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April 10, 2014

Understanding yin and yang of limb formation provides insight into how birth defects occur

TORONTO – Two new studies led by The Hospital for Sick Children (SickKids) shed light on how limbs are formed, revealing how the perfectly-timed interactions between two populations of building-block cells called progenitor cells play a role in the formation of whole limbs. The studies are published by Developmental Cell online on April 10 and in a special print edition on April 28. Congenital limb anomalies like arm or leg malformations and missing or extra fingers or toes are among the most common types of birth defects, affecting about 140 newborns each year in Canada and approximately 30,000 worldwide. These disabling defects usually require multiple operations, adaptive prosthetics or other equipment for decades to optimize function. Currently, many limb deficiencies are surgically unreconstructible. Research is inching toward eventual regeneration of limbs; however, a prerequisite is understanding how these defects occur in the first place.

Irx genes were missing link

In the first study, the research team identified two genes, Irx3 and Irx5, as critical players in limb-pattern formation and determined how these genes interact with other previously-identified genes. Scientists in the field had already discovered the genetic origin of some bones in the forearm/leg (ulna/fibula) and hand/foot (four fingers/toes excluding the thumb/great toe). However, the genetic origin of the rest of the bones in the limb – the humerus/femur, radius/tibia and thumb/great toe – had not yet been established.

“We discovered that you need these two Irx genes to program formation of the other half of the forearm/leg and hand/foot appropriately,” says Dr. Sevan Hopyan, co-senior author of the study and Orthopaedic Surgeon and Scientist in the Developmental & Stem Cell Biology program at SickKids. “We knew half the story, but now we think we know the whole story.”

The researchers learned that limb pattern would be affected by activation or inactivation of Irx genes at a very early stage, emphasizing that timing is critical. “The findings indicate that the information needed to pattern the limb is instructed genetically, even before the bud of the limb begins to grow,” says Hopyan, who is also Assistant Professor of Surgery and Molecular Genetics at the University of Toronto. “This is the strongest evidence to date of such early instruction for limb patterning.”

Timing of pathway activation is key

The second study focused on a genetic pathway known as the Sonic hedgehog (Shh) signalling pathway, which has been known for a couple of decades to play a role in the formation of the limb skeleton. They found that the exact timing at which the pathway is activated is very important.

Shh and Irx are yin and yang of limb formation

Yin-yang is a concept in Chinese philosophy that describes how opposing forces (hot and cold, light and dark, etc.) are interconnected and interdependent in nature. According to I-Ching or Book of Changes (one of the oldest Chinese classic texts), the thumb is considered to be yang and the other four fingers are yin.

Together, these studies demonstrate that Irx and Shh are mutually antagonistic and inhibit one another. The research team now understands that the Shh cells need to be suppressed at a precise moment early on to allow the Irx cells to make the humerus/femur, radius/tibia and thumb/great toe. In the second stage, Shh must be activated to inhibitIrx, leading to proper formation of the remaining parts of the limb. If either step is incorrectly timed, anomalies like missing or extra bones can result.

“This delicate balance between two populations of progenitor cells is crucial to limb formation. The Shh is the yin to the Irx’s yang and together, they form the complete limb,” says Dr. Chi-Chung Hui, co-senior author, Head & Senior Scientist in the Developmental & Stem Cell Biology program at SickKids and Professor of Molecular Genetics at the University of Toronto.

Future research may include reinterpretation of previous studies related to pattern formation, as well as more detailed work on the interaction between the Irx and Shh populations.

The first study was conducted by SickKids and the University of Toronto. The second study was a collaboration with the University of California, San Francisco and Baylor College of Medicine.

The research was funded by grants from the Canadian Institutes of Health Research, March of Dimes Canada Foundation, Pediatric Orthopaedic Society of North America, University of Toronto Fellowship and SickKids Foundation.

About The Hospital for Sick Children

The Hospital for Sick Children (SickKids) is recognized as one of the world’s foremost paediatric health-care institutions and is Canada’s leading centre dedicated to advancing children’s health through the integration of patient care, research and education. Founded in 1875 and affiliated with the University of Toronto, SickKids is one of Canada’s most research-intensive hospitals and has generated discoveries that have helped children globally.  Its mission is to provide the best in complex and specialized family-centred care; pioneer scientific and clinical advancements; share expertise; foster an academic environment that nurtures health-care professionals; and champion an accessible, comprehensive and sustainable child health system. SickKids is proud of its vision for Healthier Children. A Better World. For more information, please visit www.sickkids.ca.


For more information, please contact:

Suzanne Gold 
The Hospital for Sick Children
416-813-7654, ext. 202059

Matet Nebres
The Hospital for Sick Children