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Developmental Biology Research Highlights
Anat456 Developmental Biology Blog

Miniature Hearts

The heart is a crucial organ in the human body, without which we would be unable to survive. The circulatory system, driven by the beating heart, supplies oxygen to the tissues of the body and removes carbon dioxide and other byproducts of metabolism. When the heart isn’t performing optimally, the impact it has on the individual is detrimental.

For this reason, the development and function of the heart, as well as the treatment of cardiovascular conditions, is of great interest to the scientific community.

Human pluripotent stem cells (hPSCs), cells that have the potential to differentiate into any cell type in the human body, have been manipulated to form cardiovascular cell types in three-dimensional microtissue structures and these have become a widely-used model for studies involving cardiovascular development, cardiovascular disease, and drug toxicity studies. The current model however is a unicellular (one cell type) model, which is an issue for this research as the heart is composed of multiple communicating cell types. To make research on these hSPC-derived tissues more applicable to human physiology, developing tissue from these pluripotent stem cells that includes multiple communicating cardiovascular cell types is required.

Exciting new research published by scientists at Leiden University in Development, a prestigious journal in the field of developmental biology, does just this.

What did they do?

In this paper, Giacomelli and colleagues report the development of a method enabling the formation of three-dimensional, beating, cardiac microtissue structures that integrate both cardiomyocytes, the muscle cells of the heart, and cardiac mesoderm-derived endothelial cells, a cell type that regulates the metabolism, size, survival and contraction of cardiomyocytes.

How did they do this and what did they find?

The task for this research group was to differentiate the hPSCs into cardiomyocytes and endothelial cells from cardiac mesoderm. In this specific protocol, the two cell types were differentiated simultaneously. Two different types of hPSCs were used, human embryonic stem cells, obtained from the inner cell mass of the blastocyst of a developing embryo, and human induced pluripotent stem cells, which are generated from differentiated cells.

Figure 1: Embryonic pluripotent stem cells are obtained from the inner cell mass of the blastocyst. These cells have the ability to form all tissues of the body, including cardiomyocytes and endothelial cells, as done in this paper. Induced pluripotent stem cells can differentiate into the same range of tissues also, however, these cells are generated from differentiated adult cells.

The next thing that had to be done was isolate the two-cell types. To do this a creative technique called immunomagnetic separation was used. This technique uses magnetic beads that are coated with an antibody that binds to a primary antibody specific to an antigen of interest, where proteins specific to the cell type of interest was targeted. Using this isolation technique, they obtained isolations of approximately 95% endothelial cells and 80% cardiomyocyte cells. Endothelial cells were characterized by the co-expression of genes that are specific to endothelial cells and cardiac cells. Cardiomyocytes expressed a cell specific marker also and arranged themselves into contracting networks within 3-4 days, displaying sarcomeric structures, the unique arrangement of fibres in muscle tissue.

Figure 2: An illustration of immunomagnetic separation. A cell of interest expresses a specific cell marker which is detected by the primary antibody. This antibody is then targeted by a magnetic bead-associated secondary antibody which enables the cell to be separated through its magnetic properties.

After being isolated, cardiomyocytes and endothelial cells were combined to form the microtissue structure. Interestingly, these cells self-aggregate into these spherical structures, not requiring manual construction. Immunohistochemistry, a technique where proteins of interest are made visible through antibody staining, enabled the researchers to observe the spheroid arrangement that the cells had become organised in, and indicated that the microtissue composed of approximately 15% endothelial cells and 85% cardiomyocytes yielded the better arrangement of endothelial cells, thus being identified as the best cell combination for this protocol. Cardiomyocytes were also cultured on their own, forming microtissue structures containing only cardiomyocytes.

To see if the arrangement of the cells and the combination of cardiomyocytes and endothelial cells changed the physiology of this model, changes in the expression of a wide range of genes was investigated. After 7 days, microtissue composed of both cell types showed gene expression changes similar to those seen in the fetal to postnatal transition in human development, as well as an increase in expression of genes involved with the sarcomeric organization, electrical activity of cardiac tissue and calcium signaling (required for the contraction of cardiomyocytes). After 20 days, genes associated with these aspects of cardiovascular function and development maintained the same trend of expression.

The research group also looked at the electrical activity of these cells.
To do this they used a drug called isoprenaline (ISO) which is known to increase the spontaneous beating rate of cardiomyocytes.  Interestingly, there were no differences seen in the baseline and low ISO concentration recordings between microtissue composed of both cell types and microtissue composed of just cardiomyocytes, suggesting that the two cells have similar electrical properties at basal levels. As ISO concentrations increased, differences were seen and it was suggested that this may be because of a difference in the expression of adrenoceptors, the receptors that ISO acts on.

Why is this research important?

This is an exciting scientific development not only in cardiovascular research but also stem cell and developmental research. There are numerous potential applications for these new structures. Firstly, they provide a new platform for the study of cardiovascular development, especially in the formation of these specific cell types. In addition to this, they have the potential to form a robust and complex model of cardiovascular disease, allowing the investigation into the pathology and treatment of this condition. Considering the effects of unrelated drugs on cardiovascular function is another possible application of this new model. The use of stem cells in cardiovascular research has mainly been for transplant purposes and drug studies, whereas this model provides a unique structure that can be used to research development, physiology, and pharmacology.

Future Direction

These structures are still in the early stages of development (pardon the pun) and before considering its practical applications, a great deal of investigation into the cell model itself is needed. Further research is required to investigate cardiomyocyte maturation, focusing on aspects such as mitochondrial content and sarcomeric organization, as well as the maturation of endothelial cells. Also, there is a need to investigate the similarities and differences between the physiological function and pharmacological responses between this model and cardiac tissue in detail before it can be used to discuss human biology. Comparisons between other cardiovascular models, such as engineered heart tissue, is also required.

Further development and improvement of this model is required also. Increasing the number of different cell types included in this model would create a more complex and relevant model for human biology. Through creating a more sophisticated and elaborate architecture that better replicates human cardiac tissue, potentially using scaffold technology, a more similar arrangement of cardiomyocytes could be achieved. The differences in metabolism between the two cell types are discussed in this paper, and creating a medium that is appropriate for both cell types will mean that the cells can function optimally for a longer period of time. Outside of the realm of cardiovascular research, considering other tissues that this method could be used to create similar models may be an avenue which could provide extensive advancement in other fields also.

In summary…

Cardiac microtissue structures combining cardiomyocytes and endothelial cells provide a new model for research. This paper details an exceptional and creative protocol for the production of these structures, however, further research is required to better understand and improve this technology before it can be used as a trusted research model.

References

Giacomelli, E., Bellin, M., Sala, L., van Meer, B.J., Tertoolen, L.G., Orlova, V.V. and Mummery, C.L., 2017. Three-dimensional cardiac microtissues composed of cardiomyocytes and endothelial cells co-differentiated from human pluripotent stem cells. Development.

Lifesaving Leaves: Growing vascularized scaffolds for transplantation

 Lifesaving Leaves: Growing vascularized scaffolds for transplantation

As the global population continues to expand and the average human life expectancy increases, the demand for life-saving or life-prolonging organ and tissue transplants far exceed their availability. In the United States alone, over 100,000 patients can be found on the donor waiting list at any one time and with an average of 22 individuals passing away each day, there has never been a greater need for alternative methods of treatment. In the past decade, advancements in tissue engineering have provided hope of rectifying the shortfall, but unfortunately, clinical application has been impeded by an inability to engineer a functional vascular network. In the absence of a functional vascular network, organ viability is compromised, therefore patient recovery is jeopardised. In light of this issue, scientists have shifted their focus from engineering a vascular network to utilising those that nature has already provided. A collaboration of researchers from the United States and Slovenia have now successfully created a plant-based scaffold that is not only biocompatible with humans but can potentially be used to regenerate large volumes of any vascularized tissue (Figure 1.).

Why Plants?

