- Research article
- Open Access
Lateral motor column axons execute a ternary trajectory choice between limb and body tissues
© Luria and Laufer; licensee BioMed Central Ltd. 2007
- Received: 12 January 2007
- Accepted: 02 July 2007
- Published: 02 July 2007
Neuronal topographic map formation requires appropriate selection of axonal trajectories at intermediate choice points prior to target innervation. Axons of neurons in the spinal cord lateral motor column (LMC), as defined by a transcription factor code, are thought to innervate limb target tissues exclusively. Axons of the medial and lateral LMC divisions appear to execute a binary decision at the base of the limb as they choose between ventral and dorsal limb trajectories. The cellular logic that guides motor axon trajectory choices into non-limb tissues such as the ventral flank remains unclear.
We determined the spinal cord motor column origin of motor nerves that innervate ventral flank tissues at hindlimb level. We found unexpectedly that a subset of medial LMC axons innervates ventral non-limb mesenchyme at hindlimb level, rather than entering ventral limb mesenchyme. We also found that in a conditional BmprIa mutant where all ventral hindlimb mesenchyme is converted to a dorsal identity, all medial LMC axons are redirected into the ventral flank, while lateral LMC axons innervate the bidorsal limb.
We have found that medial LMC neurons innervate both ventral flank and limb targets. While normally only a subset of medial LMC axons innervate the flank, all are capable of doing so. Furthermore, LMC axons execute a ternary, rather than binary, choice at the base of the limb between ventral flank, ventral limb and dorsal limb trajectories. When making this choice, medial and lateral LMC axons exhibit different and asymmetric relative preferences for these three trajectories. These data redefine the LMC as a motor column that innervates both limb and body tissues.
- Motor Neuron
- Mutant Embryo
- Motor Axon
- Retrograde Label
- Genitofemoral Nerve
The precision of neural circuits requires stereotypic patterns of neuronal connectivity, which are often organized as topographic maps. Studies of how the point-to-point connections between spinal cord motor neurons and their targets are generated have revealed much about how such maps are constructed [1–3]. One principle to emerge from these studies is that functionally related neurons extend axons along shared trajectories that are precisely and accurately subdivided at intermediate choice points [4, 5]. Deciphering the logic underlying these trajectory choices is thus critical to understanding how these connectivity patterns are established.
The overall map between motor neurons and their peripheral targets is well described [3, 6–8]. The motor neurons are organized mediolaterally into medial and lateral motor columns (MMC and LMC) and their divisions. Medial MMC neurons innervate dorsal axial muscles. At thoracic levels, lateral MMC neurons innervate ventral body wall muscles, and autonomic motor neurons of the intermediolateral column (IML, column of Terni in birds) innervate sympathetic postganglionic neurons. At limb levels, lateral LMC neurons innervate dorsal limb muscles while medial LMC neurons innervate ventral limb muscles. The motor columns are further subdivided into pools, and each motor pool innervates an individual muscle . These descriptions are generally accurate, although possibly incomplete, as the identity of motor neurons that innervate many muscles, particularly outside of the limb, has not been examined in detail.
Current models suggest that shared intrinsic properties of neurons in a columnar division guide common trajectory decisions required of all axons projecting from that division . With the exception of some cervical motor populations, all motor axons exit the ventral root of the spinal cord along a ventrolateral trajectory. Divisional populations deviate from this shared path at progressively more distal choice points. At limb axial levels the final divisional decision is made in the motor plexus at the base of the limb, where LMC axons assume trajectories into either dorsal or ventral limb mesenchyme.
The discovery that combinations of LIM homeodomain transcription factors identify motor neurons within columnar divisions led to the idea that the columnar identity of a motor neuron as defined by this transcription factor code is predictive of its axonal trajectory [3, 9]. Consistent with this, neurons whose cell bodies are misplaced within morphological columns relative to their axonal trajectories nonetheless express columnar transcription factors appropriate to their trajectories . Gain and loss of function genetic experiments demonstrate that these transcription factors impose specific pathfinding behaviors on motor axons [10, 11], and do so largely by regulating expression of axon guidance receptors [12, 13].
Experiments designed to test how trajectories into limb mesenchyme are chosen by axons of LMC neurons have invariably found both medial and lateral LMC axons entering the limb. This is the case for limbs that are surgically truncated, rotated or duplicated along the dorsal-ventral axis [4, 14–17]; for mutants in which limb mesenchymal dorsoventral identity or guidance cue expression is altered [11, 18–20]; for mutants in which LMC axonal guidance receptor expression is altered [12, 19–22]; and for embryos in which spontaneous neural activity is inhibited . These results, in combination with descriptions of the neuromuscular topographic map [6–8], have led to the idea that at the base of the limb, LMC axons have only two options: they make a binary choice between trajectories into either dorsal or ventral limb mesenchyme [3, 24–26].
The contributions of both intrinsic properties of LMC motor neurons and extrinsic guidance cues to this trajectory choice have been further defined. Surgical experiments demonstrate that the choice is active, and is regulated locally by signals generated at the choice point [4, 14–16]. Molecular genetic experiments have identified primarily [12, 18, 19, 27, 28], but not exclusively , repulsive ligand:receptor combinations that influence this choice. The emerging model is that the LIM homeodomain code imparts selective insensitivity to peripheral guidance cues until axons arrive at the base of the limb [3, 13]. Differential sensitivity to guidance cues reflecting limb dorsoventral pattern then guides medial and lateral LMC axons as they choose between dorsal and ventral limb trajectories [11, 12, 18–20, 28].
While appealing, it is not clear if this model can account for the selection of trajectories by all motor axons that reach the base of the limb. Several nerves (iliohypogastric, ilioinguinal and genitofemoral) that project from the most rostral lumbar segments (L1–L2) of the spinal cord innervate lateral abdominal body wall (external oblique, internal oblique and transverse) and genital (cremaster) muscles [29, 30]. The motor axons that ultimately form these nerves extend through the plexus into ventral flank mesenchyme. These trajectories are not typical of medial MMC, IML, medial LMC or lateral LMC axons, which are all present at this axial level.
We investigated the columnar identity of the ventral flank nerves and found that they originate within the medial LMC. This result indicates there is a third trajectory, in addition to the limb trajectories, available to LMC axons, one that binary choice models do not accommodate. To investigate how peripheral cues influence LMC axon trajectory decisions, we removed the ventral limb mesenchyme using a conditional bone morphogenetic protein receptor Ia genetic model (BmprIaflox/-) , in which all hindlimb mesenchyme has a dorsal identity. When medial LMC axons are confronted with this bidorsal limb mesenchyme, they do not enter the limb, and are redirected into the ventral flank. In contrast, lateral LMC axons enter the bidorsal hindlimb and populate both dorsal and ventral nerve branches. Our results show that motor axons have a ternary, not binary, choice of trajectories at the base of the hindlimb, and reveal that medial and lateral LMC axons have different and asymmetric preferences for trajectories that enter ventral flank, ventral limb or dorsal limb mesenchyme. Our findings redefine LMC columnar identity as predictive of trajectories that include both body and limb tissues.
