June 1, 2002;
A study of mesoderm patterning through the analysis of the regulation of Xmyf-5 expression.
Xenopus laevis has been a particularly useful model organism for identifying factors involved in the induction and patterning of the mesoderm
, however, much remains to be learned about how these factors interact. The myogenic transcription factor Xmyf-5
is the earliest known gene to be expressed specifically in the dorsolateral mesoderm
of the gastrula
, a domain that is established by the interaction of dorsal and ventral
signals. For this reason, we have begun to investigate how the expression of Xmyf-5
is regulated. We have identified a 7.28 kb Xenopus tropicalis Xmyf-5
(Xtmyf-5) genomic DNA fragment that accurately recapitulates the expression of the endogenous gene. Deletion and mutational analysis has identified HBX2, an essential element, approximately 1.2 kb upstream from the start of transcription, which is necessary for both activation and repression of Xtmyf-5 expression, implying that positional information is integrated at this site. Electrophoretic mobility shift assays demonstrate that HBX2 specifically interacts with gastrula stage
embryonic extracts and that in vitro translated Xvent-1
protein binds to one of its functional motifs. Combined with gain- and loss-of-function experiments, the promoter analysis described here suggests that Xvent-1
functions to repress Xmyf-5
expression in the ventral
domain of the marginal zone
. Furthermore, the identification of HBX2 provides a tool with which to identify other molecules involved in the regulation of Xmyf-5
expression during gastrulation.
[+] show captions
Fig. 1. (A) Expression of Xmyf-5 at the gastrula and neurula stages. Expression of Xmyf-5 was assayed in albino embryos by mRNA in situ hybridisation at the early gastrula/stage 10.5 (i) and late neurula/stage 19 (ii). (B) Schematic representation of XTM1, a 7.28 kb genomic fragment containing the X. tropicalis myf-5 gene. XTM1 is composed of 3 exons of 594 bp, 76 bp and 441 bp (untranslated regions shown in grey and coding sequence in red) separated by introns of 548 bp and 1445 bp. The start of transcription is indicated by an arrow. The percent similarities of the Xtmyf-5 exons with Xlmyf-5 are shown below each exon. A X. tropicalis myf-5-specific in situ hybridisation probe was raised to the third exon. (C) The expression of XTM1 in transgenic X. laevis embryos. (iii) Non-transgenic X. tropicalis embryo at stage 10.5 stained with species-specific Xtmyf-5 probe. (iv, v) Non-transgenic albino (iv) and pigmented (v) stage 10.5 X. laevis embryos stained with the species-specific Xtmyf-5 probe; note lack of staining. (vi, vii and viii) XTM1 transgenic X. laevis embryos at stage 10.5 (vi and vii) and stage 19 (ix), stained with the species-specific Xtmyf-5 probe. Note that the embryo in vii is half-transgenic, demonstrating the specificity of the XTM1 probe. Gastrula embryos are orientated with the dorsal axis pointing up and vegetal pole facing out. The neurula stage embryos are shown with the dorsal axis pointing out and anterior to the left.
Fig. 3. (A) Schematic representation of the mCSKA minimal promoter constructs. The 785 bp MRR (yellow) was cloned in front of the mCSKA minimal promoter (101 bp shown in blue) driving the expression of GFP (shown in green) to create the transgene MRRmCSKAGFP. The numbers 1, 2 and 3 illustrate the position of the 3 putative HBX sites. (B) Expression of MRRmCSKAGFP and deletion derivatives at the gastrula stage. Expression was analysed by mRNA in situ hybridisation to GFP. All embryos are orientated with dorsal pointing up and the vegetal pole facing out.
Fig. 7. (A) Endogenous expression of Xvent-1 and Xmyf-5. (B) The effect of Xvent-1 mis-expression on Xmyf-5. Single blastomeres of 4 cell stage embryos were injected with 250 pg Xvent-1 capped mRNA along with the lineage tracer β-gal (shown in blue). Xmyf-5 expression was then assayed at stage 10-11 by mRNA in situ hybridisation (purple). Embryos are orientated with dorsal pointing up and vegetal pole facing out.
Fig. 8. The effect of Xtvent knockdown on Xtmyf-5 in X. tropicalis
embryos. (A) Percentage of embryos showing normal (white) or
expanded (black) Xtmyf-5 expression following injection with
antisense Xtvent-1 and Xtvent-2 morpholinos. Single doses were 2
ng/embryo, and double doses were 4 ng/embryo, n=number of
embryos scored. (B) Xtmyf-5 expression in wild-type embryo (i) and
embryo injected with both Xtvent-1 and Xtvent-2 antisense