Contact: dkenney@mbl.edu; 508-685-3525

C. elegans embryo imaged with the diSPIM light sheet microscopy system. The nerve ring neuropil is the bright ring structure at the top of the embryo. Credit: Mark Moyle et al., Nature, 2021.

WOODS HOLE, Mass. -- Understanding how the brain works is a paramount goal of medical science. But with its billions of tightly packed, intermingled neurons, the human brain is dauntingly difficult to visualize and map, which can provide the route toward therapies for long-intractable disorders.

In a major advance published this week in Nature, scientists for the first time report on the structure of a fundamental type of tissue organization in brains, called neuropil, as well as the developmental pathways that lead to neuropil assembly in the roundworm C. elegans. This multidisciplinary study was a collaboration between five laboratories, including scientists at the Marine Biological Laboratory (MBL), Woods Hole.

“Neuropil is a tissue-level organization seen in many different types of brains, from worms to humans,” says senior author and MBL Fellow Daniel Colón-Ramos of Yale School of Medicine. “When things are that conserved in nature, they are important.”

“But trying to understand neuropil structure and function is very challenging. It’s like looking at a spaghetti bowl,” Colón-Ramos says. “Hundreds of neurons are on top of each other, touching each other, making thousands of choices as they intermingle through different sections of the animal’s brain. How can you describe neuropil organization in a way that’s comprehensible? That is one of the contributions of this paper.”

The authors focused on the neuropil in the C. elegans nerve ring, a tangled bundle of 181 neurons that serves as the worm’s central processing unit. Through an innovative melding of network analysis and imaging strategies, they revealed that the nerve ring is organized into four layers, or strata. These strata, they showed, contain distinct domains for processing sensory information and motor behaviors. They were able to map the worm’s sensory organs and muscle quadrants onto the relevant strata.

The team also discovered unique neurons that integrate information across strata and build a type of “cage” around the layers. Finally, they showed how the layered structure of the neuropil emerges in the developing worm embryo, using high-resolution light-sheet microscopy developed by MBL Fellow Hari Shroff of the National Institute of Biomedical Imaging and Bioengineering, and MBL Investigator Abhishek Kumar.

“This is a paradigm shift where we combined two fields – computational biology and developmental biology – that don’t often go together,” says first author Mark Moyle, associate research scientist in neuroscience at Yale School of Medicine. “We showed that by using computational approaches, we could understand the neuropil structure, and we could then use that knowledge to identify the developmental processes leading to the correct assembly of that structure.”

This approach can serve as a blueprint for understanding neuropil organization in other animal brains, the authors state.

From Buildings to Boroughs to New York City

C. elegans has the best understood nervous system of all animals. More than 30 years ago, John White, Sydney Brenner, and colleagues published the worm’s “connectome” – a wiring diagram of its 302 neurons and the ~7,000 synaptic connections between them. Since that groundbreaking study, nearly every neuron in C. elegans has been characterized: its shape, functional category, the neural circuits it participates in, and its developmental cell lineage.

Daniel Colón-Ramos, Dorys McConnell Duberg Professor of Neuroscience and Cell Biology at Yale School of Medicine and Fellow of the Marine Biological Laboratory (MBL).

What was missing, though, was a picture of how these cells and circuits integrate in space and over time.

Colón-Ramos and team analyzed published data on all the membrane contacts between the 181 neurons in the nerve ring. They then applied novel network analyses to group cells into “neighborhoods” based on their contact profiles -- similar in principle to algorithms that Facebook uses to suggest friends based on people’s common contacts. This revealed the neuropil’s layered structure and enabled the team to understand cell-cell interactions in the context of functional circuits, and functional circuits in the context of higher-order neuropil structure (See video 1).

“All of a sudden, when you see the architecture, you realize that all this knowledge that was out there about the animal’s behaviors has a home in the structure of the brain,” Colón-Ramos says.

“By analogy, rather than just having knowledge of the East Side of New York and the West Side, Brooklyn and Queens, suddenly you see how the city fits together and you understand the relationships between the neighborhoods.”

“So now we could see, ‘OK, this is why these behaviors are reflex-like, because they are direct circuits that go into the muscles. And this is how they integrate with other parts of the motor program.’ Having the structure allows you to generate new models regarding how information is being processed and parceled out to lead to behaviors,” Colón-Ramos says.

Reconstructing the Birth and Development of the Nerve Ring

The brain is a product of development, starting with one embryonic cell division and ending with a complete organ. “An order emerges through time. So our next question was, how can you instruct the formation of a layered structure? How are all these decisions simultaneously occurring in hundreds of cells, but resulting in organized layers? How are the decisions coordinated through time and space?” Colón-Ramos says.

