Gonadotropin-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.

Extracellular ATP and serotonin (5-HT) are powerful triggers of nociceptive firing in the meninges, a process supporting headache and whose cellular mechanisms are incompletely understood. The current study aimed to develop, with the neurosimulator NEURON, a novel approach to explore in silico the molecular determinants of the long-lasting, pulsatile nature of migraine attacks. The present model included ATP and 5-HT release, ATP diffusion and hydrolysis, 5-HT uptake, differential activation of ATP P2X or 5-HT3 receptors, and receptor subtype-specific desensitization. The model also tested the role of branched meningeal fibers with multiple release sites. Spike generation and propagation were simulated using variable contribution by potassium and sodium channels in a multi-compartment fiber environment. Multiple factors appeared important to ensure prolonged nociceptive firing potentially relevant to long-lasting pain. Crucial roles were observed in: (i) co-expression of ATP P2X2 and P2X3 receptor subunits; (ii) intrinsic activation/inactivation properties of sodium Nav1.8 channels; and (iii) temporal and spatial distribution of ATP/5-HT release sites along the branches of trigeminal nerve fibers. Based on these factors we could obtain either persistent activation of nociceptive firing or its periodic bursting mimicking the pulsating nature of pain. In summary, our model proposes a novel tool for the exploration of peripheral nociception to test the contribution of clinically relevant factors to headache including migraine pain.