Recombinant Limbs as a Model to Study Homeobox Gene Regulation During Limb Development

Dev Biol. 1994 Nov;166(1):59-72. doi: 10.1006/dbio.1994.1296.


Recombinant limb buds constructed of an ectodermal jacket containing dissociated anterior wing bud mesoderm cells form limb-like structures lacking anteroposterior polarity. Polarity can be restored by implanting a small piece of polarizing region into the recombinant limb. We used recombinant limbs to study expression of Msx1 and Msx2 and 5' Hoxd genes in limb development. Detectable mesodermal expression of Msx1 and Msx2 was lost during preparation of the recombinants. When expression was restored both genes were expressed in a symmetrical fashion in the apical mesoderm whether or not the recombinant was polarized. The normal anterior pattern of Msx1 and Msx2 was never reestablished. The roles Msx1 and Msx2 play in limb patterning are at present not clear. In nonpolarized recombinant limbs the 5' Hoxd genes were sequentially and uniformly expressed in the subridge mesoderm across the anteroposterior axis. They exhibited nested domains of expression showing exclusively distal restriction. In polarized recombinants the 5' Hoxd genes were expressed progressively in an asymmetric, nested fashion adjacent to the implanted polarizing region; there was also a distal mesodermal region further from the implant that did not express the most 5' Hoxd genes. Both of these observations are similar to the normal limb. Our results demonstrate that the polarizing region is not necessary for the activation or maintenance of 5' Hoxd gene expression but that its presence is required in order for the normal pattern to be established. We propose that the expression of the Hoxd genes is an intrinsic property of rapidly growing limb cells and that their expression is related to the limb bud proximodistal outgrowth. Our data also support the influence of the polarizing region upon Hoxd gene expression in two ways. First there is a short-range action responsible for the normal asymmetric expression and second a long-range effect suppressing the expression of 5'-Hoxd genes by the anterior cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Chick Embryo
  • DNA Primers
  • DNA-Binding Proteins / biosynthesis
  • Ectoderm / cytology
  • Ectoderm / physiology*
  • Gene Expression Regulation*
  • Genes, Homeobox*
  • Homeodomain Proteins / biosynthesis*
  • MSX1 Transcription Factor
  • Mesoderm / cytology
  • Mesoderm / physiology*
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Transcription Factors*
  • Wings, Animal / embryology*


  • DNA Primers
  • DNA-Binding Proteins
  • Homeodomain Proteins
  • MSX1 Transcription Factor
  • MSX2 protein
  • Transcription Factors