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The basic helix-loop-helix transcription factor, Pod 1, has been shown to be expressed in the mesenchyme of many developing mouse organs, including the heart, lungs and gut. In the kidneys of developing mice, Pod 1 is highly expressed in the condensing metanephric mesenchyme, differentiating and late stromal cells and in developing podocytes. We have obtained an EST (CF270487) which contains the Xenopus laevis Pod 1 sequence. Conceptual translation of the Xenopus laevis Pod 1 sequence shows approximately 85% similarity to other vertebrate homologues. RT-PCR indicates that expression is initiated at stage 13 and increases differentially in the developing pronephros compared to the whole embryo. RT-PCR of a kidney dissection at stage 42 shows higher expression in the glomus than in the tubule or duct. In situ hybridisation analysis at tail bud stages shows the anterior-most branchial arch and pronephric glomus are intensely stained. At stage 40, staining persists in the glomus and in the epicardium region of the heart. Adult organ analysis shows expression is highest in the rectum and the spleen, with significant expression in the duodenum, heart, kidney, lungs, pancreas, skin, liver and muscle.
Fig. 2. Temporal expression pattern of Pod 1 in X. laevis. RT-PCR was
performed on unfertilised Xenopus laevis eggs (UF) and whole Xenopus
laevis embryos at stages 9, 13, 16.5, 21, 26.5, 30 and 35, using Pod 1
primers. Equalisation was carried out using ODC as a loading control and
linearity was performed with doubling dilutions of input cDNA from stage
35 embryos to reduce the risk of a PCR plateau. Negative controls were
carried out as described in the methods. RT-PCR analysis shows that Pod
1 expression is not initiated until the beginning of neurulation, stage 13
followed by a gradual increase in expression until stage 35.
Fig. 3. Whole embryo and dissected kidney temporal expression of
Pod 1 in X. laevis. (A) Pronephric tissue or presumptive pronephric tissue
was dissected from Xenopus laevis embryos at stages 12.5, 15, 20, 28 and
35 (Brennan, et al., 1998) and RT-PCR was performed using specific Pod
1 primers. The RT-PCR was equalised using ODC and linearity and
negative controls carried out as described in Experimental Procedures.
Expression of Pod 1 in whole embryos (W) was compared with that of the
kidney or kidneyprimordium dissections (K). The whole embryo temporal
expression pattern confirms the profile shown in Fig 2. The Pod 1
expression in the kidney dissections, however, is proportionally greater
than that of the whole embryo, first appearing during neurulation, stage 15
and increasing as the embryo develops. (B) The pronephros from a
Xenopus laevis stage 42 embryo was dissected into the glomus, tubules and duct regions and RT-PCR using Pod 1 primers was carried out. Equalisation was carried out using EF1α and linearity and negative controls carried
out as described in Experimental Procedures. A fine dissection of the pronephros of a stage 42 X. laevis tadpole reveals the spatial expression of Pod1
within the pronephros. Pod 1 appears to be most expressed in the glomus, less in the tubules and weakest in the duct.
Fig. 4. Whole mount in situ hybridisation of Pod 1 in X. laevis. Wholemount in situ hybridisation with a Pod 1 DIG-labelled antisense (A,C,D,F,G and I) and sense (B,E,H and J) RNA probe was performed on embryos at the stage indicated. Embryos in panels D, G, I and J have been cleared in Murray's solution to facilitate the observation of internal staining. No staining above background was present in embryos at stage 22 (A,B) as staining observed in the eye placode, ep, and somites, s, was present in the antisense and sense in situ hybridisations. (C,D) Glomus, g and anterior branchial arch, b, staining is visible in uncleared and cleared stage 28-29 embryos. (E) Stage 29 sense RNA probed embryos do not show staining in the branchial arch or the glomus. (F) Uncleared antisense stage 33 embryos show discrete staining in the branchial arches, b and the glomus, g, which is also observed in the cleared embryos (G). (H) Sense stage 33 embryos also have some weak staining in the branchial arches. The staining observed in the eye is observed in both antisense and sense stage 33 embryos and is considered to be background (F,H). The late tailbud embryos are stained in the epicardium, h, of the heart and in all three components of the pronephros, the glomus, g, tubules, t and duct, d (I). Non-specific staining is seen in the anterior region of late stage embryos in both sense and antisense hybridised embryos (J).
Fig.5. Expression pattern of Pod 1 in the adult
organs of X. laevis. RT-PCR was performed on
mRNA isolated from the organs of an adult Xenopus
laevis. The RT-PCR was equalised using EF1α
and linearity and negative controls performed as
described in Experimental Procedures. Pod 1 expression
in adult X. laevis organs appears to be
greatest in the spleen and rectum. Other, more
moderate expression is seen in the duodenum, heart, kidney, lungs, pancreas and skin. There are
also low levels in the liver, muscle, spinal cord and stomach.
