Defective jagged-1 signaling affects GnRH development and contributes to congenital hypogonadotropic hypogonadism
Ludovica Cotellessa, Federica Marelli, Paolo Duminuco, Michela Adamo, Georgios E. Papadakis, Lucia Bartoloni, Naoko Sato, Mariarosaria Lang-Muritano, Amineh Troendle, Waljit S. Dhillo, Annamaria Morelli, Giulia Guarnieri, Nelly Pitteloud, Luca Persani, Marco Bonomi, Paolo Giacobini, Valeria Vezzoli
Ludovica Cotellessa, Federica Marelli, Paolo Duminuco, Michela Adamo, Georgios E. Papadakis, Lucia Bartoloni, Naoko Sato, Mariarosaria Lang-Muritano, Amineh Troendle, Waljit S. Dhillo, Annamaria Morelli, Giulia Guarnieri, Nelly Pitteloud, Luca Persani, Marco Bonomi, Paolo Giacobini, Valeria Vezzoli
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Research Article Development Genetics

Defective jagged-1 signaling affects GnRH development and contributes to congenital hypogonadotropic hypogonadism

Abstract

In vertebrate species, fertility is controlled by gonadotropin-releasing hormone (GnRH) neurons. GnRH cells arise outside the central nervous system, in the developing olfactory pit, and migrate along olfactory/vomeronasal/terminal nerve axons into the forebrain during embryonic development. Congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome are rare genetic disorders characterized by infertility, and they are associated with defects in GnRH neuron migration and/or altered GnRH secretion and signaling. Here, we documented the expression of the jagged-1/Notch signaling pathway in GnRH neurons and along the GnRH neuron migratory route both in zebrafish embryos and in human fetuses. Genetic knockdown of the zebrafish ortholog of JAG1 (jag1b) resulted in altered GnRH migration and olfactory axonal projections to the olfactory bulbs. Next-generation sequencing was performed in 467 CHH unrelated probands, leading to the identification of heterozygous rare variants in JAG1. Functional in vitro validation of JAG1 mutants revealed that 7 out of the 9 studied variants exhibited reduced protein levels and altered subcellular localization. Together our data provide compelling evidence that Jag1/Notch signaling plays a prominent role in the development of GnRH neurons, and we propose that JAG1 insufficiency may contribute to the pathogenesis of CHH in humans.

Authors

Ludovica Cotellessa, Federica Marelli, Paolo Duminuco, Michela Adamo, Georgios E. Papadakis, Lucia Bartoloni, Naoko Sato, Mariarosaria Lang-Muritano, Amineh Troendle, Waljit S. Dhillo, Annamaria Morelli, Giulia Guarnieri, Nelly Pitteloud, Luca Persani, Marco Bonomi, Paolo Giacobini, Valeria Vezzoli

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Figure 8

Functional validation of JAG1 variants in vitro.

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Functional validation of JAG1 variants in vitro. (A) Representative West...
(A) Representative Western blot gels showing JAG1 and GAPDH expression in cell lysates of HEK293T cells transiently transfected with the empty vector (mock), with the JAG1 WT vector, or with each of the JAG1 variants. (B) Bar graph illustrates the mean ratio ± SEM JAG1 over GAPDH (n = 4 for all). Mann-Whitney U test followed by a Fisher’s exact test, *P < 0.05. a.u., arbitrary unit. (C) Confocal representative images of HEK293T cells transfected with JAG1 WT and JAG1 mutants and immunostained for JAG1 (green) and ConA (red). Nuclei were counterstained with DAPI (blue). Arrows point to the membrane JAG1 localization and arrowheads point to intracellular JAG1 retention. The experiments have been replicated 3 times. (D) Graph showing the percentage of JAG1 retention in the ER. Comparisons between treatment groups (n = 3 cultures/condition and n = 100 total cells analyzed/condition) were performed using a 1-way ANOVA followed by Tukey’s multiple-comparison post hoc test; *P < 0.05, **P < 0.005. Data represent mean ± SEM. Scale bar: 20 μm.