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Mech Dev
2001 May 01;1031-2:93-106. doi: 10.1016/s0925-4773(01)00334-3.
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Overexpression of the transcriptional repressor FoxD3 prevents neural crest formation in Xenopus embryos.
Pohl BS
,
Knöchel W
.
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Xenopus FoxD3 (XFD-6) is an intron-less gene initially expressed within the Spemann organizer and later in premigratory neural crest cells. Based upon sequence and expression pattern comparisons, it represents the Xenopus orthologue to zebrafish fkd6, chicken CWH-3 and mammalian HFH-2 (genesis). Early expression of FoxD3 is activated by the Wnt-pathway and inhibited by BMP signalling. Ectopic overexpression of FoxD3 leads to an enlargement of the neural plate concomitant with a failure in neural crest formation, loss of anterior structures, lack of closure of the neural tube and severe defects in somitogenesis. Phenotypic variation is accompanied by down-regulation of neural crest markers, including Xslug, Xtwist and Xcadherin-11. FoxD3 also inhibits its own expression, thereby acting in a negative autoregulatory loop. By injections of VP16 and engrailed fusions we can demonstrate that FoxD3 acts as a negative transcriptional regulator; this repressive function strictly requires the presence of the winged helix domain. Transplantation experiments show that FoxD3 overexpressing cells from the prospective neural crest do neither differentiate nor migrate.
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11335115
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Fig. 2. Temporal and spatial expression of FoxD3 during Xenopus development. (A) Temporal expression was determined by RT–PCR using total RNA from embryos at different developmental stages (Nieuwkoop and Faber, 1967). A control reaction was performed in the absence of reverse transcriptase. Histone H4 was used as internal control. Right side: comparative RT–PCR for FoxD3, Xslug and Xsnail transcripts in RNAs of gastrula and early neurula stages. (B–J) Spatial expression was analyzed by whole-mount in situ hybridization utilizing an antisense probe containing the complete coding region of FoxD3. Expression starts at stage 10.5 (B), is restricted at stage 11.5 to the dorsal lip (C) and is only visible in a superficial, but not the outer epithelial cell layer, as illustrated by a benzylbenzoate-treated embryo at stage 10.5 (D). During the beginning of neurulation, expression in the blastoporus region has disappeared, whereas on the lateral borders of the neural plate the prospective neural crest starts to express FoxD3 (E,F). An anterior view of a stage 16 embryo (G) which was double-stained with Krox20 (blue) and FoxD3 (red) shows expression in neural crest cells derived from rhombomeres 2, 4 and 6 (numbers mark the rhombomeres). Stage 18 embryo (H) at the onset of migration of cephalic neural crest. At stage 21, FoxD3 is predominantly expressed in migrating neural crest cells populating the hyoid arch (I). At stage 30 (J), expression is restricted to cephalic ganglia and crest cells at the posteriortrunk. (K–M) Transverse sections (50 μm) of embryos shown in H, I, and J, respectively; red arrows denote sectioning planes. bl, blastoporus; dl, dorsal lip; nc, neural crest; no, notochord; np, neural plate; nt, neural tube; rh, rhombencephalon.
