Morphogenetic movements at gastrulation require the SH2 tyrosine phosphatase Shp2 - PubMed (original) (raw)
Morphogenetic movements at gastrulation require the SH2 tyrosine phosphatase Shp2
T M Saxton et al. Proc Natl Acad Sci U S A. 1999.
Abstract
The SH2 domain-containing tyrosine phosphatase Shp2 plays a pivotal role during the gastrulation of vertebrate embryos. However, because of the complex phenotype observed in mouse mutant embryos, the precise role of Shp2 during development is unclear. To define the specific functions of this phosphatase, Shp2 homozygous mutant embryonic stem cells bearing the Rosa-26 LacZ transgene were isolated and used to perform a chimeric analysis. Here, we show that Shp2 mutant cells amass in the tail bud of embryonic day 10.5 chimeric mouse embryos and that this accumulation begins at the onset of gastrulation. At this early stage, Shp2 mutant cells collect in the primitive streak of the epiblast and thus show deficiencies in their contribution to the mesoderm lineage. In high-contribution chimeras, we show that overaccumulation of Shp2 mutant cells at the posterior end of the embryo results in two abnormal phenotypes: spina bifida and secondary neural tubes. Consistent with a failure to undergo morphogenic movements at gastrulation, Shp2 is required for embryo fibroblast cells to mount a positive chemotactic response to acidic fibroblast growth factor in vitro. Our results demonstrate that Shp2 is required at the initial steps of gastrulation, as nascent mesodermal cells form and migrate away from the primitive streak. The aberrant behavior of Shp2 mutant cells at gastrulation may result from their inability to properly respond to signals initiated by fibroblast growth factors.
Figures
Figure 1
Western blot analysis of ES cell lines. Upper, αShp2; Lower, αp120RasGAP. αShp2 antibody (UBI) does not recognize the mutant Shp2 protein. ES cell lines no. 12 and no. 5 were derived from Shp2 mutant blastocysts. R1 ES cell line lysates are shown for comparison (15).
Figure 2
Shp2 mutant cells accumulate in the tail bud and neural tissue of developing chimeric embryos. Chimeras generated with Shp2 (A_–_C) or wt (D) ES cells were dissected at E10.5 of development and stained for β-galactosidase activity with Salmon-Gal to determine the extent of ES cell contribution. Arrowheads in A_–_C point to the tail bud, which exhibits a strong accumulation of mutant cells. wt cells were able to contribute to the entire developing embryo (D). Histological sections through the trunk region of X-Gal-stained E10.0 chimeric embryos. Chimeric embryos were generated with Shp2 (E and F) or wt (G) ES cells. In medium-contribution Shp2 chimeric embryos (E), almost all of the mutant cells (blue) were found within the neural tissues of the embryo, which is ectoderm-derived. Ectoderm is the only tissue that is not generated by the progression of cells through the primitive streak at gastrulation. In high-contribution Shp2 chimeras (F), there was a strong deficiency of mutant cell contribution to the somitic mesoderm (arrows). In medium-contribution wt chimeras (G), the ES cells can contribute to all tissues of the developing embryo. drg, dorsal root ganglia; me, mesenchyme; s, somites; nt, neural tube.
Figure 3
Accumulation of Shp2 mutant cells within the caudal neuroectoderm leads to multiple defects, including spina bifida and secondary neural tubes. (A and B) Scanning electron micrograph images of E10.5 wt (A) and Shp2 mutant (B) chimeric embryos, highlighting the spina bifida, or open neural tube. (C_–_H) Transverse histological sections through the tail of an Shp2 mutant chimera depict the formation of a secondary neural tube. Rostral (C) to caudal (H) sections are shown.
Figure 4
Shp2 mutant cells show reduced contribution to the mesodermal wings of gastrulating chimeric embryos. wt (A, C, and E) and Shp2 mutant (B, D, and F) chimeras in whole-mount (A and B) or tissue sections (C_–_F). Anterior is to the left. ES cells do not contribute to the extraembryonic region and primitive endoderm of chimeric embryos and are therefore unstained. Arrow in D points to hyperaccumulation at the posterior epiblast (leading to a misshapen epiblast at this region fated to become neuroectoderm); similarly, arrowhead in F points to buildup of mutant cells within the posterior epiblast. end, endoderm; me, mesoderm; epi, epiblast; ng, neural groove; ext, extraembryonic region; ps, primitive streak.
Figure 5
Shp2 is required for cell migration. (A) Boyden chamber migration assays with wt (a and c) or Shp2 mutant (b and d) cells challenged with PDGF (a and b) or aFGF (c and d) as chemoattractive agents. Photos were taken with brightfield optics with the ×20 objective. Hematoxylin stains cell nuclei purple (arrow, a); the pores on the membrane through which the cells migrate are clearly visible (arrowhead, d). Similar results were obtained in three separate experiments by using two different Shp2 mutant cell lines. (B) Boyden chamber migration assays comparing Shp2 mutant cells infected with a control retrovirus (white bars) or a retrovirus containing the Shp2 gene (blue bars). Reexpressing Shp2 within the mutant fibroblast cells rescues their ability to respond to aFGF as a chemotactic agent. Shown is the average number of cells from five random fields of view within one membrane. Similar results were obtained in three separate experiments using two different rescued cell lines.
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