Characterization of the earliest thalamocortical interactions in vivo and in vitro

内在因素和外在因素控制大脑皮层发育的程度一直是持续研究的主题。虽然有大量证据表明模式的早期控制是新皮层固有的，但模式的主要外部来源是丘脑-皮层传入事件（TCA），它在早期阶段到达发育中的皮层，在神经发生和神经元迁移的高峰发生之前。我们感兴趣的是调节区域特异性皮质发育的复杂外部信号阵列，以及它们与皮质祖细胞和神经元内在机制的相互作用。我们特别感兴趣的是区域特异性TCA如何开始影响生发区皮质生成，并在非常早期阶段开始影响初始电路形成，当这种外在贡献可能是高阶哺乳动物皮层祖细胞和早期mytotic后神经元区域特异性发育程序的主要差异的基础时。早期进入的tca到达发育皮层的第一个区室是亚板（SP）区，这是一个短暂的胚胎皮层区室，在灵长类动物的进化过程中，其大小和复杂性大大扩大，最终在人类中达到顶峰。这种结构包含一个异质的细胞群，它有助于在皮质发生的早期阶段引导和局部靶向TCA。有趣的是，SP异常与脑瘫、儿童癫痫、精神分裂症和自闭症等人类发育障碍的发病机制有关。此外，我们实验室最近的观察表明，外室下区（oSVZ）是高等哺乳动物特有的生发区，可能是皮质发生初始阶段TCA的另一个初始靶点。这种联系的建立将更好地解释在无眼的人和去核的非人类灵长类动物模型中报告的初级视觉皮层神经元数量的急剧减少，从而进一步证实TCA在形成发育中的新皮层的早期区室中的关键作用。综上所述，这些观察结果提出了一种假设，即TCA在早期区域规范中发挥了意想不到的重要作用——尤其是在灵长类动物中——SP和oSVZ可能代表了内在和外在调节事件之间相互作用的解剖学相关性。我们将通过研究细胞类型以及在这些成分之间建立功能相互作用所涉及的特定分子机制，来解决SP和oSVZ在TCA早期到达发育中的皮质时的大量扩张。为此，我们将对人类死后胎儿组织、猕猴胚胎组织（由我们的合作者提供）以及小鼠模型进行追踪分析和免疫组织化学染色，以覆盖整个早期皮质发育的广泛时间点，并比较不同物种所涉及的主要过程。一旦我们将描述这种相互作用所涉及的细胞亚型，我们将进一步剖析其作用的分子途径，并在体外环境中验证获得的离体/体内数据。后一种方法需要首先通过有机培养在培养皿中模拟人类（和小鼠）早期皮质生成的简化版本，然后在项目的后期阶段使用更复杂的3D细胞模型（脑类器官与丘脑类器官融合）。这将使我们不仅能够确认我们将在死后人体组织（和离体小鼠样本进行直接比较）中获得的数据，而且还将提供一种新的优化的人类早期丘脑-皮层相互作用的体外模型，通过测试该系统的遗传操作和/或药物治疗，可以在未来进行进一步的分析。

The degree to which intrinsic versus extrinsic factors control the development of the cerebral cortex has been the subject of sustained research. While there is a large body of evidence for an early control of patterning that is intrinsic to the neocortex, a major extrinsic source of patterning is provided by the thalamo-cortical afferents (TCA) that reach to the developing cortex at very early stages, before the peak of neurogenesis and neuronal migration take place. We are interested in this complex array of external signals that regulate region-specific cortical development, and their interplay with the mechanisms intrinsic to cortical progenitors and neurons. We are particularly interested in how area-specific TCA begin to influence corticogenesis in the germinal zone and start to influence initial circuit formation at very early stages, when this extrinsic contribution might underlie major differences in the region-specific developmental programme of cortical progenitors and early postmytotic neurons in higher-order mammals. The first compartment of the developing cortex reached by the early-incoming TCAs is the subplate (SP) zone, a transient embryonic cortical compartment which is greatly expanded in size and complexity during primate evolution, culminating in humans. This structure contains a heterogeneous population of cells and it contributes to the guidance and areal targeting of TCA at early stages of corticogenesis. Intriguingly, SP abnormalities have been implicated in the pathogenesis of human developmental disorders including cerebral palsy, childhood epilepsy, schizophrenia and autism. Moreover, recent observations in our laboratory suggest that the outer subventricular zone (oSVZ), a unique germinal zone present specifically in higher-order mammals, might represent another initial target of TCA during the initial phases of corticogenesis. The establishment of such connection would provide a better explanation to the dramatic reduction in neuronal numbers in primary visual cortex reported in anophtalmic human and enucleated non-human primate models, thus further confirming a crucial role of TCA in shaping the earliest compartments of the developing neocortex. Together these observations raise the hypothesis of an unexpected major role of TCA in early areal specification -especially in primates-, and the SP and oSVZ might represent the anatomical correlates of the interaction between intrinsic and extrinsic regulatory events. We will address the large expansion of the SP and oSVZ in concomitance with TCA early-arrival in the developing cortex, by investigating the cell types as well as the specific molecular mechanisms involved in the establishment of a functional interaction among these components. To this aim, we will perform tracing analysis and immunohistochemical staining in human post-mortem foetal tissues, embryonic macaque tissue (provided by our collaborators), as well as in mouse models, in order to cover a wide range of time-points throughout early cortical development and compare the main processes involved in different species. Once we will characterise the cellular subtypes involved in this interaction, we will further dissect the molecular pathways underlying its effect, and validate the data obtained ex-vivo/in-vivo in an in-vitro setting. The latter approach would entail modelling a simplified version of human (and mouse) early corticogenesis in a dish by organotipic cultures in the first place, and by more complex 3D cellular models (cerebral organoids fused with thalamic organoids) at a later stage of the project. This would allow us not only to confirm the data we will obtained in post-mortem human tissues (and ex-vivo murine samples for direct comparison) but it would also provide a novel optimized in-vitro model of human early thalamo-cortical interaction where further analyses could be done in the future, by testing genetic manipulations and/or pharmacological treatments on this system.