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Developmental Biology Research Highlights
Anat456 Developmental Biology Blog

The mystery surrounding a hole in the Heart

What causes Tetralogy of Fallot?

The developmental heart disease known as Tetralogy of Fallot affects 1 in 2500 newborns. It accounts for 10% of congenital heart defects. This disease is made up of four parts which can vary in severity. At best, a patient will struggle to deliver oxygen to the body and may have to adjust their lifestyle after surgery to reduce physical exertion. At worst, lack of oxygen can cause the death of cells throughout the body and brain, resulting in brain damage or death.

The defects in Tetralogy of Fallot allow blood to bypass collecting oxygen in the lungs

 

  1. The pulmonary artery is narrow, making it difficult for blood to leave the heart to the lungs
  2. A hole in the muscle wall between the two halves of the heart, allowing blood to skip travelling to the lungs
  3. Thicker muscle around the right half of the heart, further restricting blood flow
  4. Displaced aorta from the left side to the center of the heart, allowing deoxygenated blood to enter the body

Since the 1950s, individuals born with Tetralogy of Fallot have been able to receive heart surgery in order to fix a number of these issues – in particular, the hole between the right and left ventricles, and the narrow pulmonary artery. This occurs within the first year of life, and with management can allow patients to live relatively normally.

The developmental cause of Tetralogy of Fallot is the failure of cells from a part of the embryo called the secondary heart field to migrate to the future heart, or to specialise into heart muscle cells called cardio-myocytes. This results in an impaired ability to form the cardiac outflow tract, and aorta.

While it can occur on its own, Tetralogy of Fallot is often a small part of other developmental diseases, which include defects in other organs and tissues. One of these is DiGeorge Syndrome, which also affects the face, kidneys, and immune system. DiGeorge Syndrome is also known as 22q11.2 Deletion Syndrome due to its association with the loss of area q11.2 on the 22nd chromosome. Roughly 1/3rd of DiGeorge Syndrome patients have Tetralogy of Fallot. Therefore, the loss of the 22q11.2 region is not entirely responsible the Tetralogy of Fallot.

 

22q11.2

What genes are lost when this area is deleted?

 

Deletion of this part of chromosome 22 can be inherited in an autosomal dominant fashion. This means that a parent suffering from DiGeorge Syndrome has a 50% chance of passing the disease to their child. If a child has the q11.2 area deleted on one of their chromosome 22s, this in enough for DiGeorge Syndrome to develop.

The gene Tbx1 creates a transcription factor; these are responsible for activating genes. In simple terms, when genes are activated they are read from DNA, copied into mRNA, and then these transcripts are used as the instructions for building proteins. A transcription factor enables this process to occur. By losing the gene Tbx1 from one chromosome, there is only half as much protein available.  When both copies of Tbx1 are lost in all cells, the embryo will die in the neonatal stage due to severe hear defects.

The loss of this gene, however, does not explain the variability of symptom severity. This implies that there are more mutations which increase the risk of Tetralogy of Fallot.

 

Reading Disease-causing DNA

What parts of the DNA increase the risk of Tetralogy of Fallot?

 

A Genome-wide Association Study (GWAS) uses groups of individuals known to have a condition, and evaluates what parts of DNA are changed compared to healthy individuals. The 2017 study by Guo et al. performed a GWAS on individuals with Tetralogy of Fallot who had the 22q11.2 deletion with the goal of identifying any other single-nucleotide polymorphisms (SNPs). A SNP is a mutation which changes only one of the bases which code DNA. While it might sound small, if it occurs in the worng place it can be enough to confer disease. These experiments used DNA data obtained from human 326 people with ToF and 22q11.2, and compared it to 566 people with 22q11.2 but normal cardiac anatomy.

A graphical representation of the mutations found in GWAS. The red line is the threshold for significant association with Tetralogy of Fallot.

On chromosome 5 there is a region called 15q14.3 which has three SNPs that are significantly associated with Tetralogy of Fallot. This was narrowed to a region 104.7 kb wide. In this region were a number of genes including GPR98.

The 104.7 kb region of Chromosome 5 which contains the SNPs

The GPR98 gene encodes for a G-protein coupled receptor, a type of cell surface protein that receives calcium signals from their environment. It is activated in a part of the embryo called the neural tube in mice 9 days after fertilization. This gene can also create a number of different proteins depending on how it is spliced. Guo et al. theorized that one or more of these splice variants could play a role in Tetralogy of Fallot. It is known that cells from the neural tube contribute to the formation of the cardiac septum – the muscle wall between the ventricles of the heart. The septum is damaged in Tertalogy of Fallot, a hole in it allowing for the unregulated passage of blood.

One problem with this theory is that there is no evidence to support GPR98 activity to the formation of the circulatory system, only conjecture. So why are mutations in this gene associated with Tetralogy of Fallot?

 

Folding the Genome

Genes involved in this disorder may not always be obvious

 

A model showing how two areas of DNA can be brought closer together in space to allow for interaction

There is another answer to this connundrum. Further along the DNA from GPR98 is the gene MEF2C. Although this gene is not where the mutations are occurring, it could also be a factor in Tetralogy of Fallot. The strands of DNA in a cell are known to fold, allowing pieces to come closer together in space. This is called chromatin interaction. Chromatin interaction can activate genes, so a mutation in the GPR98 region could affect the expression of MEF2C.

MEF2C is the gene Myocyte Enhancer Factor 2C, and is clearly linked to muscle synthesis and vasculature development. This is a much more likely candidate than GPR98. The chromatin interaction between these two genes has been shwon using the Hi-C technique (Dixon et al., 2015).

 

 

Conclusions from Guo et al. (2017)

Tetralogy of Fallot needs more works, especially with regards to MEF2C

 

Along with the deletion of the 22q11.2 region, there is reason to look for other mutations which increase the risk and variability of Tetralogy of Fallot. Using a GWAS to study where mutations have gathered in the genome allows for associations between genes and diseases to be made. However, it is not always obvious why a gene would be involved in a developmental disease. In Guo et al., (2017) evidence for a chromatin interaction between single-nucleotide polymorphisms in the GPR98 gene of 5q14.3 and the gene MEF2C 2 kb upstream has been put forth. If this is the case, then the process by which the heart is formed in embryogenesis become clearer – along with ways in which this process can go wrong.

Understanding the genetic causes of a disease such as Tetralogy of Fallot is vital, not only for potential treatments, but also because of the variability in the presentation of this disease. If some mutations are linked to worse prognoses, then this information is vital for those suffering from the condition. As Tetralogy of Fallot accounts for 10% of congenital heart diseases, the mystery of the hole in the heart cannot go without investigation.

