Also is ablated by pre-treatment with noggin [17]. Here, the technical aspects of the chick embryo model are presented in detail including step-by-step instructions and pitfalls. The capabilities are exemplified by a brief summary of our original experiments supplemented by new data on transplantation of nonThe Chick Embryo in Melanoma ResearchEpigenetics 
Figure 2. Transplantation of melanoma cells into three distinct niches of the chick embryo. (A) Chick embryo stage 12?3 HH before and (B) directly after transplantation of B16-F1 melanoma cells into the neural tube. The entering site of the micro-pipette is marked (asterisk in A). Note the dilated neural tube (frame in B) due to the transplanted cells when compared to (A). (C) B16-F1 cells can be detected via GFP epifluorescence in the lumen of the neural tube directly after transplantation. (D) 48 h after transplantation ventrally emigrating B16-F1 cells are clearly discernible (arrows) in lateral view; the borders of the neural tube are outlined in green. (E) Chick embryo stage 19 HH directly after transplantation of B16-F1 melanoma cell aggregates into the optic cup. (F) Aggregates were stained with nile blue sulphate before transplantation for better visibility. Higher magnification shows the aggregates behind the embryonic lens (arrow). A temporary capillary bleeding can be discerned at the injection spot at the choroid fissure in (F). (G) Macroscopically no tumor growth is visible 72 h after transplantation. (H) The former entering site of the micro-pipette (choroid fissure) is marked (arrow). (I) Chick embryo stage 12?3 HH before and (J) directly after transplantation of human melanoma cells into the ventricle of the hindbrain (rhombencephalon, frame in I). The entering site of the micro-pipette is marked with the asterisk in (J). 11967625 Note the melanoma cell-filled brain ventricle (frame in J). (K) 48 h after transplantation a growing tumor is already visible in the hindbrain (frame). (L) After 96 h a single Epigenetics condensed tumor is visible in the dorsal midline of the neural epithelium (arrow). Scale bars in A, B and E : 1 mm; scale bars in C and D: 0.5 mm. doi:10.1371/journal.pone.0053970.gtransformed primary human melanocytes into the neural crest and into the optic cup, and on malignant invasive growth of melanoma and breast cancer cells in the hindbrain as novel model for invasive brain metastasis.Materials and Methods Ethics StatementAccording to German animal care guidelines, no IACUC approval was necessary to perform the embryo experiments. According to the local guidelines, only experiments with chick embryos E18 and older need IACUC approval. However, the embryos used in this study were all in early stages of embryonic development (between E2 and E7).Preparation of Eggs and Transplantation of CellsFertilized eggs of leghorn chickens (Gallus gallus domesticus) were obtained from a hatchery (Weiss, Kilchberg, Germany) and incubated at 38uC in a temperature-controlled brooder (BRUJA Type 400a, Brutmaschinen Janeschitz, Hammelburg, Germany) (Figure 1A) without rolling. The uppermost spot of the eggshell (and thus indirectly the blastoderm, which is always oriented towards the top part of the egg) was marked on each egg with a permanent marker (Figure 1B).For transplantation of melanoma cells into the neural tube, eggs were prepared after 48 h of incubation (stage 12?3 according to Hamburger and Hamilton (HH) [18]). The equipment necessary for fenestration is shown in Figure 1C. First, the eggs w.Also is ablated by pre-treatment with noggin [17]. Here, the technical aspects of the chick embryo model are presented in detail including step-by-step instructions and pitfalls. The capabilities are exemplified by a brief summary of our original experiments supplemented by new data on transplantation of nonThe Chick Embryo in Melanoma ResearchFigure 2. Transplantation of melanoma cells into three distinct niches of the chick embryo. (A) Chick embryo stage 12?3 HH before and (B) directly after transplantation of B16-F1 melanoma cells into the neural tube. The entering site of the micro-pipette is marked (asterisk in A). Note the dilated neural tube (frame in B) due to the transplanted cells when compared to (A). (C) B16-F1 cells can be detected via GFP epifluorescence in the lumen of the neural tube directly after transplantation. (D) 48 h after transplantation ventrally emigrating B16-F1 cells are clearly discernible (arrows) in lateral view; the borders of the neural tube are outlined in green. (E) Chick embryo stage 19 HH directly after transplantation of B16-F1 melanoma cell aggregates into the optic cup. (F) Aggregates were stained with nile blue sulphate before transplantation for better visibility. Higher magnification shows the aggregates behind the embryonic lens (arrow). A temporary capillary bleeding can be discerned at the injection spot at the choroid fissure in (F). (G) Macroscopically no tumor growth is visible 72 h after transplantation. (H) The former entering site of the micro-pipette (choroid fissure) is marked (arrow). (I) Chick embryo stage 12?3 HH before and (J) directly after transplantation of human melanoma cells into the ventricle of the hindbrain (rhombencephalon, frame in I). The entering site of the micro-pipette is marked with the asterisk in (J). 11967625 Note the melanoma cell-filled brain ventricle (frame in J). (K) 48 h after transplantation a growing tumor is already visible in the hindbrain (frame). (L) After 96 h a single condensed tumor is visible in the dorsal midline of the neural epithelium (arrow). Scale bars in A, B and E : 1 mm; scale bars in C and D: 0.5 mm. doi:10.1371/journal.pone.0053970.gtransformed primary human melanocytes into the neural crest and into the optic cup, and on malignant invasive growth of melanoma and breast cancer cells in the hindbrain as novel model for invasive brain metastasis.Materials and Methods Ethics StatementAccording to German animal care guidelines, no IACUC approval was necessary to perform the embryo experiments. According to the local guidelines, only experiments with chick embryos E18 and older need IACUC approval. However, the embryos used in this study were all in early stages of embryonic development (between E2 and E7).Preparation of Eggs and Transplantation of CellsFertilized eggs of leghorn chickens (Gallus gallus domesticus) were obtained from a hatchery (Weiss, Kilchberg, Germany) and incubated at 38uC in a temperature-controlled brooder (BRUJA Type 400a, Brutmaschinen Janeschitz, Hammelburg, Germany) (Figure 1A) without rolling. The uppermost spot of the eggshell (and thus indirectly the blastoderm, which is always oriented towards the top part of the egg) was marked on each egg with a permanent marker (Figure 1B).For transplantation of melanoma cells into the neural tube, eggs were 
prepared after 48 h of incubation (stage 12?3 according to Hamburger and Hamilton (HH) [18]). The equipment necessary for fenestration is shown in Figure 1C. First, the eggs w.
