Congenital pituitary hypoplasia model demonstrates hypothalamic OTX2 regulation of pituitary progenitor cells
Ryusaku Matsumoto, Hidetaka Suga, Takashi Aoi, Hironori Bando, Hidenori Fukuoka, Genzo Iguchi, Satoshi Narumi, Tomonobu Hasegawa, Keiko Muguruma, Wataru Ogawa, Yutaka Takahashi
Ryusaku Matsumoto, Hidetaka Suga, Takashi Aoi, Hironori Bando, Hidenori Fukuoka, Genzo Iguchi, Satoshi Narumi, Tomonobu Hasegawa, Keiko Muguruma, Wataru Ogawa, Yutaka Takahashi
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Research Article Development Endocrinology

Congenital pituitary hypoplasia model demonstrates hypothalamic OTX2 regulation of pituitary progenitor cells

Abstract

Pituitary develops from oral ectoderm in contact with adjacent ventral hypothalamus. Impairment in this process results in congenital pituitary hypoplasia (CPH); however, there have been no human disease models for CPH thus far, prohibiting the elucidation of the underlying mechanisms. In this study, we established a disease model of CPH using patient-derived induced pluripotent stem cells (iPSCs) and 3D organoid technique, in which oral ectoderm and hypothalamus develop simultaneously. Interestingly, patient iPSCs with a heterozygous mutation in the orthodenticle homeobox 2 (OTX2) gene showed increased apoptosis in the pituitary progenitor cells, and the differentiation into pituitary hormone–producing cells was severely impaired. As an underlying mechanism, OTX2 in hypothalamus, not in oral ectoderm, was essential for progenitor cell maintenance by regulating LHX3 expression in oral ectoderm via FGF10 expression in the hypothalamus. Convincingly, the phenotype was reversed by the correction of the mutation, and the haploinsufficiency of OTX2 in control iPSCs revealed a similar phenotype, demonstrating that this mutation was responsible. Thus, we established an iPSC-based congenital pituitary disease model, which recapitulated interaction between hypothalamus and oral ectoderm and demonstrated the essential role of hypothalamic OTX2.

Authors

Ryusaku Matsumoto, Hidetaka Suga, Takashi Aoi, Hironori Bando, Hidenori Fukuoka, Genzo Iguchi, Satoshi Narumi, Tomonobu Hasegawa, Keiko Muguruma, Wataru Ogawa, Yutaka Takahashi

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Figure 3

Reduced expression of LHX3 and increased number of apoptotic cells in the oral ectoderm in OTX2mut-iPSCs.

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Reduced expression of LHX3 and increased number of apoptotic cells in th...
(A) Stage-specific transcription factors during pituitary differentiation in mouse development. (B) Quantitative RT-PCR analysis for representative markers of different developmental stages: non-neural placode, HESX1 and SIX1; oral ectoderm, PITX1; and pituitary progenitor, LHX3. Pituitary progenitor marker (LHX3) was significantly downregulated in OTX2mut-iPSCs at day 40. Representative data from 2 independent experiments are shown. Data show mean ± SEM; n = 3 per group. ***P < 0.001, Student’s t test (unpaired, 2-tailed). (C) LHX3 expression in oral ectoderm (indicated by dashed lines) was impaired in OTX2mut-iPSCs. Immunostaining at day 40. (D and E) Cell proliferation was evaluated by Ki-67 immunostaining. No significant difference in the number of Ki-67+ and E-cadherin+ cells was observed. Immunostaining at day 40. Representative data from 3 independent experiments are shown. Data show mean ± SEM; n = 3 per group; 1-way ANOVA (F = 0.22, df = 5, P = 0.95). (F and G) Abundance of apoptotic cells was evaluated by cleaved caspase-3 immunostaining. The number of apoptotic cells was significantly increased in oral ectoderm (E-cadherin+) derived from OTX2mut-iPSCs. Immunostaining at day 40. Representative data from 3 independent experiments are shown. Data show mean ± SEM; n = 3 per group. ***P < 0.001, 1-way ANOVA (F = 25.9, df = 5, P < 0.001) followed by post hoc Dunnett’s test. Post hoc comparison with control-iPSCs no. 1 (far left) is presented. O, oral ectoderm; H, hypothalamus progenitor. Scale bars: 100 μm.