Insights into eyesight – discovery of retina regulatory molecules

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By IMPC

Published 6th September 2018

15 novel genes related to retinal function have been found in research published by Dr. Melanie Samuel and colleagues in Cell Reports. The research reveals previously unknown retinal regulators which will help expand our understanding of the genetic landscape that regulates retinal function and uncover how genes are involved in vision loss and disease.

The retina processes visual information from the world around us and relays it to the brain. This sensory circuit is complex but highly ordered. It is comprised of distinct neural layers, and each layer has an array of different neuron types. These neurons are supported by a rich vascular system.

Vision defects globally affect around 253 million people. Such diseases can arise from disruptions to different parts of the visual circuit. For example, vascular changes can be a sign of diabetic neuropathy and defects in light-sensing photoreceptors can lead to retinitis pigmentosa. Yet many of the genes and mechanisms responsible for vision loss remain unknown.

This schematic outlines the complex circuitry in the retina. Each layer contains different neuron types that synapse onto the layer below, from the top layer that detects light to the bottom layer where axons transmit visual information to the brain.

In this research, Dr. Samuel and colleagues selected a sub-set of animal lines from the IMPC in which individual genes had been modified to eliminate their function. They then developed a specialized pipeline of high-throughput retinal screening to examine the role of these genes in more detail. The criteria for selecting candidate genes included those with human orthologues and those with expression in the retina or brain.

The availability of broad based and comprehensive phenotyping data from the IMPC helped researchers focus their search for genes relating to the retina.

In-depth analysis included examining gene expression, synapse organisation, cell morphology and the patterning of blood vessels.

Of the 102 mutant lines analysed, 16 genes were identified that regulated retinal function, 15 of these were novel and not previously associated with retina function.

A range of diverse observations were seen. Lines were found with altered vessel density and branching, disrupted synaptic organisation and neuron loss.

Here, analysis of blood vessels in the retina shows abnormalities including decreased vessel number (Rnf10) and tortuosity (Adsl) in comparison to the wild type image on the left hand side.

Here, analysis of blood vessels in the retina shows abnormalities including decreased vessel number (Rnf10) and tortuosity (Adsl) in comparison to the wild type image on the left hand side.

Many of the genes displayed a wide range of cellular functions and may also be important elsewhere in the body. Data available through the IMPC database supported this, as many of the lines had additional phenotypes, including body composition and central nervous system regulation deficits (suggested by altered body composition and open field tests, respectively).

“The results from the study highlight the kind of discoveries that are possible through harnessing the remarkable data and resources available from the IMPC.  With these efforts, we hope to continue to map the compendium of genes that regulate the retina and the brain so that we can make progress in treating human neural diseases,” said corresponding author, Dr. Samuel, assistant professor of neuroscience and the Huffington Center on Aging at Baylor College of Medicine.

To read the research click here

Interested in learning more? Dr. Samuel recently told us about her lab’s research in the video ‘What makes us unique? Exploring the role of genetics and neural function’


Other contributors to this work include Nicholas E. Albrecht, Jonathan Alevy, Danye Jiang, Courtney A. Burger, Brian I. Liu, Fenge Li, Julia Wang, Seon-Young Kim, Chih-Wei Hsu, Sowmya Kalaga, Uchechukwu Udensi, Chinwe Asomugha, Ritu Bohat, Angelina Gaspero, Mónica J. Justice, Peter D. Westenskow, Shinya Yamamoto and John R. Seavitt. The authors are affiliated with one or more of the following institutions: Baylor College of Medicine, Texas Children’s Hospital, and the Hospital for Sick Children, Toronto.

 

CRISPR/Cas9 has revolutionised genetic research, with seemingly no end to its potential applications. Until recently genome engineering relied on the use of stem cells to target new mutations in mice but now researchers can perform genome editing directly in embryos or specific tissues. As with most projects that use gene editing technology, CRISPR/Cas9 has transformed the efficiency and enhanced the applications of the IMPC.

