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The vertebrate neural crest is formed at the border between the neural plate and nonneural ectoderm during neurulation and eventually gives rise to a variety of cell types, including neurons, glia, facial chondrocytes and osteoblasts, and melanocytes. Although several secreted molecules, such as BMP, Wnts, FGF, and Noelin, have been implicated in neural crest formation, little is known about the precise intracellular mechanism underlying neural crest induction and differentiation. Here, we have identified a novel NK-1 class homeobox gene Nbx in Xenopus whose expression is correlated with neural crest formation. We also found that Nbx harbors an Eh1 domain and is a transcriptional repressor. Overexpression of Nbx suppressed neural plate makers and caused enhanced expression of the neural crest maker Slug. In contrast, the overexpression of a dominant negative form of Nbx during neurula stages suppressed the expression of the neural crest marker Slug and expanded neural markers such as Otx2 and Sox2. Taken together, we propose that Nbx is an essential transcriptional repressor required to permit neural crest induction by inhibiting the neural fate.
Fig. 2. Temporal and spatial expression of Nbx during Xenopus development. (A) Temporal expression patterns of Nbx. RNA was extracted from embryos
at the indicated stage of development. Nbx mRNA expression levels were measured by RT-PCR. The ubiquitous marker histone H4 served as a control. (B�I)
Spatial expression patterns of Nbx. Nbx expression was analyzed by whole-mount in situ hybridization. (B) Stage 12. Black arrowheads in (B) indicate outer
stripes, and white arrowheads indicate inner stripes of Nbx expression. (C) Stage 14. (D) Stage 18. (E) Stage 20. (F) Stage 25. (G) Stage 30. Arrow in (G)
indicates new expression of Nbx in mesencephalon, and a transverse section of this region is shown in (I). (H) A transverse section showing the outer stripes
at stage 14. (I) A transverse section of Nbx-expressing region in mesencephalon at Stage 30. (J�T) Comparison between recognized markers and Nbx
expression. Whole-mount double in situ hybridizations were performed with a digoxigenin-labeled Nbx probe and a fluorescein-labeled probe for another
marker. (J) Nbx (purple) and Otx2 (blue) at stage 14. (K) Nbx (purple) and Sox2 (blue) at stage 14. (L) Nbx (purple) and N-CAM (blue) at stage 16. (M, N)
Nbx (purple) and Slug (blue) at stage 16. The 1 in (M) indicates the region where only Slug was expressed; the 2 indicates where Slug and Nbx were
coexpressed (a transverse section of this region is shown in N); and the 3 indicates where only Nbx was expressed. (O) Nbx (purple) and Delta-1 (blue) at
stage 16. (P) Nbx (purple) and -tubulin (blue) at stage 16. (Q) A schematic representation of the expression of Nbx and -tubulin. (R) An enlarged view
of the outer stripe region at stage 16. Nbx (purple) and -tubulin (blue). (S, T) Nbx (purple) and - tubulin at stage 25. The transverse section of posterior
region is shown in (T).
Fig. 3. Nbx expression coincide with neural and neural crest formation. We injected the mRNAs to several genes into embryos and examined the effect of
their forced overexpression on Nbx expression by whole-mount in situ hybridization. (A) BMP4 (200 pg) reduced Nbx expression. (B) Overexpression of
tBR (500 pg) by injection into the ventral region expanded Nbx expression into the induced secondary axis. (C) Xngnr1 (100 pg) reduced Nbx expression.
(D) Xngnr1 (100 pg) induced ectopic -tubulin expression. (E, F) Xotch E (200 pg) injected embryos. (E) Xotch E reduced Nbx expression. (F) Xotch E
reduced -tubulin expression. (G) Zic3 (200 pg) caused ectopic Nbx expression (black arrowheads). The ectopic expression was limited to the posterior region
of the injected embryos (white arrowhead). (H) Zic3 (200 pg) caused ectopic Slug expression. (I) Zic5-ZFC (200 pg) reduced Nbx expression. (J) Zic5-ZFC
(200 pg) reduced Slug expression. (K) Zic5-ZFC (200 pg) reduced Sox2 expression.
