Characterization of the earliest thalamocortical interactions in vivo and in vitro

内在因素和外在因素对大脑皮层发育的控制程度一直是持续研究的主题。虽然有大量的证据表明，早期控制的图案是内在的新皮层，一个主要的外部来源的图案是由丘脑-皮层传入（TCA），达到发展中的皮层在非常早期的阶段，神经发生和神经元迁移的高峰发生之前。我们感兴趣的是这种复杂的外部信号阵列，调节特定区域的皮质发育，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，特定区域的TCA如何开始影响皮层发育的生发区，并开始影响初始电路的形成在非常早期的阶段，当这种外在的贡献可能是基于主要的差异，在特定区域的皮层祖细胞和早期postmytotic神经元的发展计划在高级哺乳动物。早期传入的TCA到达的发育皮层的第一个隔室是亚板（SP）区，这是一个短暂的胚胎皮层隔室，在灵长类动物进化过程中在尺寸和复杂性上大大扩展，最终在人类中达到顶峰。该结构包含异质细胞群，有助于在皮质生成的早期阶段指导和区域靶向TCA。有趣的是，SP异常与人类发育障碍的发病机制有关，包括脑瘫、儿童癫痫、精神分裂症和自闭症。此外，最近在我们的实验室的观察表明，外室下区（oSVZ），一个独特的germinal区存在于高级哺乳动物，可能是另一个初始目标的TCA在皮质生成的初始阶段。这种联系的建立将为无眼人类和去核非人类灵长类动物模型中报告的初级视皮层神经元数量的急剧减少提供更好的解释，从而进一步证实了TCA在形成发育中的新皮层的最早区室中的关键作用。总之，这些观察结果提出了一个意想不到的主要作用的TCA在早期区域规范的假设-特别是在灵长类动物-和SP和oSVZ可能代表的解剖学相关的内在和外在的监管事件之间的相互作用。我们将通过研究细胞类型以及这些成分之间建立功能性相互作用的特定分子机制，来解决SP和oSVZ伴随TCA早期到达发育中的皮质而大量扩增的问题。为此，我们将在人类死后胎儿组织，胚胎猕猴组织（由我们的合作者提供）以及小鼠模型中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，并比较不同物种中涉及的主要过程。一旦我们确定了参与这种相互作用的细胞亚型，我们将进一步剖析其作用的分子途径，并验证在体外环境中获得的离体/体内数据。后一种方法首先需要在培养皿中通过器官培养来模拟人类（和小鼠）早期皮质生成的简化版本，并在项目的后期阶段通过更复杂的3D细胞模型（大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中获得的数据（和用于直接比较的离体小鼠样本），而且还将提供一种新的优化的人体早期丘脑-皮质相互作用的体外模型，未来可以通过在该系统上测试遗传操作和/或药理学治疗进行进一步分析。

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.