Mutation in the Ciliary Protein C2CD3 Reveals Organ-Specific Mechanisms of Hedgehog Signal Transduction in Avian Embryos

Abstract

Primary cilia are ubiquitous microtubule-based organelles that serve as signaling hubs for numerous developmental pathways, most notably the Hedgehog (Hh) pathway. Defects in the structure or function of primary cilia result in a class of diseases called ciliopathies. It is well known that primary cilia participate in transducing a Hh signal, and as such ciliopathies frequently present with phenotypes indicative of aberrant Hh function. Interestingly, the exact mechanisms of cilia-dependent Hh signaling transduction are unclear as some ciliopathic animal models simultaneously present with gain-of-Hh phenotypes in one organ system and loss-of-Hh phenotypes in another. To better understand how Hh signaling is perturbed across different tissues in ciliopathic conditions, we examined four distinct Hh-dependent signaling centers in the naturally occurring avian ciliopathic mutant talpid² (ta²). In addition to the well-known and previously reported limb and craniofacial malformations, we observed dorsal-ventral patterning defects in the neural tube, and a shortened gastrointestinal tract. Molecular analyses for elements of the Hh pathway revealed that the loss of cilia impact transduction of an Hh signal in a tissue-specific manner at variable levels of the pathway. These studies will provide increased knowledge into how impaired ciliogenesis differentially regulates Hh signaling across tissues and will provide potential avenues for future targeted therapeutic treatments.

Keywords: C2CD3; Hedgehog signaling; ciliopathies; craniofacial; hindgut; limb; neural tube; primary cilia; talpid2.

Figure 1

Polydactylous ta² phenotype does not correlate with PTCH1 expression. (A,B) Alcian blue and Alizarin red staining of a HH39 control^+/+ (n = 5) and ta² (n = 4) hindlimbs. (C) Wholemount HH23-25 control^+/+ and ta² embryos stained with DAPI. (D–I) RNAscope in situ hybridization for (D,E) SHH, (F,G) PTCH1 or (H,I) both SHH and PTCH1 in HH24 control^+/+ (n = 9) and ta² (n = 9) hindlimbs. (J) Puncta quantification for PTCH1 expression in HH24 control^+/+ and ta² hindlimbs. A: Anterior, D1–D4: digits, Fi: fibula, Mt: metatarsal element, P: posterior, Ph: phalanges, Ti: tibia. Scale bars: (A,B) 2 mm, (C) 1 mm, (D–I) 500 µM. Error bars represent the mean data ± s.d. Statistical analysis was performed utilizing Student’s t-test (* denotes p < 0.05).

Figure 2

Expression of markers for ventral neuronal progenitors is reduced in the ta² neural tube. (A,B) RNAscope in situ hybridization for NKX2.2 in HH20 control^+/+ and ta² neural tubes. (C) Puncta quantification for NKX2.2 expression in HH20 control^+/+ and ta² neural tubes. (D,E) RNAscope in situ hybridization for OLIG2 in HH20 control^+/+ and ta² neural tubes. (F) Puncta quantification for OLIG2 expression in HH20 control^+/+ and ta² neural tubes. (G,H) RNAscope in situ hybridization for IRX3 in HH20 control^+/+ and ta² neural tubes. (I) Puncta quantification for IRX3 expression in HH20 control^+/+ and ta² neural tubes. D: dorsal, N: notochord, V: ventral. White dotted ovals outline the neural tubes. Scale bars: (A,B; D,E; G,H) 100 µm. Error bars represent the mean data ± s.d. Statistical analysis was performed utilizing Student’s t-test (* denotes p < 0.05).

Figure 3

Loss of ventral neural progenitors correlates with decreased PTCH1 expression. (A–F) RNAscope in situ hybridization for (A,B) SHH, (C,D) PTCH1, or (E,F) both SHH and PTCH1 in HH20 control^+/+ and ta² neural tubes. (G) Quantification of floor plate area in HH20 control^+/+ and ta² neural tubes. (H) Puncta quantification for PTCH1 expression in HH20 control^+/+ and ta² neural tubes. D: dorsal, FP: floor plate, N: notochord, V: ventral. White dotted ovals outline the neural tubes. Scale bars: (A–F) 100 µm. Error bars represent the mean data ± s.d. Statistical analysis was performed utilizing Student’s t-test (* denotes p < 0.05).

