Ancient gene involved in spina bifida also plays a role in motor coordination and anxiety

A/Prof Stephen Ting

by Anne Crawford

Curiosity and a compelling hypothesis prompted clinician and haematology researcher, Associate Professor Stephen Ting, to revisit the subject matter of his PhD a decade later. The findings of his recent study into the transcription factors of an ancient gene family known as the Grainyhead-like (Grhl) genes have revealed more than he expected and may have implications for future neuroscientific research.

A/Prof Ting researched the Grhl gene family for his PhD from 2000 to 2005, supervised by Professor Stephen Jane, then at the Bone Marrow Research Laboratories at Royal Melbourne Hospital. His identification of the Grhl3 gene and its role in spina bifida earned him a number of awards, including the Victorian Premier's Award in Medical Research (2005).

Grhl3 regulates multiple stages of embryonic formation. These genes play an evolutionary conserved, similar role from flies through to mammals. Using these animal models can help researchers understand the genetic causes of human disease. A/Prof Ting's Grhl3 'knockout' mice (in which the specific gene's expression is blocked, i.e. it's knocked out) exhibited spina bifida. Later, Grhl3 was also identified as a critical factor in epithelial tumour formation. During the course of his PhD, Associate Professor Ting had observed that Grhl3 was also expressed in a very specific, defined area of the mouse brain, the habenula, which he hypothesised was involved in dopamine signaling. He knew the Grhl3 antecedent gene Grainyhead (Grh) when mutated in Drosophila (fruit flies) expressed unco-ordinate movements and that biochemically, Drosophila Grh is part of the dopamine pathway.

“My thinking was that the epithelial Grhl3 function is highly conserved; if Grhl3 is expressed in the mammalian brain and the Drosophila fly Grh mutant has unco-ordinate movements, the potential extrapolation is the human condition, Parkinson’s Disease, a dopamine-deficient motor deficit disorder – people with it have a shuffling gait and trouble with their movements,” he said.

Dr Seb Dworkin, La Trobe University

A/Prof Ting collaborated with Dr Sebastian Dworkin, a post-doctoral researcher from the Jane laboratory, to investigate the hypothesis that loss of Grhl3 in the brain would lead to some form of neuromotor deficit.

Dr Dworkin identified that mouse Grhl3 was expressed specifically in the habenula, thought to modulate repressive behaviours. The researchers crossed the conditional Grhl3-mouse with a neuronal-specific expressing mouse line, Nestin-Cre to delete Grhl3 only in the brain (i.e. the habenula). Motor and behavioural testing showed that adult mice deficient in neuronal Grhl3 had defects in gait and increased levels of anxiety and inhibition.

The study, published in ‘Developmental Neurobiology’, describes for the first time a putative role for Grhl3 in the mammalian brain, suggesting a novel link between Grhl3 loss and potential disruption of neural pathways regulating both motor-coordination and inhibition of anxiety. It concluded that these mice may serve as a novel model of human conditions of impulsive behaviour or hyperactivity.  

“We’re providing evidence to the research world that Grhl3, which is not previously known to have any function in the mammalian brain, while not obviously Parkinsonian, has activity in the context of motor function and potentially behaviour relating to anxiety and risk reward,” A/Prof Ting said.

Dr Dworkin, the first author, is now a senior lecturer at La Trobe University. Professor Jane, the current Head of the Central Clinical School and the Department of Medicine, is continuing to investigate the mammalian Grhl family, elucidating the Grhl3 molecular pathways for epithelial cellular proliferation.

A/Prof Ting, senior author, switched his focus to haematopoietic stem cells some time ago and has no plans to further the Grhl3-related brain research. He is now Director of Clinical Haematology at Eastern Health and conducts haematopoietic stem cell research at the Australian Centre of Blood Diseases with A/Prof David Curtis.

However, “I still think this (Grhl3) gene is super interesting! I think with all PhD projects you leave a little bit of your heart with it,” he said.

Reference
Dworkin S, Auden A, Partridge DD, Daglas M, Medcalf RL, Mantamadiotis T, Georgy SR, Darido C, Jane SM, Ting SB. Grainyhead-like 3 (Grhl3) deficiency in brain leads to altered locomotor activity and decreased anxiety-like behaviours in aged mice. Dev Neurobiol. 2016 Dec 1. doi: 10.1002/dneu.22469. [Epub ahead of print]