Functional loss of semaphorin 3C and/or semaphorin 3D and their epistatic interaction with ret are critical to Hirschsprung disease liability

Abstract

Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.

Figure 1

Genetic Associations at the RET and SEMA3 Loci (A) Black dots represent −log₁₀ p values (y axis, left) for all SNPs studied plotted against their hg19 genomic location (with recombination rate plotted in light blue (y axis, right). The leading variants in the combined data are highlighted in red. RET and additional genes near the peak SNP rs2506030 are depicted at the bottom of the window with arrows indicating the direction of transcription; the highest peak is at the previously described enhancer variant rs2435357; the new peak is at rs2506030. (B) The 15.4 kb genomic region of high (r² > 0.8) linkage disequilibrium around SNP rs2506030 showing locations of all known common (>10%) SNPs, sequence conservation, and enhancer marks from human male fetal gut (DNase I hypersensitive sites, H3K4me1, H3K27ac). (C) The four SEMA3 genes near the peak SNPs rs12707682 and rs11766001, with legend as in (A). (D) The 219 kb genomic region of high (r² > 0.8) linkage disequilibrium around rs12707682 is depicted, with legend as in (A).

Figure 2

Expression of Sema3A, Sema3C, Sema3D, and Sema3E in the Developing Mouse Gut (A) RNA in situ hybridization analysis of Sema3A, Sema3C, Sema3D, and Sema3E in the developing mouse gastrointestinal tract relative to Ret. Expression analysis is shown for Ret, Sema3A, Sema3C, Sema3D, and Sema3E in the developing gut at stage E11.5, in E15.5 intestine sections, and in P3 intestine sections. (B) Sema3C and Sema3D gene expression patterns in wild-type and Ret^k−/k− mutant embryos. Comparisons of Sema3A, Sema3C, Sema3D, and Sema3E on intestinal sections from E15.5 wild-type and Ret^k−/k− mutant embryos are shown.

Figure 3

Synergistic Effects of Expression Knockdown of sema3c/sema3d and ret during Zebrafish Embryogenesis Morpholino co-knock-down of ret with individual sema3 orthologs to test for individual and epistatic effects on the ENS was measured at 5 dpf (days post fertilization) by immunofluorescence using the HuC primary antibody and an Alexa Fluor 568-tagged secondary antibody to observe the extent of colonization by ENS neurons. Asterisks denote posterior end of gut tube, and arrows denote position of the most posterior HuC-positive enteric neurons. The uninjected control shows complete colonization up to the cloacal end (A). The splice-blocking morpholino against sema3d had a modest effect on gut innervation at a dosage of 2.1 ng (B) but a severe impact at twice this dosage (C). A similar effect can be seen for a translation-blocking morpholino against ret at 1 (D) versus 2 (E) ng. Finally, a translation-blocking morpholino against sema3c has a strong effect on gut innervation even at a low dosage of 1.1 ng (F). Combination of morpholinos at the lower doses of sema3d (2.1 ng) and ret (1 ng) eliminates gut innervation (G) but required a higher dosage of ret (2 ng) when combined with sema3c (1.1 ng) to eliminate gut innervation (compare H and I).

Figure 4

In Silico Modeling of Semaphorin3 with Their Neuropilin and Plexin Complexes Models of the Sema3C/Neuropilin1/PlexinA2 (A, B, C) and Sema3D/Neuropilin1/PlexinA2 (D, E, F) complexes in top view (A, D), side view (B, E), and front view (C, F) are shown, with the Sema3 protomers colored in cyan, Neuropilin1 in golden brown, and PlexinA2 in green. The five residues variant in HSCR-affected subjects are colored (yellow for Ser329 and magenta for Val337 in Sema3C; dark blue for His424, magenta for Val457, and yellow for Pro615 in Sema3D) for visualization.

Figure 5

AP-SEMA Fusion Protein Accumulation and Receptor Binding (A–C) Secreted SEMA ligand concentrations were determined by measuring alkaline phosphatase (AP) activity for SEMA3C (A) and SEMA3D (B); results are shown as fold change relative to the wild-type construct and error bars represent SEM. The upper bands in (A) (for SEMA3C) and (B) (for SEMA3D) represent the AP-SEMA fusion protein of predicted molecular weight (MW) of ∼130 kDa; the lower bands of ∼90 kDa are suspected to be an abundant albumin within the medium recognized by the antibody and used as loading control. An additional band (arrowhead) was detected in only the pAPTag4 transfected supernatant at ∼63 kDa, indicating the expression of AP (MW 67 kDa) upon empty vector transfection. Mock represents conditioned medium from untransfected HEK293T cells; pAPTag4 represents the empty vector transfection. To test the transfection rate, double-transfection with AP-SEMA and FLAG-Hoxb7 on HEK293T cells followed by immunoblotting is presented in (C). (D and E) AP-SEMA binding affinity with Neuropilin1. COS-7 cells transfected with Nrp1 incubated with AP-SEMA3C- (D) or AP-Sema3D- (E) containing medium. Enzymatic detection of binding indicates that all ligands (except for empty vector) bind to the transfected cells but to different extents (scale bars represent 50 μm). Quantitative measurements of AP-SEMA binding to Nrp-1-expressing COS-7 cells were conducted. Results shown are bound AP activity averages and SEM of three independent experiments. (F and G) AP-SEMA binding affinity with Neuropilin2. Legend is the same as in (D) and (E).

Figure 6

Analysis of Gene Expression of PlexinA1, Plexin A2, PlexinA3, Plexin A4, PlexinD1, Neuropilin1, and Neuropilin2 Relative to Ret in the E15.5 Mouse Wild-Type and Ret Mutant Gut RNA in situ hybridization analysis was performed for all probes on E15.5 intestine tissue cross sections from both wild-type embryos and Ret^k−/k− mutant embryos. Ret is expressed in enteric neurons in the myenteric plexus in the outer regions of the E15.5 intestine (A, lumen denoted by asterisk). High-magnification view of boxed area of (A) is shown in (B), revealing individual Ret-expressing enteric neurons in the myenteric plexus (B, Ret panel, arrow). Ret expression is absent in corresponding cross-sections in Ret^k−/k− intestine tissue, which lack ENS neurons (B, Ret panel). PlexinA1 is expressed in population of ENS cells (B, Plxna1 panel, arrow), and this expression is absent in Ret^k−/k− gut tissues (B, Plxna1 panel), thus confirming that the expression observed in wild-type tissue corresponds to ENS expression. Expression of PlexinA2, PlexinA3, and PlexinA4 is also observed in ENS cells (B and C, Plxna2, Plxna3, Plxna4 panels, arrows), and expression is not detectable in Ret^k−/k− gut tissues (B and C, Plxna2, Plxna3, Plxna4 panels). PlexinD1 has widespread expression within the gut, but not, apparently, within the ENS (C, Plxnd1 panel). Consistent with this observation, the expression profile appears unchanged in Ret^k−/k− gut tissues (C, Plxnd1 panel). Populations of the ENS do express Neuropilin1 and Neuropilin2 (C, Nrp1, Nrp2 panels, arrows), and this expression is not observed in Ret^k−/k− gut tissues (C, Nrp1, Nrp2 panels).