Medial LMC axons project to the ventral flank mesenchyme
To determine whether these neurons exist within a single motor pool, we asked whether the retrogradely labeled neurons express additional transcription factor markers that label subsets of medial LMC neurons [33, 34]. We could readily detect labeled Nkx6.1+ and labeled Nkx6.1- neurons, and could occasionally detect labeled Er81+ neurons (Additional file 1). Together these data indicate that neurons that project along ventral flank trajectories likely derive from multiple motor pools, which is consistent with the observation that the nerves innervate multiple target muscles.
A subset of medial LMC neurons, as defined by their LIM homeodomain transcription factor code, thus unexpectedly project not to the limb, but to the ventral flank, after leaving the anterior plexus. This raises the possibility that this non-limb trajectory might be available to other medial LMC or perhaps all LMC axons. If so, then additional medial LMC neurons might also project to the ventral flank mesenchyme in appropriate mutant contexts.
BmprIaflox/-mutant embryos have completely bidorsal hindlimbs
We reasoned that a mutant lacking ventral limb mesenchyme, the predominant medial LMC axonal target, is one scenario in which additional ventral flank projections might be observed, as medial LMC axons would, of necessity, innervate an alternative mesenchymal territory. Brn4-creTg/-, BmprIaflox/-(BmprIaflox/-) mice carry a conditional null allele of the BmprIa gene that is inactivated to homozygosity by Cre recombinase expressed under the control of a transgenic Brn4 promoter . In these mice the ventral hindlimb mesenchyme is transformed to an apparently dorsal identity, while forelimb dorsoventral polarity is normal [31, 35, 36]. While BmprIaflox/-mutant hindlimbs are small, they nonetheless include proximal and intermediate limb tissue . This mutant is thus a promising candidate in which to look for additional LMC axons innervating the ventral flank.
The dorsoventral projection choice is controlled by local signals [15, 16, 37]. Thus, we first confirmed that at the time of the choice in the BmprIaflox/-mutant no residual ventral limb mesenchyme is present near the choice point, as previous studies did not establish this [31, 36]. We defined a set of molecular markers for evaluating the dorsoventral identity of the proximal limb mesenchyme. Our criteria were: (1) that each marker is expressed either throughout the dorsoventral extent of the limb or is restricted todorsal or ventral limb; and (2) that the markers share a proximal boundary adjacent to the plexus as axons begin entering the limb mesenchyme (E10.5–11.0 in hindlimb, E10.0–10.5 in forelimb) [11, 16]. The markers in combination (the 'dorsoventral code') should define the limb mesenchyme as dorsal or ventral with respect to the projection choice and account for all topography along the limb dorsoventral axis.
We used these markers to examine the dorsoventral character of BmprIaflox/-mutant limb mesenchyme. All of the Plzf+ hindlimb mesenchyme is Lmx1b+ (Figure 2b) and is also Σephrin-A- (Figure 2d). EphA4 is present throughout the dorsoventral extent of the hindlimb mesenchyme and, notably, its proximal ventral expression boundary coincides with that of Lmx1b (Figure 2c). Taken together, these results provide evidence that all of the mutant hindlimb mesenchyme is dorsal (Plzf+ Lmx1b+ Σephrin-A-) and that at the base of the limb it is proximally dorsal (EphA4+). By contrast, BmprIaflox/-mutant forelimb mesenchyme has normal dorsoventral polarity, consistent with previous reports (Additional file 2) . Thus, relative to the dorsoventral projection choice, the dorsal transformation of ventral mesenchyme in BmprIaflox/-hindlimbs is complete.
A dorsoventral axon branch point forms in BmprIaflox/-mutant hindlimbs
Medial LMC axons are redirected from ventral limb to ventral flank
While at hindlimb levels some medial LMC axons are rerouted to the ventral flank in BmprIaflox/-mutants, other medial LMC axons might assume different trajectories, or perhaps stall at the base of the limb. NF immunostaining indicates that axons do not assume random trajectories in the peripheral non-limb mesenchyme. If axons stall, growth cones should accumulate in the hindlimb plexus. To address this possibility, we examined vesicular acetylcholine transporter (VAChT) expression , which preferentially labels distal regions of axons, shortly after the dorsoventral projection choice. We did not detect significant differences in VAChT staining between forelimbs or hindlimbs of normal and BmprIaflox/-embryos (Additional file 4). These results provide evidence that axons extend through the hindlimb motor plexus of BmprIaflox/-mutants.
Extra flank nerves are present in embryos with bidorsal limbs
We also examined the trajectories of GFP+ axons that project along the lower abdominal wall in freshly dissected, intact E13.5 embryos (Figure 7). In control embryos (N = 8/8 embryos, 16/16 limbs) three nerves were detected projecting abdominally from the rostral lumbar spinal cord when viewed from a peritoneal aspect (Figure 7a). The two most rostral of these, the iliohypogastric and ilioinguinal nerves, project laterally from L1 and L2, and follow trajectories that parallel those of the more rostral subcostal and intercostal nerves. The third, the genitofemoral nerve, projects laterally upon leaving the rostral plexus in caudal L3 and curves anteriorly towards the ilioinguinal nerve. The ilioinguinal and genitofemoral trajectories together describe a roughly half-moon pattern with the ilioinguinal transcribing the meridian and the genitofemoral the perimeter. The genitofemoral leaves the plexus coincident with the femoral and obturator nerves that extend distally into the limb. More caudally no lumbar nerves follow flank trajectories after leaving the sciatic plexus, although many nerves extend into the limb (Figure 7b).
In BmprIaflox/-mutant embryos (N = 3 embryos, 6 limbs) there are differences from controls both in the distribution and number of flank projections. The genitofemoral nerve projects more directly laterally, which results in more even spacing among the three major flank nerves (Figure 7c). In addition, one to three excess nerves project from rostral lumbar segments along abdominal trajectories, although with variable projection patterns that differ even within a single embryo. These fibers are located between the ilioinguinal and genitofemoral nerves or caudal to the genitofemoral nerve. A substantial population of novel GFP+ axons also extends abdominally from the sciatic plexus (Figure 7d). Nerves projecting along the femoral and obturator trajectories are visible, although reduced in size, and sciatic projections into the limb are also present. These data provide clear evidence that the ventral flank axons normally derive from rostral lumbar regions, and that when confronted with only dorsal limb mesenchyme, motor axons originating along the length of the lumbar spinal cord aberrantly choose ventral flank trajectories.
