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Eur J Neurosci
1999 Feb 01;112:373-82. doi: 10.1046/j.1460-9568.1999.00443.x.
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Xenopus muscle-specific kinase: molecular cloning and prominent expression in neural tissues during early embryonic development.
Fu AK
,
Smith FD
,
Zhou H
,
Chu AH
,
Tsim KW
,
Peng BH
,
Ip NY
.
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A muscle-specific receptor tyrosine kinase, designated MuSK, mediates agrin-induced aggregation of acetylcholine receptors at the vertebrate neuromuscular junction. cDNAs encoding Xenopus MuSK were isolated from embryonic cDNA libraries. The full-length MuSK cDNA encodes for a polypeptide of 948 amino acids and possesses the features unique to mammalian MuSK, including four Ig-like domains, C6 box, transmembrane region and an intracellular tyrosine kinase domain. Interestingly, Xenopus MuSK also contains a kringle domain similar to that previously reported for Torpedo MuSK. The overall amino acid sequence identity of Xenopus MuSK with mammalian MuSK is approximately 65%. Northern blot analysis demonstrated the presence of three MuSK transcripts (approximately 1 kb, approximately 3 kb and approximately 7 kb) which were differentially expressed during development. The expression of the approximately 7 kb MuSK transcript remained as the predominant species in adult tissues, e.g. skeletal muscle, spleen and lung. Immunocytochemical analysis with a MuSK-specific antibody revealed that Xenopus MuSK was colocalized with AChRs at neuromuscular junctions as well as in spontaneous acetylcholine receptor hot spots of cultured muscle cells. In situ hybridization revealed prominent expression of MuSK transcripts in neural tissues and myotomal muscle during the period of neurulation and synaptogenesis. The MuSK transcript detected at abundant levels in the central nervous system (CNS) was localized to the brain, spinal cord and eye vesicles during early embryonic development. In addition, the MuSK protein in the developing eye was found to be prominently expressed during embryonic stages of 32 and 35. These findings raise an intriguing possibility that, in addition to the known function in the formation of the neuromuscular junctions, MuSK may be involved in neural development.
FIG. 1. Sequence of Xenopus MuSK. Deduced amino acid sequence of the Xenopus MuSK (upper panel). The MuSK full-length sequence encodes for 948
amino acids with a signal peptide, Ig-like domains (I–IV), C-6 box, transmembrane domain and tyrosine kinase domains (I–XI). Comparison of the amino acid
sequence of Xenopus MuSK (x-MuSK) with Torpedo MuSK (t-RTK) and mammalian MuSK (rat, r-MuSK; human, h-MuSK and mouse, Nsk2). Amino acid
differences from Xenopus MuSK are as indicated; the conserved residues are denoted by periods and the missing residues are indicated by hyphens. Lower
panel: hydropathy plot of Xenopus MuSK amino acid sequence generated using the MacVector sequence analysis program, with a window size of 19.
FIG. 2. Expression profile of Xenopus MuSK in adult tissues and embryos during development. Northern blot analysis of MuSK was performed with a cDNA fragment encoding the whole extracellular domain and part of the tyrosine kinase domain, as described in Materials and methods. The expression of Xenopus MuSK transcripts was examined in different adult tissues (A). The developmental profile of Xenopus MuSK expression was examined in embryos from stage 5 to stage 46 (B). Positions of MuSK transcripts are indicated by arrowheads on the right, and ribosomal bands are indicated on the left.
FIG. 3. Localization of MuSK mRNA by whole-mount in situ hybridization. (A–I) Anti-sense probe, (J) sense probe as control. (A–D) Lateral view: (A) stage 22, (B) stage 25, (C) stage 28, (D) stage 45. MuSK mRNA was localized in the myotomes as well as in the CNS in embryos during different stages of myotomal NMJ formation (A–C). After this period, mRNA expression is down-regulated in the myotomal musculature (D). (E–I) Dorsal view: (E) stage 22, (F) stage 28, (G) stage 40, (H, I) dorsal part of the embryo only. In this view, the expression of the MuSK mRNA in both neural tube (NT) and myotomes (MT) is clearly seen. Within the CNS, the eye vesicle is a prominent site of mRNA expression. No labelling was seen at all stages when the embryos were labelled with the sense probe (J).
