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First published online 13 August 2008
doi: 10.1242/dev.022897

Development 135, 3053-3062 (2008)
Published by The Company of Biologists 2008
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Chato, a KRAB zinc-finger protein, regulates convergent extension in the mouse embryo

María J. García-García1,2,*, Maho Shibata1 and Kathryn V. Anderson2

1 Molecular Biology and Genetics Department, Cornell University, Ithaca, NY 14853, USA.
2 Sloan Kettering Institute, New York, NY 10021, USA.


Figure 1
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  		Fig. 1. Mesoderm defects in chato embryos. Wild-type (wt) (A,C,E,G) and chato mutant (B,D,F,H) mouse embryos were assayed by in situ hybridization with markers expressed in head mesenchyme/lateral plate mesoderm/somitic mesoderm (Twist1; A,B, dorsal and ventral views, respectively), somites (Meox1; E,F, ventrolateral views) and cardiac mesoderm (Nkx2.5; G,H, ventral views). Staining for β-galactosidase activity from a Nodal-lacZ reporter labeled lateral plate mesoderm and node of wild-type (C) and chato mutant (D) embryos (lateral views). Thirty-three percent of chato mutants (n=184) had condensed somites that appeared narrow and laterally extended (F). In 52% of chato embryos (n=184), somites were not clearly discernible morphologically, but somite markers Twist1 and Meox1 marked some imperfectly shaped somites. Only 15% of chato mutants showed normal somites. Arrowheads in A,B point to head mesenchyme. Brackets in C,D highlight the different width of the lateral plate mesoderm in wild-type and chato mutant embryos. Brackets in E,F highlight the different width of the somites. Arrowheads in H mark the cardiac mesoderm in chato mutants. LPM, lateral plate mesoderm; som, somites.

 

Figure 2
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  		Fig. 2. Defects in the neural epithelium and notochord of chato embryos. Wild-type (A,C,F,I), chato (B,D,E,G,J) and Lp mutant (H) mouse embryos at E8.5 were assayed by in situ hybridization with markers expressed at rhombomeres 3 and 5 (Krox20; A,B, dorsal and ventral views, respectively), neuroepithelia (Sox2; C-E, ventral views), somites (Meox1; F-H, transverse sections) and notochord (T; F-H, transverse sections; I,J, posterior and ventral views, respectively). In some chato mutants, parts of the neuroepithelium remained open (arrowhead in D), giving the neural tube a wavy appearance. Transverse sections in F-H were hybridized with probes for both T (arrowheads) and the somitic marker Meox1 (arrows). In chato mutants, the notochord was embedded in the mesendoderm layer (arrowhead in G) and never formed an individualized rod (arrowhead in F). The notochord of Lp mutants is wider than that of wild-type embryos (F-H, arrowheads) (Greene et al., 1998Go). Expression of T in chato mutants (J) shows areas where the notochord was wider (w), thinner (t) or absent (a) as compared with wild-type embryos (I, arrowhead). NT, neural tube; not, notochord; r3, rhombomere 3; r5, rhombomere 5; som, somitic mesoderm.

 

Figure 3
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  		Fig. 3. The chato mutation disrupts Zfp568. (A) Domain structure of mouse Zfp568, containing two KRAB-A (green)/KRAB-B (yellow) domains and eleven zinc fingers (purple). The red asterisk marks the position of the chato point mutation. The red arrow points to the truncation caused by the RRU161 gene-trap. (B) Sequence comparison of the first KRAB-A domain of Zfp568/Chato with the KRAB-A consensus. Conserved residues are highlighted in green. Gray bars underline residues required for transcriptional repression (Margolin et al., 1994Go). Red letters indicate the Leu to Pro change caused by the chato point mutation. (C-F) Complementation test between chato and RRU161 gene-trap alleles. Wild-type (F) and mutant embryos of the allele combinations indicated (C-E) were assayed by in situ hybridization with T and Meox1 probes. The overall embryonic morphology, as well as defects in somites and midline, are indistinguishable between the different Zfp568 allele combinations. Notochord expression of T was irregular, showing a variable width and interruptions (arrows in D,E). (G-L) In situ hybridization with a Zfp568 probe on wild-type embryos at E7.5 (G,H) and E8.5 (J,L). RRU161 mutant embryos, which generate truncated Zfp568 transcripts, were used as negative controls (I,K). Zfp568 is expressed in all embryonic and extraembryonic tissues, as confirmed in transverse sections (H,K,L). Zfp568 was expressed at higher levels in the extraembryonic ectoderm (arrowheads). NT, neural tube; me, mesoderm; ect, ectoderm; end, endoderm; se, surface epithelia.

