Cholesterol modification restricts the spread of Shh gradient in the limb bud

Abstract

Sonic hedgehog (Shh) produced in the zone of polarizing activity is the major determinant of anteroposterior development of the amniote limb. The mature and active Shh protein is cholesterol-modified at its C terminus, and the hydrophobic nature of the modification requires the function of Dispatched (mDispA), a seven-pass transmembrane protein, for Shh release from its source. The current model suggests that the cholesterol moiety promotes the spread of Shh gradient in the limb bud. However, this model is inconsistent with findings in Drosophila and not in line with current thoughts on the role of the cholesterol moiety in Shh multimerization. Therefore, it remains unclear how the cholesterol moiety affects the postrelease extracellular behavior of Shh that relates to the shape of its activity gradient in responsive tissues. Here, we report functional analyses in mice showing that Shh lacking cholesterol modification (ShhN) has an increased propensity to spread long-distance, eliciting ectopic Shh pathway activation consistent with target gene expressions and modulating the level of Gli3 processing in the anterior limb mesoderm. These molecular alterations are reflected in the mispatterning of digits in ShhN mutants. Additionally, we provide direct evidence for the long-distance movement of ShhN across the anteroposterior axis of the limb bud. Our findings suggest that the cholesterol moiety regulates the range and shape of the Shh morphogen gradient by restricting rather than promoting the postrelease spread of Shh across the limb bud during early development.

Fig. 1.

ShhN elicits ectopic Shh pathway activation and target gene expressions in the hindlimb bud. (A–O) Whole-mount in situ hybridization of E10.5 wild-type (A–E), ShhN⁺ (K–O), and ShhN⁻ (F–J) hindlimb buds, examining the expression of Shh and its target genes Ptch1, Gli1, Gre, Hoxd13, and Fgf4, as indicated. Black arrows denote ectopic activation of Shh target genes in the anterior limb margins. Also note that the expressions of Gli1 and Hoxd13 (G and I) can be detected in a diffused pattern in the midregion of ShhN⁺ and ShhN⁻ limb-bud mesoderm (yellow arrows).

Fig. 2.

Ectopic Shh expression in the anterior margin of the limb bud at E11.5. The Shh-expressing cells and their descendants are marked by lacZ reporter gene in ShhCre;Rosa26R (A) and ShhCre/Shhfloxc;Rosa26R (B) embryos. Note the presence of ectopic Shh-expressing cells only in the anterior margins of ShhN mutant limb buds (arrow).

Fig. 3.

ShhN has increased propensity to spread across the anterior/posterior axis of the limb bud. Shh RNA (A–C) and protein (D–F, green) distributions in wild type (A and D), ShhN⁻ (B and E), and ShhN⁺ (C and F) limb buds at E10.5. The limb buds in D–F were counterstained with Hoescht's dye (red).

Fig. 4.

Long-range ShhN signal regulates Gli3 processing in the limb. (A) Protein extracts from E10.5 wild-type, ShhN⁺, and ShhN⁻ limb buds of anterior (A) and posterior (P) halves were immunoblotted and incubated with a Gli3-specific antibody recognizing full-length (Gli3–190) and repressor forms (Gli3R) of Gli3. (B) Histograms showing relative Gli3R/Gli3–190 ratio in anterior and posterior halves of wild-type and ShhN limb buds. Levels of Gli3–190 and Gli3R were normalized to an internal nonspecific control band (asterisk). Note significant reduction of Gli3R in the anterior halves of ShhN limbs compared with wild type.

Fig. 5.

The expression of ShhN causes abnormal development of digits. Skeletal analysis of E18.5 wild-type (A and D), ShhN⁻ (B and E), and ShhN⁺ (C and F) digits. (B and E) ShhN⁻ limbs develop five digits with defective digit 2. A small cartilaginous outgrowth adjacent to the presumptive forelimb digit 1 is evident (B, arrow). (C and F) ShhN⁺ limbs develop six to seven digits with complete formation of digits 2–5. Note that digit 1 in the hindlimb is replaced by digits with more posterior characteristics (F, asterisks).

Fig. 6.

Long-range signaling capacity of ShhN is independent of mDispA function. Whole-mount in situ hybridization of E10.5 wild-type (A–D) and ShhN⁺;mDispA^−/− (F–J) forelimb buds, showing the expression of Ptch1 (A and F), Gli1 (B and G), Gre (C and H), and Fgf4 (D and I). Note ectopic expressions of Shh target genes in the anterior margins of ShhN⁺;mDispA^−/− limb buds (arrows). (E and J) Skeletal analysis of E18.5 wild-type (E) and ShhN⁺;mDispA^−/− (J) forelimb digits. Note that digit numbers and patterns resemble those of ShhN⁺. (K) A model depicting ShhNp and ShhN gradients in early limb buds.