. Science 218, 348-353.
Sanchez-Garcia, I., Osada, H., Forster, A. and Rabbitts, T. H. (1993). The
cysteine-rich LIM domains inhibit DNA binding by the associated
homeodomain in Isl-1. EMBO J. 12, 4243-4250.
Schmeichel, K. L. and Beckerle, M. C. (1994). The LIM Domain Is a
Modular Protein-Binding Interface. Cell 79, 211-219.
Shawlot, W. and Behringer, R. R. (1995). Requirement for Lim1 in headorganizer function. Nature 374, 425-430.
Sheng, H. Z., Moriyama, K., Yamashita, T., Li, H., Potter, S. S., Mahon,
K. A. and Westphal, H. (1997). Multistep control of pituitary
organogenesis. Science 278, 1809-1812.
Sheng, H. Z., Zhadanov, A. B., Mosinger, B. J., Fuji, T., Bertuzzi, S.,
Grinberg, A., Lee, E. J., Huang, S., Mahon, K. A. and Westphal, H.
(1996). Specification of pituitary cell lineages by the LIM homeobox gene
Lhx3. Science 272, 1004-1007.
Staehling-Hampton, K., Hoffmann, F. M., Baylies, M. K., Rushton, E. and
Bate, M. (1994). dpp induces mesodermal gene expression in Drosophila.
Nature 372, 783-786.
Taira, M., Otani, H., Saint-Jeannet, J. and Dawid, I. B. (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.
Taira, M., Saint-Jeannet, J. and Dawid, I. (1997). Role of the Xlim-1 and
Xbra genes in anteroposterior patterning of neural tissue by the head and
trunk organizer. Proc. Natl. Acad. Sci. USA 94, 895-900.
Thor, S. and Thomas, J. B. (1997). The Drosophila islet gene governs axon
pathfinding and neurostransmitter identity. Neuron 18, 1-20.
Toyama, R., Kobayashi, M., Tomita, T. and Dawid, I. B. (1998). Expression
of LIM-domain binding proteins (ldb) genes during zebrafish
embryogenesis. Mech. Dev. 71, 197-200.
Tsuchida, T., Ensini, M., Morton, S. B., Baldassare, M., Edlund, T., Jessell,
T. M. and Pfaff, S. L. (1994). Topographic organization of embryonic
motor neurons defined by expression of LIM homeobox genes. Cell 79, 957970.
Varela-Echavarria, A., Pfaff, S. L. and Guthrie, S. (1996). Differential
Expression of LIM Homeobox Genes among Motor Neuron
Subpopulations in the Developing Chick Brain Stem. Mol. Cell. Neurosci.
8, 242-257.
Vogel, A., Rodriguez, C., Warnken, W. and Belmonte, J. C. I. (1995).
Dorsal cell fate specified by chick Lmx1 during vertebrate limb
development. Nature 378, 716-720.
Way, J. C. and Chalfie, M. (1988). mec-3, a homeobox-containing gene that
specifies differentiation of the touch receptor neurons in C. elegans. Cell 54,
5-16.
Way, J. C. and Chalfie, M. (1989). The mec-3 hene of Caenorhabditis elegans
requires its own product for maintained expression and is expressed in three
neuronal cell types. Genes Dev. 3, 1823-1833.
Wilson, T. G. (1981). Expression of phenotypes in a temperature-sensitive
allele of the apterous mutation in Drosophila melanogaster. Dev. Biol. 85,
425-433.
Xue, D., Tu, Y. and Chalfie, M. (1993). Cooperative interactions between the
Caenorhabditis elegans homeoproteins UNC-86 and MEC-3. Science 261,
1324-1328.
Yu, Y., Li, W., Su, K., Yussa, M., Han, W., Perrimon, N. and Pick, L.
(1997). The nuclear hormone receptor Ftz-F1 is a cofactor for the
Drosophila homeodomain protein Ftz. Nature 385, 552-555.
Zelzer, E., Wappner, P. and Shilo, B. (1997). The PAS domain confers target
gene specificity of Drosophila bHLH-PAS proteins. Genes Dev. 11, 20792089.