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A study of mesoderm patterning through the analysis of the regulation of Xmyf-5 expression

Wellcome Trust/Cancer Research UK Institute, Department of Zoology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
¹ Present address: Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, Australia

View larger version (51K): [in a new window]   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.

View larger version (14K): [in a new window]   Fig. 2. XTM1 deletion constructs. A schematic representation of the enzymatic deletions made to the XTM1 transgene. The exons are shaded grey and the 5', intronic and 3' sequences are shown in black. The extent of the 5' and 3' sequences from the start of transcription are shown on each transgene.

View larger version (44K): [in a new window]   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.

View larger version (45K): [in a new window]   Fig. 4. (A) Putative transcription factor binding sites within the MRR. The MRR is a 785 bp NdeI fragment ranging from –1619 bp to –834 bp from the start of transcription. Homology searches identified HBX1, 2 and 3 (blue shading) as being putative binding sites for homeodomain containing transcription factors. A TCF consensus site is shaded green. (B) Comparison of HBX2 with the consensus binding site for Xvent-2 (Trindade et al., 1999). Grey shading highlights differences from consensus sequence.

View larger version (29K): [in a new window]   Fig. 5. Mutations within HBX2. The bases within HBX2 were assigned numbers as shown. The MRR is shown in yellow, HBX2 site is in blue, individual TAAT motifs are shown in bold type and are boxed and the mutations are shown in red. The blue shading highlights the two essential elements within HBX2, named element 1 (E1) and element 2 (E2).

View larger version (46K): [in a new window]   Fig. 6. (A) EMSA of embryonic extract binding to HBX2. Binding was performed with wild type (WT) and mutant HBX2 (mE1, mE2 and mE1+2) probes. Competition was performed with 200x, 50x and 12.5x excess of unlabelled WT probe (lanes 5-7 respectively), and 200x excess of unlabelled mE1+2 competitor (lane 8). (B) EMSA of in vitro translated protein binding to HBX2. Of the proteins tested, Xcad-2, Xcad-3 and Xvent-1 were found to bind to HBX2. The lane marked Control represents the interaction between the HBX2 probe and uncharged reticulocyte lysate. *Denotes nonspecific DNA binding. Shown below is an autoradiograph of in vitro transcribed proteins, following reticulocyte transcription/translation reaction in the presence of [35S]methionine. (C) Interaction of Xvent-1 with E1 and E2. Left hand panel shows the binding of Xvent-1 to wild-type (WT) and mutant HBX2 probes (mE1 and mE2). Competition analyses are shown in the right hand panel. Competition was performed with 200x, 50x and 12.5x excess of unlabelled WT probe (lanes 9-11 respectively), and 200x excess of unlabelled mE2 competitor (lane 12). *Denotes nonspecific DNA binding.

View larger version (55K): [in a new window]   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 ß-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.

View larger version (43K): [in a new window]   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 morpholino (ii).