Study Uncovers 198 Genes Associated With Brain Morphogenesis in Mice

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By IMPC

Published 4th October 2019

With a contribution from the IMPC, recent research, published in Nature Communications, has identified 198 genes associated with brain morphogenesis in mice – 83% of these constitute genes newly implicated in brain architecture.

Brain development and morphogenesis is critical to higher-order cognition, but our knowledge of its biological basis is at best incomplete, and at worst, severely lacking. Previous studies have demonstrated that there is a significant genetic influence on brain morphology. However knowledge of which genes influence brain morphology is limited, and this presents an important problem in developmental biology. By identifying 198 genes that are associated with mouse brain morphogenesis the study provides a complementary resource to human genetic studies and predicts that many more genes could be involved in mammalian brain morphogenesis.

Binnaz Yalcin, corresponding author of the study says “This study aims at understanding the genetics of brain anatomy in the mouse. It provides a wealth of novel knowledge about which genes control the size and the shape of the brain, and a foundation on which neurobiologists can build to further study how exactly these genes control the brain anatomy”.

“I have no doubt that this resource will help medical geneticists working on ultra-rare human neurodevelopmental disorders

The researchers obtained brain samples of 1566 mutant mouse lines from the Sanger Institute Mouse Genetics Project, a partner of the IMPC. Using a histological pipeline, 118 brain morphological parameters were analysed, covering brain size, commissures, ventricles, cortex, subcortex and cerebellum. To detect neuroanatomical phenotypes (NAPs), the researchers used PhenStat, a statistical method developed by the IMPC for the identification of abnormal phenotypes. 198 genes associated with neuroanatomical phenotypes (NAP genes) were subsequently identified. The vast majority of these genes (94%) have never previously been associated with brain anatomy in mice.

Interestingly, unique human orthologs were identified for 173 of the identified mouse NAP genes. Whilst 17% of human unique orthologs of mouse NAP genes are known loci for cognitive dysfunction, 83% constitute a vast number of genes that are newly implicated in brain architecture. This dataset may therefore help in improving clinical interpretation.

Yalcin states “I have no doubt that this resource will help medical geneticists working on ultra-rare human neurodevelopmental disorders who sometimes struggle to determine the genetic mutation responsible for the underlying disease, when for example there is only one patient world-wide. So when a mutation is found in a patient’s genome and that patient exhibits the same phenotype than the mouse, a molecular diagnosis can finally be made.”

This study would not have been possible without the IMPC

The identified NAP genes converge into a small number of groups of functionally similar genes participating in shared cellular pathways. Disruption of genes within the same module can yield a similar pattern of neuroanatomical abnormalities, revealing interesting neurodevelopmental pathway/phenotype relationships. For example, the study indicates that mechanisms confined to sub-cellular compartments as subtle as dendritic spines can translate into major anatomical features.

The study represents the largest atlas of the link between genetic mutation and its associated neuroanatomical features yet, and contributes a wealth of new knowledge on the genetics of brain morphogenesis. The authors were keen to note the contribution of the IMPC towards their work, with Yalcin commenting that “This study would not have been possible without the IMPC and is a tribute to the remarkable work of the people involved in this consortium.”

The IMPC is aiming to design and produce a genome-wide mouse strain resource of human disease-associated coding variants associated with rare disease that can be used for validation of putative functional variants and insight into disease mechanism(s). To find out more, click here.


Original publication: Large-scale neuroanatomical study uncovers 198 gene associations in mouse brain morphogenesis

Members of the IMPC Consortium at CNR-Monterotondo (Italy) have used embryonic stem (ES) cells produced as part of the IMPC project, to engineer a Ccdc151 ‘knockout’ mouse model that is valuable for the study of human ciliopathies, gaining significant insight into this set of rare conditions.

Primary ciliary dyskinesia (PCD) is a rare, genetic disorder that results in chronic respiratory tract infections, abnormally orientated internal organs and infertility. The root cause of these symptoms are dysfunctional cilia and flagella, which if functioning correctly, are finger-like projections on the surface of cells that act to clear mucus and debris by coordinated beating. In addition, normal function of cilia is required for migration of egg and sperm cells.

