Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, MA 02115, USA
Author for correspondence (e-mail:
steth{at}ifm.liu.se)
Accepted 13 August 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Drosophila, drifter, islet, Lim3, Combinatorial code, Motoneurons
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In both vertebrates and invertebrates, the activity of LIM-HD proteins is
coordinated through multiple mechanisms to specify distinct neuron
subpopulations. Studies in the vertebrate spinal cord show that Isl1 (Tup
FlyBase) and Lhx3 act in concert by forming a hexameric complex with
the LIM co-factor NLI, to regulate motoneuron verses interneuron specification
(Thaler et al., 2002). By
contrast, other studies reveal that Lim1 and Isl1 exert mutual
cross-repressive interactions to control neuronal cell body position and axon
pathway selection (Kania and Jessell,
2003
). POU proteins are also required for the production and
positioning of neurons, as well as playing important roles in axon guidance.
Brn1 and Brn2, both class III POU proteins, control the initiation of radial
migration through the Cdk5 kinase pathway in neocortex neurons
(McEvilly et al., 2002
;
Sugitani et al., 2002
). The
Drosophila class III member, Drifter/Ventral veinless, controls the
distinct dendritic targeting of second order olfactory neurons
(Komiyama et al., 2003
), while
the mouse class IV POU factor Brn3.2 (Pou4f2 Mouse Genome Informatics)
regulates the pathfinding of retinal ganglion cell axons
(Erkman et al., 2000
). In
C. elegans, the POU protein UNC-86 is important for the terminal
differentiation of several neuronal subtypes, in addition to controlling axon
pathfinding in serotonergic neurons
(Duggan et al., 1998
;
Sze et al., 2002
).
Furthermore, LIM-HD and POU members have been shown to function together to
direct cell differentiation in both vertebrates and invertebrates. For
example, UNC-86 and the LIM-HD factor MEC-3 interact genetically and
physically to regulate tough sensory neuron differentiation in C.
elegans (Rockelein et al.,
2000
; Xue et al.,
1993
).
In the Drosophila embryonic ventral nerve cord (VNC), three
well-described motoneuron subtypes are distinguished by their axonal
projections sent out either through the transverse nerve (TN) or the
intersegmental nerve b (ISNb) or d (ISNd) fascicle. Previous studies have
revealed that the LIM-HD proteins, Islet (Isl/Tailup) and Lim3, act in a
combinatorial manner to dictate ISNb versus ISNd motoneuron subclass identity
(Thor et al., 1999). However,
both genes are also expressed in, and are important for, TN motoneuron
differentiation, and thus, how ISNb/d versus TN identity is determined was
unknown. Here, we demonstrate that this LIM-HD combinatorial code requires the
POU domain protein, Drifter/Ventral veinless (Dfr), to confer target
specificity between ISNb and TN motoneuron subclasses. Dfr is co-expressed
with Isl and Lim3 only in the ISNb motoneurons; when Dfr activity is
decreased, a reduction in muscle innervation similar to isl and
Lim3 mutants is observed. In addition, when we add Dfr to the
Isl/Lim3 TN combinatorial code, the TN motor axons are redirected to the ISNb
muscle target field. The retargeting of TN motor axons upon Dfr misexpression
does not occur without Isl function, suggesting cooperative actions between
these transcription factors. Our studies indicate that a specific POU protein
can modify a combinatorial LIM-HD code and act to regulate essential aspects
of target selection by distinct neuronal subgroups.
To identify possible targets of these regulators, we focused on additional
molecules that are differentially expressed between the TN and ISNb motoneuron
subclasses. A recent report described the TN motoneuron expression of the
cell-adhesion molecule (CAM) Beat Ic. beat Ic belongs to a multigene
family in Drosophila that enclodes immunoglobulin superfamily (IgSF)
proteins related to the Beat Ia axon guidance protein
(Pipes et al., 2001). The new
Beat family members, including beat Ic, have restricted neuronal
expression patterns and appear to function in a pro-adhesive manner.
Furthermore, loss of Beat Ic affects the adherence of the transverse motor
nerve and the LBD sensory neuron projection
(Pipes et al., 2001
). We
confirmed that the transverse nerve in beat Ic mutant embryos is
often bifurcated and axons explore the ventral muscle surface. These TN
defects are identical to the fasciculation defects we observe in isl
and Lim3 mutants. We also found strong genetic interactions between
isl, Lim3, and beat Ic. In addition, increasing Beat Ic
expression in isl and Lim3 mutants partly rescues the TN
axon fasciculation defects. These results indicate that the combinatorial code
of two LIM-HD proteins, Isl and Lim3, can regulate not only the trajectory of
a group of defined motor axons but also direct a single specific motoneuron
synaptic connection, and that this axon targeting may be directed through the
actions of IgSF CAMs.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The following strains were used in this study: isl37Aa
Lim3Bd6 (S.T. and J. B. Thomas, unpublished);
Lim3BD1, isl alleles tup1 and
tup2; Df(2L)OD15 removes the isl locus
and is herein noted as islDf; Df(2L)TW130 removes
the Lim3 locus (further designated as Lim3Df);
and Df(2L)E71 removes both the isl and Lim3 loci
(Thor et al., 1999;
Wright et al., 1976
). The
following dfr alleles were used: the strong hypomorph
dfrE82 (Anderson et
al., 1995
), the null allele dfrB129
(truncation mutation before the POU domain) (W. A. Johnson, unpublished) and
Df(3L)XBB70 which removes the dfr locus and is further
designated as dfrDf
(Anderson et al., 1995
). To
remove beat Ic function the deficiencies Df(2L)TE35D-GW19
(herein noted as beat IcDf) and Df(2L)RM5
(further designated as beat IcDf1)
(Pipes et al., 2001
) were
used. The hb9KK30 allele was used
(Broihier and Skeath, 2002
).
