Cdon acts as a Hedgehog decoy receptor during proximal-distal patterning of the optic vesicle

Abstract

Patterning of the vertebrate optic vesicle into proximal/optic stalk and distal/neural retina involves midline-derived Hedgehog (Hh) signalling, which promotes stalk specification. In the absence of Hh signalling, the stalks are not specified, causing cyclopia. Recent studies showed that the cell adhesion molecule Cdon forms a heteromeric complex with the Hh receptor Patched 1 (Ptc1). This receptor complex binds Hh and enhances signalling activation, indicating that Cdon positively regulates the pathway. Here we show that in the developing zebrafish and chick optic vesicle, in which cdon and ptc1 are expressed with a complementary pattern, Cdon acts as a negative Hh signalling regulator. Cdon predominantly localizes to the basolateral side of neuroepithelial cells, promotes the enlargement of the neuroepithelial basal end-foot and traps Hh protein, thereby limiting its dispersion. This Ptc-independent function protects the retinal primordium from Hh activity, defines the stalk/retina boundary and thus the correct proximo-distal patterning of the eye.

Figure 1. Expression of cdon and other Hh signalling components during P-D patterning of the optic vesicles.

(a–c) Ventral views of anterior zebrafish forebrain at optic vesicle (a is ventral to b) and optic cup (d, e) stages immunostained in toto with antibodies against Pax2 and Shh. (f–i) Dorsal (f,h) and lateral (g,i) views of zebrafish embryos hybridized in toto with probes specific for ptc1 and cdon at the 20 somites’ stage (20 ss). (j–k) Coronal sections of embryos (75–90% epiboly) hybridized for cdon and shh. The white arrows in b,c and e point to Shh immunolabelling in Pax2-positive cells. Dashed lines in a–d,j and k indicate the embryonic midline. Continuous white and black lines in f–i outline the lens and the embryonic border, respectively. a, anterior; d, dorsal; os, optic stalk; ov, optic vesicle; p, posterior; v, ventral. Scale bar, 25 μm.

Figure 2. The optic stalk is expanded in cdon morphants.

(a–j) In situ hybridization analysis for two optic stalk markers, pax2.1 (a–d) and fgf8a (f–i) at 28 hpf and 24 hpf, respectively. Embryos are shown in lateral (a,b,f,g) and frontal (c,d,h,i) views. Expression patterns of both genes are schematically represented in e and j. In cdon^ATG morphants, pax2.1 expression is expanded dorsally in the optic stalk (b,d brackets) when compared with controls (a,c brackets). Fgf8a expression is expanded caudally and laterally in the optic stalk (g,i arrowhead and dotted lines) as well as in the telencephalon (g,i) when compared with control embryos (f,h arrowhead and dotted lines). di, diencephalon; mes, mesencephalon; MHB, midbrain–hindbrain boundary; os, optic stalk; rh, rhombencephalon; tel, telencephalon. Scale bars, 100 μm.

Figure 3. Cdon acts as a negative modulator of Hh signalling.

(a–d) Lateral views of control or cdon^ATG morphants treated with DMSO (vehicle) or cyclopamine from 90% epiboly and analysed with ISH for pax2.1 expression at 28 hpf. Blocking Hh signalling abolished pax2.1 expression in the optic stalk in wt embryos (c). In cdon^ATG morphants, cyclopamine treatment counteracts the expansion of pax2.1 overexpression observed in cdon^ATGMO injected embryos (a,b,d). The lens and the body are outlined with black and white dashed lines in (a–d). Arrows indicate the extent of the pax2.1 expression in the optic stalk. DMSO, dimethyl sulphoxide; MHB, midbrain–hindbrain boundary; oc, optic cup; os, optic stalk. Scale bar, 100 μm.

Figure 4. Cdon interaction with Ptc is dispensable for its function in the optic vesicle.

(a) Schematic diagram of the design of the exon skipping MO used in the study. (b) Schema representing the zebrafish cdon mRNA (zcdon) aligned with the corresponding Cdon protein indicating the domains targeted by cdon^spl8, cdon^spl11a, cdon^spl11d and cdon^spl14 MOs. The exons that encode the 5′ and 3′ UTR regions are depicted in blue; those that, when skipped, generate a translational frame shift are indicated in red; whereas those that, when skipped, maintain the reading frame are indicated in green. (c,f,i) RT–PCR analysis of the exon skipping function of cdon^spl8, cdon^spl14 and cdon^spl11a/cdon^spl11d MOs. For detailed information about the resulting bands, noted 1–9, please refer to Supplementary Fig. 8. (d,e,g,h,j,k) ISH analysis of pax2.1 expression pattern in cdon^spl8 (d,e), cdon^spl14(g,h) and cdon^spl11a/cdon^spl11d (j,k) MO injected embryos at 26–28 h.p.f. Pax2.1 expression domain is expanded in cdon^spl8 (d,e) and cdon^spl14 morphants (g,h) in comparison with their respective controls, whereas there was no difference in the pax2.1 expression domain of control and cdon^spl11a/cdon^spl11d morphants (j,k). (l) Quantification of the optic stalk pax2.1-positive expression domain in embryos injected with the different MOs at 26–28 h.p.f. (***P< 0.001; Student’s t-test). The number of embryos analysed in each case is indicated in each column and are as follows: Cdon^ATG MO injection (control, n=28; MO, n=45); Cdon^spl8 MO injection (control, n=12; MO, n=44); Cdon^spl11a and Cdon^spl11d MOs injection (control, n=26; MO, n=65); Cdon^spl14 MO injection (control, n=22; MO, n=57). Error bars represent s.e.m. Scale bars, 100 μm. MWM, molecular weight marker.

