development

Heads or tails? Stem cells help guide the decision

/ Kevin McCormack / 1 Comment
Two cell embryo

There are many unknown elements for what triggers the cells in an embryo to start dividing and multiplying and becoming every single cell in the body. Now researchers at the Gladstone Institutes in San Francisco have uncovered one of those elements, how embryos determine which cells become the head and which the tail.

In this CIRM-funded study the Gladstone team, led by Dr. Todd McDevitt, discovered almost by chance how the cells align in a heads-to-tail arrangement.

Todd McDevitt

They had created an organoid made from brain cells when they noticed that some of the cells were beginning to gather in an elongated fashion, in the same way that spinal cords do in a developing fetus.

In a news article, Nick Elder, a graduate student at Gladstone and the co-author of the study, published in the journal Development, says this was not what they had anticipated would happen: “Organoids don’t typically have head-tail directionality, and we didn’t originally set out to create an elongating organoid, so the fact that we saw this at all was very surprising.”

Further study enabled the team to identify which molecules were involved in signaling specific genes to switch on and off. These were similar to the process previously identified in developing mouse embryos.

“This is such a critical point in the early development of any organism, so having a new model to observe it and study it in the lab is very exciting,” says McDevitt.

This is not just of academic interest either, it could have real world implications in helping understand what causes miscarriages or birth defects.

“We can use this organoid to get at unresolved human developmental questions in a way that doesn’t involve human embryos,” says Dr. Ashley Libby, another member of the team. “For instance, you could add chemicals or toxins that a pregnant woman might be exposed to, and see how they affect the development of the spinal cord.”

An inside look reveals the adult brain prunes its own branches

/ Karen Ring / Leave a comment

Did you know that when you’re born, your brain contains around 100 billion nerve cells? This is impressive considering that these nerve cells, also called neurons, are already connected to each other through an intricate, complex neural network that is essential for brain function.

Here’s how the brain does it. During development, neural stem cells produce neurons that navigate their way through the brain. Once at their destination, neurons set up shop and send out long extensions called axons and branched extensions called dendrites that allow them to form what are called synaptic connections through which they can communicate through electrical and chemical signals.

Studies of early brain development revealed that neurons in the developing brain go on overdrive and make more synaptic connections than they need. Between birth and early adulthood, the brain carefully prunes away weak or unnecessary connections, and by your mid-twenties, your brain has eliminated almost half of the synaptic connections you started out with as a baby.

This synaptic pruning process allows the brain to fine-tune its neural network and strengthen the connections between neurons that are important for brain function. It’s similar to how a gardener prunes away excess branches on fruit trees so that the resulting branches can produce healthier and better tasting fruit.

The brain can make new neurons

It was thought that by adulthood, this process of pruning excess connections between neurons was over. However, a new study from the Salk Institute offers visual proof that synaptic pruning occurs during adulthood similarly to how it does during development. The work was published today in the journal Nature Neuroscience, and it was funded in part by CIRM.

Rusty Gage, Salk Institute.

Rusty Gage, Salk Institute.

The study was led by senior author and Salk Professor Rusty Gage. Gage is well known for his earlier work on adult neurogenesis. In the late 90’s, he discovered that the adult brain can in fact make new neurons, a notion that overturned the central dogma that the brain doesn’t contain stem cells and that we’re born with all the neurons we will ever have.

There are two main areas of the adult brain that harbor neural stem cells that can generate new neurons. One area is called the dentate gyrus, which is located in the memory forming area of the brain called the hippocampus. Gage and his team were curious to know whether the new neurons generated from stem cells in the dentate gyrus also experienced the same synaptic overgrowth and pruning that the neurons in the developing brain did.

Pruning the Adult Brain

They developed a special microscope technique that allowed them to visually image the development of new neurons from stem cells in the dentate gyrus of the mouse brain. Every day, they would image the growing neurons and monitor how many dendritic branches they sent out.