 

Reference

To read in more detail about this experiment, see the paper by Guo et al. (2017)

Dixon J. R, Jung I, Selvaraj S, Shen Y, Antosiewicz-Bourget J. E, Lee A. Y, et
al. Chromatin architecture reorganization during stem cell differentiation.
Nature. 2015;518:331–336. doi: 10.1038/nature14222

Tead1: The Heart for Heart Muscle

Heart failure is a global health issue affecting millions of people worldwide. Dilated cardiomyopathy (DCM) is a form of heart failure that includes weak cardiac contractility, dilated ventricular chambers, myocyte death, and myocardial fibrosis (Fig. 1).

Figure 1: Visual comparison of components within a normal heart and a heart with dilated cardiomyopathy

DCM has a prevalence of 1 in 2,500 and a high 5-year mortality rate of 50%. The cause of DCM is still not fully known but based on past research, potential causes have been listed to be: defects in diverse cellular processes, including those resulting from injury and familial defects.

The Hippo/Tead pathway is an evolutionarily conserved pathway responsible for regulating cell proliferation, differentiation and organ size amongst various tissues and has been involved in many forms of cancers.

Figure 2: Schematic overview of the hippo signalling pathway (inactivated)

Recent research has established the role of mammalian the Hippo/Tead pathway within the cardiac development, cardiomyocyte (CM) proliferation, and regeneration. Fig. 2 provides a schematic overview of the Hippo signalling pathway. The Hippo pathway being inactivated, results in accumulation of Yap and Taz which then interact with Tead1-4 and eventually express YAP/TAZ target genes (Fig. 2). Tead1-4 are downstream transcriptional effectors of the Hippo pathway. These are related to cell proliferation, lineage differentiation and tumour formation. Tead1 is most abundant in the heart and plays an essential role in cardiac development. This was suggested based on past research; following the germline deletion of Tead1 resulting in cardiac hypoplasia and embryonic lethality. Tead1 coactivators, Yap and Taz are vital for CM proliferation during perinatal cardiac development. On the other hand, Tead1 corepressor, Vgll4, is responsible for regulating CM gene expression and inhibiting early postnatal heart growth. Therefore proving the Tead1 being highly conserved and highly expressed in cardiac muscle.

 

 

Who, What and How

The paper by Liu et al., 2017, looked at the need for Tead1 in maintaining adult cardiomyocyte function and how its loss results in lethal DCM. They carried out their experiments using mouse models, where they hypothesised that Tead1 plays a broader transcriptional regulatory role in post-proliferative CMs and is also required for normal adult cardiac function.

They tested their hypothesis by developing experiments that involved inducible CM-specific Tead1 conditional KO mouse model. They outlined the essential role of Tead1 in excitation-contraction coupling in adult CM via direct activation of SERCA2a and I-1 which are two vital components of SR calcium cycling-related genes.

Their results concluded that Tead1 is important for maintenance of adult cardiac contractility and loss of results in severe lethal DCM. The importance of Tead1 was based on its need within normal SR calcium cycling which is needed to sustain normal excitation-contraction coupling. Tead1 was able to do this by directly regulating transcription and activity of SERCA2a.
They also blocked the Tead1 activity in human induced pluripotent stem cell-derived CMs and proved the importance of Tead1 in maintaining PLN phosphorylation as well as expression of SERCA2a and I-1 which lead them to strongly suggest that this regulatory pathway is also preserved within human CMs.
Their microarray data suggested the role of Tead1 in transcriptional regulation of other genes involved in cardiac function (including mitochondrial and sarcomere encoding genes). These findings matched with the prior knowledge of Tead1 being a part of a cluster of the transcription factor that regulates cardiac-specific gene expression.

 

Why should we care?

There are multiple reasons to why study such as this one based on Tead1 and its involvement in cardiac functions is beneficial in the developmental field of research. Some of the highlighting factors include:

  • Tead1 plays an important role in the process of formation of muscular tissue (myogenesis). This is where it binds to MCAT elements in promoters of a range of muscle-specific genes.
  • Involvement of Tead1 along with its upstream regulators is shown to be important in CM proliferation during embryonic and perinatal periods.
  • Tead1 plays an important role in heart, alongside other genes and transcription factors, but recently it has shown direct targeting of myocardial during cardiovascular development.
  • Recent studies have also highlighted the role of the mammalian Hippo-Tead1 pathway during CM regeneration post-injury.

Figure 3: Overview of Tead1 regulation of adult cardiomyocyte SR-calcium cycling

So that leads us back to the question about why this paper by Liu et al., was important in relation to the reasons above. That is because this paper outlined the importance of Tead1 in the adult cardiac function via maintenance of CM calcium homeostasis, not only by direct transcriptional regulation of SERCA2a expression but also by enhancing transcription of I-1 (Fig.3).
Based on the results of this research, Tead1 is needed for normal SERCA2a expression in both mouse and human CMs. It also provides with a possible mechanism for downregulation of SERCA2a in human heart failure. This study is also important as it showed that Tead1 regulation of PLN, SERCA2a and I-1 is also conserved within human CMs. This is important as SERCA2a expression and activity is frequently decreased in heart failure as well as in I-1 regulation of phospho-PLN, which means that this study showing Tead1 as a direct transcriptional activator of SERCA2a and I-1 defines the previously unrecognised mechanism and can aid towards targeted therapy.

 

Where does this take us in future?

Following this research, possible future directions for other researchers could include looking further into following areas:

  • Efficacy of SERCA2a overexpression enhancing cardiac contractility in vivo and in initial human studies are yet to be confirmed in larger human heart failure researches. This could be a possible direction to take in future studies as this might hold a more efficient insight into findings related to SERCA2a overexpression
  • This study did not design experiments based on the tendency for decreased Tead1 protein levels in human heart failure in primary or secondary. Studies carried out in hiPS-derived CM suggest that changes in Tead1 protein levels would maintain the pathophysiology of heart failure by further reducing the SERCA2a expression and activity. Therefore, this proposes the possibility that therapeutic targets restoring Tead1 levels might be able to provide significant benefit in terms of slowing the downward spiral of a simultaneous build-up of SERCA2a expression and activity; this is usually the case in end-stage heart failure. Thus, providing a new area of interest to look into for future therapeutic targets involving altered Tead1 protein levels.

