Scientists headed by a team at the Chan Zuckerberg Biohub San Francisco (CZ Biohub SF) reported on the development of Zebrahub, a dynamic, state-of-the-art atlas of zebrafish embryonic development. Zebrahub combines high-resolution time-lapse videos of newly emerging cells in zebrafish embryos with extensive data on which genes are switched on and off as individual cells migrate to their final positions, and take up the role that they will ultimately play in the body of the adult fish.

The atlas represents the latest advancement in the goal of tracing and mapping the behavior of every cell working together to create an adult lifeform, and presenting that map in a clickable, navigable display—a sort of Google Earth for developmental biology.

“Zebrahub offers one of the first opportunities to investigate the behavior of cells in the extremely complex process of development with extremely high precision,” said Merlin Lange, PhD, a CZ Biohub SF senior staff scientist. “It’s very rare to combine both gene expression from individual cells and spatial mapping of cells over time in the same resource like this.”

Zebrahub is freely available to researchers, and includes built-in analytical tools designed for biologists. Its creation required building a suite of new instruments and software. The resource represents the most comprehensive atlas of its kind and, as the researchers write in the paper, an important step toward “ushering in a new era for developmental and evolutionary biology.”

Commented Loïc Royer, PhD, leader of the Organismal Architecture Group and director of imaging AI at CZ Biohub SF, “How a lifeform goes from a single cell to an entire body is one of biology’s biggest mysteries. With Zebrahub, we’ve created possibly the most detailed map of that process ever.”

Lange is first author and Royer is senior author of the team’s published paper in Cell, titled, “A multimodal zebrafish developmental atlas reveals the state-transition dynamics of late-vertebrate pluripotent axial progenitors.”

To form a complex adult organism such as a human or fish, a fertilized egg must split into a set of progeny that continue dividing until millions of cells have been generated and assumed their roles as parts of the skin, liver, brain, and all the other components of the body. “The development of a fertilized egg into a multicellular organism requires the coordination of various processes, including genetic regulation, cellular movements, and tissue morphodynamics,” the authors noted.

CZ Biohub SF scientists Loïc Royer (left) and Merlin Lange work on a light-sheet microscope designed and built at the Biohub. [Dale Ramos]
CZ Biohub SF scientists Loïc Royer (left) and Merlin Lange work on a light-sheet microscope designed and built at the Biohub. [Dale Ramos]
While, for the most part, all cells of an embryo contain an identical set of genes, the way that each type of cell uses these genes—switching them on and off in different combinations at different time points—is unique. Scientists have long pondered just how the “choices” regarding thousands of genes in millions of cells come together to create a fully functioning adult lifeform with many types of specialized tissues. “A major challenge for developmental biology has been understanding the lineages of individual cells as they organize at various scales to form tissues and organs,” the investigators continued. Each advancement toward solving this daunting puzzle has yielded new insights about why the process sometimes goes wrong, leading to disorders and disease. “However, the community lacks a multimodal cartography of developmental lineages incorporating spatiotemporal and transcriptomics landscapes,” Lange et al., pointed out.

A freshwater species native to South Asia, zebrafish as adults rarely exceed two inches in length, and they are a long-established model for developmental research relevant to human health. Around 70% of human genes have counterparts in zebrafish and, although visually quite different, as vertebrates we share most of the same overall body plan, in addition to the cellular and molecular processes by which various body parts initially form. Critically, zebrafish embryos are mostly transparent and—unlike those of mice, for example—develop outside the mother, making it possible for scientists to observe their early growth in detail under a microscope.

Even with powerful models such as the zebrafish, developmental biology has historically been conducted in a piecemeal fashion, limited by the complexity of examining events that are far too tiny to see, and that happen in the millions across the bodies of living organisms that can be easily damaged by the experiments that are designed to understand them. There has been no comprehensive system for considering the whole instead of just the pieces.

With Zebrahub, researchers at CZ Biohub SF hope to help change that, accelerating the field by giving researchers easy access to the breadth of these processes, all in one place. Thanks to a new set of laboratory procedures developed at CZ Biohub SF, Zebrahub is also one of the first datasets of its kind to include gene expression data specific to individual embryos. The process of collecting such data has typically required that researchers pool together DNA from multiple embryos. This advance means that Zebrahub confers the added benefit of allowing scientists to investigate the subtle expression differences that might give rise to different health outcomes among sibling fish.

