Neuroblast migration is a critical aspect of nervous system development (e.g., neural crest migration). In an unbiased forward genetic screen, we identified a novel player in neuroblast migration, the ETR-1/CELF1 RNA binding protein. CELF1 RNA binding proteins are involved in multiple aspects of RNA processing including alternative splicing, stability, and translation. We find that a specific mutation in alternatively-spliced exon 8 results in migration defects of the AQR and PQR neurons, and not the embryonic lethality and body wall muscle defects of complete knockdown of the locus. Surprisingly, ETR-1 was required in body wall muscle cells for AQR/PQR migration (i.e., it acts cell non-autonomously). Genetic interactions indicate that ETR-1 acts with Wnt signaling, either in the Wnt pathway or in a parallel pathway. Possibly, ETR-1 is involved in the production of a Wnt signal or a parallel signal by the body wall muscles that controls AQR and PQR neuronal migration. In humans, CELF1 is involved in a number of neuromuscular disorders. If the role of ETR-1/CELF1 is conserved, these disorders might also involve cell or neuronal migration. Finally, we describe a technique of amplicon sequencing to detect rare, cell-specific genome edits by CRISPR/Cas9 in vivo (CRISPR-seq) as an alternative to the T7E1 assay.
During nervous system development, neurons and their progenitors migrate to their final destinations. In Caenorhabditis elegans, the bilateral Q neuroblasts and their descendants migrate long distances in opposite directions, despite being born in the same posterior region. QR on the right migrates anteriorly and generates the AQR neuron positioned near the head, and QL on the left migrates posteriorly, giving rise to the PQR neuron positioned near the tail. In a screen for genes required for AQR and PQR migration, we identified an allele of nfm-1, which encodes a molecule similar to vertebrate NF2/Merlin, an important tumor suppressor in humans. Mutations in NF2 lead to neurofibromatosis type II, characterized by benign tumors of glial tissues. Here we demonstrate that in C. elegans, nfm-1 is required for the ability of Q cells and their descendants to extend protrusions and to migrate, but is not required for direction of migration. Using a combination of mosaic analysis and cell-specific expression, we show that NFM-1 is required nonautonomously, possibly in muscles, to promote Q lineage migrations. We also show a genetic interaction between nfm-1 and the C. elegans Slit homolog slt-1, which encodes a conserved secreted guidance cue. Our results suggest that NFM-1 might be involved in the generation of an extracellular cue that promotes Q neuroblast protrusion and migration that acts with or in parallel to SLT-1. In vertebrates, NF2 and Slit2 interact in axon pathfinding, suggesting a conserved interaction of NF2 and Slit2 in regulating migratory events.
Migration of neuroblasts and neurons from their birthplace is central to the formation of neural circuits and networks. ETR-1 is the Caenorhabditis elegans homolog of the CELF1 (CUGBP, ELAV-like family 1) RNA-processing factor involved in neuromuscular disorders. etr-1 regulates body wall muscle differentiation. Our previous work showed that etr-1 in muscle has a non-autonomous role in neuronal migration, suggesting that ETR-1 is involved in the production of a signal emanating from body wall muscle that controls neuroblast migration and that interacts with Wnt signaling. etr-1 is extensively alternatively-spliced, and we identified the viable etr-1(lq61) mutant, caused by a stop codon in alternatively-spliced exon 8 and only affecting etr-1 isoforms containing exon 8. We took advantage of viable etr-1(lq61) to identify potential RNA targets of ETR-1 in body wall muscle using a combination of fluorescence activated cell sorting (FACS) of body wall muscles from wild-type and etr-1(lq61) and subsequent RNA-seq. This analysis revealed genes whose splicing and transcript levels were controlled by ETR-1 exon 8 isoforms, and represented a broad spectrum of genes involved in muscle differentiation, myofilament lattice structure, and physiology. Genes with transcripts underrepresented in etr-1(lq61) included those involved in ribosome function and translation, similar to potential CELF1 targets identified in chick cardiomyocytes. This suggests that at least some targets of ETR-1 might be conserved in vertebrates, and that ETR-1 might generally stimulate translation in muscles. As proof-of-principle, a functional analysis of a subset of ETR-1 targets revealed genes involved in AQR and PQR neuronal migration. One such gene, lev-11/tropomyosin, requires ETR-1 for alternative splicing, and another, unc-52/perlecan, requires ETR-1 for the production of long isoforms containing 3′ exons. In sum, these studies identified gene targets of ETR-1/CELF1 in muscles, which included genes involved in muscle development and physiology, and genes with novel roles in neuronal migration.
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