Hox cluster found in Crown of Thorns starfish a surprise

A Crown of Thorns starfish, Acanthaster planci, feeding on several species of Montipora corals. The image was taken off the coast of Okinawa near Sesoko Island. Credit: Yuna Zayasu

New research published in the journal genesis, by Kenneth Baughman, Dr. Eiichi Shoguchi, Professor Noriyuki Satoh of the Marine Genomics Unit at the Okinawa Institute of Science and Technology Graduate University, and collaborators from Australia, reports an intact Hox cluster in the Crown of Thorns starfish, Acanthaster planci. This surprising result contrasts with the relatively disorganized Hox cluster found in sea urchins, which are also echinoderms, classification of animals including starfish, sea lilies, and sea cucumbers. Stanford University Professor Christopher Lowe, who studies developmental biology in echinoderms, summarizes the paper: "The translocation of the Hox cluster in echinoderms has been a major red herring for understanding their evolution. It's really good to have some hard data showing that some echinoderms exhibit some oddities that are not representative of all echinoderms."

The Hox cluster is a classic example of an 'evo-devo' genetic toolkit. The term "evo-devo" refers to the study of genetic programs that control development, which can be compared between species, and thus, across evolutionary time. 

The Hox genes coordinate segmental identity along the head to tail (anterior-posterior) axis. The Hox cluster is evolutionarily conserved and has been 

repurposed repeatedly during the evolution of the animal body plan, or how animals are shaped. Past studies have shown that Hox clusters organize the development of brain and central nervous system regions in chordates, limb bud identity in vertebrates, and, classically, antennae or wing segment identity in fruit flies.

Generally, the Hox cluster shows "colinearity," in which gene order correlates with the location of expression, or the developmental stage of expression. "For example, anterior Hox genes are expressed in regions that are closer to the head of an embryo, and are expressed sooner during development, versus the posterior Hox genes," explained Baughman. "Thus, we were surprised to see chordate-like Hox cluster organization in starfish, which have a radial body plan." Echinoderms are classical model organisms for embryology, and more recently evo-devo. Baughman added, "Interacting with the speakers and students of theOIST Winter Course 'Evolution of Complex Systems' (OWECS) allowed me to appreciate the importance of finding an intact Hox cluster in starfish."

The Crown of Thorns starfish, a predatory starfish which feeds on corals, is famous for its dramatic changes in population density on the reefs near Australia, as well as Okinawa. Over the past 50 years, this has resulted in a measurable loss of coral reefs. A recent 27-year reef monitoring study of the Great Barrier Reef estimated that the starfish accounts for 42% of the loss in coral cover, 2nd only to typhoons. While population control was the initial motivation for Crown of Thorns genome research, the Hox cluster report is one of the first to show that the species can be useful for studies in evolutionary-developmental biology. "We were excited to see the entire Hox cluster on a single genomic scaffold, a result which confirmed the remarkably high quality of the genomic data," said Prof. Satoh.

As is often the case with science, the discovery raises more questions than it answers. If starfish have a collinear Hox cluster, what accounts for their dramatic departures in body plan organization? Do starfish express Hox genes during development in a manner similar to chordates, as indicated by the organization of their Hox cluster? Baughman looks forward to addressing these questions and many more as part of his doctoral research. "I look forward to pursuing developmental biology studies that may suggest methods for mitigating damage to the coral reef caused by the Crown of Thorns starfish."

More research is being conducted at OIST on the Crown of Thorns starfish in the Marine Biophysics Unit by Masako Nakamura in cooperation with local fishermen. In addition to the research collaboration highlighted by the Hox publication, recent efforts by the Okinawa Prefectural Government and Australian Institute of Marine Science are also working on projects to protect the existing coral reefs from the Crown of Thorns starfish.

Source: Okinawa Institute of Science and Technology - OIST

Identifying gene-enhancers: New technique

Diane Dickel is the lead author of Nature Methods paper describing a new technique for identifying gene enhancers in the genomes of humans and other mammals. Credit: Roy Kaltschmidt

An international team led by researchers with the Lawrence Berkeley National Laboratory (Berkeley Lab) has developed a new technique for identifying gene enhancers -- sequences of DNA that act to amplify the expression of a specific gene -- in the genomes of humans and other mammals. Called SIF-seq, for site-specific integration fluorescence-activated cell sorting followed by sequencing, this new technique complements existing genomic tools, such as ChIP-seq (chromatin immunoprecipitation followed by sequencing), and offers some additional benefits.

