Chicken Transcription Factor AP-2: Cloning, Expression and Its Role in Outgrowth of Facial Prominences and Limb Buds

Abstract

Embryonic facial development in chick embryos involves a sequential activation of genes that control differential growth and patterning of the beak. In the present study we isolate one such gene, the transcription factor, AP-2, that is known to be expressed in the face of mouse embryos. The protein sequence of chick AP-2alpha is 94% homologous to human and mouse AP-2. Wholemount in situ hybridization with a probe for chick AP-2 identifies expression from primitive streak stages up to stage 28. The most striking expression patterns in the head are during neural crest cell migration when AP-2 transcripts follow closely the tracts previously mapped for neural crest cells. Later, expression in the facial mesenchyme is strongest in the frontonasal mass and lateral nasal prominences and is downregulated in the maxillary and mandibular prominences. Once limb buds are visible, high expression is seen in the distal mesenchyme but not in the apical ectodermal ridge. The expression patterns of AP-2 in stage 20 embryos suggested that the gene may be important in "budding out" of facial prominences and limb buds. We implanted beads soaked in retinoic acid in the right nasal pit of stage 20 embryos resulting in a specific inhibition of outgrowth of the frontonasal mass and lateral nasal prominences. AP-2 expression was completely down-regulated in the lateral nasal within 8 hr of bead application. In addition, the normal up-regulation of AP-2 in the frontonasal mass did not occur following retinoic-acid treatment. There was an increase in programmed cell death around the right nasal pit that accompanied the down-regulation of AP-2. Prominences whose morphogenesis were not affected by retinoic acid did not have altered expression patterns. We removed the apical ectodermal ridge in stage 20 limb buds and found that AP-2 expression was partially downregulated 4 hr following ridge removal and completely downregulated 8 hr following stripping. Application of an FGF-4 soaked bead to the apex of the limb bud maintained AP-2 expression. Thus AP-2 is involved in outgrowth and could be regulated by factors such as FGFs that are present in the ectoderm of both the face and limb.

Frontal nasal prominence expression driven by Tcfap2a relies on a conserved binding site for STAT proteins

“…Although an in vivo role for Stats downstream of FGFR2 has not yet been shown, mouse models of craniosynostosis containing altered FGFR2, display several phenotypes that overlap with those observed in FKO, Wnt1-Cre; Floxdel/KI, or Tcfap2a ϩ/Ϫ mice including shortened snouts, dental malocclusion, widespaced eyes, and cleft palate (Zhang et al, 1996;Nottoli et al, 1998;De Moerlooze et al, 2000;Hajihosseini et al, 2001;Eswarakumar et al, 2002Eswarakumar et al, , 2004Chen et al, 2003;Brewer et al, 2004;Nelson and Williams, 2004;Wang et al, 2005). Thus, the presence of a STAT site in the FNP/LBM-specific enhancer likely places AP-2␣ downstream of FGFR signaling in the FNP, which is consistent with other studies showing FGF-dependent changes of Tcfap2a in the craniofacial region (Shen et al, 1997;Cordero et al, 2004).…”

“…Relative intensity and domain of expression is indicated by (ϩ), while (Ϫ) indicates that no expression was observed in the transgenic embryos. The notation "nr" indicates that the number of transgenic embryos that failed to give specific staining was not recorded, but was greater than 2. mouse and chick face (Mitchell et al, 1991;Shen et al, 1997), the analysis was initiated using whole cell protein extracts derived from the chick FNP mesenchyme. Differences in the mechanisms of embryogenesis between mouse and chick, particularly the contrast between in utero versus in ovo development, make the chick a more efficient and cost-effective system for an initial analysis of the DNA binding proteins present in the early face.…”

“…Several Sox factors are known to play critical roles in craniofacial and limb skeletogenesis and are good candidates to regulate Tcfap2a (Bi et al, 1999(Bi et al, , 2001Smits et al, 2001;Sock et al, 2004). The Group E factor, Sox9, is an enticing candidate, given both its role in chondrogenesis and its responsiveness to retinoic acid (Bi et al, 1999(Bi et al, , 2001Sekiya et al, 2000;Akiyama et al, 2002Akiyama et al, , 2004MoriAkiyama et al, 2003;Sahar et al, 2005), an agent known to alter AP-2␣ expression in vitro and in vivo (Williams et al, 1988;Lü scher et al, 1989;Philipp et al, 1994;Shen et al, 1997). Another Group E candidate, Sox8, is expressed coincident with Tcfap2a in the face and limbs (Wegner, 1999;Schepers et al, 2000).…”

Identification and embryonic expression of a new AP‐2 transcription factor, AP‐2ϵ

“…Expression analyses in mammals, birds, and amphibians showed that AP-2 genes are involved in the formation of craniofacial, neuroectodermal, ectodermal structures, limb buds, and urogenital tissues (Mitchell et al, 1991;Snape et al, 1991;Moser et al, 1995Moser et al, , 1997aShen et al, 1997;Epperlein et al, 2000). The functional relevance of AP-2 proteins for mammalian development was demonstrated by the lethal phenotypes of AP-2␣, AP-2␤, and AP-2␥ mouse mutants.…”

Onset of ETS expression is not accelerated by premature exposure to signals from limb mesenchyme

“…To perturb the spatiotemporal pattern of retinoid signaling in the limb, beads (AG1-X2, formate form, Bio-Rad) were soaked in 5 mg/ml all trans-retinoic acid (Sigma) dissolved in dimethylsulfoxide and rinsed in DMEM (Gibco) plus 10% fetal calf serum (Gibco) as described in Shen et al (1997). Two or three beads, each approximately 100 m in diameter, were implanted into the proximal limb bud near the prospective crural or sciatic plexus region of St. 21.5-22.5 embryos, as illustrated in Figure 4A.…”