Abstract
ABSTRACTIn 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 forelimb bud or reduction of the forelimb field along the anterior-posterior axis. Further functional analyses revealed that expression of Hoxb genes and retinaldehyde dehydrogenase 2 (Raldh2), which are involved in limb positioning, are 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 in chicken embryos.
Highlights
Limb buds emerge as small bulges that protrude from the body trunk at discrete positions along the rostral-caudal axis
Transcripts of Cut homeobox transcription factor 2 (Cux2) were detected throughout the lateral plate mesoderm at Hamburger-Hamilton stage (HH) (Fig. S1A), and subsequently expression was increased in the forelimb-forming fields at early HH (20-somite stage; Fig. S1B)
Expression of Cux2 in the anterior part of the forelimb field gradually decreased, and Cux2 expression became restricted to the posterior part of the forelimb buds and interlimb flank region by HH 17; weak expression was detected in the posterior part of the hindlimb buds at the same stage (Fig. S1C)
Summary
Limb buds emerge as small bulges that protrude from the body trunk at discrete positions along the rostral-caudal axis. It has been proposed that the nested expression of Hox genes in the lateral plate mesoderm is related to the specification of position along the rostralcaudal axis to generate forelimb, interlimb and hindlimb regions (Burke, 2000; Burke et al, 1995; Cohn et al, 1995, 1997) Such a role of Hox genes in limb positioning is supported by mice lacking Hoxb, which show a rostral shift of the forelimb buds (Rancourt et al, 1995). Developmental analyses of chick and mouse embryos revealed that retinoic acid signaling and Hox proteins cooperatively activate Tbx transcription to induce forelimb bud formation (Nishimoto et al, 2015). Further functional analyses revealed that Cux directly activates the expression of Raldh and Hoxb genes, which are involved in the specification of the limb-forming fields These results suggest that Cux in the lateral plate mesoderm refines the forelimbforming fields via regulation of transcription of Raldh and Hoxb genes in chicken embryos
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