August 1, 1999;
Differential expression of VegT and Antipodean protein isoforms in Xenopus.
/Antipodean (Apod) gene is important for germ layer formation in Xenopus. To investigate the role of this gene at the protein level, as opposed to the RNA level, we have generated affinity purified polyclonal antibodies to Apod, and for comparison, to the other early T-box proteins Xbrachyury
. An anti-VegT
/Apod antibody reveals that there are two protein isoforms in Xenopus, one that we refer to as VegT
and a smaller molecular weight isoform that we refer to as Apod. These isoforms have different N-terminal domains resulting from developmentally regulated alternative splicing of a primary transcript arising from a single VegT
/Apod gene. VegT
is maternally expressed. Its translation is blocked during oogenesis but the protein is present from the egg
until gastrulation in the presumptive endoderm
. There is no evidence for zygotic expression of this isoform. Conversely, the Apod protein isoform is expressed only after the onset of zygotic transcription in the presumptive mesoderm
and is inducible by activin. We conclude that the developmental role of VegT
/Apod is mediated by two different proteins, with entirely different patterns of expression and response to growth factors.
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Fig. 5. Nuclear immunolocalisation of T-box proteins in sectioned Xenopus embryos revealed by affinity purified polyclonal antibodies. Embryos fixed in MEMFA and ethanol were sectioned (10 μ) and subjected to immunostaining with affinity purified polyclonal antibodies raised against Apod, Eomes, Xbra or preimmune sera. (A) stage 10.25 gastrula, anti-Apod; (B) stage 10.25 gastrula, anti-Eomes; (C) stage 10.25 gastrula, preimmune sera for anti-Apod, a similar lack of immunoreactivity was observed with preimmune sera for anti-Eomes and anti-Xbra antibodies; (D) stage 11 gastrula, anti-Xbra. Note that Xbra immunostaining cannot be detected in stage 10.25 embryos and it is not until stage 11 that it can be observed clearly. Dorsal for A to D is to the right. (E) Stage 6 cleavage stage embryo, anti-Apod immunostaining reveals VegT's presence in vegetal nuclei. The arrow indicates the position of an immunostained nucleus in the vegetal pole and the arrowhead indicates the position of an animal pole nucleus as indicated in F. Note that pigment is present in some of the cleavage planes; (F) stage 6 Hoechst staining of E; (G) stage 8 blastula, VegT immunostaining; and (H) stage 9 blastula, VegT immunostaining. (I, K) Details of the deep presumptive endoderm and presumptive mesoderm respectively of stage 10.25 embryos immunostained with anti-Apod Ab showing nuclear immunoreactivity in both these presumptive germ layers. From Western analysis we know that both VegT and Apod isoforms are expressed at this stage of development; (J, L) Nuclear staining of I and K, respectively revealed by Hoechst staining.