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First published online 21 April 2004
doi: 10.1242/dev.01112

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Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation

¹ Renal Division, Washington University School of Medicine, St Louis, MO 63110, USA
² Department of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO 63110, USA
³ Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA

View larger version (8K): [in a new window]   Fig. 1. Insertion of secretory gene trap vectors into Lama1 and Lamb1. (A) The vector pGT0TMp inserted into the third and largest intron of Lama1, which is 20.4 kb. The remaining 59 downstream exons and introns are not shown. (B) The vector pGT1TMpfs inserted into the 23rd and largest intron of Lamb1, which is 10.2 kb. The remaining exons (of the 33 total) and introns are not shown.

View larger version (61K): [in a new window]   Fig. 2. X-gal staining of Lama1 Trap and Lamb1 Trap embryos. Whole Lama1 Trap embryos are shown at E9.5 (A,C), E11.5 (D) and E13.5 (E). A control E9.5 littermate stained in X-gal is also shown (B). There is intense X-gal staining in the central nervous system and in presomitic mesoderm (arrowhead in C, black arrow in D, white arrow in E). Once formed, somites soon lose X-gal reactivity (black arrow in C). (F) The urogenital region was dissected out of an E16.5 male Lama1 Trap embryo and then stained with X-gal. Prominent staining of the vas deferens (white arrow) and punctate staining in kidneys was observed. Whole Lamb1 Trap embryos are shown at E11.5 (G) and E13.5 (H).

View larger version (104K): [in a new window]   Fig. 3. Expression of laminin chains in normal and mutant early postimplantation embryos. (A-G) Arrowheads indicate the embryonic basement membrane and the arrow indicates RM. (A) At E5.5, laminin 1 was detected in the embryonic basement membrane and in RM. (B) At E6.5, laminin 5 (red) is detected primarily in the embryonic basement membrane, though some is apparent in RM at the ectoplacental pole (asterisks). Laminin 1 (green) is abundant in RM, but its presence in the embryonic basement membrane is obscured by the 5 staining. (C,E-G) At E7.5 in wild type, laminin 1 (green) is present in both the embryonic basement membrane and in RM, but laminin 5 (red) appears confined primarily to the embryonic basement membrane. (D) This embryo is Lama5-/-, demonstrating the specificity of the 5 antibody. Note staining for 5 in the surrounding maternal tissue. (F) Note the absence of 5 in RM. (G) Nuclei (blue) are visible in the merged images. (H-J) X-gal staining of sections of Lama1+/- and Lama1-/- embryos, as indicated. Cells of the PE at the periphery of the normal embryos stain blue (H,I). In the mutant, the cells stain blue but are found in a cluster next to the small embryo (asterisk in J) owing to failure to migrate. Scale bar in A: 100 µm for A-D,H-J; 33.3 µm for E-G.

View larger version (113K): [in a new window]   Fig. 4. Analysis of basement membranes in Lama1-/- embryos. (A,C,E) Wild type; (B,D,F) Lama1-/-. Arrowheads indicate the embryonic basement membrane, and arrows indicate RM, when present. (A,B) Triple staining for laminin 1 (green), laminin 5 (red), and nuclei (blue) at E5.5. Both control and mutant embryos have a laminin 5-positive embryonic basement membrane, but the mutant lacks immunoreactivity of 1 and RM. (C,D) Double staining for laminin 5 (red) and nuclei (blue) at E6.5. Laminin 5 is present in the embryonic basement membrane in both embryos. (E,F) Double staining for collagen 1, 2(IV) (green) and nuclei (blue). Collagen IV is present in both the embryonic basement membrane and RM in the control, but only the embryonic basement membrane is present in the Lama1 mutant. Asterisk in F probably represents non-basement membrane associated collagen IV made by PE, clustered as in Fig. 3J. Note that the arrangement of nuclei in B and F suggest that cavitation occurred in Lama1 mutant embryos. Scale bar in F: 100 µm.

View larger version (80K): [in a new window]   Fig. 5. Analysis of Lamb1-/- embryos. Arrowheads indicate the embryonic basement membrane, and arrows indicate RM, when present. E5.5 control (A-C) and Lamb1-/- (D-F) embryos were stained for laminin ß1 (A,D); for laminin ß1 (red), cytokeratin 8 (green) and nuclei (blue) (B,E); and for laminin 5 (red), ctyokeratin 8 (green) and nuclei (blue) (C,F). Note the absence of basement membranes in the Lamb1-/- embryos, other than those associated with the uterine lining (UL). Anti-cytokeratin 8 stains the extraembryonic ectoderm (ExE), the PE and trophoblasts. More trophoblast invasion is apparent in the control than in the Lamb1-/- embryos. EE, embryonic ectoderm. Scale bar in F: 50 µm.

View larger version (126K): [in a new window]   Fig. 6. Analysis of basement membranes in Lama1-/-; Mr5 embryos. (A-D) Embryos were double stained for laminin 1 (green) and laminin 5 (red). Arrowheads indicate the embryonic basement membrane, and arrows indicate RM, when present. (A,B) At E6.5, the Lama1-/-; Mr5 embryo (B) has laminin 5 in the embryonic basement membrane but lacks laminin 1 and RM, when compared with the control (A). (C,D) At E7.5, Lama1-/-; Mr5 embryos appear to still be alive and to have an embryonic basement membrane containing laminin 5. (E,F) Embryos were triple stained for laminin ß1 (red), cytokeratin 8 (green) and nuclei (blue). The Lama1-/-; Mr5 embryo is much smaller than the control, but both contain embryonic ectoderm (EE), extra-embryonic ectoderm (ExE) and ectoplacental cone (EPC). Trophoblast blood sinuses (S) are present in the control but are lacking in the Lama1-/-; Mr5 embryo. Scale bars: in D, 100 µm for A-D; in F, 100 µm for E,F.

View larger version (156K): [in a new window]   Fig. 7. Analysis of Mr5 transgene expression on the Lama5-/- genetic background. Sections were double stained for laminin 1 (green) and 5 (red). The red channel is shown in A and C, and both red and green channels are shown in B and D. (A,B) At E7.5, transgene-derived laminin 5 was detected in the embryonic basement membrane and in RM (A,B), but no 5 was detected in a Lama5-/- embryo that lacked the transgene (C,D). Scale bar: 100 µm.

View larger version (65K): [in a new window]   Fig. 8. Gastrulation initiates in Lama1-/-; Mr5 embryos, as demonstrated by in situ hybridization of a digoxigenin-labeled brachyury (T) cRNA probe to frozen sections of E7.5 embryos. (AD) Two control/Lama1-/-; Mr5 embryo pairs from two different litters. Despite the aberrant shape of the Lama1-/-; Mr5 embryos, robust expression of brachyury is evident. (E,F) Brachyury expression in a Lama5-/- embryo is similar to the control.