The islH-tau-myc, Lim3A-tau-myc and Lim3A-lacZ transgenic
lines were employed. Mutations were maintained over CyO or TM6B Tb balancers
with lacZ or GFP markers.
Immunohistochemistry
Staged embryos were labeled using a modification of protocols previously
described (Certel and Johnson,
1996). The following primary antibodies were used: rabbit anti-GFP
(1:500) (Molecular Probes), rabbit anti-Glutactin (1:300)
(Olson et al., 1990
), mAb 1D4
anti-Fas2, (1:50), mAb 3A4 anti-Islet1/2, (1:20)
(Tsuchida et al., 1994
), mAb
9E10 anti-Myc (1:25) and mAb C555.6D anti-Slit, (1:50)
(Rothberg et al., 1990
). Slit
staining was performed using 0.1% Tween 20 as the detergent
(Crowner et al., 2002
).
ß-Gal protein was detected either using a rabbit polyclonal
anti-ß-gal, (1:500) (Cappel) or a mouse monoclonal mAb 40-1a
anti-ß-gal (1:5). Dfr expression was detected using preabsorbed anti-Dfr
rat sera at a 1:2000 dilution (Anderson et
al., 1995
). Monoclonal antibodies were obtained from the NIH
supported Developmental Studies Hybridoma Bank maintained by the University of
Iowa, Dept of Biological Sciences.
Secondary antibodies include biotinylated goat anti-mouse, goat anti-rabbit (1:1000) (Vector), Alexa Fluor 488-conjugated goat anti-rabbit, goat anti-mouse, goat anti-rat (Molecular Probes), Rhodamine-Red X-conjugated donkey anti-mouse, donkey anti-rat (Jackson ImmunoResearch Laboratories), Cy5-conjugated donkey anti-rat, anti-rabbit (Jackson ImmunoResearch Laboratories). All fluorescein-conjugated secondary antibodies were highly cross-adsorbed for use in multi-labeling experiments. In some figures, double-labeled images were false colored, converting red to magenta for the benefit of colorblind readers.
We generated additional Dfr rat serum by producing a glutathione S-transferase-Dfr (GST-Dfr) fusion protein containing amino acids 319-436 of the Dfr protein fused to the C-terminal end of the GST polypeptide. This region does not include the POU domain. The GST-Dfr fusion protein was expressed and purified using glutathione-agarose according to protocols provided by the manufacturer. Covance Research Products (Denver, PA) generated polyclonal rat antibodies.
Immunohistochemistry and in situ hybridization
In situ hybridization was carried out as previously described
(O'Neill and Bier, 1994). The
cDNA from EST GH22661 obtained from Research Genetics was used to generate
digoxigenin-labeled RNA probes (Boehringer Mannheim). The sense (control)
probe did not produce any specific signal. Double RNA and antibody labeling
was performed by blocking the embryos with PBS/0.1%Tween20/10%BSA following
hybridization with the RNA probe. Following blocking, the embryos were
incubated with the rabbit anti-GFP antibody (1:500 dilution, Molecular
Probes), followed by a biotinylated anti-rabbit secondary antibody (Vector
Labs). The sheep anti-DIG antibody (1:2000 dilution, Roche) and the Vectastain
Streptavidin system were added together. The antibody was detected through
standard diaminobenzidine development followed by PBS/Tween20 washes and then
the in situ signal was visualized with alkaline phosphatase development.
DNA manipulation and P element transformations
Germ-line transformants were generated as previously described
(Spradling, 1986). Multiple
insertion lines were isolated and established for each construct.
The Lim3B-Gal4 construct was constructed by inserting the 3.9 kb
EcoRI/NruI fragment from Lim3 fragment A
(Thor et al., 1999) into a
modified pCaSpeR 2/17 vector containing the Gal4-coding region [S.T.,
unpublished (details available upon request)]. Lim3B-Gal4 expression
recapitulates the Lim3A-lacZ expression in the CNS (not shown).
UAS-dsdfr transgenes were generated by producing a 1582 bp
inverted-repeat fragment through PCR amplification of the 5' end of the
dfr cDNA. The following primers were used to generate a dfr
fragment with dyad symmetry. The first product had an EcoRI site at
the 517 bp end, dfr5'RI-GTCGAATTCAAGACGGTTGCCTCACGGTTC and a
SfiI site at the 1308 end,
dfr3'SfiI-GTCGGCCATCTTGGCCTGCTGCAACTGATCGCTCGTG. The second
product had an XhoI site at the 517 end
dfr5'XhoI-GTCGAGAAGACGGTTGCCTCACGGTTC and a SfiI site at the
1308 end, dfr3'SfiI(2)-GTCGGCCTAGATGGCCTGCTGCAACTGATCGCTCGTG.
Underlined sequences denote the central nonpalindromic core of each site. This
791 bp fragment does not include the POU domain. After digestion with
SfiI, the two products were ligated together. Dimers were digested
with EcoRI and XhoI and cloned into the pUAST P-element
transformation vector provided by Andrea Brand
(Brand and Perrimon, 1993).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To verify the lack of Dfr expression in the ISNd subgroup, we focused on
the well-characterized GW/7-3M motoneuron that innervates muscle 15 via the
ISNd pathway (Dittrich et al.,
1997; Higashijima et al.,
1996
). To identify this ISNd motoneuron, we used the enhancer-trap
line, egP289, which drives lacZ expression in the
7-3M progeny (Dittrich et al.,
1997
). Although we see Dfr expression in the EW interneurons
(arrows, Fig. 1E,E'), the
ISNd GW/7-3M motoneuron does not express Dfr (arrow,
Fig. 1E,E''). The results
of these labeling experiments demonstrate that Dfr expression can be further
used to subdivide the ISNb from the TN and ISNd neuronal classes.
