Fang X et al. (2004), Vertebrate development requires ARVCF and p120 ...

XB-ART-3736

J Cell Biol 2004 Apr 01;1651:87-98. doi: 10.1083/jcb.200307109.

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Vertebrate development requires ARVCF and p120 catenins and their interplay with RhoA and Rac.

Fang X , Ji H , Kim SW , Park JI , Vaught TG , Anastasiadis PZ , Ciesiolka M , McCrea PD .

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Using an animal model system and depletion-rescue strategies, we have addressed the requirement and functions of armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF) and p120 catenins in early vertebrate embryogenesis. We find that xARVCF and Xp120 are essential to development given that depletion of either results in disrupted gastrulation and axial elongation, which are specific phenotypes based on self-rescue analysis and further criteria. Exogenous xARVCF or Xp120 cross-rescued depletion of the other, and each depletion was additionally rescued with (carefully titrated) dominant-negative RhoA or dominant-active Rac. Although xARVCF or Xp120 depletion did not appear to reduce the adhesive function of C-cadherin in standard cell reaggregation and additional assays, C-cadherin levels were somewhat reduced after xARVCF or Xp120 depletion, and rescue analysis using partial or full-length C-cadherin constructs suggested contributory effects on altered adhesion and signaling functions. This work indicates the required functions of both p120 and ARVCF in vertebrate embryogenesis and their shared functional interplay with RhoA, Rac, and cadherin in a developmental context.