Newly generated neurons (green) send out branched dendritic extensions to make connections with other neurons. (Image credit: Salk Institute)

Newly generated neurons (green) send out branched dendritic extensions to make connections with other neurons. (Image credit: Salk Institute)

After observing the neurons for a few weeks, they were amazed to discover that these new neurons behaved similarly to neurons in the developing brain. They sent out dozens of dendritic branches and formed synaptic connections with other neurons, some of which were eventually pruned away over time.

This phenomenon was observed more readily when they made the mice exercise, which stimulated the stem cells in the dentate gyrus to divide and produce more neurons. These exercise-induced neurons robustly sent out dendritic branches only to have them pruned back later.

First author on the paper, Tiago Gonçalves commented on their observations:

“What was really surprising was that the cells that initially grew faster and became bigger were pruned back so that, in the end, they resembled all the other cells.”

Rusty Gage was also surprised by their findings but explained that developing neurons, no matter if they are in the developing or adult brain, have evolved this process in order to establish the best connections.

“We were surprised by the extent of the pruning we saw. The results suggest that there is significant biological pressure to maintain or retain the dendrite tree of these neurons.”

A diagram showing how the adult brain prunes back the dendritic branches of newly developing neurons over time. (Image credit: Salk Institute).

A diagram showing how the adult brain prunes back the dendritic branches of newly developing neurons over time. (Image credit: Salk Institute).

Potential new insights into brain disorders

This study is important because it increases our understanding of how neurons develop in the adult brain. Such knowledge can help scientists gain a better understanding of what goes wrong in brain disorders such as autism, schizophrenia, and epilepsy, where defects in how neurons form synaptic connections or how these connections are pruned are to blame.

Gonçalves also mentioned that this study raises another important question related to the regenerative medicine applications of stem cells for neurological disease.

“This also has big repercussions for regenerative medicine. Could we replace cells in this area of the brain with new stem cells and would they develop in the same way? We don’t know yet.”

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A cardiac love triangle: how transcription factors interact to make a heart

/ Karen Ring / Leave a comment

 Here’s a heartfelt science story for all those Valentine’s day fans out there. Scientists from the Gladstone Institutes have identified how a group of transcription factors interact during embryonic development to make a healthy heart. Their work will increase our biological understanding of how the heart is formed and could produce new methods for treating cardiovascular disease.

The study, published today in the journal Cell, describes a tumultuous love story between cardiac transcription factors. Transcription factors are proteins that orchestrate gene expression. They have the power to turn genes on or off by binding to specific DNA sequences and recruiting other proteins that will eventually turn the information encoded in that gene into a functional protein.

Every organ has its own special group of transcription factors that coordinate the gene expression required for that organ’s development. Often times, transcription factors within a group directly interact with each other and work together to conduct a specific sequence of events. These interactions are essential for making healthy tissues and organs, but scientists don’t always understand how these interactions work.

For the heart, scientists have already identified a group of transcription factors essential for cardiac development, and genetic mutations in any of these factors can impair heart formation and cause heart defects in newborns. What’s not known, however, are the details on how some of these cardiac transcription factors interact to get their job done.

A cardiac love triangle

In the Gladstone study, the scientists focused on how three key cardiac transcription factors – NKX2.5, TBX5, and GATA4 – interact during heart development. They first proved that these transcription factors are essential for the formation of the heart in mouse embryos. When they eliminated the presence of one of the three factors from the developing mouse embryo, they observed abnormal heart development and heart defects. When they removed two factors (NKX2.5 and TBX5), the results were even worse – the heart wasn’t able to form and none of the embryos survived.

Normal heart muscle cells, courtesy Kyoto University

Normal cardiomyocytes or heart cells, courtesy Kyoto University

Next, they studied how these transcription factors interact to coordinate gene expression in heart cells called cardiomyocytes made from mouse embryonic stem cells that lacked either NKX2.5, TBX5, or both of these factors. Compared to normal heart cells, cardiomyocytes that lacked one or both of these two transcription factors started beating at inappropriate times – either earlier or later than the normal heart cells.

