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Fig. 5. Hnf1b but not osr2 and lhx1 expression is inhibited in the KF [kidney field] of Pax8 morphant at neurula stage. (A) In situ hybridization for lhx1, osr2 and hnf1b genes. Embryos were injected with MoPax8 at the 4-cell stage, in the equatorial region of both left blastomeres. At neurula stage 18, they were fixed, transversely bisected at the level of the pronephric area and analyzed by in situ hybridization with the indicated probes. For each embryo, the dotted line indicates the frontier between the injected side (on the left) and the uninjected side (on the right). Hnf1b expression is inhibited in the pronephric area (arrow) on the injected side. (B) RT-qPCR analysis of lhx1, osr2 and hnf1b gene expression in KF explants dissected at neurula stage 18 from MoC and MoPax8 injected embryos. Unlike lhx1 and osr2 expression which is not modified, hnf1b expression is downregulated in response to Pax8 depletion. Average values from four independent experiments. *P<0.05. |
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Supplementary Material. Fig. S4. Hnf1b, but notosr2 and lhx1 expression, is inhibited in the neurula KF in response to the co-injection of MoPax8 and MoPax8.B. In situ hybridization for lhx1, osr2 and hnf1b genes. Embryos were injected with MoPax8.B alone or in co-injection with MoPax8 at the 4-cell stage, in the equatorial region of both left blastomeres. At neurula stage 18, they were fixed, transversely bisected at the level of the pronephric area and analyzed by in situ hybridization with the indicated probes. For each embryo, the dotted line indicates the frontier between the injected side (on the left) and the uninjected side (on the right). Hnf1b expression is inhibited in the pronephric area (arrow) on the injected side. |
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Fig. 3. Pax8 but not Pax2 loss of function inhibits tubule marker genes expression and pronephric tubule anlage formation at the early tailbud stage. (A) Whole mount in situ hybridization for tubule marker genes. Embryos were injected with MoPax8 (ae), MoPax2 (fj) or MoC (ko) at the 4-cell stage, in the equatorial region of both left blastomeres. They were cultured until tailbud stage 25, fixed and analyzed by in situ hybridization with the indicated probes. For each embryo, injected (ao) and uninjected (a, o) sides are shown. Pax8 but not Pax2 depletion strongly reduces expression of lhx1, hnf1b, pax2, hrt1, and evi1 in the pronephric area. (B) Immunofluorescence with an anti-laminin-1 antibody on transverse sections of stage 25 embryos injected with MoPax8 (p, q) or MoPax2 (r, s) on one side. For each embryo, injected (ps) and uninjected (ps) sides are shown. DAPI staining is shown in blue (q, q, s, s). On uninjected sides and MoPax2 injected side, the pronephric tubule anlage is visualized as a group of somatic cells radially organized, surrounded by laminin-1 immunostaining (p, q, r, s, r, s). No such cell arrangement is observed on the MoPax8 inejcted side. (pq). Laminin-1 is only detected in the extracellular matrix between the epidermis and the somatic mesoderm, as well as between the splanchnic mesoderm and the endoderm. Scale bar: 140 m. |
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Fig. 4. Inhibition of lhx1 expression in response to MoPax8 is rescued by expressing Pax8. Embryos were first injected at the 4-cell stage in both left blastomeres with MoPax8. They were split into two groups. Embryos from one group were injected again with RNA Pax8-GR. Dexamethasone was added to the culture medium at the end of gastrulation (stage 13). Phenotype was assessed by studying lhx1 expression by in situ hybridization at tailbud stage 25. Phenotypes were classified into three categories: strong inhibition (a), weak inhibition (b) and normal expression (c). Inhibition of pronephric lhx1 expression caused by MoPax8 was partially rescued by inducing Pax8 function just after completion of gastrulation. Histogram shows quantitative results from three independent experiments (d). n=number of analyzed embryos in total. |
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Display additional annotations [+]
Gene |
Clone |
Species |
Stages |
Anatomy |
slc4a4
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
olfactory placode
,
otic vesicle
,
forebrain
,
pronephric duct
,
pronephric kidney
,
[+]
|
slc12a1
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
pronephric duct
,
pronephric kidney
,
pronephric tubule
,
proximal tubule
|
atp1b1
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
olfactory placode
,
otic vesicle
,
lens
,
pronephric duct
,
post-anal gut
,
[+]
|
