Dual Role of DLK1 in GnRH Neuron Ontogeny

Mutations in Delta Like Non-Canonical Notch Ligand 1 (DLK1), a paternally expressed imprinted gene, underlie central precocious puberty (CPP), yet the mechanism remains unclear. To test the hypothesis that DLK1 plays a role in gonadotropin releasing hormone (GnRH) neuron ontogeny, 75 base pairs were deleted in both alleles of DLK1 exon 3 with CRISPR-Cas9 in human pluripotent stem cells (hPSCs). This line, exhibiting More than 80% loss of DLK1 protein, was differentiated into GnRH neurons by dual SMAD inhibition (dSMADi), FGF8 treatment and Notch inhibition, as previously described, however, it did not exhibit accelerated GNRH1 expression. Activation of (WT) DLK1 with CRISPRa during dSMADi suppressed the formation of anterior neuronal precursors, and almost completely inhibited GnRH neuron fate. Instead, it induced the expression of Wnt pathway genes, markers of proliferating and non-proliferating neuronal progenitors, and dorsal spinal cord interneurons. Activation of DLK1 expression, during the FGF8 phase, significantly increased GNRH1 expression and secretion of GnRH decapeptide. In conclusion, significant loss of DLK1 protein did not accelerate GNRH1 expression, suggesting that CPP caused by paternally inherited loss-of-function mutations is due to mechanisms acting up- or downstream of GnRH neurons. Conversely, timing of DLK1 activation emerged as an important factor in determining GnRH neuron fate, with early expression disrupting GnRH lineage commitment and later activation enhancing GNRH1 expression.