By Stephanie M. McPherson

MBL Hibbitt Fellow B. Duygu Özpolat. Credit: Daniel Cojanu

Duygu Özpolat at the Marine Biological Laboratory (MBL) is tracing the lineage of regenerated reproductive cells—also called the germline—to determine the cellular programming that allows for their resurgence.

“We are trying to understand where the cells that regenerate the germline come from,” says Özpolat. “Once we know that, we may be able to understand why some organisms can regenerate them and some can’t.” Özpolat is a Hibbitt Fellow in the MBL’s Bell Center for Regenerative Biology and Tissue Engineering.

Now, Özpolat and her colleagues are a step closer to this goal. A recent study reveals that their research organism, the marine worm Platynereis dumerilii, must develop a certain number of body segments before it starts forming its original reproductive cells.

“If you're going to study the regeneration of a structure in an organism, you need to know how that original structure formed first,” says Özpolat. “And before this study, we didn't have a good understanding of when that happens.”

The paper, published in the Journal of Experimental Zoology Part B: Molecular and Development Evolution, shows that progenitor reproductive cells develop in P. dumerilii when the worms reach between 35 and 40 segments. Before this discovery, researchers working with P. dumerilii would have to conduct time-consuming assays to determine if the worm had enough original reproductive cells to begin experimentation.

Led by Özpolat and first author Emily Kuehn, a research assistant in Özpolat’s lab, the researchers used a technique called in situ hybridization to locate the germline cells in the worm’s body and track their development as segments were added. They studied hundreds of the two-centimeter worms, carried out different interventions, and generated a wealth of data that led them to their remarkably consistent conclusion.
 

The Özpolat lab’s research organism, Platynereis dumerilii. Credit Ryan Null

The team also published a first-of-kind algorithm to facilitate the use of a fluorescent in situ hybridization method in this type of experiment.

This research builds on years of work from the Özpolat lab. For example, the worms were cultured using a streamlined, scalable system developed in the lab and published in 2019. A standardized culturing system is key to understanding genetic cause and effect. If worms grow in different environments with different feeding schedules, it is nearly impossible to determine if variations observed in experiments are due to the scientists’ interventions or to growth conditions.

Özpolat and team will follow this research with investigations into what happens when germline cells are forced to regenerate. They will amputate 90 percent of the worms’ length and watch as the germlines reemerge. They will then track the function of those regenerated cells and their effect on the health of the next generation, versus control worms who reproduce without being forced to regenerate germline cells.
 

Fixed sample of the marine worm (P. dumerilii) processed for in situ hybridization to detect vasa gene expression. She is filled with oocytes, and she no longer has any internal organs. This is a part of the sexual metamorphosis process, when the worms lose their internal organs, and become mere carriers of gametes. Credit: Kuehn et al JEZ-B 2021

Understanding the cellular and molecular pathways involved in regeneration could help Özpolat and her team understand how to make it happen in humans.

“Way down the line, this could inform infertility treatments,” says Özpolat. “Our job in fundamental science is to learn how these worms do cellular reprogramming. And if they keep the mutations at the low level, how they do that? Then maybe we can apply that to human therapies for regenerating cells or reprogramming cell types from different body parts.”

Citation:

Emily Kuehn, David S. Clausen, Ryan W. Null, Bria M. Metzger, Amy D. Willis, B. Duygu Özpolat (2021) Segment number threshold determines juvenile onset of germline cluster expansion in Platynereis dumerilii. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution, DOI: 10.1002/jez.b.23100

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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.

WOODS HOLE, Mass. — For generations, scientists have relied on a handful of organisms to study the fundamentals of biology. The usual suspects—fruit flies, zebrafish, and mice, among others—all have short lifespans, small body size, can be bred through multiple generations in the laboratory, and have been developed for genetic investigations. These research organisms leave out a whole swath of biological diversity and scientists have lacked access to a cultured octopus laboratory organism—until now. Introducing the pygmy zebra octopus (O. chierchiae).

In a new paper published in the journal Frontiers in Marine Science, researchers from the Marine Biological Laboratory (MBL) introduce scientists to successful culturing methods for O. chierchiae that were developed at the MBL.

O. chierchiae adult in a shell looking at a snail. Credit: Tim Briggs

“The pygmy zebra octopus has certain biological features that make them attractive and more appropriate for laboratory research, compared to other octopuses,” says Bret Grasse, MBL’s manager of Cephalopod Operations and co-author on the paper.

Also known as the “lesser Pacific striped octopus,” the pygmy zebra octopus shares many useful similarities with other research organisms—such as small adult body size—but it also has unique features that distinguish it from other cephalopods (the group of animals that include octopus, squid, and cuttlefish).

