gene expression

Pancreatic tumor composition provides insight on treatment response

February 17, 2022 By Penquite, Cara (MU-Student) in Research Tags: bioinformatics, cancer, gene expression, mice

PanC_7 — Jing Zhou focuses the microscope through her computer. The microscope feeds its view directly to her screen so Zhou can see the pancreatic cells. | Photo by Cara Penquite, Bond LSC

By Cara Penquite | Bond LSC

Not all tumors are created equal, and potential treatments aren’t universal. When it comes to pancreatic cancer, surgery and radiation often do more harm than good due to its rapid growth and ability to spread to the liver.

Searching for alternative treatment options, Jing Zhou focused her research on immunotherapies. That led Zhou and her team to identify the sequences for over 9,000 cells from pancreatic tumors to out why some therapies don’t measure up.

“My professor said, ‘You have to do a lot of work to try and make sure your sample is ready [and] your experiments are ready. Otherwise, we [will] spend a lot of time, a lot of money, on [research] that doesn’t work,’” said Zhou, a NextGen graduate student who collaborated closely with Trupti Joshi’s bioinformatics lab at the Bond Life Sciences Center for the data analysis.

PanC_2 — Jing Zhou grabs a box with pancreatic tumor tissue samples in cover glass. Zhou can see the cancerous and normal cells by magnifying the samples in a microscope. | Photo by Cara Penquite, Bond LSC

Zhou identified differences between types of pancreatic tumors to understand why certain types of pancreatic cancer respond differently to treatment. Scientists analyzed RNA sequences for two different types of pancreatic tumors. While the project is continually evolving, her most recent findings were published online in the Journal of Translational Oncology.

Zhou focuses on a form of immunotherapy that increases the effectiveness of the immune system. Known as immune checkpoint inhibitors, the treatment turns off protein pathways that act as brakes on the immune system. In cancer patients, turning off the brakes allow immune cells, like T-cells, to attack more cancer cells.

“The cancer cells grow so fast, they expand a lot and that attracts a lot of T-cells. Normally, in a healthy tissue they don’t have so many T-cells, just a few of them to keep the body normal,” Zhou said, “But when you have a strong immune response, like in a tumor, it will have a lot of T-cells in that kind of environment.”

Not all types of pancreatic tumors respond well to immunotherapy.

“This was a very interesting project because what they were doing here is comparing four different types, [but] mainly two different types, of pancreatic cancers,” said Joshi, Bond LSC principal investigator and Assistant Professor at the Department of Health Management and Informatics in the School of Medicine and core faculty in MU’s Data Science and Informatics Institute. “One that is resistant to and one that is sensitive to this anti-PD-1 antibody treatment, what is known commonly as immunotherapy.”

PanC_4 — Jing Zhou selects a slide containing human pancreatic tumor cells to put into the microscope. While Zhou’s previous work was conducted in mice, surgeons now send human tissue samples to the lab for her to analyze as well. | Photo by Cara Penquite, Bond LSC

Tumors are composed of many types of immune cells mixed in with cancerous cells, and Joshi’s lab analyzed the composition to understand why two different types of pancreatic cancer react differently to treatment.

Unlike past analyses that relied on analyzing the tumors as a whole, the scientists used a process called single-cell RNA sequencing which allowed them to identify the various types of cells present and quantify the gene expression in individual cells. This quantification allowed the researchers to see which genes were turned on in each cell.

“The single-cell technology is the biggest advancement in sequencing technologies,” said Yuexu Jiang, a postdoc in Joshi’s lab who analyzed the single-cell RNA sequencing for the project.

Joshi suggested the analogy of a fruit smoothie to understand the concept of single-cell RNA sequencing. A fruit smoothie is a mixture of many fruits, and to understand what causes the flavor of the smoothie you must know how much there is of each fruit.

Single-cell RNA sequencing allows the researchers to quantify what types of cells are in the tumor, as well as identify the genetic makeup of the cells in the tumor.

“That really allows you to pinpoint that it is this particular gene expression in this particular type of cell which is really associated with what outcome you’re seeing in terms of these different tumors’ behavior,” Joshi said.

Pancreatic cancer is difficult to treat since surgery and radiation often do more harm than good. With its rapid growth rate and ability to easily spread throughout the body, a pancreatic cancer diagnosis comes with a 50% five year survival rate according to the American Cancer Society.

“I have been involved a lot of times in the surgery. I saw when the surgeon opened the [patient], and when they saw the metastasis, tiny things, on the liver, they just closed the [patient]. They won’t remove the cancer anymore,” Zhou said.

