Repressor activity of Headless/Tcf3 is essential for vertebrate head formation - PubMed (original) (raw)

Repressor activity of Headless/Tcf3 is essential for vertebrate head formation

C H Kim et al. Nature. 2000.

Abstract

The vertebrate organizer can induce a complete body axis when transplanted to the ventral side of a host embryo by virtue of its distinct head and trunk inducing properties. Wingless/Wnt antagonists secreted by the organizer have been identified as head inducers. Their ectopic expression can promote head formation, whereas ectopic activation of Wnt signalling during early gastrulation blocks head formation. These observations suggest that the ability of head inducers to inhibit Wnt signalling during formation of anterior structures is what distinguishes them from trunk inducers that permit the operation of posteriorizing Wnt signals. Here we describe the zebrafish headless (hdl) mutant and show that its severe head defects are due to a mutation in T-cell factor-3 (Tcf3), a member of the Tcf/Lef family. Loss of Tcf3 function in the hdl mutant reveals that hdl represses Wnt target genes. We provide genetic evidence that a component of the Wnt signalling pathway is essential in vertebrate head formation and patterning.

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Figures

Figure 1

Figure 1

Morphological analysis. a, b, huC expression at the neural plate stage shows abnormal rostral extension of trigeminal neurons (arrow). Dorsal-anterior view. c, d, 34-hour embryos show complete loss of eye, forebrain (fb) and part of the midbrain in hdl. mb, midbrain; hb, hindbrain; mhb, midbrain-hindbrain boundary; ot, otic vesicle. e, hdl larvae at 8 days show a severe head defect. f-i, Skeletal preparations of day-5 larvae stained with Alcian blue. In mutants, the ethmoid plate (ep) is reduced (asterisk), whereas ventral pharyngeal arches (p1–p7), including Meckel's cartilage (mc), are less affected. Fused pterygoid process of the palatoquadrate (pq) in hdl (arrowhead).

Figure 2

Figure 2

Gene expression analyses. Animal pole is to the top for 60–80% epiboly embryos. Anterior is to the left for tail bud (TB) and 3-somite-stage embryos (3ss). a, g, chd is relatively unaffected in the organizer. b, h, increased dkk1 expression in hdl. c, i, Widespread tcf3 expression, excluded from the ventrolateral margin in the wild type and reduced in hdl. Arrowheads indicate the organizer region. d, e and j, k, wnt8 is excluded from the dorsal margin (arrowheads) in the wild type but not in hdl. f, l, Anterior expansion of wnt1 expression in hdl. m-o and s-u, Reduced expression of anterior neural markers in hdl. p, q and v, w, Anterior expansion of MHB markers. q, w, krox20 is relatively unaffected in hdl. r, x, Graded anterior neural plate tcf3 expression, reduced in hdl.

Figure 3

Figure 3

Mapping of the hdl/tcf3 gene. a, Genetic map of linkage group 10 (LG10) showing position of hdl in relation to previously mapped genes and SSLP markers. b, Structure of the tcf3 gene. Arrowhead indicates the site of the hdl mutation. c, T-to-A transversion (green) produces an aberrant splice acceptor site (AG). The 7-bp insertion is indicated in red. d, Mse I digestion of the genomic PCR product (P2–P3), showing a polymorphism. e, Structure of Headless (Hdl). The truncated Hdl protein lacks the high mobility group (HMG) box DNA-binding motif as well as the nuclear localization signal (NLS). f, Subcellular localization of the wild-type Tcf3 and truncated Hdl proteins is changed from nuclear to cytoplasmic.

Figure 4

Figure 4

Rescue by Tcf3 expression. a, Two-day-old hdl mutant injected with wild-type Tcf3 RNA (bottom). Top, uninjected control mutant. b, An hdl mutant injected with the ΔNTcf3 plasmid DNA which is expressed after the MBT. c, ΔN-Tcf3 suppresses expression of eng2. Blue X-gal staining shows the injected side of embryo. d, Rescue of hdl by Eng-Tcf3 RNA injection. Higher doses (>50 pg) cause trunk defects. e, Opposite effects of VP16-Tcf3 on head formation. A wild-type embryo injected with VP16-Tcf3 RNA, showing a headless phenotype. f, Structure of proteins used for injection experiments.

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