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We have isolated Msx3, the third member of the murine Msxhomeoɡ gene family which is homologous to the mshgene of Drosophila. the Msx3cDNA encodes a protein of 204 amino acids which has striking regions of homology in addition to the homeodomain when compared to the other Msxfamily members.Msx3maps to the distal end of mouse chromosome 7, thus it is unlinked to either Msx1or Msx2. RNA in situ analysis of Msx3gene expression during early development revealed that it is restricted to the dorsal portion of the neural tube with a rostral boundary in the rostral rhombencephalon. This sole expression domain for Msx3overlaps with both Msx1and Msx2at early stages, but in older embryos, Msx3expression becomes restricted to the ventricular zone of the dorsal neural tube, whereas Msx1and Msx2become localized to the non-neuronal roof plate region. The absence of detectable levels of Msx3expression outside this restricted dorsal region of the developing central nervous system (CNS) is in marked contrast to Msx1and Msx2, which have extensive expression domains in other embryonic tissues, particularly craniofacial structures and the limbs. By analogy with the expression of msh/Msx genes in other organisms, it is likely that one of the ancestral functions of the msh/Msx gene family is in patterning of the CNS.