LGR4 deficiency results in delayed puberty through impaired Wnt/β-catenin signaling

Abstract

The initiation of puberty is driven by an upsurge in hypothalamic gonadotropin-releasing hormone (GnRH) secretion. In turn, GnRH secretion upsurge depends on the development of a complex GnRH neuroendocrine network during embryonic life. Although delayed puberty (DP) affects up to 2% of the population, is highly heritable, and is associated with adverse health outcomes, the genes underlying DP remain largely unknown. We aimed to discover regulators by whole-exome sequencing of 160 individuals of 67 multigenerational families in our large, accurately phenotyped DP cohort. LGR4 was the only gene remaining after analysis that was significantly enriched for potentially pathogenic, rare variants in 6 probands. Expression analysis identified specific Lgr4 expression at the site of GnRH neuron development. LGR4 mutant proteins showed impaired Wnt/β-catenin signaling, owing to defective protein expression, trafficking, and degradation. Mice deficient in Lgr4 had significantly delayed onset of puberty and fewer GnRH neurons compared with WT, whereas lgr4 knockdown in zebrafish embryos prevented formation and migration of GnRH neurons. Further, genetic lineage tracing showed strong Lgr4-mediated Wnt/β-catenin signaling pathway activation during GnRH neuron development. In conclusion, our results show that LGR4 deficiency impairs Wnt/β-catenin signaling with observed defects in GnRH neuron development, resulting in a DP phenotype.

Keywords: Endocrinology; G-protein coupled receptors; Molecular genetics; Neuroendocrine regulation; Reproductive Biology.

Figure 1. Identification of LGR4 as a candidate gene for self-limited DP with rare pathogenic variants in patients.

(A) Whole-exome sequencing (WES) was performed on 160 individuals from our cohort (125 with self-limited DP and 35 controls). Variants were filtered using filters for quality control, predicted functional annotation, minor allele frequency (MAF), and for genes with variants in multiple families. A total of 28 genes were prioritized and were targeted exome sequenced in additional 288 individuals. Further analysis identified genes significantly enriched for pathogenic variants via whole gene burden testing, and genes involved in GnRH neuronal development and puberty timing (1, 2, 10, 11). Excluded, owing to the presence of variants in multiple controls. (B) Squares and circles indicate male and female family members, respectively. Black symbols represent affected individuals, gray symbols represent unknown phenotype, and clear symbols represent unaffected individuals. “P” indicates the proband in each family, and “us” indicates unsequenced owing to lack of DNA. A black line above an individual’s symbol indicates heterozygosity for that mutation as confirmed by either WES or Fluidigm array, and verified by Sanger sequencing. (C) LGR4 extracellular domain (gold) with variants bound to R-spondin1 (blue). Variants p.I96V and p.G363C are presented (green). p.I96V and p.G363C are in the variable region of LRR2 and LRR12, respectively. p.G363C occurs in close proximity to a cysteine bond (C339-C364; orange), and this substitution introduces a steric clash. p.D844G is within the cytoplasmic domain, and no experimental structure for the LGR4 cytoplasmic domain was available. DP, delayed puberty.

Figure 2. Lgr4/Wnt/β-catenin complex is expressed in key regions for GnRH neuronal development and migration.

(A and B) Low- and high-magnification images of in situ hybridization analysis revealing Lgr4 mRNA localization in the OE at E10.5. (C and D) At E14.5, the signal is strongly visible in the VNO and immunohistochemistry reveals GnRH neurons exiting the VNO. (E and F) Low- and high-magnification images showing Lgr4 mRNA preferentially localized in the VNO, OE, and nuclei of the forebrain at E14.5. (G and H) Low- and high- magnification images showing Lgr4 expression at E17.5 in the OE and in an area of the HYP where GnRH neurons (brown) are migrating alongside. (I) Schematic showing the mouse model employed for lineage tracing. (J and K) Axin2^+/+ Rosa^YFP/YFP control mice reveal a faint and not specific staining for Axin2-GFP. (L and M) In Axin2^CreERT2/+ Rosa^YFP/YFP embryos, Axin2-GFP-positive cells are expressed in the VNO and OE, indicating the presence of Wnt/β-catenin responsive cells. (N and O) Low- and high-magnification images of same specimen showing GFP-positive cells located exclusively in the HC, whereas the MPA is negative. (P) Immunohistochemical detection of GnRH neurons followed by (Q) immunofluorescence for GFP in Axin2^CreERT2/+ Rosa^YFP/YFP embryos. A representative GnRH neuron is GFP negative. Scale bars: 25 μm (A–H), 100 μm (J–M), 250 μm (N), 50 μm (O–Q). Representative images of experiments were performed at least 3 independent times. Arrowheads point to GnRH neurons. 3V, third ventricle; E, embryonic day; HC, hippocampus; HYP, hypothalamus; MPA, medial preoptic area; MV, mesencephalic vesicle; OE, olfactory epithelium; Pir, piriform cortex; TV, telencephalic vesicle; VNO, vomeronasal organ. (A and B) Sagittal sections; (C–Q) coronal sections.

Figure 3. LGR4 mutant receptors affect Wnt/β-catenin signaling owing to defects in protein production, trafficking, and protein turnover.

