This work demonstrates a role for the Wilms tumor protein Wt1 in the specification of neurons that are involved in the control of locomotion.

The Wilms tumor suppressor gene Wt1 encodes a zinc finger transcription factor that is essential for development of multiple organs including kidneys, gonads, spleen and heart. In mammals Wt1 comprises 10 exons with two characteristic splicing events: inclusion or skipping of exon 5 and alternative usage of two splice donor sites between exons 9 and 10. Most fish including zebrafish and medaka possess two wt1 paralogs, wt1a and wt1b, both lacking exon 5. Here we have characterized wt1 in guppy, platyfish and the short-lived African killifish Nothobranchius furzeri. All fish except zebrafish show alternative splicing of exon 4 of wt1a but not of wt1b with the wt1a(-exon 4) isoform being the predominant splice variant. With regard to function, Wt1a(+exon 4) showed less dimerization but stimulated transcription more effectively than the Wt1a(-exon 4) isoform. A specific knockdown of wt1a exon 4 in zebrafish was associated with anomalies in kidney development demonstrating a physiological function for Wt1a exon 4. Interestingly, alternative splicing of exon 4 seems to be an early evolutionary event as it is observed in the single wt1 gene of the sturgeon, a species that has not gone through teleost-specific genome duplication.

Central pattern generator (CPG) networks coordinate the generation of rhythmic activity such as locomotion and respiration. Their development is driven by various transcription factors, one of which is the Wilms tumor protein (Wt1). It is present in dI6 neurons of the mouse spinal cord, and involved in the coordination of locomotion. Here we report about the presence of Wt1 in neurons of the caudoventral medulla oblongata and their impact on respiration. By employing immunohistofluorescence staining, we were able to characterize these Wt1 positive (+) cells as dB4 neurons. The temporal occurrence of Wt1 suggests a role for this transcription factor in the differentiation of dB4 neurons during embryonic and postnatal development. Conditional knockout of Wt1 in these cells caused an altered population size of V0 neurons already in the developing hindbrain, leading to a decline in the respiration rate in the adults. Thereby, we confirmed and extended the previously proposed similarity between dB4 neurons in the hindbrain and dI6 neurons of the spinal cord, in terms of development and function. Ablation of Wt1+ dB4 neurons resulted in the death of neonates due to the inability to initiate respiration, suggesting a vital role for Wt1+ dB4 neurons in breathing. These results expand the role of Wt1 in the CNS and show that, in addition to its function in differentiation of dI6 neurons, it also contributes to the development of dB4 neurons in the hindbrain that are critically involved in the regulation of respiration.

Rhythmic and patterned locomotion is driven by spinal cord neurons that form neuronal circuits, referred to as central pattern generators (CPGs). Recently, dI6 neurons were suggested to participate in the control of locomotion. The dI6 neurons can be subdivided into three populations, one of which expresses the Wilms tumor suppressor gene Wt1. However, the role that Wt1 exerts on these cells is not understood. Here, we aimed to identify behavioral changes and cellular alterations in the spinal cord associated with Wt1 deletion. Locomotion analyses of mice with neuron-specific Wt1 deletion revealed that these mice ran slower than controls with a decreased stride frequency and an increased stride length. These mice showed changes in their fore-hindlimb coordination, which were accompanied by a loss of contralateral projections in the spinal cord. Neonates with Wt1 deletion displayed an increase in uncoordinated hindlimb movements and their motor neuron output was arrhythmic with a decreased frequency. The population size of dI6, V0 and V2a neurons in the developing spinal cord of conditional Wt1 mutants was significantly altered. These results show that the development of particular dI6 neurons depends on Wt1 expression and loss of Wt1 is associated with alterations in locomotion.

Flavoproteins are key players in numerous redox pathways in cells. Flavin cofactors FMN and FAD confer the required chemical reactivity to flavoenzymes. In most cases, the interaction between the proteins and the flavins is noncovalent, yet stronger in comparison to other redox-active cofactors, such as NADH and NADPH. The association is considered static, but this view has started to change with the recent discovery of the dynamic association of flavins and flavoenzymes. Six cases from different organisms and various metabolic pathways are discussed here. The available mechanistic details span the range from rudimentary, as in the case of the ER-resident oxidoreductase Ero1, to comprehensive, as for the bacterial respiratory complex I. The same holds true in regard to the assumed functional role of the dynamic association presented here. More work is needed to clarify the structural and functional determinants of the known examples. Identification of new cases will help to appreciate the generality of the new principle of intracellular flavoenzyme regulation.