Taking a closer look, the scientists discovered that TBX5, NKX2.5 and GATA4 all hangout in the same areas of the genome in embryonic stem cells that are transitioning into cardiomyocytes. In fact, each individual transcription factor required the presence of the others to bind their DNA targets. If one of these factors was missing and the love triangle was broken, the remaining transcription factors became confused and bound random DNA sequences in the genome, causing a mess by turning on genes that shouldn’t be on.

First author on the study, Luis Luna-Zurita, explained the importance of maintaining this cardiac love triangle in a Gladstone Press Release:

Luis Luna-Zurita, Gladstone Institute

Luis Luna-Zurita, Gladstone Institute

“Transcription factors have to stick together, or else the other one goes and gets into trouble. Not only are these transcription factors vital for turning on certain genes, but their interaction is important to keep each other from going to the wrong place and turning on a set of genes that doesn’t belong in a heart cell.”

Crystal structure tells all

Protein crystal structure of NKX2.5 and TBX5 bound to DNA.

Protein crystal structure of NKX2.5 and TBX5 bound to DNA. (Luna-Zurita et al. 2016)

The last part of the study proved that two of these factors, NKX2.5 and TBX5, directly interact and physically touch each other when they bind their DNA targets. In collaboration with a group from the European Molecular Biology Laboratory (EMBL) in Germany, they developed protein crystal structures to model the molecular structure of these transcription factors when they bind DNA.

Co-author and EMBL scientist Christoph Muller explained his findings:

“The crystal structure critically shows the interaction between two of the transcription factors and how they influence one another’s binding to a specific stretch of DNA. Our detailed structural analysis revealed a direct physical connection between TBX5 and NKX2-5 and demonstrated that DNA plays an active role in mediating the interaction between the two proteins.”

Big picture

While this study falls in the discovery research category, its findings increase our understanding of the steps required to make a healthy heart and sheds light on what goes wrong in patients or newborns with heart disease.

Senior author on the paper and Gladstone Professor Benoit Bruneau explained the biomedical applications of their study for treating human disease:

DSC_0281_2

Benoit Bruneau, Gladstone Institute

“Gene mutations that cause congenital heart disease lower the levels of these transcription factors by half, and we’ve shown that the dosage of these factors determines which genes are turned on or off in a cell. Other genetic variants that cause heart defects like arrhythmias also affect the function of these factors. Therefore, the better we understand these transcription factors, the closer we’ll come to a treatment for heart disease. Our colleagues at Gladstone are using this knowledge to search for small molecules that can affect gene regulation and reverse some of the problems caused by the loss of these transcription factors.”

 

I think it’s worth mentioning that these studies were done using mouse embryos and mouse embryonic stem cells. Future work should be done to determine whether this cardiac love triangle and the same transcription factor interactions exist in human heart cells.

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UCLA Scientists Find 3000 New Genes in “Junk DNA” of Immune Stem Cells

/ Karen Ring / 2 Comments

Genes and Junk

Do you remember learning about Junk DNA when you took Biology in high school? The term was used to described 98% of the human genome that doesn’t make up its approximately 22,000 genes. We used to think that Junk DNA didn’t serve a purpose, but that was before we discovered special elements called non-coding RNAs that call Junk DNA their home. But we’re getting ahead of ourselves, so let’s take a step back.

Genes are sequences of DNA that contain the blueprints for the proteins that make your cells and organs function. Before a gene can become a protein, its transformed into a molecule called an RNA. RNAs contain messages that tell a cell’s machinery what types of protein to make and how many.

Not Junk After All

Now back to “Junk DNA”… scientists thought that because this mass of DNA sequences was never turned into protein, it served no purpose. It turns out that they couldn’t be farther from the facts.

There are actually sequences of DNA in our genomes that are blueprints for RNAs that never become proteins. Scientists call them “non-coding” RNAs, and they play very important roles in the body such as replicating DNA and regulating gene expression – deciding which genes are turned on and which are turned off.