(A) HEK293T were nontransfected (–) or transfected (+) with HA-hLGR4 plasmids (WT or mutants; 200 ng/well) and reporter vectors (TOP-Flash and Renilla [150 ng/well]). Signaling was activated with conditioned media treatment, and each transfection normalized by cotransfection with Renilla. HA-hLGR4 mutant receptors resulted in significant reduced luciferase activity. ****P < 0.0001, ***P = 0.004, *P = 0.0437; n = 4. (B) Western blot and densitometry analysis revealed reduced levels of LGR4 mutants. GAPDH was used as loading control. Molecular weight (KDa) of a protein standard is reported (left panel: WT vs. I96V **P = 0.0054; WT vs. G363C **P = 0.0039; WT vs. D844G **P = 0.0049); n = 3. (C) Representative plots and quantification of flow cytometry analysis of cell surface expression of WT and mutant LGR4 proteins expressed in HEK293T. Normalized median fluorescence intensity (nMFI) reveals reduced levels of mutant receptors at the plasma membrane compared with HA-hLGR4 WT (WT vs. p.G363C *P = 0.0228; WT vs. p.D844G *P = 0.0498). Control: HEK293T transfected with pcDNA3.1EGFP vector only. n = 3. (D) LGR4 mutants have a shorter half-life: transiently transfected HEK293T with HA-hLGR4 WT or mutant constructs treated with CHX (50 μg/mL) for different time periods (0, 3, 6, 9, and 12). Levels of LGR4 WT and mutant proteins were expressed relative to untreated LGR4 WT or mutant proteins (0 h); n = 4. Statistical analysis by 1-way ANOVA. Half-life was analyzed via a 1-phase decay equation, and degradation speed (K) compared between each mutant and WT protein using the extra sum-of-squares F test. Co, control medium; Rspo1, Rspondin-1.

Figure 4. Lgr4^–/– female mice fail to enter puberty and show reduced number of GnRH neurons during fetal and adult life.

(A) Vaginal opening for pubertal onset shows that Lgr4^+/– mice have a significant (*P = 0.01) delayed onset of puberty compared with Lgr4^+/+. Lgr4^–/– female mice fail to enter puberty completely. Lgr4^+/+ n = 10, Lgr4^+/– n = 23, Lgr4^–/– n = 7. (B) Lgr4^+/– mice are not smaller than Lgr4^+/+, as shown by the percentage relative to Lgr4^+/+. Lgr4^+/+, n = 10; Lgr4^+/–, n = 15. (C) Litter size is not affected when pairing Lgr4^+/+ with Lgr4^+/–. Crosses between Lgr4^+/+ × Lgr4^+/+ and Lgr4^+/+ × Lgr4^+/– harbor a normal number in litter size, proving that fertility is not affected in Lgr4^+/– males and females. Lgr4^+/+, n = 10; Lgr4^+/–, n = 23. (D–F) Show gross anatomy of Lgr4^+/+, Lgr4^+/–, and Lgr4^–/– reproductive tracts, respectively. (G–I) Show gross anatomy of Lgr4^+/+, Lgr4^+/–, and Lgr4^–/– ovaries, respectively. (J) Lgr4^–/– females show reproductive tracts significantly reduced in weight compared with Lgr4^+/+ females (*P = 0.0366); n = 3. Scale bars: 2.5 mm (D–F), 1 mm (G–I). (K–M) GnRH neurons number in E12.5, E16.5, and adults, respectively. In K, Lgr4^+/+ = 3, Lgr4^+/– = 3, Lgr4^–/– = 2; in L, Lgr4^+/+ = 2, Lgr4^+/– = 4, Lgr4^–/– = 3; in M, Lgr4^+/+ = 5, Lgr4^+/– = 3, Lgr4^–/– = 3. In all groups, Lgr4^–/– mice show a reduced number of GnRH neurons compared with Lgr4^+/+. (Adults, *P = 0.0265). Lgr4^+/– mice show a similar trend although not statistically significant. Unpaired 2-tailed t test was used for statistical analysis for (A–C) and (J). Kruskal-Wallis test was used for M.

Figure 5. Lgr4 impairs GnRH3-neuron development in morphants and Crispants Zebrafish.

(A) Morpholino-mediated (MO-mediated) knockdown of the lgr4 gene in zebrafish: representation of lgr4 pre-mRNA. The lgr4MO targeted the exon2-intron2 boundary (red line). (B–G) Live-imaging acquisition of GnRH3 neurons in lgr4 morphants. (B and E) normal development of GnRH3 neurons at 48 and 72 hpf. Strong GnRH3 signal is visible at the level of the OBs, AC, along the OC and Re. At 72 hpf, GnRH3 hypothalamic (Hy) projections (asterisks) are also detectable. (C and F) embryos injected with 1 pmol/embryo. (D and G) embryos injected with 1.25 pmol/embryo. Images are acquired in ventral view, anterior to the top. (H and I) Quantification of the mean fluorescence intensity (MFI) using ImageJ Software. A significant reduction in MFI was observed in morphants vs. ctrl: at 48 hpf, lgr4MO 1 pmol/embryo *P = 0.0268; lgr4MO 1.25 pmol/embryo ****P < 0.0001; at 72 hpf, lgr4MO 1.25 pmol/embryo *P = 0.0290; lgr4MO 1.25 pmol/embryo ****P < 0.0001; n = 15 (J) Crispr/Cas-mediated KO of the lgr4 gene in zebrafish: representation of lgr4 gene, including the ATG start site and regulatory regions located in the 5′ UTR (black triangles). Localization of the 2 sgRNAs used (red lines). (K–R) Live-imaging acquisition of GnRH3 neurons in lgr4 Crispants. (K and O) uninjected embryos. (L and P) Crispant-Wt. (M and Q) Crispant-Het. (N and R) Crispant-Hom (all at 48 and 72 hpf). (S and T) A significant reduction in MFI was observed in Crispants vs. ctrl. At 48 hpf, Crispant-Het ***P = 0.0003; Crispant-Hom ****P < 0.0001; at 72 hpf Crispant-Hom ****P < 0.0001; n = 15. Red squares indicate region of interest (ROI) used for GnRH3 fiber quantification. Scale bars: 50 μm (B–G and K–R). Statistical analysis by 1-way ANOVA.