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Biochem Biophys Res Commun
1999 Jan 08;2541:42-8. doi: 10.1006/bbrc.1998.9892.
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cDNA cloning and distribution of the Xenopus follistatin-related protein.
Okabayashi K
,
Shoji H
,
Onuma Y
,
Nakamura T
,
Nose K
,
Sugino H
,
Asashima M
.
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Recently, several proteins which have a follistatin module have been isolated. One of them, the follistatin-related protein (FRP), is encoded by TSC-36 (TGF-beta-stimulated clone 36) in mouse, originally isolated as a cDNA clone up-regulated by TGF-beta1 in mouse osteogenic MC3T3E1 cells. To determine the physiological role of FRP in early Xenopus embryonic development, we cloned the Xenopus FRP (xFRP) cDNA. The resulting cDNA clone was a secreted glycoprotein consisting of 299 amino acid residues with about 70% similarity to the mammalian and avian FRPs. Northern blotting analysis revealed that xFRP gene expression started at stage 10, the onset of gastrulation, gradually increased during the blastula and neurula stages and was sustained through the tail-bud stage. Whole-mount in situ hybridization analysis showed the localization of xFRP mRNAs in the Spemann organizer, notochord, neural floor plate, hypochord and somite. The similarities with the pattern of expression of Xenopus follistatin mRNA suggests that xFRP may play a role in neuralization.
FIG. 1. Nucleotide and predicted amino acid sequences derived from Xenopus FRP cDNA clone. Numbering of nucleotides starts from the A of the initiation codon. The putative signal sequence at the NH2-terminus is boxed. The follistatin module is shadowed. A potential N-linked glycosylation site is underlined. The stop codon is indicated by asterisks and the polyadenylation signal is heavily underlined.
FIG. 2. (A) Alignment of the amino acid sequence of xFRP, TSC-36, and Flik. Identical residues are indicated by dots. The dash (2) represents a gap inserted for maximal alignment. The follistatin module is shadowed, and the included cystein residues are heavily shadowed. (B) Alignment of the follistatin modules of XFS319, xFRP, and TSC-36. Identical residues are shadowed. The potential N-linked glycosylation site of XFS319 is indicated by asterisks.
FIG. 3. Expression of xFRP mRNA during Xenopus development. Northern blot anaysis was performed using one mg of stagespecific mRNA per lane. The blotting membrane was probed with 32P-labeled xFRP cDNA (upper) or Xenopus ODC cDNA (lower). Arrow indicates xFRP. Embryonic stages are indicated by numbers according to Nieuwkoop and Farber [21].
FIG. 4. Spatial distribution of xFRP mRNA during Xenopus embryogenesis. Whole-mount in situ hybridization was performed with DIG-labeled antisense xFRP RNA. (A) Stage 10 embryo, vegetal view. xFRP RNA was slightly detected in the organizer (arrowheads). (B) Stage 12 embryo, dorsal view. Signals were detected in prechordal and chordal mesoderm. (C) Stage 15 embryo, dorsal view. (D) Stage 18 embryo, dorsal view. (E) Stage 21 embryo, dorsal view. (F) Stage 21 embryo, lateral view. (G) Stage 24 embryo. (H) Stage 30 embryo.
FIG. 5. Transverse or vertical sections of the embryos stained by whole-mount in situ hybridization. Dorsal side is at the top in each figure. (A) Transverse section of stage 12 embryo. Faint signal is localized in the chordal mesoderm (arrowheads). (B, C) Transverse sections of stage 14 and 17 embryos, respectively. Notochord and predicted neural floor plate are stained. (D, E, F) Transverse sections of stage 30 embryo. The middle, anterior, and posterior region of the trunk region. xFRP mRNA is localized in notochord, neural floor plate, and hypochord. Faint signal is also observed in somites. xFRP expression fades in the middle region of notochord (D). (G) Vertical section of stage 12 embryo. Prechordal and chordal mesoderm are clearly stained (arrowheads). (H) Vertical section of stage 22 embryo. xFRP is expressed throughout the notochord. Abbreviations: not, notochord; fp, floor plate; hp, hypochord; sm, somite.
fstl1 (follistatin like 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, vegetal view, dorsal up.
fstl1 (follistatin like 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior up.
fstl1 (follistatin like 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anteriorright, dorsal up.
fstl1 (follistatin like 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 29 and 30, lateral view, anteriorright, dorsal up.