New CRISPR/Cas9-based techniques are constantly being developed, and existing systems adapted and improved, allowing increasingly sophisticated genetic changes to be made. Two papers published in the journal BMC Biology by Lanza, Gaspero, Lorenzo et al. (2018) and Codner, Mianné, Caulder et al. (2018) explore new advancements and highlight applications for these techniques.

The CRISPR/Cas9 system used in conjunction with single stranded DNA donors is revolutionising our ability to generate targeted mutations directly in the embryo. Whilst short synthetic DNA molecules facilitate this, the use of longer single-stranded DNA donors is a more recent addition to the genome editing toolbox. The two new articles summarised here compare long and short single-stranded donors in a high-throughput setting, both look at conditional knock-out mutants while also presenting advances for the generations of point mutations.

In the first study, led by researchers in Lydia Teboul’s group at the MRC Harwell institute, long single-stranded molecules are utilised to facilitate the generation of conditional alleles. They also apply the system to the introduction of point mutations remote from the recognition site of active Cas9/sgRNA complexes, which up to now has not been possible. This last technique is particularly valuable for human genomic sequencing since it enhances our ability to replicate human mutations in mice.

Alongside this breakthrough the researchers also highlight the unpredictability of this technique. As well as on-target integrations, the system can also produce an array of incorrect alleles. These include unintended point mutations, small or larger sequence rearrangements, and additional donor integrations. Such events are unpredictable by-products and therefore must be omitted in the process of validation of newly established mutant lines.

However, these by-products do not reduce the value of the new system. Instead, they illustrate the importance of a comprehensive validation of new mutants, including sequencing of the locus and copy counting of the number of copies of donor integrations. This process will likely become simplified over time, and issues overcome as new technological advancements are made. In the meantime, although unpredictable, the existing strategies remain efficient at generating desirable mutants.

Author on the paper Lydia Teboul said “An increasing body of evidence is being compiled to indicate that model validation is the newest challenge for the community. After all, the quality and reproducibility of research based on genome editing mutants depends entirely on the thorough characterisation of the mutant in question.”

The aim of the second study was to look at scaling production of conditional null alleles to create IMPC mouse lines. The research, led by Jason Heaney at Baylor College of Medicine, tested the feasibility of using CRISPR/Cas9 gene editing technology to generate conditional knockout mice using Cas9-initiated homology-driven repair (HDR), with both short oligonucleotides and longer single stranded DNA.

The results demonstrate that using pairs of short oligodeoxynucleotides can generate conditional null alleles at many loci; however, at scale there are inefficiencies with this process. On the other hand, long single stranded DNA donors may enable high-throughput production of conditional alleles. Although long single stranded DNA donors are most efficient at generating conditional alleles, pairs of short oligodeoxynucleotides are a viable alternative when use of a long single stranded donor is not feasible due to distance between loxP sites or complexity of sequence between loxP sites. Importantly, in agreement with the Teboul group, random integration of donor DNA and mutagenesis events at the target integration sites were detected when using either type of DNA donor.

 Author on the article Jason Heaney said “Single stranded DNA donors are a critical component of the CRISPR/Cas9 genome editing toolbox and are an invaluable resource for producing conditional knockout alleles in mice. But, given the preponderance for random integration and potential for mutagenesis at sites of HDR, new mouse models produced with these donor DNAs must be carefully screened.”

Both studies highlight that it is essential to screen the sequence errors to check for point mutations, rearrangements and additional donor integrations. Researchers involved in genome editing face several challenges when using CRISPR/CAS9 technology and this latest research show it is essential to understand the unpredictability of different systems. Additionally, standards for the validation and documentation of mutants would be extremely beneficial to the field, and help to ensure quality and research reproducibility.

Research articles:

Codner GF, Mianné J, Caulder A et al. (2018) Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants. BMC Biology 16: 70.

Lanza DG, Gaspero A, Lorenzo I et al. (2018) Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles. BMC Biology 16: 69.

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By IMPC

Published 12th July 2018

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