1 Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA 15261,
USA
2 Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261,
USA
* Author for correspondence (e-mail: efrank{at}pitt.edu)
Accepted 24 July 2002
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Lmo4, LIM-homeodomain proteins, Single cell cDNA libraries, Differential display, Muscle sensory neurons, Neuronal specification, Neuronal differentiation, Chick
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To identify genes that may participate in the early specification of Ia sensory neuronal identity, we constructed cDNA libraries from individual Ia sensory neurons that innervate two antagonistic limb muscles, the sartorius and the adductor longus et brevis. Differential screening identified a transcription regulatory factor, LIM domain only 4 protein (Lmo4), that is differentially expressed in sensory neurons supplying different muscles.
Members of the Lmo family of transcriptional regulatory factors lack a
DNA-binding domain but contain two protein-protein interaction LIM domains.
Lmo proteins compete for NLI (nuclear LIM domain interactor) with LIM
homeodomain transcription factors, and thereby regulate formation of LIM
homeodomain/NLI complexes and their transcriptional activity
(Bach, 2000;
Jurata et al., 2000
;
Rabbitts, 1998
).
Drosophila Lmo plays an important role in wing and peripheral nervous
system development by modulating the interaction between the LIM homeodomain
protein Apterous and Chip (Drosophila homologue of NLI)
(Milan and Cohen, 1999
;
Milan et al., 1998
;
van Meyel et al., 1999
). In
addition, Lmo proteins can interact with other transcription factors. In
Xenopus, the interaction between XLmo3 and the basic helix-loop-helix
(HLH) protein HEN1 is involved in the regulation of neurogenesis by activating
the expression of Xenopus Neurogenin 1 and NeuroD
(Bao et al., 2000
). Although
the expression of Lmo4 has been reported in mouse motoneurons
(Kenny et al., 1998
) and DRG
neurons (Sugihara et al.,
1998
) during development, the detailed expression pattern or
functional significance of Lmo4 in neurons has not been explored.
We show that Lmo4 is expressed by a subset of sensory and motoneurons shortly after these neurons are postmitotic. Moreover, Lmo4 expression does not require the presence of limb targets, suggesting that an intrinsic mechanism regulates its early expression in differentiating neurons. Because Lmo4 is co-expressed with LIM homeodomain proteins, it may regulate the functions of LIM proteins during the differentiation of sensory and motoneurons.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of single-cell cDNA libraries
Single-cell cDNA libraries were prepared by RT-PCR as described by Dulac
and Axel (Dulac and Axel,
1995). The whole cells were transferred into 4 µl cDNA-lysis
buffer [1xMMLV buffer, 0.5% NP40 (Sigma), 0.04 µl Prime RNase
inhibitor (3' 5'), 0.04 µl RNAguard (Pharmacia), 0.06 mM dATP,
dCTP, dGTP, dTTP, 0.02 OD/ml pd(T)16 (Perkin Elmer)] and the cells
were lysed at 65°C for 1 minute. The cellular RNA was annealed to Oligo dT
at room temperature for 2 minutes and reverse transcribed to synthesize the
first strand cDNA with the addition of 0.5 µl of AMV- and MMLV-reverse
transcriptases (Invitrogen) (1/1 vol/vol) at 37°C for 15 minutes. The
reverse transcriptases were inactivated at 65°C. Poly (A) was added to the
first strand cDNA with 10 units of terminal transferase (Roche) in
1xtailing buffer (1xterminal transferase buffer, 0.75 mM dATP) at
37°C for 10 minutes. Terminal transferase was inactivated at 65°C and
the cDNA was then amplified in a final 100 µl PCR buffer [1x PCR
buffer II, 2.5 mM MgCl2, 0.1 mg/ml BSA, 1 mM dNTP mixture, 0.05%
triton X-100, 0.05 µg AL1 primer
(5'-ATTGGATCCAGGCCGCTCTGGACAAAATATGAATTC(T)24-3'), 2
µl AmpliTaq (Perkin Elmer)] with 25 cycles of 94°C for 1 minute,
42°C for 2 minutes and 72°C for 6 minutes, with 10 seconds extension
time at each cycle. After the first 25 cycles of PCR amplification, an
additional 1 µl of AmpliTaq was then added directly into the tubes and
another 25 cycles were performed with the same schedule except without the
additional extension time. To ensure that only proprioceptive, and not
nociceptive, sensory neurons were selected, cDNAs of isolated neurons were
screened by PCR for the presence of trkC, a receptor for NT3 expressed in
large caliber muscle sensory neurons, and the absence of trkA, a receptor for
NGF expressed in many small caliber (both muscle and cutaneous) sensory
neurons. The trkC-specific primers were
5'-ATGCAGAGCTGCTGGCAGAGAG-3' and
5'-CCAAACTGCCTTACAGGTCGTC-3', and the trkA-specific primers were,
5'-CACGACCTGGTGGTGAAGATTG-3' and
5'-CTCTCAGCCCAGGATGTCCAGG-3'. To ensure that there was no
contamination of Schwann cells during isolation of sensory neurons, cDNAs of
isolated neurons were screened by PCR for the absence of myelin basic protein
using myelin basic protein-specific primers,
5'-GGCTCTTCTGAATTGCACTG-3' and 5'-CCACTATTACGTTGCCAAG-3.