Summary

Many of the regulatory mechanisms conserved within mouse and human heart failure still consist of many unanswered questions. I believe that this paper by Liu et al., provides strong preliminary evidence regarding Tead1 playing a critical role in cardiac development. This is based on results of Tead1 based regulation of SERCA2a, Pin phosphorylation and I-1 are seen in human hearts as well as hiPS-derived CMs. The study outlined very well that Tead1 has a cell-autonomous and essential role in adult CM excitation-contraction coupling, in addition to the prior knowledge of Yap-facilitated activation of cellular proliferation and survival.

References:

Liu, R., Lee, J., Kim, B., Wang, Q., Buxton, S., Balasubramanyam, N., Kim, J., Dong, J., Zhang, A., Li, S., Gupte, A., Hamilton, D., Martin, J., Rodney, G., Coarfa, C., Wehrens, X., Yechoor, V. and Moulik, M. (2017). Tead1 is required for maintaining adult cardiomyocyte function, and its loss results in lethal dilated cardiomyopathy. The Journal of Clinical Investigation, 2(17).

Juan, W. and Hong, W. (2016). Targeting the Hippo Signaling Pathway for Tissue Regeneration and Cancer Therapy. Genes, 7(9), p.55.

 

Are MiRNA’s the key to producing functional lungs?

New research suggests that using a microRNA therapeutic could reverse abnormal lung development observed in CDH patients

 

Figure 1: Left panel depicts normal development. Right panel depicts the herniation of abdominal contents into the thoracic cavity, resulting in mechanical compression of the lungs, contributing to their underdevelopment.

 

 

Congenital diaphragmatic hernia (CDH) occurs when the diaphragm does not close correctly, resulting in an orifice which allows abdominal structures to invade the chest cavity. The presence of abdominal organs in the thorax, as well as other underlying developmental issues, limits the normal development of the lungs, resulting in under-developed lungs which cannot function well after birth. CDH is currently managed with corrective surgery and artificial ventilation, though even with these treatments the patient will never have a well-functioning respiratory system. Due to the severe dysmorphia and associated symptoms of this condition, research efforts are focused on producing a preventative treatment rather than a curative surgery after birth.

 

Currently there is no non-surgical prenatal treatment for CDH, as the mechanisms of CDH development are still not well understood, so preventing them is near-impossible! This research team had previously discovered that a member of the microRNA family, miR-200b, is detected in higher amounts in the tracheal fluid of infants that survive with CDH. Looking further into miRNA-200b’s expression levels in the lungs has helped this team understand more about how miRNA-200b may function in the context of CDH. Upon finding that miRNA-200b is in critical regions of the lung during its development, the researchers tested utilising miRNA-200b mimics as a therapeutic for CDH-diagnosed fetuses – and have found some exciting results!

 

What did this team do?

 

This paper used nitrofen rat models of CDH to establish where miRNA-200b is present within the developing lung, and what it interacts with. Pregnant nitrofen-treated rats were administered miR-200b mimics as a proposed non-surgical prenatal treatment of the pulmonary hypoplasia associated with CDH.

The nitrofen rodent model of CDH is one of the most widely-used models to study CDH. It works on the basis that nitrofen inhibits enzymes critical for retinoic acid production, and retinoic acid is critical in normal organogenesis. Therefore, any rodent treated with nitrofen gets disrupted diaphragm and lung development, or CDH.

MiRNA-200b is part of the miRNA-200 family, and is expressed highly in epithelial tissues – like that of the lung. MicroRNA’s regulate the expression of other genes via interfering with translation of mRNAs.

 

And what did they find?

 

The researchers had 4 key findings with significant outcomes.

  1. MiRNA-200b is more highly-expressed in normal(er) lungs

Graph depicts relative levels of miRNA-200b in control and nitrofen-treated brochial BEAS-2B cells

Expression of miR-200b was not significantly different in early lung development, when normal closure of the diaphragm has not occurred. However, in later stages of development, pups with a lesser degree of hypoplasia, and that do not have CDH, have far higher miRNA-200b levels than pups with severe, CDH-related hypoplasia. In situ hybridisation was performed to identify which tissue type miRNA-200b is expressed in, in both normal and hypoplastic lungs.  MiRNA-200b was detected in the highest amounts in the elongating airway tips of both samples, yet in not as high amounts in the already differentiated proximal airway, suggesting an important role for miRNA-200b in the branching of the lungs.  Nitrofen-induced hypoplastic lungs not only had less distal tips as a whole, but within these tips, a reduced miRNA-200b expression in both the mesenchyme and epithelium of the branching tips was observed, which could explain the lesser degree of development. Rawlins et al have shown that the distal tips of the developing lung contain progenitor constructs that will give way to a fully differentiated cell in time [2], which helps to validate the thought that miRNA-200b may play a role in helping the branching of the developing lung.

  1. Inhibition of miRNA-200b via nitrofen treatment increases SMAD signalling in the lung epithelium

MiRNA-200b expression is down regulated when human bronchial epithelial cells are exposed to nitrofen in vivo, suggesting nitrofen directly inhibits miRNA-200b. The team already knew that SMAD-luciferase activity increased in response to miRNA-200b inhibition, and were able to validate that nitrofen has an inhibitory role on miRNA-200b through treating cells with nitrofen, therefore inhibiting miRNA-200b, and getting an increase in SMAD-luciferase activity. Western blotting confirmed that upon miRNA-200b inhibition, SMAD could then work with TGFbeta to increase ZEB2, through signalling in the lung epithelium. The increase in SMAD/TGFbeta signalling could be reversed by reintroducing miRNA-200b mimics. As a whole, the data suggests that miRNA-200b promotes lung branching in the distal airway tips through negatively regulating SMAD signalling.

  1. Nitrofen-induced abnormal branching can be rescued

An ex vivo lung explant culture system treated with miRNA-200b inhibitors showed a decrease in the total number of airway buds after 4 days, providing evidence that a drop in miRNA-200b levels provides a direct inhibitory effect on the lungs branching capabilities. MiRNA-200b mimics added to a nitrofen-induced hypoplastic rat lung culture increased the total number of miRNA-200b present, and as a result, the peripheral lung tissues branched at normal levels.

  1. MiRNA-200b therapy reduces CDH, and improves lung development

A) Physical comparison of negative control and miRNA-200b-treated pups. B) Graph depicts the incidence of CDH when pups are left untreated (neg. control) vs when they are treated with the miRNA-200b mimic.

