DATA RELEASE HIGHLIGHTS – JUNE 2018

Following today’s data release, the DMDD website now contains detailed phenotype data for nearly 700 embryos from 82 different knockout mouse lines. Highlights include the identification of limb defects and cysts in Col4a2 knockouts and replication of the major features of Meckel syndrome in B9d2 knockouts.

We have begun to add immunohistochemistry image data for the brain and spinal cord of some embryos at E18.5. These images give further information about lines in which the embryos appeared morphologically normal at E14.5, but were still not viable. We have also added viability data for every line at both E9.5 and E14.5.

Together with the placental phenotype data that we hold for more than 100 knockout lines, the DMDD website is a rich resource for those investigating the effect of gene mutations on embryo development, and may provide clues about the genetic basis of rare diseases.


LIMB DEFECTS SEEN IN Col4a2 KNOCKOUTS

In humans, COL4A2 mutations have been linked to porencephaly, a rare disorder with phenotypes that include the development of intracranial cysts. In the latest DMDD data, Col4a2 knockouts have a variety of nervous system disorders in line with porencephaly. However, all four embryos also show abnormal autopod morphology and cysts between the nasal septum and the oral cavity, as well as other morphological defects.

 

A Col4a2 knockout embryo has a cyst between the nasal septum and oral cavity (left) and abnormal autopod morphology (right). The individual fingers don’t diverge distally and can’t be discerned from an external view.

 


B9d2 KNOCKOUTS MODEL MECKEL SYNDROME

In humans, mutations of the gene B9D2 have been linked to Meckel syndrome, a severe disorder caused by dysfunction of the primary cilia during the early stages of embryogenesis. Meckel syndrome is characterised by multiple kidney cysts, occipital encephalocele (where a portion of the brain protrudes through an opening in the skull) and polydactyly, but it also commonly affects the brain and spinal cord, eyes, heart, lungs and bones.

B9d2 knockout mouse embryos included in our latest data release show the major features of Meckel syndrome, including polydactyly and defects in the brain, peripheral nervous system, heart and vascular system. They also display situs defects, where the left-right asymmetry of the body did not develop as expected. The image below shows a B9d2 knockout embryo with left pulmonary isomerism and symmetric branching of the principle bronchi from the trachea.

 

A B9d2 embryo showing situs defects (left). A magnified view (right) shows that both lungs have developed with a single-lobe structure. In mice the left lung usually has one lobe, while the right lung has four. In addition, the principle bronchi (red arrows) have branched symmetrically from the trachea. This branching would normally have a distinct asymmetry.

 


NEURAL IMAGE DATA NOW AVAILABLE

In around 20% of embryonic lethal lines, embryos appear morphologically normal at E14.5 but still go on to die before or shortly after birth. To understand more about why these embryos were not viable, DMDD colleagues Professor Corinne Houart and Dr Ihssane Bouybayoune at Kings College London analysed the lines at E18.5 – when embryo development is almost complete. They used immunohistochemistry to identify abnormalities in the brain and spinal cord that could not be picked up in our standard, whole-embryo morphological analyses. This data is now available for the line Trappc9, and additional lines will be added in future data releases.

 

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Immunohistochemistry analysis of the brains of two Trappc9 knockout mice. The calretinin (green) + neurofilament (red) combined stain highlights interneurons and axons, while Hoechst (blue) is a nuclear stain.

 

Neural images are available as 20-micron sections through the brain and spinal cord, and the images from different embryos can be compared side by side using the stack viewer. A separate Nissl stain was used to highlight neural death and these images can also be explored online.


 

A FULL LIST OF NEW DATA IN THE LATEST RELEASE

NEW PHENOTYPE DATA FOR EMBRYOS AND PLACENTAS

Today, DMDD has released many new images and phenotypes for embryos and placentas from embryonic lethal knockout mouse lines. We now hold data on 70 mutant lines that have been phenotyped in detail using the Mammalian Phenotype ontology. The resulting data is freely available to the scientific community and is a potential goldmine of information about the genetic basis of developmental disorders.