Aliquots of selected adductor and sartorius cDNA stocks were then subjected to
Southern blot analysis to determine the accuracy of cDNA representation after
PCR amplification. The probes used included genes expressed at high levels
(GAPDH and enolase), moderate levels (calcium-ATPase) and low levels (trkC).
Of the single cell-cDNA stocks prepared from 99 adductor and sartorius
neurons, 5 cDNA stocks that had appropriately amplified levels of marker genes
were selected for the final extension step using 1 µl AmpliTaq in 100 µl
PCR buffer: 94°C for 5 minutes, 42°C for 5 minutes and 72°C for 30
minutes. The final extended cDNAs were purified and digested with
EcoRI and cDNA fragments larger than 450 bp were separated on a 1.7%
agarose gel and purified. The size-selected cDNA was then packaged using
EcoRI-digested, dephosphorylated
Zap II phage arms according
to the manufacturer's protocol (Stratagene). The frequency of enolase-positive
plaques in each prepared single-cell cDNA library (0.4%) suggested that
representation of a given RNA was not biased during the construction of the
libraries.
Differential screening of single-cell cDNA libraries
The cDNA libraries were plated at 1000 pfu/plate and duplicate lifts were
made of each library. The first library lift was probed with the cDNA probe
for the other cell and the second lift with its own cell cDNA probe. Both cDNA
probes were prepared by re-amplifying for 10 cycles 1 µl of the original
cell cDNAs in 50 µl PCR buffer with the AL1 primer, but cold dCTP was
replaced with 100 µCi of [P]-dCTP (3000 µCi/mmole) as described by Dulac
and Axel (Dulac and Axel,
1995). Positive plaques in duplicate lifts were compared and
clones that showed differential expression were isolated. After
cross-screening 15,000 recombinant phages prepared from single-cell cDNA
stocks of three adductor and two sartorius neurons, the inserts of 95
candidate plaques were amplified using T3 and T7 primers and cell-specific
expression was confirmed using cDNA probes from the original adductor and
sartorius sensory neurons in Southern blots. Of those, five clones were
sequenced and chosen for further PCR/Southern blot analysis with single-cell
cDNA stocks prepared from 17 additional adductor and sartorius cells using
clone-specific primers. Lmo4 was confirmed to be differentially expressed in
adductor but not sartorius sensory neurons by PCR using Lmo4-specific primers,
5'-GTTCATCACAGATGGATCCCCATG-3' and
5'-GCCATGGGAAGTAGCAACATTAGG-3' (see
Fig. 1).
|
Isolation of chick Lmo4 gene
The full-length coding sequence of Lmo4 was isolated from a
random-primed E9 chick brain cDNA library (a gift from Dr W. Halfter,
University of Pittsburgh, PA). DNA was sequenced on both strands at the
University of Pittsburgh Sequencing Core Facility (GenBank Accession Number,
AF532926).
Immunohistochemistry
For cell counts of retrograde FITC-labeled sensory and motoneurons, embryos
were fixed with 4% paraformaldehyde for 2-3 hours, washed in PBS and sectioned
at 12 µm. Lmo4 protein was visualized using an Lmo4-specific goat
polyclonal antibody (Lmo4-c15, Santa Cruz Biotechnology) detected with a
Cy3-conjugated donkey anti-goat secondary antibody (Jackson Laboratories). In
other sections, Cy3- and FITC-conjugated secondary antibodies were used for
dual immunohistochemistry.
Monoclonal antibodies specific for Isl1 and Isl2 (4D5), Lim1 and Lim2 (4F2), and Lim3 (67.4E12) were obtained from the Hybridoma Bank (University of Iowa). A monoclonal antibody specific for ER81 (5B10) and a rabbit antibody specific for chick Pea3 (C115) were generously provided by Dr T. M. Jessell (Columbia University).
Limb ablation
Unilateral ablation of hindlimb bud precursors including the ectoderm and
underlying lateral plate mesoderm was performed in seven embryos at stages 15
or 18. Embryos developed to stages 27-29 and were then processed as described
above.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Unlike motoneurons that are topographically organized in the spinal cord,
functionally distinct sensory neurons that project to different muscles are
dispersed throughout the DRG. This dispersal has made it difficult to identify
pool-specific marker genes in sensory neurons. To overcome this problem, we
combined the method of differential screening of single-cell cDNA libraries
used successfully in identifying pheromone receptors
(Dulac and Axel, 1995) with
retrograde labeling of sensory neurons from identified muscle nerves. Using
this approach, we screened for genes that are differentially expressed by
proprioceptive sensory neurons (Ia afferents) innervating two different and
antagonistic limb muscles, the sartorius and the adductor. These muscles were
chosen because the Ia afferents supplying them make different sets of synaptic
connections with motoneurons. Sartorius afferents make strong connections with
sartorius motoneurons but virtually none with adductor motoneurons, while the
converse is true for Ia afferents supplying the adductor muscle
(Mendelson and Frank, 1991
).
In addition, sartorius and adductor motoneurons are located at a similar
rostrocaudal location in the cord, so sensory afferents must distinguish
between correct and incorrect synaptic partners within the same target region
(Mendelson and Frank, 1991
).
Differences in gene expression between these two groups of proprioceptive
sensory neurons therefore are likely to include genes that reflect or
influence the selection of their peripheral and central targets.
