Fig. 7

Fig. 7 (A,B) Disruption of Pld1 signaling differentially affects ISV and motor axon dorsal–ventral morphogenesis. (A) Uninjected control TG(flk1:GFP) embryos were stained with znp1 antibody to visualize primary motor neurons (red), which extend from the dorsal to the ventral side along with ISV (green). (B) Anti-acetylated tubulin antibody detects both the primary and secondary motor neuron cell body and axons (red) in the trunk region of uninjected control TG(fli1:GFP) embryos, and ISV (green) is positioned in between. Embryos with MO1-pld1 injection or 1-butanol treatment exhibit impaired ISV morphology compared with control embryos; however, all the motor neurons are able to connect from the ventral side to the dorsal side. Interestingly, the motoneurons are more branched when Pld1 signaling is disrupted (white arrowheads), compared to control embryos. (C–E,H) Transplanted wild-type cells in the notochord partially rescue the ISV defects in pld1 morphants. (C–E) Fluorescent images of TG(fli:GFP) pld1 morphants containing Rhodamine-dextran labeled cells (red) transplanted from wild-type donor embryos, and localized in the prechordal plate (C) or anterior notochord (D,E). Lateral view, anterior to the left. ISVs are visualized in green by TG(fli:GFP). White arrows point to normal ISVs in the regions of the chimeric embryos possessing transplanted wild-type cells, whereas red arrows indicate defective ISVs in segments lacking wild-type cells. (H) In chimeric embryos harboring transplanted cells in the notochord, the percentage of ISV defects in segments with wild-type cells (12%, segments number = 34, 8 embryos) compared to segments without wild-type cells (33%, segments number = 164, 15 embryos). (F,G,I) Transplanted wild-type cells in the somites cannot rescue the ISV defects in pld1 morphants. (F,G) Fluorescent images of TG(fli:GFP) pld1 morphants containing Rhodamine-dextran labeled cells (red) transplanted from wild-type donor embryos, and localized in the somites. Lateral view, anterior to the left. ISVs are visualized in green by TG(fli:GFP). White arrows with red filling point to defective ISVs in the regions of the chimeric embryos possessing transplanted wild-type cells, whereas red arrows with white filling indicate defective ISVs in segments lacking wild-type cells. (I) In the chimeras exhibiting transplanted cells in the somites, the percentage of ISV defects in segments with wild-type cells (51%, segments number = 124, 9 embryos) was comparable to segments without wild-type cells (43%, segments number = 92, 9 embryos). Scale bar represents 20 μm (A) and 30 μm (C–G).