The Met receptor tyrosine kinase prevents zebrafish primary motoneurons from expressing an incorrect neurotransmitter
© Tallafuss and Eisen; licensee BioMed Central Ltd. 2008
Received: 08 February 2008
Accepted: 29 July 2008
Published: 29 July 2008
Expression of correct neurotransmitters is crucial for normal nervous system function. How neurotransmitter expression is regulated is not well-understood; however, previous studies provide evidence that both environmental signals and intrinsic differentiation programs are involved. One environmental signal known to regulate neurotransmitter expression in vertebrate motoneurons is Hepatocyte growth factor, which acts through the Met receptor tyrosine kinase and also affects other aspects of motoneuron differentiation, including axonal extension. Here we test the role of Met in development of motoneurons in embryonic zebrafish.
We found that met is expressed in all early developing, individually identified primary motoneurons and in at least some later developing secondary motoneurons. We used morpholino antisense oligonucleotides to knock down Met function and found that Met has distinct roles in primary and secondary motoneurons. Most secondary motoneurons were absent from met morpholino-injected embryos, suggesting that Met is required for their formation. We used chemical inhibitors to test several downstream pathways activated by Met and found that secondary motoneuron development may depend on the p38 and/or Akt pathways. In contrast, primary motoneurons were present in met morpholino-injected embryos. However, a significant fraction of them had truncated axons. Surprisingly, some CaPs in met morpholino antisense oligonucleotide (MO)-injected embryos developed a hybrid morphology in which they had both a peripheral axon innervating muscle and an interneuron-like axon within the spinal cord. In addition, in met MO-injected embryos primary motoneurons co-expressed mRNA encoding Choline acetyltransferase, the synthetic enzyme for their normal neurotransmitter, acetylcholine, and mRNA encoding Glutamate decarboxylase 1, the synthetic enzyme for GABA, a neurotransmitter never normally found in these motoneurons, but found in several types of interneurons. Our inhibitor studies suggest that Met function in primary motoneurons may be mediated through the MEK1/2 pathway.
We provide evidence that Met is necessary for normal development of zebrafish primary and secondary motoneurons. Despite their many similarities, our results show that these two motoneuron subtypes have different requirements for Met function during development, and raise the possibility that Met may act through different intracellular signaling cascades in primary and secondary motoneurons. Surprisingly, although met is not expressed in primary motoneurons until many hours after they have extended axons to and innervated their muscle targets, Met knockdown causes some of these cells to develop a hybrid phenotype in which they co-expressed motoneuron and interneuron neurotransmitters and have both peripheral and central axons.
Although different subtypes of motoneurons of invertebrate species use different neurotransmitters to activate muscle [1, 2], all vertebrate motoneurons activate muscle via release of acetylcholine (ACh) . Historically vertebrate motoneurons have been considered exclusively cholinergic. However, several recent studies provide evidence that mammalian spinal motoneurons release both ACh and glutamate from collaterals within the spinal cord that synapse with inhibitory interneurons known as Renshaw cells, although ACh is still thought to be the only neurotransmitter that mediates motoneuron activation of skeletal muscle [4–6]. It is unknown how two distinct neurotransmitters are differentially regulated within these motoneurons. But the importance of appropriate regulation is underscored by a recent study showing that forced expression of neurotransmitters other than ACh in frog motoneurons causes inappropriate expression of non-cholinergic receptors at the neuromuscular junction .
Expression of the correct neurotransmitter is crucial for normal nervous system function, although the mechanisms that establish appropriate neurotransmitter expression are not well understood. Interneurons in the chick spinal cord can be induced to express ACh inappropriately by forced expression of MNR2, Lhx3, or Islet1 transcription factors [8, 9]. However, forced expression of these transcription factors causes the interneurons to initiate a program of motoneuron differentiation [8, 9] for which ACh is the appropriate neurotransmitter, suggesting that neurotransmitter expression is established by programs that specify cell fate. On the other hand, it is well-known that at least some neural crest-derived neurons of the peripheral nervous system normally change their neurotransmitter phenotypes during development, and that this is regulated by environmental signals [10, 11]. These studies show that under some conditions, neurotransmitter expression is altered in response to the environment after cell fate is specified. Consistent with this idea, changing calcium-mediated neural activity can regulate neurotransmitter expression in neurons in culture  and in vivo  without affecting expression of markers of cell fate specification . Together these studies suggest that regulation of neurotransmitter phenotype is complex and involves both intrinsic factors that regulate differentiation programs as well as responses to environmental signals.
One environmental signal known to affect neurotransmitter phenotype in motoneurons is Hepatocyte growth factor (HGF; also known as Scatter factor). Axotomy of adult hypoglossal motoneurons leads to a dramatic loss of mRNA and protein of the ACh synthetic enzyme, choline acetyltransferase (ChAT); this loss can be prevented by administration of HGF . HGF has also been shown to stimulate choline acetyltransferase activity in motoneurons in vitro . HGF acts through the Met receptor tyrosine kinase , which is expressed in motoneurons and has been shown to be important for their development. For example, HGF acts through Met as an axonal attractant and survival factor for some populations of mammalian and avian motoneurons [14, 16–22] and has also been shown to be required to recruit a subpopulation of motoneurons to a specific motor pool .
In the present study, we have taken advantage of the experimental tractability of embryonic zebrafish to investigate the role of Met in motoneuron differentiation. Development of zebrafish spinal motoneurons has been well-characterized . Zebrafish have two waves of motoneuron differentiation: primary and secondary motoneurons. Primary motoneurons (PMNs) constitute a small set of segmentally reiterated cells generated during gastrulation [38, 39]. Each PMN is individually identified based on its morphology and gene expression pattern. Within each spinal hemisegment, CaP has the most caudally located cell body, RoP has the most rostrally located cell body, and MiP has a cell body located between CaP and RoP. Some spinal hemisegments have an additional PMN, called VaP, which is essentially a duplicated CaP that typically dies . PMN axons pioneer nerve pathways followed later by axons of secondary motoneurons (SMNs) [41, 42]. SMNs are born later than PMNs and are more numerous . SMNs are born  and extend axons  over a protracted period of development; several studies suggest that there are distinct subsets of SMNs [44–47], although this has not yet been studied in detail. In addition to extensive characterization of their development, many recent studies have characterized the physiological properties of zebrafish motoneurons and how motoneurons are driven by interneurons to activate various types of behavior (see ). The neurotransmitters of zebrafish spinal interneurons have been extensively characterized. Specific subsets of interneurons have been shown to be glycinergic or glutamatergic [48, 49]. In addition, several types of interneurons have been shown to express the neurotransmitter gamma-amino butyric acid (GABA) [48–50].