Only lateral LMC axons enter the bidorsal BmprIaflox/-hindlimb
Altered LMC trajectories are due to changes in the BmprIaflox/-hindlimb
Because the Brn4-cre transgene is expressed throughout the spinal cord, in addition to the limb ectoderm [31, 35], we asked whether removing BmprIa from only the spinal cord might alter the LMC axonal trajectories. We first used molecular markers to label ventral progenitor or postmitotic motor neuron and interneuron populations at both brachial and lumbar levels of the spinal cord in BmprIaflox/-mutants. We observed no significant difference between BmprIaflox/-mutant and normal sibling progenitor or motor neuron populations at either axial level (Additional file 5). We next examined LMC motor projection patterns to BmprIaflox/-forelimbs, which have normal dorsoventral patterning. Lim1 tlz lateral LMC marking and retrograde labeling analyses from dorsal and ventral forelimb mesenchyme all show that the columnar origins of these axons are normal (Additional files 3 and 6). Thus removing BmprIa from the spinal cord does not alter the LMC dorsoventral trajectory choice at forelimb levels.
Multiple lines of evidence therefore indicate that inactivating BmprIa selectively in motor neurons does not perturb the limb motor axon projection patterns. These data include the normal projection patterns to Brn4-creTg/-, BmprIaflox/-mutant forelimbs and to Hb9cre/+, BmprIaflox/-mutant hindlimbs, as well as the normal generation of LMC neurons on both genetic backgrounds. These results provide strong support for the idea that the hindlimb projection defects observed in the Brn4-creTg/-, BmprIaflox/-mutant are caused by altered guidance cues in the limb mesenchyme.
Ventral flank mesenchyme is innervated by medial LMC neurons
We used multiple approaches to identify the motor neurons that extend axons into the ventral flank mesenchyme at hindlimb levels. All of them reveal that medial LMC neurons normally innervate both ventral limb and ventral flank mesenchyme. We therefore propose a change in the predictive value of the LIM homeodomain code regarding LMC motor axons [3, 9]: Lim1- Isl1+ Lim3- medial LMC neurons should project to both ventral flank and ventral limb.
The axons that enter the hindlimb ventral flank are likely ultimately to form the motor components of the iliohypogastric, ilioinguinal and genitofemoral nerves [29, 30]. In mammals these nerves arise from segments T12–L2 and they are the three most rostral nerves that project ventrally from the rostral hindlimb motor plexus. The motor components of the iliohypogastric and ilioinguinal nerves innervate lower abdominal muscles, while the sexually dimorphic genitofemoral nerve innervates the male cremaster muscle, which controls the position of the testes within the scrotum [48–50]. Previous experiments have not directly addressed the columnar origin of these nerves. However, retrograde labeling of the genitofemoral nerve from the cremaster muscle in rats marked a longitudinal distribution of somas in rostral lumbar regions , while ventral abdominal muscle labeling experiments consistently mark rostral lumbar motor pools [51–53]. Electrophysiological studies in humans demonstrate upper motor control of the genitofemoral nerve, and some degree of voluntary control . These results are consistent with our identification of the ventral flank-projecting axons as somatic motor axons projecting from the rostral portion of the medial LMC.
Relationship of medial LMC and lateral MMC neurons
Intriguingly, our observations support the idea that the trajectory preferences of lumbar medial LMC axons and thoracic lateral MMC axons are similarly regulated . These neurons have the same LIM homeodomain transcription factor code and similar mediolateral somatic settling positions, and the ventral flank trajectory we documented for some medial LMC axons parallels that of lateral MMC axons. These axons make a series of similar projection decisions, ignoring both the dorsal trajectory of the medial MMC and the ventromedial trajectory of the autonomic motor neurons, before assuming a distal trajectory into ventral lateral plate mesodermal derivatives [9, 13]. Our results also suggest that lateral MMC axons might perceive ventral flank and limb mesenchyme as similarly permissive, which might explain why they readily innervate ectopic thoracic limbs  or normal limbs when the axons are caudally displaced [2, 4]. However, these populations are not a simple continuum, as LMC, but not lateral MMC, neurons express FoxP1 and Raldh2, for example [32, 56]. It will be interesting to learn more about what these populations have in common, and how they diverged.
LMC axons make a three-way projection decision at the base of the limb
Our data reveal that a trajectory into ventral flank mesenchyme is an option for LMC axons, which raises the question of why its significance was not previously appreciated. One likely reason is that relatively few motor axons normally enter the ventral flank mesenchyme, and thus they have not been studied as extensively as the axons that innervate the limb. Furthermore, previous experiments investigating initial limb innervation decisions always found that LMC axons entered the limb mesenchyme [2, 11, 12, 15, 16, 19–23]. In addition, when motor innervation patterns were examined in apparently bidorsal chick hindlimbs generated surgically, they were found to be normal , in contrast to the situation in the bidorsal BmprIaflox/-mutant hindlimbs. This might reflect a bona fide difference in the behavior of chick and mouse motor axons, although more likely small amounts of ventral limb mesenchyme remained near the dorsoventral choice point following the surgical manipulations, leading to a normal projection choice . Together, these data led to the idea that at the base of the limb LMC axons make a binary choice between either dorsal or ventral limb trajectories [24, 25]. Nonetheless, our data reveal that the motor axons execute a ternary, not a binary, choice of trajectories at hindlimb axial levels.
Whether lateral LMC axons enter ventral flank mesenchyme in existing mutants is not clear. We did not find lateral LMC axons making this choice when the hindlimbs are bidorsal. Similarly, no lateral LMC axons were described entering the ventral flank in Lmx1b-/- mice, which have biventral hindlimbs , or in EphA4-/- embryos, in which apparently all LMC axons are directed to ventral limb mesenchyme . However, lateral LMC axons assuming this trajectory might have gone unnoticed, as the ventral flank nerve branch was not directly examined in the Lmx1b-/- and EphA4-/- mutants. One interesting question is why LacZ+ lateral LMC axons are not substantially redirected into the ventral flank mesenchyme of Lim1tlz/+, Lmx1b-/- embryos, as they are repulsed by ephrin-A ligands that are uniformly high across the hindlimb of this mutant [11, 12]. Perhaps attractive cues in limb mesenchyme such as glial-derived neurotrophic factor (GDNF)  can overcome these repellant signals, or an additional repulsive cue in the ventral flank makes this territory even less permissive than ventral limb mesenchyme to lateral LMC axons. Regardless, it appears that axon entry into limb mesenchyme is not a default trajectory for LMC axons, but rather involves an active choice.
