XB-ART-25590Development October 1, 1990; 110 (2): 325-30.
Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization.
We have adapted a non-radioactive technique to detect localized mRNAs in whole-mount Xenopus embryos. Synthetic antisense RNA transcribed in the presence of digoxygenin-UTP is used as a probe and is detected via an anti-digoxygenin antibody. We show that localized mRNAs can be detected from late gastrula to tadpole stages and that high as well as low abundance RNAs can be detected. The method was tested on muscle actin and alpha-globin RNAs, whose localization has previously been characterized. In addition, we used the method to determine the distribution of XA-1 RNA, an anterior ectoderm-specific RNA, which we show is expressed in the periphery of the cement gland as well as in the region of the hatching gland. The sequence of an XA-1 cDNA predicts a protein rich in proline and histidine.
PubMed ID: 1723941
Article link: Development
Species referenced: Xenopus
Genes referenced: acta4 actc1 actl6a ag1 hba1 hba3 xa-1
Article Images: [+] show captions
|Fig. 2. Detection of specific mRNAs by whole-mount in situ hybridization. (A) Detection of muscle actin at different stages of Xenopus development. Embryonic stages 13 (dorsal view), 24 and 36 (lateral views) are presented from top to bottom. The blastopore of the stage 13 embryo is arrowed (bp). In addition to somites the heart (h) of the stage 36 embryo is stained. (B) Localization of the globin mRNA in the blood islands of a stage 32 tadpole. The staining is detected on the ventral side of the embryo and is bilaterally symmetrical along the anteroposterior axis. (C) Distribution of the XAG-1 transcript in the cement gland of a stage 23 embryo. Note that under saturating conditions of staining some non-specific staining is also detected on the dorsal side of the embryo. (D-F) Localization of the XA-1 transcript. (D) and (E) represent two different views of a stage 23 embryo; (D) lateral view, (E) anterior view. (F) A stage 36 tadpole viewed in the same plane and magnification as (E). All embryos presented in this figure are albinos with the exception of (B) where a pigmented embryo was used. (A,B and C) have been cleared in benzyl-benzoate/benzyl alcohol to see deep staining; (D,E and F) have not been cleared.|