New microscope offers a 4-D view of embryonic development in living mice

For the first time, researchers can now take a look inside a living mouse embryo and observe the formation of the intestine and the first timid heartbeat of heart cells. During a critical 48-hour period – when rudimentary organs begin to emerge – scientists can track each embryonic cell and accurately determine where it's headed, what genes it has activated and which cells it has encountered in course.

Philipp Keller, a physicist and biologist at the Janelia Research Institute of the Howard Hughes Medical Institute in Ashburn, Va., Said, "The new work is literally a whole-cell solution-solving construction plan for the entire mouse." He and his colleagues report the results on October 11, 2018 in the newspaper Cell. And they make the microscope and computer tools, built at Janelia, as well as all imagery data available and publicly available.

Kate McDole, developmental biologist at Janelia and co-author of the study, says Kate McDole, developmental biologist at Janelia. These resources are essential for scientists trying to develop or regenerate organs, or someday solve development problems that occur in the womb. "To do all this, you must first understand how organs are formed," she says. "You really have to see what's going on in a real embryo."

Looking inside

Until now, the best images of live embryos came from fish and flies. Ten years ago, Keller and his colleagues developed the first "digital embryo" of zebrafish, a kind of striped shiner often studied by scientists.

The researchers scanned the fish embryos with a plate optical microscope, which projected ultra-thin samples of laser light through samples, section by incremental section. Keller has designed computer programs to make sense of all imaging data. The result gave a high resolution overview of the first 24 hours of fish development.

Keller and Janelia's colleagues followed the work with a digital fruit fly embryo reported in the newspaper Nature methods In 2014, these animals are relatively simple to represent, especially zebrafish. They are transparent and insensitive to light, making it an "easy target for microscopy".

Mice are a different story. Keeping mouse embryos alive in the laboratory – even for short periods – requires an exhaustive list of conditions. Embryos must be kept sterile, for example; they must be immersed in a kind of nutritious soup; and the gas and temperature levels must be precisely controlled. In addition, cells are incredibly sensitive to light, tissues can be dense and opaque and the embryo can not be kept under the microscope. Instead, he is anchored to a single point, so he "drifts like a little balloon," says McDole.

Finally, during the period during which the researchers wanted to observe, six and a half to eight and a half days after fertilization, the embryo was growing by more than an order of magnitude – up to a maximum of six to one and a half days after fertilization. at almost three millimeters in diameter, about the length of a sesame. seed. For the microscope, the embryo is a moving target, whose size and position change constantly. Even a human encamped in the lab, adjusting the focus every five minutes for two days, could not capture sharp images of any embryo, Keller explains.

Her team took a different approach: she designed a microscope capable of doing all the work herself.

Smarter reach

At the center of Janelia researchers' microscope, a transparent acrylic cube houses the embryo imaging chamber. Two bright leaves illuminate the embryo and two cameras record images. These components allow researchers to spy on the once unheard world of organ development, revealing dynamic events with high-resolution details never before seen.

When the gut was formed, the team found that "it's not a slow, slow process," says McDole. "The whole thing gives in and makes a huge hole." And the neural tube, the structure that later forms the brain and spinal cord, tightens like a zipper, extending over the embryo.

The brain of the microscope is equipped with a suite of algorithms to track the position and size of the embryo. These algorithms map how the light sheet moves in the sample and then determine how to get the best images while keeping the embryo focused and centered in the field of view.

Since the embryo is constantly changing, the microscope must continually adapt and make decisions in milliseconds, over hundreds of images and at hundreds of different times. "I would not say that our microscope is smarter than a human," says Keller, "but he is able to do things that a human operator could not do."

New tools

The researchers collected nearly one million images for each embryo examined. Then they built a computer toolbox to reconstruct an arc image of each embryonic cell's development. A first step was to track each cell for 48 hours of imaging data. This was based on an improved cell tracking program that the team had originally developed for fly and zebrafish embryos. Associated with a program created by the call called statistical vector flow, researchers could work backwards to determine the origin of each eight-and-a-half-day-old embryo cell, says study co-author Léo Guignard. computer scientist Janelia. It's like drawing a map of the fate and history of each cell, he says.

Without these programs, it would have taken two to three years for a human to follow each cell, says Keller.

A range of other tools allowed the team to clarify the subtleties of gastrulation, when an embryo is transformed into a multilayered structure and early organogenesis. Janelia collaborators Andrew Berger, Srinivas Turaga and Kristin Branson have built a cell division detector that automatically records which cell is divided (and where and when). And Guignard developed a program to create a virtual "middle" mouse embryo, aligning four embryos together in space and time. (Doctor Who fans will recognize the name of the program, TARDIS, a nod to the spatio-temporal machine used by the fictional doctor.)

The new microscope is the sixth that the Keller team has developed over its eight years at Janelia; each comes with new and improved software tools. According to Keller, in many cases the fields of application "allow fundamentally new types of imaging experiments", such as observing the development of whole mouse embryos .

Their latest book addresses a fundamental question in biology. He explains, "How to go from a cell to an embryo?"

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More information:
Katie McDole, Léo Guignard, Fernando Amat, Andrew Berger, Grégoire Malandain, Loïc A. Royer, Srinivas C. Turaga, Kristin Branson and Philipp J. Keller. "In vitro imaging and reconstruction of post-implantation mouse development at the single-cell level." Cell. Posted online 11th October 2018. DOI: 10.1016 / j.cell.2018.09.031