|
Display additional annotations [+]
|
|
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. |
|
Gene |
Clone |
Species |
Stages |
Anatomy |
pax2.L
|
|
laevis
|
NF stage 25
|
spinal cord
,
post-anal gut
,
optic vesicle
,
pronephric mesenchyme
,
lateral plate mesoderm
,
[+]
|
pax2.L
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
otic vesicle
,
midbrain-hindbrain boundary
,
spinal cord
,
pronephric nephrostome
,
pronephric duct
,
[+]
|
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
|
lhx1
|
|
laevis
|
NF stage 25
|
hindbrain
,
spinal cord
,
pronephric mesenchyme
|
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
,
[+]
|
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. |
|
Gene |
Clone |
Species |
Stages |
Anatomy |
pax2.L
|
|
laevis
|
NF stage 25
|
otic vesicle
,
midbrain-hindbrain boundary
,
spinal cord
,
post-anal gut
,
otic placode
,
[+]
|
pax2.L
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
otic vesicle
,
hindbrain
,
midbrain-hindbrain boundary
,
spinal cord
,
cement gland
,
[+]
|
Display additional annotations [+]
Gene |
Clone |
Species |
Stages |
Anatomy |
lhx1
|
|
laevis
|
NF stage 25
|
pronephric mesenchyme
,
lateral plate mesoderm
,
dorsal lateral plate mesoderm
|
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
,
[+]
|
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. |
|
Display additional annotations [+]
Gene |
Clone |
Species |
Stages |
Anatomy |
slc4a4
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
pronephric nephrostome
,
glomus
,
pronephric kidney
,
kidney
|
slc12a1
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
pronephric duct
,
glomus
,
pronephric kidney
,
kidney
|
atp1b1
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
forebrain
,
cement gland
,
pronephric duct
,
post-anal gut
,
eye
,
[+]
late proximal tubule
,
early distal tubule
,
early proximal tubule
,
late distal tubule
,
glomus
,
pronephric kidney
,
kidney
,
primary heart field
,
secondary heart field
,
proximal tubule
,
distal tubule
|
rgn
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
glomus
,
pronephric kidney
,
kidney
|
wt1
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
pronephric duct
,
glomus
,
pronephric kidney
,
dorsal lateral plate mesoderm
|
nphs1
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
glomus
,
pronephric kidney
,
kidney
|
cldn19
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
pronephric duct
,
glomus
|
mecom
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
pronephric duct
,
kidney
,
pronephric tubule
|
cimap1a
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
pronephric nephrostome
,
pronephric kidney
,
kidney
|
clcnkb
|
|
laevis
|
NF stage 33 and 34
to
NF stage 35 and 36
|
pronephric duct
,
late proximal tubule
,
early distal tubule
,
early proximal tubule
,
late distal tubule
,
[+]
|
|
|
Fig. 2. Pax 8 and Pax2 loss of function differentially affects tubule but not glomus marker genes expression at late tailbud stage. (A, B) Whole mount in situ hybridization for tubule marker genes. Embryos were injected with MoPax8 (ad) or MoPax2 (em) at the 4-cell stage, in the equatorial region of both left blastomeres. They were cultured until the tailbud stage 3335, fixed and analyzed by in situ hybridization with specific probes for the indicated genes. For each embryo, injected (am) and uninjected (am) sides are shown. Pax8 depletion prevents expression of marker genes all along the entire tubule expression domain. In Pax2 morphants, gene expression in the proximal part of the tubule, including nephrostomes, is affected. Expression in the distal tubule is not modified by the MoPax2 except for the terminal differentiation marker clcnkb1 whose expression is completely inhibited. Insets show higher magnification of the anterior pronephric territory. (C) Whole mount in situ hybridization for the glomus marker genes wt1 and nephrin. Embryos were injected with a mixture of MoPax8 and MoPax2 in the equatorial region of both left blastomeres at the 4-cell stage and cultured until the tailbud stage 33. Injected sides are shown in (n, o), uninjected sides in (n, o). Insets show higher magnification of the anterior pronephric territory. (D) RT-qPCR analysis of wt1 and nephrin. Embryos were injected with the indicated Mo at the 4-cell stage in all four blastomeres. They were cultured until the late tailbud stage 33 and processed for RT-qPCR analysis. Average values from three independent experiments. Neither MoPax2 nor MoPax8 injected alone or together significantly affects wt1 and nephrin expression compared to the control (MoC). |