LIFE SCIENCE

Million-cell study reveals ‘error correction potential’ for embryonic development


The path of nematode embryonic development. From the center to the outer edge of the embryonic cells continue to divide, towards maturity. Courtesy of Du Zhuo’s team

Life development is often not always smooth. Many embryos will have various types and different degrees of abnormal cell behavior at different stages of development, but this will not affect the eventual survival of the embryo. What is the reason behind this?

Using single-cell high-precision real-time tracking technology to track C. elegan embryonic cells, Du Zhuo’s team, a researcher at the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences, found that the error correction potential of embryonic development can give the biological process stability. The study was published online July 25 in the journal Cell Systems.

“This remarkable study, which used in vivo imaging to analyze the cell phenotypes of more than a million nematode embryonic cells under 750 gene knock-down conditions, highlighting the high robustness of the developmental system.” Some of the findings are highly original. One reviewer commented.

Shifting the way of thinking in “knocking and knocking”

At the beginning, Du Zhuo and his team did not study how embryonic development corrected errors, they just wanted to systematically understand the phenotype (individual morphology, function, etc.) of some genes of nematodes.

In the 1970s and 1980s, scientists revealed the cell map of the entire development process of nematodes, a multicellular animal, making it a leading model animal for high-precision analytical dynamic regulation of development. But the function of genes involved in regulating development has not been fully elucidated.

To this end, Du Zhuo’s team plans to use gene knockdown to understand the systematic phenotype of some conserved genes. “In biological research, knockout is to make the target gene completely absent, involving the construction of mutants with a relatively long cycle; Knockdown is that the target gene is still present, but the expression is reduced, the research efficiency through RNA interference technology is relatively high, and the nematodes are particularly effective and widely adopted. Du Zhuo, the corresponding author of the study, explained to China Science Daily.

Knocking down the function of hundreds of genes is not difficult for the research team. Du Zhuo said that the difficulty lies in how to carefully and accurately understand the phenotype of a gene during embryonic development. Development is a continuous, dynamic process, and continuous observation and phenotypic analysis with high spatio-temporal resolution are time-consuming and laborious tasks.

In particular, subtle changes in phenotype are complex processes that cannot be observed with the naked eye. The research team had to do an image every knockdown, knocking out hundreds of genes required thousands of images and analysis, and needed to cover a variety of cells to obtain phenotype data.

“This is also the reason why it is difficult to advance related research in the past, because there is no corresponding data to judge the phenotypic changes too subjectively.” Du Zhuo said.

To this end, the research team has established a set of high-precision cell tracking methods, using protein fluorescent labeling and four-dimensional real-time in vivo imaging technology to track the development of nematode embryos at the cellular level in situ, in real time, continuously and on a large scale. By comparing it to unclaptilized nematodes, they wanted to understand whether and how the knockdown occurred at a certain stage of development.

They spent more than three years collecting and analyzing data, and constantly found that some phenotypes after knocking down were “intermittent” – the phenotypes (i.e., cellular abnormalities) that appeared at different time nodes at the beginning disappeared later. For example, at a certain stage of development of nematodes embryos, the localization of specific cells is significantly biased, but the localization of offspring cells after division returns to normal. At the same time, nematode gene expression has a strong time specificity, and when the cell division time is delayed, it can theoretically lead to a relatively early gene expression. However, in many cases this does not happen.

“The early embryos were developed wrongly, why didn’t the late ones exist?” This made Du Zhuo and his team realize that embryonic development may have an active error correction function.

As the relevant data continued to accumulate, after doing about 750 gene knockdowns, the research team embarked on another idea: systematically studying whether embryonic development will correct errors and how to correct errors.

From “Danger” to “Turning Danger into Disaster”

“The so-called stability, that is, the ability of organisms to maintain their relatively normal state under certain genetic and environmental disturbance conditions, is of great significance for coping with genetic mutations and adapting to environmental changes.” Xiao Long, the first author of the study and a postdoctoral fellow in Du Zhuo’s team, explained.

Previous studies have shown that many molecular mechanisms can give stability to biological processes, such as random gene expression, gene function dose compensation, feedback regulation, etc. However, complex processes such as the development of multicellular organisms are regulated at multiple levels, and the manifestations and regulation methods of stability at the level of cells, tissues, organs and individuals still need to be systematically studied.

To explore the extent to which perturbations of individual gene function can induce abnormal developmental behavior of cells, the research team quantified the spatiotemporal development phenotype group of single-cell resolution under conditions of partial inactivation of about 750 genes. The results show that the embryonic development process can be described as “dangerous”: more than half of the genes are partially missing their functions, which can lead to significant abnormal cellular behavior.

