Abstract
ABSTRACTGonadotropin-releasing hormone (GnRH) neurons provide a fundamental signal for the onset of puberty and subsequent reproductive functions by secretion of gonadotropin-releasing hormone. Their disrupted development or function leads to congenital hypogonadotropic hypogonadism (CHH). To model the development of human GnRH neurons, we generated a stable GNRH1-TdTomato reporter cell line in human pluripotent stem cells (hPSCs) using CRISPR-Cas9 genome editing. RNA-sequencing of the reporter clone, differentiated into GnRH neurons by dual SMAD inhibition and FGF8 treatment, revealed 6461 differentially expressed genes between progenitors and GnRH neurons. Expression of the transcription factor ISL1, one of the top 50 most upregulated genes in the TdTomato-expressing GnRH neurons, was confirmed in 10.5 gestational week-old human fetal GnRH neurons. Among the differentially expressed genes, we detected 15 genes that are implicated in CHH and several genes that are implicated in human puberty timing. Finally, FGF8 treatment in the neuronal progenitor pool led to upregulation of 37 genes expressed both in progenitors and in TdTomato-expressing GnRH neurons, which suggests upstream regulation of these genes by FGF8 signaling during GnRH neuron differentiation. These results illustrate how hPSC-derived human GnRH neuron transcriptomic analysis can be utilized to dissect signaling pathways and gene regulatory networks involved in human GnRH neuron development.This article has an associated First Person interview with the first author of the paper.
Highlights
Hypothalamic gonadotropin-releasing hormone (GnRH) neurons are a central part of the hypothalamus-pituitary-gonadal (HPG) axis that regulates reproduction
We have recently reported a protocol for the generation of GnRHexpressing and -secreting neurons from human pluripotent stem cells, in which treatment with FGF8 at the neuronal progenitor stage leads to high expression of anterior neuronal markers such as FOXG1 and, after further differentiation, these cells give rise to GnRH-expressing neurons (Lund et al, 2016)
These results indicate that TdTomato-expressing neurons successfully mark the formation of GnRH neurons during differentiation from human pluripotent stem cells (hPSCs)
Summary
Hypothalamic gonadotropin-releasing hormone (GnRH) neurons are a central part of the hypothalamus-pituitary-gonadal (HPG) axis that regulates reproduction. GnRH secretion is periodically activated during fetal development, postnatally during ‘minipuberty’, and during adolescence (Kuiri-Hanninen et al, 2014; Young et al, 2019). GnRH neurons are gradually reactivated, which increases pituitary gonadotropin and gonadal sex steroid production. Disruption of GnRH signaling, secretion or GnRH neuron development can lead to congenital hypogonadotropic hypogonadism (CHH), which is characterized by partial or absent puberty, incomplete development of sexual characteristics and often infertility. Patients with CHH combined with anosmia/hyposmia (absent or reduced sense of smell) acquire Kallmann Syndrome (KS) diagnosis (Boehm et al, 2015; Young et al, 2019). Half of the patients can be diagnosed at the molecular level, which suggests the existence of numerous unknown genetic and biological mechanisms controlling puberty
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