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Gene found to play prominent role in central nervous system foundation and function

Researchers at the MRC Harwell Institute have gained new insights into the function of the gene Katnal1. Katnal1 is one of a small family of genes that have been linked with intellectual disability, autism and schizophrenia in humans. In mice, loss of function of the gene leads to poor learning and memory while the growth, migration and shape of neurons in the brain are all disturbed. This research highlights Katnal1 as a prime candidate for further study of the mechanisms underlying diseases of cognitive dysfunction.

We have approximately one billion nerve cells in our brain. These neurons form a complex architecture of networks, which communicate with each other and with other areas of the body through chemical signals. Very early in development, neurons migrate from their birthplace to their final destination in the brain. During this period they develop and form numerous elaborate branches enabling crucial connections to be made with many other neurons. Defects in these processes have been associated with many cognitive disorders.

Image at top shows neurons in normal mice (left) and mutant mice (right). In the mutant mice it can be seen that the neuron branches are shorter and thinner. The gene Katnal1 codes for a protein which determines the shape of microtubule structures within cells. In neurons, microtubules are important for directing neuronal migration and branching. Katnal1 and its family of genes enable the reshaping of microtubule structures at the appropriate time in developing neurons and the termination of branch growth so new ones can be formed.

This gene previously has not been well characterised, although in a small patient study loss of the gene was related to intellectual disability while one rare gene alteration has been linked to schizophrenia.

In this study, mice with a coding sequence error in Katnal1 were identified as part of a large scale genetic study. The error, or mutation, resulted in a non-functional gene – it was essentially ‘switched off’. When the behaviour of the (mutant) mice was compared to normal mice (with the correct gene) a range of behavioural abnormalities were seen including poor learning and memory.

Changes in the brain, detectable only at a microscopic level, seemed to underlie these behavioural disturbances. Analysis of different brain sections showed that the patterns of neurons in the hippocampus (a region of the brain associated with memory) and cortex (the outermost layer of the brain) were different in mutants. The cortex has well defined cell layers so anomalies are easy to spot. More neurons were seen in the outer layers of the cortex in mutants, suggesting that the neurons may have migrated too far.

Furthermore the neurons from mutants had a different shape and fewer synaptic spines – these are the structures on neurons that enable communication with other neurons.

Defects were also seen in the cilia of the mutant mice. Cilia are hair-like protrusions that stick out from all cells and are vital in early development. In the brain cilia are thought to maintain the circulation of chemicals and nutrients in the cerebrospinal fluid – a colourless fluid which maintains a healthy environment for the brain and its neurons. Defective cilia have been linked to many brain disorders including intellectual disability.

Dr Pat Nolan, one of the authors on the paper, commented:

“Our findings highlight the importance of this small group of genes in establishing the neuronal connections that are critical for precise brain functions”.  

Further study of this gene and its role in neuron growth and development may provide insight into the cognitive dysfunction underlying intellectual disability and conditions such as autism. This will increase the likelihood of being able to identify therapeutic targets and potential treatments in the future.

To read the research in Molecular Psychiatry click here

Images show neurons in normal mice (left) and mutant mice (right). In the mutant mice it can be seen that the neuron branches are shorter and thinner. 

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CRISPR/Cas9 Quality Control

Joffrey Mianné and colleagues at the MRC Harwell Institute have published new research in Elsevier Methods outlining their proposed protocols for effectively screening the results of CRISPR/Cas9 gene editing technology. 

CRISPR/Cas9 is a new gene editing technology that has revolutionised research in the field. The technology allows for faster, cheaper, and more precise gene editing than was previously possible. It is increasingly used in the field of mouse genetics to help study the relationship between genes and human disease. It is now the chosen method for the International Mouse Phenotyping Consortium (IMPC), a global project to identify the function of every gene in the mouse genome.

