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LGR4 deficiency results in delayed puberty through impaired Wnt/β-catenin signaling
Alessandra Mancini, … , Leonardo Guasti, Leo Dunkel
Alessandra Mancini, … , Leonardo Guasti, Leo Dunkel
Published June 4, 2020
Citation Information: JCI Insight. 2020;5(11):e133434. https://doi.org/10.1172/jci.insight.133434.
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Research Article Endocrinology Reproductive biology

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

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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.

Authors

Alessandra Mancini, Sasha R. Howard, Federica Marelli, Claudia P. Cabrera, Michael R. Barnes, Michael J.E. Sternberg, Morgane Leprovots, Irene Hadjidemetriou, Elena Monti, Alessia David, Karoliina Wehkalampi, Roberto Oleari, Antonella Lettieri, Valeria Vezzoli, Gilbert Vassart, Anna Cariboni, Marco Bonomi, Marie Isabelle Garcia, Leonardo Guasti, Leo Dunkel

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Figure 5

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

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Lgr4 impairs GnRH3-neuron development in morphants and Crispants Zebrafi...
(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.

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