"While ChIP-seq is very powerful in that it can query an entire genome for characteristics associated with enhancer activity in a single experiment, it can fail to identify some enhancers and identify some sites as being enhancers when they really aren't," says Diane Dickel, a geneticist with Berkeley Lab's Genomics Division and member of the SIF-seq development team. "SIF-seq is currently capable of testing only hundreds to a few thousand sites for enhancer activity in a single experiment, but can determine enhancer activity more accurately than ChIP-seq and is therefore a very good validation assay for assessing ChIP-seq results."

Dickel is the lead author of a paper in Nature Methods describing this new technique. The paper is titled "Function-based identification of mammalian enhancers using site-specific integration." The corresponding authors are Axel Visel and Len Pennacchio, also geneticists with Berkeley Lab's Genomics Division.

With the increasing awareness of the important role that gene enhancers play in normal cell development as well as in disease, there is strong scientific interest in identifying and characterizing these enhancers. This is a challenging task because an enhancer does not have to be located directly adjacent to the gene whose expression it regulates, but can instead be located hundreds of thousands of DNA base pairs away. The challenge is made even more difficult because the activity of many enhancers is restricted to specific tissues or cell types.

"For example, brain enhancers will not typically work in heart cells, which means that you must test your enhancer sequence in the correct cell type," Dickel says.

Currently, enhancers can be identified through chroma­tin-based assays, such as ChIP-seq, which predict enhancer elements indirectly based on the enhancer's association with specific epigenomic marks, such as transcription factors or molecular tags on DNA-associated histone proteins. Visel, Pennacchio, Dickel and their colleagues developed SIF-seq in response to the need for a higher-throughput functional enhancer assay that can be used in a wide variety of cell types and devel­opmental contexts.

"We've shown that SIF-seq can be used to identify enhancers active in cardiomyocytes, neural progenitor cells, and embryonic stem cells, and we think that it has the potential to be expanded for use in a much wider variety of cell types," Dickel says. "This means that many more types of enhancers could potentially be tested in vitro in cell culture."

In SIF-seq, hundreds or thousands of DNA fragments to be tested for enhancer activity are coupled to a reporter gene and targeted into a single, reproducible site in embryonic cell genomes. Every embryonic cell will have exactly one potential enhancer-reporter. 

Fluorescence-activated sorting is then used to identify and retrieve from this mix only those cells that display strong reporter gene expression, which represent the cells with the most active enhancers.

"Unlike previous enhancer assays for mammals, SIF-seq includes the integration of putative enhancers into a single genomic locus," says Visel. "Therefore, the activity of enhancers is assessed in a reproducible chromosomal context rather than from a transiently expressed plasmid. Furthermore, by making use of embryonic stem cells and in vitro differentia­tion, SIF-seq can be used to assess enhancer activity in a wide variety of disease-relevant cell types."

Adds Pennacchio, "The range of biologically or disease-relevant enhancers that SIF-seq can be used to identify is limited only by currently available stem cell differentiation methods. Although we did not explicitly test the activity of species-specific enhancers, such as those derived from certain classes of repetitive elements, our results strongly suggest that SIF-seq can be used to identify enhancers from other mammalian genomes where desired cell types are difficult or impossible to obtain."

The ability of SIF-seq to use reporter assays in mouse embryonic stem cells to identify human embryonic stem cell enhancers that are not present in the mouse genome opens the door to intriguing research possibilities as Dickel explains.

"Human and chimpanzee genes differ very little, so one hypothesis in evolutionary genomics holds that humans and chimpanzees are so phenotypically different because of differences in the way they regulate gene expression. It is very difficult to carry out enhancer identification through ChIP-seq that would be useful in studying this hypothesis," she says. 

