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Gosgnach et al. Supplementary Material In Figure 1 cell counts for motor neurons were performed on sections from lumbar spinal cord at E18.5. Wildtype versus Pax6-/- : 1644 versus 1749 cells in the lateral motor column and 335 vs 227 cells in the medial motor column, respectively. Numbers represent average cell counts from 5 sections, 3 embryos. In Figure 4d an inward current of 210 pA was required for the cell to fire action potentials following application of 10 nM allatostatin (AL), compared to 90 pA prior to drug application, and after peptide washout. Supplementary Figure S1. Analysis of ventral neuron development in Pax6-/- and En1Cre;R26-lacZflox/DTA (En1-DTA) embryos. (a-d) Cross sections through E11.5 lumbar spinal cord showing a reduction in En1-expressing V1 neurons (a) and ventrolaterally migrating Pax2+ neurons (arrowheads) that are V1 INs. Ventral Foxd3 expressing cells are largely missing in the Pax6 mutant confirming the loss of most V1 neurons. Chx10+ V2 neurons (green) and Evx1+ V0 neurons (red) are still generated in near normal numbers in the Pax6 mutant spinal cord. There is a small increase in the number of V0 neurons and a slight decrease in V2 neuron cell numbers in the Pax6-/- cord. (e-h) Cross sections through E12.5 spinal cords showing the differentiation of ventral interneurons in the En1Cre;R26-lacZflox/DTA (En1-DTA) embryonic spinal cord. While newborn V1 neurons are present in the subventricular zone in the E12.5 En1-DTA spinal cord (a, brackets), the more laterally located differentiated V1 neurons are largely missing from the 1 Gosgnach et al. ventral horn (a, arrowhead). Pax2+ neurons are generated in normal numbers (f, brackets); however, there are fewer Pax2+ cells in the ventral horn (f, arrowhead). N.B. These cells are predominantly V1 neurons. (g) Evx1+ V1 neurons are generated in normal numbers and migrate appropriately. (h) Chx10+ V2 neurons (red) and Hb9+ motor neurons are present in normal numbers in the ventral horn of En1-DTA mice. (i-j) Motor neurons in Pax6-/- and En1-DTA mice express cholinergic neurotransmitter markers. Analysis of the vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) expression at E18.5 shows no differences in their expression patterns. The columnar organization of lumbar motor neurons in these spinal cords is also normal. Supplementary Figure S2. Acute allatostatin receptor-dependent neuronal silencing in the spinal cord. (a) Ventral root ENG recordings from lL2 and lL5 of P0 AlstR192 mice. Rhythmic locomotorlike activity induced by 5 µM NMDA and 10 µM 5-HT was not affected by the addition of 5M allatostatin (AL). (b) Ventral root ENG recordings from lL2 and lL5 ventral roots of P0 nestinCre;AlstR192 mice. Rhythmic motor activity was markedly decreased after application of 1 M AL. This effect that was reversed upon wash-out of the peptide (c) Recording from a non-V1 interneuron (GFP negative) in the ventral spinal cord of an En1Cre/AlstR192; ZnG mice. An inward current step of 80 pA evoke action potentials in absence or presence of AL (10 nM). Application of 10 nM AL did not modify action potential frequency in response to a current ramp. 2 Gosgnach et al. (d) Expression of GIRK channels in the neonatal mouse spinal cord required for allatostatininduced silencing. In situ hybridization using mouse probes that detect GIRK1, 2, 3 or 4 showed that GIRK2 is expressed in interneurons throughout the spinal cord, including ventral interneurons (arrowhead). Low level expression of GIRK1 is also present throughout the spinal cord. Arrow marks motor neurons and superficial dorsal hornin 5 sections between L2 and L4; n=4 cords). Author Contributions. S.G. and G.M.L contributed equally to this work by performing the molecular and electrophysiological analyses. S.J.B. and Y.Z. participated in the electrophysiology experiments. H.S. generated and behaviorally tested the En1tlZ/tlZ mice T.V. and M.G. generated the AlstR192 transgenic mice. D.M generated the R26-DTA mice used in this study. E.M.C, O.K. and M.G. helped design and analyze experiments, and write the paper. All authors discussed and commented on the findings in the manuscript. 3