Tropepe V et al. (2006), Identification of a BMP inhibitor-responsive pr...

XB-ART-1050

Dev Biol 2006 Jan 15;2892:517-29. doi: 10.1016/j.ydbio.2005.10.004.

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Identification of a BMP inhibitor-responsive promoter module required for expression of the early neural gene zic1.

Tropepe V , Li S , Dickinson A , Gamse JT , Sive HL .

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Expression of the transcription factor zic1 at the onset of gastrulation is one of the earliest molecular indicators of neural fate determination in Xenopus. Inhibition of bone morphogenetic protein (BMP) signaling is critical for activation of zic1 expression and fundamental for establishing neural identity in both vertebrates and invertebrates. The mechanism by which interruption of BMP signaling activates neural-specific gene expression is not understood. Here, we report identification of a 215 bp genomic module that is both necessary and sufficient to activate Xenopus zic1 transcription upon interruption of BMP signaling. Transgenic analyses demonstrate that this BMP inhibitory response module (BIRM) is required for expression in the whole embryo. Multiple consensus binding sites for specific transcription factor families within the BIRM are required for its activity and some of these regions are phylogenetically conserved between orthologous vertebrate zic1 genes. These data suggest that interruption of BMP signaling facilitates neural determination via a complex mechanism, involving multiple regulatory factors that cooperate to control zic1 expression.

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Species referenced: Xenopus
Genes referenced: bmp4 csrp3 nog zic1

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	Fig. 1. The zic1 promoter is induced by BMP inhibition. (A) A kb genomic fragment was isolated from a lambda-phage library. 5VRACE revealed multiple start sites overlapping 4 initiator elements. (B) This fragment was subcloned into a luciferase reporter construct. Using the animal cap assay, 100 pg DNA and 10 pg Noggin, 800 pg Globin or 800 pg BMP4 mRNA were co-injected and luciferase activity assayed at stage 16. Mean luciferase activity T the standard deviation was plotted in a bar graph. The asterisk (*) over the Noggin bar indicates that Noggin induction is significant different ( P < 0.05) from the Globin control. The asterisk over the BMP bar and the BMP + Noggin bar indicates that Noggin induction is significantly different ( P < 0.05) from BMP alone or BMP + Noggin.
	Fig. 3. The zic1 BIRM is necessary for in vivo reporter gene expression. Embryos were examined by in situ hybridization for expression of endogenous zic1 RNA (panels AE) or for luciferase RNA in embryos transgenic for the full-length promoter construct (5045) (F), BIRM and MLP only (215) (K) and BIRM deleted from full length (5045d4) (P, Q). See Fig. 2 for further construct information. Embryos were examined at gastrula (stage 11 1.5), neurula (stage 16) or tailbud (stage 26) as whole mounts or transverse sections. Transverse sections of neurula stage embryos (whole mount neurula stage not shown) were taken at the level of the anterior notochord region and transverse sections of tailbud embryos are indicated on the whole mount tailbud images. (A) Endogenous zic1 (stage 11) shows characteristic dorsal ectoderm expression that is absent from the marginal zone. (B) Endogenous zic1 (stage 16) transverse section (dorsal at the top) showing expression in the neural plate (sensorial layer). As previously reported, strong expression can be seen in the lateral neural plate with weak expression above the notochord (dotted circle). (C) Lateral view of whole mount expression of endogenous zic1 (anterior to the right) throughout the entire neural tube (stage 26). (D) Transverse section (dorsal to the top) through the brain (anterior section plane indicated in C) of a stage 26 embryo demonstrating endogenous zic1 expression localized to the neural tube with strong mid-dorsal expression. (E) Transverse section (dorsal at the top) through the spinal cord (posterior section plane indicated in C) of a stage 26 embryo demonstrating strong dorsal neural tube expression of endogenous zic1 as well as expression within dorsal somitic tissue and tissue surrounding the dorsal somite indicative of migratory neural crest. (F) Full-length transgene (5045) expression in whole stage 11 embryo demonstrating dorsal ectoderm as well as dorsal mesoderm expression at the margin. (G) Full-length (5045) transgene expression in a transverse section (dorsal at the top) of a stage 16 embryo showing expression in the neural plate (sensorial layer), notochord and dorsomedial somite. (H) Lateral view of whole mount stage 26 embryo with expression of full-length (5045) transgene (anterior to the right) throughout most the entire neural tube, except the posterior tail region. (I) Transverse section (dorsal to the top) through the brain (anterior section plane indicated in H) of a stage 26 embryo demonstrating full-length (5045) transgene expression throughout neural tube with ectopic expression in the eye primordia and between the eye and neural tube indicative of head mesenchyme. (J) Transverse section (dorsal at the top) through the spinal cord (posterior section plane indicated in H) of a stage 26 embryo demonstrating the expression of the full-length (5045) transgene throughout the neural tube, as well as weak expression within medial somitic tissue and notochord. (K) BIRM (215) transgene expression in whole stage 11 embryo demonstrating dorsal ectoderm as well as dorsal mesoderm expression at the margin. (L) BIRM (215) transgene expression in a transverse section (dorsal at the top) of a stage 16 embryo showing expression in the neural plate (sensorial layer), notochord and dorsomedial somite. (M) Lateral view of whole mount stage 26 embryo with expression of BIRM (215) transgene (anterior to the right) in the brain and anterior spinal cord regions of neural tube. (N) Transverse section (dorsal to the top) through the brain (anterior section plane indicated in M) of a stage 26 embryo demonstrating BIRM (215) transgene expression in the neural tube with ectopic expression in the eye primordia and very weak expression between the eye and neural tube indicative of head mesenchyme. (O) Transverse section (dorsal at the top) through the spinal cord (posterior section plane indicated in M) of a stage 26 embryo demonstrating the expression of the BIRM (215) transgene weakly in the neural tube, as well as very weak expression within medial somitic tissue and notochord. Transgene expression from embryos carrying a BIRM deletion in the full-length promoter (5045d4) was not detectable in stage 11 embryos (P, whole mount dorsal view), stage 16 embryos (not shown) or stage 26 embryos (Q, whole mount lateral view anterior to the right). e, eye; nc, notochord (dotted circle); np, neural plate, nt, neural tube; s, somite.
	Fig. 4. Transcriptional modulators localized to the zic1 BIRM. Consensus binding sites for known transcription factors were identified with TRANSFAC analysis. See text for further details.
	Fig. 6. Conservation of vertebrate zic1 promoters. (A) Schematic alignment of Xenopus, fugu, chicken, mouse and human zic1 promoter sequences derived from BLAST alignments from sequences mined from the Ensembl genomic databases. Xenopus laevis sequence was from the cloned promoter fragment used in this study. Alignments were generated using the X. tropicalis zic1 locus as the base sequence (because of its high degree of homology with X. laevis) and the exonntron structure is represented by black boxes (numbered) and interconnecting lines/chevrons. Grey boxes represent UTRs. Green box represents a highly conserved promoter region identified using PIP. Sequence conservation between 50 and 100% is represented by a black dash on the plot corresponding to the relative position in the X. tropicalis locus only. (B) The zic1 promoter region in each species was aligned relative to X. tropicalis in order to visualize the relative positions of the highly conserved element (green box) and 21 bp BIRM motif (red box). Arbitrary distances (kb) are represented on the bottom. (C) Multi-species alignment of the 21 bp BIRM motif. Sequence logo representation of the consensus sequence of this conserved 21 bp motif shows that 18 out of 21 (86%) of the nucleotides match with the Xenopus sequence.