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Figure S1
Expression of motor neuron homeodomain proteins at stage 23. (A-D) At brachial levels of the spinal cord at stage 23, MNR2 (A) and Lim3 (B) are restricted to a medial subset of motor neurons, with the exception of a few cells that have migrated laterally and ventrally. HB9 (C) and Isl1/2 (D) are expressed throughout the motor neuron domain. The decrease in staining in a subset of Isl1/2+ cells (D) reflects the downregulation of Isl1 in migrating LMCl cells (Sockanathan and Jessell, 1998). At limb and thoracic levels of the spinal cord, Lim3+ V2 interneurons (B,F,J) are generated from cells immediately dorsal to the motor neuron domain. (E-H) At thoracic levels, MNR2 (E) and Lim3 (F) are restricted to a medial population of motor neurons, Isl1/2 (H) staining is more uniform throughout the motor neuron domain, and HB9 (G) is downregulated from a central population of cells within the motor neuron domain. (I-L) MNR2 (I) and Lim3 (J) label a subset of medial cells with the migration laterally of a few scattered cells at lumbar levels of the spinal cord. HB9 (K) and Isl1/2 (L) are expressed throughout motor neurons at stage 23.
Figure S2
Differentiated properties of motor neurons induced by MNR2. (A) Widespread expression of lacZ in neural cells after electroporation of a lacZ cDNA using a CMV promoter. (B) Motor neuron-restricted expression of lacZ in the nuclei of neural cells after electroporation of a LacZ cDNA using a 5¢ HB9 promoter. (C) Widespread expression of GFP expressed using a b-actin promoter. (D) Motor neuron-restricted expression of Tau-Myc using a 5¢ HB9 promoter. (E,F) To assay axonal projections we made use of a 9 kb 5¢ DNA sequence of the mouse HB9 gene (HB9p) that confers motor neuron-specific gene expression in transgenic mouse embryos Co-electroporation of MNR2 under the control of an RCAS promoter together with the HB9p-Tau-Myc construct resulted in activation of expression of the HB9p-Tau-Myc gene in dorsally positioned neurons, and led to expression of Tau-Myc in axons that projected out of the spinal cord into the periphery. (G-I) The generation of ventral interneurons is inhibited by MNR2. (G) Loss of expression of Evx1/2+ (V0 neurons), (H) loss of expression of En1+ (V1 neurons) and (I) loss of expression of Chx10+ (V2 neurons) in electroporated cells. In all cases, the repression of interneuron generation was cell-autonomous; residual V0, V1 and V2 interneurons lacked ectopic MNR2 expression. Images are representative of over 20 electroporated embryos per construct.
Figure S3
Isl1/2 expression in prospective LMC neurons after Lim3 electroporation at brachial levels. (A) Ectopic expression of Lim3 in brachial level motor neurons. (B) Most neurons that ectopically express Lim3 co-express Isl1/2 proteins. (C) Persistence of Isl1/2 expression in motor neurons that have extinguished Lim1 after Lim3 expression. All three panels show same section labelled with Isl1/2, Lim3 and Lim1/2, detected by triple-label immunochemistry. Similar observations were made in over 10 embryos.
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