Gas1 extends the range of Hedgehog action by facilitating its signaling

Abstract

Cellular signaling initiated by Hedgehog binding to Patched1 has profound importance in mammalian embryogenesis, genetic disease, and cancer. Hedgehog acts as a morphogen to specify distinctive cell fates using different concentration thresholds, but our knowledge of how the concentration gradient is interpreted into the activity gradient is incomplete. The membrane protein Growth Arrest-Specific Gene 1 (GAS1) was thought to be a negative regulator of the Hedgehog concentration gradient. Here, we report unexpected genetic evidence that Gas1 positively regulates Hedgehog signaling in multiple developmental contexts, an effect particularly noticeable at regions where Hedgehog acts at low concentration. Using a combination of in vitro cell culture and in ovo electroporation assays, we demonstrate that GAS1 acts cooperatively with Patched1 for Hedgehog binding and enhances signaling activity in a cell-autonomous manner. Our data support a model in which GAS1 helps transform the Hedgehog protein gradient into the observed activity gradient. We propose that Gas1 is an evolutionarily novel, vertebrate-specific Hedgehog pathway regulator.

Figure 1.

A genetic interaction exists between Gas1 and Shh. Neonatal control (A) and Gas1^−/− (B) mice have two nostrils (arrows), while Gas1^−/−;Shh^+/− (C) mice have one (n = 5). (D) The table shows the recovery of Gas1^−/−;Shh^−/− embryos at different stages from crosses between Gas1^+/−;Shh^+/− mice. (E–H) Gross morphology comparison between day 9.5 embryos of different genotypes (as labeled): The Gas1^−/−;Shh^−/− embryos have an inflated pericardium (arrowhead, ventral view) and a double-kinked body axis (open arrowheads, lateral view with pericardium removed). (I–L) Hematoxylin and eosin staining of transverse sections at similar levels of the heart at E9.5: For the Gas1^−/−;Shh^−/− embryos, note the failed heart tube looping compared with the other genotypes; in the right panel of L, the ventricle is not present at this level of section. (a) Atrium; (ed) edema; (ee) extra embryonic membrane; (la) left atrium; (lv) left ventricle; (nt) neural tube; (pc) pericardium; (ra) right atrium; (v) ventricle. Bar: I –L, 200 μm.

Figure 2.

Compensation provided by IHH signaling in the Shh^−/− background is eliminated in Gas1^−/−;Shh^−/− embryos. (A–D) Pax1 ISH (n = 11). (E–H) Pax3 ISH (n = 15). (I–L) PAX7 IHC (n = 9). (M–P) Dbx1 ISH (n = 5). (Q–T) Ptc1 ISH (n = 11). Arrowheads indicate the dorsal or ventral extent of gene expression; lines in E–H indicate the domain size of Pax3 and PAX7. (U,V) Shh ISH (n = 3); brackets indicate the width of Shh-expressing floorplate cells; images are at 2× higher magnification. (W,X) IHH IHC (n = 2); arrows indicate the apical localization of IHH protein. All sections are transverse at the forelimb level of E9.5 embryos. Genotypes are labeled on top of each panel. Not shown are Gas1^+/−;Shh^−/− embryos, which display a variable but intermediate phenotype between Shh^−/− and Gas1^−/−;Shh^−/−. Axis: Dorsal is up, ventral is down. Bars: all except for U,V, 100 μm; U,V, 50 μm.

Figure 3.

Gas1^−/−;Shh^+/− limbs have reduced range of SHH signaling. (A–F) Alcian blue-stained (for cartilage) and alizarin red-stained (for bone) neonatal forelimb (F.L.) and hindlimb (H.L.) autopods of Gas1^+/−;Shh^+/− (A,D) (n = 22), Gas1^−/− (B,E) (n = 22), and Gas1^−/−;Shh^+/− (C,F) (n = 19) genotypes. (G–R) Whole-mount ISH of E10.5 forelimb buds: (G–I) Fgf4 (n = 6). (J–L) Grem (n = 3). (M–O) Ptc1 (n = 7); Gli1 (not shown; n = 5); arrowheads mark the anterior boundary of expression. (P–R) Shh (n = 5). Axis: Anterior is up, posterior is down. Bars: A–F, 1 mm; G–R, 100 μm.

