To investigate when Shroom genes are expressed during embryonic development, we analyzed their mRNA levels from egg until mid-gastrula by reverse transcriptase PCR (RT-PCR). Both Shroom1 and Shroom2 mRNAs are detected in unfertilized eggs and their levels are stable during early cleavage stages. On the other hand, Shroom3 and 4 mRNAs are not detected during early development. Instead, Shroom3 mRNAs are first detected during the mid-blastula transition (MBT, around stage 9) and Shroom4 mRNAs are detected post MBT (Fig. 1).
Based on these expression patterns of Shroom1 and 2, we examined the spatial distribution of Shroom1 and 2 mRNAs in unfertilized eggs and early embryos by in situ hybridization. Surprisingly, these mRNAs appear to be localized asymmetrically along the animal-vegetal axis. mRNAs for both Shroom1 and 2 accumulated primarily in the animal hemisphere of both unfertilized eggs and cleavage stage embryos (Fig. 2A). The localization of Shroom1 and 2 mRNAs at the animal pole persisted into later cleavage stages, but the mRNA then appeared to be localized in or near the nuclei of the cells. At stage 6.5, Shroom1 and 2 mRNAs are also detected in the nucleus of some vegetal cells, although these levels are very low compared to levels seen in the animal hemisphere (Fig. 2A).
Figure 2. mRNA localization of Shroom1 and 2 during oogenesis and early development. A: In situ hybridization against Shroom1 and 2 in unfertilized egg, 1-cell, 2-cell stage, and stage 6.5. B: In situ hybridization against Shroom1 and 2 during oogenesis. C: In situ hybridization against AN2, Vg1, and Histone H3. Animal to the top in all panels.
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Several mRNAs such as AN1, AN2, AN3, and PABP have been previously reported to localize to the animal hemisphere in Xenopus (Rebagliati et al.,1985; Weeks and Melton,1987; Schroeder and Yost,1996). We, therefore, observed the distribution of Shroom1 and 2 mRNAs during oogenesis in comparison to these known, asymmetrically-localized mRNAs. Shroom1 and Shroom2 are expressed around the germinal vesicle or oocyte nucleus from the start of oogenesis, stage I (data not shown) (Dumont,1972). These mRNAs remain near the germinal vesicle during oogenesis even after it has moved towards the animal pole (Fig. 2B). The localization of AN2 mRNA, a transcript localized in the animal pole (Rebagliati et al.,1985; Weeks and Melton,1987), Vg1 mRNA, a transcript localized in the vegetal pole (Rebagliati et al.,1985; Melton,1987), and histone H3 and α-tubulin, transcripts distributed uniformly in both animal and vegetal hemispheres (Perry and Capco,1988), were examined. As expected, AN2 mRNA distribution is very similar to those of Shroom1 and 2 (Fig. 2C, top row). Vg1 mRNA localizes to the vegetal pole, as already reported (Rebagliati et al.,1985; Melton,1987) (Fig. 2C, middle row). However, though α-tubulin and histone H3 mRNA have been reported to localize in both animal and vegetal hemisphere by RNase protection assays, we unexpectedly found their localizations to be similar to that of Shroom1, Shroom2, and AN2 in our in situ hybridization data (Fig. 2C, bottom row and data not shown). Thus, while our in situ hybridizations suggest that Shroom1 and Shroom2 mRNA may be enriched animally, there remains some uncertainty. However, given that over-expression of Shroom2 or Shroom3 at blastula stages results in dramatic accumulation of pigment (Haigo et al.,2003; Fairbank et al.,2006; Lee et al.,2007), it is tempting to speculate that there is a link between animal enrichment of the Shroom family mRNAs and animal pigmentation in the Xenopus embryo.