Although plants utilise fundamentally different approaches to transport fluids, chemicals and macromolecules compared to animals and at first glance appear to be as dissimilar to humans as imaginable, their vascular network structures are surprisingly similar. Plant vasculature is arranged in the same tapered, branching configuration as that of the human cardiovascular system and consistent with their human equivalents; plant tissues also have varied functions. As plants account for a sizable proportion of the human diet and are a crucial component of the ecosystem, unsurprisingly their molecular make-up has been well researched. Plant cell walls are known to be constructed from a variety of polysaccharides, the most abundant being cellulose. Previous utilisation of cellulose as a tissue scaffold has shown the ability for mammalian cell attachment and proliferation, promote wound healing and most importantly biocompatible when implanted subcutaneously in vivo. Verification of these features is hugely advantageous when pioneering the use of a new transplantation material.

Scaffold? What is it and how are they created?

It would be easy to assume that utilising cellular material from an entirely different kingdom for the creation of vascularized human tissues may require the creation of many new techniques, but surprisingly a considerable extent of the process is conserved across kingdoms. One of the fundamental techniques used in both mammalian and plant models is called decellularization. As implied by the name, decellularization is a process which removes cellular material from a tissue or organ. The resultant outcome is an acellular scaffold of extracellular matrix (ECM) with an intact vascular network. This method formed the basis for the creation of a prevascularized scaffold as achieved by the team of United States and Slovenian researchers.

The team lead by Joshua Gershlak began with the easily acquired plants; spinach and parsley. In alignment with the generation of scaffolds constructed from mammalian tissues and using modified whole organ perfusion decellularization techniques, Gershlak and his team were able to remove unnecessary cellular material for both plant varieties.

Perfusion is a process by which fluids are delivered through the circulatory system of a given tissue. In this instance, perfusion of spinach leaves was achieved through cannulation of the petiole (the structure forming the connection between the leaf and the stem), while in parsley the stem was targeted. Using serial treatment with hexanes and phosphate buffered saline (PBS), the cuticle (protective film on the leaf surface) was removed. This was the first step of a 7-day preparation which included a 5-day perfusion with sodium dodecyl sulfate (SDS) and 48 hours of perfusion with the chemicals Triton-X-100 and sodium chlorite bleach. As a result of these decellularization procedures, Gershlak and his team were able to establish a colourless, plant-derived, pre-vascularized tissue scaffold (Figure 2.).

Behold the vascularization of the scaffold!

Although a plant derived scaffold could successfully be created, the next challenge was to test the vascular circuitry. To quantify the extent of vascular presence following decellularization, the scaffold was subjected to gravity fed perfusion of dye at a constant pressure of 152 mmHg (Figure 3.). To assess the capacity and integrity of the leaf vasculature, hollow spheres that are known as microspheres were used. By changing the size of the hollow spherical inserts (1, 10, 50 and 100 µm) while maintaining a constant perfusion pressure of 200 µl/min, the researchers were able to determine the extent of flow restriction arising from differences in vessel inner diameter.

The final test: Mammalian cell seeding

Following successful vascularization, Gershlak and his colleagues still had two major obstacles to overcome: 1. Could mammalian cells adhere to and recellularize the leaf scaffolds? 2. Once adhered, would mammalian cells be capable of maintaining their functionality?

To address these concerns leaf scaffolds were coated with the extracellular matrix glycoprotein, fibronectin to promote cellular attachment, incubated for 24 hours, and seeded with either endothelial cells or embryonic stem cell-derived cardiomyocytes. Assessment of cell viability was achieved by comparison of a fluorescent viability marker between seeded and unseeded scaffolds at 24 and 48 hours’ post incubation. Excitingly, only seeded scaffolds returned a fluorescent signal indicative of viability.

How does this discovery revolutionise biology?

Using plants to replicate vascularized tissues and organs for any part of the human body provides many benefits over traditional solutions. Typically, organs have been sourced from donors or generated from decellularization of mammal tissues, both of which are in short supply and expensive. Mammalian derived tissues also present the common complication; rejection. Although decellularization of mammalian donor tissues should render them non-immunogenic, the native biochemical and hierarchical tissue structure remain donor derived. This artefact of mammalian donor tissues combined with differences in donor age and tissue pathology confound the efficacy of transplanted tissues.

In contrast to mammalian tissues, plants are abundant and grow rapidly. These features make plants a much more cost-effective and sustainable scaffold material. Therefore, the potential for the use of plants as a scaffold for tissues and organs is enormous. This technological bioengineering advancement has opened the door to not only remedy the global donor organ shortfall created through trauma and disease but also provides the possibility to generate tissues that could be used to repair the structures affected by a range of developmental disorders.

Video sourced from Gershlak et al., (2017)

References

Gershlak, J. R., Hernandez, S., Fontana, G., Perreault, L. R., Hansen, K. J., Larson, S. A., & Rolle, M. W. (2017). Crossing kingdoms: Using decellularized plants as perfusable tissue engineering scaffolds. Biomaterials125, 13-22.

Figures 1, 2 & 3 and video adapted from Gershlak et al., (2017)

A Fresh Start – Researchers create an artificial mouse “embryo” from stem cells

Development – a sperm meets an egg, they fuse, and from this single cell an embryo is created – simple right? From IVF techniques to somatic cell nuclear transfer (the cloning method used to make Dolly the sheep), huge advances in our understanding of early development have been made over the past half-century. However, all of these techniques have required the use of an ovum – until now. Last month, researchers from Cambridge University reported in the scientific journal Science that they had created a structure resembling an early mouse embryo using only two types of stem cells and a 3D scaffold.

 

Normal development of a mouse embryo

 

What happens normally?

Before we can begin to understand how the Cambridge researchers managed this feat, we must understand what happens normally during early embryo development. Embryogenesis (or the process of developing an embryo) begins with a single cell called a zygote, which results from an ovum (egg) being fertilised by a sperm. The zygote then divides (a lot), increasing the number of cells at an exponential rate. By the 8-cell stage there are enough cells to undergo a process called compaction, where the cells bind tightly to each other to form a compact sphere. Some cells in this sphere remain in contact with the outside environment while others are entirely surrounded by other cells. It is the location of each cell in this sphere that restricts what they can become in the embryo. While this is happening a cavity also forms inside the sphere. The result of this (and a lot more cell division) is a structure known as a blastocyst. A blastocyst consists of an outer layer of cells called the trophoblast, which surrounds two types of cells, the epiblast and the primitive endoderm, and a fluid-filled cavity. The trophoblast cells will go on to form the placenta, while the epiblast will form the embryo proper.

Around this time embryos implant into the uterus, making it difficult to study what is going on. From what we understand, the next step of embryogenesis is the rearrangement of the epiblast cells into a “cup” shape. This cup, along with cells from the trophoblast forms a tubular structure known as the egg cylinder (see E5.5 in diagram below). Another cavity, the proamniotic cavity, forms in the lumen of the egg cylinder, and the third cell type (the primitive endoderm) envelops it. From here a process called gastrulation can begin. During gastrulation, the epiblast cells move again to form a three-layered structure from which the embryo will actually form. The three layers are termed “germ layers” and are made up of the ectoderm, mesoderm and endoderm, all of which contribute to different parts of the embryo. These complex steps are just the start of developing a new embryo. They provide a foundation for further events like developing a nervous system, developing organs and specifying cells called primordial germ cells (PGCs) that will go on to create the next generation.

 

Normal development of an early mouse embryo. By embryonic day 5.5 an egg cylinder has formed. Epi – epiblast, TE/ExE – trophoblast, PE/VE – primitive endoderm. From Bedzhov et al., 2014

How do you make an embryo?

Stem cells self-assemble into an embryo shape. The stem-cell embryo at 96 hours (left) compared to a normal Mouse embryo cultured in vitro for 48 hours (right). ESCs are in red and TSCs in blue. From Harrison et al., 2017.

So how do you mimic such a complex process with a few stem cells and a 3D scaffold? It turns out that it was actually fairly straightforward. The research team from Cambridge University used two types of mouse stem cell – embryonic stem cells (ESCs) that are derived from the epiblast and have the potential to make all the structures in the body, and trophoblast stem cells (TSCs) that will go on to form the placenta. Previous attempts to grow embryo-like structures using only ESCs had limited success in accurately modelling embryogenesis. However, by using both types of cells the researchers found that the cross talk between the TSCs and ESCs provided enough instruction to guide the cells into self-assembling into an embryo.

