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Fig. 2. Irx MOs causes antero-posterior neural defects. Dorsal views of Xenopus tropicalis embryos at early neurula (stg 14–15; A–R) or tadpoles (stg 42; S–X) injected with 10 ng of MOIrx1 (A, G, M, S), MOIrx2 (B, H, N, T), MOIrx3 (C, I, O, U), MOIrx4 (D, J, P, V), MOIrx5 (E, K, Q, W) or a mix of 2 ng of each MO (F, L, R, X). The MOs were co-injected with LacZ mRNA as a tracer. In all embryos, red or black arrowheads point at the injected or control side, respectively. (A–F) In Irx morphant embryos, Gbx2 is reduced and shifted caudally. (G–L). Impairment of Irx genes also caused Otx2 posterior displacement and Krox20 downregulation but does not affect Cad3 expression. (M–R) In contrast, Wnt4 expression in the spinal cord, as well as in the midbrain, is reduced. A caudal shift of the midbrain is also observed in some cases. (S–X) Later, all injected embryos show brain malformations and some of them eye defects. |
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Display additional annotations [+]
Gene |
Clone |
Species |
Stages |
Anatomy |
six3
|
|
laevis
|
NF stage 14
|
anterior placodal area
|
irx3
|
|
laevis
|
NF stage 14
|
diencephalon
,
neural plate
,
presumptive midbrain-hindbrain boundary
|
pax6
|
|
laevis
|
NF stage 16
|
diencephalon
,
hindbrain
,
pre-chordal neural plate
,
chordal neural plate
,
anterior neural fold
|
wnt4
|
|
laevis
|
NF stage 14
|
midbrain
,
spinal cord
,
chordal neural plate
,
presumptive midbrain-hindbrain boundary
|
sox2
|
|
laevis
|
NF stage 18
|
brain
,
forebrain
,
midbrain
,
hindbrain
,
spinal cord
,
[+]
|
wnt1
|
|
laevis
|
NF stage 14
|
diencephalon
,
hindbrain
,
presumptive midbrain-hindbrain boundary
|
hnf1b
|
|
laevis
|
NF stage 16
|
intermediate mesoderm
|
pax2
|
|
laevis
|
NF stage 14
|
pre-chordal neural plate
,
optic field
|
neurog2
|
|
laevis
|
NF stage 18
|
neuron
|
tubb2b
|
|
laevis
|
NF stage 18
|
anterior neural fold
,
neuron
|
rax
|
|
laevis
|
NF stage 14
|
anterior neural fold
|
en2
|
|
laevis
|
NF stage 14
|
midbrain
,
presumptive midbrain-hindbrain boundary
|
egr2
|
|
laevis
|
NF stage 14
|
chordal neural plate
,
rhombomere R3
,
rhombomere R5
|
fezf2
|
|
laevis
|
NF stage 14
|
anterior neural fold
|
|
|
Fig. 3. Irx genes are required for neural patterning. All panels, except (M) that show a stage 18 embryo, show dorsal views of stage 14–16 embryos co-injected with a mix of all five Irx MOs and LacZ mRNA. Black and red arrowheads point at the control or injected side, respectively. (A–C) Forebrain markers Rx1, Six2 and Fezf2 are expanded posteriorly (compare red with black arrowheads). (D) In addition, the anterior Pax6 domain is also shifted caudally at the expenses of the midbrain territory that lack the expression of this gene (compare red with black arrowhead). (E–F) The expression of Wnt1 and Irx3 in the posterior diencephalon is strongly downregulated (compare red with black arrowheads). (G–I) The midbrain is also reduced in the Irx morphant, as determined by the expression of En2, Pax2 and Wnt4. In addition, Wnt4 expression in the spinal cord in strongly impaired (I). (J–L) The rhomboencephalon markers Gbx2, Krox20 and Nhf1β are also downregulated in the injected embryos. (M) At stage 18, Sox2 expression shows and altered morphology of brain structures in the Irx impaired side. (N, O) The proneural gene Ngnr1 (N) and the primary neurons markers Ntubulin (O) are also downregulated in the injected side. |
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Fig. 4. Rescue of Irx MOs defects. All embryos are at stage 14–16. (A, C, E and G) are anterior and (B, D, F, H) are dorsal views, respectively. Black and red arrowheads point at control and injected sides, respectively. Embryos injected with MT-Irx1-GR (A, C, E, G) or MT-Irx3-GR (B, D, F, H) mRNAs alone (A–D) or with a mix of Irx MOs (E–H). (A–D) In the presence of Dexamethasone, the injected mRNAs caused expansion of Ngnr1 (C) and Gbx2 (D). This is not observed in the absence of the hormone (A, B). (E–H) In embryos co-injected with MT-Irx-GR mRNA and Irx MOs, in the absence of Dex, Ngnr1 (E) and Gbx2 (F) are downregulated. (G, H) This phenotype is rescued in the presence of Dex. |