According to reports, nematodes have about 20,000 genes, embryonic development produces more than a thousand cells, and this study involves half of the cells of nematode embryos. The authors say that because the RNA interference only achieved limited knockdown, and the study did not cover the full phenotype of all cells, it showed that the loss of the function of a single conserved gene can often induce developmental abnormalities in specific cells.

Life goes on and on and on. The picture shows the process of several divisions of cells in the early stages of nematodes embryos. Courtesy of Du Zhuo’s team

It is found that the relevant developmental abnormalities exist in cell cycle length, division asynchrony, asymmetric division, neural differentiation report gene expression status, cell division angle and spatial localization. “This shows that the molecular regulatory network, although solid as a whole, can often be ‘broken’ in specific cells, appear abnormal, and potentially threaten embryonic development.” Xiao Long said that this discovery also shows that the functionality and stability of molecular regulatory networks are balanced and balanced, and it is difficult to have both, and different cells have different choices.

“Surprisingly, this study found that many of the hatched embryos carried a large number of cellular-level phenotypic abnormalities.” One reviewer said. He also said that the highlight of the study is the analysis of new mechanisms for the regulation of stability, and found that a variety of abnormalities in early cells can often be “corrected” in advanced embryos in a variety of ways.

So, how do phenotypic abnormalities in embryonic development “turn the risk into ruin”? Studies have found that it can be corrected and mitigated in a number of ways. The authors mainly look at three of these approaches.

First of all, development is a dynamic process of continuous inheritance and development, and a momentary mistake does not mean a permanent mistake. For example, changes in cell division time, abnormal gene expression, and abnormal cell spatial localization, a certain proportion of behavioral abnormalities of contemporary cells can be alleviated or eliminated in their offspring cells.

Second, the potential effects of one cell behavior abnormality are mitigated by changing another. For example, when the cell division time is delayed, the time of gene expression can be kept in harmony with the delayed cell division, eliminating harmful hidden dangers to a certain extent.

In addition, the study defined clusters of cells that are related to developmental origin and function, and found that multicellularity and cell sociality help mitigate abnormalities in cell behavior. For example, even if the spatial localization of some cells is seriously abnormal, the cell cluster will make the cell arrangement tend to be normal as a whole through “internal adjustment”, keeping the cell state relatively stable.

“Each of these findings is very interesting and meaningful.” In particular, although the overall change in cell position occurs, it is relatively “normal” inside function-related cells, suggesting that function-related cells have a relatively normal mechanism to protect cell positions.

Turning “improvisation” into “proactive”

What is the significance of discovering that embryos have error correction mechanisms? Xiao Long said that although the behavior and fate of cells during the development of nematodes have long been considered highly solidified and constant, the study shows that even nematode embryonic cells using a “fixed” developmental procedure are still highly malleable and can be used to cope with developmental abnormalities.

“As with the previous work of Du Zhuo Labs, such high-quality and innovative research data provide valuable information for nematode embryonic development studies, and even broader studies on developmental stability.” Another reviewer said.

Du Zhuo hopes that by studying how embryonic development “adapts” to phenotypic errors, it can help the cognition of disease complexity and help the “initiative” of some disease treatment in the future. For example, if some individuals in populations carrying the same pathogenic mutation often do not exhibit a disease phenotype, is it that the cellular-level compensation mechanism in these individuals participates in the correction of pathogenic effects, resulting in incomplete phenotypic manifestations? Exploring these possibilities may help in the treatment of some diseases. In addition, cell clusters help alleviate abnormalities in cell behavior, is it possible to achieve some disease phenotype mitigation by reinforcing this coordination mechanism?

However, he admits that it is still far from this step. The current study only preliminarily reveals possible strategies for coping with abnormal cell developmental behavior in various forms, and expands the understanding of multi-level regulation of developmental stability. How do cells perceive behavioral abnormalities? How do you induce different types of compensatory behavior? How are molecules and cells involved? These issues still need to be further explored.

Xiao Long, a postdoctoral fellow in Du Zhuo’s research group, is the first author of the paper, Fan Du Changjiang, Qi Huan and Cong Yulin have made important contributions to the research, and researcher Du Zhuo is the corresponding author of this paper. The research was funded by the Key R&D Program of the Ministry of Science and Technology, the National Natural Science Foundation of China and the China Postdoctoral Science Foundation. (Source: China Science Daily Feng Lifei )

Related paper information:https://doi.org/10.1016/j.cels.2022.07.001



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