Neural organoids have the potential to improve our understanding of human brain development and neurological disorders. However, it remains to be seen whether these tissues can model circuit formation with functional neuronal output. Here, we have adapted air-liquid interface culture to cerebral organoids leading to improved neuronal survival and axon outgrowth. The resulting thick axon tracts display various morphologies including long-range projection within and away from the organoid, growth cone turning, and decussation. Single-cell RNA-sequencing reveals various cortical neuronal identities, and retrograde tracing demonstrates tract morphologies that match proper molecular identities. These cultures exhibit active neuronal networks, and extracortical projecting tracts can innervate mouse spinal cord and evoke contractions of adjacent muscle in a manner dependent on intact organoid-derived innervating tracts. Overall, these results reveal a remarkable self-organization of corticofugal and callosal tracts with a functional output, providing new opportunities to examine relevant aspects of human CNS development and disease.

Amyotrophic lateral sclerosis overlapping with frontotemporal dementia (ALS/FTD) is a fatal and currently untreatable disease characterized by rapid cognitive decline and paralysis. Elucidating initial cellular pathologies is central to therapeutic target development, but obtaining samples from presymptomatic patients is not feasible. Here, we report the development of a cerebral organoid slice model derived from human induced pluripotent stem cells (iPSCs) that recapitulates mature cortical architecture and displays early molecular pathology of C9ORF72 ALS/FTD. Using a combination of single-cell RNA sequencing and biological assays, we reveal distinct transcriptional, proteostasis and DNA repair disturbances in astroglia and neurons. We show that astroglia display increased levels of the autophagy signaling protein P62 and that deep layer neurons accumulate dipeptide repeat protein poly(GA), DNA damage and undergo nuclear pyknosis that could be pharmacologically rescued by GSK2606414. Thus, patient-specific iPSC-derived cortical organoid slice cultures are a reproducible translational platform to investigate preclinical ALS/FTD mechanisms as well as novel therapeutic approaches.

In terrestrial mammals, the respiratory turbinate bones within the nasal cavity are employed to conserve heat and water. In order to investigate whether environmental temperature affects respiratory turbinate structure in phocids, we used microcomputed tomography to compare maxilloturbinate bone morphology in polar seals, grey seals and monk seals. The maxilloturbinates of polar seals have much higher surface areas than those of monk seals, the result of the polar seals having more densely packed, complex turbinates within larger nasal cavities. Grey seals were intermediate; a juvenile of this species proved to have more densely packed maxilloturbinates with shorter branch lengths than a conspecific adult. Fractal dimension in the densest part of the maxilloturbinate mass was very close to 2 in all seals, indicating that these convoluted bones evenly fill the available space. The much more elaborate maxilloturbinate systems in polar seals, compared with monk seals, are consistent with a greater need to limit respiratory heat loss.

Three-dimensional neural organoids are emerging tools with the potential for improving our understanding of human brain development and neurological disorders. Recent advances in this field have demonstrated their capacity to model neurogenesis 1,2 , neuronal migration and positioning 3,4 , and even response to sensory input 5 . However, it remains to be seen whether these tissues can model axon guidance dynamics and the formation of complex connectivity with functional neuronal output. Here, we have established a longterm air-liquid interface culture paradigm that leads to improved neuronal survival and allows for imaging of axon guidance. Over time, these cultures spontaneously form thick axon tracts capable of projecting over long distances. Axon bundles display various morphological behaviors including intracortical projection within and across the organoid, growth cone turning, decussation, and projection away from the organoid. Single-cell RNAsequencing reveals the full repertoire of cortical neuronal identities, and retrograde labelling demonstrates these tract morphologies match the appropriate molecular identities, namely callosal and corticofugal neuron types. We show that these neurons are functionally mature, generate active networks within the organoid, and that extracortical projecting tracts innervate and activate mouse spinal cord-muscle explants. Muscle contractions can be evoked by stimulation of the organoid, while axotomy of the innervating tracts abolishes the muscle contraction response, demonstrating dependence on connection with the organoid. Overall, these results reveal a remarkable selforganization of corticofugal and callosal tracts with a functional output, providing new opportunities to examine relevant aspects of human CNS development and response to injury.

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