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???displayArticle.abstract??? Hepatocyte growth factor (HGF) specifically activates Met tyrosine kinase receptor, leading to mitogenic, motogenic, and morphogenic responses in a wide variety of cells. To know a role of HGF in Xenopus embryogenesis, loss-of-function mutation was introduced by dominant expression of truncated tyrosine kinase-negative Met. When tyrosine kinase-negative Met mRNA was micro-injected into two-cell to eight-cell stages Xenopus embryos, the liver development was mostly impaired and structures of pronephros and the gut were grossly underdeveloped in the restricted, late stage of development. These results strongly suggest that functional coupling between HGF and Met is essential for the development of internal organs originated from primitive gut and possibly involved in embryonic skeletogenesis. Together with developmental abnormality in mice mutated with HGF or Met gene, essential role of HGF for liver development is highly conserved from amphibian to mammalian species.
FIG. 1. Micro-injections of truncated Met mRNA in Xenopus em
bryo. (A) Schematic representation of the structure of the normal
(full-length) Met receptor and the truncated Met (DTKMet). The
indicate transmembrane domain is indicated by a closed box. The stippled
region denotes the tyrosine kinase domain. (B) Schematic structure
of pSP64 poly A vector for in-vitro synthesis of DTKMet mRNA. TM,
Transmembrane domain; TK, tyrosine kinase domain; SP6 pro, SP6
promotor.
FIG. 2. Effects of the truncated Met mRNA on Xenopus embryo. The injected embryos were allowed to develop until early tadpole stage
(A, B, C and D). Control embryos injected with 800 pg of b-galactosidase mRNA into animal (A) or vegetal (B) blastomeres at 4 cell stage,
had a normal phenotype. Embryos injected with 800 pg of DTKMet mRNA into animal blastomeres at the 4-cell stage also had a normal
phenotype (C). Embryos injected with 800 pg of DTKMet mRNA into vegetal blastomeres at the 4-cell stage had defects in ventral and
posterior regions (D).
FIG. 3. Dose-dependent increase of abnormal embryo by
DTKMet mRNA expression. Increasing doses of DTKMet mRNA
were injected into vegetal blastomeres in 4-cell stage embryos. Occurrences of typical embryos, (A) 20 pg of DTKMet mRNA; (B) 200 pg of DTKMet mRNA; (C) 400 pg of DTKMet mRNA; (D) 800 pg of
DTKMet mRNA; (E) 800 pg of b-galactosidase mRNA. (F) Frequency
of abnormal phenotypes indicated in A-E. Embryos were micro-injected
with DTKMet mRNA (closed circles) in doses ranging from 20
pg to 800 pg or b-galactosidase mRNA (open circle) in a dose of 800
pg, but the amount of the injected RNA solution were constant for
each dose. Over twenty embryos were used for each experimental
condition.
FIG. 4. External appearance and histological cross section of defected embryo injected from the vegetal side with DTKMet mRNA. Ventral
views of an typical embryo injected with 800 pg of DTKMet mRNA into vegetal blastomeres at the 4-cell stage and a normal embryo is indicated
in (A) and (B), respectively. Transverse sections of a stage 44 embryo injected with DTKMet mRNA (C, E and G) and a normal embryo (D, F
and H) are shown. Close-up views of liver of the DTKMet introduced embryo (E) and the liver of a normal embryo (F), and close-up views of the
developing kidney of the DTKMet introduced embryo (G) and a normal embryo (H) are respectively shown. Transverse sections were prepared
from the midtrunk region. e; eye, g; gut, h; heart, l; liver, l*; liver synonym, nc; notochord, nt; neural tube, p; pronephros, pt pronephros tubule.