Zebrahub features two major datasets, along with a suite of tools designed to help biologists use them. The first offers time-lapse video microscopy showing the birth and early movements of most cells in a zebrafish embryo in the first 24 hours after fertilization, during which time organs start to form. The second provides data on which genes were active in more than 120,000 zebrafish cells at 10 separate time points during the embryos’ first 10 days. “… we combine light-sheet microscopy with scRNA-seq of individual developing zebrafish embryos to construct a comprehensive multimodal atlas of cell lineages and molecular states at spatio-temporal resolution,” the scientists explained. “By combining imaging and sequencing datasets, we joined the strengths of both modalities.”

To create the time-lapse videos, Royer, Lange, and CZ Biohub SF scientists and engineers designed and built “DaXi”  (pronounced “dah-shee”), a new kind of automated microscope with a field of view large enough to capture images of entire living embryos. DaXi is a light-sheet microscope that emits and captures light in a unique way designed to protect embryos from high-intensity laser beams that would damage or even kill the embryo after a short period of time. “To image and accurately track the movement of cells in densely populated and rapidly developing embryos, we employed a single-objective light-sheet microscope (DaXi) capable of high-resolution imaging (1.0 NA) over large volumes (>1 mm3) without the need for time-consuming sample preparation protocols,” the scientists noted.

Then, to allow scientists to easily use the captured videos to study specific cells, CZ Biohub SF software engineer Jordão Bragantini, PhD, led the development of a sophisticated new program called Ultrack, which automatically identifies cell nuclei and tracks their movements in the videos over time in three-dimensional space. “We used a deep-learning-based algorithm for nuclei segmentation and cell tracking algorithm tracking (Ultrack) to reconstruct high-resolution fate maps from single-cell tracks.”

Combined, the datasets generated by these tools allow researchers to conduct virtual experiments examining where cells begin and end up during development—even running their developmental trajectory backward and forward in time.

In developing this methodology the Zebrahub team has already made some intriguing discoveries. For example, the team looked at a subset of cells—neuro-mesodermal progenitors (NMPs)—in the embryo’s tail which, at the time points they examined, it had previously been thought would only be able to give rise to one type of tissue. However, as the Zebrahub researchers analyzed the cells’ movement and expansion, they realized these cells were developing into both muscle cells and neurons that were integrating into the spinal cord.

“This was a very unexpected finding,” Lange said. “And it’s the kind of thing that would be hard to confirm without the broad view that Zebrahub provides.” The authors further stated, “Zebrahub’s combination of datasets and technologies was instrumental in refining our understanding of lineages, helping answer finer questions on the dynamics of cellular fate … Besides NMPs, this resource will shed light on other important developmental processes. Zebrahub is poised to expand with more developmental stages, multiomic datasets, and ultimately, a consensus digital lineage reconstruction of multiple embryos.”

A project five years in the making, Zebrahub required the development of multiple new technologies and relied on experts in the fields of biology, engineering, optics, physics, and data science housed under the roof of CZ Biohub SF. Every piece of technology developed in the process is open source, which will contribute to more data being added to the project as the community works together to improve our view of embryo development.

“This kind of project would never be funded through conventional channels,” said Sandra Schmid, PhD, CSO of CZ Biohub SF. “But thanks to the Chan Zuckerberg Initiative’s innovative approach to building scientific institutes, Zebrahub has not only provided millions of measurements about embryonic development that anyone in the scientific community can access, it’s also exactly the kind of data needed to power new AI initiatives that stand to take us into the future of health science.”

Zebrahub has already helped support discoveries from other labs. One team that included researchers from Ashland University in Ohio and the State University of New York in Albany used Zebrahub in concert with their own cell atlas to ask which cellular proteins might contribute to the formation of cataracts in the eye. For this, the researchers relied on Zebrahub’s gene expression database to see when the cells of the lens activate and deactivate certain genes in a way that might lead to problems.

“Zebrafish are really small, and it’s really difficult for us to peel the lens apart in order to ask questions about what genes are working in this region and how one cell might be different from another,” said Mason Posner, PhD, a professor of biology at Ashland and co-senior author of the study. Here, “that’s already been done for us and we can get these deep understandings about, for example, how this tissue even becomes transparent and functions, essentially, as biological glass.”

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