The orphan genes are required for limb bud eversion in the embryo.

Background: Unlike but similar to other arthropod and vertebrate embryos, the flour beetle develops everted limb buds during embryogenesis. However, the molecular processes directing the evagination of epithelia are only poorly understood.

Results: Here we show that the newly discovered genes and are involved in regulating the directional budding of appendages. RNAi-knockdown of results in a variety of phenotypic traits. Most prominently, embryonic limb buds frequently grow inwards rather than out, leading to the development of inverted appendages inside the larval body. Moreover, affected embryos display dorsal closure defects. The genes are evolutionarily non-conserved, and their molecular function is not evident. We further found that , a highly-conserved gene known to be involved in actomyosin-dependent cell movement and cell shape changes, shows a -like RNAi-phenotype.

Conclusions: The similarity of the inverted appendage phenotype in both the - and the RNAi gene knockdown led us to conclude that the orphan genes act in a Rho-dependent pathway that is essential for the early morphogenesis of polarised epithelial movements. Our work describes one of the few examples of an orphan gene playing a crucial role in an important developmental process.