Highlights d Afadin elimination from motor neuron progenitors causes hopping phenotype d Afadin function at the neuroepithelium controls organization of spinal midline d Miswiring of dI4-5, V0, and V2a premotor interneurons in afadin mutant mice

In the originally published version of this article, three experiments were not described in the STAR Methods section. These were the preparation of isolated spinal cords, the analysis of fictive locomotion data, and three-dimensional positional analysis. These experiments have now been described in the article online. In addition, Talpalar et al. (2011) was added to the reference list and is cited in the newly added text.

Elaborate behaviours are produced by tightly controlled flexor-extensor motor neuron activation patterns. Motor neurons are regulated by a network of interneurons within the spinal cord, but the computational processes involved in motor control are not fully understood. The neuroanatomical arrangement of motor and premotor neurons into topographic patterns related to their controlled muscles is thought to facilitate how information is processed by spinal circuits. Rabies retrograde monosynaptic tracing has been used to label premotor interneurons innervating specific motor neuron pools, with previous studies reporting topographic mediolateral positional biases in flexor and extensor premotor interneurons. To more precisely define how premotor interneurons contacting specific motor pools are organized, we used multiple complementary viral-tracing approaches in mice to minimize systematic biases associated with each method. Contrary to expectations, we found that premotor interneurons contacting motor pools controlling flexion and extension of the ankle are highly intermingled rather than segregated into specific domains like motor neurons. Thus, premotor spinal neurons controlling different muscles process motor instructions in the absence of clear spatial patterns among the flexor-extensor circuit components.

The activity of flexor and extensor motor neurons is tightly regulated by a network of interneurons in the spinal cord. The introduction of rabies retrograde monosynaptic tracing has provided a powerful method to map interneurons directly connected to motor neurons so as to visualize premotor circuits. Previous strategies have used AAV for complementing rabies glycoprotein expression in motor neurons to obtain selectivity in transsynaptic transfer to identify premotor interneurons innervating specific motor neuron pools These studies revealed differences in the location of flexor and extensor premotor interneurons. Here, we report that by using a genetic approach to complement rabies glycoprotein expression in motor neurons, we did not observe any differences in the distribution of flexor and extensor premotor interneurons. In order to identify possible causes for these paradoxical findings, we discuss advantages and caveats of the experimental designs and suggest ways forward to resolve possible ambiguities. Furthermore, to obtain a complete picture of existing approaches and results we ask for contributions from the scientific community describing the use of additional mouse models, viral constructs, and complementation methods. The aim is to generate an open, comprehensive database to understand the specific organisation of premotor circuits.