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J Neurosci
2017 Aug 30;3735:8477-8485. doi: 10.1523/JNEUROSCI.3881-16.2017.
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N1-Src Kinase Is Required for Primary Neurogenesis in Xenopus tropicalis.
Lewis PA
,
Bradley IC
,
Pizzey AR
,
Isaacs HV
,
Evans GJO
.
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The presence of the neuronal-specific N1-Src splice variant of the C-Src tyrosine kinase is conserved through vertebrate evolution, suggesting an important role in complex nervous systems. Alternative splicing involving an N1-Src-specific microexon leads to a 5 or 6 aa insertion into the SH3 domain of Src. A prevailing model suggests that N1-Src regulates neuronal differentiation via cytoskeletal dynamics in the growth cone. Here we investigated the role of n1-src in the early development of the amphibian Xenopus tropicalis, and found that n1-src expression is regulated in embryogenesis, with highest levels detected during the phases of primary and secondary neurogenesis. In situ hybridization analysis, using locked nucleic acid oligo probes complementary to the n1-src microexon, indicates that n1-src expression is highly enriched in the open neural plate during neurula stages and in the neural tissue of adult frogs. Given the n1-src expression pattern, we investigated a possible role for n1-src in neurogenesis. Using splice site-specific antisense morpholino oligos, we inhibited n1-src splicing, while preserving c-src expression. Differentiation of neurons in the primary nervous system is reduced in n1-src-knockdown embryos, accompanied by a severely impaired touch response in later development. These data reveal an essential role for n1-src in amphibian neural development and suggest that alternative splicing of C-Src in the developing vertebrate nervous system evolved to regulate neurogenesis.SIGNIFICANCE STATEMENT The Src family of nonreceptor tyrosine kinases acts in signaling pathways that regulate cell migration, cell adhesion, and proliferation. Srcs are also enriched in the brain, where they play key roles in neuronal development and neurotransmission. Vertebrates have evolved a neuron-specific splice variant of C-Src, N1-Src, which differs from C-Src by just 5 or 6 aa. N1-Src is poorly understood and its high similarity to C-Src has made it difficult to delineate its function. Using antisense knockdown of the n1-src microexon, we have studied neuronal development in the Xenopus embryo in the absence of n1-src, while preserving c-src Loss of n1-src causes a striking absence of primary neurogenesis, implicating n1-src in the specification of neurons early in neural development.
Figure 1. Xenopus n1-src elicits neurite-like processes in fibroblasts. A, Amino acid alignment of the N1-microexon in mammalian, Xenopus, and fish species. +, Basic; −, acidic; φ, hydrophobic amino acid sidechains. B, Representative COS7 cells cotransfected for 4 d with Src-FLAG and CFP constructs. Cells were stained for Src (anti-FLAG) and CFP. C, Quantification of process outgrowth in COS7 cells. Each process was defined as an extension longer than one cell soma diameter and <2 μm in diameter. Data are plotted as mean ± SEM (n = 3 independent experiments, analyzed by Kruskal–Wallis 2-tailed ANOVA; ***p < 0.001; Scale bar, 10 μm).
Figure 2. n1-src mRNA expression levels during Xenopus tropicalis development and in adult tissues. A, RT-PCR analysis of c-src and n1-src mRNA expression levels from early cleavage Stage 4 to tailbud Stage 35. rpl8 is used as a ubiquitously expressed loading control. −rt, No reverse transcriptase control; water, no template control. B, RT-PCR analysis of c-src and n1-src expression levels during (Stage 25) and after (Stage 35) primary neurogenesis, and during secondary neurogenesis (Stage 46). C, RT-PCR analysis of c-src and n1-src expression in a range of adult tissues.
Figure 3. Expression pattern of n1-src during Xenopus tropicalis primary neurogenesis. In situ hybridization analysis of n1-src mRNA expression using a 19-mer digoxigenin end-labeled antisense probe directed against n1-src-specific sequence. A–D, Early neurula Stage 14 embryos. E, F, Late neurula Stage 19 embryos. A, Dorsal view, anterior to left. B, Lateral view, anterior to the left. C, Anterior view, dorsal to the top. D, Posterior view, dorsal to the top. E, Dorsal view, anterior to the left. F, Anterior view, dorsal to the top; f+mb, presumptive forebrain and midbrain; sc, presumptive spinal cord; bp, blastopore.
Figure 4. Abnormal touch response and primary neurogenesis in n1-src-knockdown embryos. A, Diagram showing the sequences and corresponding RNA target sequences of the splice acceptor (AMO a) and donor (AMO d) splice-blocking AMOs. B, RT-PCR analysis of c-src and n1-src mRNA expression at Stage 16 in control uninjected embryos and embryos injected with a total of 20 ng AMO a, AMO d, or AMO a+d. rpl8 is used as a ubiquitously expressed loading control. −rt, No reverse transcriptase control; water, no template control. C, Representative phenotypes of embryos at larval Stage 41 bilaterally injected at the two-cell or four-cell stage with 10 ng total of a standard control MO or the AMO a+d combination. Embryos were coinjected with 100 pg of nuclear β-galactosidase and subsequently stained with X-gal (light blue) to demonstrate successful injection targeting. D, Diagram of embryo touch reflex. Touching the skin stimulates Rohon–Beard sensory neurons (s), which synapse onto commissural interneurons (i) that activate contralateral motor neurons (m), leading to muscle contraction. E, Quantitation of touch-response phenotype of the same embryos at larval Stage 28 and 41 bilaterally injected at the two-cell or four-cell stage with 10 ng total of a standard control MO or the AMO a+d combination. Data are plotted as mean ± SEM (n = 3 independent experiments, analyzed by 1-way ANOVA with Tukey's post hoc test, ***p < 0.001, control MO vs n1-Src AMO). F, In situ hybridization analysis of tubb2b expression in differentiating primary neurons of open neural plate Stage 14 embryos unilaterally injected with 5 ng total of a standard control MO or the AMO a+d combination. The injected side shows faint blue nuclear staining with the β-galactosidase lineage tracer, and is indicated with a black asterisk. Anterior is to the left. m, Motor neurons; i, interneurons; s, sensory neurons.
src (SRC proto-oncogene, non-receptor tyrosine kinase) gene expression in Xenopus tropicalis embryo, assayed via in situ hybridization, NF stage 19, dorsal view, anteriorleft.
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