The comparative behavior of medial and lateral LMC axons is informative about the regulatory logic guiding their trajectory choices. One might anticipate that peripheral guidance cues influence medial and lateral LMC axons in a similar but opposite fashion. If this were so, since lateral LMC axons populate dorsal and ventral nerve branches in equal proportion in Lmx1b-/- mutants with biventral hindlimbs , then medial LMC axons should project equally to dorsal and ventral limb in BmprIaflox/-mutants with bidorsal hindlimbs. However, medial LMC axons instead project to the ventral flank, indicating that medial and lateral LMC axonal trajectories are guided asymmetrically.
We can order the relative preference of the three trajectories from the perspective of axons in either LMC division (Figure 10b). From the perspective of medial LMC axons, the normal preference for most is to innervate ventral limb mesenchyme, with the remainder innervating the ventral flank. In the absence of ventral limb mesenchyme, ventral flank mesenchyme is strongly preferred over dorsal limb. From the perspective of lateral LMC axons, the normal preference is to innervate dorsal limb mesenchyme, and in its absence, to innervate ventral limb mesenchyme. The least favored trajectory appears to be into the ventral flank. Thus, the relative trajectory preferences for LMC axons are neither identical nor mirror images, and appear for medial LMC as: ventral limb > ventral flank > dorsal limb, and for lateral LMC as: dorsal limb > ventral limb > ventral flank.
The trajectory assumed by LMC axons might follow from one ternary choice between three options, or from sequential binary decisions. All three hindlimb LMC trajectories diverge at roughly the same proximodistal position within the hindlimb plexus (Figure 2). This contrasts with the trajectory assumed by some medial LMC axons that extend from the brachial plexus into forelimb level ventral flank mesenchyme, and that ultimately innervate the latissimus dorsi and cutaneus maximus muscles [57, 58]. The forelimb ventral flank branch clearly diverges from the ventral limb branch after the axons of both branches have entered the ventral limb mesenchyme (for example, Additional file 3). This morphology implies that the decision to assume the forelimb flank trajectory is taken after the decision to enter the ventral limb mesenchyme. In contrast, the morphology of the hindlimb plexus favors a single ternary choice.
A model for the three-way choice
The existence of both attractive and repulsive cues that guide lateral LMC axons provides a framework for thinking about how medial LMC axons are directed to ventral limb, and how a subset of them is guided to the ventral flank. In one model (Figure 10c), attractive guidance cues are present throughout the limb mesenchyme, but not in the ventral flank, and a step gradient of a repulsive cue is also present. The dorsal limb mesenchyme expresses the repellant at the highest level, the ventral limb mesenchyme at an intermediate level and the ventral flank mesenchyme at the lowest level. All medial LMC neurons express receptors for these cues, with a subset expressing the repellant receptor at higher levels. Upon arrival at the base of the limb, the growth cones integrate the attractive and repulsive cues and assume the most permissive trajectory. So long as the repellant activity in the dorsal limb mesenchyme dominates, all medial LMC axons are directed ventrally. In contrast, the moderate repellant level in ventral limb mesenchyme is permissive to most medial LMC axons, although the subset of axons with the highest level of the repellant receptor is directed more ventrally, into the flank mesenchyme. This model predicts that the limb dorsoventral patterning system controls repulsive cue levels, and thus in a completely dorsalized limb, such as in the BmprIa mutant, the entire limb mesenchyme expresses high levels of repulsive cue, and directs all medial LMC axons to ventral flank tissue.
If a subset of medial LMC neurons expresses more repulsive receptor, this leads to the question of how this subset is specified. The neurons that normally contribute to the ventral flank nerves are located in rostral segments of the lumbar spinal cord. Higher receptor expression levels therefore might be established by the system that patterns the anterior-posterior axis of the spinal cord , perhaps influenced by differential expression of pool-specific Hox genes  that act prior to overt segregation of the cell bodies into motor pools. Interestingly, the distribution of peripheral guidance cues might be established via another axial patterning system, since the dorsal limb, ventral limb and ventral flank mesenchyme are mediolaterally-arrayed derivatives of the lateral plate mesoderm . While this model can account for the observed behavior of the medial LMC axons, others are equally plausible. Whatever the mechanism, it must make all medial LMC axons competent to assume ventral flank trajectories, while compelling only a few to do so.
We found that neurons of the LMC, long thought to innervate only limb tissues, actually innervate tissues beyond the limb, such as the ventral flank. To select appropriate trajectories, LMC axons execute a three-way choice between ventral flank, ventral limb and dorsal limb targets. These findings provide new insights into the cellular logic used in guiding trajectory decisions, uncover a novel function for the LMC and illuminate the relationship between LMC axons and those of other motor columns.
Mice carried the following alleles in various combinations: BmprIa KO , Brn4-cre Tg , BmprIa flox , Hb9 cre , Hb9-GFP Tg  and Lim1 tlz . Mice were maintained on mixed C57B6.J and 129SvEv backgrounds. Brn4-creTg/-, BmprIaflox/-and Hb9 cre , BmprIaflox/-embryos were generated by intercrossing Brn4-creTg/-, BmprIaKO/+or Hb9 cre , BmprIaKO/+and BmprIaflox/floxparents. Brn4-creTg/-, BmprIaflox/-embryos were identified by hindlimb morphology . All other allelic combinations from Brn4-cre matings were indistinguishable from wild-type controls and were designated normal .
Noon of the day a mating plug was observed was designated embryonic day 0.5 (E0.5). Stage matching was based on limb bud developmental stage , since limb stage and nerve invasion are correlated [4, 64].
Retrograde and anterograde labeling of limb and ventral flank nerves
For retrograde labeling, horseradish peroxidase (HRP; 20% (w/v) in 1% lysolecithin/PBS) or tetramethylrhodamine-coupled, lysinated dextran (RDA; 50% (w/v) MW 3,000; Molecular Probes, Carlsbad, CA, USA) was injected into dorsal limb, ventral limb or ventral flank mesenchyme of explanted E13.5 embryos cultured in oxygenated (95% O2, 5% CO2) DMEM/F12 media (Invitrogen, Carlsbad, CA, USA) at 30–35°C [11, 65]. After 4–6 h tissue was fixed and cryosectioned transversely. Control sections were examined by HRP or RDA and neurofilament (NF) coimmunostaining, and evaluated at the dorsoventral branch point for targeting accuracy. Only embryos with a single labeled nerve branch were analyzed further. Ventral flank tracer injection was confirmed at caudal lumbar axial levels that lacked labeled spinal cord neurons by the presence of labeled dorsal root ganglion neurons.
