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Axial skeletal defects caused by mutation in the spondylocostal dysplasia/pudgy gene Dll3 are associated with disruption of the segmentation clock within the presomitic mesoderm

¹ Division of Mammalian Development, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
² Developmental Biology Unit, The Victor Chang Cardiac Research Institute, 384 Victoria Street, Darlinghurst, NSW 2010, Australia
³ Department of Genetics, Case Western Reserve University, and University Hospitals Cleveland, 10900 Euclid Avenue, Cleveland, OH 44106-4955, USA

View larger version (34K): [in a new window]   Fig. 1. Generation of Dll3 null mutant mice. (A) Mouse Dll3 locus, targeting vector and targeted allele, exons are boxed with coding exons in black. The neomycin resistance gene and the Herpes simplex virus thymidine kinase gene transcribed by the PGK1 promoter are shown (PGK-Neo, PGKtk). The genomic probe that identifies different HindIII fragment sizes for the wild type (14 kb) and targeted (10.5 kb) alleles is shown. PCR primers are defined by black arrowheads 1 (D3F) and 2 (D3R), and PGK-1. (B) Southern blot analysis of genomic DNA from wild-type (Dll3/Dll3), heterozygous (Dll3/Dll3neo) and homozygous null (Dll3neo/Dll3neo) mice. (C) PCR genotyping of embryos and mice from heterozygous matings. Primers D3F, D3R and PGK1 amplified 340 bp and 450 bp representing the Dll3 and Dll3neo alleles, respectively.

View larger version (68K): [in a new window]   Fig. 2. Dll3neo/Dll3neo mutants have a truncated body axis and skeletal dysplasia. (A) Dll3neo/Dll3neo mutants have a shortened body and tail compared with Dll3/Dll3neo mice. (B) Lateral view of Alcian Blue-stained embryos (14.5 dpc). The positions of vertebrae: cervical (c1 and c2), thoracic (t1), lumbar (l1), sacral (s1) and coccygeal (co1) are indicated. (C,D) Dorsal view of developing skeleton. (C) Dll3/Dll3neo embryo from left in (B). (D) Dll3neo/Dll3neo embryo from right in B. Red dots indicate centrum corresponding to the position of t1, white dots indicate centrum of thoracic vertebrae. Note that in Dll3neo/Dll3neo embryos, ossification centres lie two and three in a row instead of lying in column as seen in Dll3/Dll3neo embryo (C). Scale bar: 1.35 mm in B; 675 µm in C.

View larger version (169K): [in a new window]   Fig. 3. Skeletal dysplasia in Dll3neo/Dll3neo mutants leads to disorganisation of the peripheral nervous system. Parasagittal sections of 13.5 dpc Dll3/Dll3neo (A-C) and Dll3neo/Dll3neo (D-F) embryos. (A) Dorsal root ganglia and cartilage primordia of vertebrae are evenly spaced along the axis. (B) Enlargement of thoracic region shown in A. (C) The cartilage primordium of the bassioccipital bone and the first two cervical vertebrae are clearly identifiable as is the rostralmost dorsal root ganglion which lies caudal to cervical vertebra 2. Note the even spacing of spinal nerves (dots) in (B,C). (D) Dorsal root ganglia are irregular in size and shape and are fused. This is evident in the thoracic region (E), where the arrangement of vertebrae and rib heads is also highly disorganised. Fused dorsal root ganglia are also evident in the cervical region (F) where the cartilage primordia of cervical vertebrae 1 and 2 are fused. Note the uneven distribution of spinal nerves (dots) in (E,F). Dorsal root ganglion (g), vertebra (v), head of rib (r), cervical vertebra (c), thoracic vertebra (t), basioccipital bone (b). Scale bar: 1.8 mm in A,D; 680 µm in B,C,E,F.