Rather than beginning with a single cell equivalent to a zygote, development of the artificial embryo was started once the cells had committed to either being epiblast or trophoblast cells. To create an artificial embryo a single ESC and a small clump of TSCs are mixed together and allowed to grow within Matrigel (a gelatinous protein mixture), which provides a 3D scaffold. From here the cells self-assemble into an embryo shape with ESCs at one end and TSCs at the other. Of the ESC-TSC structures that developed, 92% of them had a characteristic cylinder shape comparable to a normally developing embryo.

Development of the proamniotic cavity in stem cell embryos at 72, 84 and 96 hours. ESCs make up the bottom half of the embryo, with TSCs above. Image from Harrison et al., 2017.

After creating these artificial “embryos” the researchers had to demonstrate how similar they were to the actual thing. By comparing their artificial embryos to normally developing mouse embryos, the research team was able to show that they followed the same pattern of development. The first critical event in embryogenesis investigated, is the formation of the pro-amniotic cavity. In the stem cell embryo, a single cavity initially developed in the ESC compartment, which was followed by a small cavity in the TSC compartment. By 96 hours after the cells had been put on the Matrigel, a single united cavity formed, accurately modelling the pro-amniotic cavity.

The next major step in embryogenesis is the specification of where the germ layers (from which the embryo will develop) will form. One way to investigate this is to look at where cells from one germ layer (such as the mesoderm) are located. In the stem cell embryos, like normal embryos, mesodermal cells were found in a distinct cluster on one side of the embryo.

Finally, the researchers investigated if primordial germ cells (PGCs) also follow a normal pattern of development in the artificial embryo. Early in development a small number of cells (the PGCs) are singled out to become precursors of ovum/sperm. Normally, PGCs form at a very specific location on the boundary between the embryo and extra-embryonic cells. PGCs can be identified by a combination of specific markers. By staining for these markers it was shown that they form in a similar location in the stem cell embryos to PGCs in normal embryos.

Overall, these experiments confirmed that stem cell are able to organise themselves to be in the right place at the right time as is needed to develop like a normal early embryo.

 

Wait, did they make baby mice?

While the artificial embryos do closely resemble normal embryo development, it is unlikely that they would be able to develop into healthy foetuses. As the stem cell embryos lack the third type of cell (the primitive endoderm) needed in development, and the embryos have not been optimised for the placenta to develop normally, they would not be able to sustain growth needed to survive.

 

So why bother?

While critics are already warning that this new discovery opens the door for genetically modified humans or even cloned babies, this is not what the researchers intend. Instead, it is hoped that this technique will provide a new method to study what actually happens during early development. For each step in embryogenesis there is crosstalk between cells, however we still don’t know what many of these signals are and which cells are sending them. Already the researchers have used the stem-cell embryos to show that a signalling molecule called nodal must be produced by the ESC compartment for the proamniotic cavity to form.

In future, a similar approach may be used to understand early human development. Because development usually takes place inside the uterus and few human embryos (from IVF) are available for scientists to study – a lot about early human development remains unknown. It is thought that two thirds of human pregnancies fail during very early embryo development. However, because early development is so difficult to study, the reasons are poorly understood. The Cambridge team’s breakthrough opens up a number of exciting opportunities to further study key events at critical stages of early development.

 

References

Harrison, S.E., Sozen, B., Christodoulou, N., Kyprianou, C., and Zernicka-Goetz, M. (2017). Assembly of embryonic and extra-embryonic stem cells to mimic embryogenesis in vitro. Science.

Images from

Mouse embryo development from http://www.pdn.cam.ac.uk/directory/watson-erica

Bedzhov, I., Graham, S.J.L., Leung, C.Y., and Zernicka-Goetz, M. (2014). Developmental plasticity, cell fate specification and morphogenesis in the early mouse embryo. Philosophical Transactions of the Royal Society B: Biological Sciences 369.

Harrison, S.E., Sozen, B., Christodoulou, N., Kyprianou, C., and Zernicka-Goetz, M. (2017). Assembly of embryonic and extra-embryonic stem cells to mimic embryogenesis in vitro. Science.

Androgens: More in Touch With Their Feminine Side Than Originally Suspected

Testosterone, a major human androgen.

In the scientific world, there are things we know: the earth is round, and there are things we don’t know: why we dream. But science is a risky business, and every now and then – if now and then meant all the time – we come across things that we thought we knew, only to be proven wrong. Case in point: the role of androgens, a typically male class of hormones, in the development of the ovaries.

It’s 2017, and you can almost hear androgen chastising the science community for assuming its gender. But in our defence, until now, Androgen seemed to be the jock of the hormone world, willing to partake in anything male-orientated. Muscle development, facial hair, chest hair, all the hair – androgen is the culprit. Considering the confidence around the role of androgen, imagine the shock when earlier this year, a team from Hokkaido university discovered that androgens play a more prominent role in the development of the ovaries than the development of the testes.

Androgens, such as testosterone, are steroid hormones that are heavily involved in development in vertebrates. Their role is typically to induce or maintain the development of male characteristics (the word androgen stems from the Greek word andro, which literally means male human being). Androgens work by binding to andgrogen receptors (AR), and are especially active during puberty and embryonic development – although their role in embryogenesis has always been slightly murky.

Ryoma Tanaka and his team from Japan’s Hokkaido University used male and female chicken embryos to study the role of androgens in embryonic development. In male chickens, androgens contribute to the development of skeletal muscles, sexually dimorphic embellishments such as the cockscomb, and courting behaviours. It is also widely acknowledged that androgens and AR signalling play crucial roles in male reproductive development, however their exact role was not entirely known.

 

Stage 1: How much, and where?

Figure 1: The AR mRNA level gradually increased in both sexes from days E7.5 to E21.5. However, AR expression was higher in the female than in the male at all developmental stages examined.

As androgens cannot be functional without androgen receptors, by assessing the amount of androgen receptors in a cell, Takana and his team could gain an indication of the functional relevance of androgens in those cells. To investigate the expression of androgen receptors in both female and male gonads, quantitative real-time PCR (qRT-PCR) was used. This process allowed researchers to quantify, in real time, the amount of AR mRNA being expressed at any one time in the tissue. In theory, more expression correlates with how integral the protein is to the developmental processes that cell is undertaking. So you would assume that a male-centric hormone would be most crucial in the development of male reproductive organs, but you would be wrong. Their results were definitive: AR expression was higher in females than in males at all developmental stages examined.

Through the use of fluorescent markers, it was found that the expression of androgen receptors was highest in the cells of lacunae and cortical cords, which suggests that these ovarian structures are most reliant on androgens during development.

 

Stage 2: The knockdown approach

So now, after finding out that AR expression is more prevalent in female reproductive organ tissue, Takana and his team needed to see if this increased expression actually meant increased functional relevance. There may be more AR expression in ovaries, but what is the functional role of androgen/AR signalling, and how significant is its contribution to development? To investigate this, a knockdown approach was used. By knocking out androgen receptor expression, they could study the physiological consequences during development of AR deficiency. Chick embryos with normal AR expression were used as controls for comparison of ovarian and testis tissue.

Takana and his team found that in the absence of AR expression (and consequently androgen function), development of the testis was normal, whereas ovarian development was compromised. Specifically, lacunae numbers were decreased. As well as this leading to an immediate disruption in ovarian structure, they suspected that it would lead to problems with ovulation later in life, which implicates both reproductive and egg laying abilities. The team hopes to extend their study through the birth and development of the chicks in order to investigate their egg-laying rates. In addition to decreased lacunae, the development of cortical cords, where egg cell precursors mature, was abnormal.

 

Figure 2: HE staining of female gonads. The knockdown embryo (ARKD Right) in comparison to the control embryo (Sc Right) is both smaller and contains lower numbers of lacunae (indicated by black arrows).

 

With that, researcher Asato Kuroiwa concluded “during embryogenesis, androgens are more important for the development of the ovaries than the development of the testes.” Levels of testosterone in male chickens increases after hatching, suggesting that androgens and AR signaling play a more prominent role in males during sexual maturity, rather than during development.

