A new study introduced an efficient interspecies blastocyst complementation system and for the first time generated functional rat forebrain tissues in mice, while revealing the autonomous and non-autonomous influences on cellular development in the context of interspecies forebrain chimeric backgrounds. It has significant implications for understanding the mechanisms of brain development in a developmental context. 

This study was published in Cell on April 25, and was conducted by YANG Hui's group and ZHOU Haibo's group from the Center for Excellence in Brain Science and Intelligence Technology (CEBSIT) of the Chinese Academy of Sciences (CAS), WU Jun's group from the University of Texas Southwestern Medical Center, and GUO Fan's group from the Institute of Zoology of CAS,  

Using stem cells to generate functional tissues or organs has always been a hot topic in stem cell research. Interspecies blastocyst complementation allows for the cultivation of tissues or organs from one species within another. This technique involves injecting pluripotent stem cells into host blastocysts that lack key developmental genes, allowing donor cells to compensate for the missing tissues or organs, thereby cultivating organs derived from another species.  

Previous studies have cultivated rat pancreas, thymus, vascular endothelial tissues, and germ cells in mice, as well as mouse pancreas, kidneys, and germ cells in rats. However, brain tissues have not yet been cultivated in interspecies embryos. Cultivating brain tissues from one species in another could help researchers study brain development and function from an evolutionary perspective. 

Conventional blastocyst complementation methods involve constructing heterozygous gene-mutated mice that affect the development of specific organs, breeding these mice to produce genetically mutated blastocysts, and injecting donor cells into these mutated blastocysts to generate blastocyst complemented chimeras. This process is time-consuming and not suitable for lethal heterozygous genes. The conventional method of validating whether a specific gene can support an organ's blastocyst complementation is lengthy.  

To overcome these limitations, researchers introduced the cocktail of targeting sgRNAs in the CRISPR/Cas9 system (C-CRISPR) technique, which achieves nearly 100% gene knockout efficiency by using multiple gRNAs with Cas9, thus avoiding the need for genetically edited animals. Combining C-CRISPR with blastocyst complementation, they created a new system called C-CRISPR-based blastocyst complementation (CCBC), which allows for rapid verification of target genes suitable for blastocyst complementation and enables the one-step generation of organ reconstruction chimeras. 

Researchers identified the gene Hesx1 as capable of supporting forebrain complementation using the CCBC system. By knocking out the Hesx1 gene in mouse zygotes to prevent host cells from developing into the forebrain, and developing the zygotes to the blastocyst stage, they then injected either mouse or rat embryonic stem cells into the gene-mutated mouse blastocoel to compensate for the missing forebrain tissues. Through these methods, they obtained mouse-mouse forebrain complementation (Hesx1^-/- +mESCs) and rat-mouse forebrain chimeras (Hesx1^-/- +rESCs). 

All Hesx1^-/- +rESCs and Hesx1^-/- +mESCs chimeras survived to adulthood and showed a weight development curve similar to traditional chimeras (WT+mESCs). In the cortical layer V and hippocampus of these chimeras, cells expressing CTIP2 were detected. In the cortex and hippocampus, whether in Hesx1^-/- +rESCs, WT+mESCs, or Hesx1^-/- +mESCs chimeras, the layer thickness and cell density were similar. 

To assess the functionality of rat neurons in the Hesx1^-/- +rESCs chimeras, researchers injected AAV8-hSyn-EGFP into the anterior lateral motor cortex of the chimera's forebrain. The experiment showed that neurons derived from rESCs were able to project axons to the thalamus, superior colliculus, and brainstem. Electrophysiological tests indicated that rat and mouse cortical neurons in the chimera forebrain could fire action potentials as the current increased. Researchers also confirmed that synaptic connections could form between rat and mouse cells, mouse and mouse cells, and rat and rat cells in the Hesx1^-/- +rESCs chimeras.  

In addition, behavioral tests including the Morris water maze, open field test, and contextual fear memory test were used to assess the forebrain function of the same-species and interspecies forebrain compensatory chimeras. The test results showed no significant differences in performance among WT+mESCs, Hesx1^-/- +rESCs, and Hesx1^-/- +mESCs chimeras, indicating that the reconstructed forebrain functioned normally. These results suggested that the Hesx1^-/- mouse embryos provided a suitable developmental environment for donor rESCs to help form functional rat forebrain tissues. 

Besides, single-cell RNA sequencing results of the cortex and hippocampus showed that rat cells in the Hesx1^-/- +rESCs chimeras formed various types of neuronal cells with types and proportions similar to those in WT rat forebrain tissues. The transcriptomes of different neuronal cell types under rat cells were closer to WT rat cells. Interestingly, early embryonic slice results showed that the rat cells formed forebrain tissues with sizes and developmental progress consistent with the host mice. This suggested that non-cell-autonomous mechanisms determined the size and developmental speed of the organ, while cell-autonomous mechanisms shaped the overall transcriptomic characteristics of the rat forebrain tissues in the chimeras. 

By comparing the transcriptomes of rat cells in the chimeras with those in WT rats, researchers identified several differentially expressed genes related to axon generation, forebrain development, and neurogenesis regulation. Through cell interaction analysis, the study observed various potential ligand-receptor interactions, which might explain how host Hesx1^-/- mouse cells influenced donor rat cells and helped them survive and differentiate in the forebrain.