Neogenin regulates Sonic Hedgehog pathway activity during digit patterning

Abstract

Background: Digit patterning integrates signaling by the Sonic Hedgehog (SHH), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) pathways. GLI3, a component of the SHH pathway, is a major regulator of digit number and identity. Neogenin (encoded by Neo1) is a cell surface protein that serves to transduce signals from several ligands, including BMPs, in various developmental contexts. Although neogenin is implicated in BMP signaling, it has not been linked to SHH signaling and its role in digit patterning is unknown.

Results: We report that Neo1 mutant mice have preaxial polydactyly with low penetrance. Expression of SHH target genes, but not BMP target genes, is altered in Neo1 mutant limb buds. Analysis of mice carrying mutations in both Neo1 and Gli3 reveals that, although neogenin plays a role in constraint of digit numbers, suppressing polydactyly, it is also required for the severe polydactyly caused by loss of GLI3. Furthermore, embryo fibroblasts from Neo1 mutant mice are sensitized to SHH pathway activation in vitro.

Conclusions: Our findings indicate that neogenin regulates SHH signaling in the limb bud to achieve proper digit patterning.

Fig. 1

Neo1^Gt/Gt mice display preaxial polydactyly. A: The right hindlimbs from control (Neo1^+/+ or Neo1^+/Gt) and Neo1^Gt/Gt mice at E17.5 or P0, as indicated. Embryos were stained with Alizarin Red and Alcian Blue to identify bone and cartilage structures. Numbers over toes represent digit identity. Note that the Neo1^Gt/Gt limb at E18.5 has an extra digit 2 and additional rudimentary metatarsal (asterisk) on the anterior side, whereas the Neo1^Gt/Gt limb at P0 has an identity shift of digit 1 to digit 2. B: Whole mount in situ hybridization of the early chondrogenesis marker Sox9 at E13.5. The red asterisk indicates an extra digit.

Fig. 2

Expression of Neo1 and limb patterning genes in control and Neo1^Gt/Gt mice. A: Expression of the Neo1-lacZ reporter in E10.5 and E11.5 hindlimb buds. B: Western blot analysis of neogenin expression in E11.5 wild-type and Neo1^Gt/Gt limb buds and in mouse embryo fibroblasts (MEFs). Blots were probed with neogenin and, as a loading control, pan-cadherin antibodies. Numbers below lanes represent the relative amounts of neogenin produced by limb buds of the indicated genotype, normalized to cadherin expression. A similar analysis was performed with MEFs, which were normalized independently from limb buds. C: Whole mount in situ hybridization of Shh (n=8), Gli1 (n=8), Ptch1 (n=10), Bmp4 (n=10), Msx1 (n=12), Msx2 (n=12) and Alx4 (n=10) expression in control (Neo1^+/+ or Neo1^+/Gt) and Neo1^Gt/Gt right hindlimb buds at E11.5. Red arrowheads indicate ectopic anterior expression of Gli1 and Ptch1.

Fig. 3

Autopod and zeugopod phenotypes of Gli3;Neo1 double mutants. A: Bone and cartilage staining of autopods of E18.5 mice of the indicated genotypes. Numbers on digits represent their identity. The asterisk indicates a nubbin. B: Bone and cartilage staining of limbs of E18.5 embryos of the indicated genotypes. The upper and the lower rows represent forelimbs and hindlimbs, respectively. Note the absence of a radius in the Gli3^Xt/Xt;Neo1^Gt/Gt embryo (black arrow) and the rudimentary and absent tibias in the Gli3^Xt/Xt;Neo1^+/+ and Gli3^Xt/Xt ;Neo1^Gt/Gt embryos, respectively (red arrowheads). The asterisk represents reduced bone formation in the anterior stylopod of the Gli3^Xt/Xt;Neo1^Gt/Gt embryo. Wt, wild-type; H, humerus; R, radius; U, ulna; Fe, femur; T, tibia; Fi, fibula.

Fig. 4

Expression of Shh and Ptch1 in Gli3;Neo1 double mutants. Whole mount in situ hybridization analysis Shh and Ptch1 expression in E11.5 embryos of the indicated genotypes. Black arrowheads indicate ectopic anterior expression of Shh, red arrowheads indicate ectopic anterior expression of Ptch1. Wt, wild-type. LF, left forelimb; RF, right forelimb; LH, left hindlimb; RH, right hindlimb; all sets of limb buds correspond to the positions identified for the Wt set in the upper left of the figure.

Fig. 5

Neo1^Gt/Gt MEFs are sensitized to SHH- and SAG-mediated Gli1 induction but neogenin does not bind SHH. A: qRT- PCR analysis of Gli1 and Ptch1 expression in Neo1^+/+ and Neo1^Gt/Gt MEFs. Two independent isolates of Neo1^+/+ MEFs and three independent isolates of Neo1^Gt/Gt MEFs were analyzed in duplicate in three separate experiments. Data were normalized to expression of Gapdh presented as fold induction over control, PBS-treated WT MEFs. Data represent means ± S.E.M. and were analyzed by Student’s t-test. *, p < 0.02; **, p < 0.005. B: Recombinant, secreted proteins comprising the ectodomains of neogenin or the SHH co-receptor, CDO, fused in-frame with the Fc region of human IgG (Neo1-Fc and CDO-Fc, respectively) were bound to protein-A sepharose. SHH-N::AP or, as a control, AP itself, were allowed to bind the Neo1-Fc and CDO-Fc matrices, which were then washed exhaustively. Bound AP activity was quantified with AP yellow liquid substrate.