View larger version (115K): [in a new window]   Fig. 4. Elements of the peripheral nervous system are disorganised in Dll3neo/Dll3neo mutants. Whole-mount immunohistochemistry with an anti-neurofilament antibody of Dll3/Dll3neo embryos (A,B,F) and Dll3neo/Dll3neo mutant embryos (C-E,G) at 10.5-11.5 dpc. Lateral view (A-E) and dorsal view (F,G). (A) Dorsal root ganglia (drg), spinal nerve (sn) and sensory chain ganglia (scg) are evenly spaced. (B) The region between the fore and hind limbs of (A) is marked with a line (anterior towards the top). Lines dorsal and ventral to the somites mark individual somitic segments and show that ventral spinal axons pass exclusively through the anterior of the somite segment. (C) dsg, sn and scg are unevenly spaced. (D) The region between the fore and hind limbs of (C) is marked with a line (anterior towards the top). Lines mark out individual somitic segments and show that the spinal axons pass through the anterior, posterior or central part of the somite segment. (E) Dll3neo/Dll3neo mutant embryo (11.5 dpc). The disarray of spinal axons and scg is more severe than in (C). (F) Dorsal view of A shows a straight neural tube, while in G the same view of E indicates that the neural tube is ‘kinked’ (anterior towards the top). Scale bar: 730 µm in A,C,E; 150 µm in B,D; 365 µm in F,G.

View larger version (158K): [in a new window]   Fig. 5. Somitogenesis is delayed and irregular with reduced mesenchymal condensation in Dll3neo/Dll3neo mutants. (A-F) Lateral view of the caudal part of 9.5 dpc embryos after RNA whole-mount in situ hybridisation with Mesp2. The black line (A-D) indicates the position of the last formed somite boundary and Mesp2 expression lies just caudal to the somite boundary that is next to form in (A,B). (A) Regularly sized and spaced epthithelial somites are present in Dll3/Dll3neo embryos. The distance between the band of Mesp2 expression and the last formed somite is equivalent to one somite width. (C) Irregularly sized epthithelial somites are formed in this Dll3neo/Dll3neo embryo. The distance between the band of Mesp2 expression and the last formed somite is equivalent to three somite widths. (E) No epithelial somites were detected in this Dll3neo/Dll3neo embryo. The band of Mesp2 expression is in an equivalent position to that in A. (B,D,F) Higher magnifications of A,C,E. (G-L) Ventral view of 8.5 dpc embryos after whole-mount in situ hybridisation with ß-spectrin 2. The arrow indicates where the next somite boundary will form. (G,H) Epithelialisation of mesenchyme to form somites is evident and marked by distinct ß-spectrin 2 gene expression in the centre of the somite in Dll3/Dll3neo embryos. (I-L) Epithelialisation of mesenchyme is poor in Dll3neo/Dll3neo mutants, with diffuse ß-spectrin 2 expression. (H,J,L) Higher magnifications of G,I,K. Scale bar: 200 µm in A,C,E; 100 µm in B,D,F; 160 µm in G,I,K; 80 µm in H,J,L.

View larger version (120K): [in a new window]   Fig. 6. The ‘segmentation clock’ and anteroposterior identity are disrupted in trunk paraxial mesoderm of Dll3neo/Dll3neo mutant embryos. (A-E) Lateral views. (A) At 9.5 dpc, Lfng expression is detected in two domains in the presomitic mesoderm of this Dll3/Dll3neo embryo: rostrally, as one or two bands (the anteriormost is just caudal to the forming somite boundary); and caudally extending to the primitive streak. (B) Lfng expression is never detected caudally in the presomitc mesoderm of Dll3neo/Dll3neo embryos and rostral expression was diffuse. Uncx4.1 expression shows clear periodicity in Dll3/Dll3neo embryos at 9.5 dpc (C). (F) Expression is clearly restricted to the posterior of epithelial somites dissected from (C). (D,E) Uncx4.1 expression is reduced in Dll3neo/Dll3neo mutants. Periodic expression is partially retained in some embryos (D) or lost (E). (G) Trunk paraxial mesoderm dissected from D; expression appears periodic but is not restricted to the posterior somite (note the lack of epithelial structure). Dorsal view showing Cer1 expression in the tail region at 10.5 dpc of Dll3/Dll3neo (H) and Dll3neo/Dll3neo embryos (I-K). (H) Cer1 is expressed anteriorly in the presumptive somite and the nascent somite. Arrows mark the site of the most recently formed somite boundary and vertical bars show expression in the anterior of the nascent somite. (I-K) Cer1 is expressed in a broad domain (vertical bar) as the bands of expression are missing. Scale bar: 125 µm in A,B; 300 µm in C-E; 60 µm in F,G; 120 µm (H-K).

