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???displayArticle.abstract??? Kidney organogenesis is initiated with the formation of the pronephric kidney and requires Pax-2 gene function. We report here the cloning and characterization of Pax-2 cDNAs from the frog Xenopus laevis, a model system suitable for the study of early kidney organogenesis. We show that expression of Xenopus Pax-2 (XPax-2) genes was confined to the nervous system, sensory organs, the visceral arches, and the developing excretory system. DNA sequencing of XPax-2 cDNAs isolated from head and pronephric kidney libraries revealed seven novel alternatively spliced Pax-2 isoforms. They all retain DNA-binding domains, but can differ significantly in their C termini with some isoforms containing a novel Pax-2 exon. We investigated the spectrum of XPax-2 splice events in pronephric kidneys, animal cap cultures and in whole embryos. Splicing of XPax-2 transcripts was found to be extensive and temporally regulated during Xenopus embryogenesis. Since all investigated tissues expressed essentially the full spectrum of XPax-2 splice variants, we conclude that splicing of XPax-2 transcripts does not occur in a tissue-specific manner.
Fig. 3. Spatial expression of XPax-2 transcripts during early Xenopus embryogenesis. Whole mount in situ hybridizations were performed on albino embryos
using digoxigenin-labeled antisense RNA probes. Hybridization events were visualized as alkaline phosphatase chromogenic reaction products. Embryos
stained in whole mount were used for sectioning and were cut at 70 mm (L, M) and 100 mM (N). (A, B) Lateral and dorsal views of a stage 13 embryo. XPax-2
expression is seen in two wedge-shaped patches of cells in the anterior 1/3 of the neural plate. Expression is absent at the midline (arrowhead). (C) Lateral
view of a stage 19 embryo. XPax-2 expression can be detected in the regions of the future midbrain-hindbrain boundary and the optic stalk (arrowhead). (D)
Dorsal view of a stage 19 embryo. Two rectangular-shaped patches of XPax-2 expressing tissues which will contribute to the midbrain-hindbrain boundary
flank the midline (arrowhead). Note that staining of XPax-2 can now also be detected in single neurons arranged in stripes on either side of the midline. (E)
Lateral view of a stage 21 embryo. XPax-2 expression is associated with the region of the future optic stalk (os) and vesicle, the midbrain-hindbrain boundary
(m-h), the otic vesicle (ov) and with cells in the spinal cord (sc). Note also the first appearance of staining in the pronephric anlage (pa). (F) Lateral view of a
stage 24 embryo. XPax-2 expression can now also be detected in the furrows of the visceral arches (va), and in both compartments of the developing
pronephric kidney, the pronephros (p) and the pronephric duct (pd). XPax-2 expression in the hindbrain (hb) is limited to the region posterior to the otic
vesicle. (G) Lateral view of a stage 28 embryo. The proctodaeum (pr) appears as a further tissue expressing XPax-2 transcripts. (H) Lateral view of a stage 32
embryo. Arrowheads indicate XPax-2 expression in the three nephrostomes of the pronephros. Note the appearance of the rectal diverticulum (rd) extending
in anterior direction (open arrow) towards the posterior end of the pronephric duct (filled arrow). (I) Lateral view of a stage 36 embryo. The pronephric duct
(filled arrow) and the rectal diverticulum (open arrow) are close to joining. Strong expression of XPax-2 is seen in the hindbrain and along the entire spinal
cord. (J) Lateral view of a stage 39 embryo. The pronephric duct has fused with the rectal diverticulum (filled arrow). XPax-2 expression is detected in the
entire excretory canal up to the point where fusion occurs with the cloaca (open arrow). The brown color in the eye is due to the onset of pigmentation. (K)
Close-up view of a stage 39 embryo to illustrate details of XPax-2 expression in the optic stalk (filled arrow), the posterior end of the midbrain (open arrow),
the otic vesicle (open arrowhead), and the hindbrain. Note also the extensive coiling of the pronephric tubules and the strong staining associated with the
nephrostomes (filled arrowheads). (L) Transversal section through a stage 32 embryo which is cut anterior to the midbrain-hindbrain boundary. XPax-2
expression is confined to the ventral portion of the optic cup (arrowheads) and to furrows of the visceral arch (arrows). (M) Transversal section through a
stained stage 32 embryo which is cut at the level of the hindbrain-spinal cord junction. Staining of the pronephroi (arrowheads) and of cells in the
ventrolateral neural tube (arrows) is indicated. (N) Horizontal section through the visceral arches of a stage 28 embryo. The plain of section is below the
eye. Arrowheads point to the expression in the furrows of the visceral arches. Embryos in shown in (A-K) were photographed uncleared, with the anterior
ends of the embryo oriented to the left.