By differentially screening cDNA libraries of adductor and sartorius
sensory neurons of stage 39 chick embryos, a stage when Ia afferents are
making synaptic connections with motoneurons
(Lee et al., 1988), we
identified a transcript of the chick homolog of the LIM domain only 4 protein
(Lmo4) that was present in most adductor but not sartorius single-cell cDNA
libraries. The adductor-specific expression of Lmo4 was further
confirmed by Southern blot analysis of a second series of single-cell cDNA
stocks by PCR using Lmo4-specific primers. Lmo4 was detected
in five out of nine adductor and none of eight sartorius cells
(Fig. 1).
The Lmo4 cDNA was used as a probe to isolate full-length clones from a random primed E9 chick brain cDNA library that contained larger inserts than those in our single-cell cDNA libraries. The full-length Lmo4 clone encodes a peptide of 165 amino acids that contains two LIM domains (Fig. 2). The Lmo4 homolog in chick is 80% conserved at the DNA sequence level with human and mouse Lmo4. At the amino acid level, however, it differs from these proteins by only two amino acids (6 and 8). Such a high degree of similarity suggests a highly conserved function in birds and mammals.
|
Lmo4 is expressed in subsets of sensory afferents
The pattern of Lmo4 expression in the DRG was determined both by RNA in
situ hybridization and by immunohistochemistry using the Lmo4-c15 antibody in
stages 35 and 39 chick embryos. The Lmo4-c15 polyclonal antibody was generated
from a C-terminal peptide of human Lmo4 located between amino acids 145 and
157 (Santa Cruz Biotechnology), a region where chicken, mouse and human Lmo4
are identical (Fig. 2A). The
antibody should therefore recognize chicken Lmo4. Direct evidence on this
point was obtained by immunohistochemistry and western blot analysis of 293
cells transiently infected with a full-length chicken Lmo4 expression
construct. Only infected cells were recognized by the antibody (data not
shown). cDNA sequences encoding chicken Lmo1 and 2 were identified in the EST
database and aligned to the human and mouse homologs
(Fig. 2A). It is unlikely that
the Lmo4-c15 antibody crossreacts with these proteins in chickens. Lmo4-c15
does not recognize human Lmo family members except Lmo4 in western blots
(Santa Cruz Biotechnology), and the partial sequences for chicken Lmo1 and
Lmo2 are virtually identical to their human homologs
(Fig. 2A). Furthermore, the
C-terminal sequences of chicken Lmo1, Lmo2 and Lmo4 are highly divergent. Only
three out of 13 amino acids in the region used to make the antibody are shared
among these family members (underlined in red in
Fig. 2B), making it improbable
that the antibody recognizes chicken Lmo1 or Lmo2. Finally, the patterns of
immunoreactivity to Lmo4-c15 and in situ labeling of Lmo4 mRNA are highly
similar in the spinal cord and DRG, including the extent of labeling of
specific sensory and motor pools (see below and data not shown).
Lmo4 protein is mainly expressed in the ventrolateral (VL) part of the DRG, where most large diameter, trkC-positive, muscle sensory cells are located. Out of all the trkC-positive neurons, 83% were Lmo4 positive. By contrast, very few of the dorsomedially (DM) located small diameter, trkA-positive, cutaneous sensory neurons express Lmo4 (Fig. 3A,B). Similar expression patterns were seen using in situ hybridization (data not shown). To investigate the profile of Lmo4 expression by sensory neurons supplying individual muscles, we combined retrograde labeling of identified peripheral nerves with immunohistochemistry or in situ hybridization for Lmo4 mRNA.
|
The difference in Lmo4 expression seen in Southern blots of single-cell cDNAs was confirmed histologically on sections of labeled neurons. Nearly 80% of sensory neurons supplying the adductor muscle are Lmo4 positive compared with only 13% of sartorius sensory neurons (Fig. 3C,D; Table 1). Similarly, 70% (153/220) of retrogradely labeled adductor neurons express Lmo4 mRNA versus 10% (nine out of 91) of sartorius sensory neurons (data not shown). To determine if Lmo4 identifies only those afferents supplying ventral (including adductor) but not dorsal (including sartorius) limb muscles, we also examined sensory neurons projecting to other dorsal limb muscles, including iliotibialis and the internal and external heads of femorotibialis. Seventy-three percent of external femorotibialis neurons are Lmo4 positive, whereas only 2-3% of internal femorotibialis and posterior iliotibialis neurons are Lmo4 positive, demonstrating that Lmo4 expression is not restricted simply according to the dorsal/ventral innervation of the limb (Fig. 3E-G; Table 1).
|
In addition to its expression in sensory neurons, Lmo4 is expressed in
satellite and Schwann cells in the DRG and spinal roots. As in neurons, the
protein is restricted to nuclei, visible throughout the DRG (small profiles in
Fig. 3C-G) and in spinal roots
(right side of Fig. 3F). Lmo4
is also expressed in small, non-neuronal cells in the ventral white matter of
the spinal cord (Fig. 4D,E).
