N1-Src Kinase Is Required for Primary Neurogenesis in Xenopus tropicalis.

Philip A Lewis,Isobel C Bradley,Alastair R Pizzey,Gareth J.O Evans,Harry V Isaacs

doi:10.1523/jneurosci.3881-16.2017

Abstract

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.

Highlights

The Src family of 11 nonreceptor tyrosine kinases evolved to regulate key signaling pathways involved in cell adhesion, migration, and cell fate in multicellular organisms (Thomas and Brugge, 1997)
The Xenopus n1-src splice variant promotes neurite outgrowth We first investigated whether the activity of N1-src isoforms has scriptase according to the manufacturer’s instructions
The activity of amphibian and mammalian n1-src is conserved The alternative splicing of neuronal src isoforms alters the ligandbinding specificity of the C-Src SH3 domain and the catalytic activity of its kinase domain (Brugge et al, 1985; Keenan et al, 2015)

Summary

Introduction

The Src family of 11 nonreceptor tyrosine kinases evolved to regulate key signaling pathways involved in cell adhesion, migration, and cell fate in multicellular organisms (Thomas and Brugge, 1997). **H.V.I. and G.J.O.E. contributed to this work. Further complexity and specificity of C-Src signaling in the brain is conferred by neuronal-specific splicing to yield N1-Src or N2Src (Brugge et al, 1985; Pyper and Bolen, 1990). The N-Src splice variants contain an additional 6 or 17 aa respectively in the SH3 domain, and are encoded by microexons situated between exons three and four of C-Src (Martinez et al, 1987). We and others have shown that N-Srcs have a higher constitutive kinase activity and an altered SH3 domain substrate specificity compared with

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