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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 used morpholino antisense oligonucleotides to knock down Met function and found that Met has distinct roles in primary and secondary motoneurons.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.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.

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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Blocking Met function. (a) RT-PCR showing that the E6I6 MO blocks met mRNA splicing. (b) Activation of Met can initiate intracellular signaling through several different downstream cascades, including MEK1/2 and PI3K [24,25]. This diagram shows the cascade and where specific inhibitors act. See text for details.
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Figure 1: Blocking Met function. (a) RT-PCR showing that the E6I6 MO blocks met mRNA splicing. (b) Activation of Met can initiate intracellular signaling through several different downstream cascades, including MEK1/2 and PI3K [24,25]. This diagram shows the cascade and where specific inhibitors act. See text for details.

Mentions: Experiments carried out using a variety of cell types have shown that activation of Met can initiate intracellular signaling through several different downstream cascades, including mitogen activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) and p38 and Akt pathways [24,25]. These cascades can act independently or can be stimulated simultaneously, and there can be crosstalk among them [26-28]. In addition to activation via Met, these intracellular signaling pathways can also be activated by other receptors [29-33]. To elucidate the roles of these pathways in cellular events, a number of specific pathway inhibitory reagents have been developed, including LY294002, which inhibits the PI3K pathway [34], U0126, which inhibits the MAPK pathway by inhibiting MEK1/2 [35], and SB203580, which inhibits Akt [36] and p38 MAP kinase [37] (Figure 1). It is currently unknown which of these signaling cascades is activated by HGF-mediated Met signaling in motoneurons, and whether different cascades affect different aspects of HGF-mediated Met function in these cells.


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

Tallafuss A, Eisen JS - Neural Dev (2008)

Blocking Met function. (a) RT-PCR showing that the E6I6 MO blocks met mRNA splicing. (b) Activation of Met can initiate intracellular signaling through several different downstream cascades, including MEK1/2 and PI3K [24,25]. This diagram shows the cascade and where specific inhibitors act. See text for details.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Blocking Met function. (a) RT-PCR showing that the E6I6 MO blocks met mRNA splicing. (b) Activation of Met can initiate intracellular signaling through several different downstream cascades, including MEK1/2 and PI3K [24,25]. This diagram shows the cascade and where specific inhibitors act. See text for details.
Mentions: Experiments carried out using a variety of cell types have shown that activation of Met can initiate intracellular signaling through several different downstream cascades, including mitogen activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K) and p38 and Akt pathways [24,25]. These cascades can act independently or can be stimulated simultaneously, and there can be crosstalk among them [26-28]. In addition to activation via Met, these intracellular signaling pathways can also be activated by other receptors [29-33]. To elucidate the roles of these pathways in cellular events, a number of specific pathway inhibitory reagents have been developed, including LY294002, which inhibits the PI3K pathway [34], U0126, which inhibits the MAPK pathway by inhibiting MEK1/2 [35], and SB203580, which inhibits Akt [36] and p38 MAP kinase [37] (Figure 1). It is currently unknown which of these signaling cascades is activated by HGF-mediated Met signaling in motoneurons, and whether different cascades affect different aspects of HGF-mediated Met function in these cells.

Bottom Line: We used morpholino antisense oligonucleotides to knock down Met function and found that Met has distinct roles in primary and secondary motoneurons.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.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.

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