Mouse embryo develops extra limbs instead of genitals

Inactivating a gene that codes for a protein involved in early mouse development results in the formation of two extra hindlimbs in the place of external genitalia.

Mice with six legs have been produced by researchers at the Gulbenkian Science Institute in Oeiras, Portugal, by inactivating the Tgfbeta receptor 1 (Tgfbr1) gene during development. Further investigations revealed that this modification changed the gene regulatory pathways controlling the decision of cells to become hindlimbs or genitals.

'The pelvic appendage (hindlimb in tetrapods) develop in the same embryonic space as the external genitalia. This implies that their development must be very precisely coordinated in order to generate hindlimbs that allow locomotion in their ecological niche, as well as compatible genitals that allow mating', Dr Moises Mallo, director of the Gulbenkian Science Institute, who led the research, said.

As development progresses, pluripotent cells will transition to become specific cell types. This is dependent on the timing and concentration of signals which activate or inactivate specific genes. The TGF-beta family of growth factors is involved in a complex network of gene regulatory signals controlling cell growth and organ development. In response to these growth factors, cells produce corresponding receptors. One key receptor, Tgfbr1, is known to be involved in the development of the hindlimbs and genitalia.

Complete inactivation of Tgfbr1 results in no hindlimbs or genitalia development, and early activation results in those structures developing earlier than usual. Research from Dr Mallo's group, published in Nature Communications, discovered that inactivation of Tgfbr1 halfway through mouse development resulted in an additional pair of hindlimbs being formed in the place of the external genitalia.

Dr Mallo and his research group further investigated how this inactivation of Tgfbr1 might have led to these changes and they found alterations in the folding conformation of DNA. The folding of DNA is a crucial aspect of gene regulation as it affects which signals can activate or inactivate genes. In the case of inactivating Tgfbr1, the changes in DNA folding resulted in increased activation of hindlimb genes and inactivation of genital genes.

'Modulation of the response to those factors provided enough plasticity to generate the required diversity in the area of the hindlimb/genital area of tetrapods adapted to different ecological niches,' explained Dr Mallo.

Flexibility in gene expression during development is important for evolution, and the results of this study could provide information on the mechanisms by which the TGF-beta family of proteins influence gene expression during organ development.