XB-ART-34678Dev Biol 2002 Aug 01;2481:118-27. doi: 10.1006/dbio.2002.0716.
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The latent-TGFbeta-binding-protein-1 (LTBP-1) is expressed in the organizer and regulates nodal and activin signaling.
The latent TGF-beta binding proteins (LTBP) are believed to control the availability of TGF-beta in the extracellular milieu. To gain insight into the potential roles of LTBP in early development, we isolated the Xenopus LTBP-1 (xLTBP-1) cDNA. The cDNA encodes a protein similar to the mammalian LTBP-1 in both size and domain structure. In addition, we found a novel longer splice isoform of xLTBP. The RNAs for both forms of xLTBP displayed temporal regulation and the shorter transcript is expressed maternally. Both transcripts also display spatial regulation and are found in the dorsal mesoderm of the organizer. In animal cap experiments, LTBP-1 potentiates the activity of activin and nodal. The activity of LTBP-1 did not appear to require covalent association with activin as the addition of medium containing activin and LTBP-1 to animal caps enhanced the activin effect. These results indicate that LTBP-1 may be part of the regulatory system that establishes the threshold of morphogen activity for activins and nodals in the dorsal side of the embryo during gastrulation.
PubMed ID: 12142025
Article link: Dev Biol
Species referenced: Xenopus
Genes referenced: chrd.1 egf gsc hoxb9 ltbp1 myod1 nodal nodal1 nppa odc1 otx2 sox17a sox17b.1 tbxt tgfb1 wnt8a
Article Images: [+] show captions
|FIG. 1. Xenopus LTBP-1 sequence and analysis. (A) Primary sequence of the short form of Xenopus LTBP-1 and its translation product. Primers used to isolate the cDNA are indicated by arrows. Red arrows indicate the RT-PCR primers. Color key: yellow boxes indicate EGF-like domains predicted by SMART with the domains separated by red boxes; red text indicates RGD cell binding site; gray box is the signal sequence; green box is the signal cleavage site; blue boxes are cysteine-rich repeats; the black box indicates the location of the 25-amino-acid deletion GTNSFGESNNDSLLNTFSPSVTHDNT. (B) Diagram of Xenopus LTBP-1 indicating the domain structure. (C) Sequence alignment of Xenopus, human, and mouse LTBP-1. Conserved residues are boxed. Blue text indicates amino acids conserved between two of the three species. Bold text indicates amino acid similarity.|
|FIG. 2. RT-PCR analysis of Xenopus LTBP-1 expression. Temporal expression of Xenopus LTBP-1. The two alternative forms are detected by using a single primer pair indicated in Fig. 1A. Transcripts are detected after 25 cycles. ODC is a loading control.|
|FIG. 3. In situ hybridization of Xenopus LTBP-1. (A–J) In situ hybridization. (A): Stage 10.5 (gastrula). Expression of xLTBP-1 is detected within the organizer region. (B) A section (depicted by the line “i” in A) shows expression is restricted to the dorsal mesoderm of the organizer (*). (C) Stage 20. Expression of xLTBP-1 in the spinal cord. No expression is seen in the anterior neural plate at this stage. Anterior/ventral is to the right. (D) Stage 25. Transcripts can be detected throughout the spinal cord region but still not in anterior domains. (E) A section through the embryo at the level of the spinal cord (indicated by the line “ii” in D); xLTBP-1 RNA expression is seen throughout the neural tube but not in the notochord at this stage. (F) Expression is also detected in the lateral plate mesoderm, Stage 28. Expression is seen in the tail, throughout the spinal cord and in the hindbrain. (G) Transverse section through the spinal cord (“iii” in F); xLTBP-1 RNA is expressed in the mesoderm lateral to the neural tube, in the notochord and in both the roof (white arrow) and floor plates (black arrow). (H) Midsagital section through the head (line “iv” in F). xLTBP-1 RNA is also expressed in anterior regions, in the hindbrain, and in the ventral forebrain. Note the expression within the notochord. (I) Stage 35. xLTBP-1 RNA is highly expressed in the muscle blocks surrounding the spinal cord. It is also expressed in the rhombomeres of the hindbrain, the eye, and the cement gland. High expression is present in the tailbud cordoneural hinge. (J) Stage 42. Additional xLTBP-1 RNA expression is observed in more anterior regions, including the heart (white arrow), the branchial arches, and around the eye. anp, anterior neural plate; cg, cement gland; ey, eye; fb, forebrain; hb, hindbrain; he, heart; nc, notochord. Locations of the sections are indicated by dashed lines. (K) xLTBP-1 RNA expression in dissected gastrula embryos: Dorsal and ventral marginal zones and animal cap explants were dissected from stage 11 gastrula embryos and analyzed by RT-PCR. xLTBP-1 was amplified by using the same primers as in Fig. 2 but for 30 cycles. The following controls are included as markers for the dissected fragments: Chordin (chd) for dorsal mesoderm, Wnt8 for ventral mesoderm, Otx for animal cap, Brachyury (xBra) as a pan mesodermal marker, and ODC as the loading control.|
|FIG. 4. Gain-of-function analysis of hLTBP-1 in animal cap explants. (A) Coexpression of hLTBP-1 with TGF- family ligands: Embryos were injected in the animal pole of both blastomeres at the two cell stage. Amounts of RNA injected were: 0.2 ng hLTBP-1; 0.5, 1.0, and 2.0 pg activin with and without hLTBP-1; 0.1, 0.2, 0.5 ng AXnr-1 with and without hLTBP-1. Animal explants were isolated at stage 9 and cultured until sibling controls reached stage 11 and processed for RT-PCR as described. (B) Coexpression of hLTBP-1 with Xenopus nodal related-1. Xnr-1 (100 pg/embryo) was injected with or without hLTBP-1 (500 pg/embryo). Animal explants were isolated at stage 9 and cultured until sibling controls reached stage 11 and processed for RT-PCR as described. xBra, brachyury (pan-mesodermal marker); Cerberus, Chordin (dorsal mesendodermal markers); goosecoid (dorsal mesoderm marker); XWnt-8 (ventral mesodermal marker); Xhox3 (ventral mesodermal marker); Sox17 (endodermal marker); EF1- , elongation factor alpha (loading control).|
|FIG. 5. Soluble hLTBP-1 can promote the effect of recombinant activin in animal cap explants. Dissected blastula-stage animal caps were exposed to conditioned media from hLTBP-1-producing CHO cells (lane 4) or from CHO cells alone (lane 3), in the absence (lanes 3 and 4) or presence of recombinant activin protein made in oocytes (lanes 5 and 6). The concentration of activin protein applied to the caps in this experiment promotes ventrolateral mesodermal fates only (lane 5). In the presence of hLTBP-1-conditioned medium, dorsal markers are expressed in the caps, indicating that hLTBP-1 and activin can functionally cooperate in solution (lane 6). xBra, brachyury (pan-mesodermal marker); XWnt-8 (ventral mesodermal marker); Hox-B9 (lateral mesodermal marker); Chordin, Cerberus (dorsal mesendodermal markers); MyoD (muscle marker); Collagen-type II (notochord marker); ODC, ornithine decarboxylase (loading control).|
|ltbp1 (latent transforming growth factor beta binding protein 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, vegetal view, dorsal right.|
|ltbp1 (latent transforming growth factor beta binding protein 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, dorsal view, anterior right.|
|ltbp1 (latent transforming growth factor beta binding protein 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35, lateral view, dorsal up, anterior right.|
|ltbp1 (latent transforming growth factor beta binding protein 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 42, lateral view, dorsal up, anterior right.|