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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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Met signaling may influence CaP axonal and neurotransmitter phenotypes through the MEK1/2 pathway. All panels show mn2Et transgenic embryos. (a-d") Embryos at 26 hpf showing GFP (green) and GABA (red); asterisks indicate CaPs or CaP/VaP pairs. (a',b',c',d') Only the green channel is shown; (a",b",c",d") only the red channel of the micrographs shown in (a,b,c,d) is shown. CaPs express GABA in U0126-treated embryos (c-c") and met MO-injected embryos (b-b") but not in controls (a-a") or in SB203580-treated embryos (d-d"). (e-h) Some CaPs have both peripheral (arrows) and central (asterisks) axons in U0126-treated embryos (g) and met MO-injected embryos (f); CaPs have only have peripheral axons in controls (e) and in SB203580-treated embryos (h). Scale bars, 10 μm.
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Figure 9: Met signaling may influence CaP axonal and neurotransmitter phenotypes through the MEK1/2 pathway. All panels show mn2Et transgenic embryos. (a-d") Embryos at 26 hpf showing GFP (green) and GABA (red); asterisks indicate CaPs or CaP/VaP pairs. (a',b',c',d') Only the green channel is shown; (a",b",c",d") only the red channel of the micrographs shown in (a,b,c,d) is shown. CaPs express GABA in U0126-treated embryos (c-c") and met MO-injected embryos (b-b") but not in controls (a-a") or in SB203580-treated embryos (d-d"). (e-h) Some CaPs have both peripheral (arrows) and central (asterisks) axons in U0126-treated embryos (g) and met MO-injected embryos (f); CaPs have only have peripheral axons in controls (e) and in SB203580-treated embryos (h). Scale bars, 10 μm.

Mentions: We used pharmacological inhibitors to learn which Met-activated intracellular signaling pathway(s) transduced the signal required to prevent GABA expression in CaP. Embryos were exposed to inhibitors of these pathways from 16–26 hpf, then fixed and examined for expression of GABA in CaPs (Table 5). Exposure to the MEK1/2 signaling inhibitor U0126 resulted in GABA-positive CaPs, similar to results observed in met MO-injected embryos (Figure 9a–c"). In addition, some CaPs in U0126-treated embryos had both peripheral and central axons, similar to met MO-injected embryos (Table 3; Figure 9e–g). Together these results suggest that the CaP axonal and neurotransmitter phenotypes seen in the absence of Met function are likely to result from lack of Met activation of MEK1/2. We also treated embryos with SB203580, which inhibits the Akt and p38 pathways and blocked SMN development. In contrast to the effect of U0126, CaPs in embryos exposed to SB203580 had the same neurotransmitter (Figure 9d–d"; Table 5) and axonal (Figure 9h; Table 3) phenotype as control CaPs, suggesting that signaling via p38 and Akt is not involved in regulating CaP neurotransmitter and axon phenotype.


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

Tallafuss A, Eisen JS - Neural Dev (2008)

Met signaling may influence CaP axonal and neurotransmitter phenotypes through the MEK1/2 pathway. All panels show mn2Et transgenic embryos. (a-d") Embryos at 26 hpf showing GFP (green) and GABA (red); asterisks indicate CaPs or CaP/VaP pairs. (a',b',c',d') Only the green channel is shown; (a",b",c",d") only the red channel of the micrographs shown in (a,b,c,d) is shown. CaPs express GABA in U0126-treated embryos (c-c") and met MO-injected embryos (b-b") but not in controls (a-a") or in SB203580-treated embryos (d-d"). (e-h) Some CaPs have both peripheral (arrows) and central (asterisks) axons in U0126-treated embryos (g) and met MO-injected embryos (f); CaPs have only have peripheral axons in controls (e) and in SB203580-treated embryos (h). Scale bars, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Met signaling may influence CaP axonal and neurotransmitter phenotypes through the MEK1/2 pathway. All panels show mn2Et transgenic embryos. (a-d") Embryos at 26 hpf showing GFP (green) and GABA (red); asterisks indicate CaPs or CaP/VaP pairs. (a',b',c',d') Only the green channel is shown; (a",b",c",d") only the red channel of the micrographs shown in (a,b,c,d) is shown. CaPs express GABA in U0126-treated embryos (c-c") and met MO-injected embryos (b-b") but not in controls (a-a") or in SB203580-treated embryos (d-d"). (e-h) Some CaPs have both peripheral (arrows) and central (asterisks) axons in U0126-treated embryos (g) and met MO-injected embryos (f); CaPs have only have peripheral axons in controls (e) and in SB203580-treated embryos (h). Scale bars, 10 μm.
Mentions: We used pharmacological inhibitors to learn which Met-activated intracellular signaling pathway(s) transduced the signal required to prevent GABA expression in CaP. Embryos were exposed to inhibitors of these pathways from 16–26 hpf, then fixed and examined for expression of GABA in CaPs (Table 5). Exposure to the MEK1/2 signaling inhibitor U0126 resulted in GABA-positive CaPs, similar to results observed in met MO-injected embryos (Figure 9a–c"). In addition, some CaPs in U0126-treated embryos had both peripheral and central axons, similar to met MO-injected embryos (Table 3; Figure 9e–g). Together these results suggest that the CaP axonal and neurotransmitter phenotypes seen in the absence of Met function are likely to result from lack of Met activation of MEK1/2. We also treated embryos with SB203580, which inhibits the Akt and p38 pathways and blocked SMN development. In contrast to the effect of U0126, CaPs in embryos exposed to SB203580 had the same neurotransmitter (Figure 9d–d"; Table 5) and axonal (Figure 9h; Table 3) phenotype as control CaPs, suggesting that signaling via p38 and Akt is not involved in regulating CaP neurotransmitter and axon phenotype.

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