Genetic interaction studies indicate that Drifter is required for the specification of ISNb motoneurons
The ISNb fascicle contains motor axons from at least eight motoneurons
innervating ventral muscles 6, 7, 12, 13, 14, 28 and 30. Wild-type innervation
of muscles 6, 7, 12 and 13 is shown in Fig.
2A. Loss of isl or Lim3 function in ISNb
motoneurons affects axon targeting resulting in a reduction of target muscle
innervation (Thor et al.,
1999; Thor and Thomas,
1997
). The most common phenotype in both isl and
Lim3 mutants is a failure to innervate the cleft between muscles 12
and 13. In Lim3 mutants, muscles 12/13 were not innervated by motor
axons in 46% of hemisegments compared with 3% in wild-type hemisegments. In
isl mutants, the lack of muscle contacts was also coupled with the
ISNb motor axons, leaving the ventral muscle field and joining the TN (26% of
hemisegments in isl mutants versus 0% in wild type)
(Thor et al., 1999
;
Thor and Thomas, 1997
).
|
|
Reducing Drifter function results in ISNb motor axons redirecting to TN neuron targets
As described above, isl and Lim3 mutants display axon
targeting defects manifested by a reduction in muscle innervation
(Fig. 4D). Results from the
trans-heterozygous genetic interaction studies suggest that Dfr is necessary
in combination with Isl and Lim3 to specify ISNb axon targeting. If this
hypothesis is correct, than a loss of Dfr function in ISNb motoneurons should
also result in a reduction in muscle innervation. To address the possible role
of Dfr in ISNb specification, we used RNA interference to reduce or eliminate
the expression of Dfr protein specifically in neurons, without affecting its
expression and function in midline glia. We generated transgenic flies
expressing double-stranded dfr RNA (UAS-dsdfr) under the
control of neuronal-specific Gal4 drivers. To further assist in the
removal of Dfr function, two independent UAS-dsdfr insertions were
recombined onto a chromosome containing a null dfr allele,
dfrB129. Although the protein produced by this allele is
detected by the Dfr antiserum, it is non-functional because of a premature
stop codon located before the DNA-binding POU domain (W. A. Johnson, personal
communication). Several Gal4 drivers were used to analyze the
effectiveness of the UAS-dsdfr transgenes. Using both the
C155(elav)-Gal4 and the Lim3B-Gal4 drivers (see Materials
and methods), Dfr protein was reduced or eliminated in the majority of ISNb
motoneurons, including the RP motoneurons
(Fig. 3A,B). As a control, the
Dfr-expressing midline glia showed no loss of Dfr protein and, accordingly, no
loss of the midline glia-specific marker Slit
(Fig. 3A,B). This showed that
UAS-dsdfr could be used together with neuronal-specific Gal4
drivers to address how loss of Dfr function affects ISNb neuron specification.
To verify that the Lim3B-Gal4 driver expression correlates with the
endogenous Lim3 expression, we labeled Lim3B-Gal4;UAS-taumycEGFP
double-transgenic embryos with the Lim3 antibody
(Broihier and Skeath, 2002).
We can visualize the TN process leaving the VNC (arrow,
Fig. 3C) and Lim3 is expressed
in these neurons (arrowhead, Fig.
3C).
|
|
Misexpression of Drifter in TN neurons results in a redirection of TN motor axons to the ISNb muscle target field
If Dfr functions as part of a LIM/POU combinatorial code to specify ISNb
motoneurons, then ectopically expressing Dfr in the Isl/Lim3-expressing TN
motoneurons would be predicted to alter TN axon pathfinding towards an
ISNb-like behavior. To test this, we misexpressed Dfr in postmitotic
Isl/Lim3-expressing TN neurons using the Lim3B-Gal4 driver. In
wild-type development, the transverse nerve forms from the fasciculation of a
sensory nerve axon (the lateral bipolar dendrite or LBD neuron) and the TMNp
neuron (Gorczyca et al., 1994;
Thor and Thomas, 1997
). At
late stage 15/16, these growth cones contact each other on the ventral
interior muscle surfaces. The TN fascicle is on a different focal plane and in
wild-type embryos does not come in contact with the ISNb fascicle
(Fig. 5A,B1).
|
We also tested the ability of the TN motoneurons to be respecified by Dfr misexpression in isl mutant embryos. Without Isl function, the retargeting of TN motor axons did not occur (n=64), suggesting possible cooperative actions between these transcription factors. Our results indicate that it is the addition of Dfr specifically to the TN Isl/Lim3 LIM-HD code that allows this motoneuron subclass to exhibit ISNb motoneuron characteristics.
Drifter, Islet and Lim3 do not regulate each other
In C. elegans touch receptor neurons, the POU protein UNC-86
directly regulates the expression of the LIM-HD gene, MEC-3
(Xue et al., 1992;
Xue et al., 1993
). And in
vertebrates, abLIM is a transcriptional target of the POU factor, Brn3.2
(Erkman et al., 2000
). To
determine whether Dfr, Isl and/or Lim3 are possible transcriptional targets of
each other, we analyzed the individual expression patterns in each mutant
background. We first tested if Isl and Lim3 are required to initiate or
maintain Dfr expression. In addition to its expression in the ISNb-projecting
RP motoneurons located at the midline, Dfr is also expressed in midline glia
(Anderson et al., 1995
). To
determine if Dfr expression remained in the ISNb neurons as well as the
midline glia, we used isl- and Lim3-null mutants carrying
the islH-tau-myc reporter construct. Dfr expression was unaffected in
isl and Lim3 single and double mutants
(Fig. 1F; not shown),
indicating that dfr is not a transcriptional target of these LIM-HD
factors. We next examined Isl and Lim3 expression in dfr mutants.
