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CRISPR activation of DLX5 drives neural progenitors to the GnRH cell fate

ABSTRACT: Gonadotropin-releasing hormone (GnRH) neurons regulate the hypothalamic pituitary-gonadal axis and are required for puberty onset and reproductive competence. However, the transcriptional regulators governing GnRH neuron specification and migration remain poorly defined. The homeodomain transcription factor DLX5 is expressed in fetal human GnRH neurons, its expression precedes that of GNRH1 in human pluripotent stem cell (hPSC)-derived GnRH neurons, and in mice it serves as a guidance cue for GnRH neuron migration. We hypothesized that DLX5 may act as an upstream regulator of human GnRH neuron fate specification and migratory capacity. Using CRISPR activation, we upregulated DLX5 during FGF8b-directed differentiation of hPSCs to GnRH neurons via dual SMAD inhibition and Notch inhibition, as previously described. DLX5 activation increased neural progenitor motility (p<0.001), upregulated FGF8 (p<0.05), and induced GABAergic markers including GAD1 and GAD2. Notably, DLX5 activation induced GNRH1 in the absence of exogenous FGF8b (p<0.05), suggesting that, in GnRH neurons, DLX5 regulates FGF8. When combined with exogenous FGF8b, DLX5 activation produced distinct neuronal patterning accompanied by upregulation of extracellular matrix genes such as SPARC, which has been implicated in neurite outgrowth. Collectively, these data indicate that activation of DLX5 promotes GnRH neurogenesis from hPSCs by driving GABAergic fate, inducing FGF8, and remodelling the extracellular matrix.

ORGANISM(S): Homo sapiens

PROVIDER: GSE337839 | GEO | 2026/07/29

REPOSITORIES: GEO

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Project description:The olfactory sensory system is formed by the coordinated morphogenesis and differentiation of the peripheral olfactory epithelium (OE) and the anterior forebrain. At early stages, immature olfactory receptor neurons (ORN) elongate their axons to penetrate the brain basement membrane, contact and form synapses with projection neurons of the olfactory bulb primordium. Axonal elongation is accompanied by migration of the GnRH+ neurons, followed by their ingression in the septo-hypothalamic area of the forebrain. This process is specifically impaired in the KallmannM-bM-^@M-^Ys syndrome (KS), a disorder characterized by anosmia and central hypogonadism. A set of transcription factors are master regulators of olfactory connectivity and GnRH neuron migration. We explored the transcriptional network underlying this process, by profiling the OE and adjacent mesenchyme at distinct embryonic ages. We also profiled the OE from embryos null for Dlx5, a homeogene essential for olfactory development, that causes a KS-like phenotype when deleted. We also applied analysis of conserved co-expression to integrate the obtained data with information on KS disease genes. The prevalent categories of genes differentially expressed during development are neuronal differentiation, extracellular remodelling and cell adhesion. From the analysis of Dlx5 mutant tissues we identify about 120 genes with a prevalence of intermediate filaments, cell signalling, epithelial and neuronal differentiation. Filtering for true OE expression and for the presence of Dlx5 binding sites, yielded twenty genes, of the following categories: 1) transmembrane adhesion/receptor molecules, 2) axon-glia interaction molecules, 3) synaptic proteins, 4) scaffold/adapter for signalling molecules. To functionally analyze these genes in vivo, we used three zebrafish fluorescent reporter zebrafish strains, in which we monitored early phases of olfactory/GnRH development upon gene downmodulation. The depletion of three (of five) Dlx5 targets affected axonal extension and targeting, while two (of two) altered GnRH neuron position and neurite organization. In one experiment we compare the olfactrory sensory epithelium from wild-type embryos, at three times of development, i.e. E11.5, E12.5 and E14.5. In a second experiment we compare the olfactory sensory epithelium from wild-type embryo with that from Dlx5 knock-out embryos, at the age E12.5

Project description:The olfactory sensory system is formed by the coordinated morphogenesis and differentiation of the peripheral olfactory epithelium (OE) and the anterior forebrain. At early stages, immature olfactory receptor neurons (ORN) elongate their axons to penetrate the brain basement membrane, contact and form synapses with projection neurons of the olfactory bulb primordium. Axonal elongation is accompanied by migration of the GnRH+ neurons, followed by their ingression in the septo-hypothalamic area of the forebrain. This process is specifically impaired in the Kallmann’s syndrome (KS), a disorder characterized by anosmia and central hypogonadism. A set of transcription factors are master regulators of olfactory connectivity and GnRH neuron migration. We explored the transcriptional network underlying this process, by profiling the OE and adjacent mesenchyme at distinct embryonic ages. We also profiled the OE from embryos null for Dlx5, a homeogene essential for olfactory development, that causes a KS-like phenotype when deleted. We also applied analysis of conserved co-expression to integrate the obtained data with information on KS disease genes. The prevalent categories of genes differentially expressed during development are neuronal differentiation, extracellular remodelling and cell adhesion. From the analysis of Dlx5 mutant tissues we identify about 120 genes with a prevalence of intermediate filaments, cell signalling, epithelial and neuronal differentiation. Filtering for true OE expression and for the presence of Dlx5 binding sites, yielded twenty genes, of the following categories: 1) transmembrane adhesion/receptor molecules, 2) axon-glia interaction molecules, 3) synaptic proteins, 4) scaffold/adapter for signalling molecules. To functionally analyze these genes in vivo, we used three zebrafish fluorescent reporter zebrafish strains, in which we monitored early phases of olfactory/GnRH development upon gene downmodulation. The depletion of three (of five) Dlx5 targets affected axonal extension and targeting, while two (of two) altered GnRH neuron position and neurite organization.

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CRISPR activation of DLX5 drives neural progenitors to the GnRH cell fate

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