Mayor R et al. (1993), Distinct elements of the xsna promoter are requ...

XB-ART-22050

Development 1993 Nov 01;1193:661-71.

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Distinct elements of the xsna promoter are required for mesodermal and ectodermal expression.

Mayor R , Essex LJ , Bennett MF , Sargent MG .

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Xsna, the Xenopus homologue of Drosophila snail, is expressed in both mesoderm and ectoderm. Expression occurs in all mesoderm initially but is down regulated in a tissue-specific fashion at the end of gastrulation in a way that reveals the subdivision of the mesoderm before its derivatives are overtly differentiated. Xsna is also expressed in the ectoderm of the prospective neural fold from stage 11, in a distinct band of cells surrounding the prospective neural plate, which we designate the neural plate border. The deep and superficial ectoderm compartments labelled by Xsna represent the prospective neural crest and the prospective roof of the neural tube, respectively. Xsna expression persists in neural crest cells during their subsequent migration. The role of the Xsna promoter in creating this pattern of expression has been investigated by injecting fertilised eggs with constructs containing the 5' upstream sequence of the gene fused to a reporter. An element of 115 base pairs (-160 to -45 relative to the transcriptional start) is sufficient to drive appropriate reporter gene expression. The promoter does not contain a TATA or CAAT box and does not have a high GC content, but RNA synthesis starts precisely at 33 bases upstream to the translational start. The start sequence can be deleted so that transcription is initiated elsewhere without affecting the expression pattern. The distribution of Xsna promoter activity within the embryo, examined using beta-galactosidase (beta-gal) fusions, is similar to that of the endogenous mRNA seen by in situ hybridisation. The contribution of elements within the 5' sequence have been assessed by comparing the expression patterns of constructs that have deletions in this region. Sequences from -112 to -97 are required for mesodermal expression and sequences from -96 to -44 are required for ectodermal expression. The behaviour of the injected promoter constructs differ in one important respect from the endogenous gene in that expression in an animal cap assay is not inducible by mesoderm-inducing factors but is inducible by cells of the vegetal pole.

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Species referenced: Xenopus
Genes referenced: cat.2 fgf2 gal.2 kcnt1 pou2f1 snai1

