Examining the influence of lineage relationships upon excitatory neocortical development

The mammalian neocortex comprises a diverse population of excitatory neurons that perform distinct roles in the neocortical circuit. These neurons are born from a heterogeneous population of progenitor cells during embryonic development and it is increasingly being recognised that individual progenitors can impart specific functional characteristics to their offspring. For example, clonally-related sister neurons in mouse neocortex are biased to form gap junctions with one another early in development, to form synaptic connections with one another as they mature, and to show similar response properties in the adult. This highlights a fundamental role for neuronal lineage in the formation of precise neocortical connectivity. However, the extent to which neuronal phenotype is determined by lineage, and the process by which this arises, is not fully understood. Nor is it known whether similar lineage relationships exist in the human neocortex. Deciphering neocortical lineage relationships has been limited by the techniques available to identify and manipulate clonally-related neurons. In this thesis I have developed novel molecular tools for the identification and manipulation of clonally-related neurons and refined their use by in utero surgery in mice. I first developed a retrovirus encoding Cre recombinase and demonstrated that this can be successfully combined with reporter mice to capitalise on optogenetics for functional studies. In an effort to establish a reliable and unequivocal method of identifying clonally-related neurons, I then constructed a retrovirus encoding genetically-distinct RNA barcodes. I confirmed that the RNA barcode could be reliably retrieved from single neurons and used to determine clonal relationships in mouse neocortex and in an in vitro model of human neocortex derived from induced pluripotent stem cells (iPSCs). By comparing the dendritic morphology of barcoded mouse neocortical neurons, I was able to demonstrate that the dendritic arbor of clonally-related neurons is more similar than control neurons derived from different progenitors. This may contribute to specific patterns of synaptic connectivity amongst clonally-related neurons. Within the iPSC system, I demonstrate the utility of the retroviral RNA barcode and revealed that clonally-related human neocortical cells exhibit a higher probability of being gap-junction coupled. These studies advance our understanding of lineage relationships in neocortical excitatory neurons in mouse and provide the first evidence that human neocortical clones exhibit similar functional relationships to those observed in the rodent neocortex.