Single-cell Transcriptome Study On The Development Of Macaque Embryonic Neocortex

The cerebral cortex is the biological basis of complex cognitive ability in mammals.The highly evolved cerebral cortex gives us far more intelligent features,higher-order perception,and thinking abilities than other species.Compared with rodent mammals,the cerebral cortex of higher primates shows multiple aspects of uniqueness.The multi-scale evolution results in a larger volume and surface area of the human neocortex and more diverse and complex types of nerve cells and synaptic connections.A series of biological events during development accompanies the generation of the embryonic cerebral cortex.Evolution strictly limits this regulation and is essential for the correct assembly and function of the neocortex.Research methods based on high-throughput transcriptomics have accelerated the understanding of gene expression programs related to brain structure and function in developmental states.Most of the research on neurodevelopment is based on mice as model animals.However,due to the considerable differences in anatomy,physiology,and nerve cell types between mouse and human brains,our understanding of the transcriptional regulation of human neocortex neurogenesis still needs to be improved.Macaque monkeys are more closely related to humans at the evolutionary level.The cortical development of the two has more standard features,including the complex composition of neural stem cells and a thicker shallow cortical hierarchy.In order to describe the temporal changes of stem cell differentiation to neuronal transcriptome during the development of advanced primate cortex represented by humans,we established an embryonic development model of macaque monkeys,covering the time point of cortical layer formation during the whole stage of embryonic neurogenesis(E40 is the beginning of neurogenesis,E50 is the formation of Layer6,E70 is the formation of Layer5,E80 is the formation of Layer4,E90 is the formation of Layer2 and Layer3).Pregnancy was terminated by cesarean section,and single-cell transcriptome sequencing was performed on the dorsal parietal cortex of the macaque fetal brain.Firstly,a single-cell transcriptome map of the parietal lobe of the neocortex of embryonic macaque monkeys was constructed using 53295 cell single-cell transcriptome data.It was confirmed that macaque monkeys mainly performed ’ neurogenesis’ in the early stage and began ’ gliogenesis ’ in the late stage of development,following a conservative cortical development model.The diversity of excitatory neuron subtypes in macaque monkeys was also found,which proved that the deep neurons of macaque monkeys were produced in the early stage of development,and the shallow excitatory neurons were produced in the late stage of development.With the LIGER analysis method,we integrated the single-cell data of macaque monkey embryonic cortical development generated in this study with some single-cell data sets of mouse and human embryonic cortical development in published studies.In the data,it was verified that the advanced primate neural stem cells oRG(marker genes HOPX,MOXD1,FAM107A,and CLU)were specific in human and mouse data.By inferring the temporal trajectory of neural precursor cells in three species,it is confirmed that the generation trajectory of intermediate precursor cells is conservative in all three species.The generation trajectory of oRG is unique to the human and macaque monkey datasets.Subsequently,by comparing the proportion of deep/upper excitatory neurons at different developmental stages between species,it was verified that the proportion of the upper layer neurons of higher primates belonging to humans and macaque monkeys in the cortical development stage was significantly higher than that of mice,a rodent model animal.By the temporal analysis of the expression of vRG(radial glial cells in the ventricle area)genes during the embryonic cortical development of the three species,the genes(DEGs)with significant differences in expression during cortical development were first screened out.According to the temporal expression trend,they were divided into five categories.Type1 and type2 were the strongest in the early stage,and then the expression intensity gradually decreased.Type 3 and type 4 were gradually increased and then decreased.The genes in type5 were gradually increased with development.We focused on the transcription factors and RNA-binding proteins.The expression of most transcription factors in 72 transcription factors has a similar temporal trend and is very conserved in vRG cells of three species,such as NEUROD1,NEUROG2,POU3F2,ETV1,ETV5,and FOS genes.It is proved that the gene expression of vRG differentiation is relatively conservative among humans,macaque monkeys,and mice.The transcription factor regulatory network of macaque monkey and mouse vRG(radial glial cells in the ventricle)was analyzed using scenic.The transcription factors with the highest regulatory activity at each time point were screened.The screened transcription factors and their regulated target genes constructed a protein interaction network.The results showed that the regulatory networks of the macaque monkey and mouse were not precisely the same.The classical developmental transcription factors,such as EOMES,NEUROD1,and EMX1,had typical roles in the regulatory networks of the two species.FOXG1 is essential in the regulation network of vRG transcription factors in macaque monkeys.FOXG1 may be related to the expansion of the primate neocortex,which provides clues for subsequent research.In summary,by sequencing and analyzing the single-cell transcriptome covering the formation process of macaque monkey embryonic cortical layers,we first analyzed the differentiation process of macaque monkey neural precursor cells from the molecular dynamics level.The results provide valuable data resources for analyzing the evolutionary characteristics of cortical development in higher primates and the application of macaque monkeys as non-human primate model animals in neurodevelopmental research species.