Neither isl nor Lim3 reporter expression was affected,
suggesting that Dfr does not regulate LIM-HD expression in ISNb neurons
(Fig. 1G; not shown).
Therefore, these results indicate Dfr, Isl and Lim3 must function at the same
hierarchical level in ISNb motoneuron specification.
Islet and Lim3 genetically interact with the IgSF CAM, Beat Ic, in TN axon targeting and fasciculation
Studies in various model systems have led to the identification of a number
of transcription factors with highly restricted expression. These factors are
important for different aspects of neuronal differentiation, including axon
pathfinding. By contrast, many other molecules, such as cell adhesion
molecules, receptor protein tyrosine phosphatases and semaphorins, also play a
crucial role in axon guidance, yet these molecules are often, at least in
Drosophila, more broadly expressed
(Dickson, 2002;
Huber et al., 2003
;
Shen, 2004
). This apparent
disjunction could indicate that the downstream genes regulated by highly
restricted transcription factors such as Dfr and Isl are be thus far
uncharacterized molecules. Therefore, to identify possible targets of the
LIM/POU or LIM-HD combinatorial codes, we looked for specific molecules that
are differentially expressed between the ISNb and TN motoneuron subclasses. A
recent study identified and described the restricted expression of 14
Drosophila Beat-like members of the immunoglobulin superfamily (IgSF)
of CAMs (Pipes et al., 2001
).
Members of the Beat family in Drosophila and the Zig family in C.
elegans contain two Ig domains and most members have been shown to be
highly restricted in their expression pattern, largely confined to subsets of
neurons (Aurelio et al., 2003
;
Aurelio et al., 2002
;
Pipes et al., 2001
).
We were particularly interested in beat Ic, because of its
expression in a small number of embryonic neurons that include the TN
motoneurons but not the ISNb neurons
(Pipes et al., 2001). We used
in situ hybridization and immunohistochemistry to verify the TN expression of
beat Ic. beat Ic transcripts are expressed in lateral cell clusters
that contain the TN neurons (arrow) but not in the ISNb RP neurons (arrowhead,
Fig. 6A). To establish the
identity of the lateral cluster cells, Lim3B-Gal4;UAS-taumycEGFP
transgenic embryos were labeled for both GFP and beat Ic expression.
We observe the GFP-expressing (brown) TN neurons also show blue (beat
Ic) staining (Fig. 6B,
arrows).
|
|
beat Ic expression can rescue the TN defects observed in islet and Lim3 mutants
At least two possible explanations can be put forwards to explain the TN
defects observed in isl and Lim3 mutants. First, the defects
observed are because the TMNp and the LBD axons cannot fasciculate or adhere
to each other. A second hypothesis is that these two axons do not recognize
each other and therefore do not grow close enough together to fasciculate
properly. If the defect lies in fasciculation, then increasing the levels of
Beat Ic, a promoter of motor axon adhesion, should reduce the TN defects in
isl and Lim3 mutants. To increase Beat Ic levels, the
pan-neuronal driver elav-Gal4 and UAS-beat Ic transgenes
were crossed into isl and Lim3 mutant backgrounds. As in
previous experiments, strong isl and Lim3 alleles were
crossed to islDf and limDf
deficiencies to create embryos null for isl and Lim3,
respectively.
Increasing the levels of Beat Ic through one copy of UAS-beat Ic and the elav-Gal4 driver significantly reduced the percentage of TN defects in isl mutants from 56% to 22% (n=66). In addition, the severity of TN adhesion defects decreased in the remaining 22% of affected hemisegments. Likewise, increasing Beat Ic levels in Lim3 mutants also significantly decreased the occurrence of TN defects from 55% to 31% (n=67). These results suggest that the TN defects observed in isl and Lim3 mutants are a result of a decrease in the adhesive properties between the TN motor axon and the LBD projection. This suggests that Beat Ic may be a direct transcriptional target of the LIM code.
At this time, we are unable to directly test this hypothesis because of the
unavailability of a Beat Ic antibody. We tried analyzing beat Ic
transcript accumulation in isl and Lim3 double mutants but
were unable to achieve cellular resolution in the mutant ventral nerve cords.
However, DNA-binding site pattern searches using previously described LIM-HD
binding motifs indicate that there are significant clusters of LIM-HD sites
surrounding and within the beat Ic locus
(Fig. 6G)
(Freeman et al., 2003;
Rebeiz and Posakony, 2004
).
The Isl1 consensus site (CTAATG) (Boam and
Docherty, 1989
; Karlsson et
al., 1990
) is found 15 times in the chromosomal region
encompassing the beat Ic locus
(Fig. 6G); the Lhx3-binding
site (AATTAATTA) (Bridwell et al.,
2001
) is found nine times (Fig.
6G) and 21 Isl 2.2 sites (YTAAGTG) (data not shown) have been
identified. Although functional analyses will be needed to determine if these
sites are necessary and/or sufficient for beat Ic expression,
searching the entire genome with the Isl 2.2 site places the beat Ic
locus seven out of the first 10 identified. Furthermore, studies in C.
elegans indicate that two LIM-HD genes, the Lmx-class gene
lim-6 and the Lhx3-class gene, ceh-14, are required
for the expression of at least four IgSF zig genes
(Aurelio et al., 2003
).
