In the past year, we have concentrated our efforts on elucidating the genetic mechanisms that direct the specification of motor neuron subtypes in the zebrafish spinal cord. Several transcription factors, including FoxP1a, HLH1, LIM-HD and LMX1B, and the gene regulatory network (GRN) they establish, are known to control the specification of the motor neuron subtypes Mauthner, Rohon Beard, and Islet cells. We have found that FoxP1a, but not HLH1, LIM-HD, or LMX1B is essential for the generation of Mauthner cells (Wang et al., 2012). Intriguingly, FoxP1a is not involved in any of the other programs in which it plays a key role. We will investigate the relationship between the specification of motor neuron subtypes and neural cell cycle (NCC) exit by determining whether FoxP1a functions downstream or upstream of the Tbr2 (Eomes) and Gfi1 transcription factors, which control the NCC exit by antagonizing the Tcf7/Wnt3a signaling pathway. A number of other transcription factors are also known to regulate Mauthner cell specification. We will determine their roles in Mauthner cell specification. Furthermore, we will assess the in vivo functions of all of the Tbr2-regulated Mauthner cell-specific genes. This will provide a molecular map of the gene network underlying the specification of Mauthner cell subtypes. To provide insight into the evolution of the molecular programs in the zebrafish and mammalian spinal cord that pattern the motor columns, we will also compare the regulatory networks established by key transcription factors that are involved in the specification of motoneurons in the zebrafish with those in other vertebrates. By conducting experiments to investigate the in vivo functions of several transcription factors that are known to control motor neuron specification in the zebrafish spinal cord, we hope to gain insight into how the transcriptional network that pattern the zebrafish spinal cord may have evolved to establish an appropriate GRN for spinal motor neurons in the zebrafish. We expect that by gaining a molecular understanding of the genetic mechanisms that pattern motor neurons in zebrafish, we will also gain insight into the genetic mechanisms that guide the development of motor neuron subtypes in the spinal cord of other organisms including humans. Thus, our studies will not only help us gain a comprehensive understanding of the genetics of motoneuron specification in the zebrafish, but also aid our understanding of how motor neuron specification and diversification evolved. The genetic programs that establish a functional connectivity between the developing central and peripheral nervous systems also undergo considerable changes over time. Thus, the mechanistic knowledge gained from our work on the development of the zebrafish motor system is likely to provide a useful guide for our understanding of the developmental basis of motor system disorders in humans. Our investigations are currently focused on studying the molecular mechanisms that control motoneuron specification, and how such mechanisms evolved and diversified to adapt to changes in the fish body plan during evolution. Motoneurons make up an important part of the nervous system of humans and are a primary cause of spinal cord injury. Because we believe our work on the zebrafish is likely to lead to a deeper understanding of motor neuron specification in humans, we will compare the transcriptional programs that specify the development of spinal motor neurons in the zebrafish with those in mice and humans. We will also investigate the gene regulatory networks in the motor column neurons of tetrapods to understand the genetic basis of the evolutionary expansion of motoneuron subtypes. Finally, we will characterize the molecular differences between zebrafish and mammalian peripheral nervous systems. This project will allow us to understand how the transcriptional networks that pattern motor neuron subtypes in the zebrafish evolved during evolution. Our comparative analyses will shed light on the genetic program that underlies spinal motor neuron development and help to identify potential therapeutic targets for spinal cord injury. PUBLIC HEALTH RELEVANCE: In the spinal cord, a region of the central nervous system, motoneurons innervate muscles, and motoneurons also play a critical role in the generation of locomotion. When a motoneuron is damaged or destroyed, there is a loss of motor function in the affected limb. The work we will conduct in the zebrafish will be applied to understanding the cause of spinal cord injury in humans. We will learn about the genetics of how motoneurons form in the zebrafish. This knowledge may provide a better understanding of spinal cord injury and, hopefully, help to improve recovery of motor functions in humans.