Immunotherapy provides another option for patients that need one.

“Getting a better understanding of the composition and what parts of the immune system are associated or involved in times when you see a cancer responding to immunotherapy […] that is really what this research was targeted towards understanding,” Joshi said.

This research published in the Journal of Translational Oncology November 9, 2021, under the title “Single-cell RNA sequencing to characterize the response of pancreatic cancer to anti-PD-1 immunotherapy.”

Lab explores link between genetic differences and domestication in kale

April 28, 2021 By Becca Wolf in Research Tags: Brassicales, gene expression, kale

Tatiana Arias and Chad Niederhuth studied the plant, kale, in this publication. | “Kale” by photofarmer is licensed under CC BY 2.0

By Becca Wolf | Bond LSC

It is said that variety is the spice of life.

When it comes to kale, much of that variation derives from domestication, and genetic differences that evolved over thousands of years resulted in different color of leaves, nutritional value, and habit and length of growth.

Understanding the links between traits and genes could one day help plant scientists create better vegetables for us.

Eight years ago, Tatiana Arias from the Chris Pires lab and Chad Niederhuth from the John Walker and Paula McSteen lab at Bond Life Sciences Center came together to research leaf differences in this species, Brassica oleracea. Brassica oleracea includes many foods from the vegetable aisle from the grocery store like kale, cabbage, and broccoli.

“In general, people do these types of studies to narrow down some specific genes, but we tried to find a bunch of different genes that could serve different purposes in the future for basic science and developmental biology studies, but also for more applied work in crop sciences,” Arias said.

She and Niederhuth’s broad approach looked at genes that they thought could have been selected during domestication of kale. As humans farmed these leafy greens over millennia, they selected for plants that grew more upright with leaves we liked to eat, developed more ornate foliage and flowered later, differentiating kale from its sisters like broccoli and cauliflower where the flowers were the valued food product.

They compared three morphotypes of Brassica oleracea plants: kale, cabbage, and TO1000 — a specific kale line.

“We brought the evolutionary history of how Kale was domesticated from the other Brassica oleracea morphotypes. Tatiana is really good at the microscope and doing really detailed sections of different parts of the plant as they’re growing,” Chris Pires said. “They sequenced using next gen sequencing, which I’d suppose you’d call it old next gen or postmodern sequencing since it’s been so long.”

While sequencing RNA, the labs found thousands of genes expressed in these different vegetable lines.

“I am a classically trained botanist, so for me it was a very new approach to think about genomics for plants,” Arias said.

While the Pires lab handled the RNA sequencing, Niederhuth in the McSteen lab handled the bioinformatics analysis, the mapping of sequence data, and the identification of the differentially expressed genes.

“It was a team effort between Tatiana and I, she was really the expert in studying these different varieties and their morphological differences and she generated a lot of the data,” Niederhuth said. “I brought to that some of my expertise in gene expression data.”

Through their work, Arias and Niederhuth found 3,958 expressed genes that separated kale from cabbage and TO1000. Some of these differences were in genes that regulate vegetative and reproductive transitions — things that create the very different look of cabbage versus kale.

Some genes they found in kale were particularly interesting. These were involved in leaf morphology, plant structure, and nutrition and were expressed differently, allowing them to identify a set of genes that may be important in kale domestication. Those genes would link, for example, to why kale is more upright and cabbage leaves curl into a head or the variation in color of the leaves.

“I didn’t necessarily know what to expect going into it,” Niederhuth said. “It was interesting to see some results that were related to some of the differences in the phenotypes we observed. Before this I had never worked with Brassica oleracea before, it was the first time I was working with genomic data from a species other than Arabidopsis. It helped me begin working with a variety of different species in many ways.”

While this research was conducted over eight years ago, Arias and Niederhuth recently published the findings.

“It’s nice to have one of many projects off my plate, and I’m glad that it’s finally gotten out there,” Niederhuth said. “This is just how life works where people in this career are oftentimes moving and things get set aside. I am glad that after all these years, we’re finally able to come together again and get it finished.”

To read more of Arias’ and Niederhuth’s research, check out the March article published on the Frontiers in Plant Science website titled, “The Molecular Basis of Kale Domestication: Transcriptional Profiling of Developing Leaves Provides New Insights Into the Evolution of a Brassica oleracea Vegetative Morphotype.”

