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Deciphering the role of C2H2 zinc finger transcription factors during primate neocortex development and evolution

Periodic Reporting for period 1 - PRIMAZINC (Deciphering the role of C2H2 zinc finger transcription factors during primate neocortex development and evolution)

Okres sprawozdawczy: 2022-04-01 do 2024-09-30

The neocortex is a fascinating brain structure, as it is the seat of mammalian, and notably primate, higher cognitive abilities. Primates have exceptional large and folded neocortices. To understand how the neocortex evolved is a formidable question, which is also linked to questions like from where do come from and where will go? Size and the degree of folding of the neocortex is controlled by the activity and behavior of the progenitor cells, which produce the neurons and glia cells which mainly build the neocortex. To understand what makes the human neocortex different from other primates, we need to compare it to the neocortex of other primate species like rhesus macaque or common marmoset. In this project, we would like to identify genetic differences, which result in different activity and behavior of progenitor cells, which ultimately result in different neocortex morphology. Moreover, we will focus on a specific class of genes which code for Zinc finger proteins (ZNFs). These ZNFs can bind DNA and modulate the activity of other genes making them ideal candidates for a regulatory role.

To achieve this goal, we would like to
(i) identify ZNFs that are differentially active between human, rhesus macaque and common marmoset
(ii) functionally study ZNFs being differentially active in humans vs. non-human primates (rhesus and marmoset) for a role in controlling progenitor activity and behavior
(iii) functionally study ZNFs being differentially active in old-world monkeys (human and rhesus) vs. new-world monkeys (marmoset) for a role in controlling progenitor activity and behavior

While for the initial analysis of gene activity differences between different primate species (objective (i)) in vivo tissue is needed. All the further functional analyses (objectives (ii) and (iii)) will be performed in vitro, mainly by using brain organoids. Brain organoids are 3D multicellular cell aggregates generated from pluripotent stem cells, which model the cytoarchitecture and the cell type composition of certain brain region(s) during a certain developmental time window.
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.
We expect to identify more such ZNFs and refining our previous dataset by the experiments proposed in objective (i). For the ZNF, which is only active in human but not in non-human primates, we expect to identify the genes which are directly and indirectly controlled by this ZNF. This will allow us to identify the underlying regulatory network, how this ZNF controls the activity and behavior of progenitor cells. Moreover, we will confirm or reject a role in the regulation of progenitor activity and behavior for the other two ZNFs which are differentially active between old- and new-world monkeys. We will then identify, which genes are controlled by these two ZNFs and therefore their underlying regulatory network.

In summary the here described project should result in a better understanding of primate neocortex development and evolution, aiding to understand the origin and the development of our own human neocortex; This could lead to novel insights into the development of cortical malformations, likely allowing future improved genetic counselling and potential strategies for modifying pathophysiological neocortex development. In addition, this will likely provide a better knowledge of the peculiarities of neocortical development of non- human primate models, allowing to assess their suitability as advanced models of human physiological and pathophysiological neocortex development.