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
LIM-HD proteins participate in a number of unique complexes through
protein-protein interactions mediated by their LIM domains
(Bach, 2000;
Gill, 2003
). For example, LIM
domains can interact with the widely expressed co-factor, NLI(Ldb1/CLIM-2) in
mice or Chip in Drosophila (Breen
et al., 1998
; Jurata and Gill,
1997
; Jurata et al.,
1998
; van Meyel et al.,
1999
). The NLI/CHIP proteins homodimerize and generate a bridge
between two LIM-HD proteins, thereby leading to the formation of tetrameric
complexes (Jurata et al.,
1998
). Although this complex is functional in vivo, it has been
found that other proteins also participate to generate further tissue
specificity. In Drosophila, the newly identified Ssdp protein
interacts with Chip to modify the activity of complexes comprising Chip and
the LIM-HD protein Apterous in the wing
(Chen et al., 2002
;
van Meyel et al., 2003
). In
vertebrates, the bHLH factors Ngn2 and NeuroM functionally interact with the
NLI, Isl1 and Lhx3 complex to initiate motoneuron differentiation
(Lee and Pfaff, 2003
). These
results suggest that the formation of further specialized combinations could
be used to confer not only tissue but also cellular specificity.
In Drosophila, Isl and Lim3 are co-expressed in a subset of CNS
neurons including two neuron subclasses, the TN and ISNb motoneurons
(Thor et al., 1999). Although
Isl and Lim3 are required in these distinct motoneurons to specific motor axon
pathway choice neuronal identity (Thor et
al., 1999
; Thor and Thomas,
1997
) these two factors alone cannot be responsible for the unique
differentiation of each subclass. In this study, we provide evidence that the
class III POU domain protein Dfr functions in combination with this Isl/Lim3
LIM code, to specify the ISNb motoneuron class. Loss-of-function analyses
indicates each of these transcription factors is required in the ISNb neurons
for the specification of motor axon target selection. Without Dfr, Isl or
Lim3, these motor axons fail to correctly innervate their designated muscle
targets. In addition, our genetic interaction studies suggest that this
phenotype indicates a common aspect of motoneuron designation has been
altered.
How might this LIM/POU code function in ISNb neurons? In C.
elegans touch receptor neurons, the LIM-HD factor, MEC-3 and the POU
protein UNC-86, physically interact to control specification
(Rockelein et al., 2000;
Xue et al., 1993
). In the
pituitary, the LIM domain of Lhx3 (P-Lim) specifically interacts with the Pit1
POU domain and is required for synergistic interactions with Pit1
(Bach et al., 1995
). We have
not determined whether Dfr, Isl and or Lim3 physically interact to regulate
ISNb motor axon target selection. However, our misexpression experiments
indicate that the re-specification of transverse motoneurons by the addition
of Dfr does require functional Isl protein. Although, this result does not
distinguish between the possibilities of direct interactions between these
proteins or the binding of a common transcriptional target, it does indicate
that a functioning `LIM code' is required for the re-specification of the TN
neurons.
A second finding of our Dfr misexpression studies is that we can robustly re-specify the target selection of postmitotic neurons. We used the Lim3B-Gal4 line to add Dfr to the LIM-only transverse motoneurons. This Gal4 line does not activate reporter construct expression until stage 14 a post-mitotic stage even for the late developing transverse motoneurons. At this stage, the TN motor axons have exited the CNS and are navigating the periphery, although the TN motor axon and LBD fascicle have not come into contact. Misexpressing Dfr even at this relatively late stage of TN motoneuron differentiation can clearly alter axon pathfinding, and in a significant percentage of hemisegments, TN motor axons actually appear to ectopically innervate ISNb muscle targets. This result shows that these motoneurons remain plastic, even after becoming postmitotic and further indicates that the LIM/POU code may be acting directly on genes involved in axon targeting.
LIM-HD factors direct target specificity through the actions of an IgSF CAM
As described above, the combinatorial expression of LIM-HD transcription
factors confers motoneuron subtypes with the ability to direct their axons to
reach distinct muscle targets. If more than one subgroup of motoneurons use a
LIM code, how does subtype-specific motor axon pathfinding occur? Presumably,
it is the downstream targets of each LIM code that confer the ability of
individual or groups of motor axons to find their correct innervation targets.
What might be the target(s) of the LIM code in Drosophila transverse
neurons?
Studies in vertebrates and invertebrates have demonstrated that members of
the IgSF class of CAMs play important roles in cell-cell recognition and
communication processes that are crucial for nervous system wiring
(reviewed by Brummendorf and Lemmon,
2001; Rougon and Hobert,
2003
). The ability of Ig-domains to form linear rods when deployed
in series, and their propensity to bind specifically to other proteins, has
made these molecules ideal for functioning as cell-surface receptors and/or
CAMs. Furthermore, IgSF molecules have dramatically increased the number of
cell-cell recognition molecules through family expansion, the generation of
multiple variants through alternative splicing, receptor multimerization and
cross-talking intracellular signaling pathways
(Brummendorf and Lemmon, 2001
;
Rougon and Hobert, 2003
).