 

Why is this relevant?

Takana and his team have made progressive, albeit somewhat surprising, discoveries in an area that until now, was relatively unchartered territory. Further study in this field could be beneficial in many areas, namely the egg industry. Understanding the role of androgens in ovarian development gives a better understanding of potential causes when hens have impaired laying abilities. As well as this, it means potential experimentation could take place around optimising the laying ability of hens – something that an industry worth almost $300 million in New Zealand alone would be very interested in.

 

 

 

Journal Reference:

Ryoma Tanaka, Hiroe Izumi, Asato Kuroiwa. Androgens and androgen receptor signaling contribute to ovarian development in the chicken embryo. Molecular and Cellular Endocrinology, 2017; 443: 114

 

 

 

A Head of Development – The Anterior Neural Tube.

The central nervous system (CNS) is highly polarized; meaning the structures that are present at either end are very different to one another. This is clearly observed in humans, with the brain located superiorly and the spinal cord running inferiorly as seen in Fig. 1 . Stepping back along the developmental timeline, we see the brain and spinal cord both originate from the same embryonic structure; the neural tube. This is formed through a process known as neurulation. Initially, neural plate specification occurs as a result of notochord signaling from below. Once this has occurred the neural crests folding back subsequently fuse in the midline, forming the tube. In order for the polarized CNS to develop from this uniform tube, a wide range of cell signaling mechanism and gene networking takes place along its length. This is observed in both dorsoventral and anteroposterior axes, of which the latter is most notable.

Figure 1: Neural Tube Development. A, the early neural tube, with the structures associated with different regions of the tube, following development, in B. 

Neural Tube Axes: The differences observed in different regions of the neural tube occur as a result of altered gene expression profiles, which leads to altered protein production, and therefore cell fate and function. Differences in gene expression are mediated through a number of mechanisms, however, concentration gradients of morphogens such as Fibroblast Growth Factor (FGF), and Bone Morphogenic Protein (BMP) play key roles. In addition to factors such as these, intrinsic factors along the tube elicit a range of downstream cellular pathways in different regions, once again resulting in the polarization of structures along the neural tube. Polarization in the dorsoventral axis has been well described, with BMP and Shh being identified as the primary dorsalizing and ventralizing signals. To comparison, regulators that mediate patterning along the anteroposterior axis are less understood. When studying development along this axis, it is important to be able to differentiate anterior and posterior structures, an often difficult task in the early undefined embryo. To so, biomarkers of anterior and posterior regions have been identified, such as OTX2 and Knox20 respectively.

Previous Findings: Morphogens such as FGF and Wnt have been shown to mediate a posterior neural fate, whereas intrinsic factors such as Dkk1 inhibit Wnt, resulting in an anterior fate. Similarly, downregulation of FGF in the anterior tube is required for the development of the forebrain, however, the underlying mechanisms that regulate this remain unclear. This article; Tbx2 regulates anterior neural specification by repressing FGF signaling pathway, by Cho et al., identified Tbx2 as a mediating factor in this inhibition of FGF, through BMP. This research used cultured Xenopus embryos, which provided an easy and accessible model to gain an understanding of processes occurring throughout embryonic development.

Figure 2: Expression of Otx2 and Krox20 following application of Tbx2.

Further Understanding: In order to gain an understanding of the involvement of TBX2 in the development of the anterior neural tube, in situ hybridization techniques were used, which simply allows the expression of a gene to be visualized. These findings indicated TBX2 expression was located in the anterior region of the developing embryo. This was further validated through an increased expression of Otx2 following the upregulation of TBX2, and vice versa, as seen in Fig. 1 . These finding indicated that TBX2 plays an important role during anterior neural development.

Having determined where TBX2 is expressed in the developing embryo, Cho et al. set out to characterize its interactions with BMP and FGF. FGF mediates posterior development through ERK phosphorylation, resulting in increased Krox20 expression. In contrast, BMP mediates anterior neural induction resulting in an increased expression of Otx2 however, this occurs through it inhibiting FGF. Similar findings were also observed with TBX2. From these findings it became clear that TBX2 and BMP were functionally similar, and therefore, Cho et al. looked to identify a relationship between the two. They found that TBX2 was in fact a molecular target of BMP, and furthermore, TBX2 mediated the inhibitory actions of BMP, the associated mechanisms of which were later investigated.

Figure 3: Expression of Otx2 and Krox20, following application of Tbx2 and/or Flrt3. 

The expression of Flrt3, a positive regulator of FGF was repressed following the inhibition of FGF by BMP, therefore raising the question as to whether TBX2 is also involved in the inhibition of Flrt3. To test this, Flrt3 expression was induced by FGF, then repressed by TBX2. Similar finding were observed when TBX2 repression in the anterior neural tube resulted in the upregulation of Flrt3, seen in Fig. 2. To summarize these findings, TBX2 and BMP, and Flrt3 and FGF are key factors that regulate the development of the neural tube along the anteroposterior axis. BMP expression results in the upregulation of TBX2, which subsequently inhibits Flrt3, resulting in the repression of FGF signaling, and therefore, mediating anterior neural development.

So far in this research Cho et al. focused on development along the neural tube however, the same factors previously mentioned have also been implicated in the development of additional structures similarly located along the anteroposterior axis of the developing embryo. For example, the eyes which develop from the anterior region of the neural tube were defective in the absence of the anterior development factor TBX2. This provides an indication that TBX2 is not only involved in the patterning along the neural tube, but also in the development of the associated structures such as those within the cranium and face.

What Does This Mean: Collectively, these results provide a further insight into the role of various signaling factors associated with neural tube polarization, primarily in the anterior region where increased neurogenesis within the ventricular walls gives rise to the primitive brain. FGF and BMP regulate the expression of posterior and anterior markers respectively, however, there are many underlying mechanisms that mediate and facilitate this. A prime example has been described in this research, which indicated increased levels of BMP attenuate the expression of FGF anteriorly resulting in anterior neural development, mediated by TBX2. Furthermore, finding such as these can also assist in our understanding of disease, what happens when things go wrong, and possible interventions.

Although these findings have been described in the Xenopus model, a species of frog with little apparent similarities to a human, the understanding gathered through this research translates accurantly to the human case of embryonic development. A common defect that occurs during the closure of the anterior neural tube, known as Anencephaly, results in a lack of brain formation. Based on the research previously described, which demonstrated the importance of BMP and TBX2 in anterior neural development, we might expect to see impairments in the signaling of these factors in humans. This is in fact the case,with altered BMP signaling previosuly being implicated in forebrain development and simialry, in associated conditions such as anencephaly.

 

 

References:

Cho, G. S., Park, D. S., Choi, S. C., & Han, J. K. (2017). Tbx2 regulates anterior neural specification by repressing FGF signaling pathway. Developmental Biology, 421(2), 183-193.

Sleeping Cells Required for Development

People have been intrigued by development for centuries, from understanding how a mother and father contribute to their offspring to characterising the genes involved in the various developmental stages. Development is a complicated process with many factors required at the right place, at the right time, in the right amount for it to occur successfully. Given this complexity and the vast number of components interacting in different ways, it seems incredible that pregnancy and development occurs much of the time without anything going wrong! Generally when we think about development, we think about growth and expansion, yet over the past decade studies have shown that, paradoxically, senescent cells are one of the multitude of factors required for correct development in mammals.

 

What is a sleepy cell?

Figure 1: Cell Cycle and Senescence

Senescence is a cellular state where the cell has lost the ability to divide and grow, so has become dormant (sleepy). However, these cells are not dead, as they are still able to produce proteins and secrete factors albeit different ones from a normal cell.

Cells can enter a short-term, transient form of senescence called quiescence (G0). If this quiescence is prolonged then it becomes senescence. Senescence occurs when the cell permanently exits the cell cycle, entering a new state where it is still metabolically active but does not replicate. This occurs during G1 of the cell cycle.

Once they become senescent, cells increase in size and become flattened. There is also chromatin and nuclear rearrangement, which accounts for the increased expression of hallmark regulatory proteins, such as p53, p21 and p16. Mitochondrial and lysosomal networks expand to keep up with secretions.