"However, because SIF-seq only requires DNA sequence from a mammal and can be used in a variety of cell types, it should be possible to compare the neuronal enhancers present in a large genomic region from human to the neuronal enhancers present in the orthologous chimpanzee region. This could potentially tell us interesting things about the genetic differences that differentiate human brain development from that of other primates."

Source DOE/Lawrence Berkeley National Laboratory

Mysteries of 'molecular machines' revealed: Phenix software uses X-ray diffraction spots to produce 3-D image

This is a membrane protein called cysZ, imaged in 3 dimensions with Phenix software using data that could not previously be analyzed. Credit: Los Alamos National Laboratory

Scientists are making it easier for pharmaceutical companies and researchers to see the detailed inner workings of molecular machines.

'Inside each cell in our bodies and inside every bacterium and virus are tiny but complex protein molecules that synthesize chemicals, replicate genetic material, turn each other on and off, and transport chemicals across cell membranes,' said Tom Terwilliger, a Los Alamos National Laboratory scientist.

'Understanding how all these machines work is the key to developing new therapeutics, for treating genetic disorders, and for developing new ways to make useful materials.'

To understand how a machine works you have to be able to see how it is put together and how all its parts fit together. This is where the Los Alamos scientists come in. These molecular machines are very small: a million of them placed side by side would take up less than an inch of space. Researchers can see them however, using x-rays, crystals and computers. Researchers produce billions of copies of a protein machine, dissolve them in water, and grow crystals of the protein, like growing sugar crystals except that the machines are larger than a sugar molecule.

Then they shine a beam of X-rays at a crystal and measure the brightness of each of the thousands of diffracted X-ray spots that are produced. Then researchers use the powerful Phenix software, developed by scientists at Los Alamos, Lawrence Berkeley National Laboratory, Duke and Cambridge universities, to analyze the diffraction spots and produce a three-dimensional picture of a single protein machine. This picture tells the researchers exactly how the protein machine is put together.

The 3-D Advance

Recently Los Alamos scientists worked with their colleagues at LBNL and Cambridge University to make it even easier to visualize a molecular machine. In a report in the journal Nature Methods this month, Los Alamos scientists and their team show that they can obtain three-dimensional pictures of molecular machines using X-ray diffraction spots that could not previously be analyzed.

Some molecular machines contain a few metal atoms or other atoms that diffract X-rays differently than the carbon, oxygen, nitrogen, and hydrogen atoms that make up most of the atoms in a protein. The Phenix software finds those metal atoms first, and then uses their locations to find all the other atoms. For most molecular machines, however, metal atoms have to be incorporated into the machine artificially to make this all work.

The major new development to which Los Alamos scientists have contributed was showing that powerful statistical methods could be applied to find metal atoms even if they do not scatter X-rays very differently than all the other atoms. Even metal atoms such as sulfur that are naturally part of almost all proteins can be found and used to generate a three-dimensional picture of a protein. Now that it will often be possible to see a three-dimensional picture of a protein without artificially incorporating metal atoms into them, many more molecular machines can be studied.

Cracking the Cascade

Molecular machines that have recently been seen in three-dimensional detail include a 'huge' molecular machine called Cascade that was reported in the journal Science this summer. The Cascade machine is present in bacteria and can recognize DNA that comes from viruses that infect the bacteria. The Cascade machine is made up of 11 proteins and an RNA molecule and looks like a seahorse, with the RNA molecule winding through the whole 'body' of the seahorse. If a foreign piece of DNA in the bacterial cell is complementary to part of the RNA molecule then another specialized machine can come by and chop up the foreign DNA, saving the bacterium from infection.

Los Alamos and Cambridge University scientists who were developing the Phenix software were part of the team that visualized this protein machine for the first time. The Phenix software has been used to determine the three-dimensional shapes of over 15,000 different protein machines and has been cited by over 5000 scientific publications.

Source: DOE/Los Alamos National Laboratory

In search of the origin of our brain

Nervous system in Nematostella vectensis embryos with different nerve cell populations, where the different neurons (here in green, blue and magenta) evidence asymmetry. Credit: Hiroshi Watanabe, Thomas Holstein / Nature Communication 5:5536, Macmillan Publishers Limited

While searching for the origin of our brain, biologists at Heidelberg University have gained new insights into the evolution of the central nervous system (CNS) and its highly developed biological structures. The researchers analysed neurogenesis at the molecular level in the model organism Nematostella vectensis. Using certain genes and signal factors, the team led by Prof. Dr. Thomas Holstein of the Centre for Organismal Studies demonstrated how the origin of nerve cell centralization can be traced back to the diffuse nerve net of simple and original lower animals like the sea anemone. The results of their research will be published in the journal "Nature Communications."

