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The Met receptor tyrosine kinase prevents zebrafish primary motoneurons from expressing an incorrect neurotransmitter.

Tallafuss A, Eisen JS - Neural Dev (2008)

Bottom Line: We found that met is expressed in all early developing, individually identified primary motoneurons and in at least some later developing secondary motoneurons.However, a significant fraction of them had truncated axons.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA. tallafuss@uoneuro.uoregon.edu

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

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Met appears unnecessary for muscle and neuromuscular junction formation. (a,b) Engrailed antibody (Eng, red) labeling showing muscle pioneer cells and znp1 antibody labeling showing motor axons (green). In met MO-injected embryos, some CaP axons are truncated (asterisk). (c,d) F59 antibody (red) labeling showing fast muscle fibers and znp1 antibody staining showing motor axons (green). F59 labeling appears the same in control (c) and met MO-injected (d) embryos, which have some truncated CaPs (asterisk). (e-f) αBTX (red) labeling showing AChRs and znp1 antibody labeling showing motor axons (green). The distribution of AChRs appears the same in control (e) and met MO-injected embryos (f) that have some truncated CaP axons (asterisks); however, it appears that the number of AChRs may be decreased at the myoseptal varicosity (arrows) by MO injection. For each experiment, 8 spinal hemisegments plus somites were examined in each of 21–33 met MO-injected embryos and 8 spinal hemisegments plus somites in each of 15 controls. Scale bar, 20 μm.
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Figure 4: Met appears unnecessary for muscle and neuromuscular junction formation. (a,b) Engrailed antibody (Eng, red) labeling showing muscle pioneer cells and znp1 antibody labeling showing motor axons (green). In met MO-injected embryos, some CaP axons are truncated (asterisk). (c,d) F59 antibody (red) labeling showing fast muscle fibers and znp1 antibody staining showing motor axons (green). F59 labeling appears the same in control (c) and met MO-injected (d) embryos, which have some truncated CaPs (asterisk). (e-f) αBTX (red) labeling showing AChRs and znp1 antibody labeling showing motor axons (green). The distribution of AChRs appears the same in control (e) and met MO-injected embryos (f) that have some truncated CaP axons (asterisks); however, it appears that the number of AChRs may be decreased at the myoseptal varicosity (arrows) by MO injection. For each experiment, 8 spinal hemisegments plus somites were examined in each of 21–33 met MO-injected embryos and 8 spinal hemisegments plus somites in each of 15 controls. Scale bar, 20 μm.

Mentions: Even though met MO-injected embryos responded significantly slower to tail touches than did control embryos, their ability to move suggested that muscle function was normal and that neuromuscular junctions were present. We verified this by labeling 26 hpf control and met MO-injected embryos with zn1 or znp1 antibodies that recognize zebrafish PMNs [69,72] and antibodies that recognize specific muscle cell types, including anti-Engrailed that recognizes a specific subset of slow muscle fibers [62,73] (Figure 4a,b), and F59, that recognizes fast muscle fibers (Figure 4c,d) [74]. PMNs were present in met MO-injected embryos, although in some cases CaP axons were truncated (see below). Engrailed and F59 labeling were both present in met MO-injected embryos and appeared similar to controls. We also examined the localization of AChR clusters using αBTX [75]. AChR clusters were present in met MO-injected embryos and had a similar distribution as in controls (Figure 4e,f). Together these observations raise the possibility that Met function is not required for formation of muscles or neuromuscular junctions and that the impaired tail touch-evoked motility of met MO-injected embryos resulted from a requirement for Met for normal differentiation of PMNs, SMNs or both.


The Met receptor tyrosine kinase prevents zebrafish primary motoneurons from expressing an incorrect neurotransmitter.

Tallafuss A, Eisen JS - Neural Dev (2008)

Met appears unnecessary for muscle and neuromuscular junction formation. (a,b) Engrailed antibody (Eng, red) labeling showing muscle pioneer cells and znp1 antibody labeling showing motor axons (green). In met MO-injected embryos, some CaP axons are truncated (asterisk). (c,d) F59 antibody (red) labeling showing fast muscle fibers and znp1 antibody staining showing motor axons (green). F59 labeling appears the same in control (c) and met MO-injected (d) embryos, which have some truncated CaPs (asterisk). (e-f) αBTX (red) labeling showing AChRs and znp1 antibody labeling showing motor axons (green). The distribution of AChRs appears the same in control (e) and met MO-injected embryos (f) that have some truncated CaP axons (asterisks); however, it appears that the number of AChRs may be decreased at the myoseptal varicosity (arrows) by MO injection. For each experiment, 8 spinal hemisegments plus somites were examined in each of 21–33 met MO-injected embryos and 8 spinal hemisegments plus somites in each of 15 controls. Scale bar, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2542365&req=5

Figure 4: Met appears unnecessary for muscle and neuromuscular junction formation. (a,b) Engrailed antibody (Eng, red) labeling showing muscle pioneer cells and znp1 antibody labeling showing motor axons (green). In met MO-injected embryos, some CaP axons are truncated (asterisk). (c,d) F59 antibody (red) labeling showing fast muscle fibers and znp1 antibody staining showing motor axons (green). F59 labeling appears the same in control (c) and met MO-injected (d) embryos, which have some truncated CaPs (asterisk). (e-f) αBTX (red) labeling showing AChRs and znp1 antibody labeling showing motor axons (green). The distribution of AChRs appears the same in control (e) and met MO-injected embryos (f) that have some truncated CaP axons (asterisks); however, it appears that the number of AChRs may be decreased at the myoseptal varicosity (arrows) by MO injection. For each experiment, 8 spinal hemisegments plus somites were examined in each of 21–33 met MO-injected embryos and 8 spinal hemisegments plus somites in each of 15 controls. Scale bar, 20 μm.
Mentions: Even though met MO-injected embryos responded significantly slower to tail touches than did control embryos, their ability to move suggested that muscle function was normal and that neuromuscular junctions were present. We verified this by labeling 26 hpf control and met MO-injected embryos with zn1 or znp1 antibodies that recognize zebrafish PMNs [69,72] and antibodies that recognize specific muscle cell types, including anti-Engrailed that recognizes a specific subset of slow muscle fibers [62,73] (Figure 4a,b), and F59, that recognizes fast muscle fibers (Figure 4c,d) [74]. PMNs were present in met MO-injected embryos, although in some cases CaP axons were truncated (see below). Engrailed and F59 labeling were both present in met MO-injected embryos and appeared similar to controls. We also examined the localization of AChR clusters using αBTX [75]. AChR clusters were present in met MO-injected embryos and had a similar distribution as in controls (Figure 4e,f). Together these observations raise the possibility that Met function is not required for formation of muscles or neuromuscular junctions and that the impaired tail touch-evoked motility of met MO-injected embryos resulted from a requirement for Met for normal differentiation of PMNs, SMNs or both.

Bottom Line: We found that met is expressed in all early developing, individually identified primary motoneurons and in at least some later developing secondary motoneurons.However, a significant fraction of them had truncated axons.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA. tallafuss@uoneuro.uoregon.edu

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus