Interspecies blastocyst complementation generates functional rat cell-derived forebrain tissues in mice

Jia Huang, Bingbing He, Xiali Yang, Xin Long, Yinghui Wei, Yanxia Gao, Yuan Fang, Wenqin Ying, Zikang Wang, Chao Li, Yingsi Zhou, Shuaishuai Li, Linyu Shi, Fan Guo, Haibo Zhou, Hui Yang, Jun Wu

Preprint posted on 13 April 2023 https://www.biorxiv.org/content/10.1101/2023.04.13.536774v1

Pinky and the Brain live action. Growing a rat brain inside a brainless mouse via interspecies blastocyst complementation.

Selected by Martin Estermann

Categories: animal behavior and cognition, cell biology, developmental biology, evolutionary biology, molecular biology, neuroscience, physiology

Background:

One of the most striking differences between mice and rats is their size. An adult mouse usually is 12 to 18 cm long and weighs 15-30 g whereas a rat is about 20 to 25 cm long and weighs 200-350 g. In addition, rat organs are typically larger than mouse organs. For example, the rat brain volume is around 4 times bigger compared to the mouse. Organ size is known to be regulated by both genetic and environmental factors, but it is not clear if there is a specie-specific growth limit or whether it is plastic and depends on the available space in the body for organs to grow.

In recent years, different methodologies have been developed to study interspecies differences. Among them, blastocyst complementation has gained much interest lately. The first step in using blastocyst complementation technology is to generate an “organless” embryo. These are usually knockout mice for a determined gene (for example Pdx1) that causes the lack of a specific organ (for Pdx1: pancreas). This can also be achieved by the deletion of specific cell types using the diphtheria toxin. The second step then is to inject embryonic stem cells into the “organless” blastocysts, which colonize the empty organ niche and form the missing organ. This technique has been applied to generate the pancreas, thymus, kidney, and heart, among others.

The generation of an organless embryo is one of the most time-consuming steps and usually relies on the development of mouse knockout models. To speed up this process, the authors of this preprint generated a screening mechanism to evaluate the effect of directly knocking out genes in the zygote, using the CRISPR/Cas9 system, eliminating the requirement of a transgenic mouse model. Furthermore, the authors combined this technique with the incorporation of rat embryonic stem cells to obtain interspecies mouse chimeras with rat-derived brains.

Key findings:

1) Development of a quick system to screen genes for organ agenesis.

As the first requirement for using the blastocyst complementation technique is the generation of an organless embryo, the authors decided to speed up the genetic screening process to generate forebrain-less embryos. To achieve a complete gene knockout in one step, they injected multiple guide RNAs (4 for each of the 7 candidate genes) together with Cas9 mRNA. This resulted in a high knockout efficiency (nearly 100%), and only 3 of the 7 genes (Dkk1, Hesx1 and Six3) resulted in forebrain agenesis (Figure 1A). When mouse embryonic stem cells (mESC) were injected in Dkk1- and Hesx1-deleted blastocysts, they rescued the phenotype and resulted in forebrain development. Among them, in the Hesx1 knock out, mESC-derived cells showed specific localization in the forebrain but not in the midbrain, whereas in the Dkk1 knockout, mESC derived cells were also present in the midbrain, but not in the hippocampus (forebrain). This suggests that the Hesx1 knock out model is suitable for forebrain blastocyst complementation experiments.

2) Rat ES cells populate and form a forebrain in the brainless mouse.

Now that the Hesx1 knock out model turned out to be the best for the blastocyst complementation of the forebrain, the authors evaluated the effect of interspecies complementation, using (red) rat embryonic stem cells (rESC) (Figure 1A). Among all born pups (414), 96% showed a partial forebrain reconstitution and 16 pups (3.84%) showed total forebrain reconstitution (Figure 1B). There were no differences in forebrain size, thickness or cell density between the wild-type mouse, and both Hesx1 knockout with mouse or rat embryonic stem cells. This suggests that the size of the organ depends on the host environment (mouse) and not the donor (rat). In addition, there were no differences in the performance of these mice in different cognitive tests, suggesting normal brain function. Interestingly, the chimeric contribution of the rat cells into the forebrain decreased throughout embryonic developmental time, from 90-100% by E12.5 to 60% by E17.5. Furthermore, rat embryonic stem cell contribution to the rest of the mouse embryo resulted in a major reduction from 60% by 12.5 to 20% by E17.5.

Figure 1: Generation of a rat ESC-derived brain in a brainless mouse. (A) Schematic view of the steps involved in rat-mouse chimeras. Briefly, Cas9 and 4 guide RNAs targeting Hesx1 were injected in a mouse zygote to generate the Hesx1 knockout blastocysts. Blastocysts were injected with red-labelled rat embryonic stem cells. (B) Whole organ (left) or section (right) photos of the resulting brains from wild-type and Hesx1 knockout mice injected with rat ESC. Red fluorescence indicates rat ESC-derived brain cells.

3) Rat-derived brain cells behave like mouse cells but their transcriptome is similar to rat brain cells

Since the mouse forebrain develops faster than the rat forebrain, the authors wanted to evaluate if the developmental pace of the rat chimeric brains was similar to the host (mouse) or if they retain their slower developmental rate (rat). By E11.5, wild-type mouse and Hesx1 knockout mice complemented with mESC and rESC showed similar development, whereas the rat control showed a 2-day delay in development (Figure 2A). This suggests that the host environment pace overcomes the developmental rate of the donor cells, resulting in synchronized development. Surprisingly, transcriptomic analysis of the rESC-derived neurons resembled rat neurons rather than mouse neurons (Figure 2B), as shown by higher Pearson correlation between samples from the same species. This suggests that the donor cellular identity is maintained autonomously, at least at the transcriptomic level.

Figure 2: Mouse vs rat identity in chimeric brains. (A) Histology of the embryonic forebrain of wildtype mouse, rat and Hesx1 mouse knockout complemented or not with rat or mouse ESC, at two developmental timepoints (E9.5 and and E11.5). (B) Heatmaps showing Pearson correlations between wild-type mouse, rat and chimeric mouse or rat brain cells.

Why I chose this preprint:

As a developmental biologist I find chimera experiments really interesting, especially when it involves two different species with different developmental timings. Before reading this preprint, I was expecting that mice with rat-derived forebrains would develop larger brain structures than the control mice. I think this research is a great example of the cells’ plasticity to respond to extrinsic signals. It is fascinating that not only the size and structure, but also the developmental timings are controlled by the host animal. What is even more interesting is that these rat-derived cells, despite showing mouse-like phenotypes, maintained their original rat transcriptomic gene expression. In the future, I would love to see if this difference in the transcriptome can regulate or control forebrain function at a molecular level.

Future directions / questions for the authors:

Q1: Why do you think there is a reduction in the donor cells in the embryo after mid-gestation? Are they attacked by the immune system?

Q2: It is interesting that the donor cells in the forebrain did not decay as the ones in the rest of the body. Do you think this has to do with the necessity of the rat cells in the empty niche? Are they losing the competition against the host cells in the non-depleted organs? Or is it because the brain is an immune privileged site?

Q3: As the rat ESC-derived neurons are transcriptomically more similar to the rat neurons than the mouse ones, do you expect to see any differences in their function or electrophysiology?

Posted on: 21 May 2023

doi: https://doi.org/10.1242/prelights.34766

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Author's response

Jia Huang, Hui Yang and Jun Wu shared

Q1: Why do you think there is a reduction in the donor cells in the embryo after mid-gestation? Are they attacked by the immune system?

A: The development of the immune system commences during the embryonic phase, further evolving and maturing postnatally. It remains unclear regarding the potential reaction of the host’s still-maturing immune system during mid-gestation in the embryo, particularly in relation to whether it would launch an offensive attack against the xenogeneic donor cells. In the conducted experiments, rat cells were introduced into mouse blastocysts prior to the host immune system’s development. Given this timing, it is plausible that these injected cells would be recognized as native to the host during the development of the host immune system. The noted reduction in the donor cells is likely attributed to cell competition or other xenogeneic barriers arising during the mid-to-late stages of prenatal development. Numerous elements constitute these xenogeneic obstacles, hindering the path to achieving robust interspecies chimerism. These include cell competition, discrepancies in ligand-receptor compatibility, variation in the timing of differentiation, and differences in cell adhesion molecules, to name a few (PMID: 34132325). These factors obstruct the smooth integration and contribution of donor cells to the host’s developmental schemes, leading to their eventual elimination or expulsion from the embryo. Future studies that delve into these barriers with the aim to mitigate them hold the promise of accomplishing full forebrain interspecies blastocyst complementation.

Q2: It is interesting that the donor cells in the forebrain did not decay as the ones in the rest of the body. Do you think this has to do with the necessity of the rat cells in the empty niche? Are they losing the competition against the host cells in the non-depleted organs? Or is it because the brain is an immune privileged site?

A: We agree that the elevated percentage of rat cells observed in the forebrain can be attributed to the emptied developmental niche created by the deletion of Hesx1 in the host embryos. This deletion, in essence, lays out a welcome mat for the donor rat cells to settle and proliferate. However, when it comes to the remaining tissues and organs, the lowered donor cell contribution is likely due to competition from the host mouse cells, or other xenogeneic barriers as mentioned above.

Q3: As the rat ESC-derived neurons are transcriptomically more similar to the rat neurons than the mouse ones, do you expect to see any differences in their function or electrophysiology?

A: Although rats and mice exhibit a number of common electrophysiological traits, discrepancies in certain specific characteristics do exist. These differences span across a range of factors, including neuronal firing patterns, action potential duration, synaptic connectivity, and inherent neuronal excitability. That being said, it’s important to note that past research has suggested that the divergences between rat and mouse electrophysiological data are typically minimal (PMID: 33679362). Consequently, it presents a challenge to distinctly differentiate between rat and mouse nerve cells in terms of functionality and electrophysiology. In this study, we provide evidence that rat-derived neuronal cells demonstrate normal projection capabilities and possess the ability to form functional connections with mouse cells. Various behavioral tests, including the Morris water maze, fear conditioning, and the open-field test, showed no compromise in functionality. The results highlight the existence of a direct connection and effective interplay between neuronal cells originating from two distinct species.

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Interspecies blastocyst complementation generates functional rat cell-derived forebrain tissues in mice

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