Because senescent cells aren’t functioning in the same way as normal cells, it looks different and secretes different factors, which are collectively called a senescence-associated secretory phenotype (SASP). Secreted factors that make up the SASP include growth factors, cytokines, chemokines and matrix remodelling proteins. The SASP can lead to inflammation, alterations in tissue microenvironment and transmission of senescence to nearby cells.

Previously, senescence was thought only to occur as a protective mechanism when a cell became stressed, through stressors including DNA damage, telomere erosion and oncogene activity. When left unchecked these stressors often lead to tumours. More recent research has expanded the current thinking to include senescent cells in development.

 

Why have sleepy cells?

Due to the stressors that can induce senescence, it can act as an anti-tumour mechanism. While senescence is helpful in this role, it can also have negative effects on our biology like ageing. Senescent cells accumulate in our tissues as we age contributing to the loss of elasticity among other things. In some specific circumstances senescent cells have been found to promote tumour formation and growth.

All of these discoveries have led scientists to wonder about the evolution of senescence, thinking that there must be some advantage. They discovered that transient senescence contributes to restriction of fibrosis in skin wounds, liver and heart as well as to assisting wound closure. Cellular senescence has more recently been described in non-pathological states, such as regeneration and development.

In 2013, two research groups (Muñoz-Espín et. al., 2013; Storer et. al., 2013) looked at senescent cells in mammalian development and found they appeared during strict developmental windows in structures such as the kidney, inner ear, and part of the developing limbs. These studies also found that the presence of senescent cells was transient, and this subpopulation of cells was cleared through an immune-mediated process. The mechanisms behind the senescence were also explored and were found to be dependent on p21, which when genetically disrupted results in loss of senescence and creates developmental abnormalities. It was therefore suggested that perhaps senescent cells have an important role in development.

The research of Davaapil and his team presented in this paper, extends our knowledge of programmed cellular senescence in development to include amphibians, suggesting that cellular senescence could occur in all vertebrates.

 

How do you find sleepy cells?

Included in a senescent cells’ SASP is β-galactosidase, which can be stained to locate senescent cells (SAβgal staining). This has previously been used for identifying senescent cells in mammalian models (Muñoz-Espín et. al., 2013; Storer et. al., 2013). Davaapil et. al., performed this staining in both axolotl (Ambystoma mexicanum) and frog (Xenopus laevis) embryos at various developmental stages.

 

Where are they?

Davaapil et. al., found senescent cells in the developing kidney (pronephros) in both the axolotl (Fig. 2) and the frog (Fig. 3). In mammals, the pronephros is a transient structure giving way to the mesonephros and the metanephros. However, in amphibians and fish the pronephros is the main form of kidney through the embryonic and larval stages and is only replaced in the functionally adult kidney later on. Senescent cells are first seen at stage 38 (Fig. 2) expanding within the pronephros until the complete degeneration of the pronephros at advanced stage 57 (not shown).

Figure 2: Programmed cellular senescence during the development of the axolotl pronephros. (A) Whole-mount senescence-associated β-galactosidase (SAβgal) staining

In addition to the frog’s pronephros, senescent cells were also found in its cement gland, the midbrain and hindbrain, and anterior cartilage (Fig. 3). Davaapil et. al., chose to focus on the cement gland as it is a transient organ that secretes mucus allowing larvae to attach to a substrate before they can swim or feed.

Figure 3: Programmed cellular senescence during Xenopus laevis development. (A-K) Representative images of whole-mounted, SAβgal stained Xenopus embryos at various developmental stages.
(A,B) Xenopus embryos, stage 46 (A) and stage 49 (B).
(C) Xenopus pronephros, stage 49.
(D-F) Xenopus brains at stage 46 (D), stage 49 (E) and stage 52 (F).
(G-I) Xenopus anterior cartilage at stage 46 (G), stage 49 (H) and stage 52 (I).
(J,K) Xenopus pronephros at stage 46 (J) and stage 49 (K).
c, cartilage; cg, cement gland; hb, hindbrain; mb, midbrain; p, pronephros.

 

The appearance and disappearance of the sleeping cell

Both the ERK and TGFβ signalling pathways have been previously linked to programmed cellular senescence. Davaapil et. al., found in the axolotl pronephros and the frog cement gland, that programmed cellular senescence was ERK-independent even though previously, senescent cells had shown high levels of phosphorylated ERK. Through TGFβ inhibition, they showed that TGFβ was necessary for programmed cellular senescence (Fig. 4). TGFβ inhibition also delayed the degeneration of the pronephros.

Figure 4: The cellular senescence in the axolotl pronephros is independent of the ERK/MAPK pathway and dependent on TGFβ signalling. Whole-mount SAβgal staining of axolotl embryos following treatment with the indicated inhibitors from stage 30 onwards. Red dotted lines highlight the pronephros.

As the structure of the pronephros disintegrates there is a striking increase in the recruitment of macrophages, which is accompanied by a low recruitment of neutrophils and other granulocytes. Macrophages are an immune cell that ‘eats’ pathogens and cellular debris, and in this instance removes the apoptotic debris of senescent cells. Davaapil et. al., were unsure as to whether the apoptosis or the macrophage infiltration occurred first but suggested that the programmed senescence promoted pronephros disintegration through the induction of apoptosis.

 

In frogs, there are senescent cells surrounding the cement gland and express high levels of both ERK and TGFβ regulators. It was found, through ERK and TGFβ inhibition, that TGFβ was also involved in the senescent cells of the cement gland (Fig. 5). Only one TGFβ inhibitor resulted in reduced senescent cell numbers.

Figure 5: TGFβ inhibition decreases cellular senescence in the Xenopus laevis cement gland, leading to structural changes in the nearby areas. SAβgal staining of whole-mount Xenopus embryos at stage 45 following treatment with the indicated inhibitors, from stage 24 onwards. cg, cement gland.

TGFβ inhibition, which reduced senescent cell numbers, resulted in smaller cement glands, elongation of the nostrils, and significant reduction in primary mouth length (Fig. 5). These additional defects were found to be specific to senescent cell loss and in turn resulted in feeding and subsequently growth defects. Davaapil et. al., concluded that senescent cell loss due to TGFβ inhibition resulted in developmental defects of the cement gland and nearby structures. All of this suggests that TGFβ is required for programmed cellular senescence in the cement gland, and for the proper morphogenesis of the cement gland and the surrounding structures.

 

How does this help us?

Other than being an exciting discovery in the world of developmental biology, the knowledge that programmed cellular senescence is essential in amphibian development further enhances our understanding of development and may have uses in a clinical setting. This is especially important as programmed cellular senescence is hypothesised to occur in all vertebrates and therefore humans. Taking this into account it is possible that developmental cellular senescence arose early during evolution and as such could be the origin of cellular senescence.

 

Where to from here?

Identifying other SASP components involved in development and senescence induction would allow us to further understand programmed cellular senescence, and development. Senescent cells, and dying cells, commonly express p53 (part of the SASP), properties which warrant further investigation of its potential role in the regulation of developmental senescence.

 


 

References

Davaapil H, Brockes JP, Yun MH. Conserved and novel functions of programmed cellular senescence during vertebrate development. Development. 2017 Jan 1;144(1):106-14.

Muñoz-Espín D, Cañamero M, Maraver A, Gómez-López G, Contreras J, Murillo-Cuesta S, Rodríguez-Baeza A, Varela-Nieto I, Ruberte J, Collado M, Serrano M. Programmed cell senescence during mammalian embryonic development. Cell. 2013 Nov 21;155(5):1104-18.

Storer M, Mas A, Robert-Moreno A, Pecoraro M, Ortells MC, Di Giacomo V, Yosef R, Pilpel N, Krizhanovsky V, Sharpe J, Keyes WM. Senescence is a developmental mechanism that contributes to embryonic growth and patterning. Cell. 2013 Nov 21;155(5):1119-30.

Figure 1 from Nakamura-Ishizu A, Takizawa H, Suda T. The analysis, roles and regulation of quiescence in hematopoietic stem cells. Development. 2014 Dec 15;141(24):4656-66.