Defense or growth: a complicated balance

April 7, 2021 By Lauren Hines in Research Tags: gene expression, plant immune system, plant science, TOPLESS gene family

By Lauren Hines | Bond LSC

The plant immune system isn’t active all the time. Plants must decide to either defend against disease or grow, but not simultaneously.

The reason behind this process is not fully understood, and the Walter Gassmann lab at Bond Life Sciences Center is searching for answers.

The lab recently found a link between members of the TOPLESS gene family, the immune receptor SNC1 and a negative regulator SRFR1. The relationship revealed when members of the gene family are absent in a plant, the immune system intensifies, and growth is stunted. The findings were published in February in the journal PLoS Genetics.

“As scientists, we try to simplify a model but sometimes the more we know, the more complicated we realize it to be,” said Jianbin Su, a research scientist in the Gassmann lab.

The study focused on TPR2 and TPR3, two genes within the TOPLESS family.

After studying these two genes, Gassmann said they did not act as he expected when partnered with SNC1. It turns out the genes act as “brakes” so then the immune system doesn’t go on forever.

“If you were to keep stimulating a plant with pathogens, you would see reduced growth over the long term,” said Gassmann, director of Bond LSC. “Plants have to balance whether they grow or if they defend, but we can artificially remove genes in the repertoire of a plant so that the immune system is on all the time from the get-go, and that manifests itself as stunted growth.”

This balance is the main obstacle facing researchers who want to increase crop yield and resistance to disease. Technically, researchers could increase the level of resistance in plants today but increasing the defense system often costs too much growth and yield.

Instead, the Gassmann lab proposes to modify the natural regulation of the plant’s immune system and bypass artificially increased resistance like pesticides.

“In the end, we would like to be able to generate crop plants that are more resistant to a wide swath of pathogens once we understand their regulatory system better,” Gassmann said.

Now, all the Gassmann lab needs to do is figure out how the system works. Easier said than done.

However, there are theories as to why the plant must choose between growth and defense. Previous theories believe there isn’t enough energy for plants to defend and grow at the same time. Gassmann said the theory doesn’t make a lot of sense to him. Even though the plants at Bond LSC are “pampered” and live in a “luxurious environment,” they stop growing whenever their immune systems are stimulated.

“I think evolutionarily, it is an advantage for plants to have a genetic circuit that makes them decide whether to grow or to defend themselves,” Gassmann said. “So, it’s not an energy limitation. It is genetic pathways that we can study to understand how that recognition works. And if there’s a genetic circuit, then we can tweak it and improve on it.”

Even with these theories, there’s more to this system than just genetics. While TPR2 and TPR3 interact with the immune receptor SNC1 and a negative regulator SRFR1 — a molecule that puts a stop or pause on a cellular process — that doesn’t mean their proteins interact in the cell.

“In terms of protein-protein interaction, many people think, ‘Oh it’s just as we interact physically,’ but actually, the cell is alive,” Su said. “In microscopes, they are moving. Sometimes the protein interaction needs a trigger to induce the protein-protein interaction.”

This interaction at the protein level still needs to be further analyzed, but it can explain why both SRFR1 and TPR2 must be absent for the plant to stop growing and be smaller.

“We think they actually work at different points in the circuit, and that’s why you get an additive effect on plant growth,” Gassmann said. “We’d be really interested to find out what those points are exactly, so there’s always more work to be done.”

While the Gassmann lab has found a new interaction revealing a bit more of the plant immunity process, there is still more to understand.

“Knocking out SRFR1 causes smaller plants, but the more accurate mechanism is still, I think, largely unknown,” Su said. “These genetic interactions with TPR2 are just one part of SRFR1 as a negative regulator in plant immunity.”

#IAmScience Dangping Luo

March 19, 2021 By Lauren Hines in #IAmScience Tags: gene expression, rice

By Lauren Hines | Bond LSC

The greenhouse isn’t a place most researchers linger. With condensation sticking to the glass windows, scientists usually don’t dawdle longer than 30 minutes in the heat and humidity. But Dangping Luo, on the other hand, doesn’t seem to mind and takes his time tending to his rice plants.

A research scientist in the Bing Yang lab at Bond Life Sciences Center, Luo, is a man of determination and conviction. For the past 18 years, Luo has been studying rice genetics and its interaction with disease.

“When I think something is of importance, I never give up,” Luo said.