The new data is accompanied by several exciting updates to our website. These include the ability to search for phenotypes by anatomy terms and the release of additional data about gene knockouts that are lethal very early in embryonic development. Highlights of the release, including examples of interesting phenotypes, can be found below.

 

SEARCH FOR PHENOTYPES USING ANATOMY TERMS

Following a major update to our search tool, users of the DMDD database can now search for phenotypes by anatomy term. This new functionality is designed to help researchers of specific organ or tissue types to quickly identify all phenotypes that are relevant to their studies. Choose from embryo and adult anatomy terms for both humans and mice.

 

Image of the DMDD search box
New search functunality gives users the option to search for phenotypes by anatomy term.

 

FIND GENE KNOCKOUTS THAT ARE LETHAL BEFORE E9.5

Around a third of the knockout lines studied by the DMDD programme have been found to cause lethality before 9.5 days of gestation. Although it is not possible for us to image and phenotype embryos from these lines, we have added them to our database and they can be found using the ‘search’ tool.

For a full list of lines that are lethal before E9.5, visit our Early lethals page.

 

EXPLORE THE NEW EMBRYO PHENOTYPE DATA

In our latest release we’ve made phenotypes available for 7 new knockout lines. These include Cfap53, which is known to be involved in left-right asymmetric patterning in humans. In mouse embryos we identified the phenotype ‘abdominal situs ambiguus’, in which the abdominal organs have neither the usual nor the mirror-image arrangement.

We have also released data on Fut8. This gene is linked to Leukocyte Adhesion Deficiency, a syndrome with symptoms including microcephaly and abnormality of the tongue and palate. In the mouse we identified various phenotypes related to the hypoglossal nerve, which controls movements of the tongue.

Some further highlights from the phenotypes released today include spinal haemorrhage in a Fut8 knockout embryo, a perimembraneous ventricular septal defect in an Arid1b knockout embryo and abnormal lens epithelium morphology in an Actn4 knockout embryo.

 

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A Fut8 knockout embryo found to have a spinal haemorrhage.

 

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Abnormal lens epithelium morphology in an Actn4 embryo. Lens epithelial cells are the parental cells responsible for growth and development of the lens.

 

 

In total, 162 distinct phenotypes were identified across 91 new mutant and wild-type control embryos. Phenotype data for a total of 81 new placentas has also been released.

 

A FULL LIST OF THE NEW DATA

HREM embryo image stacks added for Dennd4c, Dnajc8 and Pigf.

Embryo phenotypes added for Actn4, Arid1b, Cfap53, Cyp11a1, Dmxl2, Fut8 and Mfsd7c.

Placenta images and phenotypes added for B9d2, Cbx6, Commd10, Coq4, Dcx, Dhx35, Fam160a1, Gpatch1, Mfsd7c, Oaz1 and Smg1.

 

All image and phenotype data from the DMDD programme can be accessed at dmdd.org.uk. For assistance, please email contact@dmdd.org.uk.

NEW EMBRYO PHENOTYPE DATA AVAILABLE

New image and phenotype data for embryos and placentas from embryonic lethal knockout mouse lines has been made available on the DMDD website today. The knockout data includes the ciliary gene Rpgrip1l as well as Atg16l1, a gene encoding a protein that forms part of a larger complex needed for autophagy. In total we have added HREM image data for 10 new lines, embryo phenotypes for 11 lines and placenta image and phenotype data for 6 lines.

The new data was released at the same time as enhancements to our website, which have been described in a separate blog post. Keep reading to see some highlights from the phenotype data.


