Fig 2: 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.

Fig 3: Lgr4/Wnt/ß-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+/+ RosaYFP/YFP control mice reveal a faint and not specific staining for Axin2-GFP. (L and M) In Axin2CreERT2/+ RosaYFP/YFP embryos, Axin2-GFP-positive cells are expressed in the VNO and OE, indicating the presence of Wnt/ß-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 Axin2CreERT2/+ RosaYFP/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.

Fig 4: GnRH-Bmal1-/- and Kiss-Bmal1-/- produce an LH surge at lights off and have normal GnRH and kiss-10 LH responsiveness. (A) LH levels at lights off in an induced surge paradigm (for nonsurging example, please see Fig. 1A) (n = 5 to 9, one-way ANOVA not significant). (B) LH levels before and 10 min after 1 µg/kg GnRH administration (n = 3 to 9; two-way ANOVA significant for time; F1,30 = 56.89, P < 0.0001; Sidak multiple comparisons test). (C) LH levels before and 10 min after 30 nmol of kiss-10 administration (n = 3 to 8; two-way ANOVA significant for time; F1,28 = 43.61, P < 0.0001; Sidak multiple comparisons test). Different letters indicate significantly different groups (P < 0.05).

Fig 5: Chemogenetic activation of hypophysiotropic TRH neurons increases TSH release. a–c Colocalization of Trh mRNA (a, magenta) and hM3D-mCherry (b, cyan) in the PVN 2 weeks after bilateral injection of AAV-TRH-hM3D-mCherry (PVN3D). Cells expressing Trh mRNA were positive for hM3D-mCherry (yellow arrowheads; c and d). Red arrowheads label hM3D-mCherry-positive cells without Trh mRNA expression; scale bar, 50 µm. e Plasma TSH concentrations 45 min after CNO administration to Trhr1 +/+ (Bl6) and littermate Trhr1 -/- mice without (PVNCon, gray) and with injection of AAV-TRH-hM3D-mCherry into the PVN (PVN3D, red). TSH plasma concentrations are shown as percentage of Bl6 for normalization, because of a slight but non-significant increase of TSH in Trhr1 -/- compared to Bl6 mice. Two-way ANOVA for genotype: F(1/20) = 5.3, p = 0.032; *p < 0.05 (Bonferroni post test); mean ± S.E.M.; n as indicated. f Relative change in Fos mRNA expression (2-??CT) in the pituitary 45 min after CNO administration to Bl6 and Trhr1 -/- mice without (PVNCon, gray) and with injection of AAV-TRH-hM3D-mCherry into the PVN (PVN3D, red). Two-way ANOVA for genotype: F(1/19) = 4.4, p = 0.049; *p < 0.05 (Bonferroni post test); mean ± S.E.M.; n as indicated. g and h Two weeks after bilateral injection of AAV-TRH-hM3D-mCherry (PVN3D) in the PVN of C57Bl/6 mice (PVN3D-Bl6) hM3D-mCherry-positive fibers were detected in the ME. g Colocalization of hM3D-mCherry with ppTRH in fibers of the ME (red, hM3D-mCherry; green, ppTRH; blue, DAPI; scale bar, 50 µm). h Colocalization of hM3D-mCherry with CRH in fibers of the ME (red, hM3D-mCherry; green, CRH; blue, DAPI; scale bar, 50 µm). i Plasma TSH concentrations 2 h after CNO stimulation of PVNCon-Bl6 (gray) and PVN3D-Bl6 (red) mice. *p = 0.0002 (two-tailed Student’s t-test); mean ± S.E.M.; n as indicated. j Plasma ACTH concentrations 2 h after CNO stimulation of PVNCon-Bl6 (gray) and PVN3D-Bl6 (red) mice. p = 0.11 (Student’s t-test); mean ± S.E.M; n as indicated. k–m No colocalization of hM3D-mCherry with vasopressin (green, j), oxytocin (green, k), or GnRH (green, l) in fibers of the ME. Red, hM3D-mCherry; blue, DAPI; scale bar, 50 µm