“The majority of octopuses are ‘live fast, die young.’ They breed once and then immediately start to senesce and age and then die relatively quickly,” says Anik Grearson, former MBL intern and co-lead author on the paper. Unlike other octopus species, a female O. chierchiae lays several clutches of 30-90 eggs over her reproductive period.

“We can mate them and know exactly when they’ll lay their eggs. We know exactly how long they’ll incubate and we can raise offspring at a relatively high survivorship rate compared to other octopuses,” says Grasse. Add that to its small size, sexual dimorphism, and predictable breeding schedule and it’s easy to see why O. chierchiae is an ideal candidate for further exploration and research.

A pygmy zebra octopus hatchling in the MBL Cephalopod Mariculture Lab. These octopuses are about the size of a grain of rice when they hatch. They reach full size (about the size of a table grape) within six months. Credit: Tim Briggs.

Anik Grearson, co-lead author on the paper, leans over a tank in the MBL Cephalopod Mariculture Lab. She was an intern at the MBL during 2019 while she was getting her bachelor’s degree from McMaster University. She is currently in a master’s degree program at Northeastern University.

The MBL’s Cephalopod Mariculture team successfully bred O. chierchiae through multiple generations in 2019 — a global first. Breeding multiple generations in the lab is known as “closing the life cycle” and it is critical in biological research. It lets scientists study gene function and mutational effects from one generation to the next.

Being able to successfully breed octopuses in a laboratory opens up “novel science that hasn’t been possible before,” says Grasse.

Scientists at the MBL and around the world study cephalopods to learn about everything from camouflage and limb dexterity to regeneration and neurobiology. The majority of U.S. scientists studying octopuses use the California two-spot octopus (O. bimaculoides), which is local to the waters off of California. But those octopuses have yet to be successfully bred in the laboratory through multiple generations, so most scientists study wild-caught animals shipped to their labs from California.

Octopuses are also territorial, and each organism must be housed individually. An adult two-spot octopus is about the size of a softball, whereas an adult pygmy zebra octopus is only the size of a table grape, making the smaller-size species ideal for space-conscious laboratories.

“We now have this octopus species that’s really small and that can be bred regularly,” says Grearson. “The sky’s the limit for what people want to do.”

Reference:

Grearson A.G., Dugan A., Sakmar T., Sivitilli D.M., Gire D.H., Caldwell R.L., Niell C.M., Dölen G., Wang Z.Y. and Grasse B. (2021) The Lesser Pacific Striped Octopus, Octopus chierchiae: An Emerging Laboratory Model. Frontiers in Marine Science. Doi: 10.3389/fmars.2021.753483

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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.

By Bill O’Neill

We know that sharks are voracious eaters, but what do we know about the source of its microbiome? To date, not much. But a recent study from the University of Chicago and the Marine Biological Laboratory (MBL) fills a gap in understanding the microbiome associated with a close relative in the Chondrichthyes, a class of fish that includes sharks, rays and skates.

Postdoctoral Scholar Katelyn Mika and Alexander Okamoto, then an undergraduate at UChicago, were inspired to discover how the microbiome originates in the little skate in 2018, while spending the summer at MBL with UChicago Professor Neil Shubin. Also guiding them in the research was MBL Senior Scientist David Mark Welch.

“There’s a new understanding that microbiomes are very important for all multicellular organisms,” said Mark Welch, senior author on the paper. “We're essentially taking what we’ve learned from the NIH Human Microbiome Project, including the central role the microbiome plays in human health, and recognizing that microbiomes play a role in a wide variety of organisms, particularly marine organisms.”

Alexander Okamoto holds a skate egg case (Leucoraja erinacea) in the MBL Marine Resources Center. Credit: Marine Biological Laboratory

Among the ocean dwellers, corals and certain fish have had their microbiomes analyzed, but “there are huge gaps [in knowledge], and one of them has been the microbiomes of non-bony fish – sharks, rays, skates and other cartilaginous fish,” Mark Welch said.

An animal can acquire its microbiome in three different ways, the team’s study explains. First, the parent can transmit all or part of its microbiome to its offspring. Second, the animal can acquire it during social or sexual interactions with other members of its species. And third, animals can recruit microbes from their environment through contact with fluids, habitat, or diet. Humans acquire their microbiome in all three ways.

The little skate’s embryo develops in the contained environment of an egg case, commonly called a devil’s purse.

“The skate egg case is a very interesting place to study microbiome development because the egg is separate from the outside world up until a certain stage of development, when little apertures open in the egg case and there starts to be an exchange with seawater,” said Mark Welch. “So, you can ask the question more easily than in other organisms, is there vertical transmission of the microbiome? Namely, does the mother pass on part of her microbiome to the offspring?”

