The findings, published today in  journal BBA: Molecular Basis of Disease  shed new light on ocular coloboma, a rare congenital eye condition affecting around 1 in 5000 births and responsible for roughly 10% of childhood blindness.

Some of the researchers are also based at ԰������ University NHS Foundation Trust  and the Greenwood Genetic Centre in the United States.

Coloboma arises when a structure in the developing eye, the optic fissure, fails to close properly and often co‑occurs with other tissue‑fusion problems such as cleft lip and/or palate.

The research focused on YAP1, a protein that helps guide how organs form and how tissues stay healthy.

YAP1 acts like a switch inside cells, helping them decide when to grow, change, or survive based on signals they receive.

Although changes in YAP1 have been linked to coloboma, it has been unclear why some people with these changes develop severe eye defects while others remain unaffected. To address that, they tested the different variants and compared their effects.

To understand the consequences of YAP1’s inactivity during eye development, the researchers studied human retinal organoids - lab-grown miniature versions of the developing human retina grown in the lab. When they reduced the activity of YAP1, they saw effects on how early retinal cells grow and develop.

Disrupting YAP1, they found,  reduced the activity of genes needed for early retinal cells to grow and maintain their identity.

 As a result, the cells developed more slowly, providing a potential explanation for how eye formation goes wrong.

The study also showed that not all YAP1 variants have the same effect. Using computer modelling alongside experimental data, the researchers found that the precise location of each genetic change determines how strongly it disrupts YAP1 function.

This helps explain why coloboma can vary so widely between individuals, even among those carrying changes in the same gene.

Coloboma has been linked to disease causing variants in more than 40 genes, but thanks to the study, YAP1 is now identified as  an important contributor.

“These findings give us a much clearer picture of how small genetic changes can have major effects during eye development,” said the lead author from The University of ԰������.

“By pinpointing how each variant disrupts YAP1’s function, we can better interpret genetic results in patients and move closer to ways of supporting healthy eye formation.

“By combining stem‑cell models with detailed genetic testing, we’re finally beginning to understand how tiny changes in YAP1 can have such a big impact on how the eye forms.

“This work brings us a step closer to explaining why some children develop coloboma.

“Though retinal organoids cannot currently replace the use of animal models, this study shows how they can help us meet our ethical and legal obligations to replace, reduce and refine the use of animals in research wherever feasible.

“It also offers a new framework for understanding how likely YAP1 mutations are to cause disease in children with unexplained eye conditions.”

• Domain-specific mechanisms of YAP1 variants in ocular coloboma revealed by in-vitro and organoid studies is available DOI:

• Image: retinal organoid

The new tool -  now used across the world - was developed by research fellow Dr Richard Naylor who made it possible to use zebrafish larvae to easily and quickly screen new drugs for treating different kidney diseases. 

Replacement, reduction and refinement – known collectively as the 3Rs -  are an increasingly important area of biological research. 

By law scientists must demonstrate they have adhered to the principles of the 3Rs before their project license is granted by the Home Office. 

Dr Naylor will pick up his award today (14 November) at the second ever University of ԰������  3Rs symposium, organised by the University’s animal unit, where  scientists will hear about the latest advances in 3Rs science. 

Using organisms that are not able to feed independently and are therefore considered to be minimally sentient - the judges recognised the tool as a novel alternative to mammals but also for its ability to reduce animal numbers and enhance the care they receive. 

Kidney disease is a major cause of illness and accounts for 10% of all deaths in humans. 

Protein in urine is produced when kidneys do not work properly and is easily tested in humans with a simple dipstick. 

Zebrafish- which have similar genetics to humans and possess 80% of human disease-causing genes - are a popular species used by research scientists because they produce large numbers of eggs (200 to 300 per week per female), which develop externally. 

In the past it was virtually impossible to test for protein in the urine of zebra fish larvae because the tiny amounts of urine produced are immediately diluted in the fish tank. 

However, a new genetically modified zebrafish larvae model, designed and generated by Dr Naylor and his team, contains a luminescent molecule called NL-D3 in its blood. 

When kidneys are damaged, NL-D3 leaks out of the kidneys and into the urine. NL-D3 can easily be detected in embryos using a luminometer which measures the light produced by urine in the water. 

As a result, scientists can now easily test for the level of protein in high numbers of the organisms which - at less than 5 days old - are not considered to be fully sentient under the law. 

The team tested the new tool by generating a zebrafish model of Alport syndrome, a kidney disease characterized by protein in the urine, publishing their results in the prestigious journal . 

In Alport zebrafish, levels of NL-D3 increased but could be subsequently reduced using captopril, a drug that lowers blood pressure, demonstrating the efficacy of the tool. 

Dr Naylor said: “Finding 3Rs solutions to animal research is incredibly important because as scientists we care about the welfare of the animals we are privileged to work with. 

“That is why it is so exciting we have demonstrated how is possible to conduct fundamental research on kidney disease without necessarily relying on mammalian models. 

“Testament to this, we have had seven research groups in the US and Europe request embryos be sent to them. 

“And even more excitingly, we are currently collaborating with a large pharmaceutical company to model acute kidney injury and screen drugs to treat it.” 

Dr Maria Kamper, Director of the animal unit at the University of ԰������ said: “As Director of the Biological Services Facility, I am delighted to present our inaugural University of ԰������ 3Rs prize to Dr Naylor and his team. 

“Their innovative work exemplifies our commitment to advancing scientific discovery while upholding the highest standards of animal welfare. The widespread adoption of this model by kidney disease researchers worldwide proves it is an outstanding achievement in the 3Rs space.” 

Replacing mouse models
Until now, protein in the urine as a marker of kidney dysfunction in disease and in response to drug treatments was mainly used in mouse models or from human patients. But with the new tool, the ability to use zebrafish to accurately monitor kidney dysfunction increases the appeal of pre-independent feeding stage zebrafish to model kidney disease for researchers worldwide. 

Fewer numbers needed
Previously, high numbers of embryos were needed due to high variability in methods used to test kidney dysfunction in zebrafish. The new tool, however, has reduced the number of procedures needed to be performed on zebrafish embryos to zero.  As the scientists now only need to analyse embryo medium, no animals are injected or are required to be anaesthetized. And fewer larvae are needed to achieve statistical significance. 

No need to anaesthetize the embryos
Before the paper was published, the only way to measure kidney dysfunction in zebrafish was to inject fluorescent dextran directly into the animals and observe how quickly the fluorescence was lost from the blood vasculature over the subsequent days. The approach meant having to anaesthetize zebrafish embryos repeatedly, which is now lo longer necessary.

• The paper A novel nanoluciferase transgenic reporter measures proteinuria in zebrafish is published in
• Images are of zebrafish larvae