Cux2 Refines the Forelimb Field by Controlling Expression of Raldh2 and Hox genes

In vertebrates, two pairs of buds that give rise to the fore- and hindlimbs form at discrete positions along the rostral-caudal axis of the body. The mechanism responsible for the positioning of the limb buds is still largely unknown. Here we show a novel function for Cut homeobox transcription factor 2, Cux2, the ortholog of Drosophila cut, in refining the forelimb field during chick development. Cux2 is expressed in the forelimb field before the emergence of the limb buds. Knocking down the expression of Cux2 using small interfering RNA (siRNA) resulted in a caudal shift of the forelimb bud, whereas misexpression of Cux2 or the constitutively active Cux2-VP16 caused a rostral shift of the wing bud or reduction of the forelimb field along the anterior-posterior axis. Chromatin immunoprecipitation sequencing and transcriptome analyses of the lateral plate mesoderm identified potential targets of Cux2. Further functional analyses revealed that expression of retinaldehyde dehydrogenase 2 (Raldh2) and Hoxb genes, which are involved in limb positioning, is directly activated by Cux2 in the lateral plate mesoderm. Our data suggest that Cux2 in the lateral plate mesoderm refines the forelimb field via regulation of Raldh2 and Hoxb genes. Overall design: RNA-seq analysis of anterior part of limb buds or interlimb flank mesoderm from stage 19 chick embryos.