Like corals and jellyfish, the sea anemone -- Nematostella vectensis -- is a member of the Cnidaria family, which is over 700 million years old. It has a simple sack-like body, with no skeleton and just one body orifice. The nervous system of this original multicellular animal is organised in an elementary nerve net that is already capable of simple behaviour patterns. Researchers previously assumed that this net did not evidence centralization, that is, no local concentration of nerve cells. In the course of their research, however, the scientists discovered that the nerve net of the embryonic sea anemone is formed by a set of neuronal genes and signal factors that are also found in vertebrates.

According to Prof. Holstein, the origin of the first nerve cells depends on the Wnt signal pathway, named for its signal protein, Wnt. It plays a pivotal role in the orderly evolution of different types of animal cells. The Heidelberg researchers also uncovered an initial indication that another signal path is active in the neurogenesis of sea anemones -- the BMP pathway, which is instrumental for the centralization of nerve cells in vertebrates.

Named after the BMP signal protein, this pathway controls the evolution of various cell types depending on the protein concentration, similar to the Wnt pathway, but in a different direction. The BMP pathway runs at a right angle to the Wnt pathway, thereby creating an asymmetrical pattern of neuronal cell types in the widely diffuse neuronal net of the sea anemone. "This can be considered as the birth of centralization of the neuronal network on the path to the complex brains of vertebrates," underscores Prof. Holstein.

While the Wnt signal path triggers the formation of the primary body axis of all animals, from sponges to vertebrates, the BMP signal pathway is also involved in the formation of the secondary body axis (back and abdomen) in advanced vertebrates. "Our research results indicate that the origin of a central nervous system is closely linked to the evolution of the body axes," explains Prof. Holstein.

Source: Heidelberg, Universität

Mechanics of cells' long-range communication modeled by researchers

As fibrosis progresses, "bridges" of extracellular matrix appear between cells. Credit: Image courtesy of University of Pennsylvania

Interdisciplinary research at the University of Pennsylvania is showing how cells interact over long distances within fibrous tissue, like that associated with many diseases of the liver, lungs and other organs.

By developing mathematical models of how the collagen matrix that connects cells in tissue stiffens, the researchers are providing insights into the pathology of fibrosis, cirrhosis of the liver and certain cancers.

Tissue stiffness has long been know to be clinically relevant in these diseases, but the underlying changes that alter the mechanics of tissues are poorly understood. Consisting of a complex network of fibers, tissues have proven difficult to simulate and model beyond local, neighbor-to-neighbor interactions.

Developing a better understanding of the large-scale mechanical changes that occur over longer distances, specifically the process by which the extracellular matrix is pulled into compact, highly-aligned "bridges," could eventually form the basis of treatments for related diseases.

Vivek Shenoy, professor in the Department of Materials Science and Engineering in Penn's School of Engineering and Applied Science, has led an interdisciplinary research team to tackle this problem, authoring a pair of papers that were published in Biophysical Journal.

One, "Remodeling of Fibrous Extracellular Matrices by Contractile Cells: Predictions from Discrete Fiber Network Simulations" involved developing simulations that extrapolated the overall remodeling of the extracellular matrix based on the behavior of neighboring pairs of cells. The other, "Long Range Force Transmission in Fibrous Matrices Enabled by Tension-Driven Alignment of Fibers," took a more mathematical approach, producing a coarse-grained model of this remodeling that could be more broadly applied to fibrotic tissue.

"We're trying to understand how force is transmitted in tissues," Shenoy said. "Cells are the ones that generate force, and it has to be transmitted through what surrounds the cell, the extracellular matrix, or ECM. But imagine trying to model the ECM by trying to keep track of each collagen fibril in your liver; there are tens of millions of those. So we're taking what we learn from simulating those networks to turn it into a model that captures the main features with only a few parameters.

