FIGURE SUMMARY
Title

Regulation of neural crest cell fate by the retinoic acid and Pparg signalling pathways

Authors
Li, N., Kelsh, R.N., Croucher, P., and Roehl, H.H.
Source
Full text @ Development

The Pparg inhibitor GW9662 blocks adipocyte formation while enhancing osteoblast differentiation. (A,B) Ventral views of Oil Red staining showing that lipid droplet accumulation throughout the zebrafish embryo head and cardiac region is strongly diminished by GW9662 treatment for 72 hours. Droplet accumulation (arrowheads) is highest at either end of the ceratohyal cartilage element (asterisk) and around the heart. (C-F) Alizarin Red staining shows that ossification is enhanced after GW9662 treatment for 72 hours. Dermal bones (bs, branchiostegal ray 3; ps, parasphenoid; op, opercle) and a cartilage bone (hm, hyomandibula) show increased levels of ossification. Side views (C,D) and ventral views (E,F) are shown. The eyes have been removed for clarity. Quantification for this experiment is shown in Table S1 in the supplementary material. (G-J) runx2b expression is strongly upregulated after 24 hours of GW9662 treatment. The opercle, branchiostegal ray 3 and the parasphenoid primordia all show more intense staining (H,J), consistent with the increase in ossification seen by 120 hpf (D,F). Scale bars: 100 μm in F for A-F and in J for G-J.

RA signalling is required for adipocyte differentiation. (A,B) Ventral views showing that lipid droplet accumulation throughout the head and cardiac region is lost in nls-/- fish at 120 hpf. Arrowheads point to droplets present in cells near to the distal end of the ceratohyal cartilage that are present in wild-type (A) and absent in nls-/- (B) fish. (C,D) Ventral views showing that expression of the adipocyte differentiation marker cepba is strongly reduced in nls-/- fish at 60 hpf. Arrowheads point to a cluster of cepba-positive cells at the distal end of the ceratohyal cartilage (C) that is lost in nls-/- embryos (D). Arrowheads point to droplets present in cells near to the distal end of the ceratohyal cartilage that are present in wild-type (C) and absent in nls-/- (D) fish. (E,F) Ventral views showing that lipid droplet accumulation throughout the head and cardiac region is reduced after treatment with DEAB. Arrowheads point to droplets present in cells near to the distal end of the ceratohyal cartilage element (E) that are reduced in DEAB-treated fish (F). (G,H) Dorsal views showing that cebpa expression throughout the head and liver is reduced after treatment with DEAB. Brackets indicate the area of diffuse expression in the pharyngeal region (G) that is reduced in treated fish (H). The liver also shows a strong reduction in cebpa expression levels after DEAB treatment (asterisk). (I-L) Retinoic acid (RA) treatment upregulates expression of cebpa after 12 hours. Dorsal flat-mounted fish showing that cebpa expression is particularly enhanced in two clusters of cells on either side of the heart (arrowheads in I and J) and liver (asterisk). Ventral views show enhanced expression in the pharyngeal arches, especially at the distal ends of the ceratohyal cartilage (arrowheads in K and L). Scale bars: 100 μm.

RA signalling blocks early osteoblast differentiation. (A-D) Alizarin Red staining at 144 hpf shows that normal levels of ossification (A) are reduced by RA pulse treatment from 48-54 hpf (B). Alizarin Red staining at 128 hpf shows that pulse treatment with DEAB at 10 μM (D) enhances ossification when compared with control fish (C). Arrowheads point to the maxilla (mx), dentary (de) and entopterygoid (en) (which are all dermal bones). The eyes have been removed for clarity. Quantification for these experiments is shown in Tables S2 and S3 in the supplementary material. (E-P) Expression of twist2, tcf7, runx2b and osx is reduced by RA treatment (F,I,L,O) and enhanced by DEAB treatment (G,J,M,P) as compared with the control (E,H,K,N). Each panel shows side and dorsal views at low magnification (left) and a high-magnification side view of the opercle condensation (right). In the control, twist2 (E) is expressed strongly in the opercle (op), entopterygoid (not shown) and parasphenoid (arrowhead), as well as broadly in the distal region of the second arch (arrow). tcf7 (H) is expressed in the opercle and teeth (te), as well as in the brain (arrow). runx2b (K) is expressed in the opercle and parasphenoid, as well as in other skeletal elements. osx (N) is expressed in the opercle, parasphenoid, cleithrum (cl) and teeth (not shown). Scale bars: 50 μm.

