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First published online 10 July 2006
doi: 10.1242/dev.02473

Development 133, 3075-3083 (2006)
Published by The Company of Biologists 2006
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Axis specification and morphogenesis in the mouse embryo require Nap1, a regulator of WAVE-mediated actin branching

Andrew S. Rakeman1,2 and Kathryn V. Anderson1,*

1 Developmental Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA.
2 Biochemistry, Cell and Molecular Biology Program, Weill Graduate School of Medical Sciences, Cornell University, 445 East 69th Street, New York, NY 10021, USA.


Figure 1
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  		Fig. 1. Morphogenetic defects in Nap1khlo mutant embryos. (A,B) Mox1 expression marks the somites and presomitic mesoderm of wild-type E8.5 embryos (A) and reveals the small somites of E8.5 Nap1khlo embryos (B). (C-F) Brachyury (T) expression marks the primitive streak and notochord in E8.5 wild-type (C,E) and Nap1khlo (D,F) embryos. In the mutant, the primitive streak (arrow) was distended and the allantois failed to fuse with the chorion (asterisk). The notochord (arrowhead) appeared normal in Nap1khlo mutants. (G,H) The pancardiac marker Nkx2.5 was expressed in a single heart in wild-type embryos (G) and in the two lateral heart domains in Nap1khlo mutant embryos (H). Anterior is to the left in all panels.

 

Figure 2
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  		Fig. 2. Histological analysis of E8.5 Nap1khlo mutants. (A,B) The primitive streak region of wild-type embryos contained a small number of mesenchymal cells (A, arrowhead), whereas the Nap1khlo primitive streak contained a mass of disorganized mesenchymal cells (B, arrowhead). (C,D) The layered organization of myocardium (black arrows) and endocardium (white arrows) in the wild-type heart (C) was present in Nap1khlo mutant hearts (D). The foregut (asterisk) formed a closed pocket in wild-type embryos (C), but failed to close in Nap1khlo mutants (D).

 

Figure 3
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  		Fig. 3. Axis duplications in Nap1khlo mutants. (A-C) T expression. (A) Wild-type embryos have a single allantois, and a brachyury-expressing primitive streak and notochord at E8.5. Approximately one quarter of Nap1khlo homozygotes showed a partial or complete duplication of the anteroposterior axis. In some mutants, the allantois was duplicated (B, asterisk), whereas nearly the entire body axis, including the primitive streak and notochord, was duplicated in the most severely affected Nap1khlo mutants (C). (D,E) Tbx4 expression marks the allantois in a wild-type embryo (D) and in a Nap1khlo mutant with an ectopic allantois (E). Anterior is up in all panels.

 

Figure 4
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  		Fig. 4. Mapping and molecular characterization of Nap1 alleles. (A) The khlo mutation mapped between the SSLP markers D2SKI308 and D2SKI324 on chromosome 2 (http://mouse.ski.mskcc.org/). (B) Alignment of the N-terminal region of murine Nap1 (Mm) and its human (Hs), Drosophila melanogaster (Dm, kette) and C. elegans (Ce, gex-3) homologs. The leucine residue mutated in Nap1khlo is conserved in all four species (red box) and occurs in a leucine-rich region of the protein. (C,D) The Nap1GT insertion trapped the Nap1 transcript after exon 24 (C), creating a fusion of the 898 N-terminal amino acids of Nap1 with ß-geo (D). (E,F) Nap1khlo/Nap1GT mutants (F) arrested at E8.5 with multiple morphogenetic defects, including a distended primitive streak (arrows) and malformed somites (arrowheads); compare with wild type (E). Anterior is to the left in E and F.

 

Figure 5
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  		Fig. 5. Expression of Nap1 during development. (A) Nap1 protein was present in the epiblast (arrow) and visceral endoderm (arrowhead) in transverse sections of E6.5 embryos. (B) Nap1 is present in all three germ layers in transverse sections of E7.5 embryos, and is enriched in the apical region of the epiblast. (C-E) Nap1 was present in all embryonic structures in sagittal sections of E8.0 embryos (C) and was expressed in all tissues in transverse sections of E8.5 embryos (D,E). At E8.5, Nap1 was enriched in both apical and basal regions of the cells in the neural tube (D,E); based on the Nap1 mutant phenotypes, it is likely that this localization of Nap1 is required for neural tube closure. Anterior is to the left in A-C and dorsal is up in D and E. Scale bars: 100 µm.

