SMN participates in the positioning of motor neurons in the ventral neural tube

Abstract

Motor neurons extend their axons ventrally to innervate specific muscles in the hindlimb. We are examining the role of SMN, the gene that is responsible for spinal muscular atrophy (SMA), using a loss-of-function approach. First, we assessed whether SMN levels were reduced using SMN shRNAs in vitro in HEK293T cells; SMN levels were indeed decreased by SMN shRNAs but not by missense shRNAs. In vivo, at early stages when SMN levels are reduced, motor axons are growth-impaired and exhibit likely fasciculation defects. Surprisingly, when SMN levels are reduced in vivo, Islet-1-positive motor neurons appear to migrate aberrantly out of the neural tube, along the spinal nerve at later stages; this was never observed in control electroporations. We have examined the patterning of cells in the neural tube and find that it was mispatterned. Interestingly, boundary cap (BC) cells were in place, as shown by various markers (cadherin7, lingo-1 antibodies). Semaphorin6A was normally expressed by BC cells; when SMN was reduced in motor neurons, Semaphorin6A altered its expression to motor neurons. To determine if Semaphorin6A was responsible for the escape of neurons from the neural tube, Semaphorin6A was expressed by motor neurons using in ovo electroporation. Islet-1 antibody staining revealed that Semaphorin6A could account for early stages of escape of motor neurons in SMN-reduced embryos, but not at later stages. Experiments are underway to determine exactly why motor neurons leave the neural tube when SMN levels are decreased.