Fibroblast growth factor 8 (FGF8), acting through the fibroblast growth factor receptor 1 (FGFR1), has an important role in the development of GnRH neurons. We hypothesized that FGF8 regulates human GnRH neuron differentiation in a time- and dose-dependent manner via FGFR1. Human pluripotent stem cells were differentiated to GnRH neurons with ten days of dual-SMAD inhibition, followed by FGF8 (25 ng/ml, 50 ng/ml or 100 ng/ml for ten days; or 100 ng/ml for two, four, six, and ten days), and Notch inhibition for five days. FGF8 induced expression of GNRH1 in a dose-dependent fashion and the duration of FGF8 exposure correlated positively with GNRH1 expression (P<0.05, Rs=0.49). However, two days of FGF8 (100 ng/ml) induced expression of its target genes such as FOXG1, ETV5, and SPRY2, and continued FGF8 induced dynamic expression of several genes. During FGF8 exposure, FGFR1 localized to the cell surface, and its specific inhibition with PD166866 reduced GNRH1 expression (P<0.05). In neurons, FGFR1 also localized to the nucleus. Our results suggest that dose- and time-dependent FGF8 signaling via FGFR1 is indispensable for human GnRH neuron ontogeny.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons lay the foundation for human development and reproduction, however, the critical cell populations and the entangled mechanisms underlying the development of human GnRH neurons remain poorly understood. Here, by utilizing our established human pluripotent stem cells-derived GnRH neuron model, we decoded the cellular heterogeneity and differentiation trajectories at the single-cell level. We found that a glutamatergic neuron population, which generated together with GnRH neurons, showed similar transcriptomic properties with olfactory sensory neuron and provided the migratory path for GnRH neurons. Through trajectory analysis, we identified a specific gene module activated along the GnRH neuron differentiation lineage, and we examined one of the transcription factors, DLX5, expression in human fetal GnRH neurons. Furthermore, we found that Wnt inhibition could increase DLX5 expression, and improve the GnRH neuron differentiation efficiency through promoting neurogenesis and switching the differentiation fates of neural progenitors into glutamatergic neurons/GnRH neurons. Our research comprehensively reveals the dynamic cell population transition and gene regulatory network during GnRH neuron differentiation.

Auxin-inducible degron (AID) technology is powerful for chemogenetic control of proteolysis. However, generation of human cell lines to deplete endogenous proteins with AID remains challenging. Typically, homozygous degron-tagging efficiency is low and overexpression of an auxin receptor requires additional engineering steps. Here, we establish a one-step genome editing procedure with high-efficiency homozygous tagging and auxin receptor expression. We demonstrate its application in 5 human cell lines, including embryonic stem (ES) cells. The method allowed isolation of AID single-cell clones in 10 days for 11 target proteins with >80% average homozygous degron-tagging efficiency in A431 cells, and >50% efficiency for 5 targets in H9 ES cells. The tagged endogenous proteins were inducibly degraded in all cell lines, including ES cells and ES-cell derived neurons, with robust expected functional readouts. This method facilitates the application of AID for studying endogenous protein functions in human cells, especially in stem cells.