A previous study showed that zebrafish met is a specific marker of CaP and VaP . We report here that a few hours later, met is also expressed in MiP and RoP, and that even later in development met is expressed in at least some, perhaps all, SMNs. We used morpholino antisense oligonucleotides (MOs) to knock down Met function and found that this had distinct effects on SMNs and PMNs. Many SMNs required Met for their differentiation and the SMN population was significantly reduced in met MO-injected embryos. In contrast, PMN differentiation appeared normal in met MO-injected embryos. However, some PMNs had truncated peripheral axons or developed interneuron-like processes within the spinal cord. In addition, in the absence of Met many PMNs inappropriately expressed GABA. Whether vertebrate motoneurons ever normally express GABA is controversial, and we return to this point in the discussion. To learn whether distinct Met-activated signaling cascades are responsible for the different phenotypes we observed following Met knock down, we used inhibitors that affect different pathways downstream of Met activation. Our results suggest that the p38 and/or Akt cascade may be required for SMN differentiation, whereas the MEK1/2 cascade may be required for appropriate neurotransmitter expression and to prevent formation of interneuron-like axons in PMNs. Together our results suggest that Met acts through different pathways to affect different aspects of motoneuron development.
Materials and Methods
Animal husbandry and lines
Zebrafish embryos were obtained from natural spawning of AB or AB/TU wild-type or mn2Et (also referred to as parg mn2Et ) , Tg(gata2:GFP)  and Tg(pax2a:GFP)  transgenic lines. Fish were staged by hours post-fertilization at 28.5°C (hpf) .
Cloning of zebrafish chat and met
We amplified two fragments, 1,400 bp and 900 bp from cDNA from a mixture of 24 hpf and 48 hpf embryos; the 1,400 bp fragment was amplified using primers CAT3 and CAT6 and the 900 bp fragment was amplified using primers CAT5 and CAT6. The fragments were cloned into TOPO-TA vector, sequenced and tested for specific expression patterns. Primer sequences were:
We cloned a 1.1 kb fragment of zebrafish met from cDNA from a mixture of 24 hpf and 48 hpf embryos using primers zfmetE1-1 and zfmetE1-2. The fragment was cloned into PCRII-TOPO and verified by sequence and expression pattern. Primer sequences were:
Downregulation of Met
To knock down Met activity, we used three MOs (GeneTools; Philomath, OR, USA): CM1a and CM2 were designed to block met (ENSDART00000104456; NCBI Entrez GeneID 492292) translation; these are the same MOs used by Haines and colleagues , thus, we repeated their experiments, showing the absence or reduction of myoD RNA expression in fin myoblasts in MO-injected embryos at 48 hpf, to verify that these MOs worked. CM1a was designed to anneal to ATG-containing sequences and CM2 was targeted against the 5' untranslated region of met. We also used an additional MO, E6I6, which was designed to block met mRNA splicing, leading to a deletion of exon 6 and a truncation of the Met protein. We determined that the E6I6 MO blocked met mRNA splicing by RT-PCR (Figure 1a). As a control, we used an MO similar to CM1a, but containing a 5 bp mismatch (CM1a-5 mm). About 2–4 nl of MO, diluted in water, were injected into the cytoplasm of one-cell-stage embryos. For most experiments, CM1a and CM2 were injected together, at concentrations at which each MO alone had no effect (CM1a, 0.6 mM; CM2, 0.8 mM); throughout this paper, embryos injected with a combination of CM1a and CM2 are referred to as met MO-injected embryos. Injection of E6I6 resulted in essentially the same phenotypes as injection of CM1a, CM2 or a mixture of the two translation blockers. MO sequences and concentrations were:
CM1a, 5'-ATAGTGAATTGTCATCTTTGTTCCT-3', 0.7–1.0 mM;
CM2, 5'-CTGTAAAATAAAGACACCTGTCGGA-3', 0.9–1.2 mM;
E6I6, 5'-GATTTGTGATGACTCTTACCACAAA-3', 0.7–1.0 mM;
CM1a-5 mm, 5'-ATAC TC AATTC TCATG TTTC TTCCT-3', 1.0 mM;
underlines represent mismatches.
In some experiments we co-injected mouse Met mRNA (mMet in pSP64; generous gift of G. Vande Woude, Van Andel Research Institute, Grand Rapids, MI, USA)  together with the MOs. As a further control to test whether RNA diluted the MO to a non-effective concentration, we performed a set of control experiments in which we injected a similar amount of lacZ mRNA, mMet RNA, together with the MOs. Injection of lacZ mRNA alone did not affect neuronal development. In contrast, injection of lacZ mRNA plus MOs resulted in a phenotype similar to injection of MOs alone, showing that any effects of mMet RNA plus MO injections were specific to the mMet mRNA.
Blocking Met downstream effectors with pharmacological inhibitors
Met activates a number of different signaling pathways [15, 57, 58] and inhibitors that block these pathways have been previously used to test Met function (see Figure 1b) . We used the following inhibitors to test Met function in zebrafish: U0126 (InvivoGen; San Diego, CA, USA), which blocks MEK1 and MEK2 , LY294002 (InvivoGen), which blocks PI3K , and SB203580 (InvivoGen) which blocks p38 and Akt [36, 37]. Because LY294002 had severe effects on overall development, we did not pursue its effects on motoneuron differentiation. For the inhibitor studies, embryos were dechorionated and incubated in embryo medium. Cell permeable inhibitor was added at 16 hpf; embryos remained in the inhibitor solution until further processed at either 26 hpf or 48 hpf. We performed dose-response experiments to determine the optimal inhibitor concentrations to use for experiments. We tested concentrations between 10 and 120 μM. In both cases we found that concentrations below 50 μM had no effect and concentrations above 80 μM were deleterious to embryonic development. Therefore, we used both U0126 and SB203580 at 60 μM.
RNA in situ hybridization and immunohistochemistry
RNA in situ hybridization and immunohistochemistry were carried out according to standard protocols .
RNA in situ hybridization
The following antisense RNA probes were used: islet2 (isl2) , glutamate decarboxylase 1 (gad1, also known as gad67 ), met (1.1 kb fragment spanning 1–1,165 bp of NCBI sequence AY687384; Entrez GeneID: 492292) and chat (900 bp fragment spanning 1,099–1,967 of NCBI sequence XM682602; Entrez GeneID: 559274).
The following primary antibodies were used: JL-8 mouse anti-GFP (Chemicon; Temecula, CA, USA) was used at 1:200; zn1 (University of Oregon) was used at 1:150; znp1 (University of Oregon) was used at 1:750; rabbit anti-GABA (Sigma; St. Louis, MO, USA) was used at 1:1,000; anti-Alcam (Alcam was previously known as DM-GRASP, Neurolin, zn5 antigen, and zn8 antigen; University of Oregon) was used at 1:4,000; F59  was used at 1:10; 4D9  was used at 1:50. Primary antibodies were revealed using secondary antibodies coupled to Alexa Fluor568 (goat anti-rabbit 1:1,000; Invitrogen-Molecular Probes; Eugene, OR, USA); Alexa Fluor488 (goat anti-mouse 1:1,000; Invitrogen-Molecular Probes); Alexa Fluor546 (goat anti-mouse IgG1, 1:1,000; Invitrogen-Molecular Probes); Alexa Fluor488 (goat anti-mouse IgG2a, 1:1,000; Invitrogen-Molecular Probes); Alexa Fluor546 (goat anti-mouse IgG2b, 1:1,000; Invitrogen-Molecular Probes).
