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First published online 25 February 2004
doi: 10.1242/dev.01048

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Maspin plays an essential role in early embryonic development

¹ Baylor College of Medicine, Department of Molecular and Cellular Biology, One Baylor Plaza, Houston, TX 77030, USA
² Harvard Medical School, Department of Genetics, 200 Longwood Avenue, Boston, MA 02115, USA

View larger version (23K): [in a new window]   Fig. 1. Targeted disruption of maspin. (A) Targeting strategy. Homologous recombination replaces the seventh exon with the neomycin resistance (neo) gene. Note the change of SacI (S1) into BamHI in the construct. Restriction site are (S1) SacI; (Xh1) XhoI; (K1) KpnI; (B1) BamHI; (Xb) XbaI; (Sa1) SalI. (B) Southern blot analysis of Mp+/+ and Mp+/– ES cells. The genomic DNAs were digested with a SacI enzyme and the blot was probed with 32P-labeled SacI-SacI fragment (3.6 kb). The 3.6 kb band was from the wild-type allele. A novel 12 kb band indicated the presence of the targeted allele. (C) RT-PCR analysis of maspin mRNA expression in Mp+/+ and Mp–/– embryoid bodies.

View larger version (84K): [in a new window]   Fig. 4. Defective embryoid body formation in the Mp–/– ES cells. (A-D) Phase contrast micrographs of Mp+/+ and Mp–/– embryoid bodies. Arrowheads show the lumen formed in the Mp+/+ embryoid body and arrows show the outer layer of cells detaching from the Mp–/– embryoid bodies. (B) Maspin expression in Mp+/+ and Mp–/– embryoid bodies analyzed by immunostaining. Arrowhead shows the lumen in the Mp+/+ EB; arrow shows the endodermal cells which express maspin. Note no maspin protein is detected in Mp–/– EB. (C) GATA4 immunostaining of Mp+/+ and Mp–/– EBs. Arrows indicate the endodermal cells and arrowhead indicates the lumen of the Mp+/+ EBs. (D) HNF4 expression (fast-red labeled cells) of Mp+/+ and Mp–/– EBs analyzed by in situ hybridization. (E) Oct4 immunostaining of Mp+/+ and Mp–/– EBs.

View larger version (173K): [in a new window]   Fig. 2. Histological analysis and localization of maspin in early embryonic development. (A) Histological analysis of Mp+/+ and Mp–/– embryos at 5.5 dpc. (a) Mp+/+ embryo. (b,c) Abnormal Mp–/– embryos undergoing resorption. (d) Empty decidua from the implantation of presumed Mp–/– embryos. Arrows indicate the debris of the embryos. (B) Localization of maspin (a,c,e,g) and GATA4 (b,d,f,h) in early embryonic development from E4.5 to E7.5. Note that maspin is specifically expressed in the visceral endoderm from E5.5 to E7.5. GATA4 is present in the ve, pre and pe. exe, extra-embryonic endoderm; ve, visceral endoderm; ect, ectoderm; Tb, trophoblast cell; icm, inner cell mess; pe, paretial endoderm; pre, primitive endoderm.

View larger version (174K): [in a new window]   Fig. 3. Phase-contrast micrographs of Mp+/+ (left) and Mp–/– (right) embryonic outgrowths at different stages. Arrows indicate the inner cell mass and arrowheads indicate the trophoblast giant cell. icm, inner cell mass.

View larger version (47K): [in a new window]   Fig. 5. Apoptosis and proliferation analysis of Mp+/+ and Mp–/– EBs. (A) TUNEL analysis of the Mp+/+ and Mp–/– EBs. (a) TUNEL staining. Fluorescent green cells are TUNEL-positive apoptotic cells. Left panels are TUNEL staining; right panels are the overlapped images of DAPI and TUNEL staining. Note the pattern of apoptosis in Mp+/+ (below) and Mp–/– (above) EBs. Arrows indicate the apoptotic cells. (b) Analysis of TUNEL-positive apoptotic cells in Mp+/+ and Mp–/– EBs. N, the number of cells counted. No significant difference is observed between the two classes of EBs. (B) Mp–/– EBs have a reduced rate of cell proliferation. (a) The mitotic index of EBs was determined by immunostaining with anti-phosphorylated histone 3 (H3P). Arrows indicate the proliferating cells. (b) Statistical analysis of the percentage of H3P-positive cells in Mp+/+ and Mp–/– EBs. N, the number of cells counted. Note that there is a significant difference between the two classes of EBs.

View larger version (80K): [in a new window]   Fig. 6. Gain and loss of maspin function in embryoid body formation. (A-C) Rescue of defective EBs from Mp–/– ES cells using adenovirus-maspin. Note that the adenovirus control has no effect on lumen formation (arrows). (b) GATA4 staining of the adenovirus-maspin treated EBs. Arrows indicate GATA4-positive endodermal cells and arrowheads indicate the small lumen formed after adenovirus-maspin infection. Inset is Oct4 immunostaining of the adenovirus-maspin treated Mp–/– EBs. (C) Blocking of EB formation using a maspin antibody. Note that the pre-immune serum has no effect on Mp+/+ EBs. Arrows indicate the layer of cells that are detaching.

View larger version (73K): [in a new window]   Fig. 7. (A) Expression of fibronectin (a,c,e,g) and laminin (b,d,f,h) in Mp+/+ EBs, Mp+/+ E7.5 embryos, and Mp–/– EBs. (a,b) Mp+/+ EB sections after 3 days of cell culture. (c,d) Mp+/+ EBs after 8 days of cell culture. (e,f) Mp+/+ embryos at E7.5; (g,h) Mp–/– EBs after 8 days of cell culture. (B) Maspin affects endodermal cell adhesion. (a) Endodermal cell adhesion to fibronectin. (b) Endodermal cell adhesion to laminin matrix. Mp+/+ endodermal cells were treated with either preimmune serum or anti-maspin antiserum. Significant differences in cell adhesion were observed when the Mp+/+EN, +anti-Mas and Mp+/– EN cells to Mp+/+ EN cells (indicated by an asterisk) were compared. (c) Decreased rate of cell proliferation in the Mp+/– EN cells. Note the difference in the cell doubling times for the Mp+/+ and Mp+/– cells.