FIG. 4. Immunohistochemical analysis reveals the colocalization of MuSK and AChR in skeletal muscles of adult Xenopus. Frozen adult muscle sections were stained with anti-MuSK antibody (MuSK) or rhodamine-conjugated a- bungarotoxin (AChR). Scale bar, 10 mm.
FIG. 5. Localization of MuSK at the NMJ and AChR hot spots. (A) Phase contrast of a neuronmuscle coculture; (B) MuSK labelling with the anti-MuSK antibody; (C) rhodamine-conjugated a-bungarotoxin labelling of AChRs. Arrowheads indicate the neuron–muscle contacts as well as the AChR aggregates. MuSK was colocalized with AChR aggregates at the NMJ. The nerve process can be seen in phase contrast as indicated by the arrow in (A). (A–C) represent the same view but with different optics. (D) and (E) shows the colocalization of MuSK and AChR hot spots in uninnervated muscle cells. (D) MuSK labelling; (E) rhodamine-conjugated a-bungarotoxin labelling. (D) and (E) represent the same view. Scale bar, 10 mm.
FIG. 6. Expression of MuSK in developing Xenopus eyes. (A) Reverse transcription was used to prepare the first strand of cDNA from total RNAs. The cDNAs were amplified by PCR with two sets of primers flanking different regions of Xenopus MuSK (TK, tyrosine kinase domain; EC, extracellular domain). Southern blot analysis was performed using the appropriate MuSK cDNA probe. PCR fragments obtained with primers specific for EF1 were used as control for equal loading. 1 , cDNAs from stage 32 eyes; –, RNAs from stage 32 eyes (without reverse transcription); C, water control; and Mu, adult muscle cDNA. (B) Membrane fractions from eyes (stage 32, 35 or 57) or adult muscle (Mu) were obtained, and proteins (µ 2 mg) were fractionated by 6% SDS–PAGE. Electrophoresed proteins were transferred onto nitrocellulose membrane, and detected by anti-MuSK antibody (C-19) and peroxidaseconjugated secondary antibody. Positions of molecular markers (97 kDa and 200 kDa) are as indicated.
FIG. 7. Immunohistochemical analysis reveals the expression of MuSK protein in the developing eye of stage 35 Xenopus embryos. Whole-mount embryos were stained with anti-MuSK antibody (C-19). (A) Control serum, (B) C-19 followed by secondary antibody, (C) higher magnification of (B). Scale bar, 100 mm.
FIG. 8. Functional expression of Xenopus MuSK in chick muscle cells. (A) Western blot analysis of crude cell lysates from mock-transfected chick muscle cells or cells over-expressing Xenopus MuSK using the MuSK-specific antibody (upper panel). Induction of Xenopus MuSK tyrosine phosphorylation by agrin in chick myotubes overexpressing Xenopus MuSK (lower panel). Molecular weight markers are in kDa. (B) Overexpression of Xenopus MuSK enhanced the aggregation of AChRs in primary chick muscle culture. Chick myotubes were mock transfected or transfected with Xenopus MuSK construct, and then treated with active agrin for 12 h. Aggregation of AChRs was assayed by staining with rhodamine-conjugated a-bungarotoxin and counted as described in Materials and methods. Data are presented as the number of AChR aggregates per myotube, mean 6 SEM (n 5 5). The asterisk indicates that the number of agrininduced AChR aggregates differed significantly from mock-transfected cultures (P , 0.05, unpaired t-test).
musk (muscle, skeletal, receptor tyrosine kinase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anteriorleft, dorsal up.
musk (muscle, skeletal, receptor tyrosine kinase) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