Figure 4.

Gli3 is epistatic to Gas1 in digit patterning, and Gas1 inhibits GLI3 processing. (A) Alcian blue- and alizarin red-stained E16.5 forelimb (F.L.) and hindlimb (H.L.) autopods of Gli3^−/− and Gas1^−/−;Gli3^−/− limbs (n = 5). Bar, 1 mm. (B) Diagram depicting how E10.5 limb buds were dissected. (C) Western blot analysis for GLI3 on indicated genotype and limb bud halves. (A) Anterior; (P) posterior. GLI3-190 and GLI3-R bands are as labeled. A Gli3^−/− limb bud was used for a control. Blots were stripped and reprobed for α-tubulin for a loading control. Asterisks indicate nonspecific bands. (D) Western blot repeated for three embryos of each genotype and band intensity quantified. GLI3-R:GLI3-190 ratios are indicated.

Figure 5.

GAS1 facilitates SHH signaling activity. (A) SHH IHC on horizontal sections of E10.5 limb buds (n = 10) of labeled genotypes; arrowheads mark the anterior boundary of detectable SHH. For color development, Gas1 mutant limbs were developed for approximately half as long to compensate for the doubled Shh dosage. (B) Fold induction (Y-axis) of NIH3T3-GLI-Luc cells transfected with control siRNA or Gas1 siRNA by various concentrations of SHH-N (X-axis); (inset) Western blot for GAS1, then reprobed for α-tubulin as a loading control. (C) Fold induction (Y-axis) of NIH3T3 cells transiently cotransfected with GLI-Luc reporter and plasmids expressing various effector genes and/or shRNA against Gas1 (as labeled) in the presence or absence of 10 nM SHH-N. (D) SHH-N-AP-binding assay of COS cells expressing Gas1 and/or Ptc1, as well as other control genes are as labeled. They were assayed for binding to SHH-N-AP or AP alone, as determined by the picomoles bound (Y-axis) (input = 1.25 nM). In B–D, all assays were performed in triplicate; error bars = 1 standard deviation.

Figure 6.

Gas1 expression alters the position of neuronal progenitors in the neural tube. Chick trunk neural tube was electroporated with Gas1N/A-IRES-GFP (A–D) or Gas1-IRES-GFP (E–L) plasmids and analyzed for GFP (B,D,F,H,J,L), PAX7 (A,E,I), and NKX2.2 (C,G,K) by double immunofluorescence (green for GFP, and red for PAX7 or NXK2.2) on the same sections. Each pair of images (A and B, C and D, etc.) is from the same section; n ≥ 10 for each pair. I–L are at 2× higher magnification than A–H. Brackets indicate altered domains of PAX7 or NKX2.2, arrows indicate corresponding PAX7-negative and GFP-positive domains, arrowheads denote NKX2.2 and GFP doubly positive cells, and open arrowheads indicate the ventral midline.

Figure 7.

HH signaling negatively regulates Gas1 expression. Gas1 expression was determined by X-gal staining in E9.5 neural tubes at forelimb level (A,B), E10.5 limb buds viewed dorsally (C,D), and E8.0 nodes viewed ventrally (E,F). Genotypes are labeled on top of each panel. (G–I) Model for the role of Gas1 in transforming a hypothetical HH concentration gradient to an activity gradient. The Y-axis represents the magnitudes of three gradients: HH concentration (black line), HH activity (red dashed line), and GAS1 levels (blue dashed line). The X-axis represents a HH source and the distance from that source. The shape of the HH protein gradient is based in part on mathematical modeling (Saha and Schaffer 2006). GAS1 levels have direct implications for HH activity gradients and the distance from the HH source (green line) where the activation threshold (dashed gray line) for a hypothetical target gene is reached. For simplification, the contributions of other known HH-modifying proteins are not included in this model.