Figure 5. Localized interference with Cdon expression in the optic vesicles expands the distal optic stalk domain.

(a–f) Lateral (a,b) and ventral (c) views of the control (a) and experimental (b) optic vesicles (ov) of HH14 chick embryos with unilateral focal electroporation of a carboxyfluorescein conjugated cCdon^ATG MO at HH8. Embryos were hybridized for pax2 (blue signal) and immunostained with anti-fluorescein antibodies (brown signal) to detect MO distribution (a–d,f). Pax6 immunohistochemistry was performed in cryostat sections of electroporated embryos (e,g). Pax2 expression is expanded to the entire optic vesicle of cCdon^ATG MO-treated embryos (b,c) in comparison with the non-electroporated control eye (a,c). Pax2 expansion is associated with a reduction of Pax6 distribution in the retina (f,g) in contrast to a wild-type condition (d,e). The optic vesicles are outlined with black (a–c) or white (g) dashed lines. Scale bar, 50 μm.

Figure 6. Cdon overexpression modifies Shh protein distribution in the optic stalk neuroepithelium.

(a–p) Confocal analysis of coronal sections at the level of the optic vesicle of HH14 chick embryos electroporated at HH8 with a construct carrying mCherry (a–d), Cdon (e–h) or its deleted derivatives lacking the Ptc (CdonΔFnIII(1-2)) (i–l) or Shh (CdonΔFnIII(3)) (m–p) binding domains. Sections were immunostained with antibodies against mCherry (a), Cdon (e,i,m) and Shh (b,f,j,n). Note the localization of Shh protein in Cdon-positive cells—particularly in their basal regions (f,h white arrow)—falling outside of the Shh-expression domain (e–h). A similar accumulation is observed in the presence of the CdonΔFnIII(1-2) construct (i–l white arrow) but was hardly detectable in mCherry or CdonΔFnIII(3) expressing cells falling outside of the Shh domain (a–d and m–p). The midline is indicated with a yellow dotted line. The normal extent of Shh distribution is indicated with a yellow line, whereas extended Shh localization is indicated in red. In p the arrowhead points to a Cdon-positive cell within the Shh domain, whereas the arrow points to the first Cdon-positive cell falling outside the Shh domain. fp, floor plate; os, optic stalk. Scale bar, 50 μm.

Figure 7. Cdon and Boc promote morphological changes of the neuroepithelial basal side where Shh preferentially accumulates.

(a–r) Confocal analysis of coronal sections at the level of HH14 chicken optic stalk (a–l) and HH10 neural tube (m–r) electroporated at HH8 with a construct carrying mCherry (a–c) Cdon (d–f), CdonΔFnIII(1-2) (g–i), CdonΔFnIII(3) (j–l), EGFP-GPI (m–o) or Boc-EGFP (p–r). Sections in (a–l) were immunostained with antibodies against Cherry (a) Cdon (d,g,j) and Shh (b,e,h,k). Images in (n,o) and (q,r) are high magnification views of cells shown in (m,p) respectively. Note how cells electroporated with Boc-EGFP (q,r, arrows), Cdon (d) or CdonΔFnIII(1-2) (g) present an enlarged basal end-foot when compared with EGFP, mCherry or CdonΔFnIII(3) neuroepithelial cells (n,o and a,j arrows). This enlarged end-foot is a preferential site of Shh accumulation (e,f,h,i). Note also the absence of Shh signal in the cells immediately surrounding the Cdon-positive cells (e,h, dotted line arrows). This distribution is not observed n cells expressing mCherry (b,c) or CdonΔFnIII(3) (k,l). (s) Quantification of the basal end-foot width of neuroepithelial cells electroporated with Cdon and its deleted versions. The number of cases analysed for each data set is indicated in the respective column. The numbers of quantified cells are indicated in the graph labels and are as follows: Cherry, n=17; Cdon, n=14; CdonΔFnIII(1-2), n=16 and CdonΔFnIII(3), n=13. Error bars represent s.e.m. (**P<0.01, ***P<0.001; Student’s t-test). There is no statistical difference in the basal end-foot width between Cdon and CdonΔFnIII(1-2) expressing cells (P=0.066, Student’s t-test) or between Cherry and CdonΔFnIII(3) expressing cells (P=0.998; Student’s t-test). Scale bar, 10 μm.