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Fig. 1
DSCAM expression in the developing Xenopus visual system and morpholino oligonucleotide-mediated knockdown. Immunostaining reveals patterns of DSCAM expression in the retina and tectum of developing Xenopus tadpoles. (a, b) DSCAM immunoreactivity (green) localizes to the midbrain (a) and retina (b) of stage 40 tadpoles. In the midbrain optic tectum DSCAM immunoreactivity is localized to postmitotic cell bodies (white arrow and insert in a) and neuropil (np). In the developing retina (b), DSCAM immunoreactivity localizes to the inner nuclear layer (inl), ganglion cell layer (gcl) and optic nerve head (ONH). c Western blot analysis of whole brain lysates confirms DSCAM expression in stage 38, 41, and 47 tadpoles. Whole-embryo lysates at stage 30 show a 40% decrease in DSCAM expression after microinjection of DSCAM MO at the 2-cell stage. d Microinjection of lissamine-tagged DSCAM or Control MO into a light-shaded blastomere of 4-cell or 8-cell stage embryos localized the MO to cells in the eye and brain of developing tadpoles unilaterally. e, f Lissamine-tagged Control MO (red) did not alter DSCAM expression (green) in stage 38 tectum (e) or stage 45 retina (f; see magnified insert) by injection at the 8-cell stage. g-i Decreased DSCAM expression (green) is observed in the tectal hemisphere of stage 45 tadpole (g, h; yellow arrowheads) and portion of retina of stage 40 tadpole (i; see magnified insert; yellow arrowheads) with DSCAM MO lissamine tag (red). j DSCAM immunostaining of stage 45 tadpole brain lipofected with plasmids coding for Xenopus Dscam and tdTomato. Note the increased levels of DSCAM immunoreactivity in tdTomato-labeled neuron (yellow arrow). The white arrowheads denote endogenous DSCAM expression. np, neuropil; v, ventricle; MO, morpholino; inl, inner nuclear layer; gcl, ganglion cell layer; onl, outer nuclear layer; ONH, optic nerve head, cm, ciliary margin. Scale bars: 100 μm in (a); 50 μm in (f, g, i); 30 μm in (j) |