Expression of Lmo4 mRNA in non-neuronal cells in murine DRGs has been reported
previously (Sugihara et al.,
1998).
|
Lmo4 is expressed in subsets of motoneurons
To determine whether Lmo4 is also differentially expressed in motoneurons
supplying limb muscles, we examined its expression in the cord at lumbosacral
(LS) levels LS1-LS3 at stage 39. Within LS1, most motoneurons (identified by
Isl1/Isl2 expression) within the lateral motor column (LMC) do not express
Lmo4. But within LS2 and LS3, Lmo4 is expressed in approximately half of LMC
motoneurons (Fig. 4A-C). To
determine if the expression is specific for motor pools, we labeled individual
pools using retrograde fills from muscle nerves
(Fig. 4D-H;
Table 1). Several motor pools
have the same fraction of Lmo4-positive neurons as the corresponding sensory
pools. For example, 80-90% of adductor and external femorotibialis motoneurons
are Lmo4 positive, compared with 75-80% for the corresponding sensory neurons.
By contrast, only 15% of sartorius motoneurons express Lmo4, compared with 13%
for sartorius sensory neurons. Expression of Lmo4 mRNA, determined by in situ
hybridization, is similar for each of these motor pools (91%, 92% and 10%,
respectively; data not shown). Sensory and motoneurons supplying the same
muscle do not always share similar Lmo4 expression patterns, however.
Virtually all posterior iliotibialis motoneurons express Lmo4, while only 2%
of the sensory pool is Lmo4 positive. Furthermore, the expression of Lmo4 is
not strictly organized with respect to motor pools; an intermediate fraction
(40%) of internal femorotibialis motoneurons is Lmo4 positive.
Neurons with common expression patterns of LIM homeodomain proteins
can differ in their Lmo4 expression during development.
LIM homeodomain transcription factors are expressed early during sensory
and motoneuron development and are important for their functions. Null
mutation of Isl1 prevents the development of sensory and motor neurons
(Pfaff et al., 1996). In the
spinal cord, combinatorial expression of the LIM homeodomain proteins Isl1,
Isl2 and Lim1 defines subclasses of motoneurons that segregate into columns
and select distinct projection pathways in the periphery
(Kania et al., 2000
;
Tsuchida et al., 1994
).
Co-expression of Lmo4 can modulate the transcriptional activity of LIM
proteins and may thereby influence neuronal differentiation.
The observation that Lmo4 is expressed in only some sensory pools at stage
39 raised the possibility that Lmo4 might be differentially expressed in
sensory neurons at earlier developmental stages as well. At stage 26, when
muscle sensory neurons are establishing their first peripheral projections and
before many cutaneous (DM) neurons are born, Lmo4 is expressed in many
Isl1/2-positive muscle sensory neurons
(Fig. 5A). By contrast, Lmo4
expression is excluded from DM neurons from the outset, even before they
develop a trkA-positive/trkC-negative phenotype (area outlined by dots in
Fig. 5B). By stage 27, when
Pea3 expression begins in sensory neurons, Lmo4 is not expressed in a subset
of Pea3-positive cells (14%, Fig.
6A). Pea3 is expressed only in proprioceptive sensory neurons
(Lin et al., 1998), so the
expression of Lmo4 in some but not all of these cells suggests that, even at
this stage, only a subset of muscle sensory neurons are Lmo4 positive.
Similarly, at stage 35, when trkC provides a good marker for proprioceptive
neurons, 17% of trkC-positive cells are Lmo4 negative
(Fig. 6B). Thus, Lmo4 has a
restricted expression pattern in sensory neurons at a time when they are
making their peripheral and central connections.
|
|
|
Lmo4 expression does not predict expression of ER81 or Pea3
The ETS proteins ER81 and Pea3 are expressed in subsets of sensory and
motoneurons in a pool-specific manner. We therefore determined whether Lmo4
expression was correlated with either of these ETS family members. Expression
in some sensory pools did match that of ER81. The adductor and external
femorotibialis pools, which have high levels of Lmo4 expression, also express
ER81, whereas sartorius and internal femorotibialis sensory neurons, most of
which do not express Lmo4, are also not ER81 positive. But the correlation for
all sensory neurons is not strong. Only 65% of ER81-positive sensory neurons
are Lmo4 positive at stage 35. The fraction of ER81-positive cells that are
also Lmo4-positive is similar in each of LS1 through LS4. Lmo4 expression also
does not correlate with the presence or absence of Pea3; 71% of Pea3 sensory
neurons are Lmo4 positive (Fig.
6C,D; Table 2)
A similar situation is found for Lmo4 and ETS expression in motoneurons.
The expression of Lmo4 and ER81 is correlated in the adductor, sartorius and
external femorotibilas motor pools. But posterior iliotibialis motoneurons,
which are Lmo4 positive, are located mainly in LS4 where there is virtually no
expression of ER81 (Lin et al.,
1998). In addition, internal femorotibialis motoneurons are also
ER81 negative, but 40% of them express Lmo4
(Table 2). A substantial
portion of Pea3-positive motoneurons, which do not express ER81, also express
Lmo4 (Fig. 6E,F;
Table 2). These results suggest
that although the expression of Lmo4 precedes that of ETS genes in sensory and
motor neurons, it does not predict the later expression of these genes.
