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Fig. 4. FoxD1 knockdown by the FoxD1-MO eliminates both neural and dorsal mesoderm cell fates. (A) One-cell stage embryos (left panel) were injected animally with the FoxD1-MO (20 ng, right panel). Using in situ hybridization, cell fates along the neural AP axis were examined at neurula stage 17. Three AP markers were examined; otx2 for the mid-forebrain region, krox20 for the hindbrain, and hoxb9 for the spinal cord. In FoxD1
is restricted to the forebrain region. This region is characterized by a strong inhibition of canonical Wnt and BMP signaling (Niehrs et al., 2001). Thus under normal physiological conditions, FoxD1 is primed to induce anterior neural tissue. In AC explants permissive for BMP and canonical Wnt activities, intrinsic FoxD1 neural caudalizing is unmasked, which is not in the correct cellular context of normal FoxD1 protein function and activity in vivo. These results morphants, otx2 expression was reduced in 100% of the embryos (n=19); krox20 expression was reduced in either one or both rhombomeres in 69% of the embryos (n=13), and hoxb9 expression was inhibited in 77% of the embryos (n=13). (B) One-cell stage embryos were injected marginally with the FoxD1-MO (25 ng). Total RNA was isolated from pools of five control (lane 2) or FoxD1 morphant embryos (lane 3) at late neurula stages. sqRT-PCR was carried out to mesoderm markers expressed along the dorso-ventral axis: xnot (dorsal notochord), myod and muscle actin/MA (dorsal-lateral muscle) and vent1, vent2 and wnt8 (ventral). odc serves as a control for quantitating RNA levels. (C) One-cell stage embryos were injected marginally with two concentrations of the FoxD1-MO (25 and 40 ng). In situ hybridization was performed to early gastrula embryos, stage 11-11.5. Expression of the dorsal chd marker (upper panels) was reduced at both FoxD1-MO concentrations in 78% of the embryos (n=23). Expression of the dorsal-lateral myod marker was also reduced in 93% of the FoxD1 morphant embryos (n=15). (D) One-cell stage embryos were injected marginally with the two concentrations of the FoxD1-MO (25 and 40 ng). In situ hybridization was performed to neurula embryos, stage 14-15. Expression of the notochord chd marker (upper panels) was reduced at both FoxD1-MO concentrations in 100% of the embryos (n=25). Expression of the muscle MA marker was also reduced in 68% of the morphant embryos (n=19). Two other notochord and muscle markers, xnot and myod were also inhibited to similar levels in the same experiment (not shown). (E) One-cell stage embryos were injected marginally with the mRNA (800 pg) encoding the foxd1-vp16 antimorph protein. Total RNA was isolated from pools of five control (lane 2) or FoxD1-VP16 antimorph protein expressing (lane 3) embryos at late neurula stage 16. sqRT-PCR was carried out to the mesodermal markers: xnot, chd, and MA. (F) One-cell stage embryos (lane 2) were injected either marginally (left panel) or animally (right panel) with the FoxD1-MO (20 ng, lane 3), mRNA encoding the foxd1 (25pg) protein (lane 5) or both (lane 4). Total RNA was isolated from pools of seven embryos in each group. In the left panel, sqRT-PCR was performed on RNA isolated from gastrula stage 11 embryos to address the rescue of myod expression by ectopic FoxD1 protein over expression (compare lanes 3-5). Xhis serves as a control for quantitating RNA levels. In the right panel, sqRT-PCR was performed on RNA isolated from neurula stage 16 embryos to address the rescue of ncam expression by FoxD1 (compare lanes 3-5). Image published in: Polevoy H et al. (2017) Copyright © 2017. Reproduced with permission of the Publisher, University of the Basque Country Press.
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