To reveal ACh receptor (AChR) clusters, embryos were fixed at 4°C for 4 h, rinsed in phosphate-buffered saline, incubated in 5 μg/ml α-bungarotoxin (αBTX-546; Invitrogen-Molecular Probes) in incubation buffer [phosphate buffered saline plus 0.1% Tween/20 (PBT) + 1% dimethyl sulfoxide (DMSO) + 5% normal goat serum (NGS)] for 30 minutes at room temperature, rinsed in PBT, and then followed by a regular immunohistochemistry protocol (adapted from  with minor modification).
Embryos were scored and photographed with a Zeiss Axioplan microscope and photographed using a Nikon Coolpix 995 digital camera or imaged using a Zeiss LSM5 confocal microscope.
Acridine orange staining
For characterization of cell death, embryos were stained according to Williams and Holder , with minor modifications. Briefly, embryos were incubated for 20 minutes in 5 mg/ml Acridine Orange (Sigma) in embryo medium, washed three times for 5 minutes in embryo medium and observed under fluorescence microscopy with an fluoro-iso-thio-cyanate (FITC) filter.
We used high-speed imaging to monitor trunk movements evoked by touch . In our experiment, the head of the embryo was embedded in agarose in such a way that the trunk was not restricted in its movements. The embryo was stimulated with an insect pin mounted on a micromanipulator (Narishige; East Meadow, NY, USA). The stimulus was repeated 5 times at 1 s intervals and recorded using a high-speed digital video camera (Pixelink; Ottawa, ON, Canada) at 100 frames per second. Movies were analyzed using Quicktime (Apple) and individual frames were assembled for presentation in Adobe Photoshop.
met is expressed in a subset of zebrafish spinal motoneurons
Met is required for normal touch-evoked behavior
Expression of met in PMNs and at least some SMNs prompted us to ask whether Met function is necessary for proper regulation of motoneuron-mediated behaviors. Zebrafish embryos begin to exhibit spontaneous muscle contractions that result in coiling movements shortly after PMN axons first extend out of the spinal cord [69–71]; spontaneous coiling requires functional PMNs . met MO-injected embryos had normal spontaneous coiling, suggesting that some PMNs were present and functional.
Met plays a role in formation of ventral motor nerves
By 48 hpf, SMNs had formed both dorsal and ventral motor nerves (Figure 5c–d'). In met MO-injected embryos the ventral nerves appeared thinner than in control embryos (Figure 5c,d). Because znp1 labels both PMNs and SMNs, and in 75% of hemisegments CaP axons had extended normally, this result suggests that at least some SMN ventral axons were truncated or failed to extend when Met was knocked down. In addition, there were some truncated ventral nerves. Previous studies showed that CaP is unnecessary for extension of SMN ventral axons . Thus, the truncated ventral nerves could represent truncated CaP axons in segments in which SMN axons failed to extend. Alternatively, they could represent a combination of truncated CaP and truncated SMN axons. Together these observations suggest that Met is important for normal ventral axon extension by both PMNs and SMNs. We provide further tests of this hypothesis below.
Normal secondary motoneuron differentiation requires Met signaling
The number of second motoneurons is reduced following Met knock down
Number of embryos
Percent of segments with ventral nerve
GFP+ somata within 3 cell diameters of floor plate
20 ± 0.47
9 ± 0.61
6 ± 0.40
GFP+ somata 3 cell diameters dorsal of floor plate
5 ± 0.39
10 ± 0.52
8 ± 0.68
Met may mediate secondary motoneuron development through activation of p38 and/or Akt
SB203580 affects SMN development
Number of embryos
Percent of segments
with reduced SMN
Surprisingly, although SB203580-treated embryos lacked most SMNs, at 30 hpf their tail touch-evoked motility was essentially indistinguishable from control embryos (data not shown). Only a few SMNs have projected axons by this stage ; thus, this result suggests that at this stage most of the touch response is mediated by activity of PMNs, rather than by activity of SMNs, although this has not yet been tested directly. If this is the case, then the movement defects we observed at 30 hpf in met MO-injected embryos would have to arise from a requirement for Met in PMN differentiation, rather than from the decrease in SMN number. To test whether this was the case, we examined the effects of Met knock down on PMN development.
Met prevents CaPs from extending interneuron-like axons within the spinal cord
Met knockdown results in CaPs with both peripheral and central axons
Number of CaPs with
only a peripheral axon
Number of CaPs with
peripheral and central axons
Whether CaP has an interneuron-like axon appears uncorrelated with whether it has a truncated peripheral axon
23 CaP axons in 6 embryos
MO 33 CaP axons in 10 embryos
55 CaP axons in 8 embryos
Ventral edge of muscle
Just out of neural tube
Met prevents CaPs from expressing an interneuron-specific neurotransmitter
Based on their morphology, many CaPs in met MO-injected embryos appeared to have a hybrid identity that combined features of both motoneurons and interneurons. To learn whether this hybrid identity extended to molecular features, we assayed two aspects of CaP: expression of Islet proteins and neurotransmitter phenotype. We found that CaPs in met MO-injected embryos had normal expression of Islet proteins (Figure 7b,c), suggesting that they retained motoneuron identity. To learn whether CaPs expressed normal cholinergic properties or expressed interneuron-specific neurotransmitters, we tried several antibodies to ChAT, the ACh synthetic enzyme, but none of them worked in our hands. Because the chat sequence is highly conserved within vertebrates, we used the goldfish chat sequence  to blast zebrafish databases (NCBI) and found a hypothetical sequence with high homology to all vertebrate chat sequences. We amplified chat from zebrafish cDNA, verified the sequence and found that it was expressed in cells with the correct position and morphology to be PMNs (Figure 7d). We confirmed chat expression in CaPs using double fluorescent in situ hybridization with islet2 (Figure 7e), and also confirmed that CaPs co-express met and chat (Figure 7g). We analyzed chat expression in CaPs in met MO-injected embryos at 24 hpf and found that it was unaltered relative to controls (Figure 7e,f), showing that this aspect of CaP identity did not depend on Met function.
CaPs express GABA in embryos in which Met is knocked down
GABA expression in CaP may be regulated by the MEK1/2 but not by the p38 or Akt pathways
We report two key findings about the role of the Met receptor tyrosine kinase in motoneuron development. First, Met is required for formation of some zebrafish SMNs. Our experiments suggest that this role of Met acts through the p38 and/or Akt signaling cascade. Second, Met is required to prevent CaP motoneurons from co-expressing features of motoneurons and interneurons, including axon pathway and neurotransmitter phenotype. Our experiments suggest that this role of Met acts through the MEK1/2 signaling cascade. We discuss each of these observations in turn.