Luo was working as a teacher in Southern China when he found his fascination for rice genetics. Luo went on to complete his Ph.D. at the South China Agricultural University in 2013. He then moved to Iowa with his family for his post-doctorate at Iowa State University, where he joined the Yang lab. Luo followed Yang to Bond LSC in 2018.

Although he was offered a professor position at a university in China, he turned it down in lieu of finishing his work at Bond LSC. The position is still open for him.

Part of that work continues to be published this year. Luo published a paper in January describing how the Xa7 resistance gene in rice protects the SWEET14 gene that is susceptible to a major disease for rice, bacterial blight.

“We hope that we can elucidate the mechanism and explain the resistance,” Luo said. “[This gene] may help us understand other genes better and can usually help us breed good rice.”

Luo found that Xa7 is an adaption in rice that has emerged to counteract efforts by blight bacteria pathogen infection.

“In the world, rice is very important, even in the U.S.,” Luo said. “Actually, almost half of the world’s population depends on rice.”

Luo’s 20 years of knowledge about rice comes in handy in the lab.

“I regularly ask him basic questions. I think he knows everything,” said Chenhao Li, visiting student in the Yang lab. “He’s really an expert on rice development and pathology, so it’s very nice because he has much more knowledge than me since I’m a student.”

Li met Luo three years ago at Iowa State University and has seen for himself Luo’s dedication.

“If you keep those plants healthy, they will give you great results,” Li said. “He just takes care of rice like it’s his baby. We usually take soil from the field, there are many impurities, but he would just totally clean the soil to prepare for the rice.”

Luo is proud that he’s been able to uncover many important rice genes and won’t stop until all his questions are answered.

“I really enjoy doing such work, even when it’s very hard,” Luo said.

ACCase: The Gatekeeper of Plant Oil

February 23, 2021 By Becca Wolf in Research Tags: gene expression, plant oil, plant science

Jay Thelen sitting amongst Liquid Chromatography-Tandem Mass Spectrometers in the lab. | photo by Becca Wolf, Bond LSC

By Becca Wolf | Bond LSC

Two decades ago Jay Thelen speculated an unknown protein anchored acetyl-CoA carboxylase (ACCase), an important enzyme complex, to the chloroplast membrane. He even published a paper about it, not knowing exactly what the membrane protein was.

Flash forward and Thelen, a professor of biochemistry and principal investigator at Bond Life Sciences Center, now knows exactly what it is, which is detailed in a recent paper in Nature Communications. And this finding could potentially lead to crops with oil in their leaves that could be harvested.

“The finding was very gratifying, it was like an itch I was finally able to scratch,” Thelen said.

The previously uncharacterized membrane protein belongs to a novel class of proteins in his lab that are now referred to as Carboxyltransferase Interactors (CTIs), based upon the screen used to identify them. Yajin Ye, a former postdoctoral scientist in his lab, discovered that once CTIs are silenced, the amount of triacylglycerol storage oil in plant leaves increased. Oil is not typically found in leaves, so this finding was surprising.

“Normally, plants accumulate oil in the seed or the mesocarp, as carbon for the germinating embryo or a reward for seed dispersal, respectively,” Thelen said. “But when Yajin knocked out the gene he found that the plant leaves began to accumulate oil at rates four-fold higher than normal.”

Building off this discovery, Thelen and his lab continued to do more experiments on ACCase. In plants, ACCase acts as the gatekeeper of fatty acid biosynthesis. And fatty acids are the principal component of the storage lipid triacylglycerol.

“The regulation of this enzyme is quite sophisticated, particularly in plants due to its modular, multi-subunit nature,” Thelen said.

They soon found that the CTI, which anchors ACCase to the envelope membrane, serves as a negative regulator, suppressing ACCase function during light. When the three CTI genes were knocked out by CRISPR, it permitted ACCase to accumulate oil in plant leaves. Thelen first showed this in the model plant Arabidopsis, but is also investigating this in other plants.

“This has potential to make an impact in crops,” Thelen said. “The long-term objective of my lab is to try to increase oil accumulation in plants. This is an interesting discovery that led us to think that maybe leaves could be an alternative location for oils. If we can raise the levels in the leaves, they could be harvested and extracted for their oils and used for industrial food and feedstock purposes instead of discarded.”

Thelen and the University of Missouri patented this discovery and licensed it to a Boston-area company Yield10 Bioscience to commercialize this idea.

Thelen plans to continue looking at ACCase to look for its limits and processes.

“We’re still focused on making basic discoveries regarding the regulation of this first step in fatty acid synthesis,” Thelen said. “There certainly are more discoveries to be made.”

