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Dev Dyn
2011 Dec 01;24012:2680-7. doi: 10.1002/dvdy.22765.
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ARVCF depletion cooperates with Tbx1 deficiency in the development of 22q11.2DS-like phenotypes in Xenopus.
Tran HT
,
Delvaeye M
,
Verschuere V
,
Descamps E
,
Crabbe E
,
Van Hoorebeke L
,
McCrea P
,
Adriaens D
,
Van Roy F
.
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The 22q11.2 deletion syndrome is a common dominant genetic disorder characterized by a heterozygous deletion of a cluster of genes on chromosome 22q11.2. TBX1, a transcription factor belonging to the T-box gene family, is a key player in the syndrome. However, heterozygosity of Tbx1 in mouse models does not fully recapitulate the phenotypes characteristic of the disease, which may point to the involvement of other genes in the deleted chromosomal region. Hence, we investigated the contribution of the catenin ARVCF, another gene that is deleted in 22q11.2DS. During Xenopus development, ARVCF mRNA is expressed in the pharyngeal arches and depleting either ARVCF or Tbx1 results in delayed migration of the cranial neural crest cells and in defects in the craniofacial skeleton and aortic arches. Moreover, double depletion of ARVCF and Tbx1 revealed that they act cooperatively, indicating that decreased ARVCF levels may also contribute to 22q11.2DS-associated phenotypes.
Figure 1. Determination of the expression pattern of ARVCF by whole-mount in situ hybridization. X. laevis embryos at different developmental stages were examined using an antisense probe directed against ARVCF. a: Lateral view of stage-18 embryo with dorsal side at the top, anterior to the left. The arrowhead shows enriched transcripts in the neural plate and bordering neural crest. b: Lateral view of a stage-26 tailbud with the anterior towards the left. The line shows the position of the section shown in panel c. c: Horizontal section through the head region of a stage-26 tailbud showing the presence of ARVCF transcripts in the cranial neural crest cells (yellow arrowhead). d: A similar section of a stage-26 embryo stained with the neural crest marker Twist as a reference. e: Stage-31 early tadpole showing enriched ARVCF expression in the head region and the heart (yellow arrowhead).Download figure to PowerPoint
Figure 2. Defects in the aortic arches and the craniofacial skeleton after depletion of ARVCF or Tbx1. A: To evaluate aortic arch formation, 50 ng of ARVCF MO or control MO was injected at the eight-cell stage (d–f), or 50 ng Tbx1 MO (g–i) or control MO (a–c) at the one-cell stage. To visualize the aortic arches, embryos were fixed at stage 48 and stained with the von Willebrand factor antibody. This antibody stains mainly the aortic arches (aa) connected to the heart (arches are numbered). At this stage, three pairs of aortic arches are normally present: arches 3, 4, and 5. Depletion of ARVCF frequently results in total loss of aortic arch 5 and less developed arches 3 and 4 (d–f). Knockdown of Tbx1 also results in defects in aortic arch development (g–i). Again, aortic arch 5 is often missing while the others are less developed. B: Embryos were injected at the four- or eight-cell stage in two dorsal blastomeres with 50 ng ARVCF MO (c,d), or at the one-cell stage with 50 ng Tbx1 MO (e,f) or control MO (a,b). Tadpoles are shown at stage 48 either alive (left) or fixed and stained with Alcian Blue to reveal the cartilage structures (right). Knockdown of ARVCF or Tbx1 results in malformation of the cartilage structures of the head. Meckel's cartilage (MC) and the ceratohyalcartilage (CH) are malformed. The ceratobranchial cartilage (CB) is also affected in tadpoles injected with ARVCF MO or Tbx1 MO. C: The heads of embryos injected with ARVCF MO or Tbx1 MO were significantly narrower than those of control embryos (P < 0.0001), and this phenotype could be rescued by coinjection with 20 pg of ARVCF RNA that lacks the 5′UTR and can not be targeted by the morpholino (P < 0.0001).Download figure to PowerPoint
Figure 3. Three-dimensional reconstructions of CT-scanning images of embryos injected with ARVCF or Tbx1 MO (scale bar = 0.25 mm). Embryos at the four-cell stage were injected in one dorsal blastomere with 40 ng of Control MO, ARVCF MO, or Tbx1 MO, together with a fluorescent tracer. Tadpoles were fixed at stage 48 and subjected to CT-scanning. All reconstructions are shown in ventral view. Structures visible are the eyes (open arrowhead), the olfactory placode and vomeronasal organ (asterisk), the cranial muscles (closed arrowheads), and the branchial basket (arrows). The 3D-reconstructions reveal that injection of ARCVF MO or Tbx1 MO results in the reduction of the gill apparatus (arrows).Download figure to PowerPoint
Figure 4. Effects of ARVCF or Tbx1 depletion on neural crest cell specification and migration. Embryos were injected unilaterally with 50 ng ARVCF MO, Tbx1 MO, or control MO, or with a combination of the two MOs at a subphenotypic dose (20 ng). RNA encoding β-galactosidase was coinjected as a tracer. Embryos were fixed at stage 20, 27, and 32 and processed for in situ hybridization with a probe directed against Twist. Injection of ARVCF MO or Tbx1 MO does not interfere with the induction of neural crest cells (NCC) and the mandibular (m), hyoid (h), and branchial (br) streams can be easily discerned at stage 20. The injected side is marked by an asterisk. However, at stages 20 and 27, the migration of Twist-positive NCCs is delayed in the side injected with ARVCF MO or Tbx1 MO compared to the non-injected side. The delay is even greater in embryos injected with a combination of ARVCF and Tbx1 MO at a sub-phenotypic dose (20 ng). At stage 32, Twist patterns are largely identical in the injected and non-injected sites, indicating that all CNCCs have ultimately reached their final destination.
Figure 5. Cooperative effect of the depletion of ARVCF and Tbx1. A: Fifty nanograms of control MO (a–c) or combined subphenotypic doses of ARVCF MO and Tbx1 MO (d–f) were injected at the one-cell stage. Tadpoles are shown alive at stage 48 (a,d), fixed and stained with Alcian Blue to reveal the cartilage structures (b,e), or stained with the von Willebrand factor antibody to reveal the aortic arches (c,f). Simultaneous depletion of ARVCF and Tbx1 results in malformation of cartilage, as seen in a living embryo (d) and after Alcian Blue staining (e), compared with the control embryos (a, b). Depletion of both ARVCF and Tbx1 also results in defects in aortic arch formation and frequent absence of arch 5 (f). B: The cooperative effect was also manifested in the width of the head. Embryos injected with a combination of subphenotypic doses of ARVCF and Tbx1 MO had significantly narrower heads than tadpoles injected with either control, Tbx1, or ARVCF MO (P = 0.0017).Download figure to PowerPoint
arvcf (ARVCF, delta catenin family member) gene expression in Xenopus laevis embryos, NF stage 18, as assayed by in situ hybridization, dorsal view, anteriorleft.
arvcf ( ARVCF, delta catenin family member ) gene expression in Xenopus laevis embryos, NF stage 26/27, as assayed by in situ hybridization, lateral view, anteriorleft, dorsal up.
arvcf (ARVCF, delta catenin family member ) gene expression in Xenopus laevis embryos, NF stage 31, as assayed by in situ hybridization, lateral view, anteriorleft, dorsal up.