Met is necessary for formation of some zebrafish secondary motoneurons
In chick, mouse, and rat, Met is expressed in a subset of spinal motoneurons and the Met ligand HGF is important for the differentiation of these cells [14, 16, 17, 19]. Met appears to be expressed primarily in limb-innervating lateral motor column motoneurons and HGF acts as a chemoattractant for the axons of these cells in vitro and in vivo, as well as promoting their survival through the period of normal programmed cell death when tested in vitro [16, 17, 19].
In zebrafish, met is also expressed in a subset of spinal motoneurons, in this case in all primary and at least some secondary motoneurons of the medial motor column. In the case of SMNs, Met appears to be required for formation of these cells, as their number is significantly reduced when Met activity is knocked down; whether or not this is the case for the HGF-dependent limb-innervating motoneurons of chick, mouse, or rat has not been reported. The decrease in SMN number when Met is knocked down might result from death of SMNs or their progenitors. We did not see increased cell death, suggesting an alternative possibility that SMNs differentiated as interneurons, as we have previously seen in the absence of Islet1  or Nkx6 proteins [65, 83]. Consistent with this possibility, at 48 hpf there were more interneuron-like axons within the spinal cord and somata in positions consistent with spinal interneurons in Tg(gata2:GFP) embryos injected with met MOs than in Tg(gata2:GFP) control embryos.
To begin to learn which intracellular signaling pathways may be involved in Met-mediated SMN formation, we exposed embryos to inhibitors that act on specific pathways downstream of Met activation. Our results suggest that the p38 and/or Akt pathways are required for normal development of SMNs. However, a caveat to this interpretation is that the phenotype was much more severe when these pathways were blocked than when Met was knocked down using MOs. One possible explanation is that Met was incompletely knocked down by our MOs but completely blocked by the pharmacological inhibitor. Alternatively, because these pathways are activated by other receptors in addition to Met, pharmacological blockade may lead to more widespread effects, including effects that are non cell-autonomous (see below). In the future it will be important to learn which other receptors activate these pathways in SMNs and how this is related to activation of these pathways by Met. Finally, the expression of both zebrafish p38 genes is widespread throughout early development , raising the possibility that p38 activated pathways could have both cell-autonomous and non cell-autonomous effects on SMN development.
Met prevents primary motoneurons from expressing interneuron-like properties
We have previously found that knocking down function of several transcription factors expressed in PMNs, and in some cases in their progenitors, results in these cells expressing interneuron-like properties. Thus, in the absence of Islet1, PMNs develop interneuron-like axons within the spinal cord, rather than peripheral axons, and many of these cells express the interneuron neurotransmitter GABA . Similarly, in the absence of Nkx6 transcription factors, MiPs develop a hybrid phenotype in which they have both peripheral axons that innervate muscle and central axons that extend within the spinal cord, although these cells do not express GABA . Here we report that knocking down Met function causes CaPs to express a hybrid phenotype in which many of them have both a peripheral axon innervating muscle and a central axon extending within the spinal cord. In addition, these cells co-express cholinergic and GABAergic properties. Despite expression of GABA, PMNs are still able to activate muscle in the absence of Met function. However, the touch response is significantly slower than in control embryos at developmental stages at which this behavior is likely to be mediated primarily by PMNs. Thus, this slower response is probably a result of impairment in PMN function.
The ability of PMNs to develop a hybrid phenotype in the absence of Met reveals a degree of plasticity not previously reported for motoneurons. Previous studies showed that the absence of specific transcription factors in motoneuron progenitors [9, 84–89] or in newly post-mitotic motoneurons  allows these cells to co-express motoneuron and interneuron properties. These studies reveal the importance of specific transcription factors in preventing motoneurons from developing interneuron properties early in their development. Other studies have shown that postmitotic motoneurons can change their identity from one motoneuron subtype to another in response to environmental cues  and that environmental signals can override genetic programs and cause motor axons to extend along aberrant pathways . These studies reveal plasticity in motoneuron subtype specification. In contrast, here we show that motoneurons are able to express interneuron-like properties at late stages of development. Zebrafish met is expressed in PMNs about 6–8 hours after they initially extend growth cones and after their axons have extended to and innervated their specific target muscles. Thus, our studies raise the question of whether the ability to develop interneuron characteristics long after their peripheral axons innervate their muscle targets is a general feature of motoneurons. It is well-known that at least some neural crest-derived peripheral neurons have long-lived phenotypic plasticity [10, 11], but it is not typically believed that this is the case for central neurons. This issue is particularly important because a recent study has shown that forcing motoneurons to release neurotransmitters other than ACh causes their muscle targets to express receptors to the motoneuron-expressed neurotransmitters , potentially leading to inappropriate muscle responses to motoneuron activation.
The late expression of met in PMNs raises the interesting question of what prevents CaPs from extending interneuron-like axons and expressing GABA at stages prior to met expression. Although we do not have an answer to this question, we hypothesize that many different factors are required to prevent motoneurons from expressing interneuron-like properties. Consistent with this hypothesis, several transcription factors, including Islet1 , Nkx6 [85, 88, 65, 83], Lhx3 , Hb9  and AML1/Runx1  have been shown to prevent various types of motoneurons from adopting interneuron-like properties. It is not yet clear, but will be exciting to learn, the identities of the downstream targets of these transcription factors and how they regulate different aspects of interneuron development. We predict that different downstream targets prevent motoneurons from expressing different interneuron properties at different developmental stages. We have proposed that zebrafish PMNs have a high propensity to develop into motoneuron/interneuron hybrids because, as has been postulated from studies in mammals , zebrafish motoneurons are closely-related to specific types of interneurons . In zebrafish and chick, both motoneurons and interneurons can arise from a single progenitor [92–94]; whether this is also the case in mammals is unknown because single progenitor labeling experiments have not been reported.
GABA expression in developing motoneurons
Although vertebrate motoneurons are generally considered exclusively cholinergic, several recent studies provide evidence that mammalian spinal motoneurons can release both ACh and glutamate at central synapses on Renshaw cells [4–6]. However, ACh is still thought to be the only neurotransmitter that mediates motoneuron activation of skeletal muscle [4–6]. Thus, it is surprising that during early development, frog muscles express not only AChRs at the nascent neuromuscular junction (NMJ) but also several other types of neurotransmitter receptors, including glutamate receptors, glycine receptors and GABA receptors . From experiments in which they altered motoneuronal neurotransmitter expression, Borodinsky and Spitzer  have argued that the final complement of receptors at the NMJ results from matching the neurotransmitter released by motoneurons with the receptors on muscle cells. In their studies, they never saw GABA expression by motoneurons under control conditions. However, several earlier studies reported transient expression of GABA in motoneurons in chick, monkey  and rat . GABA may act not only as a neurotransmitter, but also as a trophic factor during development [97, 98], and it may be important for integrating developing neurons into circuits . These features might explain early transient expression in neurons that do not normally use GABA as a neurotransmitter. However, neither we nor others have reported GABA expression in zebrafish spinal motoneurons, and the issue of whether transient GABA expression is a common feature of vertebrate spinal motoneurons remains unresolved.
