Virtual Embryo Predicts Real Gene Expression Patterns, Cell by Cell

By the time an embryo starts to show form and structure, every cell in the embryo “knows” its fate, whether it is to become a brain cell, a heart cell, or any other kind of cell. It already has, at this early stage of development, a unique transcriptional identity, one that reflects the cell’s position within the embryonic whole, one that follows a transcriptional blueprint.

Now scientists are finally getting a peek at transcriptional blueprints, starting with the spatial gene expression patterns for Drosophila melanogaster. The scientists, based at the Max Delbrück Center (MDC), have analyzed the unique gene expression profiles of thousands of single cells and reassembled the embryo from these data using a new spatial mapping algorithm. The result is a virtual fly embryo showing exactly which genes are active where at a crucial stage in morphogenesis, stage 6, the onset of gastrulation.

The D. melanogaster embryo, the MDC scientists explained, has been an exquisite model for the patterning principles that shape cellular identities: “The fertilized egg undergoes 13 rapid nuclear divisions resulting in a syncytial embryo of about 6000 nuclei. By developmental stage 5, nuclei have moved to the embryo periphery, become surrounded by cell membranes, and spatial gene expression patterns emerge as cells translate anteroposterior and dorsoventral positional information into transcriptional responses. Stage 6 is marked by the first morphogenetic movements after cellularization completes.”

To capture patterning principles, the MDC scientists devised a computational mapping strategy called DistMap. Using this strategy, the scientists reconstruct the embryo to predict spatial gene expression approaching single-cell resolution. Details of this work appeared August 31 in the journal Science, in an article entitled “The Drosophila Embryo at Single-Cell Transcriptome Resolution.”

“We produce a virtual embryo with about 8000 expressed genes per cell,” the article’s authors wrote. “Our interactive “Drosophila-Virtual-Expression-eXplorer” (DVEX) database generates three-dimensional virtual in situ hybridizations and computes gene expression gradients.”

Essentially, the MDC team computed genome-wide spatial gene expression patterns from single-cell transcriptome data alone. In their paper, the MDC researchers describe a dozen new transcription factors and many more long noncoding RNAs that have never been studied before. Also, they propose an answer to a question that has puzzled scientists for 35 years: How does the embryo break synchronicity of cell divisions to develop more complex structures?

During gastrulation, distinct germ layers form and cells become restricted with regard to which tissues and organs they may differentiate into. “We believe that the Hippo signaling pathway is at least partly responsible for setting up gastrulation,” said Nikolaus Rajewsky, Ph.D., one of the study’s senior authors. This pathway controls organ size, cell cycles, and cell proliferation, but had never been implicated in the development of the early embryo.

“We not only showed that Hippo is active in the fly, but we could even predict in which regions of the embryo this would lead to a different onset of mitosis and therefore break synchronicity,” he continued. “And that is just one example for how useful our tool is to understand mechanisms that have escaped traditional science.”

When the researchers started creating the virtual embryo, they did not know whether it would be possible. A key pillar of their eventual success is the Drop-Seq technology, a droplet-based, microfluidic method that allows the transcriptional profiling of thousands of individual cells at low cost. To process the data collected via Drop-Seq, the MDC team deployed the DistMap algorithm, which mapped transcriptomic data of cells back to their original position in the virtual embryo.

The construction of the virtual embryo allowed the scientists to predict the expression of thousands of genes, an almost impossible task by traditional experimental means. Finally, the scientists validated these predictions by visualizing the gene expression profiles at the bench with a traditional approach: In situ hybridization allows visualizing patterns of gene expression with colorful dyes that are visible under the microscope.

It is the first time that it has been possible to look at the about 6000 cells of the embryo individually, assess their gene expression profiles—and understand what determines their behavior in the embryo. “The most important technological advance of this study is that we don’t lose the spatial information that is required to understand how embryonic cells act in concert,” commented the scientists. “This really is unchartered territory and requires new bioinformatics approaches to make sense of the collected data.”

The scientists are already planning follow-up projects. One example would be to map the cells at different time points to see how they work together to form organs and tissues. Another would be to check whether the mapping approaches are applicable to more complex tissues.