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

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.

Unique transcriptomes of sensory and non-sensory neurons: insights from Splicing Regulatory States

“…The functional importance of microexons is attested by multiple single-case studies (Quesnel-Vallières et al, 2015 ; Parras et al, 2018 ; Laurent et al, 2015 ; Ohnishi et al, 2014 ; Lewis et al, 2017 ; Gonatopoulos-Pournatzis et al, 2020 ; Gonatopoulos-Pournatzis and Blencowe, 2020 ; Poliński et al, 2023 ), as well as by the numerous neurodevelopmental defects of Srrm4 mutant mice. Homozygous Srrm4 mutant mice die after birth and undergo dramatic CNS alterations (Quesnel-Vallières et al, 2015 ), while heterozygous mutant mice have altered synaptic transmission and neuronal excitability resulting in autism-like behavior (Quesnel-Vallières et al, 2016 ).…”

Epithelial-Mesenchymal Transition: A Groundless Skyscraper

“…Expression of other genes were detected in Xenopus embryos. Expression patterns of cdh1, cdh2, vim, snai1, sox2, cdk1, plk1, birc5, ezh2, lsd1, akt1 and ptk2 were from Zhang et al ( 2017) (Zhang et al, 2017); sox9 was from Lee and Saint-Jeannet (2011) (Lee and Saint-Jeannet, 2011); mcl1 from Sena et al (2020) (Sena et al, 2020); h2ax from Lee et al (2010) (Lee et al, 2010); src from Lewis et al (2017) (Lewis et al, 2017); and snai2 and twist1 were from Wang et al (2015) (Wang et al, 2015). Dorsal view is shown for Xenopus embryos, with the anterior of embryos to the left.…”

MicroExonator enables systematic discovery and quantification of microexons across mouse embryonic development

“…From the STRING analysis, we found evidence for Srcdependent phosphorylation of Git1, Ctnnd1 and Ptk2 [64][65][66], though the impact of neuronal microexon alternative splicing for these phosphorylation events remains unknown. Moreover, recent studies show that n-Src microexon inclusion is required for normal primary neurogenesis and L1cam-dependent neurite elongation [52,67], implying a strong phenotype. Another central node in the PPI network that is known to undergo microexon alternative splicing changes that are important for axon growth is L1cam, a founding member of the L1cam protein family.…”