It has been known for many years that environmental signals can alter subtype specification in newly post-mitotic motoneurons . Here we show that motoneurons retain the ability to develop interneuron-like characteristics, including both axon trajectory and neurotransmitter phenotype, long after they have innervated their muscle targets. In zebrafish, motoneurons and some types of interneurons are generated from the same progenitor domain [92–94], and previous studies showed that in the absence of Notch signaling motoneurons are the preferred fate of cells within that domain [94, 100]. Here we suggest that despite this, motoneurons may require continuous signaling to prevent them from developing interneuron-like properties. Our current results also show that motoneurons that co-express interneuron-like properties can still innervate target muscle. In addition, we suggest that the Met receptor tyrosine kinase acts through different intracellular signaling cascades to affect distinct aspects of development in different motoneuron subtypes.
Gamma-amino butyric acid
Green fluorescent protein
Hepatocyte growth factor
Mitogen activated protein kinase
Morpholino antisense oligonucleotide
We thank Keith Beadle for help with motility assays, G Vande Woude for the mouse Met mRNA, Peter Currie for a met MO sample and a partial met DNA, Shin-Ichi Higashijima and Joe Fetcho for gad1 mRNA, Steve Ekker for the mn2Et line, Michael Brand for the Tg(pax2a:GFP) line, Shuo Lin for the Tg(gata2:GFP) line, Chris Doe, Phil Washbourne and Monte Westefield for critical reading of the manuscript, and Amanda Lewis, Joy Murphy, Jacob Lewis and the staff of the UO Zebrafish Facility for animal husbandry. Supported by NIH grants NS23915 and HD22486 and AHA postdoctoral fellowship 0420027Z.
- Marder E, Eisen JS: Transmitter identification of pyloric neurons: electrically coupled neurons use different transmitters. J Neurophysiol. 1984, 51 (6): 1345-1361.PubMedGoogle Scholar
- Clarac F, Pearlstein E: Invertebrate preparations and their contribution to neurobiology in the second half of the 20th century. Brain Res Rev. 2007, 54 (1): 113-161.View ArticlePubMedGoogle Scholar
- Eisen JS: Genetic and molecular analyses of motoneuron development. Curr Opin Neurobiol. 1998, 8 (6): 697-View ArticlePubMedGoogle Scholar
- Herzog E, Landry M, Buhler E, Bouali-Benazzouz R, Legay C, Henderson CE, Nagy F, Dreyfus P, Giros B, El Mestikawy S: Expression of vesicular glutamate transporters, VGLUT1 and VGLUT2, in cholinergic spinal motoneurons. Eur J Neurosci. 2004, 20 (7): 1752-1760.View ArticlePubMedGoogle Scholar
- Nishimaru H, Restrepo CE, Ryge J, Yanagawa Y, Kiehn O: Mammalian motor neurons corelease glutamate and acetylcholine at central synapses. Proc Natl Acad Sci U S A. 2005, 102 (14): 5245-5249.PubMed CentralView ArticlePubMedGoogle Scholar
- Mentis GZ, Alvarez FJ, Bonnot A, Richards DS, Gonzalez-Forero D, Zerda R, O'Donovan MJ: Noncholinergic excitatory actions of motoneurons in the neonatal mammalian spinal cord. Proc Natl Acad Sci U S A. 2005, 102 (20): 7344-7349.PubMed CentralView ArticlePubMedGoogle Scholar
- Borodinsky LN, Spitzer NC: Activity-dependent neurotransmitter-receptor matching at the neuromuscular junction. Proc Natl Acad Sci U S A. 2007, 104 (1): 335-340.PubMed CentralView ArticlePubMedGoogle Scholar
- Tanabe Y, William C, Jessell TM: Specification of motor neuron identity by the MNR2 homeodomain protein. Cell. 1998, 95 (1): 67-80.View ArticlePubMedGoogle Scholar
- Thaler JP, Lee SK, Jurata LW, Gill GN, Pfaff SL: LIM factor Lhx3 contributes to the specification of motor neuron and interneuron identity through cell-type-specific protein-protein interactions. Cell. 2002, 110 (2): 237-249.View ArticlePubMedGoogle Scholar
- Black IB, Patterson PH: Developmental regulation of neurotransmitter phenotype. Curr Top Dev Biol. 1980, 15 Pt 1: 27-40.View ArticlePubMedGoogle Scholar
- Landis SC: Target regulation of neurotransmitter phenotype. Trends Neurosci. 1990, 13 (8): 344-350.View ArticlePubMedGoogle Scholar
- Borodinsky LN, Root CM, Cronin JA, Sann SB, Gu X, Spitzer NC: Activity-dependent homeostatic specification of transmitter expression in embryonic neurons. Nature. 2004, 429 (6991): 523-530.View ArticlePubMedGoogle Scholar
- Okura Y, Arimoto H, Tanuma N, Matsumoto K, Nakamura T, Yamashima T, Miyazawa T, Matsumoto Y: Analysis of neurotrophic effects of hepatocyte growth factor in the adult hypoglossal nerve axotomy model. Eur J Neurosci. 1999, 11 (11): 4139-4144.View ArticlePubMedGoogle Scholar
- Wong V, Glass DJ, Arriaga R, Yancopoulos GD, Lindsay RM, Conn G: Hepatocyte growth factor promotes motor neuron survival and synergizes with ciliary neurotrophic factor. J Biol Chem. 1997, 272 (8): 5187-5191.View ArticlePubMedGoogle Scholar
- Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF: Met, metastasis, motility and more. Nat Rev Mol Cell Biol. 2003, 4 (12): 915-925.View ArticlePubMedGoogle Scholar
- Ebens A, Brose K, Leonardo ED, Hanson MG, Bladt F, Birchmeier C, Barres BA, Tessier-Lavigne M: Hepatocyte growth factor/scatter factor is an axonal chemoattractant and a neurotrophic factor for spinal motor neurons. Neuron. 1996, 17 (6): 1157-View ArticlePubMedGoogle Scholar
- Yamamoto Y, Livet J, Pollock RA, Garces A, Arce V, deLapeyriere O, Henderson CE: Hepatocyte growth factor (HGF/SF) is a muscle-derived survival factor for a subpopulation of embryonic motoneurons. Development. 1997, 124 (15): 2903-PubMedGoogle Scholar
- Caton A, Hacker A, Naeem A, Livet J, Maina F, Bladt F, Klein R, Birchmeier C, Guthrie S: The branchial arches and HGF are growth-promoting and chemoattractant for cranial motor axons. Development. 2000, 127 (8): 1751-PubMedGoogle Scholar
- Novak KD, Prevette D, Wang S, Gould TW, Oppenheim RW: Hepatocyte growth factor/scatter factor is a neurotrophic survival factor for lumbar but not for other somatic motoneurons in the chick embryo. J Neurosci. 2000, 20 (1): 326-PubMedGoogle Scholar
