Kuerner KM and Steinbeisser H (2006), Expression analysis of IGFBP-rP10, IGFBP-like a...

XB-ART-65

Dev Dyn 2006 Oct 01;23510:2861-7.

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Expression analysis of IGFBP-rP10, IGFBP-like and Mig30 in early Xenopus development.

Kuerner KM , Steinbeisser H .

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To date, five members of the insulin-like growth factor-binding protein (IGFBP) superfamily have been described in Xenopus laevis. Here, we report the isolation of two new IGFBPs: xIGFBP-rP10, and xIGFBP-like. The proteins share the same domain architecture, and together with Mig30, form a subgroup within the IGFBP superfamily. Temporal expression analysis shows that they are expressed differentially during early development. xIGFBP-rP10 is continuously expressed, whereas Mig30 expression peaks during gastrulation. IGFBP-like is expressed from neurulation onward. The three genes have characteristic spatial expression domains, which overlap in some regions. Both xIGFBP-rP10 and Mig30 are expressed on the dorsal side of the embryo during gastrulation. Later, xIGFBP-rP10 is expressed in the notochord, the floor plate, the somites, and the fin. xIGFBP-like expression is seen primarily in the developing central nervous system and overlaps with Mig30 expression at the end of neurulation in the developing somites and in tail bud stages in the eyes.

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Species referenced: Xenopus laevis
Genes referenced: aen chrd igfbpl1 ins kazald1 mapre2 mig30 nog nppa odc1 otp rp1

