QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 12 September 2007
doi: 10.1242/dev.007799

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Range, R. Articles by Lepage, T. Search for Related Content PubMed PubMed Citation Articles by Range, R. Articles by Lepage, T.

Cis-regulatory analysis of nodal and maternal control of dorsal-ventral axis formation by Univin, a TGF-ß related to Vg1

Ryan Range^*, François Lapraz^*, Magali Quirin, Sophie Marro, Lydia Besnardeau and Thierry Lepage

UMR 7009 CNRS, Université Pierre et Marie Curie (Paris 6) Observatoire Océanologique, 06230 Villefranche-sur-mer, France.

View larger version (49K): [in this window] [in a new window]   Fig. 1. Phylogenetic footprinting and regulatory element expression analysis of nodal. (A) Diagram showing the position of the P. lividus nodal gene in the BAC (top) and the region used for the phylogenetic footprinting analysis (brackets). (B) 15 kb of sequence surrounding the P. lividus nodal gene was aligned with 15 kb of S. purpuratus (Sp) and 9 kb of L. variegatus (Lv) genomic sequence using Vista. The two exons are labeled in blue and non-coding sequence showing more than 75% homology in pink. (C) Enlargement of the two non-coding conserved regions used for the expression and deletion analyses. Broken lines delineate the sequences used for the deletion analysis in D. (D) P. lividus genomic DNA sequence was used to drive GFP expression in P. lividus embryos. Merged GFP-fluorescent and DIC images of pluteus-stage embryos illustrating the results of the deletion analysis.

View larger version (35K): [in this window] [in a new window]   Fig. 2. Spatial GFP expression driven by the R-module and intron GFP reporters. (A) Diagram of the five regulatory elements from sea urchin nodal used for the spatial expression analysis. (B) Expression data summary for GFP constructs containing the various regulatory elements. Embryos with expression clones just in the oral ectoderm were scored as oral only; clones in either aboral ectoderm or endoderm were scored as ectopic only; and embryos that contained clones in both oral and ectopic territories were scored as oral+ectopic. (C-E) Examples of embryos that fit into these categories: (C) oral only, (D) oral+ectopic, (E) ectopic only.

View larger version (22K): [in this window] [in a new window]   Fig. 3. The R-module and intron have different temporal expression profiles and are both influenced by autoregulation by Nodal signaling. (A) Kinetics of luciferase expression levels driven by the R-module EpGluc, intron EpGluc and control EpGluc constructs. (B) Comparison at the hatched blastula stage of the transcriptional activities of R-module, intron, R-module Smad(-) (i.e. lacking the binding sites for Smads) and intron Smad(-) in normal sea urchin embryos and embryos injected with the Nodal morpholino or treated with the Alk4/5/7 inhibitor SB431542. The data are presented as the ratio of luciferase expression between EpGluc and the wild-type module.

View larger version (84K): [in this window] [in a new window]   Fig. 4. Homeodomain, bZIP, TCF-like, Oct, Sox, Smad and Myb-like binding sites are important for R-module transcriptional activity. (A) Alignment of the P. lividus R-module with the corresponding S. purpuratus and L. variegatus sequences. The conserved sites mutated in the functional analysis are underlined in black and the colored boxes indicate putative transcription factor binding sites identified using MatInspector. Arrowheads indicate the limit of the R-module. (B) Sequence comparisons of predicted binding sites within the R-module with known consensus sequences for transcription factors. (C) Effects of Smad, homeodomain, bZIP, TCF-like, Oct and Sox site mutations on the transcriptional activity of the R-module at the hatched blastula stage. The homeodomain site mutation data are from a triple mutant, whereas TCF-like, bZIP, Oct and Sox correspond to single mutations within the R-module. (D) Mutation of a putative Myb-like site increases the transcriptional activity of the R-module and R-module Smad(-) constructs. The data are presented as the ratio of luciferase expression between EpGluc and the wild-type module. (E) Effect of the Myb-like binding site mutation on the spatial expression of a GFP reporter driven by the R-module.

View larger version (52K): [in this window] [in a new window]   Fig. 5. nodal expression in the sea urchin relies early on autoregulation and Univin signaling and requires SoxB1 and TCF function. (Aa-h) Effects of SoxB1 morpholino and dnTcf mRNA on the expression of the endogenous nodal and univin genes. (d,e) Injection of SoxB1 morpholino diminishes SoxB1 protein expression. Anti-SoxB1 in green (arrow). (Ba-t,Ca-j) Effects of the Nodal, Alk4/5/7 and Univin morpholinos on the expression of the endogenous nodal and univin genes. (D) Effects of treatment with SB431542, of microinjection of dnTcf mRNA and of Nodal, SoxB1 and Univin morpholinos on the transcriptional activity of the R-module at the hatched blastula stage. The data are presented as the ratio of luciferase expression between EpGluc and the wild-type module. Two different morpholino oligonucleotides directed against univin and soxB1 were used and gave similar results. (E) QPCR analysis of nodal expression in SB431542-treated or MoUnivin-injected embryos. VEB, very early blastula; EB, early blastula; PHB, pre-hatching blastula.