Despite the acceleration of the technology – the results obtained with CRISPR/Cas9 can often be unpredictable. Frequently the genetic change made is not present uniformly throughout the organism (known as mosaicism) and other unwanted changes can be found at the site where the gene has been altered. It is essential that mice taken forwards carry only the desired change so that any physiological changes seen are because of that and not something else on the genome. The ability to correctly select mice with the desired mutation requires robust and accurate methods.

The technology allows many types of genetic changes to be made, including deletions and even swapping the individual molecules making up the code of the DNA. Here the researchers have proposed a framework to analyse the results of CRISPR/Cas9 activity according to the type of genetic alteration intended. They have ascertained that due to the high level of unpredictability in the first generation it is better to definitively characterise the following generation and establish the mutant mouse line from there.

This research will contribute to the current debate on best practice for the use of CRISPR/Cas9 in biomedical research.

How CRISPR/Cas9 works

The CRISPR-Cas9 system is made up of two key molecules – the enzyme Cas9 and a piece of guide RNA. In brief, the guide RNA locates and binds to the target DNA where the change is going to be made. Its sequence is complementary to that of the target DNA. The Cas9 then acts as molecular scissors and makes a cut across both strands of DNA in the double helix. The cell then recognises that the DNA is damaged and attempts to repair it. Scientists have been able to harness the cell’s own DNA repair machinery to introduce changes into the genome. 

PhD opportunities in 2017

 There are four vacancies available at the MRC Harwell Institute for 4 year MRC funded PhD/DPhil Studentships starting in October 2017.

The MRC Harwell Institute is an International Centre for Mouse Genetics at the forefront of studies in mouse functional genomics and mouse models of human disease. We are engaged in lifetime studies – from developmental abnormalities through to diseases of ageing.

There are several research themes offering PhD projects at the MRC Harwell Institute for 2017:

  • Genetic basis of type 2 diabetes
  • Genetics of circadian rhythms and sleep in health and disease
  • The genetics and pathology of deafness
  • The role of cilia in development and disease
  • Disorders of sex development
  • Bioinformatics of mouse models of disease
  • Statistical genomics
  • Investigating Novel Stress Response Pathways in Neurological Disease
  • Novel and bespoke mouse models for dissecting neurodegenerative disease

Click here for more information and details about how to apply.

Closing date Tuesday 7th February

Royal Society hosts tribute to Mary Lyon

         

In tribute to the eminent geneticist Mary Lyon and her role in developing the theory of X chromosome inactivation, a process implicated in disease inheritance, 100 researchers from nine countries attended a scientific meeting hosted by the Royal Society. As part of the event researchers attended a one day meeting at the MRC Harwell Institute where Mary developed her theory. The event brought together worldwide experts in this dynamic field to discuss the latest research advances and reflect on the life and work of Mary Lyon, who first proposed the theory of X chromosome inactivation 55 years ago. ​

The meeting marked the opening of a brand new Mary Lyon exhibition at Harwell. The exhibition includes a timeline of Mary’s life surrounded by panels exploring her education, career at Harwell, and major discoveries. A map of the world pinpoints the many places she visited during her career, bringing to life her global network of researchers.

Mary’s career at Harwell spanned a period of more than 50 years and it was during this time that she made many remarkable discoveries. Mary developed the theory of X chromosome inactivation in 1961 while studying mice with different coloured patches of fur. She hypothesised that one of the two X chromosomes in the cells of female mammals is randomly inactivated during early development so that females don’t have twice the number of X chromosome gene products as males, a potentially toxic double dose. Her hypothesis, now accepted and supported by subsequent research, has had profound implications in understanding the genetic basis of X-linked diseases as well as being one of the first descriptions of epigenetic phenomena.

Mary went to Cambridge in the early 1940s, at a time when women were not official members of the university. Despite taking the same courses as men women were awarded a ‘titular’ degree. In 1998 Mary and other women from her era were officially awarded a full undergraduate degree. WW2 served to change the position of women in the world and had a strong influence on Mary’s career. Much of her research involved looking at the effects of radiation in mice as result of the events of WWII. Most of the important discoveries Mary made were offshoots of studying radiation induced mutations in mice.