Another important function that non-coding RNAs control is cell differentiation, or the maturation of immature cells into adult cells. Differentiation is a complicated process, and because non-coding RNAs are relatively new to the scientific world, we haven’t figured out their exact roles in the differentiation of stem cells into adult cells.

Understanding Immune Cell Development

In a study published this week in Nature Immunology, UCLA scientists reported the discovery of 3000 new genes that make a type of non-coding RNA called a long non-coding RNA (lncRNA) that regulates the differentiation of stem cells into mature immune cells like B and T cells, which play a key role in fighting infection. This important study was funded in part by CIRM.

UCLA scientists David Casero and Gay Crooks with the sequencing machine that separated the genetic information within the bone marrow and thymus gland tissue stem cells. (Image credit: Mirabai Vogt-James, UCLA Broad Stem Cell Research Center)

UCLA scientists David Casero and Gay Crooks with the sequencing machine used to identify the 3000 new genes. (Image credit: Mirabai Vogt-James, UCLA Broad Stem Cell Research Center)

Using sequencing technology and bioinformatics, they mapped the RNA landscape (known as the transcriptome) of rare stem cells isolated from human bone marrow (hematopoietic stem cells) and the thymus (lymphoid progenitor cells). They identified over 9000 genes that produced lncRNAs that were important for moderating various stages of immune cell development. Of this number, over 3000 were genes whose lncRNAs hadn’t been found before.

First author, David Casero explained the importance of their discovery in a UCLA press release:

Our findings are exciting because they provide a huge and unique resource for the whole immunology community. We will now be able to drill down on the specific LncRNA genes that seem to be most important at each stage of immune cell development and understand how they function individually and together to control the process.

 

Co-senior author and UCLA professor Gay Crooks explained that the goal of their work was to gain a better understanding of how the immune system develops in order to battle serious diseases that affect it and open up avenues for generating better cell therapies.

If we can understand how the immune system is generated and maintained during life, we can find ways to improve production of immune cells for potential therapies after chemotherapy, radiation and bone marrow transplant, or for patients with HIV and inherited immune deficiencies. In addition, by understanding the genes that control this process we can better understand how they are changed in cancers like leukemia and lymphoma.

 

Final Words

While this study focused on the role of lncRNAs in the development of the immune system and the differentiation of immune stem cells, the technology in this study can be used to understand the development of other systems and organs.

Scientists are already publishing papers on the role of lncRNAs in the differentiation of stem cells in the brain and heart, and further work in this field will undoubtedly uncover many new and important lncRNA genes. If the pace keeps up, the term “Junk DNA” will need to be retired to the junk yard.

junk-dna-series2

Image source www.biocomicals.com

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A Glimpse Inside the Cellular Universe: Scientists Track the Growth of an Organism, One Cell at a Time

/ cirmweb / Leave a comment

Trying to keep tabs on how an organism grows from a single fertilized egg into an embryo, cell by cell, is hard work. So hard in fact, that no one’s quite figured out how to do it.

Digital fruit fly embryo, reconstructed from live imaging data recorded with a SiMView light-sheet microscope. Each colored circle in the image shows one of the embryo's cells, and the corresponding tail indicates that cell's movement over a short time interval at around 3 hours post-fertilization [Credit: Kristin Branson, Fernando Amat, Bill Lemon and Philipp Keller (HHMI/Janelia Research Campus)]

Digital fruit fly embryo, reconstructed from live imaging data recorded with a SiMView light-sheet microscope. Each colored circle in the image shows one of the embryo’s cells, and the corresponding tail indicates that cell’s movement over a short time interval at around 3 hours post-fertilization
[Credit: Kristin Branson, Fernando Amat, Bill Lemon and Philipp Keller (HHMI/Janelia Research Campus)]

The problem, as researchers have lamented, is that there’s just too much happening—all at the same time—for the human eye to parse through all the data, even with the aid of the most powerful microscopes.

But now, scientists at the Howard Hughes Medical Institute (HHMI) have devised a high-tech shortcut: a new computational program that measures in real-time the three-dimensional development of each individual cell in a developing fetus.