For anterograde labeling, explanted E12.5 embryos were dorsally laminectomized, and a pulled glass needle was introduced from a dorsomedial aspect towards the LMC. Approximately 10–40 nl (empirically determined to label specifically the most medial LMC neurons) of HRP or RDA was injected into every second lumbar spinal cord segment. Hb9-GFP Tg transgene  GFP fluorescence at E12.5 is weaker in medial than in lateral LMC neurons, and some injections were guided under UV illumination to this GFP low region. Labeling accuracy was determined in cryosections co-immunostained for HRP or RDA and NF or LMC division markers. Of 67 injected embryos, 10 (5 normal, 5 mutant) were further analyzed, because the vast majority of HRP+ cells were medial LMC neurons. In some embryos a few labeled axons appeared to be from sensory or medial MMC neurons.
Immunostaining, ephrin detection and in situ hybridization
Antibody stains on cryosections were performed following standard methods [9, 11]. Primary antibodies, dilutions and sources: rabbit anti-EphA4 1:1,000 (Zymed, Carlsbad, CA, USA); goat anti-HRP 1:1,000 (Jackson ImmunoResearch Labs, West Grove, PA, USA); mouse anti-Lim3 (61-8C10) 1:50, mouse anti-NF (2H3) 1:100 and mouse anti-Isl1/2 (4D5) 1:100 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA); rabbit anti-Lim1/2 1:4,000, guinea pig anti-Lim3 1:4000, rabbit anti-Isl1/2 (K4) 1:2,500, guinea pig anti-Isl1/2 1:16,000, guinea pig anti-murine Lmx1b 1:16,000, guinea pig anti-FoxP1 1:500 and guinea pig anti-Scip 1:4,000 [9, 12, 44]. Secondary antibody conjugates, all raised in donkey: Alexa-488 1:2,000 (Molecular Probes), Cy3 1:2,000 or Cy5 1:1,000 (Jackson ImmunoResearch Labs). EphA4-AP fusion protein  was used to detect ephrin-A protein in limb cryosections .
Neurons retrogradely labeled by HRP or RDA and colabeled with LMC markers were counted . Four or more sections from three or more embryos encompassing at least 150, and typically more than 250, retrogradely labeled cells were counted for each condition and marker combination. Statistical comparisons were performed using a two-tailed heteroscedastic Student's t-test, with a significance limit of P < 0.05. Values are reported as mean ± standard error of the mean (SEM).
Imaging and morphometric analysis
Expression patterns were compared using multicolor immunofluorescence imaging or overlaying of RNA in situ hybridization or EphA4-AP staining patterns from adjacent cryosections . For Lim1 tlz nerves, immunofluorescence intensity of NF+ and LacZ+ nerve branches were compared using the ImageJ program as described [11, 23]. The rostrocaudal extent of motor columns, pools and nerve branches was determined by scoring alternate transverse sections for marker presence. The length of the region was then calculated by multiplying the number of sections between the first and last positive section by the section thickness. Typically >80% of internal sections were positive for a given nerve branch.
We thank Bryan Crenshaw and Thomas Jessell for mice, Jeremy Dasen, Susan Brenner-Morton and Thomas Jessell for antibodies, Tyler Cutforth for EphA4-AP reagent, and Jonathan Licht and Randy Johnson for RNA in situ probe templates. We thank Amy Daniel and Jason Wu for technical support. We thank Thomas Jessell, Artur Kania, Julia Kaltschmidt, Bryan Crenshaw, Susan Brenner-Morton and members of the Laufer and Jessell Labs for insightful discussions and advice. We thank Daniel Vasiliauskas, Lori Zeltser, Oliver Hobert, Zaven Kaprielian, Thomas Jessell, Artur Kania and Julia Kaltschmidt for critical comments on the manuscript. EL acknowledges support from the NIH, the Muscular Dystrophy Association, the New York State Spinal Cord Injury Research Program and the Howard Hughes Medical Institute Research Resource Program for Medical Schools. The funding bodies were not involved in study conception, design or execution, or the decision to submit the manuscript for publication.
- Jessell TM: Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat Rev Genet. 2000, 1 (1): 20-29. 10.1038/35049541.View ArticlePubMedGoogle Scholar
- Landmesser LT: The acquisition of motoneuron subtype identity and motor circuit formation. Int J Dev Neurosci. 2001, 19 (2): 175-182. 10.1016/S0736-5748(00)00090-3.View ArticlePubMedGoogle Scholar
- Shirasaki R, Pfaff SL: Transcriptional codes and the control of neuronal identity. Annu Rev Neurosci. 2002, 25: 251-281. 10.1146/annurev.neuro.25.112701.142916.View ArticlePubMedGoogle Scholar
- Lance-Jones C, Landmesser L: Pathway selection by embryonic chick motoneurons in an experimentally altered environment. Proc R Soc Lond B Biol Sci. 1981, 214 (1194): 19-52.View ArticlePubMedGoogle Scholar
- Tosney KW, Landmesser LT: Specificity of early motoneuron growth cone outgrowth in the chick embryo. J Neurosci. 1985, 5 (9): 2336-2344.PubMedGoogle Scholar
- Romanes GJ: The Motor Pools of the Spinal Cord. Prog Brain Res. 1964, 11: 93-119.View ArticlePubMedGoogle Scholar
- McHanwell S, Biscoe TJ: The localization of motoneurons supplying the hindlimb muscles of the mouse. Philos Trans R Soc Lond B Biol Sci. 1981, 293 (1069): 477-508. 10.1098/rstb.1981.0082.View ArticlePubMedGoogle Scholar
- Hollyday M, Jacobson RD: Location of motor pools innervating chick wing. Journal of Comparative Neurology. 1990, 302 (3): 575-588. 10.1002/cne.903020313.View ArticlePubMedGoogle Scholar
- Tsuchida T, Ensini M, Morton SB, Baldassare M, Edlund T, Jessell TM, Pfaff SL: Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes. Cell. 1994, 79 (6): 957-970. 10.1016/0092-8674(94)90027-2.View ArticlePubMedGoogle Scholar