 

Speaking at the event Dr Sohaila Rastan, one of Mary’s PhD students commented: “Now 55 years after the hypothesis was first described, Mary Lyon would have found it very gratifying to see how much research it has spawned. Although the X inactivation field has advanced so significantly some basic questions still remain unanswered.

Professor Steve Brown, Director of the MRC Harwell Institute, said: “Mary would have relished the cut and thrust of the scientific discussion at the meeting, and would have joined in the excitement of the many new developments that were reported. She laid the groundwork for all that has followed, and the meeting was a fitting tribute to her scientific legacy.”

11/10/2016

Genes essential for life discovered in mouse embryos ​

Scientists at the MRC Harwell Institute and a team spanning eight different centres across the world have collaborated in an effort to identify and decipher the function of genes that are essential for life. The animal study, published in Nature, is the first of its kind to use high-throughput phenotyping, and provides novel insights into a variety of gene functions, many of which are known to be involved in human diseases.


Gene expression patterns in E12.5 embryos captured after lacZ wholemount staining. Targeted genes are from left to right, top row: Ngfr, Eomes, Adam11, Col9a2; bottom row: Fgf8, Atp1a3, Trpm2, Casz1.

The goal of the International Mouse Phenotyping Consortium (IMPC) is to elucidate the function of every gene in the mouse genome (~20,000 genes). Centres across the world have collaborated to do this by breeding mouse lines where the gene of interest has been inactivated or “knocked out”, this gives us insights into gene function by seeing what happens when the gene is rendered inactive. To date, almost 5,000 new knockout mouse lines have been created by the IMPC. Mice share 85% of their genes with humans. Studying the genes of mice therefore is crucial for helping us to understand human gene function. 

Approximately one third of all mammalian genes are essential for life, mice which have had these genes knocked out are not able to survive beyond an embryo stage or for very long after birth. Many of these genes have not been well characterised. Abnormalities in essential genes have been found to be involved in many human conditions, particularly developmental and rare diseases. Improving our understanding of these genes is therefore vital.

What was done

The IMPC has already developed a phenotyping pipeline for mice that survive to adulthood, this is a range of procedures to identify and quantify the characteristics or “phenotypes” seen when a gene has been knocked out. As mice with essential genes knocked out generally don’t survive to adulthood, the IMPC have developed a pipeline specifically to look at these essential genes, thus allowing us to see what might be going on at specific developmental stages. Features of the pipeline include establishing a window of lethality (working out the time of embryo death) and an analysis of gross morphology to observe phenotypes in freshly dissected embryos.

High resolution, high throughput 3D imaging was also used for the first time in the pipeline. A high throughput system means that images can be analysed quickly and on a large scale, this is important considering the number of genes being looked at (there are around 7,000 essential genes). Imaging techniques are also amenable to automated computational analysis, allowing for the identification of aberrant anatomical phenotypes – which may not have been possible to notice or picked up by manual inspection.

What was found

A strong correlation was found between genes causing lethality in mice and genes causing diseases in humans. Here, scientists found that of 593 essential mouse genes also shared with humans, that 183 of these were associated with human diseases.

Identification of novel phenotypes

The pipeline has enabled novel phenotypes that had not previously been seen to be reported for 86 genes. In all cases the 3D imaging revealed additional phenotypes that may have been missed by gross inspection.

Surprising findings

One of the most surprising findings was that many of the phenotypes seen with essential genes were seen in some embryos and not others. This was unexpected as the mice are almost genetically identical to each other and reared in identical environments. In addition, as these genes have such core functions usually very little variation is seen between organisms when they are disrupted. These findings have opened up a new avenue for further exploration in future studies.

Why is this study important?