A recent genomic analysis indicates the Drosophila IgSF repertoire
consists of about 150 proteins; in C. elegans, 80 IgSF molecules are
predicted (Aurelio et al.,
2002; Hutter et al.,
2000
; Hynes and Zhao,
2000
; Vogel et al.,
2003
). Members of the Beat family in Drosophila and the
zig family in C. elegans are located in gene clusters, have
restricted expression patterns and share the same domain architecture
(Aurelio et al., 2002
;
Fambrough and Goodman, 1996
;
Pipes et al., 2001
;
Vogel et al., 2003
). Each
protein is comprised exclusively of two Ig modules with either a transmembrane
domain [beat Ic, beat Ib, beat IIa, beat VI (GPI), zig-1] or secreted
signals (10 Beat genes: zig-2 to zig-8)
(Aurelio et al., 2002
;
Hutter et al., 2000
;
Hynes and Zhao, 2000
;
Vogel et al., 2003
). The Beat
family members that have been functionally characterized appear to control
fasciculation through anti- and pro-adhesive properties
(Fambrough and Goodman, 1996
;
Pipes et al., 2001
).
Furthermore, unlike many previously described CAMs, several members of these
families are expressed in subsets of CNS neurons.
beat Ic is transcribed in a small number of cells in the embryonic
nerve cord, including the transverse motoneurons
(Pipes et al., 2001) (this
study). Loss-of-function experiments indicate Beat Ic is required in a
pro-adhesive manner for the proper recognition/or fasciculation of the TMNp
motor axon and the LBD fascicle. These TN defects are identical to the
fasciculation defects we observe in isl and Lim3 mutants.
Our trans-heterozygous combinations reveal strong genetic interactions between
isl, Lim3 and beat Ic, and furthermore, we found that
increasing Beat Ic expression in isl and Lim3 mutants
significantly rescues these TN axon fasciculation defects. Using in situ
hybridization, we are unable to determine if beat Ic TN expression is
dependent upon Isl and Lim3 function. However, DNA-binding site pattern
searches (Freeman et al.,
2003
; Rebeiz and Posakony,
2004
) using the described LIM-HD-binding motifs indicate that
there are significant clusters of LIM-HD sites surrounding and within the
beat Ic locus.
The downstream targets of combinatorial codes that control motor axon
pathway selection in vertebrates and invertebrates, as well as the
subtype-specific axon guidance cues remain poorly understood. Our experiments
suggest that the LIM-HD code in Drosophila may specify a subset of
axon target selection through the actions of IgSF CAMs. Previous studies have
suggested that Beat Ic does not function as a homophilic adhesion molecule
(Pipes et al., 2001). This
leads to the hypothesis that the function of Beat Ic is mediated by
heterophilic binding to an unknown Beat partner protein. Therefore, the
further characterization of both secreted and transmembrane IgSF molecules
that exhibit restricted neuronal expression might provide a mechanism with
which to refine subtype-specific signals through restricted cues or altering
adhesive properties.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson, M. G., Perkins, G. L., Chittick, P., Shrigley, R. J. and Johnson, W. A. (1995). drifter, a Drosophila POU-domain transcription factor, is required for correct differentiation and migration of tracheal cells and midline glia. Genes Dev. 9,123 -137.[Abstract]
Aurelio, O., Hall, D. H., Hobert, O. and Boulin, T.
(2002). Immunoglobulin-domain proteins required for maintenance
of ventral nerve cord organization. Identification of spatial and temporal
cues that regulate postembryonic expression of axon maintenance factors in the
C. elegans ventral nerve cord. Science
295,686
-690.
Aurelio, O., Boulin, T. and Hobert, O. (2003).
Identification of spatial and temporal cues that regulate postembryonic
expression of axon maintenance factors in the C. elegans ventral nerve cord.
Development 130,599
-610.
Bach, I. (2000). The LIM domain: regulation by association. Mech. Dev. 91, 5-17.[CrossRef][Medline]
Bach, I., Rhodes, S. J., Pearse, R. V., 2nd, 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 92,2720 -2724.[Abstract]
Boam, D. S. and Docherty, K. (1989). A tissue-specific nuclear factor binds to multiple sites in the human insulin-gene enhancer. Biochem. J. 264,233 -239.[Medline]
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.
Breen, J. J., Agulnick, A. D., Westphal, H. and Dawid, I. B.
(1998). Interactions between LIM domains and the LIM
domain-binding protein Ldb1. J. Biol. Chem.
273,4712
-4717.
Bridwell, J. A., Price, J. R., Parker, G. E., McCutchan Schiller, A., Sloop, K. W. and Rhodes, S. J. (2001). Role of the LIM domains in DNA recognition by the Lhx3 neuroendocrine transcription factor. Gene 277,239 -250.[CrossRef][Medline]
Broihier, H. T. and Skeath, J. B. (2002). Drosophila homeodomain protein dHb9 directs neuronal fate via crossrepressive and cell-nonautonomous mechanisms. Neuron 35, 39-50.[Medline]
Brummendorf, T. and Lemmon, V. (2001). Immunoglobulin superfamily receptors: cis-interactions, intracellular adapters and alternative splicing regulate adhesion. Curr. Opin. Cell Biol. 13,611 -618.[CrossRef][Medline]
Campos-Ortega, J. A. and Hartenstein, V. (1985). The Embryonic Development of Drosophila melanogaster. Heidelberg, Germany: Springer-Verlag.
Certel, S. J. and Johnson, W. A. (1996). Disruption of mesectodermal lineages by temporal misexpression of the Drosophila POU-domain transcription factor, Drifter. Dev. Genet. 18,279 -288.[CrossRef][Medline]
Certel, K., Hudson, A., Carroll, S. B. and Johnson, W. A.
(2000). Restricted patterning of vestigial expression in
Drosophila wing imaginal discs requires synergistic activation by both Mad and
the drifter POU domain transcription factor.
Development 127,3173
-3183.