- Sharma K, Sheng HZ, Lettieri K, Li H, Karavanov A, Potter S, Westphal H, Pfaff SL: LIM homeodomain factors Lhx3 and Lhx4 assign subtype identities for motor neurons. Cell. 1998, 95 (6): 817-828. 10.1016/S0092-8674(00)81704-3.View ArticlePubMedGoogle Scholar
- Kania A, Johnson RL, Jessell TM: Coordinate roles for LIM homeobox genes in directing the dorsoventral trajectory of motor axons in the vertebrate limb. Cell. 2000, 102 (2): 161-173. 10.1016/S0092-8674(00)00022-2.View ArticlePubMedGoogle Scholar
- Kania A, Jessell TM: Topographic motor projections in the limb imposed by LIM homeodomain protein regulation of ephrin-A:EphA interactions. Neuron. 2003, 38 (4): 581-596. 10.1016/S0896-6273(03)00292-7.View ArticlePubMedGoogle Scholar
- Shirasaki R, Lewcock JW, Lettieri K, Pfaff SL: FGF as a target-derived chemoattractant for developing motor axons genetically programmed by the LIM code. Neuron. 2006, 50 (6): 841-853. 10.1016/j.neuron.2006.04.030.View ArticlePubMedGoogle Scholar
- Ferguson BA: Development of motor innervation of the chick following dorsal-ventral limb bud rotations. J Neurosci. 1983, 3 (9): 1760-1772.PubMedGoogle Scholar
- Whitelaw V, Hollyday M: Neural pathway constraints in the motor innervation of the chick hindlimb following dorsoventral rotations of distal limb segments. J Neurosci. 1983, 3 (6): 1226-1233.PubMedGoogle Scholar
- Tosney KW, Landmesser LT: Pattern and specificity of axonal outgrowth following varying degrees of chick limb bud ablation. J Neurosci. 1984, 4 (10): 2518-2527.PubMedGoogle Scholar
- Lance-Jones CC: Motoneuron projection patterns in chick embryonic limbs with a double complement of dorsal thigh musculature. Dev Biol. 1986, 116 (2): 387-406. 10.1016/0012-1606(86)90141-7.View ArticlePubMedGoogle Scholar
- Eberhart J, Barr J, O'Connell S, Flagg A, Swartz ME, Cramer KS, Tosney KW, Pasquale EB, Krull CE: Ephrin-A5 exerts positive or inhibitory effects on distinct subsets of EphA4-positive motor neurons. J Neurosci. 2004, 24 (5): 1070-1078. 10.1523/JNEUROSCI.4719-03.2004.View ArticlePubMedGoogle Scholar
- Huber AB, Kania A, Tran TS, Gu C, De Marco Garcia N, Lieberam I, Johnson D, Jessell TM, Ginty DD, Kolodkin AL: Distinct roles for secreted semaphorin signaling in spinal motor axon guidance. Neuron. 2005, 48 (6): 949-964. 10.1016/j.neuron.2005.12.003.View ArticlePubMedGoogle Scholar
- Kramer ER, Knott L, Su F, Dessaud E, Krull CE, Helmbacher F, Klein R: Cooperation between GDNF/Ret and ephrinA/EphA4 Signals for Motor-Axon Pathway Selection in the Limb. Neuron. 2006, 50 (1): 35-47. 10.1016/j.neuron.2006.02.020.View ArticlePubMedGoogle Scholar
- Helmbacher F, Schneider-Maunoury S, Topilko P, Tiret L, Charnay P: Targeting of the EphA4 tyrosine kinase receptor affects dorsal/ventral pathfinding of limb motor axons. Development. 2000, 127 (15): 3313-3324.PubMedGoogle Scholar
- Eberhart J, Swartz ME, Koblar SA, Pasquale EB, Krull CE: EphA4 Constitutes a Population-Specific Guidance Cue for Motor Neurons. Dev Biol. 2002, 247 (1): 89-101. 10.1006/dbio.2002.0695.View ArticlePubMedGoogle Scholar
- Hanson MG, Landmesser LT: Normal patterns of spontaneous activity are required for correct motor axon guidance and the expression of specific guidance molecules. Neuron. 2004, 43 (5): 687-701. 10.1016/j.neuron.2004.08.018.View ArticlePubMedGoogle Scholar
- Landmesser L: The development of motor projection patterns in the chick hind limb. J Physiol. 1978, 284: 391-414.PubMed CentralView ArticlePubMedGoogle Scholar
- Tosney KW, Landmesser LT: Development of the major pathways for neurite outgrowth in the chick hindlimb. Dev Biol. 1985, 109 (1): 193-214. 10.1016/0012-1606(85)90360-4.View ArticlePubMedGoogle Scholar
- Hollyday M: Specificity of initial axonal projections to embryonic chick wing. Journal of Comparative Neurology. 1990, 302 (3): 589-602. 10.1002/cne.903020314.View ArticlePubMedGoogle Scholar
- Ohta K, Iwamasa H, Drescher U, Terasaki H, Tanaka H: The inhibitory effect on neurite outgrowth of motoneurons exerted by the ligands ELF-1 and RAGS. Mech Dev. 1997, 64 (1-2): 127-135. 10.1016/S0925-4773(97)00056-7.View ArticlePubMedGoogle Scholar
- Eberhart J, Swartz M, Koblar SA, Pasquale EB, Tanaka H, Krull CE: Expression of EphA4, ephrin-A2 and ephrin-A5 during axon outgrowth to the hindlimb indicates potential roles in pathfinding. Dev Neurosci. 2000, 22 (3): 237-250. 10.1159/000017446.View ArticlePubMedGoogle Scholar
- Altman J, Bayer S: Atlas of prenatal brain development. 1995, Boca Raton , CRC PressGoogle Scholar
- April EW: Clinical Anatomy. The National Medical Series for Independent Study. 1997, New York , Williams & Wilkins, 393-399. 3rdGoogle Scholar
- Ahn K, Mishina Y, Hanks MC, Behringer RR, Crenshaw EB: BMPR-IA signaling is required for the formation of the apical ectodermal ridge and dorsal-ventral patterning of the limb. Development. 2001, 128 (22): 4449-4461.PubMedGoogle Scholar
- Tamura S, Morikawa Y, Iwanishi H, Hisaoka T, Senba E: Expression pattern of the winged-helix/forkhead transcription factor Foxp1 in the developing central nervous system. Gene Expr Patterns. 2003, 3 (2): 193-197. 10.1016/S1567-133X(03)00003-6.View ArticlePubMedGoogle Scholar
- Lin JH, Saito T, Anderson DJ, Lance-Jones C, Jessell TM, Arber S: Functionally related motor neuron pool and muscle sensory afferent subtypes defined by coordinate ETS gene expression. Cell. 1998, 95 (3): 393-407. 10.1016/S0092-8674(00)81770-5.View ArticlePubMedGoogle Scholar
