Calcium dynamics tune timing of developmental transitions to generate evolutionarily divergent axon tract lengths

ABSTRACT The human brain has undergone an evolutionary expansion in size, both in terms of cell numbers and the size of cellular structures, including axon tracts. Human brain development also progresses slowly and takes particularly long. However, the functional relevance of slowed timing, and whether it is responsible for these changes in size, remains unknown. Here, we investigate this by studying axon tract development in human and mouse brain organoids. We demonstrate that human axon tracts grow ∼2x more slowly than those of mice, reflecting their slowed tempo, but that this actually leads to shorter human axons, not longer. To overcome the effect of slowed tempo, human axons have a more prolonged growth duration that enables them to project farther despite their slower growth rate. Using a combination of single-cell RNA sequencing and live imaging, we demonstrate that the prolonged duration involves a different mechanism to that controlling tempo and is driven by calcium dynamics. Human axons exhibit a reduced calcium influx compared to mouse, mediated by L-Type voltage-gated calcium channels. Stimulating this calcium influx in human neurons triggers earlier cessation of growth, leading to shorter axon tracts similar to those of mouse. We further show that increasing calcium speeds up the transition to the synaptogenesis stage. Thus, calcium regulation sets the timing of transitions to disproportionately extend developmental duration, thereby enabling evolutionary expansion of human neurons. Competing Interest Statement MAL is an inventor on patents covering cerebral organoids, and is co-founder and advisory board member of a:head bio. Footnotes Updated with new data, including scRNA-seq, new data analysis including pseudotime, and analysis of synaptogenesis.