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Dev Dyn
2011 Dec 01;24012:2601-12. doi: 10.1002/dvdy.22721.
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Kazrin, and its binding partners ARVCF- and delta-catenin, are required for Xenopus laevis craniofacial development.
Cho K
,
Lee M
,
Gu D
,
Munoz WA
,
Ji H
,
Kloc M
,
McCrea PD
.
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The novel adaptor protein Kazrin associates with multifunctional entities including p120-subfamily members (ARVCF-, delta-, and p0071-catenin). Critical contributions of Kazrin to development or homeostasis are indicated with respect to ectoderm formation, integrity and keratinocyte differentiation, whereas its presence in varied tissues suggests broader roles. We find that Kazrin is maternally loaded, is expressed across development and becomes enriched in the forming head. Kazrin's potential contributions to craniofacial development were probed by means of knockdown in the prospective anterior neural region. Cartilaginous head structures as well as eyes on injected sides were reduced in size, with molecular markers suggesting an impact upon neural crest cell establishment and migration. Similar effects followed the depletion of ARVCF (or delta-catenin), with Kazrin:ARVCF functional interplay supported upon ARVCF partial rescue of Kazrin knockdown phenotypes. Thus, Kazrin and its associating ARVCF- and delta-catenins, are required to form craniofacial tissues originating from cranial neural crest and precordal plate.
Figure 1. xKazrin expression during Xenopus development. A–C: xKazrin spatio-temporal expression was assessed by means of in situ hybridization of late gastrula (A), neurula (B), and tadpole stage embryos (C). The probe consisted of Dig-labeled anti-sense RNA of full-length xKazrinA, while the negative control was the sense counterpart. D: Kazrin mRNA expression in the head region of tadpoles shown in (C) were visualized using microtome sectioning. Brain neural tube (NEU) and eye (Eye) are indicated. E: Extensive Kazrin mRNA signal was obtained upon prolonged substrate reaction. Right panel shows enlarged image of the head region. Pharyngeal arch (PA) is indicated by an arrowhead. F:Xenopus embryo lysates taken from varying stages as indicated (0.5 embryo/ lane) were subject to SDS-PAGE and immuno-blotted for xKazrin (polyclonal antibody directed against carboxyl 147 amino acids of xKazrinA and IgG purified), or for GAPDH (glyceraldehyde-3-phosphate dehydrogenase; loading control (bottom panel). G: Immuno-blot of Xenopus adult tissues (SDS-soluble fraction) as indicated. xKazrin was detected as 47 kDa (KazrinA or B) and 40 kDa (KazrinC/D) isoforms. Equal total protein was loaded per lane, and actin used as a loading control. Molecular weights are indicated in kilodaltons (kDa).Download figure to PowerPoint
Figure 6. xKazrin or xARVCF depletion perturbs the expression of neural crest cell markers. A: The indicated morpholinos (20 ng) were injected into the animal region of one dorsal blastomere (animal region) of four-cell embryos. At early neurula stages (stage 15), embryos were fixed and neural crest cells visualized using whole-mount in situ hybridization for two neural crest markers: Twist and FoxD3. B,C: Embryos were fixed at early tail bud stages, stages 20 and 22 (respectively B and C) and whole-mount in situ hybridization for twist was performed. Unseparated branchial neural crest cell streams are indicated by brackets. Asterisks (*) indicate the injected side. Br, Branchial neural crest cells; aBR, anterior branchial neural crest cells; pBR, posterior branchial neural crest cells; H, Hyoid neural crest cells; M, Mandibular neural crest cells.Download figure to PowerPoint
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