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	Fig. 1. Xsna expression in the mesoderm and ectoderm. Distribution of Xsna mRNA shown by in situ hybridisation. Scale bar, 400 mm in A-C and E; 200 mm in D and F. Stage numbers shown in top left (A) Stage 9, almost sagittal section. Xsna transcripts in most-vegetal tier of marginal zone on dorsal side. bc, blastocoel; d, dorsal; v, ventral (B) Stage 11. Oblique section between horizontal and parasagittal, with the most-dorsal side on the left. Superficial layer of marginal zone cells, suprablastoporal endoderm (se), as well as mesoderm (m), expresses Xsna during involution through blastopore (b). Arrows indicate first appearance of transcripts in ectoderm (ec). (C) Stage 12. Transverse section approximately one third from the anterior. Xsna expression (arrows) in both superficial (sf) and deep (dp) ectoderm (ec). (D) Stage 18. Transverse section at the level of the head somites. Xsna transcripts are present in lateral plate mesoderm (lp) but not prospective myotome (my). The labelled dorsolateral and ventromedial somite compartments are probably prospective dermatome (dm) and sclerotome (s). Premigratory cephalic neural crest (c) and superficial ectoderm cells, on the tips of the neural folds (f) are also labelled. (E) Stage 23. Transverse anterior trunk section. Absence of Xsna transcripts from notochord (n), myotome (my) and pronephros anlage (pn) in the dorsal lateral plate, while transcripts are still detected in the dermatome (dm), sclerotome (s) and the rest of the lateral plate (lp). Transcripts present in roofplate and trunk neural crest (rp + c) in and above the closed neural tube, respectively. (F) Stage 39. Horizontal head section showing branchial arches (ba) with branchial arch cartilages (bc) containing Xsna transcripts. a, anterior; p, posterior.
	Fig. 2. Organisation of the Xenopus snail gene. (A). Sequence of 5¢ region of Xsna. Sequences in bold are possible sites for Oct1, Sp-1, B2 in TF111A promoter and AP4 (a-d) respectively (e) Translational start of endogenous Xsna. Numbers relate to transcriptional start (0). Lower case letters, sequence of CAT leader. (B). Sequences at splice sites. Upper case letters, Xsna cDNA sequence with numbers indicating position in cDNA of bold-lettered bases.
	Fig. 3. Start site of transcription. Extension products of an oligonucleotide complimentary to bases 77-96 of Xsna were obtained by reverse transcription using total RNA from (a) stage 12 embryos (b) stage 20 heads (c) stage 20 posterior parts. A DNA sequence using the same primer is shown in parallel (NB, the sequence tracks show the complement of the Xsna promoter). The translational start is at +33.
	Fig. 4. Effect of promoter sequences on expression of the CAT reporter gene. CAT constructs containing the Xsna promoter are shown (left) with black shading in the region affecting the quantitative expression most strongly. Corresponding CAT assays are shown on the right. Open triangle, labelled substrate; closed triangle, product. Assays were made on 4 stage 11 embryos. ID- 45/+75, internal deletion; ui, uninjected.
	Fig. 5. Patterns of b-gal expression in embryos obtained from fertilized eggs injected with promoter constructs. Fertilised eggs were injected with the full-length promoter (2 kb) except D and E which were injected with D -160. No significant difference was observed between the two constructs. b, blastopore; d, dorsal midline; e, endoderm; v, ventral. (A) Stage 10.25. b-gal expression in mesoderm(m). (B) Stage 10.5, cut through dorsal midline. Expression in mesoderm(m) and in animal cap ectoderm. (C) Stage 11. b-gal expression in prospective neural folds (n) on lateral aspect of embryo. (D) Stage 13. b-gal expression seen in thin sections in the prospective neural folds(n) and lateral plate mesoderm (lp). Black triangles, expression in superficial ectoderm; white triangles expression in deep ectoderm although some superficial layer cells are also labelled. (E) Stage 13. b-gal expression in neural fold but not in neural plate (np) or ventral ectoderm. (F) Stage 14. Transverse section through middle of embryo showing b-gal expression in lateral plate (lp) and in the left side somite. (G) Stage 19. b-gal expression in roof of neural tube (n) and in the cephalic neural crest (cn); (bleached embryo). (H) Stage 25. Lateral view of expression in cephalic (cn) and trunk neural crest (tn). (I) Stage 33/34. b-gal expression in somites (s) and in the branchial arches (ba).
	Fig. 6. b-gal expression in mesoderm and ectoderm of embryos obtained from fertilized eggs injected with truncated promoter constructs. b-gal constructs injected into embryos are on the left. Frequency of occurrence of expression in mesoderm and neural folds at stage 18 is shown on the right. Embryos were scored as positive if any b-gal-positive cells were found in mesoderm or neural folds by dissection. ID- 45/+75, internal deletion. Bar on histogram, s.e.m.
	Fig. 7. Patterns of b-gal expression in embryos obtained from fertilized eggs injected with E-promoter constructs. Fertilised eggs were injected at the 2-cell stage in both blastomeres with construct D -96. b, blastopore; d, dorsal midline; m, mesoderm; n, neural folds; v, ventral. (A) Stage 11. Thin section, b-gal expression in prospective neural folds and not mesoderm. (B) Stage 11.5, expression in the presumptive neural folds (bleached embryo). d, dorsal midline; a, anterior; p, posterior. (C) Stage 13, expression in neural folds (n).
	Fig. 8. Effect of Injecting E-promoters into specific blastomeres. The C1, A2, and A4 blastomeres at the 32-cell stage (Dale and Slack,1987) and at the 1-cell stage were injected with either the Epromoter (D -96) or the complete short promoter (D -160). *Occurrence of b-gal-stained cells in neural folds, epidermis or mesoderm were scored as in Fig. 6.
	Fig. 9. Effect of mesoderm-inducing factors and the vegetal pole cells on expression of promoter-CAT fusions. Animal cap explants obtained from embryos injected with the full-length WTCAT construct were cultured in media containing growth factors or placed in a conjugate with vegetal pole cells. CAT activity was measured in the explants at the equivalent of stage 10.5. Caps (30- 45) were homogenised and the equivalent of 4 caps were used per assay. (A) Embryos at stage 10.5. (B) Caps alone. (C) Caps + bFGF 9 u per ml. (D) Caps + activin 20 u per ml. (E) Caps + bFGF and activin. (F) Caps + vegetal pole. (G) Vegetal pole alone. Acetylated product is at the top of the figure.