Lmo4 expression does not require signals from the limb
Some aspects of sensory neuronal phenotype, such as the specification of
muscle versus cutaneous targets, are determined before peripheral projections
are established (Honig et al.,
1998; Ma et al.,
1999
; Oakley et al.,
2000
). Motor neuronal phenotype is also largely specified before
target innervation (Lance-Jones and
Landmesser, 1980
). For both sensory and motor neurons, expression
of LIM homeodomain proteins occurs shortly after exit from the cell cycle and
is not blocked by removal of their peripheral targets
(Ensini et al., 1998
;
Lin et al., 1998
;
Sockanathan and Jessell,
1998
). By contrast, the pool-specific factors ER81 and Pea3 that
are expressed at later times require peripheral targets, as limb ablation
blocks their expression. Lmo4 is expressed relatively early, as are the LIM
proteins, yet its expression in sensory neurons is correlated with peripheral
target identity, as are the ETS proteins. It was therefore of interest to
learn if the expression of Lmo4 required peripheral targets.
To investigate the influence of peripheral targets on Lmo4 expression, we
unilaterally ablated hindlimb buds at stage 15. No obvious difference in Lmo4
protein or mRNA expression was seen in DRG after ablation at either stage
(Fig. 7A; data not shown).
Lmo4-positive sensory neurons were restricted to the large cell population and
were located in the ventrolateral portion of the DRG as in normal embryos. By
contrast, Pea3 expression was reduced, as reported previously
(Lin et al., 1998)
(Fig. 7B).
|
The observed loss of Pea3 expression is complicated by the fact that limb
bud ablation induces an early peak of cell death in DRG
(Caldero et al., 1998). This
peak occurs at the stage when we assessed Lmo4 and Pea3 expression after
ablation, and it is confined largely to the VL (proprioceptive) population.
Early death of VL neurons is unlikely to influence our conclusions about Lmo4
expression, however, because there is no obvious reduction in expression.
Furthermore, the persistence of Lmo4 expression provides additional support
for the conclusion by Lin and co-workers that loss of Pea3 expression is not
due to cell death (Lin et al.,
1998
). Most Pea3-positive neurons co-express Lmo4 at stage 27, so
the persistence of normal numbers of Lmo4-positive cells implies that the
cells have not died.
The effect of limb bud deletion on Lmo4 and Pea3 expression in motoneurons
is similar to that in sensory neurons. The number and location of
Lmo4-positive motoneurons are unchanged after ablation. Pea3 expression in
motoneurons, however, is largely abolished
(Fig. 7D,E)
(Lin et al., 1998). Thus, for
both sensory and motor neurons, expression of Lmo4 is determined
intrinsically; it does not require peripheral targets.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lmo4 expression in developing sensory neurons
The results described here show that Lmo4 is expressed in muscle
pool-specific patterns in sensory neurons. We initially isolated Lmo4
because of its differential expression in sensory neurons supplying the
adductor versus sartorius muscles. In addition to sartorius sensory neurons,
neurons supplying the internal femorotibialis and posterior iliotibialis
muscles are also Lmo4 negative, while most sensory neurons projecting to
external femorotibialis are Lmo4-positive. Despite the fact that Lmo4 is
expressed in most (83%) trkC-positive sensory neurons, its absence from those
neurons supplying specific muscles suggests that it may contribute to muscle
pool specificity of these cells.
Lmo4 is already expressed by stage 26, when sensory neurons are establishing their first peripheral projections and before many cutaneous (DM) neurons are born. The exclusion of Lmo4 from DM neurons, clearly visible at stage 35 after these cells have developed their distinctive trkA-positive phenotype, is apparent by stage 28. Even within the population of muscle sensory neurons, early Lmo4 expression appears to be selective. Based on co-expression with Pea3, Lmo4 is selective for a subset of muscle sensory neurons by stage 27. A similar fraction of neurons co-express trkC and Lmo4 at stage 35. Although there is no direct evidence that Pea3-positive sensory neurons at stage 27 represent the same population as trkC-positive sensory neurons at stage 35, the similarity in fractions of Lmo4-negative neurons at these two stages suggests that Lmo4 expression may be determined already by stage 27, before most muscle sensory neurons have reached their peripheral targets.
A separate line of evidence supporting the idea that Lmo4 expression does
not require peripheral targets is provided by the limb ablation experiments.
When hind limb buds are removed at stage 15, there is still extensive
expression of Lmo4 within the DRG at stage 28. The persistence of Lmo4
expression implies that Lmo4, like Isl1/Isl2, is an intrinsic signaling factor
in sensory neurons. Instead of decreasing expression, limb ablation might
actually increase expression of Lmo4. Relatively few proprioceptive muscle
sensory neurons (15%) normally do not express Lmo4, so expression by all
proprioceptive neurons would only result in a small fractional increase, which
could be difficult to observe. According to this interpretation, certain
peripheral targets, such as the sartorius, internal femorotibialis and
posterior iliotibialis muscles, could modulate the phenotype of their sensory
innervation by inhibiting expression of Lmo4. Muscle targets of proprioceptive
sensory neurons are believed to specify the synapses these neurons make within
the spinal cord (Frank and Wenner,
1993
), and inhibition of Lmo4 expression could then be one
mechanism contributing to this specification.
