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Int J Dev Biol
2013 Jan 01;5711-12:829-36. doi: 10.1387/ijdb.130109sc.
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Essential role of AWP1 in neural crest specification in Xenopus.
Seo JH
,
Park DS
,
Hong M
,
Chang EJ
,
Choi SC
.
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The neural crest (NC) comprises a transient and multipotent embryonic cell population, which gives rise to a wide variety of cell types, including craniofacial cartilage, melanocytes, and neurons and glia of the peripheral nervous system. The NC is induced by the integrated action of Wnt, FGF, and BMP signaling, and its cell fates are subsequently specified by a genetic cascade of specific transcription factors. Here we describe a critical role of AWP1 in NC induction during Xenopus early development. Xenopus AWP1 (XAWP1) was found to be expressed in the presumptive preplacodal ectoderm, neural tissue, and posterior dorsal mesoderm, but was absent in the neural fold along the anterior-posterior axis of the neurulae. Notably, XAWP1 was induced by FGF8a in naïve ectodermal tissue. XAWP1-depleted embryos exhibited defects in pigmentation, craniofacial cartilage, and in the dorsal fin. A knockdown of XAWP1 impaired both endogenous and the FGF8a or Wnt8-induced expression of NC markers without affecting mesoderm formation. Furthermore, NC induction inhibited by XAWP1 depletion was rescued by co-expression of activating forms of beta-catenin or TCF3. In addition, overexpression of XAWP1, in concert with BMP inhibition, induced the expression of neural plate border specifiers, Pax3 and Msx1, and these regulatory factors recovered NC induction in the XAWP1-depleted embryos. Beta-catenin stability and Wnt-responsive reporter activity were also impaired in AWP1-depleted cells. Taken together, these results suggest that XAWP1 functions as a mediator of Wnt signaling to regulate NC specification.
Fig. 1. Expression of the
XAWP1 gene. (A) RT-PCR
analysis showing the temporal
expression pattern of AWP1 in
Xenopus early development.
Ornithine decarboxylase (ODC)
serves as a loading control. –RT,
a stage 27 control embryo in the
absence of reverse transcriptase.
(B-I) Spatial expression
pattern of XAWP1. (B) Animallateral
view with the vegetal pole
to the bottom. (C) Vegetal-lateral
view with dorsal to the right.
(D) Sagittal section of a stage
10.5 gastrulae. Arrows in (C,D)
denote the dorsal blastopore
lip. Arrowheads indicate the
involuting dorsal mesoderm.
(E) Dorsal view with anterior to
the top. (F) Anterior view of the
embryo shown in (E) with dorsal
to the top. ne, neural ectoderm;
pe, preplacodal ectoderm. (G,H)
Transverse sections of the embryo in (E) at the levels indicated by the dashed lines. Arrows in (G) denote the preplacodal ectoderm. SM, somitic mesoderm;
nc, notochord; ov, otic vesicle; ba, branchial arch; kt, kidney tubule; sm, somites. (J) Four-cell stage embryos were injected in the animal pole
region as indicated with FGF8a (1 ng), XWnt8 (400 pg) and Xnr-1 (100 pg) and then animal caps were excised at stage 9 and cultured to stage 10.5 for
RT-PCR analysis. WE, a stage 10.5 whole embryo. Con. AC, uninjected control animal caps.
Fig. 2. A knockdown of XAWP1 disrupts neural crest derivatives. (A) Western blotting analysis of the specificity and efficiency of MOs. Four-cell stage embryos were injected in the animal pole region with XAWP1-Myc (200 pg) alone or with Co MO (30 ng), AWP1 MO1 (20 ng) or AWP1 MO2 (30 ng) and animal cap explants were then dissected at stage 9 and cultured to stage 11 for western blotting with an anti-myc antibody. ��-actin was used as a loading control. (B-G) Embryos were injected in the dorso-animal region of two blastomeres at the 8-cell stage with Co MO (10 ng), AWP1 MO1 (10 ng for C and D, 5 ng for E and G), AWP1 MO2 (30 ng) and hAWP1 (5 pg) as indicated and cultured to stage 38. Arrows and arrowheads indicate defects in the dorsal fin and pigmentation, respectively. sub, suboptimal. (H,I) Fourcell stage embryos were injected in the dorso-animal region of one blastomere with AWP1 MO1 (5 ng) or Co MO (10 ng) and then subjected to paraffin sectioning and eosin staining at stage 40. An arrow indicates the defective eye in the injected side. (J,K) One blastomere of a four-cell stage embryo was injected in the dorso-animal region with AWP1 MO1 (5 ng) or Co MO (10 ng) and then stained with alcian blue at stage 45. Arrows indicate severely disrupted ceratobranchial and Meckel’s cartilage in the injected side. cb, ceratobranchial cartilage; mc, Meckel’s cartilage; ch, ceratohyalcartilage.
