Reverse Signaling by Glycosylphosphatidylinositol-LinkedManducaEphrin Requires a Src Family Kinase to Restrict Neuronal MigrationIn Vivo

Abstract

Reverse signaling via GPI-linked Ephrins may help control cell proliferation and outgrowth within the nervous system, but the mechanisms underlying this process remain poorly understood. In the embryonic enteric nervous system (ENS) of the moth Manduca sexta, migratory neurons forming the enteric plexus (EP cells) express a single Ephrin ligand (GPI-linked MsEphrin), while adjacent midline cells that are inhibitory to migration express the cognate receptor (MsEph). Knocking down MsEph receptor expression in cultured embryos with antisense morpholino oligonucleotides allowed the EP cells to cross the midline inappropriately, consistent with the model that reverse signaling via MsEphrin mediates a repulsive response in the ENS. Src family kinases have been implicated in reverse signaling by type-A Ephrins in other contexts, and MsEphrin colocalizes with activated forms of endogenous Src in the leading processes of the EP cells. Pharmacological inhibition of Src within the developing ENS induced aberrant midline crossovers, similar to the effect of blocking MsEphrin reverse signaling. Hyperstimulating MsEphrin reverse signaling with MsEph-Fc fusion proteins induced the rapid activation of endogenous Src specifically within the EP cells, as assayed by Western blots of single embryonic gut explants and by whole-mount immunostaining of cultured embryos. In longer cultures, treatment with MsEph-Fc caused a global inhibition of EP cell migration and outgrowth, an effect that was prevented by inhibiting Src activation. These results support the model that MsEphrin reverse signaling induces the Src-dependent retraction of EP cell processes away from the enteric midline, thereby helping to confine the neurons to their appropriate pathways.

Amyloid Precursor Proteins Are Dynamically Trafficked and Processed during Neuronal Development

“…After 1–14 days, neurons were fixed with 4% PFA, permeabilized with PBST for 10 min, and immunolabeled with anti-cAPPL and anti-nAPPL antibodies. Manduca GV1 cells are an ectoderm-derived cell line (Lan et al, 1999; Hiruma and Riddiford, 2004) that endogenously expresses APPL and other neuronal proteins (Lan et al, 1999; Hiruma and Riddiford, 2004; Swanson et al, 2005; Coate et al, 2009). GV1 cells were grown on Lab-Tek tissue culture chamber slides (Nunc #177445) in Grace's complete medium plus 10% FBS to 50% confluence, then fixed and immunolabeled, as previously reported (Coate et al, 2009).…”

“…Manduca GV1 cells are an ectoderm-derived cell line (Lan et al, 1999; Hiruma and Riddiford, 2004) that endogenously expresses APPL and other neuronal proteins (Lan et al, 1999; Hiruma and Riddiford, 2004; Swanson et al, 2005; Coate et al, 2009). GV1 cells were grown on Lab-Tek tissue culture chamber slides (Nunc #177445) in Grace's complete medium plus 10% FBS to 50% confluence, then fixed and immunolabeled, as previously reported (Coate et al, 2009). Replicate cultures were co-immunolabeled with anti-cAPPL plus each of the following antibodies against cytoplasmic compartment markers, based on published evidence and epitope predictions that these antibodies recognize homologous proteins in Drosophila cells: rabbit-anti-Rab4 (Cell Signaling #2167, 1:500; and Abcam #ab87802, 1:200); rabbit-anti- Drosophila Rab5 (Abcam #ab31261, 1:500; Wang et al, 2014); rabbit-anti-Rab7 (Abcam #ab77993, 1:500) and rabbit-anti-dRab7 (1:1000; gift of Dr. Patrick Dolph; 1:1000; Chinchore et al, 2009; Wang et al, 2014); rabbit-anti-Rab9 (Abcam #ab179815, 1:200); rabbit-anti-Rab10 (Abcam #ab113947, 1:500); goat-anti-Rab10 (Santa Cruz #sc-6564, 1:200); rabbit-anti-Rab11a (1:1000; gift of Dr. Don Ready; Satoh et al, 2005); mouse-anti-Rab11 (BD Transduction # 610657, 1:200; Wang et al, 2014); Rabbit-anti- Drosophila LAMP1 (Abcam #ab30687, 1:500); rabbit-anti- Drosophila Lava Lamp (gift of Dr. John Sisson, 1:250; Papoulas et al, 2005); rabbit anti- Drosophila VPS4 (rabbit anti- Drosophila dVPS4, 1:100; gift of Dr. Harald Stenmark; Rodahl et al, 2009); and rabbit-anti-Evi (Evenless Interrupted, 1:400; gift of Dr. Vivian Budnik; Koles et al, 2012).…”

Signaling from Axon Guidance Receptors

“…Recruitment and activation of SFKs have been documented downstream of reverse signaling via GPI-anchored ephrinA ligands (Davy et al 1999). Work in the moth Manduca sexta has shown that repulsion of migratory enteric neurons at the enteric midline is controlled by reverse signaling via the single GPI-anchored ephrin and that this process requires SFK function (Coate et al 2009). The link between GPI-anchored ephrinAs and SFKs may be provided by transmembrane proteins such as p75 NTR and TrkB Marler et al 2008).…”

Otic Mesenchyme Cells Regulate Spiral Ganglion Axon Fasciculation through a Pou3f4/EphA4 Signaling Pathway

“…In particular, Eph receptors, the largest family of vertebrate receptor tyrosine kinases, are known to interact with ephrin ligands to generate both “forward” and “reverse” signals and have been linked extensively with axon guidance (Coate et al, 2009; Huai and Drescher, 2001; Pasquale, 2005; Wilkinson, 2001). While several studies have documented the presence of Ephs and ephrins in the inner ear (Bianchi and Gale, 1998; Zhou et al, 2011), a role for fasciculation and/or radial bundle formation has not been described.…”