Figure 4

The frontonasal ectodermal zone (FEZ) is expanded and dispersed in ta² embryos. (A–F) RNAscope in situ hybridization for (A,B) SHH, (C,D) FGF8 or (E,F) both SHH and FGF8 in HH20 control^+/+ and ta² craniofacial complexes. Arrowheads in (A,B) demarcate boundaries of SHH expression, arrowheads in (C,D) demarcate boundaries of FGF8 expression, and arrowheads in (E,F) demarcate the FEZ. (G,H) RNAscope in situ hybridization for FGF8 in HH20 control^+/+ and ta² brains. (I,J) RNAscope in situ hybridization for OTX2 in HH20 control^+/+ and ta² brains. fb: forebrain, hb: hindbrain, mb: midbrain. Scale bars: (A–F) 250 μm, (G–J) 500 µm.

Figure 5

Expanded FEZ correlates with increased PTCH1 expression. (A–F) RNAscope in situ hybridization for (A,B) SHH, (C,D) PTCH1 or (E,F) both SHH and PTCH1 in HH20 control^+/+ and ta² brain and craniofacial complex. Asterisks (*) denote ectopic expression of (B) SHH, (D) PTCH1, or (F) both SHH and PTCH1 in the developing ta² brain and craniofacial complex. d: diencephalon, fb: forebrain, hb: hindbrain, mb: midbrain, mn: mandibular arch, or: optic recess, t: telencephalon, tp: tuberculum posterius. Scale bars: (A–F) 500 µm.

Figure 6

ta² embryos present with shortened gastrointestinal (GI) tracts accompanied with ectopic enteric neural crest cell (ENCC) migration. (A,B) Dissected gastrointestinal tracts from HH39 control^+/+ (A, n = 3) and ta² (B, n = 3) embryos. (C–E) Length measurements of HH39 control^+/+ and ta² (C) gastrointestinal tracts, (D) small intestines, and (E) colons. (F–I) Immunostaining for (F,G) P75 and (H,I) VERSICAN in transverse sections of HH34 control^+/+ and ta² hindguts. Dotted black circles in (F,G) indicate the intestinal epithelium. c: colon, ce: cecum, cl: cloaca, e: esophagus, Ep: epithelium, g: gizzard, pv: proventriculus, si: small intestine. Scale bars: (A,B) 6 mm. Error bars represent the mean data ± s.d. Statistical analysis was performed utilizing Student’s t-test (* denotes p < 0.05).

Figure 7

Hypoplastic GI phenotype does not correlate with increased PTCH1 expression. (A–F) RNAscope in situ hybridization for (A,B) SHH, (C,D) PTCH1 or (E,F) both SHH and PTCH1 in the hindgut epithelium of transverse sections of HH29 control^+/+ and ta² hindguts. (G) Puncta quantification for PTCH1 in HH29 control^+/+ and ta² hindguts. e: epithelium, m: mesenchyme. Scale bars: 100 µm (A–F). Error bars represent the mean data ± s.d. Statistical analysis was performed utilizing Student’s t-test (* denotes p < 0.05).

Figure 8

Summary of phenotypes, associated Hh activity, and PTCH1 expression across Hh-dependent signaling centers in control and ta² embryos. (A–H) Schematics of (A,B) hindlimb, (C,D) neural tube, (E,F) FEZ, and (G,H) GI tract phenotypes in (A,C,E,G) control^+/+ and (B,D,F,H) ta² embryos. (I–I’’’) Level of Hh activity associated with ta² phenotypic presentations. (J–J’’’) Summary of PTCH1 expression in ta² organs. di: diencephalon, Ep: epithelium, FEZ: frontonasal ectodermal zone, FNP: frontonasal prominence, Mes: intestinal mesenchyme, NCC: neural crest cell, ne: neuroepithelium, pMN: motor neuron progenitors, te: telencephalon.