DETAILED EMBRYO PHENOTYPES REVEALED

The comprehensive and detailed nature of DMDD embryo phenotyping means that we are able to identify a wide range of abnormalities. In the data released today, a total of 423 phenotypes were scored across 78 embryos. These included gross morphological defects such as exencephaly and edema, but also abormalities on a much smaller scale such as an unusually small dorsal root ganglion, absent hypoglossal nerve and narrowing of the semicircular ear canal.

In the image below, a Trim45 embryo at E14.5, was found to have abnormal optic cup morphology and aphakia (a missing lens).

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HREM imaging of a Trim45 knockout embryo reveals abnormal optic cup morphology and aphakia on the left side.

3D modelling of the exterior of an Rpgrip1l knockout embryo at E14.5 revealed a cleft upper lip, as well as polydactyly.

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A 3D HREM model of an Rpgrip1l embryo shows a cleft upper lip.

All phenotypes are searchable on the DMDD website, highlighted on relevant images, and the full-resolution image data is available to explore online.


SYSTEMATIC PLACENTAL ANALYSIS

DMDD also carries out systematic phenotyping of the placentas from knockout lines. The image below shows a Cfap53 knockout placenta at E14.5, which was found to have an aberrant fibrotic lesion. The density of fetal blood vessels was also considerably reduced, the overall effect being to reduce the nutrient flow from mother to embryo.

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Placental histology for the line Cfap53 shows a fibrotic lesion (large arrow) and several regions of reduced blood vessel density (small arrows).

 


GENE EXPRESSION PROFILES

Work is underway to measure the gene expression profiles for embryos from embryonic lethal knockout lines, a study that complements the morphological phenotype data we are gathering. One of our ultimate goals is to allow data users to explore correlations between gene, morphological phenotype and gene expression profile. The first part of this dataset was released recently – a temporal baseline gene expression profile for wild type embryos.

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Example expression profiles of Nacad and Pdzk1 with increasing somite number. The data shows that, at this depth of sequencing, Nacad is switched on during somitogenesis and Pdzk1 is switched off.

 

The expression data is now accessible via a dedicated wild type gene expression profiling page on the DMDD website, which also gives background information about the analysis. Mutant expression data will follow in the new year.


LINKS BETWEEN DMDD GENES AND HUMAN DISEASE

Many of the genes studied by the DMDD programme are known to have links to human disease, including several new lines that have been made available in this release.

Rpgripl1: in humans, mutations in RPGRIPL1 are known to cause Joubert Syndrome (type 7) and Meckel Syndrome (type 5), a rare disorder affecting the cerebellum.

Cfap53: the human ortholog of this gene is known to be associated with visceral heterotaxy-6, in which organs have an abnormal placement and/or orientation.

Arhgef7: in humans the ortholog is associated with Borjeson-Forssman-Lehmann Syndrome.

Arid1b: in humans, mutations in ARID1B are associated with Coffin-Siris Syndrome.

Embryonic lethal lines with no known links to human disease may also be novel candidate genes for undiagnosed genetic disorders. Visit the DMDD website to explore the phenotype data.


A FULL LIST OF NEW DATA

HREM embryo image data has been added for Actn4, Arid1b, Cfap53, Crim1, Cyp11a1, Dmxl2, Fut8, Gas2l2, Mfsd7c, Rala.

Embryo phenotype data has been added for Atg16l1, Capza2, Coro1c, Crim1, Cyfip2Gas2l2, Gm5544Rala, Rpgrip1l, Syt1, Trim45.

Placenta image and phenotype data has been added for Arhgef7, Arid1b, Fam21, Fut8, Med23, Timmdc1.

If you have questions about the DMDD programme or our data, please email contact@dmdd.org.uk.

DATA RELEASE – AUGUST 2016

New embryonic-lethal knockout mouse lines are now available on the DMDD database.

If you haven’t previously taken a look at our data (or even if you have) now would be a good time to explore our website.  We’ve added new embryo phenotype data and HREM images for many knockout lines, taking our total dataset to more than 4 million images of 550 embryos. We also have placental histology images and phenotypes available for over 100 mutant lines.