The only other proteins known to be expressed in sensory neurons in a
pool-specific fashion are the ETS transcription factors ER81 and Pea3. Like
Lmo4, ER81 is expressed in the majority of sensory neurons supplying the
adductor and external femorotibialis muscles, but in few sartorius or internal
femorotibialis sensory neurons (Lin et
al., 1998). Because Lmo4 is expressed before sensory neurons
attain their definitive ETS phenotype, these results suggest that Lmo4
expression might predict the later expression of ER81. In this regard, it is
interesting that Lmo4 expression does not require that sensory neurons have
peripheral targets. By contrast, the definitive ETS phenotype of sensory
neurons is expressed only after they contract their peripheral targets, and
ETS gene expression is dependent on these targets. A possible scenario is that
expression of Lmo4 could be permissive for ER81, but the ultimate expression
of ER81 also depends on contact with the appropriate peripheral target.
According to this idea, only a fraction of Lmo4-positive neurons will also be
ER81 positive, which would explain why many sensory neurons in LS1 are Lmo4
positive but few express ER81. A weakness of this idea, however, is that many
ER81-positive neurons do not express Lmo4, at least by stage 35. Throughout
LS1 to LS4, 35% of ER81-positive neurons are Lmo4 negative, and the fraction
of Lmo4-negative/ER81-positive cells is similar in each of these segmental
ganglia. It is therefore unlikely that Lmo4 expression plays a major role in
determining the ultimate ETS phenotype of developing sensory neurons.
Lmo4 expression in developing motoneurons
Many functional subsets of motoneurons within the LMC can be defined by
co-expression of LIM homeodomain proteins and ETS proteins
(Lin et al., 1998). The
differential expression of LIM homeodomain proteins by medial and lateral LMC
neurons is established early, soon after neurons exit the cell cycle and
migrate to the LMC (Tsuchida et al.,
1994
; Ensini et al.,
1998
; Sockanathan and Jessell,
1998
). As early as stage 26, when motor axons are making selective
projections to their peripheral targets, Lmo4 is already expressed in subsets
of motoneurons that otherwise share the same pattern of LIM and Isl
expression. As Lmo4 could modify the transcriptional activity of LIM
homeodomain proteins, differential expression of Lmo4 in motoneurons may serve
to direct otherwise similar motoneurons along different developmental
pathways. Like LIM proteins that are important in the early determination of
motor pool identity, expression of Lmo4 in motoneurons does not require
signals from limb tissue. It is possible that early differential expression of
Lmo4 seen in motoneurons at stage 26 may just reflect their developmental
status rather than definitive functional diversity. However, Lmo4 is expressed
in some but not all Pea3-positive neurons (that are all Lim1/Lim2-positive) at
stage 35, when all motoneurons have already exited the cell cycle, arguing for
a role of Lmo4 in motoneuron differentiation.
Motoneurons can also be divided into functional subsets, e.g. extensors and
flexors. These two classes receive distinct sets of interneuronal inputs
(Fedirchuk et al., 1999;
Landmesser and O'Donovan,
1984a
; O'Donovan,
1989
). These distinctive patterns of inputs are already apparent
at stage 25, when motor axons are still growing to their muscle targets.
Furthermore, the identity is intrinsic to motoneurons; anteroposterior limb
bud rotation does not change interneuronal inputs
(Landmesser and O'Donovan,
1984b
; Milner et al.,
1998
; Vogel, 1987
)
(for a review, see Landmesser,
2001
). Interestingly, of the five motor pools we studied, Lmo4 is
expressed predominantly in those supplying extensor muscles. Approximately 90%
of motoneurons supplying adductor, external femorotibialis and posterior
iliotibialis muscles (all extensors) express Lmo4. Similarly, the
Lim3-positive LMC neurons in the brachial cord that supply the rhomboideus
muscle, another extensor, are also Lmo4 positive (data not shown). By
contrast, the two flexor pools we labeled, sartorius and internal
femorotibialis, have lower levels of Lmo4 expression (15% and 40%,
respectively). It will be interesting to be determine if Lmo4 is an intrinsic
marker for motoneurons supplying extensor versus flexor muscles.
Function of Lmo4
Lmo4 can regulate the transcriptional activities of LIM homeodomain factors
in several ways. Lmo transcriptional regulatory factors lack a DNA binding
domain but contain two protein-protein interaction LIM domains. Lmo proteins
can compete for NLI with LIM homeodomain transcription factors, and thereby
regulate the formation of LIM homeodomain/NLI complexes and their
transcriptional activity. A recent study has shown that Drosophila
Lmo can bind to Chip with higher affinity than the LIM homeodomain of Apterous
and thereby regulate Apterous activity levels in vivo
(Weihe et al., 2001). Whether
there is a differential affinity to NLI between Lmo4 and other LIM homeodomain
proteins is not yet known.
Lmo4 may also compete for other co-factors besides NLI that are specific
for individual LIM homeodomain proteins and could thus regulate the expression
of downstream target genes. For example, by expressing chimeric LIM domains
derived from different Islet family members (i.e. Isl1, Isl2 and ISL3) in
zebrafish, Okamoto and his colleagues concluded that Isl2 probably forms a
transcriptional complex with an Isl2-specific co-factor, in addition to NLI.
Interaction with an Isl2-specific co-factor could contribute to the role of
Isl2 in the differentiation of primary motoneurons, neuronal positioning,
peripheral axonal outgrowth and neuronal transmitter expression in zebrafish
(Segawa et al., 2001).