- Sun W, Funakoshi H, Nakamura T: Localization and functional role of hepatocyte growth factor (HGF) and its receptor c-met in the rat developing cerebral cortex. Brain Res Mol Brain Res. 2002, 103 (1-2): 36-View ArticlePubMedGoogle Scholar
- Hanson MG, Landmesser LT: Characterization of the circuits that generate spontaneous episodes of activity in the early embryonic mouse spinal cord. J Neurosci. 2003, 23 (2): 587-600.PubMedGoogle Scholar
- Hayashi Y, Kawazoe Y, Sakamoto T, Ojima M, Wang W, Takazawa T, Miyazawa D, Ohya W, Funakoshi H, Nakamura T, Watabe K: Adenoviral gene transfer of hepatocyte growth factor prevents death of injured adult motoneurons after peripheral nerve avulsion. Brain Res. 2006, 1111 (1): 187-195.View ArticlePubMedGoogle Scholar
- Helmbacher F, Dessaud E, Arber S, deLapeyriere O, Henderson CE, Klein R, Maina F: Met signaling is required for recruitment of motor neurons to PEA3-positive motor pools. Neuron. 2003, 39 (5): 767-View ArticlePubMedGoogle Scholar
- Segarra J, Balenci L, Drenth T, Maina F, Lamballe F: Combined Signaling through ERK, PI3K/AKT, and RAC1/p38 Is Required for Met-triggered Cortical Neuron Migration. J Biol Chem. 2006, 281 (8): 4771-View ArticlePubMedGoogle Scholar
- Xiao GH, Jeffers M, Bellacosa A, Mitsuuchi Y, Vande Woude GF, Testa JR: Anti-apoptotic signaling by hepatocyte growth factor/Met via the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways. Proc Natl Acad Sci U S A. 2001, 98 (1): 247-252.PubMed CentralView ArticlePubMedGoogle Scholar
- Zimmermann S, Moelling K: Phosphorylation and regulation of Raf by Akt (protein kinase B). Science. 1999, 286 (5445): 1741-1744.View ArticlePubMedGoogle Scholar
- Rane MJ, Coxon PY, Powell DW, Webster R, Klein JB, Pierce W, Ping P, McLeish KR: p38 Kinase-dependent MAPKAPK-2 activation functions as 3-phosphoinositide-dependent kinase-2 for Akt in human neutrophils. J Biol Chem. 2001, 276 (5): 3517-3523.View ArticlePubMedGoogle Scholar
- Westermarck J, Li SP, Kallunki T, Han J, Kahari VM: p38 mitogen-activated protein kinase-dependent activation of protein phosphatases 1 and 2A inhibits MEK1 and MEK2 activity and collagenase 1 (MMP-1) gene expression. Mol Cell Biol. 2001, 21 (7): 2373-2383.PubMed CentralView ArticlePubMedGoogle Scholar
- Rivas MA, Carnevale RP, Proietti CJ, Rosemblit C, Beguelin W, Salatino M, Charreau EH, Frahm I, Sapia S, Brouckaert P, Elizalde PV, Schillaci R: TNFalpha acting on TNFR1 promotes breast cancer growth via p42/P44 MAPK, JNK, Akt and NF-kappaB-dependent pathways. Exp Cell Res. 2007Google Scholar
- Cui H, Cai F, Belsham DD: Leptin signaling in neurotensin neurons involves STAT, MAP kinases ERK1/2, and p38 through c-Fos and ATF1. Faseb J. 2006, 20 (14): 2654-2656.View ArticlePubMedGoogle Scholar
- Nelson JM, Fry DW: Akt, MAPK (Erk1/2), and p38 act in concert to promote apoptosis in response to ErbB receptor family inhibition. J Biol Chem. 2001, 276 (18): 14842-14847.View ArticlePubMedGoogle Scholar
- Porter AC, Vaillancourt RR: Tyrosine kinase receptor-activated signal transduction pathways which lead to oncogenesis. Oncogene. 1998, 17 (11 Reviews): 1343-1352.View ArticlePubMedGoogle Scholar
- Chen JH, Liu TY, Wu CW, Chi CW: Nonsteroidal anti-inflammatory drugs for treatment of advanced gastric cancer: cyclooxygenase-2 is involved in hepatocyte growth factor mediated tumor development and progression. Med Hypotheses. 2001, 57 (4): 503-505.View ArticlePubMedGoogle Scholar
- Vlahos CJ, Matter WF, Hui KY, Brown RF: A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem. 1994, 269 (7): 5241-5248.PubMedGoogle Scholar
- Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, Van Dyk DE, Pitts WJ, Earl RA, Hobbs F, Copeland RA, Magolda RL, Scherle PA, Trzaskos JM: Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem. 1998, 273 (29): 18623-18632.View ArticlePubMedGoogle Scholar
- Lali FV, Hunt AE, Turner SJ, Foxwell BM: The pyridinyl imidazole inhibitor SB203580 blocks phosphoinositide-dependent protein kinase activity, protein kinase B phosphorylation, and retinoblastoma hyperphosphorylation in interleukin-2-stimulated T cells independently of p38 mitogen-activated protein kinase. J Biol Chem. 2000, 275 (10): 7395-7402.View ArticlePubMedGoogle Scholar
- Cuenda A, Rouse J, Doza YN, Meier R, Cohen P, Gallagher TF, Young PR, Lee JC: SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett. 1995, 364 (2): 229-233.View ArticlePubMedGoogle Scholar
- Lewis KE, Eisen JS: From cells to circuits: development of the zebrafish spinal cord. Prog Neurobiol. 2003, 69 (6): 419-449.View ArticlePubMedGoogle Scholar
- Eisen JS, Myers PZ, Westerfield M: Pathway selection by growth cones of identified motoneurones in live zebra fish embryos. Nature. 1986, 320 (6059): 269-View ArticlePubMedGoogle Scholar
- Eisen JS, Pike SH, Romancier B: An identified motoneuron with variable fates in embryonic zebrafish. Journal of Neuroscience. 1990, 10 (1): 34-PubMedGoogle Scholar
- Myers PZ, Eisen JS, Westerfield M: Development and axonal outgrowth of identified motoneurons in the zebrafish. Journal of Neuroscience. 1986, 6 (8): 2278-PubMedGoogle Scholar
- Pike SH, Melancon EF, Eisen JS: Pathfinding by zebrafish motoneurons in the absence of normal pioneer axons. Development. 1992, 114 (4): 825-PubMedGoogle Scholar
- Myers PZ: Spinal motoneurons of the larval zebrafish. J Comp Neurol. 1985, 236 (4): 555-561.View ArticlePubMedGoogle Scholar
- Westerfield M, McMurray JV, Eisen JS: Identified motoneurons and their innervation of axial muscles in the zebrafish. Journal of Neuroscience. 1986, 6 (8): 2267-PubMedGoogle Scholar
- Uemura O, Okada Y, Ando H, Guedj M, Higashijima S, Shimazaki T, Chino N, Okano H, Okamoto H: Comparative functional genomics revealed conservation and diversification of three enhancers of the isl1 gene for motor and sensory neuron-specific expression. Dev Biol. 2005, 278 (2): 587-606.View ArticlePubMedGoogle Scholar