Fig. 6. Temporal regulation of alternative splicing of XPax-2 transcripts during early Xenopus embryogenesis. Schematic representations of the XPax-2
protein are shown in panels A. Conserved sequence motifs (PD, paired domain; OP, octapeptide; HD, partial homeodomain) are outlined, and arrows mark
the relative positions of the sequences recognized by the primers used for RT-PCR (see also Fig. 4). Total RNA was extracted from unfertilized eggs (E) and
embryos of the indicated stages, and assayed for the expression of XPax-2 splice variants by RT-PCR. Amplification products were separated on agarose gels,
transferred onto nylon membranes and hybridized with appropriate radiolabeled XPax-2 probes. The migration positions of size standards are shown on the
left of the autoradiographs. On the right, selected amplification products are identified by arrowheads. Their exon composition was determined by comparison
with XPax-2 cDNAs of known exon composition (autoradiographs on the right of C and D) and by sequencing of the cloned amplification products. The
numbering of exons is based on the model in Fig. 4. (A) Alternative splicing in the paired domain region. Primers XP-25 and XP-31 directed against
sequences in exon 1 and 3, respectively, were used for the RT-PCR analysis. A single amplification product with a structural organization consisting of exons
1, 2 and 3 was detected. (B) Alternative splicing in the region of the octapeptide motif. Primers XP-59 and XP-11 are directed against sequences in exon 4
and 7, respectively. The autoradiograph displays the temporal expression profile of alternatively spliced XPax-2 transcripts in the early embryo. (C)
Alternative splicing between exons 4 and 10. Primers XP-59 and XP-63 are used for PCR amplification. The autoradiograph on the left displays the
spectrum of alternative splice variants in the developing embryo. The autoradiograph on the right compares the amplification products generated with XPax-2
cDNA templates of defined exon composition to those detected in the developing embryo. (D) Alternative splicing in the C-terminal region. Primers XP-9
and XP-24 are directed against sequences in exon 8 and 13, respectively. The autoradiograph on the left displays the spectrum of alternative splice variants in
the developing embryo. Amplification products generated by alternate use of 3splice acceptors present in intron 11 or exon 13 are marked by asterisks or
filled circles, respectively. The autoradiograph on the right compares the amplification products generated with XPax-2 cDNA templates of defined exon
composition to those detected in the developing embryo. (E) Control for equal RNA amounts. RT-PCR experiments were carried out in parallel with
ornithine decarboxylase (ODC) specific primer pair ODC-1/ODC-2. Amplification products were separated by agarose gel electrophoresis and visualized by
staining with ethidium bromide. M, lane with size markers.
Fig. 7. Alternative splicing of XPax-2 transcripts in the developing pronephric kidney. (A) Outline of the dissection strategy. To illustrate the distribution of
XPax-2 expressing tissues, embryos of stage 24 and 36, respectively, stained by in situ hybridization are shown. The dissection planes are indicated with
black lines. Heads (h) and explants enriched in pronephric tissue (p) were used for RNA isolation, and subsequent RT-PCR analysis. Head explants contain
brain tissues, sensory organs, and visceral arch material. (B) Identification of alternative splice variants using primer pair XP-59/XP-63. Head (h) and
pronephric (p) explants were analyzed by RT-PCR. The autoradiograph is shown with the size markers on the left. Amplification products verified by DNA
sequencing are identified by arrowheads and their exon compositions are shown. (C) Identification of alternative splice variants using primer pair XP-9/XP-
24. (D) Control for equal RNA amounts. RT-PCR experiments were carried out in parallel with histone H4 specific primer pair H4-1/H4-2. Amplification
products were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. M, lane with size markers. The limitations of
histone H4 as a control for pronephric RNA preparations are discussed in Section 2.7.
Fig. 8. Induction of XPax-2 splice variants in animal cap explants. Embryos were cultured until stage 8, at which the upper part of the animal hemisphere
(animal cap) was removed. The animal caps were treated for 12 h at 22 with either activin (10 ng/ml), retinoic acid (RA, 10 mM), basic FGF (bFGF; 100
ng/ml) or a combination of activin and RA. Control explants were cultured in the absence of any factors. Expression of XPax-2 splice variants was assayed by
RT-PCR (panel A-C) and by whole mount in situ hybridization (panel D) once reference embryos reached stage 28. In panels A and B, schematic
representations of the XPax-2 protein are shown to illustrate the RT-PCR strategy. Autoradiographs is shown with the size markers on the left. (A)
Identification of alternative splice variants using primer pair XP-59/XP-63. (B) Identification of alternative splice variants using primer pair XP-9/XP-24.
(C) Control for equal RNA amounts. RT-PCR experiments were carried out in parallel with elongation factor-1a (EF-1a) specific primer pair EF1a-1/
EF1a-2. Amplification products were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. M, lane with size markers.
(D) Induction of XPax-2 expressing tissues in animal cap explants. Animal caps were incubated either under control conditions or in presence of added
factors (activin alone; activin and RA) as described above. Note the absence of XPax-2 transcripts in control animal caps.