View larger version (100K): [in a new window]   Fig. 7. Hes5, Hes1 and Hey1 expression is altered in Dll3neo/Dll3neo mutants. Gene expression determined by whole-mount RNA in situ hybridisation. Dorsal view of tail region from 10.5 dpc embryos, caudal boundary of most recently formed somite marked with an arrow. (A-F) Hes5 expression; (A-D) four distinct patterns of Hes5 expression identified in Dll3/Dll3 and Dll3/Dll3neo embryos. A tight band of Hes5 expression is marked with a horizontal line; broader caudal domains of expression are marked with a vertical line. Hes5 is also strongly expressed in the neural tube. (E,F) Hes5 is not detected in the presomitic mesoderm of Dll3neo/Dll3neo mutants, despite being strongly detected in the neural tube. (G-I) Three distinct patterns of Hes1 expression are detected in the presomitic mesoderm of Dll3/Dll3 and Dll3/Dll3neo embryos. Vertical lines indicate bands/domains of expression. Hes1 expression is also detected in the caudal somite. (J,K) Only a single, relatively narrow band of Hes1 expression is detected in the presomitic mesoderm of Dll3neo/Dll3neo mutant, this is located rostrally. Tail regions showing Hes5 (A-D) and Hes1 (G-I) expression are arranged in a hypothetical progression, according to the oscillatory expression of Hes1 (Jouve et al., 2000). Hey1 expression is clearly detected in the caudal somite in Dll3/Dll3 and Dll3/Dll3neo embryos (L,M) but not Dll3neo/Dll3neo mutants (N). Dynamic Hey1 expression is represented in (L,M) with a broad band of expression in the presomitic mesoderm (vertical line, L) that condenses (vertical line, M) in Dll3/Dll3 and Dll3/Dll3neo embryos. Only a single narrow band of expression (vertical line) is detected in Dll3neo/Dll3neo mutants (N). Scale bar: 200 µm in A-F; 250 µm in G-K.

View larger version (74K): [in a new window]   Fig. 8. Dll3neo and Dll3pu are equivalent alleles with respect to skeletal dysplasia. Lateral views of Alcian Blue-stained 14.5 dpc embryos. (A) Dll3neo/Dll3neo, (B) Dll3neo/Dll3pu, (C) Dll3pu/Dll3pu. The skeletal primordia appear to be in greater disarray and there are fewer coccygeal vertebrae in A than in C. Scale bar: 1.25 mm.

View larger version (18K): [in a new window]   Fig. 9. A comparison of gene expression in paraxial mesoderm of normal and Delta mutants. Black and grey areas represent localisation of transcript and the wavy line represents dynamic gene expression. The most recently formed somite is SI, the forming somite is S0, and the block of presomitic mesoderm cells of one somite length (caudal to S0) is S-I according to (Dale and Pourquie, 2000; Ordahl, 1993). Gene expression was determined by RNA in situ hybridisation. This study examined expression in wild-type and Dll3 mutant embryos. Wild-type expression patterns were in accordance with those previously reported: Mesp2 (Saga et al., 1996), Cer1 (Biben et al., 1998), Uncx4.1 (Mansouri et al., 1997; Neidhardt, 1997), Lfng (Forsberg et al., 1998; Johnston et al., 1997), Hes1 (Jouve et al., 2000), Dll1 (Bettenhausen et al., 1995; Dunwoodie et al., 1997) and Dll3 (Dunwoodie et al., 1997). For Hes5, we identified four distinct patterns of expression in presomitic mesoderm (Fig. 7). Gene expression in Dll1 mutants is based on previous reports (del Barco Barrantes et al., 1999; Jouve et al., 2000). The levels of expression of Dll1 in Dll3 mutant embryos was low with diffuse boundaries and is indicated by grey shading.

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