Realizing that addition of miRNA-200b can induce lungs to branch normally, the researchers decided to test whether addition of miRNA-200b could be a therapeutic for CDH sufferers. MiRNA-200b mimics were administered to pregnant nitrofen-treated rats at day 9 of gestation. When the pups were born, 90% of the miRNA-200b treated rats were active and pink (suggesting oxygenation), while 70% of the control rats, who were only treated with nitrofen, died within 10 minutes of birth. CDH was detected at lower rates in the miRNA-200b treated group (23%) than the untreated (60%), demonstrating a protective effect of miRNA-200b. taken together, all of the findings suggest hopes for inducing lung branching in fetuses diagnosed with CDH, meaning some of the effects of the condtition can have their effect lessened.

 

What do their findings mean?

 

This whole study validates this research group’s previous findings that CDH infants with less severe hypoplasia presented with higher miRNA-200b levels in their tracheal fluid than those infants with severe hypoplasia. It has been shown that miRNA-200b plays a crucial role in the development of the lung, through inhibiting SMAD signalling. As a whole, the studies show that increasing miRNA-200b levels in those with CDH can rescue abnormal branching that is characteristic of the condition. It is now a matter of pursuing these findings in the hope of creating a prenatal medical therapy to reverse the effects of CDH in human patients.

 

What can be done to further this progress?

 

Moving forward from these findings, it is key to test the safety of this method of therapeutic. Wang et al demonstrated that miRNA-200’s effect on ZEB2 transcription can induce stem cell generation [3], so future studies should evaluate miRNA-200b’s role in this process. The role of miRNA-200b should not only be confirmed in a pluripotent-inducing state but in general, to ascertain any potential side effects that could occur through administering this as a therapeutic.

 

Who new something so little could potentially save someones life…

 

 

 

 

 

 

 

[1] Khoshgoo, N., Kholdebarin, R., Pereira-Terra, P., Mahood, T. H., Falk, L., Day, C. A., … & Correia-Pinto, J. (2018). Prenatal microRNA miR-200b Therapy Improves Nitrofen-induced Pulmonary Hypoplasia Associated With Congenital Diaphragmatic Hernia. Annals of surgery.

 

[2] Rawlins, E. L., Clark, C. P., Xue, Y., & Hogan, B. L. (2009). The Id2+ distal tip lung epithelium contains individual multipotent embryonic progenitor cells. Development, 136(22), 3741-3745.

 

[3] Wang, G., Guo, X., Hong, W., Liu, Q., Wei, T., Lu, C., … & Wang, J. (2013). Critical regulation of miR-200/ZEB2 pathway in Oct4/Sox2-induced mesenchymal-to-epithelial transition and induced pluripotent stem cell generation. Proceedings of the National Academy of Sciences, 110(8), 2858-2863.

Huntington’s Disease: It starts at at fertilisation

Figure 1. Normal brain (left) compared to a degraded Huntington’s disease brain (right). Image source

Huntington’s disease (HD), like many other neurodegenerative diseases has been a hot topic of discussion for many years but there is little knowledge about what really causes it. While we know that HD is caused by one gene, little is known about why it has such a wide range of symptoms and large variability in age of onset. Scientists have yet to come up with an effective treatment for the condition and currently treatment involves treating the symptoms with limited success. Contrary to the current gain-of-toxic-function hypothesis, HD may be caused by chromosomal instability from day one. Researchers from the Rockefeller University in New York have discovered that signs of HD start within a few months of development.

What is Huntington’s Disease?

Huntington’s disease is a neurodegenerative disease characterised by ongoing neuron death. The condition is mostly genetic in nature and originates in the Huntingtin (HTT) protein gene, caused by increased numbers of CAG repeats within the gene. HD is an autosomal dominant condition, meaning that if someone has the mutation they will develop HD, regardless of whether their other copy of normal or not. Traditionally HD has been described as neuron dysfunction and cell death occurring as an adult, but this may just be when symptoms start to show. Symptoms of HD include progressive worsening of movement, cognition and psychosis as the individual ages until they become bedridden. WE can see in Figure 1 that there is a significant amount of brain shrinkage, with a significant reduction in white matter volume caused by dying neurons which are characteristic of HD. There are currently no successful therapies for HD, and treatment is limited to treating any symptoms that the person has. By understanding how HD is brought on, hopefully viable treatments can be developed.

The severity of HD depends on the number of CAG repeats they have, it is accepted that less than 35 repeats will not lead to HD, 36-39 repeats has an increased risk and they may or may not develop HD and 40+ repeats guarantees that they will develop HD. The HTT gene is characterised as having a polyQ tract, meaning that it has multiple CAG repeats within it, creating a chain of glutamine amino acid within the HTT protein. This chain of glutamine is what causes the signs and symptoms of HD, and in this article, they explore the possibility that this is what causes chromosome instability right from the get go.

What did they do?

The aim of this study was to find out if neurogenesis is indeed impaired during development and the sort of impact this could have on the adult. This study expands on the idea that HTT protein has an important role in development around the entire body, not just the brain. The study was also based around the fact that the severity of HD has a linear positive relationship with the number of CAG repeats. To complete this study the researchers developed the first ever human embryonic stem cell line (hESC) that models HD. They used an accepted hESC line and used a relatively new technology called Crispr-Cas to edit the genome and add as many CAG repeats as they wanted. They were able to model a normal gene with 22 repeats, with increasing numbers of 45, 50, 58, 67 and 74 with 67 and 74 being extremely severe juvenile onset cases.

A lot of this study was focused around the organisation of the brain and the disorganisation that occurs in HD. To do this they focused on particular ares of the brain called rosettes. Rosettes are essentially rings of neurons that form with stem cells in the centre, called the lumen, where they can be protected. It is well documented that rosettes and lumens are essential for neuron development and without them neuron growth is significantly retarded2.

What did they find?

Figure 2. Angle of the mitotic plane in relation to the lumen. The angle has become randomised and less organised with more polyQ repeats. Centrosomes indicated by green fluorescence are located within the lumen and dividing DNA is shown in white.

Ordinarily the mitotic plane would be at a ~90° angle to the lumen, this allows the cells to divide and grow the lumenal size but in figure 2 we can see this isn’t the case. This suggests that HTT plays a role in orienting the mitotic plane and controlling the direection that the cells divide. Without this regulation the cells divide in random planes due a loss of control over the mitotic spindles, this prevents growth of the lumen, which was also found. HTT plays a role in the formation of neural rosettes and lumen, which  are essential for neurogenesis. They also saw that there was a significant difference in the nearest-neighbor distances between rosettes with randomised organisation of neural rosettes. All of these results point towards the fact that HTT mutations affect the earliest stages of human neurodevelopment, rather than acting only in adults, as commonly assumed.

