XB-ART-42563Development February 1, 2011; 138 (3): 475-85.
Rapid differential transport of Nodal and Lefty on sulfated proteoglycan-rich extracellular matrix regulates left-right asymmetry in Xenopus.
The spatiotemporally dynamic distribution of instructive ligands within embryonic tissue, and their feedback antagonists, including inherent stabilities and rates of clearance, are affected by interactions with cell surfaces or extracellular matrix (ECM). Nodal (here, Xnr1 or Nodal1 in Xenopus) and Lefty interact in a cross-regulatory relationship in mesendoderm induction, and are the conserved instructors of left-right (LR) asymmetry in early somitogenesis stage embryos. By expressing Xnr1 and Lefty proproteins that produce mature functional epitope-tagged ligands in vivo, we found that ECM is a principal surface of Nodal and Lefty accumulation. We detected Lefty moving faster than Nodal, with evidence that intact sulfated proteoglycans in the ECM facilitate the remarkable long distance movement of Nodal. We propose that Nodal autoregulation substantially aided by rapid ligand transport underlies the anteriorward shift of Nodal expression in the left LPM (lateral plate mesoderm), and speculate that the higher levels of chondroitin-sulfate proteoglycan (CSPG) in more mature anterior regions provide directional transport cues. Immunodetection and biochemical analysis showed transfer of Lefty from left LPM to right LPM, providing direct evidence that left-side-derived Lefty is a significant influence in ensuring the continued suppression of right-sided expression of Nodal, maintaining unilateral expression of this conserved determinant of asymmetry.
PubMed ID: 21205792
PMC ID: PMC3014634
Article link: Development
Species referenced: Xenopus
Genes referenced: actl6a clstn2 fn1 lefty myc nodal nodal1 sdc2 tjp1
Antibodies: Cdh1 Ab3 Cspg4 Ab1 Ctnnb1 Ab4 Fn1 Ab1 Itgb1 Ab1 Sdc2 Ab1
Article Images: [+] show captions
|Fig. 1. Bilayered LPM is LR symmetrical from tailbud-tadpole stages with splanchnic-somatic structural differences beginning at stage 23. (A,B) Diagrams indicate stage/length and sectional planes. Analysis every 0.1 mm (dashed red frame) was between anterior-posterior LPM extremes indicated by red/blue frames. Representative mid-embryo sections (purple frame) are shown. (A) Stage 17 (10×, 40×), left and right LPM each comprising two layers. β-Catenin (green); DAPI (blue). Fibronectin (red) flanks epidermal/endodermal faces of left and right LPM. (B) Stage 23: maintenance of bilayered left and right LPM. (C) Left/right LPMs are structurally similar during these stages, but somatic/splanchnic layers become distinct, symmetrically, from stage 23; somatic cells are more squamous, splanchnic are more columnar. (D-F) Somatic and splanchnic LPM show different basal lamina compositions. Somatic: strong fibronectin, HSPG and Laminin signal; splanchnic: much weaker HSPG/Laminin signal, especially laterally. Scale bars: 100 μm, in top images A,B; 20 μm in bottom images A,B; 20 μm in C-F.|
|Fig. 2. LPM undergoes symmetric epithelial polarization after Xnr1/Lefty expression. Anterior, middle and posterior transverse cryosections showing F-actin (Phalloidin, red), ZO1 (green) and nuclei or ECM [blue, DAPI (A,B), Laminin (C)]; 40× images. ZO1 alone (left side shown) is in grayscale, chained lines indicate LPM epidermal/endodermal boundaries. (A) Stage 17, LPM is not yet polarized. ZO1 puncta indicate tight junctions in polarized epidermal layer; no puncta are apparent within LPM at stages before asymmetric Xnr1/Lefty expression. (B) Stage 23, unpolarized LPM during peak Xnr1/Lefty expression. (C) Stage 25, punctate ZO1 signal appears at somatic/splanchnic interface in anterior left and right LPM; at this stage, asymmetric Xnr1/Lefty expression is waning. Arrowheads in C indicate ZO1 in epithelial archenteron. Scale bars: 20 μm.|