- McWhorter ML, Monani UR, Burghes AH, Beattie CE: Knockdown of the survival motor neuron (Smn) protein in zebrafish causes defects in motor axon outgrowth and pathfinding. J Cell Biol. 2003, 162 (5): 919-931.PubMed CentralView ArticlePubMedGoogle Scholar
- Pineda RH, Svoboda KR, Wright MA, Taylor AD, Novak AE, Gamse JT, Eisen JS, Ribera AB: Knockdown of Nav1.6a Na+ channels affects zebrafish motoneuron development. Development. 2006, 133 (19): 3827-3836.View ArticlePubMedGoogle Scholar
- Higashijima S, Mandel G, Fetcho JR: Distribution of prospective glutamatergic, glycinergic, and GABAergic neurons in embryonic and larval zebrafish. J Comp Neurol. 2004, 480 (1): 1-18.View ArticlePubMedGoogle Scholar
- Higashijima S, Schaefer M, Fetcho JR: Neurotransmitter properties of spinal interneurons in embryonic and larval zebrafish. J Comp Neurol. 2004, 480 (1): 19-37.View ArticlePubMedGoogle Scholar
- Bernhardt RR, Patel CK, Wilson SW, Kuwada JY: Axonal trajectories and distribution of GABAergic spinal neurons in wildtype and mutant zebrafish lacking floor plate cells. J Comp Neurol. 1992, 326 (2): 263-272.View ArticlePubMedGoogle Scholar
- Segawa H, Miyashita T, Hirate Y, Higashijima S, Chino N, Uyemura K, Kikuchi Y, Okamoto H: Functional repression of Islet-2 by disruption of complex with Ldb impairs peripheral axonal outgrowth in embryonic zebrafish. Neuron. 2001, 30 (2): 423-436.View ArticlePubMedGoogle Scholar
- Balciunas D, Davidson AE, Sivasubbu S, Hermanson SB, Welle Z, Ekker SC: Enhancer trapping in zebrafish using the Sleeping Beauty transposon. BMC Genomics. 2004, 5 (1): 62-PubMed CentralView ArticlePubMedGoogle Scholar
- Meng A, Tang H, Ong BA, Farrell MJ, Lin S: Promoter analysis in living zebrafish embryos identifies a cis-acting motif required for neuronal expression of GATA-2. Proc Natl Acad Sci U S A. 1997, 94 (12): 6267-6272.PubMed CentralView ArticlePubMedGoogle Scholar
- Picker A, Scholpp S, Bohli H, Takeda H, Brand M: A novel positive transcriptional feedback loop in midbrain-hindbrain boundary development is revealed through analysis of the zebrafish pax2.1 promoter in transgenic lines. Development. 2002, 129 (13): 3227-3239.PubMedGoogle Scholar
- Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF: Stages of embryonic development of the zebrafish. Dev Dyn. 1995, 203 (3): 253-310.View ArticlePubMedGoogle Scholar
- Haines L, Neyt C, Gautier P, Keenan DG, Bryson-Richardson RJ, Hollway GE, Cole NJ, Currie PD: Met and Hgf signaling controls hypaxial muscle and lateral line development in the zebrafish. Development. 2004, 131 (19): 4857-4869.View ArticlePubMedGoogle Scholar
- Ma PC, Tretiakova MS, Nallasura V, Jagadeeswaran R, Husain AN, Salgia R: Downstream signalling and specific inhibition of c-MET/HGF pathway in small cell lung cancer: implications for tumour invasion. Br J Cancer. 2007, 97 (3): 368-377.PubMed CentralView ArticlePubMedGoogle Scholar
- Maina F, Pante G, Helmbacher F, Andres R, Porthin A, Davies AM, Ponzetto C, Klein R: Coupling Met to specific pathways results in distinct developmental outcomes. Mol Cell. 2001, 7 (6): 1293-View ArticlePubMedGoogle Scholar
- Hauptmann G, Gerster T: Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos. Trends Genet. 1994, 10 (8): 266-View ArticlePubMedGoogle Scholar
- Appel B, Korzh V, Glasgow E, Thor S, Edlund T, Dawid IB, Eisen JS: Motoneuron fate specification revealed by patterned LIM homeobox gene expression in embryonic zebrafish. Development. 1995, 121 (12): 4117-PubMedGoogle Scholar
- Crow MT, Stockdale FE: Myosin expression and specialization among the earliest muscle fibers of the developing avian limb. Dev Biol. 1986, 113 (1): 238-254.View ArticlePubMedGoogle Scholar
- Patel NH, Martin-Blanco E, Coleman KG, Poole SJ, Ellis MC, Kornberg TB, Goodman CS: Expression of engrailed proteins in arthropods, annelids, and chordates. Cell. 1989, 58 (5): 955-968.View ArticlePubMedGoogle Scholar
- Downes GB, Granato M: Acetylcholinesterase function is dispensable for sensory neurite growth but is critical for neuromuscular synapse stability. Dev Biol. 2004, 270 (1): 232-245.View ArticlePubMedGoogle Scholar
- Williams JA, Holder N: Cell turnover in neuromasts of zebrafish larvae. Hear Res. 2000, 143 (1-2): 171-181.View ArticlePubMedGoogle Scholar
- Cheesman SE, Layden MJ, Von Ohlen T, Doe CQ, Eisen JS: Zebrafish and fly Nkx6 proteins have similar CNS expression patterns and regulate motoneuron formation. Development. 2004, 131 (21): 5221-5232.View ArticlePubMedGoogle Scholar
- Zeller J, Schneider V, Malayaman S, Higashijima S, Okamoto H, Gui J, Lin S, Granato M: Migration of zebrafish spinal motor nerves into the periphery requires multiple myotome-derived cues. Dev Biol. 2002, 252 (2): 241-256.View ArticlePubMedGoogle Scholar
- Carrel TL, McWhorter ML, Workman E, Zhang H, Wolstencroft EC, Lorson C, Bassell GJ, Burghes AH, Beattie CE: Survival motor neuron function in motor axons is independent of functions required for small nuclear ribonucleoprotein biogenesis. J Neurosci. 2006, 26 (43): 11014-11022.View ArticlePubMedGoogle Scholar
- Beattie CE, Carrel TL, McWhorter ML: Fishing for a mechanism: using zebrafish to understand spinal muscular atrophy. J Child Neurol. 2007, 22 (8): 995-1003.View ArticlePubMedGoogle Scholar
- Melancon E, Liu DW, Westerfield M, Eisen JS: Pathfinding by identified zebrafish motoneurons in the absence of muscle pioneers. Journal of Neuroscience. 1997, 17 (20): 7796-PubMedGoogle Scholar
- Saint-Amant L, Drapeau P: Time course of the development of motor behaviors in the zebrafish embryo. J Neurobiol. 1998, 37 (4): 622-632.View ArticlePubMedGoogle Scholar