“Layered structures are a fundamental unit of brain organization – the retina is a layer, and the cortex is a layer. If we could understand it for the worm, it would allow us to create models that might help us understand the development of layers in other vertebrate organs, like the eye,” Colón-Ramos says.

This part of the research began in 2014 when Colón-Ramos and Moyle began collaborating with microscope developers Shroff and Kumar at the MBL. “We started by building a microscope (the diSPIM) that let us look at the embryo with better spatial and temporal resolution than the tools of the time,” Shroff says.

They then identified every cell in the C. elegans embryo using lineaging approaches developed by co-author Zhirong Bao of Sloan Kettering Institute (these findings are catalogued at WormGUIDES.org). “This was a painful process, but very important to do,” Shroff says.

After years of sharing a lab at the MBL, numerous adjustments to the diSPIM system, integrations with other critically important technology, and plenty of frustration, the collaborators succeeded in resolving the developmental sequence of the C. elegans neuropil and revealing principles that guide its stratified organization (see video 2).

“This would have been impossible without the long-term, gentle imaging of the diSPIM,” Colón-Ramos says. “In developing the technology, many changes seemed incremental but in fact were very enabling, allowing us to do something we couldn’t do before. Often the changes we needed fell between two disciplines with different vocabularies, and it required prolonged, focused, exhaustive conversations to identify them. That is what our collaboration at the MBL enabled.”

In addition to Colón-Ramos, Shroff, and Bao, co-corresponding authors on the paper include Smita Krishnaswamy of Yale School of Medicine and William A. Mohler of University of Connecticut Health Center.

Researchers taking a beach break in Woods Hole. Front row: Hari Shroff. Middle row (left to right): Anthony Santella, Zhirong Bao, Titas Sengupta, Ryan Cristensen, Pavak Shah, Lin Fan. Back row (left to right): Mark Moyle, Abhishek Kumar, Bill Mohler. Credit: Daniel Colón-Ramos

Co-authors Hari Shroff and Bill Mohler in Woods Hole in 2014, testing ways of looking at diSPIM volumes in 3D with a program Mohler wrote. Photo courtesy of Hari Shroff

 
Citation: Mark W. Moyle et. al (2021) Structural and developmental principles of neuropil assembly in C. elegans. Nature DOI: 10.1038/s41586-020-03169-5

Homepage image: Adult C. elegans, 5 days old. Credit: Coleen Murphy, Princeton University / NIH Image Gallery

Funding for this research was provided by the National Institutes of Health, the National Institute of Biomedical Imaging and Bioengineering, the Marine Biological Laboratory Whitman and Fellows programs, and Howard Hughes Mewdical Institute.

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The Marine Biological Laboratory (MBL) is dedicated to scientific discovery – exploring fundamental biology, understanding marine biodiversity and the environment, and informing the human condition through research and education. Founded in Woods Hole, Massachusetts in 1888, the MBL is a private, nonprofit institution and an affiliate of the University of Chicago.

The Marine Biological Laboratory is pleased to announce that registration is open for LSFM 2021, a conference that will bring together innovators in microscopy from academic institutes, industry, and federal laboratories to share and brainstorm the latest developments taking place in light-sheet research and its biological applications.

The conference will provide a combination of virtual and in-person lectures and training activities. The program will begin with a pre-conference tutorial for participants to learn about the anatomy of LSFM on May 8, 2021 followed by a virtual conference held May 9-11, with an optional in-person workshop May 12-14.

Visit mbl.edu/lsfm for more information and to register.

Contact: dkenney@mbl.edu; 508-685-3525

Mollusks such as the eastern oyster, sea scallop, and Atlantic surf clam are the most vulnerable among shellfish to ocean acidification. Southeastern Massachusetts has the highest mollusk harvest revenues of any coastal area in the United States, according to NOAA Fisheries.

Ocean acidification causes shellfish to form weaker, smaller shells, making them more vulnerable to predation and their offspring more likely to perish. This environmental threat has dire economic implications for Massachusetts, whose seafood economy in 2016 alone supported 87,000 jobs and generated $7.7 billion in sales, the vast majority coming from scallops and lobsters.

“The two major drivers of ocean acidification are very different, but both are important,” said Anne Giblin, director of the Ecosystems Center at the Marine Biological Laboratory (MBL) and one of the commission members charged with identifying the science behind ocean acidification.

In open offshore waters, such as the Gulf of Maine, the main driver of acidification is excess carbon dioxide (CO₂) in the atmosphere due to human activities. Up to 30 percent of this CO₂ is absorbed by the ocean, which makes it more acidic (lower pH).