Find out more about this study in, “Docking of acetyl-CoA carboxylase to the plastid envelope membrane attenuates fatty acid production in plants” published in the December 2020 edition of Nature Communications.

Seed size matters: searching for a gene to make a bigger soybean

November 2, 2020 By Becca Wolf in Research Tags: CRISPR, gene expression, soybean

Bing Stacey | photo by Mariah Cox, Bond LSC

By Becca Wolf | Bond LSC

Patience is a virtue, at least it is for Bing Stacey.

Stacey recently completed a project that took her a total of eight years. It took her five years to develop a fast neutron mutant population and it took an additional three years to screen the population to identify a mutant that showed increase soybean seed size and then identifying the causative gene.

This gene, GmKIX8-1, and the seed size QTL, qSW17-1, can potentially be exploited for increasing yield in soybeans. Being able to alter these to increase seed size is important to improve the economic traits of soybeans such as yield and seed quality.

“The most important thing for farmers is the yield,” said Stacey, assistant professor of plant sciences at Bond Life Sciences Center. “For farmers, they plant soybeans, and the value or the profit they get is based on yield.”

There are two components of yield: seed size and seed quality. Stacey went to work finding what genes contribute to these components.

Mutants are very important for gene study because they have a new DNA sequence for genes. While it sounds like a sci-fi term, mutants are just plants manipulated using chemicals, radiations, or genetic modifications to change their characteristic traits in comparison to the norm. And the DNA changes to the GmKIX8-1 gene Stacey found is exactly the mutation she was looking for.

When she began this research, there were not many mutants for soybeans regarding seed size, so she had to develop her own mutant population using fast neutron irradiation.

After taking several years to develop this population, Stacey eventually found several mutants showing altered seed traits, including one showing increased seed size.

“So, we have the mutant, and then we utilized a fast and cost-effective way to genotype for changes in the DNA sequence of the mutant that is associated with the increased seed size. The genotyping method is called Comparative Genome Hybridization (CGH) which can be completed within one week and specifically detects missing DNA sequences in the mutant genome,” Stacey said.

Next, Stacey used CRISPR/Cas9 mutagenesis, which is a very powerful way to cut out specific DNA sequences in targeted genes in an organism. Before CRISPR, plant scientists could only create random mutations in plants. For example, using processes involving mutagenic chemicals and radiations, which meant one had to look at a very large number of mutants to screen for induced changes in the DNA sequence of specific genes.

Using fast neutron mutagenesis which creates deletions in bases, combined with CRISPR/Cas9, Stacey was able to characterize the role of the GmKIX8-1 gene in controlling seed size in soybeans.

“In a way, we got lucky because there is a QTL overlapping GmKIX8-1,” Stacey said. “And once you know the genetics behind a trait, then it is possible to define a possible mechanism of how the gene works, for example, how it makes soybean plants produce bigger seeds.” GmKIX8-1 controls the ability of cells to multiply. It also increases the size of the organs and the seeds of soybeans, which is exactly what Stacey was looking for.

An additional new and important thing Stacey found in her work was that GmKIX8-1 controls seed size, but not leaf size, in a dosage-dependent manner indicating that this gene is a major player in regulating seed size but not leaf size.

“This gene dosage effect on seed size and the identification of GmKIX8-1 as the causative gene behind a major seed size QTL are the novel aspects of this work,” Stacey said.

These novel aspects have been rewarding for Stacey.

“I’m quite happy, and it’s more fulfilling discovering something novel, something that hasn’t been reported before in other plants, instead of confirming what has been already reported,” Stacey said, “For example, based on what is already well-known in maize, or in rice, or in Arabidopsis, I can ask the question, ‘Does this gene also work in soybean?’ Answering this question is still worthwhile to me because maybe the gene will not work at all, or maybe it works in a different way, and that would also be novel, but to discover new genes or new explanations on how a gene works is more fulfilling.”

For more information on Bing Stacey’s work, check out the September 2020 edition of the New Phytologist Foundation article, “GmKIX8‐1 regulates organ size in soybean and is the causative gene for the major seed weight QTL qSw17‐1”

Using improved technology to create an Immune Cell Atlas

October 28, 2020 By Becca Wolf in Research Tags: agriculture, gene expression, RNA

Ashley Meyer | photo by Mariah Cox, Bond LSC

By Becca Wolf | Bond LSC

When given an opportunity to use the newest technology, one has to take it.

