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First published online August 18, 2003
doi: 10.1242/10.1242/dev.00673

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Dissimilar regulation of cell differentiation in mesencephalic (cranial) and sacral (trunk) neural crest cells in vitro

¹ Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
² Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA

View larger version (28K): [in a new window]   Fig. 1. Explants of dorsal neural tube containing premigratory neural crest cells were used to generate primary cultures of cranial and trunk neural crest cells. After 24 hours of incubation, explants were removed and the media were replaced. Five different cell types were detected using morphology and specific antibodies: chondrocytes, glial cells, smooth muscle cells, pigment cells and neurons.

View larger version (66K): [in a new window]   Fig. 2. Survival of cranial and trunk neural crest cell cultures is differentially affected by secreted factors. Bright-field photograph of cranial neural crest culture treated with recombinant FGF2 protein for 36 hours reveals migrating cells (A). The same culture stained with DAPI to reveal nuclei (B) and with anti-p75 nerve growth factor (NGF) receptor antibody that recognizes neural crest cells (C). A histogram of survival rates for cranial and trunk neural crest cells cultures shows that the cranial and trunk neural crest cell cultures had different survival rates under similar culture conditions (t-test: P<0.001, except for TGFß1 for which P<0.03) (D). % survival=(number of cultures after 7 days in culture/number of explants displaying cell outgrowth on 1st day)x100. (E-G) Apoptosis is not detected in 48 hour cranial neural crest cultures treated with FGF2 (E), whereas many cranial neural crest cells treated with BMP2 for 18 hours (F) and 36 hours (G) undergo cell death as detected with TUNEL and HRP-conjugated antibody.

View larger version (87K): [in a new window]   Fig. 3. Immunohistochemical analysis of the cranial and trunk neural crest cells cultured in the presence of FGF2 and in combination with BMP2 and TGFß1. The bottom row shows cranial crest cultures infected with the virus containing the constitutively active (stabilized) ß-catenin. Panels show some of the most representative cultures for each culture condition. Specialized cell types are arranged in columns, whereas rows depict different growth conditions. Note that chondrogenesis is induced in the presence of exogenous FGF2 but is suppressed when BMP2 or TGFß1 are also added. Scale bars: 100 µm.

View larger version (37K): [in a new window]   Fig. 5. The effect of FGF2 and other cytokines on cell differentiation of the cranial and trunk neural crest cells in vitro. This histogram of the results described in Fig. 3 compares effects of the cytokines on generation of pigment, glial, smooth muscle, neuronal cells and chondrocytes. As most of the cultures were stained with only two or three different cell-specific antibodies, the histogram represents percentage of independent cultures displaying a particular differentiated cell type from the total number of cultures stained for that marker. Chondrogenesis is inhibited by both BMP2 and TGFß1 proteins even in the presence of FGF2/8. The total number of cultures tested for each of the cell types is indicated above the representative bars. Note that all cultures were scored for melanogenesis.

View larger version (93K): [in a new window]   Fig. 4. Mis-expression of a stabilized version of ß-catenin using RCAS virus in cranial neural crest cells. The 'exclusion' function of Adobe PhotoShop was used to demonstrate the overlap (red) between pigments cells (black) and smooth muscle cells (blue) in non-infected crest cell cultures (A) or cultures infected (B) with RCAS::ß-catenin virus, both treated with FGF2. Note that the relative ratios of smooth muscle cells and pigmented cells are dramatically different.

View larger version (17K): [in a new window]   Fig. 6. Individual variation of cell differentiation in the cranial neural crest cultures treated with FGF2, FGF2+BMP2 and FGF2+TGFß1 based on double immunostaining with antibodies against COL II and SMA. Each color bar represents an average fraction of a particular cell type in ten individual primary cultures. Error bars represent average deviation for the sample for each of the cell types. Color bars indicate different types of cells: red, chondrocytes; orange, smooth muscle cells; green, pigment cells.

View larger version (69K): [in a new window]   Fig. 7. Immunohistochemical analysis of the cranial neural crest cultures infected with the RCAS::Hoxa2 and RCAS::Hoxd10 viruses and comparison with trunk neural crest culture in medium containing the exogenous purified FGF2 or BMP2 protein. All antibodies used are the same as shown in Fig. 3. In RCAS::Hoxa2-infected cells, the clusters of melanocytes, glial and smooth muscle cells form similarly to the FGF2 cultures; however, the ColII-positive cells form significantly smaller clusters and in fewer cultures. No chondrocytes are detected in Hoxd10-infected cultures, a condition that mimics the trunk neural crest cultures. Scale bars: 100 µm.