This post explores some of the phenotypes observed in the new data, and highlights new lines that could be relevant for clinicians researching rare diseases and developmental disorders. But there isn’t enough space here to include every interesting feature of the data – the best thing to do is to explore it yourself.


EMBRYO PHENOTYPES

Our phenotypers at the Medical University of Vienna have observed many interesting phenotypes in the new data.

Embryos from the line Adamts3 display both subcutaneous edema and bifid ureter. A bifid ureter is the most common malformation of the urinary system, [1] in which there is a duplex kidney drain into separate ureters. This observation highlights the incredible resolution of HREM images, which allow detailed phenotypes to be scored for each embryo.

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Bifid ureter (left side) observed in an Adamts3 mutant embryo. The red arrows highlight a single ureter on the right side, but two branches on the left side.

 

Embryos from the line H13 suffered from severe abnormalities in heart morphology, and had an abnormal heart position within the body. The stomach situs was also inverted, as shown in the image below. Note that severely malformed embryos often have different tissue characteristics, which can result in reduced image clarity.

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Comparison between a H13 mutant embryo (left) and its wild-type litter-mate (right). The yellow arrows indicate situs invertus of the stomach.

 

Embryos from the line Brd2 exhibited a profound ventricular septal defect, as shown in the video below.

 


PLACENTAL PHENOTYPES

Our placental image and phenotype dataset is growing rapidly and now contains more than 100 lines.

H13 knockout placentas were smaller than their wild-type counterparts and showed reduced vascularisation in the placental labyrinth, the region of the placenta that allows nutrient and gas exchange between the mother and the developing embryo.

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A comparison of the placenta from a H13 mutant embryo and that of its wild-type litter-mate.

Vascularisation of the labyrinth is crucial to allow the embryo to receive the oxygen and nutrients needed for normal development. This is just one example, but many more placental phenotypes are available on our website.


LINKS TO CLINICAL STUDIES

Systematic knockout mouse screens can offer a wealth of information about the genetic basis of rare diseases. Many DMDD lines have human orthologues known to be associated with developmental disorders, and the nature of our study means that it would not be possible to derive equivalent systematic data from human patients.

New knockout lines of potential clinical interest include:

Brd2: the human ortholog of this gene is associated with epilepsy, generalised, with febrile seizures plus, type 5.

Cog6: in humans, COG6 is linked to Shaheen Syndrome and congenital disorder of glycosylation, type IiI.

Npat: the human ortholog is associated with Ataxia-telangiectasia, a rare inherited disorder affecting the nervous and immune systems.

Nsun2: in humans, NSUN2 is linked to mental retardation, autosomal recessive 5 and Dubowitz syndrome.


A FULL LIST OF NEW DATA

Embryo phenotype data added for: Adamts3, Brd2, Cog6, Cpt2, Dhx35, H13, Mir96, Npat, Nsun2, Pdzk1.

HREM embryo images added for: Atg16l1, Capza2, Cog6, Coro1c, Cyfip2, Dhx35, Gm5544, Nadk2, Nrbp1, Rab21, Rpgrip1l, Syt1.

Placenta image and phenotype data added for: 1110037F02Rik, Actn4, Atg16l1, Camsap3, Capza2, Cfap53, Coro1c, Crim1, Crls1, Cyfip2, Dmxl2, Gm5544, Gtpbp3, H13, Nsun2, Rab21, Rala, Rpgrip1l, Syt1, Trim45.


REFERENCES

[1] Obstructed bifid ureteric system causing unilateral hydronephrosis, A. Bhamani1 and M. Srivastava2, Rev Urol. 2013, 15(3) p.131–134, PMC3821993.

1 Department of General Medicine, The Ipswich Hospital, Ipswich, UK.
2 Department of Radiology, Barking, Havering and Redbridge NHS Trust, Romford, Essex, UK.