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Fig. 2. Localisation and activity of XtSulf2. RNAs coding for tagged proteins were injected into the animal hemisphere of both blastomeres of a two-cell X. laevis embryo and processed for immunohistochemistry at NF stage 10. (A) Embryos injected with 1 ng of Nuc-GFP. (B and C) Embryos injected with 2 ng of XtSulf2–GFP. (D–F) Embryos co-injected with RNA coding for XtSulf1-Myc (D) and XtSulf2–GFP. (E) The yellow regions in (F) show where XtSulf1 and XtSulf2 expression overlap. Embryos were co-injected with mRNA coding for FGFR1-Myc (G) and XtSulf2–GFP (H) proteins. While both proteins appear associated with the membrane, they do not completely overlap (I). Western analysis shows in (J) that XtSulf2 while injection of mRNA coding for XtSulf2 inhibits activation of dpERK by FGF4 protein, it has no effect on dpERK activation by the intracellularly drug induced iFGFR1. (K) BMP4 activity was assayed by Western blot analysis using antibodies against phospho-SMAD 1/5/8. The phosphorylation of SMAD 1/5/8 stimulated in animal caps by injection of mRNA coding for BMP4 is down-regulated in animal caps co-expressing XtSulf2. However, animal caps expressing a constitutively active BMP4 receptor, Alk3 continue to express phospho-SMAD 1/5/8 in the presence of XtSulf2, indicating that XtSulf2 acts upstream of the BMP4 receptor. (L) Neuralisation of animal caps was assayed using RT-PCR to detect NCAM expression. Whole embryos (WE) express NCAM at NF stage 18, while control animal caps do not. Injection of mRNA coding for XtSulf2 results in the activation of NCAM expression. Samples with water and without AMV Reverse Transcriptase (RT) were used as negative controls; L8 was used as a loading control. |
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| Gene |
Clone |
Species |
Stages |
Anatomy |
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sulf2
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tropicalis
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NF stage 18
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midbrain-hindbrain boundary
,
neuroectoderm
,
anterior neural tube
,
anterior neural fold
,
neural tube
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sulf2
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tropicalis
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NF stage 20
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midbrain
,
hindbrain
,
midbrain-hindbrain boundary
,
pineal gland
,
ventral
,
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sulf2
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tropicalis
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NF stage 25
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midbrain
,
hindbrain
,
midbrain-hindbrain boundary
,
head region
,
mandibular arch
,
[+]
|
|
sulf2
|
|
tropicalis
|
NF stage 28
|
retina
,
midbrain
,
hindbrain
,
midbrain-hindbrain boundary
,
spinal cord
,
[+]
|
|
sulf2
|
|
tropicalis
|
NF stage 35 and 36
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olfactory placode
,
retina
,
midbrain
,
hindbrain
,
midbrain-hindbrain boundary
,
[+]
|
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Fig. 1. Zygotic expression of XtSulf2. Zygotic transcripts for XtSulf2 are detected by whole mount in situ hybridisation in X. tropicalis embryos. (A–B) At stage 18, zygotic transcripts for XtSulf2 are detected in the anterior neural tube most promimently at the mid-hindbrain junction (asterisk in B). (C–D) At stage 20, XtSulf2 is expressed in the ventral part of anterior neural tube, in the ventral midbrain and hindbrain and at the midbrain–hindbrain junction (asterisk), and in the mandibular mesendoderm underlying the presumptive cement gland (arrow in D and E). (E) At stage 25, XtSulf2 is expressed in the pineal gland (arrowhead), the mandibular mesendoderm subjacent to the cement gland (arrow), the MHJ (asterisk) and the pineal gland (arrowhead). Strong expression continues in the ventral midbrain and hindbrain. (F–J) At stage 28, XtSulf2 is expressed in the mandibular arch, pineal gland and neural retina. Expression is detected in the ventricular zone (VZ) of the midbrain and ventrally in the neural tube. (K) At stage 35, XtSulf2 is expressed in the MHJ (asterisk), pineal gland (arrowhead) olfactory placodes (arrows), and neural retina. (G–J, L–O) 50 µm vibratome transverse sections through respectively (F) and (K). (G, I, L, N) 10× magnification, (H, J, M, O) 40× magnification. R is retina, VZ is ventricular zone, NT is neural tube, and Nc is notochord. |