Fig. 4. Nbx acts as a transcriptional suppressor. (A) Schematic representation
of Nbx, VP-Nbx, and EnR-Nbx constructs. (B) Phenotypes of Nbx
(200 pg)-injected embryos. The Nbx injection into the dorsal side caused a
head defect. (C) Uninjected embryos developed normally. (D, E) Wholemount
in situ hybridization analysis of Nbx (200 pg)- and -gal (100
pg)-injected embryos at stage 11. Nbx suppressed the organizer markers
Otx2 (D) and goosecoid (E). (F, G) Whole-mount in situ hybridization
analysis of -gal (100 pg)-injected embryos. -gal injection did not affect
the expression of Otx2 (F), goosecoid (G). (H) Phenotype of VP-Nbx (100
pg)-injected embryo. The VP-Nbx injection into the ventral side caused
ectopic cement gland formation (arrowhead in H). (I:) Uninjected embryos
developed normally. (J�M) Whole-mount in situ hybridization analysis of
VP-Nbx (100 pg)- and -gal (100 pg)-injected embryos. VP-Nbx injection
into the dorsal side led to the expanded expression of Otx2 (J) and
goosecoid (K). VP-Nbx injection into the ventral side resulted in the
ectopic expression of Otx2 (L) and goosecoid (M) at the injected region.
(N) Phenotype of EnR-Nbx (100 pg)-injected embryo. The injection caused
a head defect. (O:) Uninjected embryos developed normally.
Fig. 5. Overexpression of Nbx in lateral neural plate suppressed neural
plate makers and caused enhanced neural crest formation. (A) Schematic
representation in the Nbx construct. (B�I) Whole-mount in situ hybridization
analysis of Nbx-injected embryos. Each panel shows two embryos with
the same treatment. (B�D) Expression of neural plate makers. Overexpression
of Nbx suppressed neural plate makers Otx2 (B), N-CAM (C), and
Sox2 (D). (E) Overexpression of Nbx expanded the expression of epidermal
keratin into the neural plate. (F�I) Expression of neural crest maker Slug.
Slug was reduced at stage 15 (F), but enhanced Slug expression was
observed at injected region at stage 20 (G, I) and stage 25 (H), (J) RT-PCR
analysis of gene expression in the animal caps at stage 20 equivalent that
were injected with 300 pg tBR mRNA (lane 1), coinjected with 300 pg tBR
and 100 pg Nbx mRNA (lane 2), uninjected (lane 3), and injected with 100
pg Nbx mRNA alone (lane 6). The expression of makers in sibling whole
embryo (lane 4). The control reactions with no reverse transcription step
(lanes 5 and 7). (K) Animal caps coinjected with 300 pg tBR and 100 pg
Nbx contained melanophores at stage 40 equivalent (Black spots in K).
(Blown pigments were not melanophores, they were pigment granules
derived from the egg.) (L) Uninjected animal caps at stage 40 equivalent.
Fig. 6. Overexpression of VP-Nbx-GR with DEX addition from stage 13
suppressed neural crest induction and expanded neural plate markers.
(A) Schematic representation of the VP-Nbx-GR construct. (B�I) Wholemount
in situ hybridization analysis of VP-Nbx-GR-injected embryos with
DEX addition from stage 13 (B, D, F, H) and without DEX addition (C, E,
G, I). Each panel shows two embryos with the same treatment. (B) Overexpression
of VP-Nbx-GR with DEX addition from stage 13 suppressed
induction of neural crest maker Slug. (C) The VP-Nbx-GR injection without
DEX addition did not affect Slug induction. (D, F, H) Overexpression
of VP- Nbx-GR with DEX addition from stage 13 expanded the expression
of the neural plate marker Sox2 (D) and the anterior neural plate marker
Otx2 (F) at the injected region and suppressed the epidermal marker
epidermal keratin (H). (E, G, I) The VP-Nbx-GR injection without DEX
addition did not affect Sox2 (E), Otx2 (G), or epidermal keratin (I). (J)
Coinjection of VP-Nbx-GR (100 pg) and Nbx (200 pg) and -gal (100 pg)
with DEX addition from stage 13. Nbx rescued Sox2 expansion by VPNbx-
GR.
nkx1-2 (NK1 homeobox 2) gene expression in a Xenopus laevis embryo, as assayed by in situ hybridization, NF stage 14. Dorsal view, anterior up.
nkx1-2 (NK1 homeobox 2) gene expression in a Xenopus laevis embryo, as assayed by in situ hybridization, NF stage 20. Dorsal view, anterior up.
nkx1-2 (NK1 homeobox 2) gene expression in a Xenopus laevis embryo, as assayed by in situ hybridization, NF stage 30. Lateral view, anteriorleft, dorsal up.