- Brustein E, Saint-Amant L, Buss RR, Chong M, McDearmid JR, Drapeau P: Steps during the development of the zebrafish locomotor network. J Physiol Paris. 2003, 97 (1): 77-86.View ArticlePubMedGoogle Scholar
- Trevarrow B, Marks DL, Kimmel CB: Organization of hindbrain segments in the zebrafish embryo. Neuron. 1990, 4 (5): 669-679.View ArticlePubMedGoogle Scholar
- Hatta K, Bremiller R, Westerfield M, Kimmel CB: Diversity of expression of engrailed-like antigens in zebrafish. Development. 1991, 112 (3): 821-832.PubMedGoogle Scholar
- Devoto SH, Melancon E, Eisen JS, Westerfield M: Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation. Development. 1996, 122 (11): 3371-3380.PubMedGoogle Scholar
- Liu DW, Westerfield M: Clustering of muscle acetylcholine receptors requires motoneurons in live embryos, but not in cell culture. J Neurosci. 1992, 12 (5): 1859-1866.PubMedGoogle Scholar
- Beattie CE, Melancon E, Eisen JS: Mutations in the stumpy gene reveal intermediate targets for zebrafish motor axons. Development. 2000, 127 (12): 2653-2662.PubMedGoogle Scholar
- Fashena D, Westerfield M: Secondary motoneuron axons localize DM-GRASP on their fasciculated segments. J Comp Neurol. 1999, 406 (3): 415-424.View ArticlePubMedGoogle Scholar
- Robu ME, Larson JD, Nasevicius A, Beiraghi S, Brenner C, Farber SA, Ekker SC: p53 activation by knockdown technologies. PLoS Genet. 2007, 3 (5): e78-PubMed CentralView ArticlePubMedGoogle Scholar
- Krens SF, He S, Spaink HP, Snaar-Jagalska BE: Characterization and expression patterns of the MAPK family in zebrafish. Gene Expr Patterns. 2006, 6 (8): 1019-1026.View ArticlePubMedGoogle Scholar
- Moon SJ, Fujikawa Y, Nishihara T, Kono S, Kozono K, Ikenaga T, Esaka M, Iijima N, Nagamatsu Y, Yoshida M, Uematsu K: Partial cloning and expression of mRNA coding choline acetyltransferase in the spinal cord of the goldfish, Carassius auratus. Comp Biochem Physiol B Biochem Mol Biol. 2005, 141 (3): 253-260.View ArticlePubMedGoogle Scholar
- Hutchinson SA, Eisen JS: Islet1 and Islet2 have equivalent abilities to promote motoneuron formation and to specify motoneuron subtype identity. Development. 2006, 133 (11): 2137-2147.View ArticlePubMedGoogle Scholar
- Martin SC, Heinrich G, Sandell JH: Sequence and expression of glutamic acid decarboxylase isoforms in the developing zebrafish. J Comp Neurol. 1998, 396 (2): 253-266.View ArticlePubMedGoogle Scholar
- Hutchinson SA, Cheesman SE, Hale LA, Boone JQ, Eisen JS: Nkx6 proteins specify one zebrafish primary motoneuron subtype by regulating late islet1 expression. Development. 2007, 134 (9): 1671-1677.PubMed CentralView ArticlePubMedGoogle Scholar
- Arber S, Han B, Mendelsohn M, Smith M, Jessell TM, Sockanathan S: Requirement for the homeobox gene Hb9 in the consolidation of motor neuron identity. Neuron. 1999, 23 (4): 659-674.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.PubMed CentralView ArticlePubMedGoogle Scholar
- Shirasaki R, Pfaff SL: Transcriptional codes and the control of neuronal identity. Annu Rev Neurosci. 2002, 25: 251-281.View ArticlePubMedGoogle Scholar
- Thaler J, Harrison K, Sharma K, Lettieri K, Kehrl J, Pfaff SL: Active suppression of interneuron programs within developing motor neurons revealed by analysis of homeodomain factor HB9. Neuron. 1999, 23 (4): 675-687.View ArticlePubMedGoogle Scholar
- Vallstedt A, Muhr J, Pattyn A, Pierani A, Mendelsohn M, Sander M, Jessell TM, Ericson J: Different levels of repressor activity assign redundant and specific roles to Nkx6 genes in motor neuron and interneuron specification. Neuron. 2001, 31 (5): 743-755.View ArticlePubMedGoogle Scholar
- Stifani N, Freitas AR, Liakhovitskaia A, Medvinsky A, Kania A, Stifani S: Suppression of interneuron programs and maintenance of selected spinal motor neuron fates by the transcription factor AML1/Runx1. Proc Natl Acad Sci U S A. 2008, 105 (17): 6451-6456.PubMed CentralView ArticlePubMedGoogle Scholar
- Eisen JS: Determination of primary motoneuron identity in developing zebrafish embryos. Science. 1991, 252 (5005): 569-View ArticlePubMedGoogle Scholar
- Sharma K, Leonard AE, Lettieri K, Pfaff SL: Genetic and epigenetic mechanisms contribute to motor neuron pathfinding. Nature. 2000, 406 (6795): 515-519.View ArticlePubMedGoogle Scholar
- Kimmel CB, Warga RM, Kane DA: Cell cycles and clonal strings during formation of the zebrafish central nervous system. Development. 1994, 120 (2): 265-276.PubMedGoogle Scholar
- Park HC, Shin J, Appel B: Spatial and temporal regulation of ventral spinal cord precursor specification by Hedgehog signaling. Development. 2004, 131 (23): 5959-View ArticlePubMedGoogle Scholar
- Shin J, Poling J, Park HC, Appel B: Notch signaling regulates neural precursor allocation and binary neuronal fate decisions in zebrafish. Development. 2007, 134 (10): 1911-1920.View ArticlePubMedGoogle Scholar
- Philippe E, Gaulin F, Delagrave C, Geffard M: Expression of GABA-immunoreactivity by spinal motoneurons of some vertebrates. Neurosci Lett. 1990, 116 (1-2): 12-16.View ArticlePubMedGoogle Scholar
- Ma W, Behar T, Barker JL: Transient expression of GABA immunoreactivity in the developing rat spinal cord. J Comp Neurol. 1992, 325 (2): 271-290.View ArticlePubMedGoogle Scholar
- Nguyen L, Rigo JM, Rocher V, Belachew S, Malgrange B, Rogister B, Leprince P, Moonen G: Neurotransmitters as early signals for central nervous system development. Cell Tissue Res. 2001, 305 (2): 187-202.View ArticlePubMedGoogle Scholar
- Owens DF, Kriegstein AR: Developmental neurotransmitters?. Neuron. 2002, 36 (6): 989-991.View ArticlePubMedGoogle Scholar
- Akerman CJ, Cline HT: Refining the roles of GABAergic signaling during neural circuit formation. Trends Neurosci. 2007, 30 (8): 382-389.View ArticlePubMedGoogle Scholar
- Appel B, Eisen JS: Regulation of neuronal specification in the zebrafish spinal cord by Delta function. Development. 1998, 125 (3): 371-PubMedGoogle 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.