Profiling data on the developing olfactory/GnRH system reveal cellular and molecular pathways potentially relevant for the Kallmann syndrome

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. In one experiment we compare the olfactory 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嗅觉感官系统由外周嗅觉上皮（peripheral olfactory epithelium, OE）与前脑前端协同完成形态发生与分化过程共同构成。在发育早期，未成熟的嗅觉受体神经元（olfactory receptor neurons, ORN）会延伸轴突以穿透脑基底膜，与嗅球原基的投射神经元建立接触并形成突触。轴突延伸过程伴随促性腺激素释放激素阳性（GnRH+）神经元的迁移，随后这些神经元会迁入前脑的隔下丘脑区。该发育过程在卡尔曼综合征（Kallmann's syndrome, KS）患者体内会出现特异性损伤，卡尔曼综合征是一种以嗅觉缺失与中枢性性腺功能减退症为特征的疾病。一系列转录因子是嗅觉连通性与GnRH神经元迁移的主调控因子。本研究通过对不同胚胎发育阶段的外周嗅觉上皮及其邻近间充质进行转录谱分析，解析了该过程背后的转录调控网络。我们还对Dlx5基因敲除胚胎的外周嗅觉上皮进行了转录谱分析；Dlx5是嗅觉发育必需的同源框基因，其缺失会引发类似卡尔曼综合征的表型。此外，我们通过保守共表达分析，将所得实验数据与卡尔曼综合征疾病基因的相关信息进行整合。发育过程中差异表达基因的主要功能类别包括神经元分化、细胞外重塑与细胞黏附。通过对Dlx5突变组织的转录谱分析，我们鉴定出约120个差异表达基因，其富集的功能类别集中于中间丝、细胞信号转导、上皮与神经元分化。进一步筛选真正在外周嗅觉上皮中表达且带有Dlx5结合位点的基因，最终得到20个候选基因，可分为以下类别：1）跨膜黏附/受体分子；2）轴突-胶质细胞相互作用分子；3）突触相关蛋白；4）信号分子的支架/衔接蛋白。为在体内对上述候选基因进行功能验证，我们使用了三种斑马鱼荧光报告品系，通过基因下调技术监测嗅觉与GnRH神经元发育的早期阶段。实验结果显示，5个Dlx5靶标基因中有3个的敲低会影响轴突延伸与靶向定位，而2个靶标基因的敲低则会改变GnRH神经元的位置与神经突组织形态。本研究包含两组对照实验：第一组比较了野生型胚胎在三个发育时间点（即胚胎日E11.5、E12.5与E14.5）的嗅觉感官上皮；第二组则比较了胚胎日E12.5时，野生型胚胎与Dlx5基因敲除胚胎的嗅觉感官上皮。