( X) Co-expression of Rybp and Cbx under Mnx1 fails to induce Hoxc9 in brachial MNs. Bolt symbol indicates electroporated side of spinal cord. ( V) Misexpression of Rybp in postmitotic MNs under Mnx1 in chick leads to ectopic Hoxc9 and Hoxc10 expression in brachial MNs. ( U) Schematic of Cbx and Rybp interactions in PRC1. ( S–T) Co-labeling of Hoxc6/Hoxc9 and Hoxc4/Hoxc10 in Ring1 MNΔ mice, showing ectopically expressed caudal Hox proteins and loss of rostral Hox expression in brachial segments. ( P–R) Loss of Hoxc8 and ectopic Hoxc10 and Hoxd10 expression in brachial MNs of Ring1 MNΔ mice. ( O) Summary of changes in MN Hox expression of Ring1 MNΔ mice. ( N) Quantification of MNs: 73☓ Mnx1 + MNs in controls, versus 68☒ in Ring1 MNΔ mice, n = 6 sections, p = 0.1879, unpaired t-test.

( L–M) Hoxc6 is lost from brachial MNs and Hoxc9 is ectopically expressed in Ring1 MNΔ mice. ( J–K) Ring1 MNΔ mice express Mnx1 and Slc18a3. ( I) Rnf2 (Ring1B) is selectively removed from progenitors (boxed region) and post-mitotic MNs (oval) in E12.5 Ring1 MNΔ ( Ring1 -/-::Rnf2 flox/flox, Olig2 Cre) mice. ( G) Quantification of MNs: 54☒ Mnx1 + MNs per section in brachial controls, versus 60☑ in Ezh MNΔ mice, n = 6 sections, p = 0.0342, unpaired t-test. ( E–F) Brachial Hoxc6 expression is normal in Ezh MNΔ mice, and no ectopic Hoxc9 is detected. ( C–D) MNs express Mnx1 and Slc18a3 (Vacht) in Ezh MNΔ mice.

( B) Brachial spinal sections showing H3K27me3 is depleted from progenitors (boxed region) and post-mitotic MNs (oval) in E11.5 Ezh MNΔ ( Ezh1 flox/flox::Ezh2 flox/flox, Olig2 Cre) embryos.

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