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FGF2 promotes skeletogenic differentiation of cranial neural crest cells

¹ Developmental Biology Unit, Institute of Child Health, University College London, London WC1N 1EH, UK
² Neural Development Unit, Institute of Child Health, University College London, London WC1N 1EH, UK

View larger version (18K): [in a new window]   Fig. 1. The size of neural crest explant cultures increases in response to FGF2 in a dose-dependent manner. In the presence of 0.1 ng/ml (n=10) or 1 ng/ml FGF2 (n=10), neural crest cultures undergo enhanced proliferation compared with cultures grown in the absence of FGF2 (n=12) or in the presence of 10 ng/ml FGF2 (n=10). Explant growth is expressed as the increase in the percentage of the explant diameter with time. Values are mean±s.e.m. (for 5 and 10 days only for clarity of presentation).

View larger version (139K): [in a new window]   Fig. 2. Effect of FGF2 on neural crest cell morphology. Phase contrast photomicrographs illustrate that after 5 days (A-C) neural crest cultures grown in the absence of FGF2 have flattened considerably (A) whereas cells in 1 ng/ml FGF2 retain a more compact cell shape (B) and those in the presence of 10 ng/ml FGF2 have begun to form small nodules resembling cartilage (C, arrowheads). Following 10 days in culture (D-F), cells in control cultures have flattened even further with intercellular spaces appearing (D), whereas in 1 ng/ml FGF2, a concentration that enhances proliferation, there is an increase in cell density (E), while the nodules formed in 10 ng/ml FGF2 have increased markedly in size compared with 5 day cultures (F, arrowheads indicate perimeter of nodule). Scale bars: 100 µm.

View larger version (137K): [in a new window]   Fig. 3. Expression of PCNA in 10 day neural crest cultures. (A,C,E) Fluorescent images showing PCNA staining. (B,D,F) Corresponding phase contrast micrographs (n=4 of each treatment type). (A,B) Most cells in untreated cultures are negative for PCNA with very few cells staining positively (arrow). (C,D) A large proportion of cells in 1 ng/ml FGF2 express PCNA. (E,F) Very few cells within a differentiated nodule in 10 ng/ml FGF2 express PCNA (arrow), while the proliferative non-differentiated cells at the edge of the nodule are intensely immunoreactive (arrowheads). Scale bars: 100 µm.

View larger version (62K): [in a new window]   Fig. 4. Characterisation of cartilage in cellular nodules developing in neural crest cultures. (A,B) Brightfield in situ hybridisation for type II collagen mRNA. (A) Type II collagen mRNA is intensely expressed in a nodule. (B) Individual cells in the areas surrounding the nodule begin to express type II collagen at intermediate levels (arrows), with intensity of expression increasing as differentiation proceeds (arrowheads). (C-F) Confocal images of cultures stained immunohistochemically for type II collagen during progression of chondrogenic differentiation. Type II collagen, green; nuclei, red. (C) Exposure to 10 ng/ml FGF2 leads to clusters of cells beginning to express intracellular type II collagen. (D) These cells then aggregate to form nodules of cartilage, and (E) eventually type II collagen is secreted into the extracellular compartment (arrowheads). (F) Single cells expressing type II collagen appear in areas surrounding nodules. (G,H) Histological staining for cartilage. (G) Cartilage nodules stain positively with Alcian Blue and (H) sections through such nodules display metachromasia when stained with Toluidine Blue, indicating a cartilaginous matrix. (I,J) Examination of nodules by transmission electron microscopy, shows cellular ultrastructure typical of cartilage. (I) A typical chondroblast within the nodule has swollen ER (red asterisks), a scalloped plasma membrane profile and is surrounded by large areas of extracellular matrix. (J) A representative mature chondrocyte is enclosed within its own lacuna (arrowheads) and embedded in the extracellular matrix. Scale bars: 50 µm in A,B,G,H; 10 µm in C-F; 5 µm in I,J.

View larger version (20K): [in a new window]   Fig. 5. Critical exposure times to 10 ng/ml FGF2 for chondrogenic differentiation of neural crest cells. Although continuous exposure to FGF2 is not required, the initial 24 hour exposure period is critical in promoting skeletogenic differentiation. The number of cultures containing cartilage nodules after 10 days in vitro is expressed as percentage of the total number of cultures analysed under each experimental condition (indicated above each bar).

View larger version (18K): [in a new window]   Fig. 6. Influence of different factors on the level of chondrogenic differentiation achieved by neural crest cells when cultured for 10 days in the presence of 10 ng/ml FGF2. (A) Explants of varying sizes display no significant differences in the number of cultures undergoing chondrogenesis (t-test: P<0.4). (B) Neural crest obtained from embryos which were of approximately 6 hours difference developmentally, exhibit no significant difference in incidence of chondrogenesis (t-test: P<0.4). (C) The cranial axial region from which the neural crest originates determines skeletogenic response to FGF2 signalling: explants from the fore- and hindbrain region show a markedly reduced capability of differentiation when compared with those from the midbrain (t-test: P<0.001 for both comparisons).

View larger version (74K): [in a new window]   Fig. 7. Evidence for osteogenic and chondrogenic differentiation in neural crest cultures grown for 28 days. (A) Culture stained with both Alcian Blue (c) and von Kossa (asterisk), indicating the existence of cartilage matrix and calcium phosphate mineralisation respectively. (B) Transverse section through a von Kossa-stained nodule containing hypertrophic chondrocytes (arrows) and areas of new mineralisation (arrowheads). (C,D) Confocal images of cultures stained immunohistochemically for osteonectin. Osteonectin is expressed (C) in the extracellular matrix and (D) intracellularly. (E) Transmission electron microscopy showing ultrastructure of a chondrocyte undergoing hypertrophy, a pale cell with swollen endoplasmic reticulum cisternae (arrowheads). (F) Osteoid matrix formation in association with mineralised areas of nodule. Mineralised matrix stains with von Kossa, and is bounded by regions of hypertrophying cartilage (h) and osteoid matrix (arrowheads). (G,H) Bone formation in 28 day cultures. (G) A sheet of alizarin red positive cells (b) is lying away from Alcian Blue-positive cartilage nodules (c) suggesting endochondral bone formation. (H) An Alizarin Red-positive area (arrowheads) within an Alcian Blue-stained region of cartilage showing endochondral bone formation. Scale bars: 10 µm in A,C,D; 50 µm in B,F; 5 µm in E; 1 mm in G; 0.1 mm in H.

View larger version (65K): [in a new window]   Fig. 8. Immunohistochemical detection of FGFRs in vivo and in cultured neural crest cells. In transverse sections of mesencephalic region of stage 9 quail embryos (n=3 for each category) FGFR1 (A) FGFR2 (B) and FGFR3 (C) are expressed ubiquitously but staining is particularly intense in the apices of the neural folds (arrows in A). (D) Control section stained with non-immune serum shows no staining. Scale bars, 100 µm. (E-H) Neural crest cells in culture (n=4 for each category). FGFR1 (E), FGFR2 (F) and FGFR3 (G) are all expressed by neural crest cells in culture. (H) Control culture stained with non-immune serum is negative. Scale bar: 50 µm. ect, ectoderm; end, endoderm; m, mesoderm; n, notochord.