An adult little skate (Leucoraja erinacea) in the MBL Marine Resources Center. Credit: Andres Pruna

A little skate embryo in its case, commonly called a devil’s purse. Credit: Laurel Hamers

Mika and Okamoto got on the trail of this question when they heard a talk by Andrew Gillis, who is joining the MBL next year as an associate scientist. Gillis’s description of the skate egg case opening to the environment about two-thirds of the way through development caught their attention.

“We began wondering what happens with the microbiome, given the egg’s fairly dramatic life history changes,” said Okamoto.  “It’s basically dumped on the bottom of the ocean, abandoned by its parent for many months, and then it opens up suddenly and seawater rushes in.”

“Alexander and I looked at each other and said, ‘That's weird,’ because most eggs are fully sealed until the time of hatching, and these are not,” said Mika.

They conducted the research during the summer of 2019, while Okamoto was working as a research assistant in MBL Director Nipam Patel’s lab. Using next-generation sequencing to characterize the microbial community at six points in the skate’s embryonic development, their efforts yielded the first evidence of vertical transmission of the microbiome from parent to offspring in the Chondrichthyes.

Katelyn Mika and Alexander Okamoto by the Rachel Carson statue in MBL’s Waterfront Park. Credit: Alexander Okamoto

“This was the first microbiome study Alexander and I had ever done,” said Mika. “I'm a geneticist and he's worked on evolutionary and organismal biology, so we were both a bit out of our depth. We had to learn as we went.”

Their findings are a useful step in the study of microbiomes, which can be indicators of how well an organism is doing in its environment, said Mark Welch.

“We know that a lot of marine species are under stress because of anthropogenic changes, global warming,” he said. “One of our broader goals is to be able to study the microbiomes of these organisms and determine very quickly, how healthy are they?”

There’s plenty of room for more research on animal microbiomes, said Mika – including on sharks.

“There's a chain catshark at the MBL whose eggs look incredibly similar to the skates' eggs,” she said. “They go through the same sort of development where they're in that egg case for the first two thirds, then they have slits that open, and then they finish developing with that open waterflow through the eggs. I'm desperate to know if their microbiomes are similar to the skates' or if they're totally different.” Sounds like a good reason to come back to the MBL!

This research was funded by a grant from the Microbiome Center, a collaboration between University of Chicago, Marine Biological Laboratory, and Argonne National Laboratory; and by a National Science Foundation Graduate Research Fellowship to Alexander Okamoto.

Citation:

Katelyn Mika, Alexander Okamoto, Neil Shubin and David Mark Welch (2021) Bacterial community dynamics during embryonic development of the little skate (Leucoraja erinacea). Animal Microbiome, DOI: 10.1186/s42523-021-00136-x

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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; 508-289-7139

WOODS HOLE, Mass. – Since artificial intelligence pioneer Marvin Minsky patented the principle of confocal microscopy in 1957, it has become the workhorse standard in life science laboratories worldwide, due to its superior contrast over traditional wide-field microscopy. Yet confocal microscopes aren’t perfect. They boost resolution by imaging just one, single, in-focus point at a time, so it can take quite a while to scan an entire, delicate biological sample, exposing it to light dosages that can be toxic.

To push confocal imaging to an unprecedented level of performance, a collaboration at the Marine Biological Laboratory (MBL) has invented a “kitchen sink” confocal platform that borrows solutions from other high-powered imaging systems, adds a unifying thread of “Deep Learning” artificial intelligence algorithms, and successfully improves the confocal’s volumetric resolution by more than 10-fold while simultaneously reducing phototoxicity. Their report on the technology, called “Multiview Confocal Super-Resolution Microscopy,” is published online today in Nature (print publication Dec. 9).

“Many labs have confocals, and if they can eke more performance out of them using these artificial intelligence algorithms, then they don’t have to invest in a whole new microscope. To me, that’s one of the best and most exciting reasons to adopt these AI methods,” said senior author and MBL Fellow Hari Shroff of the National Institute of Biomedical Imaging and Bioengineering.

Among its innovations, the new confocal platform uses three objective lenses, allowing one to image a wide variety of sample sizes, from nuclei and neurons in the C. elegans embryo to the whole adult worm. Multiple specimen views are rapidly captured, registered and fused to yield reconstructions with improved resolution over single-view confocal microscopy. The platform also introduces innovative scan heads for the three lenses, allowing line-scanning illumination to be easily added to the microscope base.

Moreover, the team added “super-resolution” capacity to the platform (enhanced resolution beyond the diffraction limit of light) by adapting techniques from structured illumination microscopy.

