The chicken talpid3 gene encodes a novel protein essential for Hedgehog signaling

Abstract

Talpid3 is a classical chicken mutant with abnormal limb patterning and malformations in other regions of the embryo known to depend on Hedgehog signaling. We combined the ease of manipulating chicken embryos with emerging knowledge of the chicken genome to reveal directly the basis of defective Hedgehog signal transduction in talpid3 embryos and to identify the talpid3 gene. We show in several regions of the embryo that the talpid3 phenotype is completely ligand independent and demonstrate for the first time that talpid3 is absolutely required for the function of both Gli repressor and activator in the intracellular Hedgehog pathway. We map the talpid3 locus to chromosome 5 and find a frameshift mutation in a KIAA0586 ortholog (ENSGALG00000012025), a gene not previously attributed with any known function. We show a direct causal link between KIAA0586 and the mutant phenotype by rescue experiments. KIAA0586 encodes a novel protein, apparently specific to vertebrates, that localizes to the cytoplasm. We show that Gli3 processing is abnormal in talpid3 mutant cells but that Gli3 can still translocate to the nucleus. These results suggest that the talpid3 protein operates in the cytoplasm to regulate the activity of both Gli repressor and activator proteins.

Figure 1.

Localization of Shh protein, effects of reducing Shh activity in wild-type and talpid³ limb buds, and status of Gli3 protein. (A,B) Localization of Shh protein in stage 19 HH wild-type (A) and talpid³ (B) limb buds. Dotted lines indicate limit of staining. (C) Western blot analysis of Shh protein in different regions (anterior, middle, posterior) of stage 20 HH wild-type (wt) and talpid³ (ta³) limbs. (D,E) Hoxd13 expression in wild-type (D) and talpid³ (E) right limbs treated with cyclopamine compare with control left limbs. (F,G) Hoxd13 expression in wild-type (F) and talpid³ (G) right limbs after removal of posterior mesenchyme; compare with control left limbs. (H,I) Status of Gli3 protein in different regions (anterior, a; middle, m; posterior, p) of stage 24 wild-type and talpid³ limb buds. (H) Immunoblot shows levels of Gli3A and Gli3R in wild-type and talpid³ legs (n > 3). (I) Histograms show levels of Gli3 proteins in wild-type and talpid³ wing buds; standard deviations shown, n = 3. (J) Immunoblot directly compares levels of Gli3 protein in wild-type and talpid³ limbs and trunk tissue. Numbers at top of each lane show ratios of Gli3A/Gli3R. H and J also illustrate tubulin loading controls.

Figure 4.

Rescue of talpid³ neural tube with activated Gli constructs. (A–H) Stage 20 HH wild-type and talpid³ embryos electroporated with Gli3A^HIGH (activated Gli construct). Green indicates expression of GFP in transfected cells; red indicates protein expression detected by immunohistochemistry. A1–H1 are overlays of GFP and immunostaining; A2–H2 are immunostaining alone. (A–D) Wild-type neural tube. Induction of Nkx2.2-positive cells (arrows, A1,A2); Islet1-positive cells (arrows, B1,B2). Down-regulation of Pax6 (arrow, C1,C2); Pax7 (D1,D2). (E–H) talpid³ neural tube. Induction of Nkx2.2-positive cells (E1,E2) and Islet1-positive cells (upper arrow, F1,F2). Down regulation of Pax6 (arrow, G1,G2); Pax7 (arrow, H1,H2).