Ashley Meyer, the lab supervisor of the Wes Warren lab at Bond Life Sciences Center, recently started using the improved technology of single cell RNA sequencing to create an Immune Cell Atlas for chickens. This search browser of sorts allows people to search for cells and gene expression in chickens.

“It would be a platform for researchers to have a baseline of what should be normal, and then be able to run comparative experiments to say ‘Okay, if we challenge this chicken with this disease, what biological response are we seeing?’ Which can help you then understand the disease better,” Meyer said. “Like, ‘How is it being transmitted? How is this chicken reacting to it?’”

Poultry is one of the largest agricultural industries in Missouri, so it is important to keep chickens healthy.

“The Immune Cell Atlas will help us analyze tissues and will help us perform the single cell RNA sequencing on them to ask what’s normal and what’s diseased so that we can see what’s happening,” Meyer said. “It will also allow us to see which chickens are more susceptible than others or how the progression diseases happen.”

Meyer and the Warren lab are some of the first researchers to apply this technology to chicken based research.

Single cell RNA sequencing (RNAseq) allows Meyer to sequence the RNA of an individual cell which provides in-depth data. This can be done using single cell RNAseq which uses the entire cell, or it can be done using single nuclei RNAseq which uses just the nucleus of a cell. This profiles gene expression in cells that make up a tissue to get data on a cellular level to understand the composition of cells.

It is important to target the RNA because it is the messenger that brings instructions from the DNA for controlling the synthesis of proteins. RNA is commonly synthesized in the nucleus of a cell.

“We’re some of the first people to get our hands on some avian single cell data, specifically looking at nuclei. My job is a lot of optimizing these protocols to isolate these nuclei,” Meyer said.

Using nuclei allows Meyer to use samples that have been frozen, collected in other locations, etc., which provides more flexibility.

Isolating the nuclei through RNAseq allows Meyer to identify different cell types based on genes being expressed in a cell in an organ such as a spleen or lung. This information can then be added to the Immune Cell Atlas.

Meyer is a part of a team working on this project including two master’s students, a bioinformatics analyst, and principal investigator, Wes Warren, at Bond LSC, as well as a bioinformatics analyst who works remotely from Santa Cruz, California.

While the team is working from around the country, they are all connected by a common goal.

“Things have been pretty hectic, the break with the COVID quarantine kind of paused everything and then when we got back it just kind of took off, we’ve generated a couple data sets just in the last month or so,” Meyer said.

Having started this research about six months ago, they still have a way to go. Meyer and her collaborators are currently focused on analyzing five to six immune system organs, and plan on expanding in the future.

Since single cell RNAseq is so new, it can be challenging being one of the first labs to use it at MU and in the avian community.

“Since it’s still newer, everyone’s still figuring out how it works,” Meyer said. “Everybody’s determining their own way of analyzing the data. We’re working through the hiccups of everything, and not having years and years of research to guide us can be challenging; however, also exciting because then we can find what works and what doesn’t work which is the whole point of science.”

While challenging, it is rewarding. Meyer hopes that in the future once they have created the chicken Immune Cell Atlas, they are able to expand to other animals.

“It’s exciting to be part of something that’s newer and having something that could be beneficial for other researchers,” Meyer said. “I am a huge advocate for sharing science and teaching science so creating something to aid other research is super empowering for me.”

The taming of the brain

January 27, 2020 By Roger Meissen in Research Tags: animal science, Cheryl Rosenfeld, gene expression

Bond LSC scientist delves into how domestication alters the fox brain

Russian silver foxes that have been tamed show changes in their brains. | photo provided by Anna Kukekova

By Roger Meissen | Bond Life Sciences Center

You might be familiar with the idiom “don’t bite the hand that feeds you,” but when it comes to a certain lineage of tame Russian silver foxes it’s quite literal.

After more than 50 generations of breeding, these tame foxes likely offer insight into how selective breeding leads to domestication, and scientists dove deeper to look at what this trait might mean in terms of changes in the brain and the genes behind them.

“These silver foxes act like domesticated dogs and are assumingly a good model to look at the process of domestication, tracing what happened as the wolves became domesticated into what we recognize today as the domesticated dog. Wouldn’t that be fascinating to know all the things that coincide with that?” said Cheryl Rosenfeld, a Bond Life Sciences Center principal investigator at the University of Missouri and co-lead author on the study. “Presumably, to become domesticated the one biggest organ that would have to change is the brain, because that’s where fearfulness, aggression, and social behaviors emerge from, so my interest is in the brain and how various factors might affect it.”

