As a first step, we established a unified protocol allowing the generation of human, rhesus macaque and common marmoset brain organoids using the same components and protocol steps. Moreover, for the functional analysis of the candidate genes we established the electroporation of primate brain organoids and validated this protocol by electroporation of the previously characterized human-specific gene ARHGAP11B in chimpanzee brain organoids. We found that ARHGAP11B, similar to other model systems, also in brain organoids increases the type of progenitors which is thought to be key for the large and strongly folded human brain. We therefore established that the electroporation of primate brain organoids can be used to study the effects of genes on the activity and behavior of progenitor cells.
As the experiments for the identification of genes that are differentially active between human, rhesus macaque and common marmoset got delayed, we used a previously generated dataset, which allowed us to identify genes which are present in human but not in non-human primates and present in old-world monkey but not in new-world monkeys, which per definition means that these genes are differentially active between human and non-human primates and between old-world monkeys and new-world monkeys, respectively. This allowed us to study a ZNF, which is only active in human but not in non-human primates. We could show that this gene, when artificially being active in chimpanzee brain organoids, leads to increased cell divisions of progenitor cells while, when deactivated in human brain organoids, leads to reduced cell divisions of progenitors indicating a likely important role of this gene in human neocortex expansion.
Two other ZNF genes are currently in our analysis pipeline. These genes are differentially active between old- and new-world monkeys and could be important players for the pronounced neocortex size and folding differences between these two primate families.