Figure 3. Dysfunction during cell division, with the chromosomes indicated in white. 22Q is normal and splitting in two, 50Q is splitting into four and 58Q is splitting into three.

Figure 4. Abnormal neuron from 58Q cell line at day 45, note the enlarged body, multiple nuclei and thick processes compared to outlying cells

Dysfunction in cell division such as multiple origins of division seen in figure 3 will cause neurons with abnormal number of cells and may lead to apoptosis of the resulting cells. The neurons may also become incorporated as a functional cell seen in Figure 4 but it likely will not function as effectively as it should and is clearly abnormal. This means that sufferers are likely at a disadvantage due to abnormal cells and decreased neuron count. Previous research has been focused on older individuals who have already developed symptoms, therefore already showing a dramatically decreased brain volume. It would be interesting to do a study on HD brains to see whether there is fewer neurons in young individuals.

What does this mean?

This is a revolutionary study and changes the way we look at Huntington’s forever. They have been able to prove that the disease starts right at the beginning stages of development. The creation of these stem cell line is invaluable to further HD research to better understand the disease. Because we now know that HD is indeed development and starts as soon as neurons develop we can now concentrate on hopefully developing more targeted therapies. Therapies such as gene editing technology, Crispr-Cas may potentially utilised in the future as the technology develops, they have shown in this paper that it is possible to edit these genes and current studies have shown that editing these gene in a patient may soon be possible. Further research in animal models into neuron number during early development and how it progresses as they age would be invaluable to furthering our understanding. Also expanding the hESC lines would be beneficial to do comparisons between gender and genetic background. Another step they could take is looking at the scarring or inflammation caused by dying cells and investigating the connections that abnormal cells make and whether they are fully functional.

References

  1. Ruzo, A., Croft, G. F., Metzger, J. J., Galgoczi, S., Gerber, L. J., Pellegrini, C., … Brivanlou, A. H. (2018). Chromosomal instability during neurogenesis in Huntington’s disease. Development, 145(2), doi: 10.1242/dev.156844.
  2. Hříbková, H., Grabiec, M., Klemová, D., Slaninová, I., Sun, Y. (2018). Five steps to form neural rosettes: structure and function. Journal of Cell Science, doi: 10.1242/jcs.206896.

Germline Mosaicism causes high rates of disease in family

Germline Mosaicism causes high rates of disease

 

Campomelic dysplasia is a rare disease which effects approximately 1:200,000 births worldwide. Campomelic is Greek for ‘bent limb’1,2. It is an extremely lethal disorder. 90% of patients with the disorder die within the first 2 years of life due to respiratory distress2–5. There is a range of symptoms and some patients will survive into adulthood. Campomelic dysplasia effects the skeletal and reproductive systems. Although other body systems such as the central nervous system can also be affected6,7. Symptoms include bowed (bent) long bones in the legs (and occasionally the arms)8. Short torso with only 11 pairs of ribs instead of the normal 12. Weakened cartilage of the upper respiratory tract resulting in respiratory distress. Club feet, cleft palate, small chin and a flat face along with other facial symptoms9. An interesting feature of the disorder is complete or partial sex reversal this is seen in 75% of 46, XY individuals1. A genotypic XY individual may present with a completely female phenotype. Individuals may also present with a mixed phenotype, internal female organs (ovaries) and external male genitals or internal male organs (testes) and external female genitals.

 

Figure 1. Bowing of the tibia and fibular can be seen, this is characteristic of Campomelic dysplasia patients (10).

 

 

Campomelic Dysplasia

Figure 2. A mother with a mild phenotype sue to mosaicism passes on the disease allele in an autosomal dominant fashion (10)

Campomelic Dysplasia is an autosomal dominant disorder. This means an individual only needs one copy of the mutated allele to develop the disorder. Normally, Campomelic dysplasia is caused by new (DeNovo) mutations in the SOX9 gene. This means individuals with the disease generally have no family history of it. Occasionally it can be passed down from a parent if they have a mild phenotype caused by a mutation in only some of their cells. In this case we would say the parent is mosaic for the mutation. If the mutation is only present in some of the germline cells then the parent will likely not present with any symptoms of the disease. If they are mosaic in their other cells as well they will often show mild symptoms of the disorder. A parent that has a mosaic mutation is likely to have a higher risk of having a child with Campomelic Dysplasia than normal10.

 

 

SOX9

SOX9 is a transcription factor which recognises the sequence CCTTGAG11,12. Transcription factors work to regulate the expression of other genes. Transcription factors can work with or without the help of other transcription factors or genes to regulate development. SOX9 is known to be involved in both the skeletal and reproductive pathways. It is involved with cell differentiation13, changing a cell from one cell type into another. SOX9 is expressed in a variety of tissue types such as chondrocytes, brain, testes, kidneys & liver14. SOX9 is abundantly expressed in chondrocytes making it important for bone development. Chondrocytes are cells which eventually become cartilage and bone15.

 

SOX9 is also involved in the reproductive pathway. It is expressed in the testes during development. The male reproductive pathway is turned on by SOX9 interacting with a range of genes. The sex determining region (SRY) on the Y chromosome id the first to be expressed16. SOX9 is upregulated in response to SRY17. SOX9 then works with steroidogenic factor 1 to regulate transcription of the anti-Muellerian hormone (AMH) gene. This gene is important in the reproductive system especially in helping cells change from one cell type to another.

 

 

Mosaicism

A paper published in Congenital Anomalies in March 2018 by Higeta Daisuke et al. was the first to use molecular technology to look at Mosaicism as a possible cause for Campomelic dysplasia. They confirmed that the Mother and her two affected children had the same mutation in the exon 2 of the SOX9 gene. The mother presented with mild symptoms while the oldest daughter had reasonably severe symptoms and the younger brother had severe symptoms only living to 9 months of age. When looking at the genotype of the mother they found that the peaks in sequencing (below) mere much smaller for the mutated allele in the mother than would be expected for a heterozygous phenotype. This along with her mild phenotype of the disease suggested that she was mosaic (only some of her cells contained the mutation) for the disease allele.

 

Figure 3. Sanger sequencing reads for the family showing deleted C (indicated by *) in mother, oldest daughter and youngest son (10).

 

To look at the suspected mosaicism they first sequenced the father, mother and three siblings. Shown in figure 3 is the sequencing peaks from the region of the gene where the mutation is present. The mutation is indicated by the *. There are peaks two peaks of the same height for S1 and S3 at this site but for the mother the mutated peak is lower. This suggested to the researchers that it was possibly present at a lower rate than the expected 50%.

