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First published online September 9, 2005
doi: 10.1242/10.1242/dev.02021

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Cardiac and CNS defects in a mouse with targeted disruption of suppressor of fused

Yale University School of Medicine, 333 Cedar Street, Box 208005, New Haven, CT 06520-8005, USA

View larger version (44K): [in a new window]   Fig. 1. Targeted disruption of the Sufu gene. (A) The Sufu gene consists of 12 exons and contains a putative PEST sequence. The carboxy terminus is required for Gli binding (Kogerman et al., 1999; Dunaeva et al., 2003). In the targeting vector (middle), the intron 6-exon 7 junction and most of exon 7 was replaced with the neo gene. The fragment used as an external probe in Southern blotting for screening and genotyping is indicated. (B) Southern blot analysis of genomic DNA from 9.5 dpc wild-type, heterozygous and homozygous embryos. DNA was digested with BamHI and hybridized with an intron 8 probe that detects the 9 kb normal allele and the 11 kb targeted allele. (C) Northern blot analysis of 9.5 dpc total whole embryo RNA using a full-length Sufu cDNA probe detected a 5.5 kb transcript in all genotypes, but aberrant 8.5 kb and 1.5 kb transcripts were detected in the homozygous mutant. A 3' probe did not detect any transcript in the homozygous mutant, but the expected 5.5 kb transcript was detected in wild-type and heterozygous embryos. Gapdh transcript is equal in all genotypes. (D) Western blot analysis of 9.5 dpc whole embryo lysates using an anti-Sufu antibody that detects the carboxy terminus. No Sufu protein expression was detected in the homozygous mutant, but the expected 54 kDa band was present in wild-type and heterozygous embryos. ß-Actin expression was equal in all genotypes.

View larger version (71K): [in a new window]   Fig. 2. Sufu-/- mutants exhibit an open neural tube and abnormal cardiac looping. Gross morphology of wild-type (A) and Sufu-/- (B,C) embryos at 9.5-10.5 dpc. Cross-sections of 9.5-10.5 dpc wild-type (D) and Sufu-/- (E,F) embryos stained with Hematoxylin and Eosin. Sufu-/- embryos had an open neural tube and failed to complete turning. The descending aorta was dilated (E,F). Some Sufu-/- embryos had a normal D-looped heart (B,E) whereas others displayed an inverted L-looped heart (C,F). Higher magnification of the cardiac region is shown in G (normal looping) and H (inverse looping). Red arrows display the direction of cardiac looping. da, descending aorta; ht, heart; nt, neural tube.

View larger version (77K): [in a new window]   Fig. 3. Nodal and Pitx2 expression in wild-type and Sufu-/- mouse embryos. (A,B) Whole-mount RNA in situ using a digoxigenin-labeled Nodal riboprobe on 7.5 dpc embryos shows Nodal expression in the region of the node in a wild-type embryo (A) and marked expansion of the region of Nodal expression in a Sufu-/- embryo (B). (C,D) Whole-mount in situ hybridization using a Pitx2 digoxigenin-labeled riboprobe on 8.5 dpc embryos (posterior views) shows Pitx2 expression in the head folds and left lateral plate mesoderm in a wild-type embryo (C); Pitx2 expression is absent in the left lateral plate mesoderm of a Sufu-/- embryo (D).

View larger version (124K): [in a new window]   Fig. 4. Abnormal node morphology in Sufu-/- mouse embryos. Immunofluorescent staining with anti-acetylated tubulin antibodies was performed on wild-type (A,D) and Sufu-/- (B,C,E,F) mouse embryos at 7.75 dpc. In wild-type embryos, the node is composed of tightly packed, monociliated cells and is roughly the shape of an isosceles triangle with the base oriented toward the posterior (yellow outlining in A). The cytoskeletons of cells surrounding the node are visible in A, which shows the normal size difference between node cells and their larger neighbors. Variable node dysmorphology (B,C) is seen in the Sufu-/- embryos. Five out of five 7.75 dpc Sufu-/- embryos examined displayed abnormal node architecture, ranging from mild changes in the shape of the node to irregular distribution of nodal cells. B shows an ovoid to diamond-shaped node in a Sufu-/- embryo. C shows three or more separate irregular islands of nodal tissue with flattened cells, atypical for a normal node, visible between the islands. (D-F) Higher magnification views show normal monocilia in a wild-type embryo (D), and in Sufu-/- embryos (E,F). Boxes in A-C outline the areas shown at higher magnification in D-F. All panels are oriented with anterior at the top and posterior at the bottom. Ant, anterior; Post, posterior. Scale bars: 50 µm in A-C; 10 µm in D-F.

View larger version (107K): [in a new window]   Fig. 5. Derepression of Shh RNA expression in Sufu-/- mutants. Whole-mount in situ hybridization using a Shh digoxigenin-labeled riboprobe on wild-type (A) and Sufu-/- (B) embryos at 9.5 dpc. Sufu-/- (B) embryos exhibited a dramatic upregulation of Shh expression throughout the brain and developing spinal cord when compared with wild type. Cross-section of a wild-type embryo (C) demonstrates that Shh is normally expressed in the floor plate; the Sufu-/- embryo (D) displays a dorsal expansion of Shh expression throughout the neural tube. Cross-sections are of the thoracic region. Scale bars: 100 µm.

View larger version (47K): [in a new window]   Fig. 6. Excess Hh signaling and ventralization of the neural tube in Sufu-/- mutants. Cross-sections of the thoracic region stained with Hematoxylin and Eosin show the morphology of a normal neural tube of wild type (A) and the open neural tube of Sufu-/- mutants (B) at 9.5 dpc. Immunohistochemistry with markers of neural progenitor cells (anti-PTCH, anti-Foxa2, anti-Nkx2.2 and anti-Pax6) in wild-type (C,E,G,I) and Sufu-/- (D,F,H,J) 9.5 dpc embryos. Ptch1 (a Hh target gene product), Foxa2 and Nkx2.2 were expressed in the floor plate of wild-type embryos (C,E,G), with Foxa2 expression being restricted to the ventral midline. In Sufu-/- embryos (D,F,H), expression of ventral markers expanded to dorsal regions of the neural tube. Pax6 was expressed in lateral regions of the ventricular zone of the wild type (I), but its expression was repressed in Sufu-/- mutants (J). Cross-sections are of the thoracic region. Scale bars: 50 µm.

View larger version (121K): [in a new window]   Fig. 7. Pax7 expression in wild type and Sufu-/- mutants. Whole-mount in situ hybridization using a Pax7 digoxigenin-labeled riboprobe on wild-type (A) and Sufu-/- (B) embryos at 9.5 dpc. The Sufu-/- embryo exhibited virtually no Pax7 expression throughout the brain and developing spinal cord compared with wild type. Cross-section of a wild-type embryo (C) demonstrates that Pax7 is normally expressed in the dorsal neural tube; a Sufu-/- embryo (D) displays virtually no expression throughout the neural tube. Cross-sections are of the thoracic region.

View larger version (58K): [in a new window]   Fig. 8. Ptch1 expression in wild type and Sufu-/- mutants. Whole-mount in situ hybridization using a Ptch1 digoxigenin-labeled riboprobe on wild-type (A) and Sufu-/- (B) embryos at 9.5 dpc. The Sufu-/- embryo exhibited an expansion of the expression domain and gross overexpression of Ptch1.

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