Ccdc151 is a gene that is known to be associated with PCD, and here, researchers have engineered a mouse model, using embryonic stem cells from the IMPC, in which the gene is deleted. Common features of PCD in human patients such as left-right body asymmetry and dysfunctional spermatogenesis were detected in these Ccdc151-knockout mice. “The Ccdc151-knockout mouse model faithfully recapitulates several features of human PCD disease” remark Francesco Chiani and Tiziana Orsini, co-first authors of the paper. “The availability of this animal model will allow researchers to further dissect the mechanisms by which pathological conditions develop in different organs”.

This animal model will be useful for studying mechanisms underlying hydrocephalus, a condition whose treatment has not changed for decades.

The researchers also observed that Ccdc151-knockouts develop severe hydrocephalus – a condition in which cerebrospinal fluid accumulates in the brain, causing increased pressure inside the skull and potentially leading to brain damage and other complications. This is the first example of hydrocephalus caused by loss of function in the Ccdc151 gene. Although hydrocephalus is rarely seen in human patients with PCD, having a mouse model that exhibits all of the features of hydrocephalus could be useful for researchers. “This animal model will be useful for studying mechanisms underlying hydrocephalus, a condition whose treatment has not changed for decades”, said the lead authors. It is hoped that further studies may help to uncover other genes that interact with Ccdc151 that lead to the development of hydrocephalus.

this micro-CT imaging methodology could be applied to facilitate studies on gene expression directly in the intact brain

The researchers made extensive use of X-ray micro-CT 3D imaging to study the hydrocephalic brains of Ccdc151 knockout mice – a technique that uses X-rays to create a virtual 3D model of a target object. Additionally, a novel micro-CT method was used to study expression of the Ccdc151 gene in the brain. This novel method is based on the generation of a molecular signal within the mouse brain. The authors remarked that “this micro-CT imaging methodology could be applied to facilitate studies on gene expression directly in the intact brain carrying the lacZ reporter gene, which is widely used as a reporter gene in mouse models.”

The link between Ccdc151 and ciliary function is now clear, but the mechanism by which the Ccdc151 protein contributes is less so. “The precise mechanism by which Ccdc151 protein accomplishes its function is unknown and will be addressed in future research”, says Chiani. The Ccdc151-knockout mouse model, generated via IMPC resources, could be “instrumental to dissect the role of the motile cilia in diverse physiological processes during development, adult life and aging”.

In addition to providing biological resources that can contribute to research such as this, the IMPC is curating a catalogue of mammalian gene function, with phenotyping data for knockout mouse models such as Ccdc151.

Research paper: Functional loss of Ccdc151 leads to hydrocephalus in a mouse model of primary ciliary dyskinesia

Quotes taken from interview with Disease Models & Mechanism: First person – Francesco Chiani and Tiziana Orsini

In collaboration with NC3Rs, IMPC member institution MRC Harwell has launched a new citizen science project that aims to advance medical research and mouse welfare.

The mouse is a vital model organism as we seek to understand the function of genetic variation. The analysis of genetic variants in the mouse has provided crucially important insights for the biomedical and clinical sciences through hypothesis-driven discovery research. When researchers make changes to the genetic make up of mice, they can subsequently observe them to deduce the effect of the introduced changes. Mice are sociable animals and are housed in groups, however, in order for scientists to observe them, they may previously have been removed from their cages and into an unfamiliar environment.

Scientists at MRC Harwell have worked together with Actual Analytics in order to develop the Home Cage Analysis system (HCA), which has the potential to change the way that mice are studied, improve their welfare and drastically change the way that we collect data from mouse models by allowing the public to contribute.

Home Cage Analysis System

The challenge proposed by the NC3Rs CRACK IT initiative was to ‘develop an automated, minimally-invasive or non-surgical system to assess activity, behaviour and interaction of at least two mice in the cages and setting the animals were reared in’. The Home Cage Analysis System was the result. The system is able to track the movement of three individual mice without removing them from their social group, only requiring the minimally invasive insertion of a microchip. Through high grade video recording, it is now possible to observe the activity of mice 24/7.