 

Figure 4
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  		Fig. 4. Convergent extension in wild-type definitive endoderm. Whole-mount in situ hybridization with Ttr probes of wild-type mouse embryos at E7.5 (A,B), E8.0 (C-F) and E8.5 (G-J). Lateral and posterior views illustrate how definitive endoderm (white) grows along the anterior-posterior axis (length) and narrows laterally (width). Ttr highlights the extraembryonic visceral endoderm (VE). The white tissue covering the embryonic region corresponds to definitive endoderm (DE). Arrows point to white extraembryonic tissue (ext). All images are at the same magnification. At E7.5, some VE cells (arrowhead in A,B) still overlay the exterior of the embryonic region; the line in A,B delimits embryonic-extraembryonic parts. Gut closure prevented visualization of all the definitive endoderm in I and J. (b,d,f,h,j) Representative transverse sections of the embryos in the columns above, counterstained with Fast Red. Sections correspond to intermediate levels along the anterior-posterior axis. Only half of each section is shown (midline at the right edge of panel). (K) Plot of length (blue) and width (green) definitive endoderm measurements in wild-type embryos of different stages; data in µm. Dimensions of the endoderm were taken as exemplified by dashed lines in G,H. Note that measurements were taken in non-Ttr-stained embryos, in which transparency of the tissue allowed for accurate measurements of the whole definitive endoderm. Error bars indicate s.d. See Fig. 5H for primary data.

 

Figure 5
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  		Fig. 5. Failure of convergent extension in the definitive endoderm of chato mutants. (A-D) Wild-type (A,B) and chato mutant (C,D) 8-somite stage mouse embryos hybridized with Ttr probes to highlight extraembryonic visceral endoderm (blue) and definitive endoderm (exterior layer of embryonic tissues in white). ext, white extraembryonic tissue. (A,C) Lateral views; (B,D) anterior views. (E-G) Plots of (E) wild-type (blue) and chato (red) definitive endoderm length (µm), (F) wild-type (green) and chato (red) definitive endoderm width (µm), and (G) definitive endoderm length-to-width ratio (LWR) in wild-type (gray) and chato mutant (red) embryos. Error bars indicate s.d. *P<0.05, **P<0.01. (H) Length and width average measurements ±s.d. in µm. The number of embryos analyzed for each stage is indicated (# embryos); na, not assayed.

 

Figure 6
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  		Fig. 6. Cell number changes across the width of the definitive endoderm. (A) Plot of the average definitive endoderm cell number in wild-type (gray) and chato mutant (red) mice. Cells were counted in sections of the definitive endoderm stained with Fast Red at medial levels along the anterior-posterior axis (see Fig. 4). Error bars indicate s.d. (B) Average number of cells ±s.d. The total number of sections counted for each condition is indicated (# sections); na, not assayed. ***P<0.0001.

 

Figure 7
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  		Fig. 7. Proliferation in definitive endoderm. Cryosections of wild-type (A,C) and chato mutant (B,D) mouse embryos at different embryonic stages were labeled with anti-E-cadherin (red) and phospho-histone H3 (green) antibodies. Mitotic cells (green) in the definitive endoderm (highlighted in red by localization of E-cadherin) are indicated by arrowheads. E-cadherin is also present in E7.5 epithelia (ep), embryonic surface ectoderm (se) and extraembryonic visceral endoderm (VE, dashed line). Proliferation of mesoderm and epithelial tissues was not significantly different between wild-type (n=21 embryos/380 sections) and chato mutant (n=3 embryos/56 sections) embryos at these stages.

 

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© The Company of Biologists Ltd 2008