The neuromuscular junction has been extensively employed in order to identify crucial determinants of synaptogenesis. At the vertebrate neuromuscular synapse, extracellular matrix and signaling proteins play stimulatory and inhibitory roles on the assembly of functional synapses. Studies in invertebrate species have revealed crucial functions of early morphogens during the assembly and maturation of the neuromuscular junction. Here, we discuss growing evidence addressing the function of Wnt and Bone morphogenetic protein (BMP) signaling pathways at the vertebrate neuromuscular synapse. We focus on the emerging role of Wnt proteins as positive and negative regulators of postsynaptic differentiation. We also address the possible involvement of BMP pathways on motor neuron behavior for the assembly and/or regeneration of the neuromuscular junction.

It has long been thought that the same molecules guide both trunk neural crest cells and motor axons as these cell types grow and extend to their target regions in developing embryos. There are common territories that are navigated by these cell types: both cells grow through the rostral portion of the somitic sclerotomes and avoid the caudal half of the sclerotomes. However, these cell types seem to use different molecules to guide them to their target regions. In this review, I will talk about the common and distinct methods of migration taken by trunk neural crest cells and motor axons as they grow and populate their target regions through chick embryos at the level of the trunk.

Bidirectional signaling between Eph receptor tyrosine kinases and their cell-surface protein signals, the ephrins, comprises one mechanism for guiding motor axons to their proper targets. During projection of motor axons from the lateral motor column (LMC) motor neurons of the spinal cord to the hindlimb muscles in chick embryos, ephrin-A5 has been shown to be expressed in the LMC motor axons until they reach the base of the limb bud and initiate sorting into their presumptive dorsal and ventral nerve trunks, at which point expression is extinguished. We tested the hypothesis that this dynamic pattern of ephrin-A5 expression in LMC motor axons is important for the growth and guidance of the axons to, and into, the hindlimb by knocking down endogenous ephrin-A5 expression in the motor neurons and their axons. No perturbation of LMC motor axon projections was observed in response to this treatment, suggesting that ephrin-A5 is not needed for LMC motor axon growth or guidance.

INTRODUCTION: In ovo electroporation of half of the avian neural tube is a simple procedure in which one places the electrodes parallel to the neural tube, flanking the intended axial region of transfection. It is possible to modify this technique to target the ventral quadrant of the neural tube that contains motor neurons in the lateral motor column (LMC) and their axons by positioning the electrodes in an offset dorsal/ventral configuration, instead of the standard parallel position. If the electroporation is performed in the neural tube of stage 15 chick embryos, the medial portion of the LMC is targeted primarily; however, if neural tubes of stage 17 embryos are electroporated, the entire LMC will be transfected. This technique can be used to examine the behavior of motor axons as they project into the developing limb when genes are misexpressed, overexpressed, or knocked down via RNAi (using short hairpin RNA [shRNA]). The un-electroporated half of the neural tube serves as an internal control, or an enhanced green fluorescent protein (EGFP) reporter construct (pCAX) serves as a control for the electroporation and for EGFP expression. By electroporating a DNA construct that contains EGFP, or co-electroporating the DNA of interest with a GFP reporter construct, it is possible to verify the success of the electroporation in ovo.

INTRODUCTION: In ovo electroporation is an efficient approach for manipulating gene expression in multiple tissues of the chicken embryo, which greatly facilitates functional analysis of the role of specific genes during development. A successful protocol to deliver plasmid DNA into a specific tissue has to meet three basic criteria: high efficiency of transfection, low mortality of embryos, and localization of transfected cells. In this protocol, we describe an in ovo electroporation procedure for targeting muscle precursor cells in the lateral dermomyotome and axial skeletal precursor cells in the sclerotome of the somites of stage 15 chicken embryos. At this stage, the microinjection of DNA solution into individual somites is relatively easy, and the orientation of the electrodes directs the negatively charged DNA into the target tissues. The range of electrical current used is adjusted to maximize the transfection but minimize the mortality of the embryos.