October 1, 2001;
Molecular targets of vertebrate segmentation: two mechanisms control segmental expression of Xenopus hairy2 during somite formation.
Vertebrate hairy genes are expressed in patterns thought to be readouts of a "segmentation clock" in the presomitic mesoderm
. Here we use transgenic Xenopus embryos to show that two types of regulatory elements are required to reconstitute the segmental pattern of Xenopus hairy2
. The first is a promoter element containing two binding sites for Xenopus Su(H), a transcriptional activator of Notch
target genes. The second is a short sequence in the hairy2
3'' untranslated region (UTR), which most likely functions posttranscriptionally to modulate hairy2
RNA levels. 3'' UTRs of other hairy-related, segmentally expressed genes can substitute for that of hairy2
. Our results demonstrate a novel mechanism regulating the segmental patterns of Notch
target genes and suggest that vertebrate segmentation requires the intersection of two regulatory pathways.
[+] show captions
Figure 1. Pattern of hairy2 in Tailbud Stage Embryos(A) Whole-mount in situ hybridization for hairy2a RNA in a cleared stage 28 embryo. Expressing tissues include: PSM Stripe (the anterior presomitic mesoderm stripe), FP (neural tube floorplate), RP (neural tube roofplate), Br.A (branchial arches), PN (pronephros), FM (fin mesenchyme), as well as the brain and eye.(B) Similar in situ in an uncleared embryo.(C) In situ for hairy1.(D) In situ for hairy2a in a stage 26 embryo (plastic embedded and imaged with DIC optics). Arrowheads indicate somite borders.(E–H) Proposed order of hairy2a PSM stripe patterns (dorsal is top, anterior is left).(I) hairy2a PSM stripes in three adjacent presumptive somites (magenta, marked by arrows).(J) (left) and (K) (right) sides of the same embryo showing different patterns for hairy2a (the left side has been flipped to be in the same orientation as the right side). A similar pair of images from another embryo is shown in (L) and (M)
Figure 3. Reconstitution of the hairy2a Pattern in Transgenic Xenopus Embryos(A) Whole-mount in situ hybridization for eGFP using the H2pm transgene.(B) H2pmH2G transgenes express eGFP in the PSM stripe (arrowheads).(C) The arrow indicates hairy2a expression in the floorplate.(D) The arrow indicates no transgene expression in the floorplate, while the arrowhead indicates expression in the roofplate.(E and F) H2pmH2G transgenes mimic the variability of the hairy2a PSM stripe.(G and H) The H2mpH2G transgene mimics the left-right asymmetry of hairy2a. The left side image has been flipped to be in the same orientation as the right side.(I) H2pm3U transgene expresses eGFP in the anterior PSM stripe (arrowhead)