The work so far has helped to identify novel human genes that are associated with diseases. This will be crucial going forwards for helping to improve our understanding of the biological mechanisms underlying these disorders. It will reveal unique insights into how things can go wrong at very early stages of development, before an organism is even born, to better understand the process of development. 

Performing these studies on a large scale using standardised and quality controlled procedures helps to ensure accurate and reliable data. All data and knockout mice produced by IMPC are freely available, thus reducing the need for replication and the number of animals used in research.

Age-related disease genes discovered

 In the first ever study of its size, scientists at the MRC Harwell Institute, led by Paul Potter, have conducted a large-scale genetic screen in mice to discover genes involved in
age-related disease. The findings so far have been published in Nature Communications

Advancing age is a risk factor for many diseases. As we get older the risk of getting dementia, diabetes, and cardiovascular disease increases, and we are also more likely to experience other health problems such as age-related hearing loss. As the age of the UK population continues to rise (1 in 3 babies born in the UK in 2013 are expected to celebrate their 100th birthday) it is increasingly important to devise new therapies and approaches to treatments. Our genetic makeup is known to play a significant part in susceptibility to age-related disease – yet very little is known about these underlying genes.

What was done

In order to identify novel genes and biological pathways associated with age-related disease, changes or "mutations" were introduced into the genomes of mice. The mice were then aged and regularly screened throughout their lives to find any effects of the genetic mutations. Phenotypes or characteristics detected after 6 months were identified as late-onset phenotypes and therefore may be related to ageing. Once a phenotype was found whole genome sequencing was carried out to pinpoint the gene responsible.

What has been found so far  

To date, 27 late-onset phenotypes have been identified across a wide disease spectrum. Of these, the responsible defective genes have been found in 12 cases. Already this research has led to some interesting findings. Ageing the mice has revealed phenotypes and genes which would not have been seen otherwise.

Slc4a10 – a novel late-onset hearing loss gene

One example of a novel gene that has been uncovered, and a highlight of the screen so far, is the gene Slc4a10. Late-onset hearing loss was seen in mice which had a mutation in Slc4a10. In humans, very little is known about what causes this type of hearing loss. Impaired hearing was seen in mice at 9 months, it was then further impaired at 12 months, suggesting a progressive late-onset phenotype.

The expression of the Slc4a10 gene was localised to a specific part of the inner ear. On closer examination, it was found that the surface area of the stria vascularis was significantly reduced in mice with the mutation. The stria vascularis is important for maintaining ion concentration in the fluid of the inner ear, and this ionic balance is critical for auditory transduction – the process of turning sound vibrations into electrical signals. This gene had not been previously related to hearing loss in mice or humans and may provide a new insights into how this gene is involved in hearing.

Why is this study important?

The Slc4a10 findings illustrate how a large-scale screen can be used to uncover and characterise novel genes related to ageing. Many other genes have been found and further investigations begun.

The genomes of mice and humans are remarkably similar, sequencing of the mouse genome so far has found that we share 99% of our genes with mice. This study is a vital springboard for a better understanding of the genes in humans which may be involved in these diseases. It will enable new and more accurate preclinical animal models of late-onset human disease to be developed, which more closely resemble diseases in human patients. Several of the genes identified in this programme are now being studied in humans.

This study has also prompted a late-onset screen to be done by the International Mouse Phenotyping Consortium (IMPC). The IMPC aims to remove (knock out) every single gene in the mouse genome and phenotype the mice to produce a comprehensive catalogue of gene function.

Professor Steve Brown, Director of the MRC Harwell Institute, commented: “For the first time, we have been able to use the mouse to shed light on the diverse set of genes involved with late-onset disease in the human population. The work demonstrates that there is much that we don’t know about the genetic basis of late-onset disease, but the models that we have generated and the genes that we have identified are providing a powerful insight into disease mechanisms that will ultimately improve the prospects for new therapeutic interventions.”