 

Figure 4. The normal (wild type) allele is shown at the top marked by a W. The disease causing mutant allele is marked by an M. These alleles were amplified by PCR using allele specific primers. The PCR products were run on an electrophoresis gel, bands in both W and M means the individual has the disease causing allele (10). C = control, F = father, M= mother, S1 = sibling one (affected), S2 = sibling 2, S3 = sibling 3 (affected).

 

They confirmed what they were seeing in sequencing by amplifying a small region of the gene where the mutation was present. They then ran the amplified (PCR) product on a gel which separates the fragments by size. There will be a band present under W if they have a normal allele and there will be a band present under M if they have a mutated allele. You can see in figure 4 a phenotypically normal individual (control, father, sibling 2) will only have one band on the gel. Whereas S1 and S3 have 2 bands on the gel showing they are heterozygous for the mutation. The Mother also has two bands although the mutant band is not as strong as the wild type band this shows that she is heterozygous in at least some of her cells.

 

 

Figure 5. Proportion of mutant allele present in mother and two affected offspring. And unaffected individual should have 100% W (10).

Next they used something called AS-qRT-PCR to look at the amount of the normal and mutated allele present in an individual. As you can see in figure 5 an unaffected individual © has 100% wild type allele present. The two effected children have 50% wild type, 50% wild type which is what you would expect in normal circumstances. The mother has approximately 66% normal allele and 34% mutated. This indicates that of the cells that were sampled approximately 68% have a mutated allele.

 

 

 

Why was this important?

Higeta et al. 201810 were the first research group to use molecular techniques such as Sanger sequencing and AS-qPRT-PCR to confirm a mosaic mutation was the cause of Campomelic dysplasia in this family. In the past a few other research groups such as Smyk et al. 2007. have used other techniques such as CGH analysis and FISH to confim mosicism in the father of two affected children18. Understanding the phenotype of mosaic people could help parents of affected children understand the real recurrence rate of Campomelic dysplasia in their family and prevent having more children with the disease by early screening and genetic testing

 

 

 

References

 

  1. Păvăloaia, O. et al. Campomelic dysplasia with dextrocardia and without sex-reversal. Arch. Clin. Cases Vol 4 No 1 2017DO – 1022551201714040110088 (2017).
  2. Vineet, J. & Biswaroop, S. Campomelic dysplasia. Jourmal Pediatr. Orthop. 23, 485–488 (2014).
  3. Bohlen, A. E. et al. A mutation creating an upstream initiation codon in the SOX9 5′ UTR causes acampomelic campomelic dysplasia. Mol. Genet. Genomic Med. 5, 261–268 (2017).
  4. Mansour, S., Hall, C. M., Pembrey, M. E. & Young, I. D. A clinical and genetic study of campomelic dysplasia. J. Med. Genet. 32, 415 (1995).
  5. Eger, K. J. Campomelic Dysplasia. J. Diagn. Med. Sonogr. 21, 343–349 (2005).
  6. Iravani S, Debich-Spicer D & Gilbert-Barness E. Pathological case of the month. Arch. Pediatr. Adolesc. Med. 154, 747–747 (2000).
  7. Yao, B. et al. The SOX9 upstream region prone to chromosomal aberrations causing campomelic dysplasia contains multiple cartilage enhancers. Nucleic Acids Res. 43, 5394–5408 (2015).
  8. Gopakumar, H. et al. Acampomelic Form of Campomelic Dysplasia with SOX9 Missense Mutation. Indian J. Pediatr. 81, 98–100 (2014).
  9. Wunderle, V. M., Critcher, R., Hastie, N., Goodfellow, P. N. & Schedl, A. Deletion of long-range regulatory elements upstream of <em>SOX9</em> causes campomelic dysplasia. Proc. Natl. Acad. Sci. 95, 10649 (1998).
  10. Higeta, D. et al. Familial campomelic dysplasia due to maternal germinal mosaicism. Congenit. Anom. 37, (2018).
  11. De Santa Barbara, P. et al. Direct Interaction of SRY-Related Protein SOX9 and Steroidogenic Factor 1 Regulates Transcription of the Human Anti-Müllerian Hormone Gene. Mol. Cell. Biol. 18, 6653–6665 (1998).
  12. Wang, J. M., Irwin, R. W. & Brinton, R. D. Activation of estrogen receptor α increases and estrogen receptor β decreases apolipoprotein E expression in hippocampus in vitro and in vivo. PNAS 103, 16983–16988 (2006).
  13. Gentilin B. et al. Phenotype of five cases of prenatally diagnosed campomelic dysplasia harboring novel mutations of the SOX9 gene. Ultrasound Obstet. Gynecol. 36, 315–323 (2010).
  14. Tonni, G., Ventura, A., Pattacini, P., Bortolini, M. C. & Baffico Ave, M. p.His165Pro: A novel SOX9 missense mutation of campomelic dysplasia. J. Obstet. Gynaecol. Res. 39, 1085–1091 (2013).
  15. Hinton, R., Jing, Y., Jing, J. & Feng, J. Roles of Chondrocytes in Endochondral Bone Formation and Fracture Repair. J. Dent. Res. 96, 23–30 (2017).
  16. Wagner, T. et al. Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 79, 1111–1120 (1994).
  17. Knower, K., Kelly, S. & Harley, V. Turning on the male – SRY, SOX9 and sex determination in mammals. 101, (2003).
  18. Smyk M. et al. Recurrent SOX9 deletion campomelic dysplasia due to somatic mosaicism in the father. Am. J. Med. Genet. A. 143A, 866–870 (2007).

A big, fat and new discovery

A big, fat and new discovery

 

Adipocytes are now acknowledged as having roles outside of being local stores of energy providing both mechanical and thermal insulation, including roles in numerous tissues in regards to health and disease including immune, growth and metabolism regulation, tissue development and cancer progression. However, there is a lack of knowledge on the probable function adipocytes plays in wound healing.

Drosophila fat body cells (FBCs) are the equivalent to vertebrate adipocytes and liver, with established roles in regulating metabolism, regulating systemic growth and regulating systemic immunity, with activation innate immune pathways resulting in systemic expression and secretion of numerous antimicrobial peptides (AMPs). Despite being accepted as having several roles, the FBCs have always been considered immobile. However, prior to this study, FBCs role in would repair had not been explored.

Cross-section view of your skin. Can see adipocytes labelled as fat cells.

So, what’d they do?

To explore the potential functions of FBCs during wound healing, the researchers used a laser to make small sterile wounds in the epithelia of Drosophila pupae and then used live imaging to capture the role of FBCs.