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	Figure 3. In situ hybridization of Mig30. Positions of sections are indicated by dotted lines, and letters mark corresponding panels. A-C: Sagittal sections; dorsal to the left. A: Stage (st.) 8.5; asterisk marks the blastocoele chordin- and noggin-expressing center. B,C: Black arrowheads mark the dorsal lip. B: St. 10.25. C: St. 11.5. D: St. 14; anterior view. E-G: St. 17. E: Anterior view. F: Dorsal view, anterior to the left. G,H: Transverse sections. I,J: St. 20. I: Anterior view. J: Dorsal view, anterior to the left. K,L: St. 31. K: Lateral view. L: Transverse section. M,M prime : St. 39. M: Lateral view. M prime : Transverse section. bt, branchial tissue; e, eye; he, heart; le, lens; lp, prospective lens placode; no, notochord; nt, neural tube; otp, prospective otic placode; otv, otic vesicle; re, retina; so, somite.
	Figure 4. In situ hybridization of xIGFBP-rP10. Positions of sections are indicated by white dotted lines and letters mark corresponding panels. A,B: Sagittal sections. A: Stage (st.) 4. B: St. 10.5; black arrowheads mark dorsal lip; insert: vegetal view. C,D: St. 15. C: Anterior view. D: Dorsal view, anterior to the left; insert: transverse section. E,F: St. 21. E: Anterior view. F: Dorsal view, anterior to the left. G-I: St. 24. G: Lateral view; insert: vegetal view of head. H: Transverse section. I: Crude horizontal section at the level of the notochord. J-M: St. 30. J: Lateral view; insert: vegetal view of head. K-M: Transverse sections. N,O: St. 35; lateral view. O: Magnification of head region. P,Q: St. 40; lateral view. O: Magnification of head region; reversed saturation image of P, to visualize pattern of strongly stained areas. cg, cement gland; cnh, chordoneural hinge; df, dorsal fin; fp, floor plate; hm, head mesenchyme; no, notochord; otv, otic vesicle; so, somite; vf, ventral fin; VII and IX/X, cranial ganglia.
	Figure 5. Whole-mount in situ hybridization of xIGFBP-like. Positions of sections are indicated by white dotted lines, and letters mark corresponding panels. A,B: Stage (st.) 15. A: Anterior view. B: Dorsal view, anterior to the left. C: St. 18; anterior view. D-G: St. 21. D: Anterior view. E: Dorsal view, anterior to the left. F: Reversed saturation image of D, to visualize pattern of strongly stained areas. G: Transverse section. H-J: St. 25. H: Lateral view. I: Dorsal view, anterior to the left. J: Transverse section. K-M: St. 32. K: Lateral view. L,M: Transverse sections. N-Q: St. 39. N: Lateral view. O: Magnification of head region, dorsal view, anterior to the left. P: Transverse section. Q: Transverse section. aen, archencephalic region; anp, anterior neural plate; er, prospective eye region; fb, forebrain; hb, hindbrain; le, lens; mb, midbrain; nr, neural retina; no, notochord; otv, otic vesicle; ov, optic vesicle; pnr, prospective neural retina; psm, presomitic mesoderm; sc, spinal cord; so, somite.
	mig30 (mig30) gene expression in Xenopus laevis embryo, as assayed by in situ hybridization, NF stage 39, lateral view, anterior left, dorsal up.
	igfbpl1 (insulin-like growth factor binding protein-like 1) gene expression in a Xenopus laevis embryo, as assayed by in situ ybridization, NF stage 41, lateral view, anterior left, dorsal up.
	Figure 1. A: Schematic diagram of the protein domain architecture. Black, signal peptide (SP); yellow, insulin growth factor-binding homologues (IB, SMART accession no. 00121); blue, kazal-type serine protease inhibitors (KAZAL, SMART accession no. 00280); green, overlap of IB and KAZAL; orange, immunoglobulin C-2 type (IGc2, SMART accession no. 00408). B: Sequence alignment of hIGFBP-rP1 (GenBank accession no. NP_001544), xIGFBP-like (GenBank accession no. DQ515890), and xIGFBP-rP10 (GenBank accession no. AAH72910, Xenopus laevis MGC80370 protein) together with their human homologues (hIGFBP-like, GenBank accession no. NP_001007564; hIGFBP-rP10, GenBank accession no. NP_112191) and Mig30 (GenBank accession no. BAB84702). Note: The xIGFBP-like is not a full-length sequence. Conserved amino acids are shaded in gray. Dashes indicate gaps introduced to maximize the alignments. Protein domains are highlighted with same colors as in A. Note: The white box in the overlap of IB and KAZAL of hIGFBP-rP10 and white box in IB of xIGFBP-rP10 mark single amino acid substitutions (see the Results section). C: Cladogram of human and Xenopus laevis IGFBPs and IGFBP-rPs (see Table 1). The proteins characterized in this study (marked with a dot) together with their human homologues and hIGFBP-rP1 form a group (shaded in gray). For the putative Xenopus homologues of hIGFBP-1 and -2, GenBank accession numbers are AAH60008 and AAH84133, respectively. The tree was generated using ClustalW.
	Fig. 2. Expression of Mig30, xIGFBP-like, and xIGFBP-rP10 during early Xenopus laevis embryogenesis determined by reverse transcription- polymerase chain reaction. Developmental stages after Nieuwkoop and Faber (N.F.; Nieuwkoop and Faber, 1967) are indicated. Ornithine decarbox- ylase (ODC) served as the loading control.
	mig30 (mig30) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 8.5, saggital section, anterior up.
	mig30 (mig30) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, saggital section, anterior up.
	mig30 ( mig30) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, anterior view, dorsal up (E) and dorsal view, anterior right (G).
	mig30 (mig30) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up (K), and a transverse section through trunk section, dorsal up (L).
	kazald1 (Kazal-type serine peptidase inhibitor domain 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 3 (4-cell), lateral view, anterior left, dorsal up.
	kazald1 (Kazal-type serine peptidase inhibitor domain 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, lateral view, anterior left, dorsal up.
	kazald1 (Kazal-type serine peptidase inhibitor domain 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.


	kazald1 (Kazal-type serine peptidase inhibitor domain 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 40, lateral view, anterior left, dorsal up.
	igfbpl1 ( insulin-like growth factor binding protein-like 1) gene expression in a Xenopus laevis embryo, as assayed by in situ ybridization, NF stage 15 lateral view, anterior left, dorsal up.
	igfbpl1 ( insulin-like growth factor binding protein-like 1) gene expression in a Xenopus laevis embryo, as assayed by in situ ybridization, NF stage 21, anterior view, dorsal up.
	igfbpl1 ( insulin-like growth factor binding protein-like 1) gene expression in a Xenopus laevis embryo, as assayed by in situ ybridization, NF stage 21, lateral view, anterior left, dorsal up (H), and dorsal view (I).
	igfbpl1 ( insulin-like growth factor binding protein-like 1) gene expression in a Xenopus laevis embryo, as assayed by in situ ybridization, NF stage 21, lateral view, anterior left, dorsal up (H), and dorsal view (I).