This story has also been reported on by the MRC

Diabetes gene mechanism discovered

Dr Roger Cox and colleagues at MRC Harwell have uncovered a new mechanism for how the diabetes gene SOX4 may be working, revealing a potential new therapeutic target for diabetes therapy.

Normally, after you have eaten a meal or sugary food the levels of sugar or glucose in your blood increase, this stimulates release of the hormone insulin which allows cells in the body to absorb this glucose and use it for energy, or store it for future use. Type 2 diabetes can occur when there is reduced insulin secretion in response to these increased glucose levels. Type 2 diabetes typically affects older people, but it is increasingly becoming common in younger people and has been associated with obesity.

Large scale genomic studies looking for common gene variations across lots of people have helped to identify many regions in our genome which may be involved in increasing the risk of type 2 diabetes, one of the genes in these regions is SOX4. In new research published in Diabetes, scientists at MRC Harwell and the Oxford Centre for Diabetes, Endocrinology & Metabolism have revealed a potential molecular mechanism underlying one aspect of how SOX4 may be involved in the pathology of type 2 diabetes.

How insulin is normally released from cells

Ordinarily, insulin and other materials that need to be transported out of the cell are packaged into ‘granules’. When the granule makes contact with the outer surface of the cell, the two fuse together and a fusion gap or pore forms allowing the contents of the granule to exit the cell – this process is known as exocytosis. For effective ‘full fusion’ exocytosis the pore initially opens, rapidly expands, then after a short delay collapses after the cargo has been released. Sometimes this process does not work properly and the pore expansion is halted during the initial opening, it may then eventually close, this is known as ‘kiss-and-run’ exocytosis.

Investigating Sox4 in mice

The SOX4 gene codes for a transcription factor, a protein that regulates whether other genes are activated or not. Scientists compared exocytosis in mice which had the typical or ‘wild type’ gene with mice that had a mutation in the gene, an incorrect version. Cells with wild type Sox4 followed the typical pattern suggestive of full fusion exocytosis taking place. In comparison, in cells with mutant Sox4 the pattern suggested kiss-and-run exocytosis, in other words that fusion pore expansion was impaired.  

Scientists then carried out a gene expression microarray to see what genes Sox4 is involved in regulating, most notably the gene Stxbp6, which has been previously linked to faulty fusion pore expansion in other cells. Analysis in rat cells found that in both mutant and wild type Sox4 cells there was also increased expression of Stxbp6, but that the effect was stronger in the mutant.

Investigating SOX4 in humans

Scientists then extended these findings to human cells. There was higher SOX4 expression in cells from donors who had type 2 diabetes.  

Why are these findings important?

These findings together suggest that increased SOX4 expression leading to increased STBP6 expression may be causing impaired expansion of the fusion pore, and consequently be involved in reduced insulin secretion in type 2 diabetes. Uncovering this mechanism paves the way for new therapeutic targets to be explored, for example to promote full fusion and release of insulin. 

MRC Festival 2016

As part of the MRC’s inaugural Festival of Medical Research, both of our units at MRC Harwell opened their doors to the general public on Saturday 18th June for a ‘Your genes and you’ open day.

“I have enjoyed expanding my knowledge on genes and getting involved with hands on activities” – just one of the comments we received at our first MRC Festival. The rain just about held off for a jam packed day of activities and tours. Members of staff from all areas of our institution got involved on the day and ran activities. Visitors had the opportunity to use complex microscopes, extract DNA from a strawberry, watch a laser in action, learn about the latest research at MRC Harwell, and much more!  

 

 

The Mary Lyon Centre (MLC) ran tours of its world-class animal facility throughout the day giving visitors a rare chance to see how an animal facility is run. MLC staff also showcased a range of phenotyping equipment to visitors and talked to them in more detail about mouse welfare.

 

Visitors were able to learn about the techniques and procedures used to analyse genes and proteins during tours of our working laboratories, as well as speak to scientists in our histology and clinical chemistry laboratories. 