“The hardware summit that gets climbed in this platform is the multiple lenses around the sample, and then the super-resolution trick, which takes a combination of hardware and computation to achieve. It’s a tour de force, but it’s a pretty phototoxic recipe. There’s a lot of light being delivered to the sample,” said co-author and MBL Fellow Patrick La Rivière of the University of Chicago.

One way to address phototoxicity is to lower the light coming from the microscope’s laser. But then you begin having problems with “noise” in the image -- background graininess that can obscure fine details of the object you want to image (the “signal”). This is where artificial intelligence comes in.
 

Mouse esophageal tissue slab (XY image), immunostained for tubulin (cyan) and actin (magenta), imaged in triple-view SIM mode. On right, anatomical regions are highlighted. Credit: Yicong Wu and Xiaofei Han et al, Nature, 2021.

 
The team trained a Deep Learning computer model, or neural network, to distinguish between poorer-quality images with a low signal-to-noise ratio (SNR) and better images with a higher SNR. “Eventually the network could predict the higher SNR images, even given a fairly low SNR input,” Shroff said.

“Deep Learning allows you to take this hardware summit as the gold standard for resolution and then train a neural network to achieve similar results with much lower SNR data, many fewer acquisitions, and so much less light dose to the sample,” La Rivière said.

The team demonstrated the platform’s capabilities on more than 20 different fixed and live samples, targeting structures that ranged from less than 100 nanometers to a millimeter in size. These included protein distributions in single cells; nuclei and developing neurons in C. elegans embryos, larvae and adults; myoblasts in Drosophila wing imaginal disks, and mouse renal, esophageal, cardiac, and brain tissues. They also see potential applications for imaging human tissue in histology and pathology labs.

Shroff, La Rivière and co-author and cell biologist Daniel Colón-Ramos of Yale School of Medicine have been collaborating at MBL for nearly a decade to develop imaging technologies with higher speed, resolution and longer duration. Collaborators on this confocal platform also included Applied Scientific Instrumentation, a company they worked with both at MBL and at the National Institutes of Health.

Yicong Wu, first author on the paper, built the new confocal platform and deployed its Deep Learning approaches. Wu learned how to use Deep Learning at the MBL in the pilot version of a new course launched this year, DL@MBL: Deep Learning for Microscopy Image Analysis. (La Rivière is a faculty member in the course.)

“It’s a testament to the course that Yicong could learn Deep Learning methods in 4 days and quickly innovate with them, so we can now apply them in our lab,” Shroff said. “That’s a short feedback scheme, right? It was great that MBL catalyzed it.”

Citation:

Yicong Wu and Xiaofei Han, et al. (2021) Multiview Confocal Super-Resolution Microscopy. Nature, DOI: 10.1038/s41586-021-04110-0.

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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.

WOODS HOLE, Mass. – ResilientWoodsHole, a public-private partnership readying for the impacts of climate change in Woods Hole, will hold a free, public symposium on Thursday, November 18, 6:30-8:30 PM, in Clapp Auditorium, Marine Biological Laboratory, 7 MBL Street, Woods Hole.

 Registration for virtual or in-person attendance is here.

ResilientWoodsHole’s mission is to secure the future of the vibrant, blue-economy village of Woods Hole in the face of major challenges and threats -- both to village infrastructure and the natural habitat -- associated with climate change.

Jump-started by three local science organizations (Woods Hole Oceanographic Institution, Marine Biological Laboratory, and NOAA Northeast Fisheries Science Center), ResilientWoodsHole has now expanded to include key local stakeholders, including the business community, the homeowner community, and the Town of Falmouth.

The three science organizations have developed assessments of potential impacts of sea-level rise and coastal storms, which were delivered in a September 2020 symposium, “Rising Tides: Phase I.”

In the upcoming symposium (Phase II), attendees will get a look inside the climate science work happening in Woods Hole; be introduced to local, regional, and statewide climate resiliency planning efforts; and become engaged around local challenges, ongoing work, and potential solutions.

The symposium will include three panel discussions:

• I. Climate Science Panel with scientists from Woods Hole Oceanographic Institution, Marine Biological Laboratory, and NOAA Northeast Fisheries Science Center
• II. Planning and Resources Panel with representatives from the state’s Office of Coastal Zone Management; the Cape Cod Commission; and the Town of Falmouth
• III. ResilientWoodsHole Initiative Panel presenting results to date and expected work to come in Phase III. A key outcome of Phase III will be a comprehensive, phased strategy for increasing coastal resiliency, including village-wide design concepts and planning.
• IV. Q & A with audience members

In-person attendance will be limited to the first 225 registrants to accommodate social distancing, and attendees must be masked. Parking will be available in any MBL lot. For more information, please contact: info@resilientwoodshole.org.

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