In coastal waters, the main driver of acidification is nutrient pollution from sources such as sewerage and septic systems, and fertilizer for crops and lawns. Nutrient pollution leads to excessive growth of algae and marine plants, which starves the coastal habitat of oxygen and can suffocate the marine organisms living there. When they die and decompose, they release CO₂, leading to a sharp, localized increase in ocean acidification.

The commission proposes several actions to prevent and manage the damage of ocean acidification, which has increased rapidly since the Industrial Revolution.

“The state legislature has proposed a climate plan that would reduce state CO₂ emissions, and they are now in negotiations with the governor over the details,” Giblin said. “Massachusetts alone can’t solve the global CO₂ problem, but I hope the plan will be passed because we really need to start going down that path.”

Along with shellfish, other marine species may be impacted as the ocean acidifies (the pH of the water decreases). MBL Ecosystems Center Director Anne Giblin comments.

“I think controlling local nutrient pollution is the step we can take to have the biggest impact right away,” Giblin added, especially “because of the double whammy of oxygen depletion and acidification” that nutrient pollution presents.

The first meeting of the state’s Ocean Acidification Commission was held at the MBL in 2019.

The commission was co-chaired by State Senator Julian Cyr (D-Truro) and State Rep. Dylan Fernandes (D-Barnstable, Dukes and Nantucket).

Scientists at the MBL Ecosystems Center have been pioneering research on coastal systems, particularly the impacts of nutrient pollution, for more than 40 years at sites along the Massachusetts coast, including Plum Island Ecosystems, Boston Harbor, and on Cape Cod.

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The Marine Biological Laboratory (MBL) is dedicated to scientific discovery – exploring fundamental biology, understanding marine biodiversity and the environment, and informing the human condition through research and education. Founded in Woods Hole, Massachusetts in 1888, the MBL is a private, nonprofit institution and an affiliate of the University of Chicago.

Contact: Diana Kenney
dkenney@mbl.edu; 508-685-3525

By Hank Hogan

Egg cells start out as round blobs. After fertilization, they begin transforming into people, dogs, fish, or other animals by orienting head to tail, back to belly, and left to right. Exactly what sets these body orientation directions has been guessed at but not seen. Now researchers at the Marine Biological Laboratory (MBL) have imaged the very beginning of this cellular rearrangement, and their findings help answer a fundamental question.

“The most interesting and mysterious part of developmental biology is the origin of the body axis in animals,” said researcher Tomomi Tani. An MBL scientist in the Eugene Bell Center at the time of the research, Tani is now with Japan’s National Institute of Advanced Industrial Science and Technology.

The work by Tani and Hirokazu Ishii, reported this week in Molecular Biology of the Cell, shows that both parents contribute to the body orientation of their offspring. For the animal species studied in the research (sea squirts), input from the mother sets the back-belly axis while that of the father does so for the head-tail axis.

“Both the maternal and the paternal cues are required to establish the body plan of the developing animal embryo,” stated Tani.

This research addresses fundamental questions in developmental biology and may also provide clues as to why things sometimes go wrong. Such knowledge could benefit fields as diverse as medicine and agriculture.
 

Left: Propagation of calcium ion waves in fertilized Ciona egg. (Selective Plane Illumination Microscopy [SPIM] movie showing fluorescence of calcium ion indicator, Rhod-dextran). Right: Autofluorescence image of the same egg to show the cytoplasmic movement. Each frame was taken every 2s. Replay speed, 10 frames/s. Credit: Hiro Ishii and Tomomi Tani.

 
The prevalent theory of how the body axis is set has been that actin filaments inside the egg, which are involved in cell motion and contraction, power the rearrangement of cytoplasmic material in the egg after it has been fertilized. But seeing this happen has been a challenge because the onset of the process takes place rapidly and over very small distances within living cells.

To overcome these hurdles, Tani and Ishii used a fluorescence polarization microscope, a technology developed a few years ago at MBL by Tani, Shalin Mehta (now at Chan Zuckerberg Biohub) and MBL Senior Scientist Rudolf Oldenbourg, along with scientists at other institutions. This technology makes it possible to image events taking place at distances measured in nanometers, or thousands of times smaller than the diameter of a human hair. The methodology is also a familiar one to Tani and others.

“Using polarized light for looking at dynamics of molecular order is a tradition of MBL imaging,” Tani noted, one that began with pioneering live-cell studies by Shinya Inoué in the 1950s.

When polarized, light waves oscillate either partially or completely in only one direction: up/down, left/right, clockwise/counterclockwise, and so on. That’s why a filter will let polarized light through in one orientation, but block it when rotated.

Tani and Ishii attached fluorescent probe molecules, which glow when illuminated with the right light, to the actin in eggs of sea squirts (Ciona), a marine species often studied by researchers as a model for animal development. The probe-actin link was very rigid, Tani said, allowing the microscope to detect the orientation of the actin molecules by working with polarized light.
 