Our colleagues from the Research Complex at Harwell (RCaH) joined us with some very sophisticated equipment, including a 3D printer. Visitors had a go at protein crystallisation and mapping the positions of molecules in a cell using super-resolution.

We were lucky to have some VIP guests attend, including the Mayor and Deputy Mayor of Didcot Steve Connel and Jackie Billington and the Didcot and Wantage MP Ed Vaizey. It was fantastic to have them attend and engage with them about the important research we are doing at MRC Harwell. In addition, Agnes Jawara from the organisation Understanding Animal Research attended, Agnes interviewed people before and after their tour of the MLC to see what their opinions of animal research were. 

 

Watch this video to see some of the action from the day! 

Cross word answers for lab book: 

Scientists shed new light on organ positioning in early embryos

An international team of scientists from the UK, the USA, and Japan have revealed new aspects of how developing embryos establish a left and a right hand side. 

While we are externally mirror symmetrical between left and right, our internal organs are asymmetrically positioned and patterned (the heart lies towards the left and the liver towards the right side of the body). This asymmetry is established between 19 and 22 days of development in humans, often before the mother even knows that she is pregnant. If this process goes wrong, it can lead to birth defects and is particularly associated with congenital heart disease. New research, published in the journal PLoS Genetics provides insight into how this process happens and the ways in which it can go wrong.

The earliest known event in mammalian left-right (L-R) patterning, surprisingly, is not an asymmetry in where a gene functions, but a physical flow of fluid (from right to left) within a short-lived pit in the embryo. This tear-shaped pit is known as the node. The direction of this ‘nodal flow’ determines which side of the embryo will develop as the left. This leftward 'nodal flow' is driven by the action of motile cilia, hair-like structures protruding from the cell surface within the node. Exactly how nodal flow is detected by the embryo remains unclear: we know that the cells directly surrounding the node (the crown cells) are required to detect nodal flow; that the crown cells must each have an immotile cilium and that they must contain the putative calcium channel protein Polycystin-2 (PKD2). Previously the research team implicated a second Polycystin protein, PKD1L1, in this pathway.

Describing these new findings, Dominic Norris, Programme Leader here at MRC Harwell explains, “Firstly, we have been able to define a genetic pathway in which each element represses the next. This controls the early steps of L-R patterning: ‘nodal flow’ represses the gene Pkd1l1, which in turn represses Pkd2; this represses Cerl2 which encodes a known repressor of NODAL signalling (an important pathway in development of the embryo). Secondly, we demonstrate that PKD1L1 (the protein which is encoded by the Pkd1l1 gene) can mediate a response to flow.  Finally, we have shown that a portion of the structure of PKD1L1 that sits outside of the cell is critical both for detecting flow and for proper L-R patterning. Together, these exciting findings reveal a genetic pathway operating at the level of flow sensation and demonstrate that PKD1L1 is able to act to elicit flow-induced chemical signals, thereby supporting the ‘mechanosensation model’ of nodal flow sensation (i.e. that the force of fluid flow itself can be directly detected by the node crown cells during the establishment of L-R asymmetry)”.

This research has emerged from an international collaboration that has made it possible to combine genetics, biophysics, and structural biology. In the UK, Rohannah Hussain and colleagues at Diamond Light Source, the UK synchrotron science facility, made it possible to understand the nature of structural changes caused by a point mutation in PKD1L1. This work took place on Diamond’s Circular Dichroism beamline, B23Surya Nauli and colleagues at Chapman University, USA, allowed the role of PKD1L1 in flow detection to be assessed in cells. Hiroshi Hamada and colleagues in Osaka, Japan, provided novel techniques that allowed the team to more precisely analyse ‘nodal flow’.

This work provides a greater understanding of how genes interact with a physical flow of fluid to reproducibly establish a left and right hand side in the developing embryo. This knowledge underpins our ability to understand how the process of L-R patterning can go wrong, leading to congenital diseases, including congenital heart disease.

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