"The key here is the mechanics," he said. "In particular, how does ECM, as a fibrous material, differ from solids, gels and other materials that are better studied."

Rebecca Wells, an associate professor in Penn's Perelman School of Medicine and a co-author on the latter paper, provided insight into the clinical relevance of the mechanics that characterize ECM-related disorders.

"Fibrosis occurs when you have an injury and the tissue responds by depositing ECM, forming scar tissue," Wells said. "In liver fibrosis, the liver can stiffen by up to an order of magnitude, so measuring stiffness is a common diagnostic test for the disease. Increased stiffness also occurs in cancer, where tumors are typically stiffer than the surrounding tissue."

Existing experimental evidence showed that mechanical forces were at play in the changes in both fibrosis and cancer and that these forces were important to their development and progression but could not explain the long-ranging changes cells were able to produce to change their environments. When put in tissue-simulating gels, cells can deform their immediate surroundings but are unable to pull on more distant cells. In real, ECM-linked tissue, however, cells' range of influence can be up to 20 times their own diameter.

"If you look at a normal tissue," Shenoy said, "you see the cells are more rounded, and the network of ECM fibers is more random. But as cancer progresses, you see more elliptical cells, more ECM, and you see that the ECM fibers are more aligned. The cells are the ones generating force, so they're contracting and pulling the fibers, stretching them out into bridges."

"That's also the pathology of cirrhosis," Wells said. "My group had been looking at the early mechanical changes associated with liver fibrosis, which progresses to cirrhosis, but then, by collaborating with Vivek, we started to wonder if these large scale changes in the architecture of the liver could have a mechanical basis and if something similar to what is seen in gels might be occurring in the liver. This is a new way of approaching the problem, which has largely been thought of as biochemical in origin. And there are other tissues where it is probably the same thing, the lung, for example."

The researchers found that the critical difference between the existing models and ECM's long-range behavior was rooted in its elastic properties. Materials with linear elasticity cannot transmit force over the distances observed, but the team's simulations showed that nonlinear elasticity could arise from the ECM's fibrous structure.

"In our model, every component is linearly elastic," Shenoy said, "but the collective behavior is nonlinear; it emerges because of the connectivity. When you deform the network, it's easy to bend the 'sticks' that represent collagen fibers but hard to stretch them. When you deform it to a small extent, it's all the bending of the fibers, but, as you deform further, it can't accommodate bending any more and moves over to stretching, forming the bridges we see in the tissue."

Such simulations can't predict which fibers will end up in which bridge, necessitating the coarser-grained model the researchers described in their second paper. By showing the point at which linear elasticity gives way to its nonlinear counterpart, the team produced a more complete picture of how the alignment of collagen bridges under tension transmit force between distant cells.

Further studies are needed to elucidate the feedback loops between ECM stiffening and cell contraction strength. The team is conducting physical experiments to confirm and refine their in silico findings.

"Right now," Wells said," we're hypothesizing that the mechanical interactions modeled by the Shenoy lab explain aspects of cancer and fibrosis, and we're developing the experimental systems to confirm it with real cells."

Source: University of Pennsylvania

Birth of four foals from genotyped, cryopreserved embryos: A first in Europe

Genotyping allows scientists to choose the embryos they want to use based on different criteria: sex, like in this experiment, the absence of known genetic disorders, or, perhaps in the future, other traits that are tied to behavior, such as emotivity or sociability. Credit: INRA/C. Maitre

IFCE and INRA announced that, for the first time ever in Europe, four foals were successfully born as the result of the transfer of genotyped and cryopreserved embryos. The goal of this work is to better understand embryonic development, control livestock reproduction, and maintain breed genetic diversity. Furthermore, it is advantageous for the horse industry to be able to determine the traits of a future foal.

The INRA Val de Loire center at Nouzilly is where the technology to maintain embryo viability following genotyping and cryopreservation was honed, and then, last summer, the transfer of several embryos took place at the IFCE Haras du Pin Stud Farm, located in the French department of Orne. The partnership between the two institutions has now been cemented by the birth of the healthy foals.

What were the steps leading up to the birth of these foals?