Left: Ciona egg before and after fertilization (Movie of Total Internal Reflection Fluorescence Microscopy [TIRFM] images; F-actin probe is AF488-phalloidin). Right: Transient changes of F-actin alignments from the time before fertilization to the first cell division of Ciona egg. Positions of AF488-phalloidin particles bound to F-actin are shown as yellow dots, and the orientation of F-actin is shown as yellow bars. Frame intervals, 10s. Replay speed, 15 frames/s. Credit: Hiro Ishii and Tomomi Tani

 
So, if the actin all pointed in one orientation, the researchers spotted it. If the actin was jumbled, they could see that too. When Tani and Ishii looked at unfertilized eggs, they saw a mostly random arrangement of actin. After fertilization, a calcium ion wave passed through the egg and the actin filaments lined up and contracted along the orientation that was at a right, or 90^o, angle to the future back/belly axis. The cytoplasm then moved. This body plan formation process began just after fertilization.

The fertilized egg orientation research is being followed up with other investigations. One of the long-term goals of such imaging is to detect and understand the force in the developing embryo that shape its morphology, its form and structure.

“We hope that the molecular orders in the cytoskeleton tell us something like ‘field lines’ of mechanical forces that organize the morphology of multicellular organisms,” Tani said in discussing future efforts.

Citation: Hirokazu Ishii and Tomomi Tani (2021) Dynamic organization of cortical actin filaments during the ooplasmic segregation of ascidian Ciona eggs. Mol. Biol. Cell, DOI: 10.1091/mbc.E20-01-0083

Homepage photo: Screen capture from Movie 1. Left: Propagation of calcium ion waves in fertilized Ciona egg. Right: Autofluorescence image of the same egg to show the cytoplasmic movement. Credit: Hiro Ishii and Tomomi Tani

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The Marine Biological Laboratory (MBL) is dedicated to scientific discovery – exploring fundamental biology, understanding marine biodiversity and the environment, and informing the human condition through research and education. Founded in Woods Hole, Massachusetts in 1888, the MBL is a private, nonprofit institution and an affiliate of the University of Chicago.

“Why Study Biology by the Sea?” (University of Chicago Press, 2020) is a fascinating collection of essays on the historical emergence of marine biological stations, the diversity of work they pursue, and their significant contributions to scientific discovery and knowledge.

The volume sprang from discussions at the 29th annual MBL-ASU History of Biology Seminar at the Marine Biological Laboratory (MBL) in 2016, organized by Jane Maienschein of Arizona State University and Karl Matlin of University of Chicago.

Several essays explore MBL history, including a profile of the seaside lab that inspired its formation (the Stazione Zoologica in Naples, Italy) and the MBL's inspiration, in turn, of the first marine lab in China. Bookending the volume are a foreword by MBL Director Nipam Patel and an epilogue by Alejandro Sánchez Alvarado of the Stowers Institute, both of which situate the MBL in the present day.

As essays on the MBL’s early years reveal, the lab remains remarkably true, in many respects, to the ethos on which it was founded. The MBL’s first director, C.O. Whitman, felt strongly that the lab's scientific integrity depended on integrating research and education, a topic MBL McDonnell Fellow Kate MacCord explores in the book. (Whitman even fended off a proposed merger with the Carnegie Institution during a dire period in MBL’s financial history, MacCord writes, because a condition of the merger was scuttling the MBL’s educational mission.) And in an era of increasing specialization in biology, Whitman firmly believed the lab should investigate a wide diversity of organisms and questions, a strategy that both Patel and Sánchez Alvardo pick up as being essential today, with particular reference to marine biodiversity.

In “Microscopes and Moving Molecules: The Discovery of Kinesin at the MBL,” Matlin probes how a culture of intense collaboration and competition at the MBL, along with new, enabling technologies in light microscopy, played crucial roles in a major new finding. In another essay, MBL McDonnell Scholar Kathryn Maxson Jones focuses on research using the squid giant axon in the 1940s and 1950s, including at the MBL. Her essay, writes Fabio De Sio in a review of the volume, argues for “the importance of marine stations for the birth of the neurosciences, as frameworks for novel experimental systems, and as niches for the cultivation in parallel of both model-based and comparative approaches."

Both longtime fans and new members of the MBL community will find much to discover in “Why Study Biology By the Sea?” The volume, co-edited by Matlin, Maienschein, and Rachel A. Ankeny, is available for purchase through the MBL Gift Shop.

This book is the third volume in a series edited by Maienschein called "Convening Science: Discovery at the Marine Biological Laboratory." The next volume, "Nature Remade: Engineering Life, Envisioning Worlds," will be published in July 2021.