- Sander M, Paydar S, Ericson J, Briscoe J, Berber E, German M, Jessell TM, Rubenstein JL: Ventral neural patterning by Nkx homeobox genes: Nkx6.1 controls somatic motor neuron and ventral interneuron fates. Genes Dev. 2000, 14 (17): 2134-2139. 10.1101/gad.820400.PubMed CentralView ArticlePubMedGoogle Scholar
- Heydemann A, Nguyen LC, Crenshaw EB: Regulatory regions from the Brn4 promoter direct LACZ expression to the developing forebrain and neural tube. Brain Res Dev Brain Res. 2001, 128 (1): 83-90. 10.1016/S0165-3806(01)00137-7.View ArticlePubMedGoogle Scholar
- Soshnikova N, Zechner D, Huelsken J, Mishina Y, Behringer RR, Taketo MM, Crenshaw EB, Birchmeier W: Genetic interaction between Wnt/beta-catenin and BMP receptor signaling during formation of the AER and the dorsal-ventral axis in the limb. Genes Dev. 2003, 17 (16): 1963-1968. 10.1101/gad.263003.PubMed CentralView ArticlePubMedGoogle Scholar
- Ferns MJ, Hollyday M: Motor innervation of dorsoventrally reversed wings in chick/quail chimeric embryos. J Neurosci. 1993, 13 (6): 2463-2476.PubMedGoogle Scholar
- Barna M, Pandolfi PP, Niswander L: Gli3 and Plzf cooperate in proximal limb patterning at early stages of limb development. Nature. 2005, 436 (7048): 277-281. 10.1038/nature03801.View ArticlePubMedGoogle Scholar
- Riddle RD, Ensini M, Nelson C, Tsuchida T, Jessell TM, Tabin C: Induction of the LIM homeobox gene Lmx1 by WNT7a establishes dorsoventral pattern in the vertebrate limb. Cell. 1995, 83 (4): 631-640. 10.1016/0092-8674(95)90103-5.View ArticlePubMedGoogle Scholar
- Chen H, Lun Y, Ovchinnikov D, Kokubo H, Oberg KC, Pepicelli CV, Gan L, Lee B, Johnson RL: Limb and kidney defects in Lmx1b mutant mice suggest an involvement of LMX1B in human nail patella syndrome. Nat Genet. 1998, 19 (1): 51-55. 10.1038/ng0598-51.View ArticlePubMedGoogle Scholar
- Iwamasa H, Ohta K, Yamada T, Ushijima K, Terasaki H, Tanaka H: Expression of Eph receptor tyrosine kinases and their ligands in chick embryonic motor neurons and hindlimb muscles. Dev Growth Differ. 1999, 41 (6): 685-698. 10.1046/j.1440-169x.1999.00468.x.View ArticlePubMedGoogle Scholar
- Flanagan JG, Vanderhaeghen P: The ephrins and Eph receptors in neural development. Annu Rev Neurosci. 1998, 21: 309-345. 10.1146/annurev.neuro.21.1.309.View ArticlePubMedGoogle Scholar
- Monuki ES, Weinmaster G, Kuhn R, Lemke G: SCIP: a glial POU domain gene regulated by cyclic AMP. Neuron. 1989, 3 (6): 783-793. 10.1016/0896-6273(89)90247-X.View ArticlePubMedGoogle Scholar
- Dasen JS, Tice BC, Brenner-Morton S, Jessell TM: A Hox regulatory network establishes motor neuron pool identity and target-muscle connectivity. Cell. 2005, 123 (3): 477-491. 10.1016/j.cell.2005.09.009.View ArticlePubMedGoogle Scholar
- Wilson JM, Rempel J, Brownstone RM: Postnatal development of cholinergic synapses on mouse spinal motoneurons. J Comp Neurol. 2004, 474 (1): 13-23. 10.1002/cne.20089.View ArticlePubMedGoogle Scholar
- Wichterle H, Lieberam I, Porter JA, Jessell TM: Directed differentiation of embryonic stem cells into motor neurons. Cell. 2002, 110 (3): 385-397. 10.1016/S0092-8674(02)00835-8.View ArticlePubMedGoogle Scholar
- Yang X, Arber S, William C, Li L, Tanabe Y, Jessell TM, Birchmeier C, Burden SJ: Patterning of muscle acetylcholine receptor gene expression in the absence of motor innervation. Neuron. 2001, 30 (2): 399-410. 10.1016/S0896-6273(01)00287-2.View ArticlePubMedGoogle Scholar
- Nagy JI, Senba E: Neural relations of cremaster motoneurons, spinal cord systems and the genitofemoral nerve in the rat. Brain Res Bull. 1985, 15 (6): 609-627. 10.1016/0361-9230(85)90211-4.View ArticlePubMedGoogle Scholar
- Zempoalteca R, Martinez-Gomez M, Hudson R, Cruz Y, Lucio RA: An anatomical and electrophysiological study of the genitofemoral nerve and some of its targets in the male rat. J Anat. 2002, 201 (6): 493-505. 10.1046/j.1469-7580.2002.00112.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Calguner E, Erdogan D, Elmas C, Bahcelioglu M, Gozil R, Ayhan MS: Innervation of the rat anterior abdominal wall as shown by modified Sihler's stain. Med Princ Pract. 2006, 15 (2): 98-101. 10.1159/000090911.View ArticlePubMedGoogle Scholar
- Gerrits PO, Boers J, Holstege G: The lumbar cord location of the motoneurons innervating psoas and iliacus muscles: a single and double labeling study in the female Syrian golden hamster. Neurosci Lett. 1997, 237 (2-3): 125-128. 10.1016/S0304-3940(97)00842-2.View ArticlePubMedGoogle Scholar
- Nicolopoulos-Stournaras S, Iles JF: Motor neuron columns in the lumbar spinal cord of the rat. J Comp Neurol. 1983, 217 (1): 75-85. 10.1002/cne.902170107.View ArticlePubMedGoogle Scholar
- Tani M, Kida MY, Akita K: Relationship between the arrangement of motoneuron pools in the ventral horn and ramification pattern of the spinal nerve innervating trunk muscles in the cat (Felis domestica). Exp Neurol. 1994, 128 (2): 290-300. 10.1006/exnr.1994.1139.View ArticlePubMedGoogle Scholar
- Ertekin C, Bademkiran F, Yildiz N, Ozdedeli K, Altay B, Aydogdu I, Uludag B: Central and peripheral motor conduction to cremasteric muscle. Muscle Nerve. 2005, 31 (3): 349-354. 10.1002/mus.20247.View ArticlePubMedGoogle Scholar
- Turney BW, Rowan-Hull AM, Brown JM: The innervation of FGF-induced additional limbs in the chick embryo. J Anat. 2003, 202 (1): 83-92. 10.1046/j.1469-7580.2003.00131.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Sockanathan S, Jessell TM: Motor neuron-derived retinoid signaling specifies the subtype identity of spinal motor neurons. Cell. 1998, 94 (4): 503-514. 10.1016/S0092-8674(00)81591-3.View ArticlePubMedGoogle Scholar