Seven days after fertilization, embryos were collected from Welsh B ponies that are part of INRA's livestock. The embryos were genotyped: scientists sampled some of the embryos' cells to analyze their genomes. In this experiment, embryos were selected based on sex, the idea being to use sex-based selection to test the technique's feasibility. The embryos were then cryopreserved in liquid nitrogen (at -196°). Last summer, they were transferred into saddlebred mares at the Haras du Pin center. After an 11-month gestation period, the foals were born in May. They were of the expected sex: two females and two males. This is the first time that such an event has taken place in Europe, and it is the product of more than 10 years of various types of embryonic research carried out by INRA and IFCE scientists.

What made it so difficult to apply this technique to horses?

Although embryo preservation techniques are already well developed for bovines, small ruminant species, and even humans, preserving horse embryos is a very complex process. For instance, horse embryos vary greatly in size: 7-day-old embryos range in diameter from 200 to 700 micrometers. It is very difficult to cryopreserve the largest embryos because the liquid inside them forms ice crystals when the embryos are frozen at very cold temperatures. What's more, horse embryos are surrounded by a capsule that interferes with successful cryopreservation.

What is the significance of this event, which is the first of its kind in Europe?

There are several reasons why being able to successfully cryopreserve embryos is important. For example, it allows us to maintain breed genetic diversity, particularly that of breeds with small population sizes, such as the Landais or the Poitevin Mulassier. Furthermore, the factor that currently limits the use of embryo transfer is its cost: the transfer center has to maintain a team of recipient mares that are reproductively synchronized with the donor mares. Cryopreservation means that the transfer doesn't have to take place immediately; it can wait until a recipient mare becomes available to receive the embryo. Finally, it may now be possible to directly repopulate horse herds that have experienced losses as a result of various issues, such as disease-related problems, instead of having to use the indirect technique of crossbreeding.

Why genotype the embryos?

Genotyping allows us to choose the embryos we want to use based on different criteria: sex, like in this experiment, the absence of known genetic disorders, or, perhaps in the future, other traits that are tied to behavior, such as emotivity or sociability. It is advantageous for the horse industry to be able to determine the traits of a future foal. We will next aim to simplify the process -- to make this technology more accessible and user friendly for those in the horse industry.

Source: INRA-France

Researchers develop a system to reconstruct grape clusters in 3D, assess quality

Antonio José Sánchez Salmerón, researcher at the Instituto ai2 of the UPV, explains that, today, grape classification is based on an inspection by a panel of experts, that award it score depending on a series of parameters that determine its quality. Credit: Image courtesy of Asociación RUVID

Researchers of the Universitat Politècnica de València (UPV) have developed software to help reconstruct grape clusters with three-dimensional computer vision techniques. The system helps to automatically assess different parameters that define the quality of the wine grape during harvest time.

During the work, the researchers of the UPV collaborated with the Research Centre of Vine and Wine related Sciences of the University of La Rioja, the Spanish National Research Council (CSIC, in Spanish) and the Government of La Rioja. The results of this work were released last September in the journal Food Control.

Antonio José Sánchez Salmerón, researcher at the Instituto ai2 of the UPV, explains that, today, grape classification is based on an inspection by a panel of experts, that award it score depending on a series of parameters that determine its quality. Moreover, different tests are performed in the laboratory in order to estimate the quantity of sugar, the pH, the total acidity and the phenolic quality.

"Among the factors that define the quality of a wine, one of the most important is the quality of the grape as the raw material, but this concept is difficult to assess, due to problems such as subjective parameters, the short period of time available in the field to do the analysis during harvest time, the lack of measuring instruments and their high price, as well as the mixing of good quality and bad quality grape in the trucks. The introduction of this 3D grape reconstruction system helps assess different quality parameters for a wine grape cluster avoiding these problems. One of these parameters is the average size of the grape, which is a very important factor as it establishes the ratio between the quantity of skin and pulp," explains the researcher.

"Increasing the objectivity and automating the grape quality monitoring tasks would be a technological breakthrough with regard to the traditional evaluation system of the grape, based on the knowledge of an expert, and it would have a great impact on the wine industry," adds Sánchez.

Source: Asociación RUVID