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Protease nexin-1 (PN-1)/glia-derived nexin (GDN) is a member of the Serpin (serine proteinase inhibitor) family, and can inhibit thrombin, plasmin, and plasminogen activators. PN-1 has been shown to be a neuroprotective factor in a number of assay systems, and this activity has been assumed to be a function of its protease inhibitory function. Here, we report cloning and characterization of a Xenopus orthologue of PN-1 (xPN-1). xPN-1 was isolated in a functional screen of an egg cDNA library for factors that modify early axial patterning. xPN-1 is expressed maternally through late tadpole stages, and is expressed preferentially in the notochord, the pharyngeal endoderm, the otic vesicle, and the ventral region of the brain in tailbud embryos. Over-expression of xPN-1 causes defective gastrulation, inhibits convergent extension movements in activin induced animal caps, and inhibits expression of a distinct subset of activin induced mesendodermal markers. Interestingly, expression of point or deletion mutation of the Reactive Center Loop of xPN1,which is essential for the protease inhibitory activity of all serpins, had effects on Xenopus development indistinguishable from those of wild type xPN-1. These observations suggest the possibility that xPN-1 has a novel activity in addition to its established function as an inhibitor of serine proteases.
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16797167
???displayArticle.link???Mech Dev
Fig. 2. Spatial expression pattern of xPN-1 in embryogenesis. (A) RT-PCR of the animal region (A), the marginal region (M), the vegetal region (V), the mixture of the dissected three parts (+++) and intact whole embryos (W) at Stage 9. (B–Q) in situ hybridization of xPN-1. (B) Stage 13 in dorsal view. The anterior side is up. (C) A transverse section of a Stage 13 embryo. (D) Stage 15 in dorsal view. The anterior side is up. (E) Transverse section of a Stage 20 embryo. (F–H) Stage 25. Red lines in F indicate the planes of the section shown in H and I. The embryo was cleared in G. (J–M) Stage 30. Red lines in J indicate the planes of the section shown in L and M. The embryo was cleared in K. (N–Q) Stage 35. Red lines in N and P indicate the planes of the section shown in P and Q, respectively. The embryo was cleared in O. br, brain; no, notochord. np, neural plate; ov, otic vesicle; ph, pharynx; pe, pharynxendoderm.
Fig. 5. xPN-1 inhibits expression of a subset of mesendoderm genes in whole embryos. Embryos were injected with 2 ng of each RNA into two dorsal blastomeres at the four-cell stage, and fixed at Stage 10.5 for in situ hybridization analysis. Embryo is in vegetal view and the dorsal side is up. (A–D) in situ hybridization of Xbra. Embryos were injected with EGFP (A), xPN-1 (B), xPN-1 pm (C) or xPN-1 δC RNA (D). xPN-1 and xPN-1 δC inhibited Xbra expression on the dorsal side (red arrows). xPN-1 pm caused weak Xbra expression (red arrows). (E–H) in situ hybridization of chordin (chd). Embryos were injected with EGFP (E), xPN-1 (F), xPN-1 pm (G) or xPN-1 δC RNA (H). chordin expression was not inhibited in each condition. (I–L) in situ hybridization of cerberus (cer). Embryos were injected with EGFP (I), xPN-1 (J), xPN-1 pm (K) or xPN-1 δC RNA (L). xPN-1 and xPN-1 δC inhibited cerberus expression on the dorsal side (red arrows). (M–P) in situ hybridization of goosecoid (gsc). Embryos were injected with EGFP (M), xPN-1 (N), xPN-1 pm (O) or xPN-1 δC RNA (P). goosecoid expression was not inhibited in each condition. (Q–T) in situ hybridization of Xnr1. Embryos were injected with EGFP (Q), xPN-1 (R), xPN-1 pm (S) or xPN-1 δC RNA (T). Xnr1 expression was not inhibited in any condition.
Fig. 3. Dorsal side overexpression of xPN-1 caused gastrulation defect. (A) Schema of constructs: xPN-1 pm has mutations at Arg362 and Ser363 of the process site
(P110) in the Reactive Center Loop (blue box). Amino acid sequences of intact and the processing mutant (pm) of the RCL are indicated. xPN-1 DC encodes
protein terminated at 348Gly (P15). (B) Uninjected control embryos at stage 16. (C) Embryos injected with 1 ng of xPN-1 RNA into the dorsal side at the four-cell
stage, photographed at stage 16. (D) Embryos injected with 1 ng of xPN-1 RNA into the ventral side at the four-cell stage, photographed at stage 16. (E) Uninjected
control embryos at stage 40. (F) Embryos injected with 1 ng of xPN-1 RNA into the dorsal side at the four-cell stage, photographed at stage 40. (G) Embryos injected
with 2 ng of xPN-1 pm RNA into the dorsal side at the four-cell stage, photographed at stage 40. (H) Embryos injected with 1 ng of xPN-1 DC RNA into the dorsal
side at the four-cell stage, photographed at stage 40. (I) Embryos injected 1 ng of xPN-1 RNA into the ventral side at the four-cell stage, photographed at stage 40. (J)
Embryos injected with 2 ng of xPN-1 pm RNA into the ventral side at the four-cell stage, photographed at stage 40. (K) Embryos injected with 1 ng of xPN-1 DC
RNA into the ventral side at the four cell stage, photographed at stage 40. (L) Graph of numbers of embryos, which showed normal gastrulation (open bar) and
gastrulation defect (solid bar) in injection of three xPN-1 RNAs.
Fig. 4. xPN-1 inhibits elongation of animal caps and a subset of mesendodermal genes expression induced by activin. (A) Animal caps at stage 20. Embryos were
injected with 1 pg of activin RNA plus 2 ng of EGFP (A), xPN-1 (B), xPN-1 pm (C) or xPN-1 DC RNA (D), or injected with 2 ng of EGFP (F), xPN-1 (G), xPN-1 pm
(H) or xPN-1 DC RNA (I) alone, and were dissected for animal cap assay at Stage 9. Uninjected caps were used as a control (E). (J) RT-PCR of animal caps
co-injected with 1 pg of activin RNA plus 2 ng of EGFP, xPN-1, xPN-1 pm or xPN-1 DC RNA at Stage 10.5. xPN-1 and both mutants of xPN-1 inhibited expression
of Xbra, chordin (chd) and cerberus (cer), but not goosecoid (gsc) or Xnr1, induced by activin. xPN-1, xPN-1 pm or xPN-1 DC alone did not induce any
mesendoderm gene expression. (K) RT-PCR of animal caps co-injected with 1 pg of activin RNA plus 2 ng of EGFP, xPN-1, xPN-1 pm or xPN-1 DC RNA at Stage
30. xPN-1 and both mutants of xPN-1 inhibited expression of muscle specific actin (m-actin), but not collagen type II (Col II) or endodermin (edd), induced by
activin. xPN-1 and xPN-1 DC induced expression of NCAM, Otx2 and XAG-1 when injected alone, while xPN-1 pm induced expression of Otx2 and XAG-1 when
injected alone. Neither wild type nor mutants xPN-1 expression induced mesendodermal genes in animal caps. WE, whole embryo positive control; RT-, reverse
transcriptase minus negative control in EF-1 a PCR.