|Fig. 3. Xnr16MYC-CS and Lefty6MYC-CT move substantially from AC grafts. (A) Xnr1 and Lefty constructs: blue box, pro-domain; CS1/CS2, cleavage sites liberating mature ligands; 6MYC tag was inserted just downstream of CS1 (Xnr1) or C-terminally (Lefty). (B) AC-grafting schematic. (C-L′) Transverse cryosections were used to detect Myc (red; grayscale in C′-L′), laminin (blue) and nuclei (DAPI, white); dorsal panels focus axially/paraxially, lateral panels on LPM. Membrane-bound GFP (mGFP, green) marks engrafted cells; 2.5 μm optical sections. Open arrowheads, Myc; closed arrowheads, nonspecific epidermal haze. (C-F′) Xnr16MYC-CS, (G-J′) Lefty6MYC-CT, (K,K′) Xnr1UNTAGGED and (L,L′) LeftyUNTAGGED. (C,C′,D,D′) Representative section ∼110 μm anterior of graft margin; Xnr16MYC-CS signal in basal lamina surrounding notochord/neural tube. Dorsal and left LPM Xnr16MYC-CS signal colocalized with laminin. (E,E′,F,F′) Representative images, dorsal and lateral Xnr16MYC-CS signal within/near graft. Note absence of endoderm signal. (G,G′,H,H′) Lefty6MYC-CT signal colocalized with laminin in dorsal and lateral views, ∼340 μm anterior of graft. (I,I′,J,J′) Dorsal and lateral images of Lefty6MYC-CT signal. Note Lefty6MYC-CT signal is within endoderm, not colocalized with laminin. (K-L′) AC grafts with Xnr1UNTAGGED or LeftyUNTAGGED reveal artefactual hazy epidermal signal (closed arrowheads). Scale bars: 25 μm.|
|Fig. 6. Xyloside decreases left LPM-restraint of Xnr1 signal and alters the distance traveled. (A,B) Endogenous Xnr1 expression in left LPM of controls but complete absence from 80% of xyloside-treated embryos. (C,D) CSPG is found periaxially (around notochord) and at the somite/dorsal endoderm interface in DMSO-treated (n=9) embryos but is absent from all xyloside-treated embryos (n=11). (E,F) Xnr16MYC-CS grafts display dorsal periaxial signal, which is markedly reduced with xyloside treatment. (G,H) Dorsolateral left LPM signal on ECM in controls and lack of endodermal signal (inset; graft, green outline). Xyloside-treated Xnr16MYC-CS engrafted embryos showed increased endoderm signal (inset), interstitial and intracellular. Note increased relative signal at splanchnic:endodermal ECM. Open arrowheads, epidermal:somatic ECM; yellow arrowheads, splanchnic:endodermal ECM. (I) Xyloside treatment reduces distance traveled by Xnr16MYC-CS along lateral and periaxial routes (Fig. 4). *Non-parametric Mann-Whitney test: P≤0.05. Scale bars: 25 μm.|
|Fig. 7. Model for effect of sulfated glycosaminoglycans (sGAGs) during asymmetric gene expression. (A) Asymmetrically-produced Xnr1 in the left LPM beginning at stage 18/19 begins to move anteriorwards, concentrated over ECM of left LPM/periaxial tissue surfaces, and begins to induce Lefty. Lefty travels anteriorly along left LPM/periaxial ECM more rapidly than does Xnr1, and into endoderm. Lefty catches up to Xnr1, shutting down Xnr1 autoregulation. Lefty stability may prevent a second Xnr1 wave from initiating. (B) sGAGs (stippling) within LPM and periaxial ECM (yellow line) help retain a significant fraction of Xnr1 in proximity to left LPM, while Lefty (not shown) moves more freely to right LPM either directly through endoderm or `up-and-over' the dorsal axial midline. sGAG removal allows lateral travel of Xnr1 into endoderm, reducing LPM signal and planar movement. This orthogonal transfer reduces the strength of Xnr1 autoregulation within the LPM.|
|Fig. S1. Left and right LPM persist as bilayers following mesoderm formation. (A-F) Examination of the left and right LPM before (stage 17), during (stage 20-25) and after (stage 28 and 34) asymmetric Xnr1 expression. (C-F) The LPM is thicker in anterior regions, becoming thinner more posteriorly, beginning at stage 23. The somatic LPM cells are more squamous, splanchnic LPM cells more columnar (see inset in D-F). Scale bars: 100 in top 10images of A-F; 20 in all 40images of A-F.|
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