View larger version (51K): [in this window] [in a new window]   Fig. 6. Sea urchin Alk4/5/7 is required for dorsal-ventral axis formation and Nodal signaling. (Aa-d) MoAlk4/5/7 disrupts dorsal ventral axis formation and blocks the response to nodal overexpression. (Ba-h) Rescue experiment to demonstrate the specificity of the Alk4/5/7 morpholino. Embryos were injected with Alk4/5/7 morpholino (b,e) or alk4/5/7 mRNA (c,f) alone, or successively injected with the Alk4/5/7 morpholino then with a synthetic alk4/5/7 mRNA containing eight mismatches over the region recognized by the morpholino (d,g,h). Whereas embryos injected with the Alk4/5/7 morpholino alone developed with a radialized phenotype indicative of inhibition of Nodal signaling, embryos co-injected with the Alk4/5/7 morpholino and the modified wild-type alk4/5/7 mRNA developed into normal pluteus larvae.

View larger version (117K): [in this window] [in a new window]   Fig. 7. Univin functions in the Nodal signaling pathway. (Aa-j,Ba-j) Functional analysis of sea urchin univin. Inhibition of Univin function phenocopies the loss-of-function of Nodal, whereas overexpression of Univin causes ectopic expression of nodal.

View larger version (124K): [in this window] [in a new window]   Fig. 8. Univin is related to vertebrate Vg1/Dvr1. (A) Molecular phylogeny of GDF/Vg1/Univin family members. (B) Synteny between sea urchin univin/bmp2/4 and zebrafish bmp2a/dvr1.

View larger version (25K): [in this window] [in a new window]   Fig. 9. Expression territory comparisons of Vg1/Univin signals with Nodal and nuclear ß-catenin in sea urchin, chick, zebrafish and Xenopus. Embryos are depicted before gastrulation. (A) In the early sea urchin embryo, nuclear ß-catenin is present in the vegetal pole region while zygotic univin and nodal are expressed in the overlying presumptive ectoderm. (B) In the chick embryo, Vg1 and Wnt8c are expressed in the posterior marginal zone and cooperate to induce nodal expression in the adjacent epiblast and primitive streak. (C) In the zebrafish embryo, maternal Vg1 transcripts are expressed ubiquitously, while ß-catenin accumulates in nuclei of the dorsal marginal zone and zygotic nodal transcripts at the blastoderm margin. (D) In Xenopus embryos, ß-catenin is stabilized on the dorsal side while Xnr transcripts and Vg1 are expressed in a dorsal-to-ventral gradient. Data taken from the published literature (Skromne and Stern, 2001; Agius et al., 2000; Helde and Grunwald, 1993; Schier and Talbot, 2005) and this study.

View larger version (40K): [in this window] [in a new window]   Fig. 10. Model of nodal regulation and dorsal-ventral axis specification in the sea urchin embryo. (A) Integration of signaling and maternal transcription factor inputs by cis-regulatory elements of the nodal gene. (B) Starting at the 32-64 cell-stage, nodal expression is initiated throughout most of the presumptive ectoderm by combinatorial maternal inputs from p38 MAP kinase and maternal Univin, as well as from signals emanating from the vegetal pole. Unidentified repressors prevent expression of nodal and zygotic expression of univin in the animal pole domain. These signals are transduced by maternal transcription factors such as Smad, homeodomain, Oct, bZIP families and require SoxB1 and TCF, resulting in a broad initial expression of nodal in the ectoderm. Expression of nodal and zygotic expression of univin at the vegetal pole is prevented by the ß-catenin-mediated downregulation of SoxB1. (C) Starting at the very early blastula stage, an endogenous ventral-dorsal redox gradient, possibly related to an asymmetric distribution of mitochondria in the egg and/or early embryo and acting on bZIP and other redox-sensitive transcription factors, results in a slightly increased expression of nodal on the presumptive ventral side, thereby reinforcing the Nodal autoregulatory loop. This slight asymmetry in the expression of nodal is translated into a corresponding asymmetry in the expression of Lefty, which starts downregulating Nodal signaling on the presumptive dorsal side. (D) During blastula stages, Nodal autoregulation and Lefty-mediated lateral inhibition then establish a robust reaction diffusion system which results in the sharp restriction of nodal expression to the ventral territory.