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doi: 10.1242/10.1242/dev.00176

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Node and midline defects are associated with left-right development in Delta1 mutant embryos

Gerhard K. H. Przemeck^1,*, Ulrich Heinzmann^2,*, Johannes Beckers¹ and Martin Hrabé de Angelis^1,

¹ GSF, Institute of Experimental Genetics, Ingolstaedter Landstr. 1, D-85764 Neuherberg, Germany
² GSF, Institute of Pathology, Ingolstaedter Landstr. 1, D-85764 Neuherberg, Germany

View larger version (116K): [in a new window]   Fig. 1. Tail placement in E9.5 and heart morphology in E10.5 embryos. (A) As a result of an anti-clockwise rotation, the tail lies at the right body side of the wild-type embryo (+/+). In half of the homozygous Dll1lacZ mutants (-/-), the tail was left-sided or abnormal (three out of 33), due to the randomisation of the direction of axial rotation. (B) Expression of Hand1 in the wild-type heart (+/+) is seen in the future systemic ventricle (sv) and on the outside of the future pulmonary ventricle (pv). In mirror-imaged mutant hearts (-/-), Hand1 expression was detected in the appropriate position, although in reversed locations. (C-E) Scanning electron micrographs of a normal, fully looped wild-type heart (C), an incomplete looped mutant heart (D) and of a mirror-imaged heart (E), where the future pulmonary ventricle is on the left and the future systemic ventricle on the right. The arrow in E indicates the beginning bifurcation into branchial arteries. a, atrium; bc, bulbus cordis; L, left body side; pv, future pulmonary ventricle; R, right body side; sv, future systemic ventricle.

View larger version (146K): [in a new window]   Fig. 2. Analysis of LR-asymmetric gene expression at E8.5 (A-N). (A-E) Expression of Nodal at the 0-6 somite stage. In wild-type embryos (A) Nodal is expressed in the left lateral plate mesoderm (LPM) and lateral to the node (arrow). (B) Cross-section of the embryo in A; arrows indicate Nodal expression in the left LPM and lateral to the node. In homozygous Dll1lacZ mutant embryos Nodal expression in the LPM was either left-sided (4/21), right-sided (4/21; E), bilateral (3/21; arrows in D) or not detectable (10/21). Nodal expression in the node region was either normal (4/21), changed [3/21 with a right bias (arrow in E) or equal to both sides] or absent (14/21). (C) Cross-section of the embryo shown in D; arrows indicate the bilateral expression domains of Nodal in the LPM. (F-H) Leftb expression at the four to seven somite stage. Leftb expression in wild-type embryos (F) was exclusively in the left LPM, whereas in homozygous Dll1lacZ mutant embryos Leftb expression was either left-sided (5/16, H), right-sided (3/16), bilateral (4/16; arrows in G) or not detectable (4/16). (I-L) Pitx2 expression at the six to 10 somite stage. Expression in wild-type embryos (I) was found in the head mesenchyme and the left LPM. In homozygous Dll1lacZ mutant embryos, Pitx2 expression was found in the head mesenchyme and either in the left LPM (5/25), right LPM (1/25; arrow in K), bilateral in the left and right LPM (15/25; arrows in L) or absent from the LPM (4/25). (M,N) Floorplate-specific expression of Ebaf at the two to six somite stage. In wild-type embryos (M), expression of Ebaf was found in the left half of the floorplate, whereas in homozygous Dll1lacZ embryos (N), Ebaf expression was not detectable (0/11). (O,P) Ventral view of brachyury (T) expression at the six to 10 somite stage; pictures were taken in a transmitted-light mode. A continuous expression of T was found in the notochord of wild-type embryos (arrowheads in O), whereas notochordal expression of T in homozygous Dll1lacZ embryos (P) was strongly reduced or absent.

View larger version (77K): [in a new window]   Fig. 3. Analysis of midline morphology. (A,B) Hnf3b expression at E8.5. Sections of plastic embedded embryos after whole-mount RNA in situ hybridisation. The floorplate specific expression domain (green brackets in A and B) in homozygous Dll1lacZ mutant embryos (B) was enlarged in comparison with wild-type embryos (A). Note the sheet-like notochord morphology in B, which appeared regionally along the AP axis. (C-H) Comparison of cell numbers between wild type and mutant floorplate and notochord at E9.5 and E10.5. (C-F) Semi-thin cross-sections of the neural tube just posterior to the forelimb-bud level of wild-type (C,E) and homozygous Dll1lacZ mutant embryos (D,F). (G,H) Quantitative analysis of serial cross sections of five individuals each per age and genotype. Homozygous Dll1lacZ mutant embryos have significantly more cells in the floorplate (G) and fewer cells in the notochord (H) when compared with wild-type embryos (P<0.0007 for the floorplate, P<0.0005 for the notochord). Broken line indicates that parts of the picture were removed due to space restrictions.

View larger version (88K): [in a new window]   Fig. 4. Expression analysis of Notch-signalling pathway genes at TS11 (E7.5). Each panel shows a view of the embryo from the top (the approximate position of the node is marked by red circles) and below a lateral view of the same embryo (the position of the allantois is marked by asterisks). Anterior and posterior are marked by a and p, respectively. (A,B) X-gal staining to demonstrate the activity of ß-galactosidase (lacZ) in heterozygous (Dll1+/lacZ; A) and homozygous (Dll1lacZ/lacZ; B) mutant embryos. (C,D,E,G) Expression of delta 3 (Dll3, C), jagged 1 (Jag1, D), Notch1 (E), Notch2 (G) and lunatic fringe (Lfng, I) in wild-type embryos. (F,H,K) Although expression of Dll3 and Jag1 is not changed in homozygous Dll1lacZ mutant embryos at this stage, expression of Notch1 (5/18), Notch2 (4/18) and Lfng (4/18) among the offspring from Dll1lacZ heterozygous crosses was abnormal.

View larger version (177K): [in a new window]   Fig. 5. Scanning electron micrographs (SEM) of the mouse node from TS10 to TS12 (E7.5-E8.5). Insets show the corresponding embryo at lower magnification. Anterior and posterior are marked by a and p, respectively. (A,B) At TS10, the node is established at the apex of the embryonic cone. The wild-type node (A) consists of small mesendodermal cells each with a prominent single cilium. In homozygous Dll1lacZ mutants (B), morphological disturbances are evident as rupturing of the surface, bulging of cells and loss of monociliated cells. (C,D) The node at TS11. Wild-type embryos (C) have a club-shaped node with evenly distributed ciliated cells. Homozygous Dll1lacZ mutants have a deformed node, where abnormal cell masses adjacent to the node disrupt the node symmetry (arrow in D). (E,F) SEM of the ventral mouse node from embryos at the six-somite stage (TS 12). The wild-type node (E) consists of microvilli-lined mesendodermal cells each with a cilium, whereas the homozygous Dll1lacZ mutant node contained in addition enlarged cells without cilia (arrows in F).

View larger version (38K): [in a new window]   Fig. 6. Scheme of Notch-signalling pathway gene expression pattern at TS11 in wild-type (A) versus mutant (B) embryos. Details are given in the text. Hypothesised molecular boundaries are indicated as broken green lines, morphological boundaries of the node and embryonic midline as broken black lines.