Combinatorial interactions of Lmo4 with other transcription factors might
provide additional mechanisms for the regulation of transcription during
neuronal development. In enkaphalin-producing neurons, Lmo4 interacts with the
transcription factor DEAF1 (deformed epidermal autoregulatory factor 1)
(Sugihara et al., 1998). DEAF1
has been implicated in opioid production by regulating enkaphalin
transcription through a retinoic acid-responsive element. Interestingly, in
the fetal and adult mouse brain, Lmo4 expression is region specific: high
levels of expression are present in the limbic system and in regions involved
in autonomic, motor and neuroendocrine regulation
(Huggenvik et al., 1998
).
Recently, studies in breast cancer cell lines have demonstrated that Lmo4
expression is upregulated and forms a multiprotein complex with CtIP and BRCA1
(Sum et al., 2002
;
Visvader et al., 2001
). A role
for BRCA1 in neurons has not been explored.
Different LIM homeodomain proteins are known to activate different
downstream target genes (Hobert and
Westphal, 2000). The pattern of neuronal generation in the ventral
neural tube is achieved primarily by the spatially restricted expression of
transcriptional repressors (Muhr et al.,
2001
). By modulating the transcriptional activity of LIM
homeodomain proteins, Lmo4 is likely to be involved in the specification of
motor neuronal identity. Its restricted expression in subsets of muscle
sensory neurons suggests that it contributes to the specification of sensory
neurons as well.
![]() |
ACKNOWLEDGMENTS |
---|
H.-H. C. is supported by an NRSA fellowship.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bach, I. (2000). The LIM domain: regulation by association. Mech. Dev. 91, 5-17.[CrossRef][Medline]
Bao, J., Talmage, D. A., Role, L. W. and Gautier, J.
(2000). Regulation of neurogenesis by interactions between HEN1
and neuronal LMO proteins. Development
127,425
-435.
Caldero, J., Prevette, D., Mei, X., Oakley, R. A., Li, L.,
Milligan, C., Houenou, L., Burek, M. and Oppenheim, R. W.
(1998). Peripheral target regulation of the development and
survival of spinal sensory and motor neurons in the chick embryo.
J. Neurosci. 18,356
-370.
Dulac, C. and Axel, R. (1995). A novel family of genes encoding putative pheromone receptors in mammals. Cell 83,195 -206.[Medline]
Ensini, M., Tsuchida, T. N., Belting, H. G. and Jessell, T.
M. (1998). The control of rostrocaudal pattern in the
developing spinal cord: specification of motor neuron subtype identity is
initiated by signals from paraxial mesoderm.
Development 125,969
-982.
Fedirchuk, B., Wenner, P., Whelan, P. J., Ho, S., Tabak, J. and
O'Donovan, M. J. (1999). Spontaneous network activity
transiently depresses synaptic transmission in the embryonic chick spinal
cord. J. Neurosci. 19,2102
-2112.
Frank, E. and Wenner, P. (1993). Environmental specification of neuronal connectivity. Neuron 10,779 -785.[Medline]
Hamburger, V. and Hamilton, H. C. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88,49 -92.
Hobert, O. and Westphal, H. (2000). Functions of LIM-homeobox genes. Trends Genet. 16, 75-83.[CrossRef][Medline]
Hollyday, M. (1980a). Motoneuron histogenesis and the development of limb innervation. Curr. Top. Dev. Biol. 15,181 -215.[Medline]
Hollyday, M. (1980b). Organization of motor pools in the chick lumbar lateral motor column. J. Comp. Neurol. 194,143 -170.[Medline]
Honig, M. G., Frase, P. A. and Camilli, S. J.
(1998). The spatial relationships among cutaneous, muscle sensory
and motoneuron axons during development of the chick hindlimb.
Development 125,995
-1004.
Huggenvik, J. I., Michelson, R. J., Collard, M. W., Ziemba, A.
J., Gurley, P. and Mowen, K. A. (1998). Characterization of a
nuclear deformed epidermal autoregulatory factor-1 (DEAF-1)-related (NUDR)
transcriptional regulator protein. Mol. Endocrinol.
12,1619
-1639.
Jurata, L. W., Thomas, J. B. and Pfaff, S. L. (2000). Transcriptional mechanisms in the development of motor control. Curr. Opin. Neurobiol. 10, 72-79.[CrossRef][Medline]
Kania, A., Johnson, R. L. and Jessell, T. M. (2000). Coordinate roles for LIM homeobox genes in directing the dorsoventral trajectory of motor axons in the vertebrate limb. Cell 102,161 -173.[Medline]
Kenny, D. A., Jurata, L. W., Saga, Y. and Gill, G. N.
(1998). Identification and characterization of LMO4, an LMO gene
with a novel pattern of expression during embryogenesis. Proc.
Natl. Acad. Sci. USA 95,11257
-11262.