The path away from aggression

While this story eventually focuses on the brain, the path to a modern model of tameness starts in Soviet Russia in the 1950s.

Russian geneticist Dmitry K. Belyaev bred hundreds of foxes in Siberia, initially selecting them based on their willingness to approach humans in their cage. In its beginning only 10 percent of those tested showed a weakened wild response, and those foxes were bred together to reinforce this docile nature. In later generations, their behavior was further evaluated based on their likelihood to interact with humans approaching their cage, their territorial behavior, response to attempts to pet them and reaction to the experimenter leaving the interaction. Lyudmila Trut, his intern now in her 80s and an author on this study, continues the breeding project.

“Those that showed a muted wild response would get bred together, so with each passing generation, the foxes showed diminished fear response. Once they established this tame fox population, they eventually developed the aggressive ones doing just the opposite in that they paired those foxes showing the most aggressive responses to an experimenter approaching the cage,” Rosenfeld said. “They repeated this over time and, ultimately, what they have now, going out 50 generations, are tame foxes that act and even look in some ways like pet dogs.”

The tame silver foxes love human interaction from attention to belly rubs, and exhibit traits like tail wagging, gazing into the faces of humans and a change in their barks. Physical changes accompanied these behavioral changes, with coat color shifting from silvered-black fur to a more mottled, piebald pattern, and tails becoming curly despite these traits not being intentional selected for by breeders. In contrast, those bred for aggression showed hyper aggressive responses to humans, as indicated by lunging at the cage.

The taming of the brain

A change in behavior as observed in these silver foxes likely comes with unseen shifts in genetics and brain function. Rosenfeld decided to look at these changes in the hypothalamus region of the brain with the help of researchers from the University of Illinois at Urbana-Champaign, Cornell University, the Broad Institute of MIT and Harvard and the Institute of Cytology and Genetics in Novosibirsk, Russia.

“We are interested in changes in gene expression associated with the hypothalamic-pituitary-adrenal axis, the main hormonal system involved in stress-response and it was very exciting and productive to collaborate with Dr. Rosenfeld and her colleagues on the analysis of gene expression in the hypothalamus of tame and aggressive foxes” said Anna Kukekova, the senior author and associate professor in the Department of Animal Sciences at the University of Illinois at Urbana-Champaign.

Rosenfeld compared genes in the hypothalamus between tame foxes and aggressive foxes as well as comparing them to genes in two other brain regions — the prefrontal cortex and the basal forebrain — significant in terms of learning and memory.

“The hypothalamus was a good target organ, because it controls social behaviors like getting along or gregariousness and usually hormones like the pro-social hormone oxytocin tends to increase in animals who are social,” Rosenfeld said. “We basically did a global approach, looking at every gene we could identify and how they differ in terms of expression between the tame and aggressive foxes.”

Among genes with differential hypothalamic expression in tame and aggressive foxes they found seven identical genes with the same pattern of expression between all three brain regions. It suggests that when you select for tameness that some genes will change regardless of brain region.

“It was interesting to us because these genes were involved in processes including cell division and making more neurons, differentiation, adhesion like how the cells contact each other, and the most surprising, carbohydrate processing.”

Rosenfeld commented that some of these involved — such as, carbohydrate processing — suggest potential changes to accommodate domestication between the tame and aggressive foxes. This falls in line with how scientists believe dogs became domesticated 10,000 years ago, growing ever closer to human populations and developing to scavenge their scraps and garbage, although in this study both tame and aggressive foxes were maintained on the same diet.

“As animals get domesticated, they have to also accommodate different diets than what they’ve been eating in the wild because now they’re getting scraps from humans,” Rosenfeld said. “They’re selecting for genes that are involved in carbohydrate process, like metabolism processing, so it’s sort of telling us when you consider domestication that we might have to think about how the animal may have to then turn on a whole set of other genes to accommodate this new diet.”

But there were other brain differences as well.

“We also found genes suppressed in tame foxes associated in inflammation processes we call interleukin signaling, cytokines production and communication between cells to each other, and this gene expression linked to whether they were tame versus aggressive.”

The researchers’ original idea that genes related to hormones like oxytocin ended up not panning out when they saw little change between tame and aggressive foxes. But, she said it was interesting that the prevalent changes they did see involved more genes down-regulated or suppressed in tame than aggressive foxes.