What did they see?

Surprisingly, the once considered immobile fat body cells were actually mobile! The FBCs moved towards and accumulated at the wound rapidly, remaining at the wound site until it closed up. The larger the wound size the more FBCs accumulated, and the longer they stayed as the healing process is greater than smaller wounds. For a small wound, a single FBC could plug the wound, up to 5 FBCs would be present at a larger wound site.

Schematic of FBCs migration to a wound.

 

The video shows FBC Migration to varying sized wounds, the FBC response to wounding (first movie) followed by FBC recruitment to small (second), medium (third), and large (fourth) wounds. Epithelial nuclei in red; FBCs in green; wound as dotted circle. Shows an FBC approaching, contacting, and leaving a repairing epithelial wound. Other FBCs appear later and remain patrolling in the vicinity. The second, third, and fourth movies show increasing numbers of FBCs recruited to larger wounds.

But is this just a big fat coincidence?

To determine if their observations were true migration rather than just catching a wave of passive flow of the hemolymph (a fluid equivalent to blood in most invertebrates) to the wound site they tracked individual cells in wounded and non-wounded pupae. This analysis suggested that the FBCs accumulated at the wounds in response to the wound with purpose.

But how are the fat body cells getting to the wound?

Believe it or not, the fat body cells “swim”. 

Looking at the actin cytoskeleton, which provides the cell a basis for movement, they captured with live imaging that fat cells during migration are not attached to an epithelial surface but dwell in the hemolymph and are continually going through actin-based contractile waves pushing the cells forward by means of contracting and relaxing (peristaltic movement). 

Schematic of peristaltic migration.

In the unwounded pupae these waves are happening constantly, but once wounded the waves get directed towards the wound. Inhibition of myosin-ll ultimately prevented the peristaltic movement the migration of FBCs to the wound site. This rules out passive movement by body contractions being the cause of the fat cells accumulating at wound sites, but rather suggestive of an actin-myosin-dependent FBC migration.

Swim, fat body cell, swim

A previously described models of adhesion-independent migration describe a mode that fits the observations made in this study. This model has been illustrated in numerous cell types which states that cells migrate by swimming. Like the FBCs in this study, neutrophils have been demonstrated to “swim” when in a viscous environment, as have lymphocytes, which are known to migrate in suspension by contraction waves. Although, how these swimming cells produce an internal force and how this produces the forward motion observed remains a mystery.

Cool, but do they do once there?

Drosophila hemocytes are equivalent to our macrophages and are drawn to wound sites in a similar fashion to how our immune cells are drawn to wounds.  Previously it has been shown that larval hemocytes work together and communicate with FBCs during bacterial infections.  In this scenario, hemocytes phagocytose (envelop and destroy bacteria and other foreign material) while the FBCs generate antimicrobial peptides (AMPs), proteins which can directly kill bacteria, yeasts, fungi and virus cells. The levels of AMPs is significantly lower when hemocytes are not present.

This research team set out to determine if there is an interaction between FBCs and hemocytes during the wound healing process. To achieve this, they wounded pupae with stained FBCs and hemocytes. Live imaging showed that hemocytes reach the wound first, but as FBCs arrive they sweep the hemocytes out of the way. By genetically altering just the hemocytes to undergo cell death they investigated if the presence of hemocytes is necessary for the recruitment of FBCs. However, the loss of the hemocytes did not change the frequency of FBC recruitment to the wound site, therefore it is not a signal released by the hemocytes that are attracting the FBCs.

However, even when lacking the hemocytes and their phagocytosing function, the FBCs still managed to sweep aside the bulk of the cellular debris and engulf debris at the wound site. Therefore, FBCs have a critical function in wound repair acting to physically clear the wound where hemocytes then phagocytose the majority of debris, and FBCs have a minor role in also phagocytosing the debris.

But wait.. there’s more!

Just when you thought that FBCs couldn’t possibly have more functions, they were also shown to plug the wound to help prevent infection. Light and transition microscopy showed that the FBCs form a ridiculously tight seal between the wound-associated FBCs and the epithelial wound margin, a gap so tiny that even bacteria couldn’t squeeze past.  When threatened with infection, the FBCs secreted an AMP, showing they also act to fight off microbial invaders. To show this labelled E.coli was added to a live image of pupae expressing a reporter of the AMP Atticin. In the non-effected wound, no up-regulation of Atticin was observed, however, in response to infection Atticin is unregulated in FBCs that were plugging the wound and those nearby. Indicating that FBCs can both detect bacteria at the wound site but also act accordingly by delivering AMPs.

Schematic depicting the collaborative clearance of cell debris from wound site by FBCs and hemocytes.

Where to next?

This study has shown astonishing results in non-vertebrates, these results were unexpected as until this point adipocytes were not known to be motile in any organism. The key next step is to determine if mammalian adipose cells are also motile and function in aiding in wound repair and preventing infection. Interestingly, mouse adipocytes have been shown to secrete AMPs, similar to Drosophila FBCs.

Why should you care?

This study also opens doors for more genetic studies to deepen our understanding of adipocytes in tissue repair and regeneration.The communication between adipocytes and immune cells seem to be critical in multiple diseases, such as type 2 diabetes, so further study into this could uncover key insights into these links.

References:

Franz, A., Wood, W. and Martin, P., 2018. Fat body cells are motile and actively migrate to wounds to drive repair and prevent infection. Developmental cell44(4), pp.460-470.

The people turning to stone – and the new discovery that could help them

The skeleton of Harry Eastlack – perhaps the best-known sufferer of Fibrodysplasia ossificans progressiva. Eastlack donated his skeleton to further FOP research following his death in 1973, and it is currently displayed at the Mütter Museum at The College of Physicians of Philadelphia.

Around 1 in 2 million people worldwide suffer from a debilitating condition where their body turns to bone. Fibrodysplasia ossificans progressiva, or FOP, is a condition which causes the soft tissue within the body, such as muscles, ligaments and tendons, to be irreversibly replaced with bone. The name of the condition literally translates to “soft connective tissue progressively turns to bone”, and it is even colloquially known as Stone Man Syndrome, or Human Mannequin Disease.

Current therapies are limited, but a new discovery published in February of this year brings new hope to finding a cure.

 

Life with FOP

It is estimated that around 3,500 individuals suffer from FOP, however only around 800 cases have been confirmed – making it one of the rarest conditions known to medicine. FOP patients are normal at birth, with the exception of malformed big toes which are commonly angled towards the midline of the foot and missing a bone. Individuals usually experience an episode of bone deposition by age 5, which can be brought on by trauma to the soft tissue. Even minor bumps, dental work or injections can induce an event. Simply being careful isn’t enough either as the new bone can be deposited spontaneously with absolutely no cause at all.