Figures 2-5 from Davaapil H, Brockes JP, Yun MH. Conserved and novel functions of programmed cellular senescence during vertebrate development. Development. 2017 Jan 1;144(1):106-14.

Of Mosquitoes and Mice – New Insights into the Zika Virus

Recently the World Health Organisation declared that Zika virus was a public health emergency and concern after an increase of incidences in Brazil. Zika virus itself is nothing new but this outbreak produced disastrous effects that had not been associated with the virus until now. This sparked world interest in the virus and the need to produce ways to model the viral infection and how it infiltrates and bypasses the immune system to produce its damaging effects. In March 2017 Shashank Tripathi released a paper that investigated a new mouse model for Zika virus that gave insight into strain specific differences and the immune responses of the host.

What is Zika and Why Should We be Concerned

Zika virus is a type of Flaviviridae virus similar to that of Dengue, Yellow fever or West Nile virus. These viruses produce symptoms that span from mild rashes to critical organ system breakdowns but Zika isn’t this extreme to those that are infected. Zika produces mild symptoms that last 2-7 days that include mild rashes, headache and muscle aches and pains. One method it does this through inflammation of the central nervous system including brain and other organs such as liver and reproductive organs.

Zika infects people through mosquito bites that pass the virus into the bloodstream, it has also been seen that in humans the Zika virus can pass through unprotected sexual contact as well as from mother to child in pregnancy. When Zika virus gets into a developing embryo it can cause some major developmental issues, a major one being microcephaly. Microcephaly is when the affected child has an unusually small head, this can lead to a host of other problems that come with having a small and underdeveloped brain such as seizures, learning disabilities and developmental delays in things such as walking and talking. Because infection with Zika virus can lead to some disastrous consequences there needs to be some way to study it and understand how it causes these symptoms.

Why We Need a Model to Study

It is difficult to study diseases in humans in a controlled scientific manner and in a way that is ethical and safe. It is important to have study diseases so that we gain an understanding of how it works and so that we can develop and test new therapies. This leads to the need to have a animal or cell model so that we can look at how infection with Zika virus can cause inflammation and other symptoms such as microcephaly. The recent paper was released by Shashank Tripathi looked into the use of a mouse model to look at inflammation from the immune system in response to Zika virus and believes to have found one.

The Creation of a Mouse Model

The paper “A novel Zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses” was built on previous knowledge that the Zika virus targets specific areas of the human immune system and stops them to signal for antiviral response, specifically, Signal Transducer and Activator of Transcription 2 or STAT2. This sits in a cell and when activated by cytokines, a type of immune cell, it produces new products to be made from DNA that form proteins to launch an immune attack against invading viruses. When Zika virus infects a person it targets it and degrades it meaning the virus escapes from the clutches of some of the immune system. This then leads to the effects that are seen including inflammation. The issue with using a mouse model is that their immune system works slightly differently and in the earlier efforts to produce a mouse model it was found that Zika does not target STAT2 in mice as it does in humans so mice do not have the same infection process as humans do. This proved difficult for our model but they tried to find a way. They tried knocking out and removing the STAT2 gene in mice to get around this problem creating Stat2-/- mice.

Figure 1. This shows the stat2-/- mice getting infected ad having symptoms earlier than the other mouse model and wild type (wt). Much more like a human infection

Shashank and team found that the Stat2-/- mice were now able to be infected by Zika after giving a pinprick to the foot much like a mosquito bite. They found that these mice became sickly and losing weight 3 days after they gave the infection, much like the time believed it takes in humans for symptoms to occur. They compared another mouse that has been previously used for Zika and found a delayed infection. They looked into the RNA levels in different organs to show that the virus was being produced and replicated. They found the most RNA in total in the mice were found on day 2 however in the brain and central nervous system there was an increase in RNA found between day 2 and day 6 post infection. In the reproductive organs there was most RNA around day 4. These results showed that these mice were able to be infected in a similar manner to human infection with similar outcomes.

Zika has multiple stains, these stains are slightly different in their genetic makeup and originated in different areas of the world, they also cause slightly different symptoms. There is an African Zika strain and an Asian Zika strain, the African strain is not known to be as aggressive in its attack and more sporadic cases have been recorded. The Asian strain however became much more prevalent currently and linked to the severe symptoms such as microcephaly. Outlined in this paper there was investigation into how these strains are different and how this changes their pathogenicity. Due to the creation of the disease animal model this was able to be looked at in the Stat2-/- mice.

Zika Strain Differences

Figure 2. Phylogenic tree showing separation of the African and Asian strains

Using the mouse model, the researchers compared the pathogenicity of African strains and Asian strains in Stat2-/- mice, another mice used for Zika studies and a wild type mice. There were differences found in when the infection symptoms showed and the severity of these symptoms between the African strains and the Asian strains on the different mouse types. The authors concluded from this that pathogenicity was different between the two strains. To further this they looked at the amount of Zika RNA found in different organs. It was found that all mice infected with an African strain had symptoms and that these had higher mortality and more severe symptoms than those infected with the Asian strains, however didn’t necessarily have the most RNA. The Asian strains showed a more delayed infection but infection was longer and more sustained but less sever than those infected with the African strain. It was concluded that the levels of Zika RNA do not correlate with the severity and pathogenicity of the different strains.

To test the different strains and how they relate to inflammation they tested different proteins of the immune system and how they were deferentially expressed. It found that there higher levels of immune system proteins such as interferons and more activation of cytokines in the mice infected with the African strain rather than the Asian strain. Markers involved with T cells which are cell of the immune system were also upregulated. What is important is that all Stat2-/- mice had inflammation in the central nervous system which is the same as the human response to infection with Zika virus.

Where does this lead us?

By having a mouse model to study Zika virus infection in humans that has similar inflammatory responses we can gain a better understanding of how the virus works and what makes its symptoms so variable between people, strains and at different stages of development. We can also gain insight into the strain differences as this recently published paper has done, this could allow us to understand the differences in infections types and could give us insight into how to treat different strains of the virus.

 

 

References

Tripathi S, Balasubramaniam VRMT, Brown JA, Mena I, Grant A, et al. (2017) A novel Zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses. PLOS Pathogens 13(3): e1006258. https://doi.org/10.1371/journal.ppat.1006258

 

Why the change of heart? –FGF and ventricular development.

Regenerative medicine has been an area of ‘hot topic’ research in recent years. It is providing a promising beginning to new treatments for cardiac conditions such as heart disease, which in itself, is on the rise due to the obesity epidemic facing modern Western societies. However, in order to put this treatment into action in a clinical setting, timing of the transition of cardiomyocytes (heart cells) from being plastic to becoming their definitive cell type and the processes that control this transition during development must be understood. Recent research by Pradhan and colleagues investigates the role of fibroblast growth factor (FGF) in determining the fate of the cells that make up the ventricle in the developing zebrafish heart and therefore, provides further insight into the understanding needed for development of regeneration techniques.

Development of the zebrafish heart- let’s take a step back.

It is important to note that cardiac cell fate is controlled by a multitude of intrinsic and environmental signals at different time points throughout development (FGF is not the only one!). In all vertebrates, including the zebrafish, the cardiac tissue is derived from an early layer of cells in the embryo called the mesoderm. With the guidance of many signals from surrounding tissues, cells of the mesoderm come together to form the early structure of the heart called the heart tube (see article by Stainier et al., 1993). This heart tube is then divided into separate chambers called the atria and ventricle and this is where FGF comes in (all will be explained later). Unlike a human heart where there are two atria and two ventricles, in the zebrafish, there is only one atria and one ventricle. Although there is this structural difference between humans and zebrafish, most of the molecular pathways are similar, therefore, making the zebrafish a great research tool for the development of the human heart.

What was known before their study?

It is known that Nkx is a transcription factor that increases expression of Irx4 and Hey2 (transcription factors that maintain ventricular myocardium (heart tissue) by stopping ectopic or abnormal expression of atrial genes in this area). This has previously been seen in mouse, chick and Xenopus (frog) models as well as zebrafish.