This program stands to revolutionize how scientists understand the microscopic cellular ‘universe.’ As lead author, HHMI Group Leader Dr. Philipp Keller, explained in a news release:

“We wanted to reconstruct the elemental building plan of animals, tracking each cell from very early development until the late stages, so that we know everything that has happened in terms of cell movement and cell division.”

This technique, which is described in the latest issue of the journal Nature Methods, was built upon Keller’s 2012 development of a something called SiMView, a one-of-a-kind microscope that can capture precise 3D images of cells over a period of hours or even days.

But this was only the first step. Since the development of SiMView, Keller has been working on improving the system so that it could be used more broadly and over the course an organism’s development as an embryo. Specifically, Keller had sought to use this technique to look at how specific parts of the body develop—cell by cell. As Keller elaborated:

“In particular, we wanted to understand how the nervous system forms. Ultimately, we could like to collect the developmental history of every cell in the nervous system and link that information to the cell’s final function.”

In collecting and analyzing these vast datasets, researchers would then be well-poised to understand underlying molecular mechanisms of nervous system diseases.

Keller and his team have been looking for ways to both capture and analyze the vast amount of data hidden within each cell as it grows, matures and divides, with limited success—even the SiMView system was only active at a much smaller scale than what the team desired. One of the main issues is that as the cells in the embryo grow and divide, they become densely packed. They also shift around constantly, making tracking incredibly difficult to view.

The solution, Keller said, was to simplify the data. First, they clustered groups of 3D pixels called ‘voxels’ together into larger units, called ‘supervoxels.’ Next, they programmed the software to recognize the nuclei of each cell within the supervoxels. Then, using high-speed microscopy, they could capture images in a very quick sequence—so quick that individual cells wouldn’t be able to move out of the frame.

In this way, they are able to gather about 95% of all available data, a far higher number than that achieved by traditional methods. For the remaining 5%, the team employed even more complex algorithms to sort through the data. The end result, Keller says, is a wealth of knowledge that reveals more than many ever thought possible. According to Keller:

“You know the path, you know where it is at a certain time point. You know it divided from a certain point, you know the daughter cells, you know what mother cell it came from.”

In the early tests, the team studied the cellular ‘lineages’ of 295 early-stage nerve cells, called neuroblasts. Interestingly, they were not only able to trace these lineages in their entirety, but they could also predict their behavior later in their lifespan based on how they behaved early on.

The software, which is free and readily available to interested researchers, can be applied to a wide variety of data types—including different organisms and different microscopes.

This development stands to potentially become highly valuable to the stem cell research community. Increasingly, stem cell scientists are finding that in order to drive stem cells towards a desired adult tissue efficiently and completely, they need to try to recreate the stem cells’ natural environment. This should make it easier to build the right cellular “Neighborhood,” and help foster the transition from basic research into effective therapies.

Confining Cells within Geometric Structures Key to Replicating Embryonic Development

/ cirmweb / Leave a comment

It’s like trying to capture, and then recreate, a moment in time: the exact instant after fertilization when a small group of dividing cells begin to organize themselves into the various cellular layers that will one day make up the skin, the heart, the liver and the brain. But for all the advances in our understanding of how an embryonic stem cell grows, matures and differentiates—scientists still can’t replicate that very important process in the lab.

Forty-two hours after they began to differentiate, embryonic cells are clearly segregating into the various layers that will one day become specific tissues and organs. Researchers say the key to achieving this patterning in culture is confining the colonies geometrically. [Credit: The Rockefeller University]

Forty-two hours after they began to differentiate, embryonic cells are clearly segregating into the various layers that will one day become specific tissues and organs. Researchers say the key to achieving this patterning in culture is confining the colonies geometrically. [Credit: The Rockefeller University]

But now, scientists at The Rockefeller University have tried something new, and in so doing have finally found a way to stimulate this organization, thus mimicking in a petri dish what happens in the human embryo. The missing ingredient, the researchers found, wasn’t a molecule or chemical compound. Rather, the team just had to use a bit of geometry.