Fig. 3. XAWP1 is required for neural crest induction. (A-I)
One blastomere of a four-cell stage embryo was injected
in the dorso-animal region with Co MO (5 ng), AWP1 MO1
(5 ng for B,C,E,F; 2 ng for G,I), AWP1 MO2 (20 ng) and
hAWP1 (2.5 pg) as indicated and then subjected to in situ
hybridization against Slug or Sox10 at stage 16. The injected
side was traced by lacZ staining. All embryos are shown in
dorso-anterior view with posterior to the top. (J) Four-cell
stage embryos were injected in the animal pole region with
the indicated combination of noggin (100 pg), XWnt8-CSKA
(100 pg), Co MO (40 ng), AWP1 MO1 (20 ng) and AWP1
MO2 (40 ng), and the animal caps were dissected at stage
9 and cultured to stage 16 for RT-PCR analysis. (K-P) Two blastomeres of a four-cell stage embryo were injected
in the dorsal-marginal region with Co MO (10 ng) or AWP1 MO1 (10 ng) and subsequently subjected to in situ
hybridization against Xbra at stage 10.5 or MyoD or Xnot at stage 14. Embryos are shown in vegetal view with
dorsal to the top (K,L) or in dorsal view with anterior to the top (M-P).
Fig. 4. XAWP1 regulates neural crest induction upstream of Msx1 and
Pax3. (A) Four-cell stage embryos were injected in the animal pole region
with DN BMP4 receptor (1 ng) with or without XAWP1 (0.5, 2 ng), and
the animal cap explants were excised at stage 9 and cultured to stage 11
for RT-PCR analysis. (B-D) Four-cell stage embryos were injected in the
dorso-animal region of one blastomere with AWP1 MO1 (5 ng) with or
without Msx1 (100 pg) or Pax3 (70 pg) as indicated and then subjected to
in situ hybridization against Sox10 at stage 16.
Fig. 5. Neural crest induction by FGF or Wnt signal requires XAWP1
function. (A-K) One blastomere of a eight-cell stage embryo was injected
in the dorso-animal region as indicated with FGF8a (20 pg), XWnt8-CSKA
(100 pg), DN XWnt8 (400 pg), XAWP1 (300 pg), pt ��-catenin (100 pg), VP16-
Tcf3 (50 pg), Co MO (5 ng) and AWP1 MO1 (5 ng) and then subjected to in
situ hybridization against Sox10 at stage 16. Arrows indicate the expanded
expression of Sox10.
Fig. 6. AWP1 is involved in Wnt/-catenin signaling. (A) Four-cell stage
embryos were injected in the animal pole region with the indicated combinations
of -catenin-myc (100 pg), Co MO (40 ng), AWP1 MO1 (40 ng),
hAWP1 (400 pg) and XAWP1 (400 pg), and the animal caps were excised
at stage 9 and cultured to stage 11 for western blotting. (B) HEK 293T
cell were transfected as indicated with N LRP6 (450 ng), XDsh (450 ng),
AWP1 siRNA (50 nM) and Co siRNA (50 nM). Three independent experiments
were performed and a single representative result is shown. Error
bars denote standard deviations.
zfand6 (zinc finger, AN1-type domain 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3, lateral view, animal up.
zfand6 (zinc finger, AN1-type domain 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 16, dorsal view, anterior up.
zfand6 (zinc finger, AN1-type domain 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, dorsal up, anteriorleft.