How Will This Be Of Benefit?

Not only do these developments have the potential to improve the welfare of mice involved in research, but it also has exciting implications for the science itself. It will now be possible to collect more data on mouse behaviour, potentially providing us with vital information on the early stage of diseases. Additionally, the more data that is collected, the greater the statistical power of testing. It is also important not to forget that mice are naturally nocturnal, and the Home Cage Analysis system will allow information to be captured on mouse behaviour at night, a time when previously this important information would have been missed. Perhaps most excitingly, the Rodent Little Brother project allows the public to get involved and to supporting cutting-edge research on mouse models.

What is the Role of the Public?

The rise of machine learning is very exciting, and could have lots of positive implications in research. The end goal of this citizen science project is to have a computer algorithm to track and annotate mouse behaviour for us. However, this algorithm first needs to learn what different mouse behaviours are. By allowing people to manually annotate these mouse behaviours, we will be able to feed the algorithm enough information to be able to collect data 24/7 in the future!

Overall, it is hoped that this project will advance understanding of how genes cause disease and aid the development of new therapies, all whilst improving mouse welfare.

To get involved in the project, visit the Rodent Little Brother Home Page.

To read about mouse welfare within the IMPC, click here.

CLOVES syndrome is a rare condition that is characterised by tissue overgrowth and vascular abnormalities, caused by mosaic gain-of-function mutations in the PIK3CA gene. The way in which CLOVES syndrome manifests itself is highly variable but common features include fatty overgrowths, vascular anomalies, kidney problems and spinal-related symptoms. The condition has no specific treatment and a low survival rate. It is one of a number of conditions that can be grouped under the umbrella of PIK3CA-related overgrowth syndromes (PROS).

By initially undertaking research on a mouse model, and subsequently with human patients, it has been shown by Dr Guillaume Canaud and his team at the Necker-Enfants Malades Hospital in Paris, that BYL719 (an inhibitor of PIK3CA, currently undergoing clinical trials for treating PIK3CA dependent tumours) can prevent and improve organ dysfunction and can improve disease symptoms in patients suffering from CLOVES syndrome.

A mouse model of CLOVES syndrome

The researchers generated mice that express a PIK3CA transgene upon the administration of tamoxifen, mimicking the activity of human CLOVES syndrome. These mice showed similar symptoms to human sufferers of CLOVES, with MRI revealing scoliosis, kidney cysts and muscle abnormalities. Subsequent histological examination revealed further organ abnormalities including additional kidney problems, and abnormalities in the liver and spleen. Furthermore, a high level of cell proliferation was observed in all of the affected organs.

Rapamycin or BYL719?

Rapamycin has previously shown evidence of improving vascular malformations, and when tested on the mouse model of CLOVES syndrome it improved survival rate. However, it did not improve organ abnormalities and did not significantly reduce tumour growth. In contrast, mice treated with BYL719 were found to have preserved tissues and normal vessels. Importantly, BYL719 administration strongly reduced cell proliferation in all affected organs. Withdrawal of BYL719 led to the recurrence of tumours within four weeks, suggesting that continuous administration of BYL719 could relieve the symptoms of CLOVES syndrome.

BYL719 leads to huge improvements in patients with CLOVES syndrome

BYL719 was initially administered to two patients suffering with CLOVES syndrome, who both, after being treated with BYL719, showed dramatic and rapid improvement in their condition. There was a major reduction of vascular tumour abnormalities and overgrowths in addition to improved renal function and a significantly increased quality of life in both patients. The only observed side-effect was hyperglycaemia, which was able to be controlled by a controlled diet.

On the basis of these initial results, Canaud and his team were given permission to treat 17 additional patients with CLOVES syndrome by administering BYL719. The 14 children and 3 adults all showed substantial clinical improvement. A reduction in size of vascular tumours was observed in all of the patients, as well as a drastic reduction in metabolic activity of affected areas. In addition to an improvement to skin capillary abnormalities and scoliosis, all patients reported decreased tiredness. The growth of the children was not affected during the 6 months of treatment and the only side-effects seen were discrete mouth ulcerations in 3 patients (that ultimately disappeared spontaneously) and the aforementioned hyperglycaemia.