It is also known that FGF has a role in very early embryonic heart development in producing the correct number of cardiomyocytes, both in the atria and ventricle. By 18 hours post-fertilization, amhc and vmhc (genes coding for proteins specific to the atria and ventricle) are being expressed, indicating that from this time point onwards, the atrial and ventricular cell types are starting to differentiate into their final fate.

Knowing these controlling factors of heart development raises further questions. What is controlling Nkx expression? Does FGF have a role in maintaining cardiac tissue in later stages of embryonic development or is its role restricted to early stages only? Pradhan and colleagues set out to answer these questions in their study looking at the role of FGF signalling in later stages of development (at different time points after 18 hours post-fertilization) by inhibiting FGF signalling using one of two methods which sound complicated but achieve the same thing in different ways:

  1. Inserting a transgene containing a heat-activated negative form FGF (referred to as Tg(hsp70:dnfgfr1)).
  2. Exposure to an FGF antagonist called SU5402.

 

What did they find?

  • Inhibiting FGF signalling caused ectopic expression of amhc in the ventricle (figure 4) at decreasing levels from 18, 24 and 28 hours post-fertilization but by 29 hours post-fertilization, there was no more ectopic expression of amhc. This initial finding demonstrates a number of things about FGF signalling in heart development:
  1. FGF signalling prevents ectopic expression of amhc in the ventricle.
  2. FGF has a more general role in heart development before 18 hours-post fertilization (seen in previous studies) and a more specific role in maintaining ventricular cell fate in development after 18 hours post-fertilization.
  3. FGF signalling is only needed in ventricular maintenance until about 28 hours post-fertilization. After this, it is no longer needed as either the cells have reached their definitive fate and are no longer able to change into another cell type OR some other unknown factor takes over the role of ventricular maintenance.
  • Inhibition of FGF signalling also caused vmhc expression to be REPLACED by amhc expression in the ventricle. This indicates that FGF signalling not only stops the ectopic expression of amch in the ventricle but also maintains the expression of vmhc in the developing heart.
  •  Also seen after inhibition of FGF signalling was an increase in atrium size and decrease in ventricle size due to an increase and decrease in the number of respective cells (figure 4). This demonstrates that FGF not only has a role in stopping ectopic expression of atrial-specific genes/proteins in the ventricle but also has a role in maintaining relative amounts of cells in the atria and ventricle to produce the morphology of the heart.
  • Lastly, they found that inhibiting FGF signalling caused a decrease in cardiac expression of two Nkx genes, nkx2.5 and nkx2.7. This result confirmed that FGF acts upstream of Nkx to maintain and enhance its function in maintenance of cardiac cell fate.

Figure 1: (A-D) in situ hybridisation showing expression of amhc in only the atrium in the control (A) and expression of amhc in both the atrium and ventricle (white arrows in B-D) after inhibition of FGF signalling. (E-H) Immunofluorescence with MF20 (red- marks myocardium) and S46 (green- marks Amhc protein) showing ectopic expression of Amhc protein in the ventricle (white arrows in F-H) and also enlargement of the atrium and compaction of the ventricle in inhibition of FGF signalling (F-H) compared to the control (E). (Pradhan et al., 2017).

 Concluding ideas:

FGF signalling over the course of cardiac development in the zebrafish maintains ventricular cell fate and ultimately suppresses myocardial plasticity so the cells of one chamber can’t suddenly change and become cells of the other chamber. FGF does this by enhancing the action of Nkx and stopping the ectopic expression of atrial-specific genes in the ventricle within a specific time window (18hpf to 28hpf). As in all scientific research, with emergence of new evidence comes the emergence of new questions. For example, now that we know that FGF acts upstream of Nkx, what are the factors, if any, that act downstream of Nkx and what would be the effect on cardiac cell fate if these were inhibited? It is these types of questions that need to be answered and understood before the use of cardiac cells in regenerative medicine can be used in the human body in a clinical setting. Animal models such as zebra fish are an extremely useful tool to gain understanding of these concepts and move us a step in the right direction.

References:

Pradhan, A., Zeng, X. I., Sidhwani, P., Marques, S. R., George, V., Targoff, K. L., . . . Yelon, D. (2017). FGF signaling enforces cardiac chamber identity in the developing ventricle. Development, 144(7), 1328-1338. doi:10.1242/dev.143719

Stainier, D. Y., Lee, R. K., & Fishman, M. C. (1993). Cardiovascular development in the zebrafish. I. Myocardial fate map and heart tube formation. Development, 119(1), 31-40.

 

Supersize Sox: The contribution of SOX6 to obesity

 

Unknown-1Due to the dramatic change in the lifestyle and diet of the western world in the last 500years, we are faced with challenges our ancestors would never have thought possible. Food was once a sought after and scarce entity and the lives of ancient humans were focused around producing sufficient food with which to survive, however today it has become a commodity which is abused and wasted. Our bodies are simply not adapted to cope with the high sugar and high fat diet with which we live off giving rise to health issues associated with this dramatic change in lifestyle and diet. Obesity is one of the most common pathologies to affect the western world today and recent data has shown that it is likely to affect close to 31% of adults in New Zealand. This issue is undeniably a growing one, and is costing the nation millions in healthcare, not to mention greatly affecting the quality of life of those affected. Obesity is often a family disease, with multiple members of the same family frequently affected, however the reason for this linkage within families is often put down to similar environments rather than genetic factors. It is undeniable that environmental factors play a key role in the development of obesity, in particular lifestyle choices and diet, however evidence is increasingly being presented in support that developmental factors also contribute.
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Each organism has only one genome, but due to the differential and cell specific
expression of genes within this genome many different epigenomes are created. These differences arise through epigenetic mechanisms such as histone modifications, acetylation and methylation, which act to make different sections of the genome more or less accessible to expression. The investigation of such mechanisms and how and where
they are acting during development is becoming increasingly relevant in fields of biological sciences. Epigenetics are responsible for cell differentiation and specification and in addition are being recognised as having a role in many disease states.

In relation to obesity, evidence is emerging to suggest that epigenetic changes as a result of an individuals prenatal environment can contribute to the development of an obese phenotype later in life. More specifically infants with low birth weight as a result of impaired fetal growth and the resulting accelerated growth in early postnatal life are thought to cause epigenetic chances, which predispose and individual to becoming obese (Varvarigou, 2010). Although the association between impaired fetal development and obesity is well supported there is less knowledge of the molecular mechanisms and specific genetic and epigenetic pathways involved. This however is exactly what Walter Stünkel and his colleagues set out to investigate in their recent paper.

Their paper looked at adipocytes, which are cells that are specialized in storing fat in the form of triglycerides within lipid droplets. Stünkel et al took adipocytes differentiated from mesenchymal stem cells (cells capable of multiple different fates) of both normal and growth restricted newborns and compared the two. From this they were able to demonstrate that a specific gene, SOX6 was upregulated in the adipocytes developed from growth-restricted infants. Essentially, through the enrichment of H3K27 acetylation sites in the promoter region of SOX6 a pathway is activated which acts to promote fat cell formation. The key role of SOX6 in adipocyte differentiation was shown through the silencing of the SOX6 gene in numerous cell lines the resulting reduction in the formation of lipid droplets supported the role of SOX6 in adipogenesis. Further, they also showed that expression of SOX6 activated genes downstream all of which are known to play a role in adipogenesis. Interactions between SOX6 and the MEST promoter also act to inhibit WNT signaling which has in the past been shown to inhibit adipogenesis. This is the first time these genetic pathways have been demonstrated experimentally and it highlights the key role of SOX6 in promoting adipogenesis differentially in mesenchymal stem cells from growth restricted and normal neonates. Through the enrichment of H3K27 acetylation sites in the promoter region of SOX6 a pathway is activated which acts to promote fat cell formation.

Screen Shot 2016-03-25 at 2.08.59 pm

Fig 1: Suggested role of SOX6 in the regulation of adipogenesis

Also of interest was the investigation of SOX6 and its role in lipid metabolism in vivo, in a mouse model. SOX6 expression was reduced specifically in white adipocytes and resulted in a reduction in cholesterol levels and liver triglyceride levels. Further, factors positively correlated with levels of fat mass were also reduced significantly when SOX6 expression was decreased. These results provide further evidence of the role of SOX6 in the promotion of fat associated factors.

