Role of transketolase-like1 in human cortical neurogenesis

Transketolase-like 1 (TKTL1) is one of the few proteins with a single amino acid substitution found in almost all present-day humans but absent from extinct archaic humans, Neandertals and Denisovans, and other primates. This amino acid substitution in TKTL1 is a lysine in archaic humans but an arginine in modern humans. Modern human TKTL1 (hTKTL1), but not archaic TKTL1 (aTKTL1), increases the abundance of basal radial glia (bRG), the subtype of neural progenitor cells that is most efficient to generate neurons. The techniques presented in this chapter have been pivotal to understand the implication of TKTL1 in the development of the neocortex. The techniques are the following: (i) Mouse and ferret in utero electroporation of plasmids to induce TKTL1 expression in the neocortex and study its implication in progenitor cell behaviour; (ii) incubation of electroporated mouse hemispheres with pharmacological inhibitors of metabolic pathways (ex-vivo rotation culture) to decipher the implication of TKTL1 in the pentose phosphate pathway; (iii) incubation of human foetal neocortical tissues with these inhibitors (free floating tissue culture) to confirm the physiological role of these metabolic pathways in human; (iv) knocking-out hTKTL1 in human foetal neocortical tissue using ex vivo electroporation and CRISPR/Cas9 to study the physiological role of hTKTL1 in neocortical development; and (v) ancestralization of the hTKTL1 sequence to aTKTL1 in human embryonic stem cells, used to generate cerebral organoids.