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. Author manuscript; available in PMC: 2023 Mar 2.
Published in final edited form as: Science. 2022 Sep 2;377(6610):eabq4515. doi: 10.1126/science.abq4515

Fig. 3. GnRH transcriptional machinery disequilibrium underlies cognitive impairments.

Fig. 3

(A) Putative microRNA-transcription-factor network regulating hypothalamic GnRH promoter activation during postnatal development (adapted from (18)). (B-D) RT-PCR analysis of expression levels of microRNAs located on chromosome 16 (B) and miR-200 family members (C), as well as hypothalamic GnRH promoter modulators (D) in the preoptic region (POA) of adult WT and Ts65Dn littermates (B: n=11,10,10,11,10 WT mice; n=9,8,8,9,8 Ts65Dn mice; C: n=8,9,7,9,9 WT mice; n=6,6,6,6,6 Ts65Dn mice; D: n=11,11,9,10,11,11,7,11,11,8 WT mice; n=9,9,9,9,9,9,6,9,9,6 Ts65Dn mice). (E) Generation of Gnrh::Gfp;Ts65Dn reporter mice, which express GFP under an ectopic Gnrh promoter. GnRH-GFP neurons were FACS-isolated from the POA of Gnrh::Gfp and Gnrh::Gfp;Ts65Dn littermates at P12. (F) RT-PCR analysis of gene expression in FACS-sorted GnRH-GFP cells (n=10,10,11,11,10,11 WT mice; n=9,7,9,9,9,10 Ts65Dn mice). (G) Experimental design to evaluate the functional involvement of miR-200 family members in odor discrimination and novel object recognition in Ts65Dn mice. Red dot: viral injection site; LV, lateral ventricle; MePO, median preoptic nucleus; OVLT, organum vasculosum laminae terminalis. (H) Effect of viral overexpression of miR-200b in the POA on miR-200 family member expression (n=4,5,5 mice). (I-M) Effect of viral miR-200b overexpression in the POA on the number of neurons expressing Gnrh transcripts in the OVLT (I,J) and the proportion expressing Otx2 (I,K), as assessed by fluorescent in situ hybridization, as well as odor discrimination (L) and novel object recognition (M) in Ts65Dn mice (J,K; n=4,4,4 mice; L,M: n=5,5,6 mice). (N,O) Odor discrimination (N; n=6 per group) and novel object recognition (O; n=6 per group) in 12-month old male mice selectively lacking Dicer in GnRH neurons.

Values represent means ± SEM. *P<0.05; **P<0.01; ***P<0.001. Unpaired Student’s-test or Mann-Whitney U test (B-D,F), paired Student’s-test or Wilcoxon matched-pair test (L,M) and Kruskal-Wallis test (H) for comparisons between 2 conditions, one-way (J,K) or two-way repeated measures (N,O) ANOVA followed by Tukey’s and Sidak’s post hoc tests for ≥ 3 conditions.