rgn
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
glomus
,
pronephric kidney
|
wt1
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
glomus
,
pronephric tubule
,
proximal tubule
|
wt1
|
|
laevis
|
NF stage 25
|
glomeral mesenchyme
,
pronephric mesenchyme
,
glomus
|
hnf1b
|
|
laevis
|
NF stage 25
|
posterior neural tube
,
pronephric mesenchyme
|
hey1
|
|
laevis
|
NF stage 25
|
brain
,
forebrain
,
midbrain
,
hindbrain
,
neural crest
,
[+]
|
pax2
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
otic vesicle
,
midbrain-hindbrain boundary
,
spinal cord
,
pronephric nephrostome
,
pronephric duct
,
[+]
|
pax2
|
|
laevis
|
NF stage 25
|
spinal cord
,
post-anal gut
,
optic vesicle
,
pronephric mesenchyme
,
lateral plate mesoderm
,
[+]
|
nphs1
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
glomus
|
cldn19
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
pronephric nephrostome
,
pronephric duct
|
mecom
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
forebrain
,
midbrain
,
hindbrain
,
pronephric duct
,
cranial neural crest
,
[+]
|
mecom
|
|
laevis
|
NF stage 25
|
forebrain
,
telencephalon
,
mandibular crest
,
neural crest
,
pronephric mesenchyme
,
[+]
|
cimap1a
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
epidermis
,
pronephric nephrostome
|
clcnkb
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
pronephric duct
,
pronephric tubule
,
proximal tubule
,
distal tubule
|
|
|
Supplementary Material. Fig. S2 Pronephric phenotype induced by injection of MoPax2.2. Whole mount in situ hybridization for tubule and glomus marker genes. Embryos were injected with MoPax2.2 at the 4-cell stage, in the equatorial region of both left blastomeres. They were cultured until the tailbud stages 3335 (A) or 25 (B) and analyzed by in situ hybridization with specific probes for the indicated genes. For each embryo, injected (aq) and uninjected (aq) sides are shown. Pax2 depletion does not lead to any change in the expression of pronephric marker genes at stage 25. At the late tailbud stage, gene expression in the proximal part of the tubule, including nephrostomes, is affected by MoPax2.2 while expression in the distal tubule is not modified except for the terminal differentiation marker clcnkb1 whose expression is completely inhibited. Expression of the glomus marker genes wt1 and nephrin is not significantly affected in MoPax2.2 injected embryos. Insets show higher magnification of the anterior pronephric territory. Numbers indicate how many embryos showed the phenotype among the total number of injected embryos. |
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Display additional annotations [+]
Gene |
Clone |
Species |
Stages |
Anatomy |
rgn
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
glomus
,
pronephric kidney
|
wt1
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
glomus
,
pronephric kidney
,
primary heart field
|
hnf1b
|
|
laevis
|
NF stage 25
|
pronephric mesenchyme
,
lateral plate mesoderm
,
glomus
,
dorsal lateral plate mesoderm
|
hey1
|
|
laevis
|
NF stage 25
|
forebrain
,
telencephalon
,
presomitic mesoderm
,
somite
,
pronephric mesenchyme
,
[+]
|
pax2
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
otic vesicle
,
hindbrain
,
midbrain-hindbrain boundary
,
spinal cord
,
cement gland
,
[+]
|
pax2
|
|
laevis
|
NF stage 25
|
otic vesicle
,
midbrain-hindbrain boundary
,
spinal cord
,
post-anal gut
,
otic placode
,
[+]
|
nphs1
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
glomus
,
pronephric kidney
|
mecom
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
forebrain
,
midbrain
,
hindbrain
,
pronephric duct
,
cranial neural crest
,
[+]
|
mecom
|
|
laevis
|
NF stage 25
|
forebrain
,
midbrain
,
hindbrain
,
mandibular crest
,
hyoid crest
,
[+]
|
cimap1a
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
pronephric nephrostome
,
pronephric kidney
|
|
|
Supplementary Material. Fig. S3 Pronephric phenotype induced by co-injection of MoPax8 and MoPax8.B. Whole mount in situ hybridization for tubule and glomus marker genes. Embryos were injected with MoPax8.B alone or in co-injection with MoPax8 at the 4-cell stage, in the equatorial region of both left blastomeres. They were cultured until the tailbud stages 3335 (A), early tailbud stage 2325 (B) and analyzed by in situ hybridization with specific probes for the indicated genes. For each embryo, injected (av) and uninjected (av) sides are shown. A strong reduction of the tubule marker genes (pax2, odf3, smp30, lhx1, hnf1b, pax2, hrt1, and evi1) expression is observed in response to the injection while the glomus markers wt1 and nephrin are still expressed. Insets show higher magnification of the anterior pronephric territory. |