Chen, L., Segal, D., Hukriede, N. A., Podtelejnikov, A. V.,
Bayarsaihan, D., Kennison, J. A., Ogryzko, V. V., Dawid, I. B. and Westphal,
H. (2002). Ssdp proteins interact with the LIM-domain-binding
protein Ldb1 to regulate development. Proc. Natl. Acad. Sci.
USA 99,14320
-14325.
Crowner, D., Madden, K., Goeke, S. and Giniger, E. (2002). Lola regulates midline crossing of CNS axons in Drosophila. Development 129,1317 -1325.[Medline]
Dickson, B. J. (2002). Molecular mechanisms of
axon guidance. Science
298,1959
-1964.
Dittrich, R., Bossing, T., Gould, A. P., Technau, G. M. and
Urban, J. (1997). The differentiation of the serotonergic
neurons in the Drosophila ventral nerve cord depends on the combined function
of the zinc finger proteins Eagle and Huckebein.
Development 124,2515
-2525.
Duggan, A., Ma, C. and Chalfie, M. (1998).
Regulation of touch receptor differentiation by the Caenorhabditis elegans
mec-3 and unc-86 genes. Development
125,4107
-4119.
Erkman, L., Yates, P. A., McLaughlin, T., McEvilly, R. J., Whisenhunt, T., O'Connell, S. M., Krones, A. I., Kirby, M. A., Rapaport, D. H., Bermingham, J. R. et al. (2000). A POU domain transcription factor-dependent program regulates axon pathfinding in the vertebrate visual system. Neuron 28,779 -792.[Medline]
Fambrough, D. and Goodman, C. S. (1996). The Drosophila beaten path gene encodes a novel secreted protein that regulates defasciculation at motor axon choice points. Cell 87,1049 -1058.[Medline]
Freeman, M. R., Delrow, J., Kim, J., Johnson, E. and Doe, C. Q. (2003). Unwrapping glial biology: Gcm target genes regulating glial development, diversification, and function. Neuron 38,567 -580.[Medline]
Gill, G. N. (2003). Decoding the LIM development code. Trans. Am. Clin. Climatol. Assoc. 114,179 -189.[Medline]
Gorczyca, M. G., Phillis, R. W. and Budnik, V.
(1994). The role of tinman, a mesodermal cell fate gene, in axon
pathfinding during the development of the transverse nerve in Drosophila.
Development 120,2143
-2152.
Grenningloh, G., Rehm, E. J. and Goodman, C. S. (1991). Genetic analysis of growth cone guidance in Drosophila: fasciclin II functions as a neuronal recognition molecule. Cell 67,45 -57.[Medline]
Higashijima, S., Shishido, E., Matsuzaki, M. and Saigo, K.
(1996). eagle, a member of the steroid receptor gene superfamily,
is expressed in a subset of neuroblasts and regulates the fate of their
putative progeny in the Drosophila CNS. Development
122,527
-536.
Huber, A. B., Kolodkin, A. L., Ginty, D. D. and Cloutier, J. F. (2003). Signaling at the growth cone: ligand-receptor complexes and the control of axon growth and guidance. Annu. Rev. Neurosci. 26,509 -563.[CrossRef][Medline]
Hutter, H., Vogel, B. E., Plenefisch, J. D., Norris, C. R.,
Proenca, R. B., Spieth, J., Guo, C., Mastwal, S., Zhu, X., Scheel, J. et
al. (2000). Conservation and novelty in the evolution of cell
adhesion and extracellular matrix genes. Science
287,989
-994.
Hynes, R. O. and Zhao, Q. (2000). The evolution of cell adhesion. J. Cell Biol. 150,F89 -F96.[Medline]
Inbal, A., Levanon, D. and Salzberg, A. (2003).
Multiple roles for u-turn/ventral veinless in the development of Drosophila
PNS. Development 130,2467
-2478.
Jessell, T. M. (2000). Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1,20 -29.[CrossRef][Medline]
Jurata, L. W. and Gill, G. N. (1997). Functional analysis of the nuclear LIM domain interactor NLI. Mol. Cell. Biol. 17,5688 -5698.[Abstract]
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
-3157.
Kania, A. and Jessell, T. M. (2003). Topographic motor projections in the limb imposed by LIM homeodomain protein regulation of ephrin-A: EphA interactions. Neuron 38,581 -596.[Medline]
Karlsson, O., Thor, S., Norberg, T., Ohlsson, H. and Edlund, T. (1990). Insulin gene enhancer 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.[CrossRef][Medline]
Komiyama, T., Johnson, W. A., Luo, L. and Jefferis, G. S. (2003). From lineage to wiring specificity. POU domain transcription factors control precise connections of Drosophila olfactory projection neurons. Cell 112,157 -167.[Medline]
Landgraf, M., Bossing, T., Technau, G. M. and Bate, M.
(1997). The origin, location, and projections of the embryonic
abdominal motorneurons of Drosophila. J. Neurosci.
17,9642
-9655.
Lee, S. K. and Pfaff, S. L. (2003). Synchronization of neurogenesis and motor neuron specification by direct coupling of bHLH and homeodomain transcription factors. Neuron 38,731 -745.[Medline]
Lindsley, D. L. and Zimm, G. G. (1992). The Genome of Drosophila melanogaster. San Diego, CA: Academic Press.
McEvilly, R. J., de Diaz, M. O., Schonemann, M. D., Hooshmand,
F. and Rosenfeld, M. G. (2002). Transcriptional regulation of
cortical neuron migration by POU domain factors.
Science 295,1528
-1532.
O'Neill, J. W. and Bier, E. (1994). Double-label in situ hybridization using biotin and digoxigenin-tagged RNA probes. Biotechniques 17,874 -875.