Lance-Jones, C. and Landmesser, L. (1980). Motoneurone projection patterns in the chick hind limb following early partial reversals of the spinal cord. J. Physiol. 302,581 -602.[Abstract]
Landmesser, L. (1978). The distribution of motoneurones supplying chick hind limb muscles. J. Physiol. 284,371 -389.[Abstract]
Landmesser, L. T. (2001). The acquisition of motoneuron subtype identity and motor circuit formation. Int. J. Dev. Neurosci. 19,175 -182.[CrossRef][Medline]
Landmesser, L. T. and O'Donovan, M. J. (1984a). Activation patterns of embryonic chick hind limb muscles recorded in ovo and in an isolated spinal cord preparation. J. Physiol. 347,189 -204.[Abstract]
Landmesser, L. T. and O'Donovan, M. J. (1984b). The activation patterns of embryonic chick motoneurones projecting to inappropriate muscles. J. Physiol. 347,205 -224.[Abstract]
Lee, M. T., Koebbe, M. J. and O'Donovan, M. J. (1988). The development of sensorimotor synaptic connections in the lumbosacral cord of the chick embryo. J. Neurosci. 8,2530 -2543.[Abstract]
Lin, J. H., Saito, T., Anderson, D. J., Lance-Jones, C., Jessell, T. M. and Arber, S. (1998). Functionally related motor neuron pool and muscle sensory afferent subtypes defined by coordinate ETS gene expression. Cell 95,393 -407.[Medline]
Ma, Q., Fode, C., Guillemot, F. and Anderson, D. J.
(1999). Neurogenin1 and neurogenin2 control two distinct waves of
neurogenesis in developing dorsal root ganglia. Genes
Dev. 13,1717
-1728.
Mendelson, B. and Frank, E. (1991). Specific monosynaptic sensory-motor connections form in the absence of patterned neural activity and motoneuronal cell death. J. Neurosci. 11,1390 -1403.[Abstract]
Milan, M. and Cohen, S. M. (1999). Regulation of LIM homeodomain activity in vivo: a tetramer of dLDB and apterous confers activity and capacity for regulation by dLMO. Mol. Cell 4,267 -273.[Medline]
Milan, M., Diaz-Benjumea, F. J. and Cohen, S. M.
(1998). Beadex encodes an LMO protein that regulates Apterous
LIM-homeodomain activity in Drosophila wing development: a model for LMO
oncogene function. Genes Dev.
12,2912
-2920.
Milner, L. D., Rafuse, V. F. and Landmesser, L. T.
(1998). Selective fasciculation and divergent pathfinding
decisions of embryonic chick motor axons projecting to fast and slow muscle
regions. J. Neurosci.
18,3297
-3313.
Muhr, J., Andersson, E., Persson, M., Jessell, T. M. and Ericson, J. (2001). Groucho-mediated transcriptional repression establishes progenitor cell pattern and neuronal fate in the ventral neural tube. Cell 104,861 -873.[Medline]
O'Donovan, M. J. (1989). Motor activity in the isolated spinal cord of the chick embryo: synaptic drive and firing pattern of single motoneurons. J. Neurosci. 9, 943-958.[Abstract]
Oakley, R. A., Lefcort, F. B., Plouffe, P., Ritter, A. and Frank, E. (2000). Neurotrophin-3 promotes the survival of a limited subpopulation of cutaneous sensory neurons. Dev. Biol. 224,415 -427.[CrossRef][Medline]
Pfaff, S. L., Mendelsohn, M., Stewart, C. L., Edlund, T. and Jessell, T. M. (1996). Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell 84,309 -320.[Medline]
Rabbitts, T. H. (1998). LMO T-cell
translocation oncogenes typify genes activated by chromosomal translocations
that alter transcription and developmental processes. Genes
Dev. 12,2651
-2657.
Segawa, H., Miyashita, T., Hirate, Y., Higashijima, S., Chino, N., Uyemura, K., Kikuchi, Y. and Okamoto, H. (2001). Functional repression of Islet-2 by disruption of complex with Ldb impairs peripheral axonal outgrowth in embryonic zebrafish. Neuron 30,423 -436.[CrossRef][Medline]
Sockanathan, S. and Jessell, T. M. (1998). Motor neuron-derived retinoid signaling specifies the subtype identity of spinal motor neurons. Cell 94,503 -514.[Medline]
Sugihara, T. M., Bach, I., Kioussi, C., Rosenfeld, M. G. and
Andersen, B. (1998). Mouse deformed epidermal autoregulatory
factor 1 recruits a LIM domain factor, LMO-4, and CLIM coregulators.
Proc. Natl. Acad. Sci. USA
95,15418
-15423.
Sum, E. Y., Peng, B., Yu, X., Chen, J., Byrne, J., Lindeman, G.
J. and Visvader, J. E. (2002). The LIM domain protein LMO4
interacts with the cofactor CtIP and the tumor suppressor BRCA1 and inhibits
BRCA1 activity. J. Biol. Chem.
277,7849
-7856.
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]
Visvader, J. E., Venter, D., Hahm, K., Santamaria, M., Sum, E.
Y., O'Reilly, L., White, D., Williams, R., Armes, J. and Lindeman, G. J.
(2001). The LIM domain gene LMO4 inhibits differentiation of
mammary epithelial cells in vitro and is overexpressed in breast cancer.
Proc. Natl. Acad. Sci. USA
98,14452
-14457.
Vogel, M. W. (1987). Activation patterns of embryonic chick lumbosacral motoneurones following large spinal cord reversals. J. Physiol. 389,491 -512.[Abstract]
Weihe, U., Milan, M. and Cohen, S. M. (2001).
Regulation of Apterous activity in Drosophila wing development.
Development 128,4615
-4622.