 

Figure 6
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  		Fig. 6. Behavior of WAVE complex components in Nap1khlo mutants. (A) Western blot analysis of WAVE1 protein in E8.5 embryos, normalized to {gamma}-tubulin levels. (B-E) Immunofluorescent staining shows that Sra1 (B,C) and Abi1 (D,E) are localized to the surface of wild-type (B,D) but not Nap1khlo (C,E) primary mesodermal cells. Scale bars in B-E: 25 µm. (F-I) Actin structures in wild-type (F,H) and Nap1khlo (G,I) epithelial (F,G) and mesodermal (H,I) cells, visualized by phalloidin staining. (J,K) Localization of the actin-binding protein cortactin (green) and DAPI (blue) in wild-type (J) and Nap1khlo (K) mutant mesodermal cells. Scale bars in F-K: 50 µm.

 

Figure 7
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  		Fig. 7. Cell migration defects in Nap1khlo explants and embryos. (A,B) Wild-type (A) and Nap1khlo (B) primitive streak explants stained with DAPI (blue) and antibodies against E-cadherin (red) to distinguish the E-cadherin-expressing epithelial cells from E-cadherin-negative mesodermal cells. (C-F) Transverse sections of wild-type (C,E) and Nap1khlo (D,F) E7.5 embryos stained for expression of E-cadherin. Brackets mark the layer of mesenchymal cells adjacent to the primitive streak; whereas a single layer of mesoderm cells lie adjacent to the primitive streak in wild type, 3-4 layers of mesoderm cells underlie the Nap1khlo primitive streak. Scale bars: 100 µm.

 

Figure 8
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  		Fig. 8. Behavior of nascent mesoderm and endoderm. (A-D) Cripto (A,B) and Lim1 (C,D) expression in nascent mesoderm cells; these markers occupied their normal domains in Nap1khlo mutants (B,D) compared to wild-type embryos (A,C) at E7.5. (E,F) By E7.5, Cerl-expressing cells of the foregut migrated to the anterior of wild-type embryos (E), but failed to reach the anterior of Nap1khlo mutant embryos (bracket, F), and instead accumulated adjacent to the primitive streak (arrowhead). (G,H) Foxa2 expression in wild-type (G) and Nap1khlo mutant embryos (H) at E7.5. The Foxa2-expressing foregut domain failed to reach the anterior of Nap1khlo embryos (bracket). Anterior is to the left in all panels.

 

Figure 9
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  		Fig. 9. Expanded domain of expression of primitive streak markers in Nap1khlo mutants. (A-C) Posterior views of T expression in E7.5 wild-type (A) and Nap1khlo embryos (B,C). These Nap1khlo mutants expressed T ectopically at the embryonic-extraembryonic boundary (B) or in two distinct primitive streaks (C). (D,E) Wnt3 is expressed in the posterior of E6.5 wild-type embryos (D), but is expressed around the circumference of Nap1khlo mutants (E) at E6.5. Anterior is to the left in D and E.

 

Figure 10
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  		Fig. 10. AVE defects in Nap1khlo mutants. (A-D) Cells of the AVE, marked by expression of Cerl at E6.5 (A,B) and Hex at E7.5 (C,D), were present at the embryonic-extraembryonic boundary of wild-type embryos (A,C), but failed to migrate completely in half of the Nap1khlo mutants examined (B,D, brackets). Anterior is to the left. (E-H) All Hex-GFP-expressing cells migrated towards the anterior of wild-type embryos at E6.0 (E,G), but some Hex-GFP cells remained distal (F, arrow) or migrated to the posterior (H, arrows) in half of the Nap1khlo embryos examined (green, Hex-GFP; red, phalloidin). Anterior is out in E and F, and to the left in G and H. (I,J) High magnification anterior views of the Hex-GFP-expressing cells in E6.0 wild-type (I) and Nap1khlo (J) embryos. Vertical arrows in I and J represent the orientation of the proximodistal axis. Note that that although the wild-type AVE cells are elongated along the proximodistal axis, the Nap1khlo cells are not. Scale bars in E-J: 50 µm.

 

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© The Company of Biologists Ltd 2006