Reporting in the June 29 issue of the journal Nature Methods, the Rockefeller team—led by Dr. Ali Brivanlou—describes how they constructed microscopic circular patterns on glass plates that confined embryonic stem cells inside, similar to a hedge maze.

To their amazement, the cells confined within these patterns soon began to go through gastrulation, the process by which embryonic stem cells begin to form highly organized layers that eventually mature into the body’s various organs and tissues. A second group of cells not confined within these patterns, however, did not.

The next question they had to figure out, according to the researchers, was why.

To solve this mystery, Brivanlou and his team next monitored specific chemical signals between the cells as they matured. In so doing they uncovered a delicate arrangement of chemical cues—molecular ‘on-and-off-switches’—that guided each cell down one developmental path as opposed to another. What were crucial to these cues going off without a hitch, the researchers found, were the geometric patterns.

As Dr. Aryeh Warmflash, one of the paper’s lead authors, stated in this week’s news release:

“At the fundamental level, what we have developed is a new model to explore how human embryonic stem cells first differentiate into separate populations with a very reproducible spatial order just as in an embryo. We can now follow individual cells in real time in order to find out what makes them specialize, and we can begin to ask questions about the underlying genetics of the process.”

Added Brivanlou:

“Understanding what happens in this moment, when individual members of this mass of embryonic stem cells begin to specialize for the very first time and organize themselves into layers, will be key to harnessing the promise of regenerative medicine.”

Confining Cells within Geometric Structures Key to Replicating Embryonic Development

/ cirmweb / Leave a comment

It’s like trying to capture, and then recreate, a moment in time: the exact instant after fertilization when a small group of dividing cells begin to organize themselves into the various cellular layers that will one day make up the skin, the heart, the liver and the brain. But for all the advances in our understanding of how an embryonic stem cell grows, matures and differentiates—scientists still can’t replicate that very important process in the lab.

Forty-two hours after they began to differentiate, embryonic cells are clearly segregating into the various layers that will one day become specific tissues and organs. Researchers say the key to achieving this patterning in culture is confining the colonies geometrically. [Credit: The Rockefeller University]

Forty-two hours after they began to differentiate, embryonic cells are clearly segregating into the various layers that will one day become specific tissues and organs. Researchers say the key to achieving this patterning in culture is confining the colonies geometrically. [Credit: The Rockefeller University]

But now, scientists at The Rockefeller University have tried something new, and in so doing have finally found a way to stimulate this organization, thus mimicking in a petri dish what happens in the human embryo. The missing ingredient, the researchers found, wasn’t a molecule or chemical compound. Rather, the team just had to use a bit of geometry.

Reporting in the June 29 issue of the journal Nature Methods, the Rockefeller team—led by Dr. Ali Brivanlou—describes how they constructed microscopic circular patterns on glass plates that confined embryonic stem cells inside, similar to a hedge maze.

To their amazement, the cells confined within these patterns soon began to go through gastrulation, the process by which embryonic stem cells begin to form highly organized layers that eventually mature into the body’s various organs and tissues. A second group of cells not confined within these patterns, however, did not.

The next question they had to figure out, according to the researchers, was why.

To solve this mystery, Brivanlou and his team next monitored specific chemical signals between the cells as they matured. In so doing they uncovered a delicate arrangement of chemical cues—molecular ‘on-and-off-switches’—that guided each cell down one developmental path as opposed to another. What were crucial to these cues going off without a hitch, the researchers found, were the geometric patterns.

As Dr. Aryeh Warmflash, one of the paper’s lead authors, stated in this week’s news release:

“At the fundamental level, what we have developed is a new model to explore how human embryonic stem cells first differentiate into separate populations with a very reproducible spatial order just as in an embryo. We can now follow individual cells in real time in order to find out what makes them specialize, and we can begin to ask questions about the underlying genetics of the process.”

Added Brivanlou:

“Understanding what happens in this moment, when individual members of this mass of embryonic stem cells begin to specialize for the very first time and organize themselves into layers, will be key to harnessing the promise of regenerative medicine.”