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Species referenced: Xenopus laevis
Genes referenced: actl6a akt1 arvcf cdh3 ctnnd1 inhba myc rac1 rhoa rhoa.2
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	Figure 3. xARVCF and Xp120 are essential to embryonic development. Embryos were depleted for xARVCF or Xp120 (20 ng morpholino injected into one dorsal cell at the 4-cell stage) or injected with 20 ng of standard control morpholino. Substantial rescues were effected via coinjection of morpholino with xARVCF or Xp120 in vitro transcribed mRNA (0.01 ng), indicating specificity of the depletion phenotypes. Embryos were scored at stage 12 (gastrula) for defects (histogram), which included partial/improper closure of the blastopore. As depicted, depleted embryos at later stages (tailbud and tadpole) displayed a bowed appearance characteristic of reduced dorso-axial mesoderm elongation, which was likewise rescuable to the standard control (normal embryo) phenotype. (A) xARVCF depletions and rescue upon coinjection of xARVCF mRNA. (B) Xp120 depletions and rescue upon coinjection of Xp120 mRNA. See also combined depletion/rescue data presented in Tables I and II.
	Figure 5. C-Cadherin rescues xARVCF or Xp120 depletion. Embryos were injected with 20 ng of standard control morpholino or depleted for xARVCF or Xp120 (20 ng of respective morpholino injected into one dorsal cell at the 4-cell stage). Rescues were effected by coinjection of xARVCF or Xp120 morpholino with C-cadherin in vitro transcribed mRNA (2 ng) and scored at stage 12 (gastrula) for defects including partial/improper closure of the blastopore. Resulting gastrulation phenotypes are shown in A with percentages presented in B. Depletion phenotypes and C-cadherin rescues were apparent at both gastrula and later developmental stages (tailbud and tadpole).
	Figure 7. DN-RhoA rescues xARVCF or Xp120 depletion. Embryos were injected with standard control morpholino or depleted for xARVCF or Xp120 (20 ng respective morpholino injected into one dorsal cell of 4-cell embryos). Rescues were effected by coinjection of xARVCF or Xp120 morpholino with xARVCF or Xp120 in vitro transcribed mRNA (0.02 ng, self-rescue positive control) or with DN-RhoA mRNA (0.5 pg) and scored at gastrula stages 102. (A) DN-RhoA rescues xARVCF depletion to almost self-rescue and standard control injection levels. (B) DN-RhoA rescues Xp120 depletion to self-rescue and standard control injection levels. As depicted, depletion phenotypes and DN-RhoA rescues were apparent at both gastrula and later developmental stages (tailbud and tadpole). See also aggregate depletion/rescue data presented in Tables I and II.
	Figure 8. DA-Rac rescues xARVCF or Xp120 depletion. Embryos were injected with standard control morpholino or depleted for xARVCF or Xp120 (20 ng respective morpholino injected into one dorsal cell of 4-cell embryos). Rescues were effected by coinjection of xARVCF or Xp120 morpholino with 0.5 pg DA-Rac and scored at gastrula stages 102. DA-Rac rescues xARVCF (A) or Xp120 (B) depleted embryos to substantial extents relative to standard control injections. As depicted, depletion phenotypes and DA-Rac rescues were apparent at both gastrula and later developmental stages (tailbud and tadpole). See also combined depletion/rescue data presented in Tables I and II.
	Figure 1. Morpholino-directed depletion of xARVCF or Xp120. xARVCF and Xp120 protein from total embryo extracts isolated at the indicated developmental stages were detected via SDS-PAGE/Western blotting using anti-xARVCF or anti-Xp120 polyclonal antibodies. Embryos at the 1-cell stage were injected with a combination of either xARVCF morpholinos (5 ng each of xARVCF MO I and MO II, total delivery) or of Xp120 morpholinos (40 ng each of Xp120 MO I and MO II, total delivery), while the standard control morpholino was injected at matched doses. Protein loads were assessed by Western blotting samples for actin. The anti-Xp120 antibody recognizes three major Xp120 isoforms. Developmental stages presented include 6 or 7 (blastula, before zygotic transcription/mid-blastula transition), 11 or 12 (late gastrula), and 20 (early tailbud). When comparing experimental samples with their stage-matched standard control, depletion efficiencies ranged from 20â90% depending on factors including embryo batch variation, the morpholinos used, and morpholino dose. xARVCF displayed an SDS-PAGE mobility of â¼100 kD, whereas the Xp120 isoforms migrated between 92â98 kD; p120's lowermost band/isoform may arise as a consequence of protein degradation given its appearance in some but not all experiments.
	Figure 2. xARVCF or Xp120 depletion results in reduced C-cadherin levels. X. laevis C-cadherin protein in total embryo extracts was detected using antiâC-cadherin polyclonal antibody. Embryos were injected at the 1-cell stage with 20 ng each of xARVCF morpholino, Xp120 morpholino, or standard control morpholino and harvested at developmental stages 11 (gastrula) or 20 (early tailbud). Protein loads were assessed by Western blotting samples for actin. C-Cadherin reduction after embryonic depletion of xARVCF or Xp120 ranged from 10â50% depending on factors including embryo batch variation, the morpholinos used, and morpholino dose.
	Figure 4. Xp120 partially rescues depletion of xARVCF, and xARVCF largely rescues codepletion of xARVCF and Xp120. (A) Gastrulation defects in embryos depleted for xARVCF (20 ng morpholino injected into one dorsal blastomere at the 4-cell stage) were largely rescued upon coinjection of xARVCF in vitro transcribed mRNA (0.01 ng, positive control for rescue) as well as substantially cross-rescued upon coinjection of Xp120 mRNA (0.01 ng). (B) Gastrulation defects in embryos codepleted for xARVCF and Xp120 (20 ng of respective morpholino coinjected into one dorsal blastomere at the 4-cell stage) were largely rescued upon coinjection of xARVCF and Xp120 in vitro transcribed mRNAs (0.02 ng of respective mRNA, positive control for codepletion rescue) or upon coinjection of xARVCF mRNA alone (0.02 ng).
	Figure 6. xARVCF and/or Xp120 depletion inhibits convergent-extension of naive ectoderm explants. Embryos were injected with standard control morpholino or depleted for xARVCF and/or Xp120 (40 ng of respective morpholino injected into animal hemisphere of each cell of 2-cell embryos). Animal caps were subsequently isolated from stage 8 (blastula) embryos and incubated in the absence or presence of 20 ng/ml human activin A. Elongations were scored on a scale of 0â3, respectively reflecting no, slight, moderate, or full extension relative to activin-treated caps injected with the standard control morpholino. Total scores were calculated by multiplying the number of caps receiving a particular score by that score, and then summing such products across the four score categories. Depletion of xARVCF and/or Xp120 significantly reduced the fraction of caps displaying strong elongations. The chart displays aggregate data from two experiments. The asterisk notes that the score distribution of the xARVCF + Xp120 double depletion resembles that of xARVCF or Xp120 single depletions, although fewer total caps were examined.
	Figure 9. Overexpression of xARVCF produces a dendritic phenotype in NIH-3T3 cells that is rescuable by DA-RhoA. NIH-3T3 cells were transfected with xARVCF, together with pcDNA3 (control vector) or pcDNA3-RhoA-(V12)-Myc (DA-RhoA). After 24 h, cells were fixed in PFA and double labeled for immunofluorescent microscopy using anti-xARVCF (polyclonal) and anti-Myc (monoclonal) antibodies to detect xARVCF and RhoA, respectively. (a and c) Cells transfected with xARVCF and stained for xARVCF. (b and d) The same cells stained for the presence of RhoA (Myc epitope-tag). The dendritic phenotype is observed in xARVCF-transfected cells (a), while the reversal of this phenotype upon coexpression of V12-RhoA (DA-RhoA) is evident in panel c.

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