Big toe abnormalities characteristic of patients with Fibrodysplasia ossificans progressiva.

FOP patients experience a specific type of bone growth called heterotopic ossification, or HO. Heterotopic refers to something occurring in an abnormal location, and ossification is the process of new bone being laid down. HO usually begins on the spine, ribcage and shoulders, permanently locking these parts of the body in place. Other muscles including the tongue, eye and digestive muscles manage to escape solidification. HO works through a bone production mechanism known as endochondral ossification, whereby a cartilage template is first laid down which the bone progressively replaces.

Ashley Kurpiel was misdiagnosed with a severe case of bone cancer as a child which lead doctors to unnecessarily amputate her right arm before realising five months later that she suffered from FOP.

An FOP ‘flare-up’ is characterised by large, suddenly appearing soft tissue swellings which are often mistaken for cancerous tumours. In one case, doctors misdiagnosed an American woman named Ashley Kurpiel with a severe form of cancer and amputated her right arm, before realising five months later that she suffered from FOP and the amputation was not necessary.

Sometimes, these swellings can regress, but most commonly they are replaced by cartilage and finally bone. The formation of this secondary skeleton causes joints to fuse in place and significantly restricts the mobility of sufferers. Many lose the ability to walk and feed themselves, eventually become wheelchair bound or confined to their bed, and require full time care.

Individuals with FOP usually reach adulthood, though the average life expectancy is only 40 years. The most common cause of death is respiratory failure by virtue of a condition known as thoracic insufficiency syndrome – whereby the bone growths on the thorax fix the ribcage in place and impair the ability to breathe.

FOP is an autosomal dominant condition, meaning that it is genetic and can be passed on from parents to children. An individual with the condition has a 50% chance of passing it on to their children, but even so, FOP can arise spontaneously during development meaning even those with no family history of the disease can be born with it.

 

Is there a cure?

Unfortunately, there is no cure for FOP. Surgery to remove the bone growths only makes the condition worse. Additionally, simple procedures associated with surgery such as intubation or inserting an IV can cause a flare-up. Therapies currently available only treat the symptoms of the disease such as pain and inflammation.

However, there is currently a significant amount of work going in to finding a treatment. Much of this work centres around a drug called Palovarotene which has shown promising results in preventing HO. The drug is in phase 3 of clinical trials, and results are expected to be seen in 2020.

 

So, what causes this devastating condition?

Dr. Eileen Shore and Frederick Kaplan, M. D. are heavily involved in FOP research and headed the team responsible for discovering the FOP gene.

Scientists have been investigating FOP for many years and in 2006, a group of researchers lead by Dr. Eileen Shore and Frederick Kaplan, M.D., finally uncovered its cause: a single-point mutation affecting just one of the 3 billion base pairs that make up the human genome. The mutation is found in chromosome 2, within a gene known as the activin A type I receptor, or ACVR1 for short. ACVR1 is a receptor for the bone morphogenic pathway (BMP), which as the name suggests, plays a significant role in the formation and patterning of the skeleton.

The single-point mutation that is responsible for FOP is a base substitution at position 617 of the ACVR1 gene, simply swapping a guanine base to an adenine base. Consequently, this changes the codon it produces from CAC to CGC. Codons are blueprints for amino acids, and amino acids are the building blocks of proteins. CAC is the recipe for an amino acid known as arginine, while CGC produces histidine. This change alters the structure, and consequently the function, of the protein they build.

When the ACVR1 gene is subjected to this mutation, it causes the receptor that is built from its genetic blueprint to become hyperactive and signal to its downstream targets without the initiation signal it normally requires. Thus, the BMP pathway which causes the development of bone tissue, is turned on when it shouldn’t be.

Further, the area of the gene that the mutation affects is a highly-conserved sequence that is found even in the fruit fly. When a genetic sequence is preserved across such a wide range of species, it can be inferred that the gene has a critical biological function that is necessary to beings of all walks of life. Non-critical genes can tolerate mutations that accumulate over time, and can even contribute to the evolution of new genetic function; while mutations in critical genes rarely even make it to the gene pool as they are fatal to the organisms carrying it, so the gene remains in its unaltered state.

 

The new discovery that could help FOP patients

This image of a mouse embryo from the study shows that the developing skeletal muscle contains the mutant FOP gene. It is linked to a reporter gene called green fluorescent protein, or GFP, which makes it glow as shown.

A new study published in February of this year has uncovered an extra piece of the puzzle and provides new hope for a potential cure.The study’s aim was to discover which cells form the bony depositions in FOP patients. They used cell lineage tracing methods and found that the new bone formed by HO is almost entirely created by cells known as “fibro/adipogenic progenitor cells”, or FAPs.

FAPs are typically involved in tissue repair, though their function evidently becomes erroneous when carrying the FOP mutation. FAPs are proven to be involved in both the spontaneous and injury-induced types of bone deposition that FOP patients experience.

FAPs require a protein complex called Activin A in order to function. In the days following injury, Activin A levels rise to about 9 times their normal levels. This causes a similar explosion in FAP numbers, ACVR1 receptor numbers, and causes the inflammation seen in early FOP flare-ups.

Normally, this inflammation would be followed by normal muscle repair mechanisms. Instead, when FAP cells contain the FOP mutation in the ACVR1 gene, they respond by migrating into the injury area and turning into cartilage, and soon calcifying to become bone only two weeks later.

 

How does this relate to curing FOP?

Inhibiting Activin A prevents new bone deposition. Figure (d) shows a FOP mouse with bone growths indicated by the arrows. Figure (e) shows a FOP mouse treated with an Activin inhibitor – notice the absense of new bone growths.

Most significantly, this study revealed that HO can be prevented by inhibition of these Activin A protein complexes. In the absence of Activin A, FAP cell numbers drop significantly. Without these cells which cause the bone growths, the bone growths of course cannot form. The treatment has been tested in a mouse FOP model with promising results, as shown in the panel to the left. A single dose of an Activin A inhibitor at 2-6 weeks of age protected the mice from new bone deposition until 16 weeks of age – a significant length of time considering the short lifespan of a mouse.

It is the hope that development of a therapeutic Activin A inhibitor may finally be the answer to FOP and bring hope to those diagnosed with this debilitating condition.

 

 

 

 

Read the FAP research article here, or read about the ACVR1 mutation discovery here.

 

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.