View larger version (32K): [in a new window]   Fig. 8. The effect of FGF2, other cytokines and Hox genes on cell differentiation of the cranial neural crest in vitro. A histogram summarizing the results of the experiments from Fig. 4 showing how Hox genes control cell differentiation of the cranial neural crest cells. For comparison, we included data on differentiation of anterior (somite level 10-12) and posterior (somite level 32-33) trunk neural crest cells. Both Hoxa2 and Hoxd10 can suppress chondrogenesis in cranial crest cells although to a different extent. Hoxa2 limits the rate of chondrogenesis by about 30%, whereas Hoxd10 completely suppresses it. Other cell types are also affected differently: Hoxa2 does not alter the overall rate production of smooth muscle or glial cells but Hoxd10 strongly limits myogenesis and gliagenesis relative to uninfected condition. The total number of cultures tested for each of the cell types is indicated above the representative bars. Note that all cultures were scored for melanogenesis.

View larger version (26K): [in a new window]   Fig. 9. Comparison of survival in cultures infected with RCAS::Hoxa2 and RCAS::Hoxd10 and treated with FGF2 or BMP2. Survival of Hoxa2- expressing cells is increased relative to uninfected cranial neural crest cells whereas survival of Hoxd10- expressing cells increased almost fivefold and became markedly more similar to that of trunk neural crest (t-test: P<0.001 for all pairwise comparisons with a control). All are significantly different except Hoxa2+FGF2.

View larger version (80K): [in a new window]   Fig. 10. Analysis of the trunk neural crest cells in long-term culture. RT-PCR amplification of cranial crest-specific markers from cranial and trunk neural crest cultures. Id2 and Noelin are highly expressed in both cranial explants and 14-day-old cranial crest but not in the new trunk crest cultures (A). A histogram showing the number of cultures positive for Alcian Blue staining, i.e. undergoing chondrogenesis (B). Note that on the second week of incubation (D14), about 80% trunk neural crest cultures undergo chondrogenesis and RCAS::Hoxd10 infection can suppress chondrogenesis in these cultures. (C-J) In situ hybridization on trunk neural crest cultures incubated for 7 or 14 days. Hoxb4 (C) but not ColII (D) signal is detectable in most cells of the week-old trunk neural crest cultures. ColII expression (E,F) is detectable in chondrocytes present throughout the 2-week-old cultures. Double in situ hybridization (G-J) revealed that very none or few Hoxb4-expressing cells are also positive for ColII transcript. (K-W) Immunochemistry with antibodies revealing chondrocytes (COLII), smooth muscle cells (SMA), neuronal cells (NF200) and 3C2 antibody (RCAS-infected cells) demonstrates the effect of the ectopic trunk Hox expression on chondrogenesis in long-term trunk crest cultures. (K,O,T) Trunk neural crest culture grown for 7 days, analyzed for presence of chondrocytes (K), smooth muscle cells (O) and neurons (T). Note that no COL2-positive cells are detected in these cultures. (L,P,U) Similar cultures grown for 2 weeks have many chondrocytes that appear throughout the culture, whereas smooth muscle cells and neurons are still present. (M,R,V) Trunk neural crest cultures infected with RCAS::Hoxd10 virus and grown for 2 weeks display a much lower level of chondrogenesis with no effect on myogenesis and neurogenesis. (N,S,W) To ensure that the cultures were indeed expressing the RCAS construct, 3C2 antibody was used to reveal the extent of the retroviral infection.

View larger version (30K): [in a new window]   Fig. 11. Distinct modes of regulatory mechanisms of differentiation of the cranial and trunk neural crest cells. BMP2/4, GGF, TGFß1 and WNT1 act to induce neurogenesis, gliagenesis, myogenesis and melanogenesis, respectively, from the multipotent trunk neural crest stem cells (A) (Shah et al., 1996; Anderson, 1997; Sieber-Blum and Zhang, 1997; Zhang et al., 1997; Francis-West et al., 1998; Dunn et al., 2000). Inductive and repressive roles of the FGF2/8, BMP2/4, SHH, TGFß1 and WNT pathways on the cranial neural crest differentiation (B). The positive regulators are shown in red and the negative ones are in blue. FGF2/8 appears to be generally required for normal proliferation and survival of the cranial but not trunk neural crest. FGF2/8 also seem to be a positive regulator/survival factor for the melanogenic cells in both cranial and trunk crest cultures. Some of the markers used to identify the different cell types are shown in brackets. Some cell types, particularly smooth muscle cells and pigment cells, differentiate in the surviving cultures grown with media containing no purified exogenous proteins. It is not clear whether these cells rely on the residual factors present in the serum, such as a clearly detectable BMP-like activity, or represent a default state of the cranial neural crest differentiation.