PiggyBac transgenic strategies in the developing chicken spinal cord

The chicken spinal cord is an excellent model for the study of early neural development in vertebrates. However, the lack of robust, stable and versatile transgenic methods has limited the usefulness of chick embryos for the study of later neurodevelopmental events. Here we describe a new transgenic approach utilizing the PiggyBac (PB) transposon to facilitate analysis of late-stage neural development such as axon targeting and synaptic connection in the chicken embryo. Using PB transgenic approaches we achieved temporal and spatial regulation of transgene expression and performed stable RNA interference (RNAi). With these new capabilities, we mapped axon projection patterns of V2b subset of spinal interneurons and visualized maturation of the neuromuscular junction (NMJ). Furthermore, PB-mediated RNAi in the chick recapitulated the phenotype of loss of agrin function in the mouse NMJ. The simplicity and versatility of PB-mediated transgenic strategies hold great promise for large-scale genetic analysis of neuronal connectivity in the chick

Two Notch Ligands, Dll1 and Jag1, Are Differently Restricted in Their Range of Action to Control Neurogenesis in the Mammalian Spinal Cord

Notch signalling regulates neuronal differentiation in the vertebrate nervous system. In addition to a widespread function in maintaining neural progenitors, Notch signalling has also been involved in specific neuronal fate decisions. These functions are likely mediated by distinct Notch ligands, which show restricted expression patterns in the developing nervous system. Two ligands, in particular, are expressed in non-overlapping complementary domains of the embryonic spinal cord, with Jag1 being restricted to the V1 and dI6 progenitor domains, while Dll1 is expressed in the remaining domains. However, the specific contribution of different ligands to regulate neurogenesis in vertebrate embryos is still poorly understood.In this work, we investigated the role of Jag1 and Dll1 during spinal cord neurogenesis, using conditional knockout mice where the two genes are deleted in the neuroepithelium, singly or in combination. Our analysis showed that Jag1 deletion leads to a modest increase in V1 interneurons, while dI6 neurogenesis was unaltered. This mild Jag1 phenotype contrasts with the strong neurogenic phenotype detected in Dll1 mutants and led us to hypothesize that neighbouring Dll1-expressing cells signal to V1 and dI6 progenitors and restore neurogenesis in the absence of Jag1. Analysis of double Dll1;Jag1 mutant embryos revealed a stronger increase in V1-derived interneurons and overproduction of dI6 interneurons. In the presence of a functional Dll1 allele, V1 neurogenesis is restored to the levels detected in single Jag1 mutants, while dI6 neurogenesis returns to normal, thereby confirming that Dll1-mediated signalling compensates for Jag1 deletion in V1 and dI6 domains.Our results reveal that Dll1 and Jag1 are functionally equivalent in controlling the rate of neurogenesis within their expression domains. However, Jag1 can only activate Notch signalling within the V1 and dI6 domains, whereas Dll1 can signal to neural progenitors both inside and outside its domains of expression

EPHA4 and V2 interneurons in the mammalian locomotor network

Central pattern generators (CPGs) are neural networks that can execute halfautomated movements without supraspinal or sensory input. Hindlimb locomotion in mammals is dependent upon such a CPG which is located ventrally in the spinal cord lumbar enlargement. The key features of mammalian locomotion are ipsilateral excitatory interneurons which execute rhythm generation, as well as commissural inhibitory interneurons which execute left-right coordination and flexor-extensor coordination. Little is known about the ipsilateral excitatory interneurons in the mammalian CPG, and our goal was to identify and describe the role of such excitatory, ipsilateral interneurons. First we identified the EphA4-positive neurons as a key component of the mammalian CPG, a component which normally is restricted to one side by the signaling induced by the interaction of the axon guidance molecules ephrinB3 and EphA4. We found that in EphA4 and ephrinB3 null mice, CPG neurons aberrantly cross the midline. This results in an abnormal synchronized bilateral coordination ( hopping ) due to an increased crossover of excitatory, normally EphA4-expressing neurons, which make connections to the CPG and thus override the normal alternating cross-coordination in the locomotion system. Next, we directly demonstrated that EphA4-positive neurons are rhythmically active and that a subset of these are ipsilaterally projecting, excitatory interneurons with projections onto motor neurons. This positions them as an identified excitatory interneuron group in the mammalian CPG and suggests that the group may be defined by the combined expression of EphA4 and glutamate release. We then went on to look for a possible overlap between EphA4-positive interneurons and the developmentally defined group of ventral V2 interneurons in the spinal cord. We found that the vast majority of V2 interneurons express EphA4 and further characterized the V2 interneurons as segmental ipsilaterally projecting, excitatory (V2a) or inhibitory (V2b) interneurons located in a position which suggests that they are members of the mammalian CPG. Furthermore, we show that there are more EphA4-expressing interneurons than what can be accounted for by V2 interneurons and motor neurons, and the aberrant crossing of processes from EphA4-positive neurons in ephA4LacZ-/- mice does not originate from the V2a population. Finally, we used a mouse model where excitatory ipsilaterally projecting, Chx10- positive (V2a), interneurons are specifically ablated. In the absence of Chx10 neurons, the locomotor burst activity displayed increased variation, but flexor-extensor coordination was unaffected while left-right alternation was disrupted. Evidence for a direct excitatory input of V2a interneurons onto commissural interneurons was provided by anatomical tracing studies. Among the commissural interneurons contacted were the V0 (Evx1 positive) interneurons which are involved in left-right alternation. These observations point to an essential role for V2a interneurons in the control of left-right alternation. Together, work presented in this thesis has identified essential components of the mammalian CPG for walking, leading us towards a better understanding of the fundamental principles for the organization of the mammalian locomotor network. Our hope is that our contribution will help to improve clinical neuro-rehabilitation of spinal cord injured patients

EphA4 defines a class of excitatory locomotor-related interneurons

Relatively little is known about the interneurons that constitute the mammalian locomotor central pattern generator and how they interact to produce behavior. A potential avenue of research is to identify genetic markers specific to interneuron populations that will assist further exploration of the role of these cells in the network. One such marker is the EphA4 axon guidance receptor. EphA4-null mice display an abnormal rabbit-like hopping gait that is thought to be the result of synchronization of the normally alternating, bilateral locomotor network via aberrant crossed connections. In this study, we have performed whole-cell patch clamp on EphA4-positive interneurons in the flexor region (L2) of the locomotor network. We provide evidence that although EphA4 positive interneurons are not entirely a homogeneous population, most of them fire in a rhythmic manner. Moreover, a subset of these interneurons provide direct excitation to ipsilateral motor neurons as determined by spike-triggered averaging of the local ventral root DC trace. Our findings substantiate the role of EphA4-positive interneurons as significant components of the ipsilateral locomotor network and describe a group of putative excitatory central pattern generator neurons

Role of EphA4 and EphrinB3 in local neuronal circuits that control walking.

Local circuits in the spinal cord that generate locomotion are termed central pattern generators (CPGs). These provide coordinated bilateral control over the normal limb alternation that underlies walking. The molecules that organize the mammalian CPG are unknown. Isolated spinal cords from mice lacking either the EphA4 receptor or its ligand ephrinB3 have lost left-right limb alternation and instead exhibit synchrony. We identified EphA4-positive neurons as an excitatory component of the locomotor CPG. Our study shows that dramatic locomotor changes can occur as a consequence of local genetic rewiring and identifies genes required for the development of normal locomotor behavior