Odden, J. P., Holbrook, S. and Doe, C. Q.
(2002). Drosophila HB9 is expressed in a subset of motoneurons
and interneurons, where it regulates gene expression and axon pathfinding.
J. Neurosci. 22,9143
-9149.
Olson, P. F., Fessler, L. I., Nelson, R. E., Sterne, R. E., Campbell, A. G. and Fessler, J. H. (1990). Glutactin, a novel Drosophila basement membrane-related glycoprotein with sequence similarity to serine esterases. EMBO J. 9,1219 -1227.[Abstract]
Pipes, G. C., Lin, Q., Riley, S. E. and Goodman, C. S. (2001). The Beat generation: a multigene family encoding IgSF proteins related to the Beat axon guidance molecule in Drosophila. Development 128,4545 -4552.[Medline]
Rebeiz, M. and Posakony, J. W. (2004). GenePalette: a universal software tool for genome sequence visualization and analysis. Dev. Biol. 271,431 -438.[CrossRef][Medline]
Rockelein, I., Rohrig, S., Donhauser, R., Eimer, S. and
Baumeister, R. (2000). Identification of amino acid residues
in the Caenorhabditis elegans POU protein UNC-86 that mediate UNC-86-MEC-3-DNA
ternary complex formation. Mol. Cell. Biol.
20,4806
-4813.
Rothberg, J. M., Jacobs, J. R., Goodman, C. S. and Artavanis-Tsakonas, S. (1990). slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains. Genes Dev. 4,2169 -2187.[Abstract]
Rougon, G. and Hobert, O. (2003). New insights into the diversity and function of neuronal immunoglobulin superfamily molecules. Annu. Rev. Neurosci. 26,207 -238.[CrossRef][Medline]
Schmid, A., Chiba, A. and Doe, C. Q. (1999).
Clonal analysis of Drosophila embryonic neuroblasts: neural cell types, axon
projections and muscle targets. Development
126,4653
-4689.
Shen, K. (2004). Molecular mechanisms of target specificity during synapse formation. Curr. Opin. Neurobiol. 14,83 -88.[CrossRef][Medline]
Shirasaki, R. and Pfaff, S. L. (2002). Transcriptional codes and the control of neuronal identity. Annu. Rev. Neurosci. 25,251 -281.[CrossRef][Medline]
Skeath, J. B. and Thor, S. (2003). Genetic control of Drosophila nerve cord development. Curr. Opin. Neurobiol. 13,8 -15.[CrossRef][Medline]
Spradling, A. C. (1986). P element-mediated transformation. In Drosophila, A Practical Approach (ed. D. B. Roberts), pp. 175-197. Oxford, UK: IRL Press.
Sugitani, Y., Nakai, S., Minowa, O., Nishi, M., Jishage, K.,
Kawano, H., Mori, K., Ogawa, M. and Noda, T. (2002). Brn-1
and Brn-2 share crucial roles in the production and positioning of mouse
neocortical neurons. Genes Dev.
16,1760
-1765.
Sze, J. Y., Zhang, S., Li, J. and Ruvkun, G. (2002). The C. elegans POU-domain transcription factor UNC-86 regulates the tph-1 tryptophan hydroxylase gene and neurite outgrowth in specific serotonergic neurons. Development 129,3901 -3911.[Medline]
Thaler, J. P., Lee, S. K., Jurata, L. W., Gill, G. N. and Pfaff, S. L. (2002). LIM factor Lhx3 contributes to the specification of motor neuron and interneuron identity through cell-type-specific protein-protein interactions. Cell 110,237 -249.[Medline]
Thor, S. and Thomas, J. B. (1997). The Drosophila islet gene governs axon pathfinding and neurotransmitter identity. Neuron 18,397 -409.[CrossRef][Medline]
Thor, S., Andersson, S. G., Tomlinson, A. and Thomas, J. B. (1999). A LIM-homeodomain combinatorial code for motor-neuron pathway selection. Nature 397, 76-80.[CrossRef][Medline]
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 79,957 -970.[Medline]
van Meyel, D. J., O'Keefe, D. D., Jurata, L. W., Thor, S., Gill, G. N. and Thomas, J. B. (1999). Chip and apterous physically interact to form a functional complex during Drosophila development. Mol. Cell 4,259 -265.[Medline]
van Meyel, D. J., Thomas, J. B. and Agulnick, A. D.
(2003). Ssdp proteins bind to LIM-interacting co-factors and
regulate the activity of LIM-homeodomain protein complexes in vivo.
Development 130,1915
-1925.
Vogel, C., Teichmann, S. A. and Chothia, C.
(2003). The immunoglobulin superfamily in Drosophila melanogaster
and Caenorhabditis elegans and the evolution of complexity.
Development 130,6317
-6328.
Wright, T. R., Hodgetts, R. B. and Sherald, A. F.
(1976). The genetics of dopa decarboxylase in Drosophila
melanogaster. I. Isolation and characterization of deficiencies that delete
the dopa-decarboxylase-dosage-sensitive region and the
alpha-methyl-dopa-hypersensitive locus. Genetics
84,267
-285.
Xue, D., Finney, M., Ruvkun, G. and Chalfie, M. (1992). Regulation of the mec-3 gene by the C. elegans homeoproteins UNC-86 and MEC-3. EMBO J. 11,4969 -4979.[Abstract]
Xue, D., Tu, Y. and Chalfie, M. (1993). Cooperative interactions between the Caenorhabditis elegans homeoproteins UNC-86 and MEC-3. Science 261,1324 -1328.[Medline]
|