- Haase G, Dessaud E, Garces A, de Bovis B, Birling M, Filippi P, Schmalbruch H, Arber S, deLapeyriere O: GDNF acts through PEA3 to regulate cell body positioning and muscle innervation of specific motor neuron pools. Neuron. 2002, 35 (5): 893-905. 10.1016/S0896-6273(02)00864-4.View ArticlePubMedGoogle Scholar
- Livet J, Sigrist M, Stroebel S, De Paola V, Price SR, Henderson CE, Jessell TM, Arber S: ETS gene Pea3 controls the central position and terminal arborization of specific motor neuron pools. Neuron. 2002, 35 (5): 877-892. 10.1016/S0896-6273(02)00863-2.View ArticlePubMedGoogle Scholar
- Liu JP, Laufer E, Jessell TM: Assigning the positional identity of spinal motor neurons: rostrocaudal patterning of Hox-c expression by FGFs, Gdf11, and retinoids. Neuron. 2001, 32 (6): 997-1012. 10.1016/S0896-6273(01)00544-X.View ArticlePubMedGoogle Scholar
- Nelsen OE: Comparative Embryology of the Vertebrates. 1953, New York , The Blackiston Company, Inc., 508-511:Google Scholar
- Mishina Y, Suzuki A, Ueno N, Behringer RR: Bmpr encodes a type I bone morphogenetic protein receptor that is essential for gastrulation during mouse embryogenesis. Genes Dev. 1995, 9 (24): 3027-3037. 10.1101/gad.9.24.3027.View ArticlePubMedGoogle Scholar
- Mishina Y, Hanks MC, Miura S, Tallquist MD, Behringer RR: Generation of Bmpr/Alk3 conditional knockout mice. Genesis. 2002, 32 (2): 69-72. 10.1002/gene.10038.View ArticlePubMedGoogle Scholar
- Wanek N, Muneoka K, Holler-Dinsmore G, Burton R, Bryant SV: A staging system for mouse limb development. Journal of Experimental Zoology. 1989, 249 (1): 41-49. 10.1002/jez.1402490109.View ArticlePubMedGoogle Scholar
- Wang G, Scott SA: The "waiting period" of sensory and motor axons in early chick hindlimb: its role in axon pathfinding and neuronal maturation. J Neurosci. 2000, 20 (14): 5358-5366.PubMedGoogle Scholar
- Lance-Jones C, Landmesser L: Motoneurone projection patterns in the chick hind limb following early partial reversals of the spinal cord. J Physiol. 1980, 302: 581-602.PubMed CentralPubMedGoogle Scholar
- Feldheim DA, Kim YI, Bergemann AD, Frisen J, Barbacid M, Flanagan JG: Genetic analysis of ephrin-A2 and ephrin-A5 shows their requirement in multiple aspects of retinocollicular mapping. Neuron. 2000, 25 (3): 563-574. 10.1016/S0896-6273(00)81060-0.View ArticlePubMedGoogle Scholar
- Laufer E, Dahn R, Orozco OE, Yeo CY, Pisenti J, Henrique D, Abbott UK, Fallon JF, Tabin C: Expression of Radical fringe in limb-bud ectoderm regulates apical ectodermal ridge formation. Nature. 1997, 386 (6623): 366-373. 10.1038/386366a0.View ArticlePubMedGoogle Scholar
- Avantaggiato V, Pandolfi PP, Ruthardt M, Hawe N, Acampora D, Pelicci PG, Simeone A: Developmental analysis of murine Promyelocyte Leukemia Zinc Finger (PLZF) gene expression: implications for the neuromeric model of the forebrain organization. J Neurosci. 1995, 15 (7 Pt 1): 4927-4942.PubMedGoogle Scholar
- Naiche LA, Papaioannou VE: Loss of Tbx4 blocks hindlimb development and affects vascularization and fusion of the allantois. Development. 2003, 130 (12): 2681-2693. 10.1242/dev.00504.View ArticlePubMedGoogle Scholar
- Rallis C, Bruneau BG, Del Buono J, Seidman CE, Seidman JG, Nissim S, Tabin CJ, Logan MP: Tbx5 is required for forelimb bud formation and continued outgrowth. Development. 2003, 130 (12): 2741-2751. 10.1242/dev.00473.View ArticlePubMedGoogle Scholar
- Gibson-Brown JJ, Agulnik SI, Chapman DL, Alexiou M, Garvey N, Silver LM, Papaioannou VE: Evidence of a role for T-box genes in the evolution of limb morphogenesis and the specification of forelimb/hindlimb identity. Mech Dev. 1996, 56 (1-2): 93-101. 10.1016/0925-4773(96)00514-X.View ArticlePubMedGoogle Scholar
- Mercader N, Leonardo E, Azpiazu N, Serrano A, Morata G, Martinez C, Torres M: Conserved regulation of proximodistal limb axis development by Meis1/Hth. Nature. 1999, 402 (6760): 425-429. 10.1038/46580.View ArticlePubMedGoogle Scholar
- Nagai T, Aruga J, Takada S, Gunther T, Sporle R, Schughart K, Mikoshiba K: The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation. Dev Biol. 1997, 182 (2): 299-313. 10.1006/dbio.1996.8449.View ArticlePubMedGoogle Scholar
- Rincon-Limas DE, Lu CH, Canal I, Calleja M, Rodriguez-Esteban C, Izpisua-Belmonte JC, Botas J: Conservation of the expression and function of apterous orthologs in Drosophila and mammals. Proc Natl Acad Sci U S A. 1999, 96 (5): 2165-2170. 10.1073/pnas.96.5.2165.PubMed CentralView ArticlePubMedGoogle Scholar
- Retaux S, Rogard M, Bach I, Failli V, Besson MJ: Lhx9: a novel LIM-homeodomain gene expressed in the developing forebrain. J Neurosci. 1999, 19 (2): 783-793.PubMedGoogle Scholar
- Ramesh V: Merlin and the ERM proteins in Schwann cells, neurons and growth cones. Nat Rev Neurosci. 2004, 5 (6): 462-470. 10.1038/nrn1407.View ArticlePubMedGoogle Scholar
- Wine-Lee L, Ahn KJ, Richardson RD, Mishina Y, Lyons KM, Crenshaw EB: Signaling through BMP type 1 receptors is required for development of interneuron cell types in the dorsal spinal cord. Development. 2004, 131 (21): 5393-5403. 10.1242/dev.01379.View ArticlePubMedGoogle Scholar
- Novitch BG, Chen AI, Jessell TM: Coordinate regulation of motor neuron subtype identity and pan-neuronal properties by the bHLH repressor Olig2. Neuron. 2001, 31 (5): 773-789. 10.1016/S0896-6273(01)00407-X.View ArticlePubMedGoogle Scholar
- McMahon AP: Neural patterning: the role of Nkx genes in the ventral spinal cord. Genes Dev. 2000, 14 (18): 2261-2264. 10.1101/gad.840800.View ArticlePubMedGoogle Scholar
- Briscoe J, Pierani A, Jessell TM, Ericson J: A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell. 2000, 101 (4): 435-445. 10.1016/S0092-8674(00)80853-3.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.