Cephalic neural crest gives rise to adipocytes and osteoblasts. (A-E) cebpa and sox10:gfp are co-expressed in the same cells at 54 hpf. (A) Ventral view of a zebrafish embryo with cebpa detection by chromogenic substrate. The boxed area indicates the region shown in B-E. cebpa (B) and sox10:gfp (C) are co-expressed in the same cells (overlay in E). The nuclear dye DAPI is used as a counterstain to show that not all cells express each marker and that the optical sections are less that one cell diameter (D). (F-J) osx and sox10:gfp are co-expressed in the same cells at 54 hpf. (F) Side view of a fish with osx detection by chromogenic substrate. The boxed area indicates the region shown in G-J. osx (G) and sox10:gfp (H) are co-expressed in the same cells (overlay in J). (I) Staining with the nuclear dye DAPI. Scale bars: 10 μm.

GW9662 enhances runx2b expression within 10 hours of treatment. (A-D) The opercle (op), cleithrum (cl) and the parasphenoid (ps) all show more intense staining (C,D versus A,B), consistent with the increase in ossification seen by 120 hpf. Scale bar: 100 μm.

RA signalling affects gene expression within 2 hours of treatment. (A-I) Whereas expression of tcf7 (A-C) and runx2b (D-F) is reduced after 2 hours of RA treatment, osx shows little change (G-I). osx is not expressed in the opercle at this time point. We do not see precocious expression of osx in the opercle after DEAB treatment, nor do we see affects on the cleithrum and parasphenoid expression after DEAB or RA treatments. (J-L) After 7.5 hours of treatment, osx shows a strong response to treatment. Panels are arranged and annotated as in Fig. 2.

RA signalling simultaneously increases bone matrix synthesis while inhibiting runx2b expression. (A,B) Alizarin Red staining at 144 hpf shows that a late pulse treatment with RA results in increased ossification, particularly in the dentary (de), opercle (op) and notochord (nc). Low-magnification ventral views are shown to the left and high-magnification images of the opercle are shown to the right. (C-H) col1a1 is expressed around the developing bony skeleton including the cleithrum (cl) and opercle in untreated fish (C,F). This expression is increased after 2 hours (D) and 4 hours (G) of RA treatment and decreased by similar treatment with DEAB (E,H). Low-magnification side views are shown to the left and high-magnification images of the opercle are shown to the right. (I-K) runx2b is expressed around the developing bony skeleton, including the ceratohyal (ch), fifth ceratobranchial and pharyngeal teeth (cb5) and opercle in untreated fish (I). This expression is diminished by RA treatment (J), and is increased by DEAB treatment (K). Each panel shows low-magnification side and ventral views on the left and high-magnification images of the ceratohyal, fifth ceratobranchial and pharyngeal teeth and opercle on the right. Scale bars: 25 μm.

RA and DEAB treatments commencing at 48 hpf do not have a strong affect on the differentiation of other cephalic neural crest derivatives or of muscle. (A-C) Ventral views of sox9a (SRY box containing gene 9a) expression showing that treatments from 48-60 hpf do not affect chondrocyte specification. sox9a marks chondrocytes and is a key regulator of chondrogenesis. (D-F) Dorsal views of myoD (myogenic differentiation 1) expression showing that treatments from 48-60 hpf do not affect myocyte specification. myod marks muscle precursors and is a key regulator of myogenesis. (G-I) Ventral views of Alcian Blue-stained fish showing that treatments do not affect the cartilaginous skeleton. (J-L) Ventral views of antibody staining (mf20, Developmental Studies Hybridoma Bank) for muscle showing that treatments do not affect cephalic muscle. (M-O) Side views of antibody staining (anti-HU, Molecular Probes) for neurons showing that treatments do not affect cranial ganglia (tg, trigeminal ganglion). (P-R) Side views of mbp (myelin basic protein) expression showing that glial differentiation is not affected by treatments. (S-U) Dorsal views of live zebrafish showing the presence of melanophores (black cells) and iridophores (silver cells indicated by yellow arrows). (V-X) Side views of live zebrafish showing the presence of xanthophores (blue cells). Zebrafish were raised in 0.0001% Methylene Blue in order to visualize xathophores (Le Guyader and Jesuthasan, 2002).

Acknowledgments
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