The six-layered neocortex is a neuroanatomical hallmark of all extant mammals. The massively enlarged human neocortex is thought to be largely responsible for the cognitive and behavioral abilities that set us apart from other animals. The evolution of this important structure is thus an enduring source of interest for neuroscientists and nonscientists alike. The brains of reptiles and birds, the closest living relatives of mammals, completely lack a morphologically defined neocortex. It has therefore been extremely challenging to determine whether reptiles and birds possess any brain structures that share an evolutionary relationship with the neocortex through common descent with modification. Harvey Karten first proposed that the dorsal telencephalon (DT) of reptiles and birds contains cells homologous to neocortical neurons found in particular layers. These conserved cell types include “input” cells found in neocortical layer 4 (L4) and “output” cells located in neocortical L5. In birds, these cells are found in clustered neuronal aggregates, or nuclei, that bear little morphological resemblance to neocortical layers. The Ragsdale laboratory previously tested Karten’s cell-type homology hypothesis in comparative molecular studies. They found that the input and output cells of mammals, birds, and reptiles share specific expression of molecular marker genes. These findings support the hypothesis of cell-type homology and suggest that the last common ancestor of amniotes had input and output cells in its DT. Over evolutionary time, these cell types were reorganized into very different structures in extant amniote groups. I took advantage of the unique nuclear morphology of avian DT for an unbiased, forward RNAseq search for genes that are conserved across amniote DT cell types. For these experiments, embryonic day 14 tissue from seven distinct districts was harvested from the DT of the chicken (Gallus gallus). I first sought to identify novel marker genes for avian DT input nuclei. Markers specific to input nuclei were tested for conservation in mouse neocortical L4. In addition to the previously known gene RORB, three additional transcription factors—RORA, NR0B1, and SATB1—were found to be enriched in avian DT input nuclei as well as in mouse neocortical L4. In contrast, almost all non-transcription factor molecules were found to be divergent in their expression patterns. These results strengthen the case for homology of DT input cells and identify a candidate gene regulatory network for DT input cell identity. In contrast, the extensive molecular expression differences between chick and mouse DT input territories may contribute to the massive divergence in their DT architectures. Guided by the observation that transcription factor genes are likely to be conserved at the cell-type level, I tested whether the avian mesopallium, an enigmatic DT structure, shares homology with any cell population in mammalian neocortex. Five transcription factor genes were identified that are highly enriched in both the chicken mesopallium and mouse neocortical intratelencephalic (IT) neurons, cell populations that were known to share extensive connectional similarities. I propose the novel hypothesis that IT cells are ancestral to amniotes and function in a conserved circuitry with input and output cells. These molecular findings on input cells and IT cells were extended to a reptile, the American alligator (Alligator mississippiensis). Transcription factor marker genes for input and IT cells were found to be expressed in both the crocodilian dorsal ventricular ridge and the cerebral cortex. I identified a clear reptilian mesopallium. The surprising finding that neocortical cell-type homologs are organized into neocortex-like layers in the alligator dorsal cortex indicates a previously unappreciated cellular and architectural complexity to reptile cerebral cortex.




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