Abstract
BackgroundExpression 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.ResultsWe 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.ConclusionWe 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.
Highlights
Expression of correct neurotransmitters is crucial for normal nervous system function
Our results suggest that the p38 and/or Akt cascade may be required for Secondary motoneuron (SMN) differentiation, whereas the MEK1/2 cascade may be required for appropriate neurotransmitter expression and to prevent formation of interneuron-like axons in Primary motoneuron (PMN)
For each condition we examined eight spinal hemisegments per embryo. n = 56 met morpholino antisense oligonucleotide (MO)-injected embryos and 32 control embryos. (d) chat is expressed in somata in the normal location of PMNs; gray lines show segment boundaries. (e,f) Expression of chat and the CaP and VaP-specific marker islet2 shows that in met MOinjected embryos (f) CaPs express chat, as they do in controls (e)
Summary
Expression of correct neurotransmitters is crucial for normal nervous system function. 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 [12] and in vivo [7] without affecting expression of markers of cell fate specification [7] 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
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