Conserved and divergent mechanisms of cortical development across mammalian brain evolution

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During vertebrate evolution, the mammalian brain underwent significant changes, such as the emergence of a six-layered neocortex and an expansion and diversification of interhemispheric connectivity. The subsequent emergence of the corpus callosum exclusively in eutherian mammals, after they diverged from marsupials and monotremes, was another notable evolutionary innovation that allowed the expansion of neocortical connectivity in large-brained species, such as humans. The correct development of the neocortex and its circuits is essential for sensory-motor and cognitive functions, and requires a precise orchestration of myriad events including the generation, migration and differentiation of neurons, as well as elongation and guidance of axons to their appropriate targets. Impairments of any of these processes can result in neurodevelopmental disorders in humans, hence the importance of understanding the basic mechanisms regulating these developmental steps to ensure healthy brain formation. Due to the absence in fossil records of soft tissue anatomical detail, such as the nervous system, many questions regarding the evolution and development of the brain remain unanswered. One way to investigate how the mammalian neocortex originated, expanded and evolved, is to adopt a comparative approach in extant species. This allows the identification of features that have been conserved or that have diverged across lineages.This thesis focuses on identifying key mechanisms regulating the development and evolution of the neocortex and its connections, combining experimental insights from mice, as a well-established model of eutherian brain development, and a similarly-sized marsupial, the Australian fat-tailed dunnart (Sminthopsis crassicaudata, Dasyuridae). By comparing the development of the neocortex and its circuits between therian mammals (marsupials and eutherians), this thesis also explores the molecular processes involved in the evolution of new brain structures, such as the corpus callosum. In order to address these questions, several methods from molecular, developmental and comparative neurobiology were employed, including gene transfection of specific neuronal populations via in pouch and in utero electroporation, as well as stereotaxic brain injections, assays of neurogenesis, immunofluorescence and advanced microscopy analyses.The first part of this thesis describes the characterisation of the fat-tailed dunnart as an innovative model system to study brain development and evolution. A developmental staging system, with a detailed characterisation of maturation features of dunnart brain and body, is presented in relation to mouse and human development. Then, cortical neurogenesis is investigated in dunnarts and compared to mice, concluding that, despite the postnatal and protracted development of the neocortex in marsupials, the main steps and patterns of neocortical formation are broadly conserved across therian mammals. Following this, the establishment of corticocortical connectivity in dunnarts is explored, including axonal elongation and targeting of long-range projection neurons. Finally, an analysis of the expression of major transcription factors known to direct projection fate in eutherians (i.e. SATB2 and CTIP2), as well as manipulatory experiments that altered their expression in dunnarts and mouse, reveals the existence of a broadly conserved and ancient program of molecular specification of commissural and subcerebral axonal fates during neocortical development of therian mammals.In summary, this thesis not only introduces the fat-tailed dunnart as an innovative model to investigate brain evolution and development, but also provides mechanistic insights into the main principles guiding mammalian brain formation and evolution. By comparing developmental processes in marsupial and eutherian species, this thesis concludes that key aspects of neocortical neurogenesis, neuronal projection fate determination, and targeting, many of which were previously assumed to be novel traits of eutherians related to the evolution of the corpus callosum, show remarkable conservation between mammalian lineages with different commissural routes. This evidence suggests that these conserved mechanisms play a critical role in the formation of functional and healthy cortical circuits in therian mammals, regardless of commissural route.