Stünkel et al have provided robust and convincing evidence of the presence of epigenetic changes to the expression of SOX6 associated with impaired fetal growth. In addition they give evidence of the conserved evolutionary role of SOX6 in adipogenesis across vertebrate groups (Humans, mice and zebra fish). These changes, which occur very early on in development, have the potential to affect an individual for many years to come. It is known that epigenetic changes can be long lasting and span an individuals lifetime, in addition the strong association between growth impairment in utero and obesity later in life supports that the effects are long lasting in this case. However before we can state this definitively the longevity of this modification and its causative role in obesity later in life must still be investigated further.

Not only is obesity a huge issue due to the health conditions that arise from it, but also it carries a significant stigma that people who suffer are lazy, unhealthy and unmotivated and that it is their fault that they are affected. It is only relatively recently that evidence in support that genetics and epigenetics may play a role has begun to surface. Obesity is complex issue, and due to this strategies aimed at overcoming it need to target not just one cause, but all of them. The key role of life style choices and diet should remain a focus, along with continuing to push for legislation that will aid in encouraging healthy food choices. However as our knowledge of genetic and epigenetic factors and the environmental conditions that drive these increases, the possibility that these could also be applied in an attempt to target obesity becomes a reality. This knowledge has the potential completely change the public opinion of obesity and also the way in which we are able to treat it.Unknown


References

Leow SC, Poschmann J, Too PG, Yin J, Joseph R, McFarlane C, Dogra S, Shabbir A, Ingham PW, Prabhakar S, Leow MK. The transcription factor SOX6 contributes to the developmental origins of obesity by promoting adipogenesis. Development. 2016 Mar 15;143(6):950-61.

Varvarigou, A.A., 2010. Intrauterine growth restriction as a potential risk factor for disease onset in adulthood. Journal of Pediatric Endocrinology and Metabolism23(3), pp.215-224.

Snailed it, knowing our left from right

Externally, most organisms including us humans look bilateral. That being, if we cut a human being directly in half (theoretically, murder is not endorsed by the author) down the middle each half would have one eye, one arm, one leg and so on, a mirror image of itself. However, the internal composition is very different. If we were to inspect the inside of our half human, we would find only the left side would contain a heart. The asymmetry of the internal organs of most bilateral organisms has previously not been extensively studied, the genetic basis largely unknown.

A team of researchers lead by Angus Davison investigated the origins of this asymmetry using Lymnaea stagnalis, a snail which shows naturally inherited difference in the coiling of their shell, known as chirality. Unlike humans and other bilateral organisms, these snails wear their asymmetry on their backs, making it very easy to observe the phenotype. The coil of the shell is controlled by a single maternally inherited gene. Naturally, most have a shell that coils clockwise (two dominant alleles DD), those with the recessive alleles (dd) exhibit an anti-clockwise shell.

Using genetic mapping (finding a genes location) and genomic approaches they were able to determine the chirality locus. This method narrowed the candidate genes down to six, of which only one, Ldia2, showed significant difference in expression associated with genotype. Ldia2 is one copy of tandemly-duplicated diaphanous-related formin genes (aka formin), the other being Ldia1. Whilst that sounds complicated, tandem duplication only means that a single gene has been duplicated beside itself and diversified in function to give Ldia1 and Ldia 2. The Ldia2 gene in dd individuals was found to have a single base pair deletion resulting in a frameshift, and a non-functional protein. Formin are a group of proteins involved in the formation of actin filaments (also known as microfilaments) the support structure or scaffold of the cell.

In DD individuals (clockwise shells) there was high levels of Ldia2 transcripts compared to the dd (anticlockwise) where its expression was negligible. The frameshift mutation is not lethal to the dd snails, owing the to paralog Ldia1 which likely has similar roles in embryonic development allowing the embryo to continue developing.

Asymmetrical inheritance of Ldia2 during cell division. At the two cell stage it has localised to one cell, and at the four cell stage is again only localised in one of the cells

Figure 1

When a fertilized egg begins to divide, it initially divides into two, then four, then eight and so forth. Interestingly, when investigating the expression of Ldia2 mRNA during these divisions, they found its expression was already asymmetrical by the two cell stage only being expressed in one of the cells (Figure 1), and further confined to only one cell by the four-cell stage. This demonstrates that asymmetry is established very early in development, and occurs before any morphological features appear.

Taking this finding one step further, they decided to look at how inhibition of the Ldia2 formin gene would affect phenotype of DD individuals. They found that they could partially convert those with clockwise shells to being anti-clockwise using formin inhibitor drugs, those snails, however, unfortunately did not survive. That aside, it showed that formin was acting in the way they suspected in chirality, and disruption of its function resulted (as found in those individuals with the mutant Ldia2) an anti-clockwise shell.

The question then arises? Is this solely the case in L. stagnalis or is this asymmetry of the cytoskeleton which provides the blue-print for development a conserved mechanism across other groups

Using the the model organism Xenopus laevis (a frog, not some kind of warrior princess) they conducted two experiments to investigate the left-right patterning of this vertebrate. Firstly, they applied the same formin inhibiting drugs as used on the snails to embryos of various stages of development. They found that exposure to these drugs caused heterotaxia, where organs develop on the incorrect side of the body. To further this, they injected into the embryo the mouse formin gene dia1 into Xenopus embryos to stimulate a gain of formin function. Again, they observed heterotaxia, with disorganization of organs occurring which can be seen in Figure 2. From this figure we can see that over expression of formin leads to a pretty messed up tadpole. And so it appears that formin is not restricted to the asymmetrical patterning of snails but also of vertebrate.

Figure 2: Embryos were injected into the animal pole with mRNA encoding mouse dia1 formin and scored for visceral organ situs at stage 45. Images: Examples of organ situs for experimental microinjection with wild-type mouse dia1 mRNA. The control shows a wild-type (situs solitus) tadpole, ventral view, demonstrating the normal arrangement of the stomach (yellow arrowhead), heart apex (red arrowhead), and gall bladder (green arrowhead). Heterotaxic tadpoles (ventral view) resulting from formin overexpression show reversal of all three organs, i.e., situs inversus; the gut position and looping and gall bladder; or the heart.

Figure 2: Embryos were injected  with mRNA encoding mouse dia1 formin Images:  The control shows a wild-type (situs solitus) tadpole, ventral view, demonstrating the normal arrangement of the stomach (yellow arrowhead), heart apex (red arrowhead), and gall bladder (green arrowhead). Heterotaxic tadpoles (ventral view) resulting from formin overexpression show reversal of all three organs, i.e., situs inversus; the gut position and looping and gall bladder; or the heart.

But what does it all mean? And why should you care?

While which way a snail shell coils may seem insignificant to some, the same mechanism likely underpins why we are the way we are. This research shows the asymmetry is likely an ancient basal trait of animals, showing that formin is the earliest “symmetry-breaking” determinant in snails and frogs, and possibly even all bilateral animals. Now knowing the role formin plays in asymmetry means new research could explore formins function in other organisms. This may be of particular interest for human disease and those individuals who suffer from Heterotaxy Syndrome, where organs are not where they are suppose to be (for example, the heart on the right instead of the left). It’s hard to believe that those slow often overlooked critters in our gardens could hold the key to understanding the evolution of bilateral asymmetry.

“You have to know the past to understand the present” – Carl Sagan

Check out the video below to see Angus talk about his research

 


References:

Davison, A., McDowell, G. S., Holden, J. M., Johnson, H. F., Koutsovoulos, G. D., Liu, M. M., … & Yang, F. (2016). Formin is associated with left-right asymmetry in the pond snail and the frog. Current Biology, 26(5), 654-660.

Figures 1 & 2 adapted from Davison et al., (2016)

Snail and tapole picture © Esther de Roij and Gary McDowel

University of Nottingham (25 February 2016). Snail shells offer clue in unravelling origins of body asymmetry [File video]. Retrieved from https://www.youtube.com/watch?v=Mh8-QyJsDVo