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<p>Mutations in Delta Like Non-Canonical Notch Ligand 1 (<i>DLK1</i>), a paternally expressed imprinted gene, underlie central precocious puberty (CPP), yet the mechanism remains unclear. To test the hypothesis that <i>DLK1</i> plays a role in gonadotropin releasing hormone (GnRH) neuron ontogeny, 75 base pairs were deleted in both alleles of <i>DLK1</i> 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 <i>GNRH1</i> expression. Activation of (WT) <i>DLK1</i> 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 <i>DLK1</i> expression, during the FGF8 phase, significantly increased <i>GNRH1</i> expression and secretion of GnRH decapeptide. In conclusion, significant loss of DLK1 protein did not accelerate <i>GNRH1</i> 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 <i>DLK1</i> activation emerged as an important factor in determining GnRH neuron fate, with early expression disrupting GnRH lineage commitment and later activation enhancing <i>GNRH1</i> expression.</p> Graphical Abstract <p></p>

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Dual Role of DLK1 in GnRH Neuron Ontogeny

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年9月9日

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  • Nazli Eskici,
  • Celia Gomez-Sanchez,
  • Shrinidhi Madhusudan,
  • Kristiina Pulli,
  • Salla Keskitalo,
  • Tanja Turunen,
  • Kirsi Vaaralahti,
  • Yafei Wang,
  • Markku Varjosalo,
  • Taneli Raivio

摘要

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.

Graphical Abstract