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First published online 10 October 2006
doi: 10.1242/jcs.03205

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BMP-2-dependent integration of adult mouse subventricular stem cells into the neural crest of chick and quail embryos

Anatomisches Institut, Oesterbergstr. 3, D-72074 Tübingen, Germany

View larger version (115K): [in a new window]   Fig. 1. (A) SVZ neurospheres stained with Nile Blue sulphate immediately before injection. (B) Paraffin section of BMP-2 pre-treated neurosphere: nestin expression was observed in 70% of the cells evaluated by microscopical counting of ten sections of neurospheres using image processing software (Analysis, SIS, Germany). (C) Untreated neurosphere: nestin expression was observed in 70% of the cells. (D) Neurosphere after hybridization with the mL1 probe. Strong HNK-1 reactivity was observed in both BMP-2-treated (E) and untreated (F) neurosphere cells.

View larger version (107K): [in a new window]   Fig. 2. (A) Neurosphere transplant (BMP-2 pre-treated) in the chick embryo neural tube 12 hours post transplantation (p.t.) (stage 13, HH) with visible fluorescence signal of the neurosphere. Transplant is located between the 18th pair of somites. (B) Enlargement of the same embryo as in A in paraffin section stained with HNK-1 antibody (brown reaction). SVZ neurosphere cells appear HNK-1 positive, whereas autochthonous chick neural crest cells are HNK-1 negative at this stage in the lower trunk region. (C) Same embryo as in A and B in parallel section. Anti-GFP staining shows integration of SVZ cells into the dorsal neuroepithelium and the roof plate, but transplanted mouse cells and host cells do not intermingle yet. (There is some nonspecific staining in somites.) (D) BMP-2 pre-treated neurosphere 24 hours p.t. in the chick embryo neural tube (stage 19, HH, total incubation time 72 hours). The transplant is located at the region of the upper appendage. GFP-epifluorescence shows bilateral emigration of single cells of the transplant laterally above the projected border of the neural tube. (E) The same embryo as in D. The tangentially cut cryostat section (see insert at lower left for orientation) stained with GFP antibody shows intermingling of mouse and chick cells and single GFP-positive mouse cells in the region of the roof plate (upper right insert). (F) Paraffin section of a different embryo of the same experiment. Emigrated SVZ cells visualized by in situ hybridization with the mL1 probe. Reactive cells can be seen in the dorsal neural tube and at the ventral root.

View larger version (33K): [in a new window]   Fig. 3. Untreated neurosphere in the chick embryo neural tube 24 hours after transplantation (stage 19, HH). (A) GFP epifluorescence is restricted to the lumen of the neural tube. (B) The same embryo as in A, in transversally cut paraffin section. Neurosphere cells remaining in the lumen of the chick embryo neural tube can be seen with no or little attachment to the neural tissue (arrow). Part of the neurosphere fell out of the neural tube lumen during the histological processing (insert).

View larger version (106K): [in a new window]   Fig. 4. BMP-2 pre-treated neurosphere 24 hours after transplantation into the quail embryo neural tube (equal to stage 19, HH, total incubation time 72 hours). (A) Fluorescence image. (B) Enlargement of the boxed region in A. Emigrating SVZ neurosphere cells are visible by their typically stretched cellular morphology. Bilaterally emigrating cells are contained within the neuroepithelium (black borders of the neural tube). (C) In situ hybridization of the same embryo. Emigrated mouse SVZ cells can be seen in the roof plate of the neural tube when visualized using the mL1 probe. (D) Enlargement of C shows that only cell nuclei are stained. (E) Parallel section of embryo shown in C. Quail nuclei stained with the QCPN antibody, which recognises a species-specific antigen carried by all quail cells (brown colour), but not by mouse cells. A region of dorsal neural tube remains unstained. Immunohistochemistry for anti-GFP (F) shows that transplanted cells in the dorsal neural tube are GFP positive (mouse cells). Comparison with parallel sections in C, D, and E reveals that there is no cross-reactivity between mL1, QCPN and anti-GFP.

View larger version (56K): [in a new window]   Fig. 5. (A) Stage 18 chick embryo (3 days total incubation time) stained with HNK-1 antibody as whole mount. Emigrating neural crest cells show a typical pattern for this specific stage. (B) An enlargement of the boxed region in A showing the area of the upper limb bud (site of transplantation of neurospheres in other embryos). An emigrating population of neural crest cells can be observed outside the dorsal neural tube. These cells do not follow a segmental migration pattern. (C) Corresponding patterns of bilaterally emigrating neurosphere cells after BMP-2 pre-treatment can be seen in the same trunk location of a transplanted embryo.