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A novel pathway for MuSK to induce key genes in neuromuscular synapse formation.

Lacazette E, Le Calvez S, Gajendran N, Brenner HR - J. Cell Biol. (2003)

Bottom Line: Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes.The same pathways are used to regulate synaptic expression of AChR epsilon.We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChR epsilon. We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

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A 2.1-kb nsk2/musk promoter fragment confers spatio-temporal expression patterns to reporters comparable to those of endogenous musk. (A) Examples of endplates marked by histochemical staining for AChE and expressing colocalized β-Gal–positive nuclei upon transfection with p(1.6nlslacZ-N). Nuclei are in blue and AChE is in brown. Bar, 40 μm. (B) The percentage of β-Gal–positive fibers with colocalizations of β-Gal–positive nuclei and synaptic AChE observed (as illustrated in A) is significantly higher than expected for a random process. Means ± SE are given (n = 4 muscles, 17–56 β-Gal–positive fibers [170 fibers total] examined per muscle). (C) The level of firefly luciferase (luc) activity expressed upon transfection with p(1.6luc-N) increases linearly with the activity of a cotransfected standard, p(RL-TK), of Renilla luciferase, indicating that normalization to RL-TK expression to account for differences in transfection efficiency between muscles as performed in D is appropriate. (D) The 2.1-kb nsk2/musk promoter fragment responds to muscle denervation (4 d) by increasing expression of luciferase activity (means ± SE, n = 5). In the same experiments, the levels of RL-TK were not significantly different in innervated control and in denervated fibers (data not depicted). (E) Activation of p1.6luc-N and p1.6luc-Nmut in C2C12 myoblasts and in differentiating myotubes (n = 3 parallel cultures for each transfected plasmid). *P < 0.05 and **P < 0.01 in two-tailed t test. Bars ± SEM.
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fig2: A 2.1-kb nsk2/musk promoter fragment confers spatio-temporal expression patterns to reporters comparable to those of endogenous musk. (A) Examples of endplates marked by histochemical staining for AChE and expressing colocalized β-Gal–positive nuclei upon transfection with p(1.6nlslacZ-N). Nuclei are in blue and AChE is in brown. Bar, 40 μm. (B) The percentage of β-Gal–positive fibers with colocalizations of β-Gal–positive nuclei and synaptic AChE observed (as illustrated in A) is significantly higher than expected for a random process. Means ± SE are given (n = 4 muscles, 17–56 β-Gal–positive fibers [170 fibers total] examined per muscle). (C) The level of firefly luciferase (luc) activity expressed upon transfection with p(1.6luc-N) increases linearly with the activity of a cotransfected standard, p(RL-TK), of Renilla luciferase, indicating that normalization to RL-TK expression to account for differences in transfection efficiency between muscles as performed in D is appropriate. (D) The 2.1-kb nsk2/musk promoter fragment responds to muscle denervation (4 d) by increasing expression of luciferase activity (means ± SE, n = 5). In the same experiments, the levels of RL-TK were not significantly different in innervated control and in denervated fibers (data not depicted). (E) Activation of p1.6luc-N and p1.6luc-Nmut in C2C12 myoblasts and in differentiating myotubes (n = 3 parallel cultures for each transfected plasmid). *P < 0.05 and **P < 0.01 in two-tailed t test. Bars ± SEM.

Mentions: To test for synapse-specific activation, the proximal 1.6-kb upstream region was linked to nlslacZ as a reporter, followed by 0.5 kb of intronic sequence containing the N-box (Fig. 1 C, p1.6nlslacZ-N). Innervated, i.e., electrically active rat soleus muscles were then transfected in vivo by electroporation with p1.6nlslacZ-N, and synapse-specific expression of nlslacZ was tested 10–14 d later in the microscope by examining the location of muscle nuclei expressing β-galactosidase (β-Gal)–positive nuclei with respect to the location of synapses. The latter were marked by histochemical staining for acetylcholinesterase (AChE). Indeed, in some fibers β-Gal–positive nuclei were tightly colocalized with synapses (Fig. 2 A); in other fibers they were also seen in nonsynaptic regions. To see whether the AChE/β-Gal colocalization reflected preferential activation of the nsk2/musk promoter fragment at the synapse, we compared the number of β-Gal/AChE colocalizations expected for a random process with the number of colocalizations actually observed. For this purpose, fiber bundles were dissected, and the lengths of fiber segments occupied by β-Gal–positive nuclei relative to the length of the dissected fibers were determined. This ratio multiplied by the number of β-Gal–positive fibers examined gives the number of β-Gal–positive synapses expected for random colocalization. Comparison with the number of β-Gal/AChE colocalizations actually observed showed that β-Gal–positive synapses occurred five to six times more frequently than expected by chance (Fig. 2 B).


A novel pathway for MuSK to induce key genes in neuromuscular synapse formation.

