In an aim to try and identify novel factors expressed in the brain during development and associated with the nigrostriatal dopaminergic system, we analysed protein extracts from developing and adult rat brains (removed under terminal anaesthesia) using Western blotting and a monoclonal antibody to GDF-5.
GDF-5 is a member of the TGF-b superfamily, playing a key role in correct limb formation. It has also been previously shown to be a potent dopaminergic neurotrophin in vitro and in vivo. This is the first study on GDF-5 protein expression in the developing and adult rat brain. We have found that GDF-5 expression in the rat brain begins on embyronic day (E) E12 and increases up to E14 before decreasing to its lowest level by postnatal day (P) P1. Its expression then increases with age, reaching maximal levels in the adult brain. It is expressed in the developing ventral mesen-cephalon (VM) and in the adult striatum and substantia nigra, indicating its association with the nigrostriatal pahway. Receptors for this growth factor are also expressed by dopaminergic neurones in the adult rat substantia nigra (Charytoniuk et al. Neurosci. 100, 1997). Of the soft tissues we examined from adult rat, GDF-5 immunoreactivity was only detected in extracts from brain and heart.
GDF-5 reaches maximal levels in the rat VM on E14 when dopaminergic neurones are beginning to differentiate, suggest-ing that it may be involved in specifying dopaminergic cell fate. We examined the effects of GDF-5 (10 ng/ml and 100 ng/ml doses) on the differentiation of E14 rat VM primary cultures and neural progenitor cells (NPCs) expanded using the mitogens EGF (20 ng/ml) and FGF (20 ng/ml). Treatment of E14 VM primary cultures with GDF-5 (10 ng/ml and 100 ng/ml) resulted in a significant increase in both the number of glia and total cell number after 7 d in vitro and also significantly reduced the number of cells displaying features of apoptosis. This is the first report of GDF-5 protein expression in the developing and adult rat brain and its association with the nigrostriatal dopaminergic system. Its function in the adult rat heart remains unknown.