“Most of the genes in the hypothalamus actually we found were suppressed in tame relative to hyper aggressive foxes, you know, squashing a hyperactive response, Rosenfeld said. “So, it’s an interesting question to consider that, as dogs became more domesticated, did they become more prone to disease because they don’t have such a heightened immune response? We can’t really answer that question just yet.”

Read more about this work in the journal article “Hypothalamic transcriptome of tame and aggressive silver foxes (Vulpes vulpes) identifies gene expression differences shared across brain regions.” This research published in October 2019 in the journal Genes, Brain and Behavior.

Cheryl Rosenfeld is a principal investigator in MU’s Bond Life Sciences Center and a professor of biomedical sciences at the University of Missouri.

MU researchers find key gene in spinal locomotion, yield insight on paralysis

March 21, 2014 By Paige Blankenbuehler in Research Tags: Bond Life Sciences Center, gbx1, gbx2, gene expression, paralysis, research, scicomm, science, science communication, University of Missouri

Samuel Waters and graduate researcher Desiré Buckley review stages of embryonic development. — BLANKENBUEHLER

The difference between walking and being paralyzed could be as simple as turning a light switch on and off, a culmination of years of research shows.

Recently, University of Missouri Assistant Professor of biology Samuel T. Waters isolated a coding gene that he found has profound effects on locomotion and central nervous system development.

Waters’ work with gene expression in embryonic mouse tissue could shed light on paralysis and stroke and other disorders of the central nervous system, like Alzheimer’s disease.

Waters works extensively with two coding genes called “Gbx1” and “Gbx2”. These genes — exist in the body with approximately 20,000 other protein-coding genes — are essential for development in the central nervous system.

“To understand what’s going wrong, it’s critical that we know that’s right,” Waters said.

Coding genes essentially assign functions for the body. They tell your fingernail to grow a certain way, help develop motor control responsible for chewing and, as shown in Waters’ research, help your legs work with your spinal cord to facilitate movement.

Waters and his researchers, including graduate student Desiré Buckley, investigated the function of the Gbx1 by deactivating it in mouse embryos and observing their development over a 18.5-day gestation period — the time it takes a mouse to form.

The technology could eventually contribute to developing gene therapies for paralysis that happens at birth or from a direct result of blunt trauma, like a car accident.

“Understanding what allows us to walk normally and have motor control, allows us to have better insight for developing strategies for repairing neural circuits and therapies,” Waters said.

Technology for isolating genes and their functions

Waters studies embryonic mouse development. To understand certain gene functions, he inactivates different genes using a technology called “Cre-loxP.”

Genes can be isolated, then inactivated throughout embryonic tissue. Many of Waters’s studies inactivate genes to harness a better understanding of which genes are responsible for what.

“The relevance of it to the well-being of humans, is apparently relevant to development and more importantly to the development of the central nervous system,” Waters said. “Now it’s taking me to the point where we’re getting a bird’s eye view of what’s actually regulating our ability to have locomotive control.”

No Gbx1, no regular locomotion

Mice that Waters uses in his lab, “display a gross locomotive defect that specifically affects hind-limb gait,” according to their article published in Plos One, February, 2013.

In contrast to its family member Gbx2, when Gbx1 is inactivated, Waters concluded, the anterior hindbrain and cerebellum appear to develop normally. But neural circuit development in the spinal cord —- what allows us to walk normally —- is compromised, he said. According to an article published by

Waters, November 2013, in Methods in Molecular Biology, this occurs despite an increase in the expression level of its family member, Gbx2, in the spinal cord.

A video recording from the research, which was funded by the National Science Foundation and start-up funds from MU, show the mouse with the Gbx1 held back, with an abnormal hind-limb-gait.

Mice with this inactivated gene were otherwise normal, Waters said.

“If they were sitting there without moving, you wouldn’t know anything was wrong with them,” Waters said.” They’re able to mate, eat and appear to function normally.”

Photographs taken during the research that show the hind limb gait defect in specimen with Gbx1 held back.

No Gbx2, no jaw mobility

When Gbx2 function is impaired in the mouse, Waters observed that development of the anterior hindbrain, including the cerebellum, a region of the brain that plays an important role in motor control, didn’t form correctly.

The mice, as a result, cannot suckle, so they die at birth, Waters said.

“We’re getting a better insight into the requirements for suckling — another motor function required for our survival,” Waters said.

The research has paved the way for investigating other coding genes and their responsibilities and roles in development, Waters said.

“We have a lot to do still,” Waters said. “So, why am I so excited about it? That’s part of the reason.”