Lacazette E, Le Calvez S, Gajendran N, Brenner HR - J. Cell Biol. (2003)

A 2.1-kb nsk2/musk promoter fragment confers spatio-temporal expression patterns to reporters comparable to those of endogenous musk. (A) Examples of endplates marked by histochemical staining for AChE and expressing colocalized β-Gal–positive nuclei upon transfection with p(1.6nlslacZ-N). Nuclei are in blue and AChE is in brown. Bar, 40 μm. (B) The percentage of β-Gal–positive fibers with colocalizations of β-Gal–positive nuclei and synaptic AChE observed (as illustrated in A) is significantly higher than expected for a random process. Means ± SE are given (n = 4 muscles, 17–56 β-Gal–positive fibers [170 fibers total] examined per muscle). (C) The level of firefly luciferase (luc) activity expressed upon transfection with p(1.6luc-N) increases linearly with the activity of a cotransfected standard, p(RL-TK), of Renilla luciferase, indicating that normalization to RL-TK expression to account for differences in transfection efficiency between muscles as performed in D is appropriate. (D) The 2.1-kb nsk2/musk promoter fragment responds to muscle denervation (4 d) by increasing expression of luciferase activity (means ± SE, n = 5). In the same experiments, the levels of RL-TK were not significantly different in innervated control and in denervated fibers (data not depicted). (E) Activation of p1.6luc-N and p1.6luc-Nmut in C2C12 myoblasts and in differentiating myotubes (n = 3 parallel cultures for each transfected plasmid). *P < 0.05 and **P < 0.01 in two-tailed t test. Bars ± SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199368&req=5

fig2: A 2.1-kb nsk2/musk promoter fragment confers spatio-temporal expression patterns to reporters comparable to those of endogenous musk. (A) Examples of endplates marked by histochemical staining for AChE and expressing colocalized β-Gal–positive nuclei upon transfection with p(1.6nlslacZ-N). Nuclei are in blue and AChE is in brown. Bar, 40 μm. (B) The percentage of β-Gal–positive fibers with colocalizations of β-Gal–positive nuclei and synaptic AChE observed (as illustrated in A) is significantly higher than expected for a random process. Means ± SE are given (n = 4 muscles, 17–56 β-Gal–positive fibers [170 fibers total] examined per muscle). (C) The level of firefly luciferase (luc) activity expressed upon transfection with p(1.6luc-N) increases linearly with the activity of a cotransfected standard, p(RL-TK), of Renilla luciferase, indicating that normalization to RL-TK expression to account for differences in transfection efficiency between muscles as performed in D is appropriate. (D) The 2.1-kb nsk2/musk promoter fragment responds to muscle denervation (4 d) by increasing expression of luciferase activity (means ± SE, n = 5). In the same experiments, the levels of RL-TK were not significantly different in innervated control and in denervated fibers (data not depicted). (E) Activation of p1.6luc-N and p1.6luc-Nmut in C2C12 myoblasts and in differentiating myotubes (n = 3 parallel cultures for each transfected plasmid). *P < 0.05 and **P < 0.01 in two-tailed t test. Bars ± SEM.
Mentions: To test for synapse-specific activation, the proximal 1.6-kb upstream region was linked to nlslacZ as a reporter, followed by 0.5 kb of intronic sequence containing the N-box (Fig. 1 C, p1.6nlslacZ-N). Innervated, i.e., electrically active rat soleus muscles were then transfected in vivo by electroporation with p1.6nlslacZ-N, and synapse-specific expression of nlslacZ was tested 10–14 d later in the microscope by examining the location of muscle nuclei expressing β-galactosidase (β-Gal)–positive nuclei with respect to the location of synapses. The latter were marked by histochemical staining for acetylcholinesterase (AChE). Indeed, in some fibers β-Gal–positive nuclei were tightly colocalized with synapses (Fig. 2 A); in other fibers they were also seen in nonsynaptic regions. To see whether the AChE/β-Gal colocalization reflected preferential activation of the nsk2/musk promoter fragment at the synapse, we compared the number of β-Gal/AChE colocalizations expected for a random process with the number of colocalizations actually observed. For this purpose, fiber bundles were dissected, and the lengths of fiber segments occupied by β-Gal–positive nuclei relative to the length of the dissected fibers were determined. This ratio multiplied by the number of β-Gal–positive fibers examined gives the number of β-Gal–positive synapses expected for random colocalization. Comparison with the number of β-Gal/AChE colocalizations actually observed showed that β-Gal–positive synapses occurred five to six times more frequently than expected by chance (Fig. 2 B).

Bottom Line: Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes.The same pathways are used to regulate synaptic expression of AChR epsilon.We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Basel, CH-4056 Basel, Switzerland.

ABSTRACT
At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChR epsilon. We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

Show MeSH
Related in: MedlinePlus