ABSTRACT
The neuregulins (NRGs) are a family of alternatively spliced factors that play important roles in nervous system development and disease. In motor neurons, NRG1 expression is regulated by activity and neurotrophic factors, however, little is known about what controls isoform-specific transcription. Here we show that NRG1 expression in the chick embryo increases in motor neurons that have extended their axons and that limb bud ablation before motor axon outgrowth prevents this induction, suggesting a trophic role from the developing limb. Consistently, NRG1 induction after limb bud ablation can be rescued by adding back the neurotrophic factors BDNF and GDNF. Mechanistically, BDNF induces a rapid and transient increase in type I and type III NRG1 mRNAs that peak at 4h in rat embryonic ventral spinal cord cultures. Blocking MAPK or PI3K signaling or blocking transcription with Actinomycin D blocks BDNF induced NRG1 gene induction. BDNF had no effect on mRNA degradation, suggesting that transcriptional activation rather than message stability is important. Furthermore, BDNF activates a reporter construct that includes 700bp upstream of the type I NRG1 start site. Protein synthesis is also required for type I NRG1 mRNA transcription as cycloheximide produced a super-induction of type I, but not type III NRG1 mRNA, possibly through a mechanism involving sustained activation of MAPK and PI3K. These results reveal the existence of highly responsive, transient transcriptional regulatory mechanisms that differentially modulate NRG1 isoform expression as a function of extracellular and intracellular signaling cascades and mediated by neurotrophic factors and axon-target interactions.
Subject(s)
Axons/physiology , Gene Expression Regulation, Developmental/physiology , Motor Neurons/metabolism , Neuregulin-1/metabolism , Protein Isoforms/metabolism , Age Factors , Animals , Cells, Cultured , Chick Embryo , Embryo, Mammalian , Enzyme Inhibitors/pharmacology , Female , Gene Expression Regulation, Developmental/drug effects , Limb Buds/injuries , Motor Neurons/drug effects , Nerve Growth Factors/genetics , Nerve Growth Factors/metabolism , Nerve Growth Factors/pharmacology , Neuregulin-1/genetics , Pregnancy , Protein Biosynthesis/drug effects , Rats , Rats, Sprague-Dawley , Signal Transduction/physiology , Spinal Cord/cytology , Time FactorsABSTRACT
BACKGROUND: The chicken embryo is an important model organism for developmental biology studies. At present, many techniques on this model have been set up, from surgical procedures to molecular biology methods, to answer capital questions of cell biology. The study of the genes involved in motoneurons (MNs) survival and cell death is critical for a better understanding of the molecular mechanisms leading to MNs degenerative diseases, such as amyothophic lateral sclerosis (ALS) and motor peripheral neuropathies. NEW METHOD: Here, we describe the combination of a well known surgical procedure able to induce MNs cell death, the limb-bud removal (LBR), with a very popular method used in molecular biology to test gene function in living organisms, the in ovo electroporation (IOE). The aim of this work is to provide an effective method for the investigation of genes involved in MNs survival and cell death under lesion conditions. RESULTS: Our method allows the successful electroporation of the 40-50% of MNs on the side of LBR with a high survival rate early and late after procedure. COMPARISON WITH OTHER METHODS: This modified LBR technique combined with IOE allows a higher MN expression efficiency compared to an already published method. CONCLUSIONS: Our work opens the possibility of screening a multitude of genes involved in MNs survival or cell death in vivo with high reproducibility and efficiency on a flexible and inexpensive animal model. The LBR/IOE technique opens a new way for the optimization of subsequent studies on mammalian models of diseases affecting MNs survival.