The IMPC is aiming to design and produce a genome-wide mouse strain resource of human disease-associated coding variants associated with rare disease that can be used for validation of putative functional variants and insight into disease mechanism(s). To find out more, click here


Research paper: Targeted therapy in patients with PIK3CA-related overgrowth syndrome

Research published in Nature identifies SET domain protein 3 (SETD3) as a physiological actin methyltransferase, and uncovers SETD3’s crucial role in the regulation of smooth muscle contractility and its link to primary dystocia in mammals.

For many years it has been known that actin, essential for a large number of cellular processes such as cell motility and the regulation of DNA transcription, is methylated at the amino acid histidine 73 (His73). His73 methylation is found in several model organisms, but its function for many years had remained unclear. After identifying SETD3 as the methylator of actin His73, researchers sought to discover the purpose of actin His73 methylation, and with the help of some mice, they were successful.

Identifying the function of SETD3

To identify the enzymatic function of SETD3, the researchers performed in vitro methylation assays, which showed that the only potential substrate methylated by SETD3 was β-actin. The researchers were then able to identify the exact location of methylation on actin by SETD3 using tandem mass spectrometry. This turned out to be His73. In order to analyse the catalytic specificity of SETD3, the scientists compared methylation events in human cells both with and without the presence of SETD3. Of the 180 histidine methylated peptides, actin-His73 methylation was the only modification that was altered in the absence of SETD3 – identifying actin-His73 methylation as the primary physiological function of SETD3.

Studying SETD3 deficient mice

To confirm the physiological function of SETD3 in vivo, the researchers obtained mice with one copy of their Setd3 gene knocked out (Setd3+/-) from the Canadian Mouse Mutant Repository. The mouse strain was made at the Toronto Centre for Phenogenomics, an IMPC member institution, using embryonic stem cells. From this strain, it was possible to generate Setd3 null homozygote mice (Setd3-/-) i.e. mice with both of their copies of Setd3 knocked out. The methylation of actin wasn’t detected in any tissues obtained from SETD3-deficient mice, however, in tissues that expressed SETD3, the majority of actin was methylated – confirming the role of SETD3 as the actin His73 methyltransferase.

Actin methylation regulates actin polymerisation

Previous studies suggest that the methylation of His73 influences actin polymerization dynamics, and here the researchers observed that methylation promoted actin polymerization kinetics in vitro. To explore this idea further, they obtained mouse embryonic fibroblasts that were positive for actin-His73me and compared them with fibroblasts from Setd3-/- mice that lacked methylation. Cells containing methylated actin were more efficient at migrating than cells without methylated actin – consistent with the idea that methylation of actin by SETD3 positively regulates the polymerisation of actin.

Uterine cell contraction and primary dystocia

Mice without functioning SETD3 protein are able to survive, despite the cell migration defect observed in their embryonic fibroblasts. The researchers were able to infer therefore, that actin-His73me must have a specialised role that isn’t a necessity for survival. IMPC data for Setd3 identifies several phenotypes associated with Setd3 knockout mice including short tibia, decreased body length and decreased lean body mass. In addition to this, the researchers noticed that litter sizes in female Setd3-/- mice were significantly smaller than expected. After mating Setd3-/- females with wild type males, dystocia, normally a rare phenotype, was noted in 8 out of 9 Setd3-/- mice. The lack of obvious pelvic abnormalities in the Setd3-/- females mean that the cause of dystocia is most likely genetic. Unlike with wild type mice, early labour could not be induced in Setd3-/- mice with oxytocin, suggesting a specific requirement for SETD3 in the contraction of the uterus during labour, a process which relies upon correctly functioning actin. This led to the proposal that actin-His73me is linked to uterine cell contraction in the primary dystocia of SETD3 deficient mice.


More IMPC related research: Research Reveals Novel Genetic Influences On Osteoporosis


Research paper covered in this article: SETD3 is an actin histidine methyltransferase that prevents primary dystocia

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