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First published online 11 February 2004
doi: 10.1242/dev.01030

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Specification of vertebral identity is coupled to Notch signalling and the segmentation clock

Institut für Molekularbiologie OE5250, Medizinische Hochschule, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany

View larger version (89K): [in a new window]   Fig. 1. Transgene expression and phenotypic outcome. (A) Schematic representation of the wild-type and truncated Dll1 proteins, and structure of the msd::Dll1dn transgene. In Dll1dn all but 12 amino acids of the intracellular domain proximal to the transmembrane domain have been removed. For details of the construction of msd::Dll1dn see Materials and methods. (B) Expression of msd::Dll1dn in a homozygous day 9.5 transgenic msd::Dll1dn line 19 (b) and wild-type control (a) embryo visualized by whole-mount in situ hybridisation with an antisense probe specific for the SV40pA sequence. Transgene expression is restricted to the posterior psm and recently formed somites. No expression is detected in the anterior psm corresponding to somitomeres S-1 and S0; psm, presomitic mesoderm. (C) External phenotypes and skeletal preparations of 3-week-old wild-type (a-e) and transgenic (f-v) mice. Dorsal (c,h,o,t) and ventral (d,i,p,u) view of the cervical region, lateral view of the whole vertebral column (b,g,n,s) and thoracic region after removal of the ribs (e,j,q,v). Hemizygous transgenic mice (m-q) show kinky tails (m,n), reduced laminae (black arrow in o), split vertebral bodies (white arrows in p) and reduced or missing pedicles (asterisks in q). Expressivity and penetrance of these defects are significantly increased and also fusions of laminae were observed (arrowheads in h,t) in homozygous animals of independent transgenic lines 13, 19 and 25 (f-l) and in hemizygous msd::Dll1dn line 19 mice that carry only one functional copy of Dll1 (r-v).

View larger version (24K): [in a new window]   Fig. 2. Distribution and frequency of skeletal malformations. Percentage of observed malformations along the vertebral column in transgenic msd::Dll1dn19 and mutant Dll1lacZ mice. Heterozygous Dll1lacZ mice (C) show a low frequency of all four types of malformations analysed. In hemizygous msd::Dll1dn mice (A) most prominent phenotypes are split vertebral bodies in the cervical and lumbar region, missing or reduced pedicles mainly found in the central thoracic region as well as fusions or reductions of laminae. Increasing the dose of Dll1dn in homozygotes (B) as well as reducing endogenous Dll1 levels in double heterozygous msd::Dll1dn/Dll1lacZ mice (D) increased expressivity and penetrance of all phenotypes. Wild-type control animals (n=11) did not show any of the defects observed in transgenic or mutant mice (data not shown). n, number of analysed animals.

View larger version (46K): [in a new window]   Fig. 3. Reduced Notch activity and AP patterning defects in msd::Dll1dn homozygous embryos. Consistent with reduced Notch activity in msd::Dll1dn embryos, expression of the Notch target Hes5 was not detected in the presomitic mesoderm of day 9.5 and 11.5 msd::Dll1dn embryos (C,D) in contrast to wild type (asterisks in A,B). Uncx4.1 expression (E-H) in posterior somite halves was significantly downregulated in transgenic embryos (arrowheads in H), whereas expression of the anterior somite compartment marker Tbx18 (I-L) was expanded into posterior somite halves (bracket in L) of day 9.5 embryos. Differential expression of the sclerotome marker Pax9 in anterior and posterior somite compartments (M-P) was less distinct in msd::Dll1dn day 9.5 embryos particularly in the cervical region (N,P).

View larger version (131K): [in a new window]   Fig. 8. Shifted position of anterior limb buds in mice with impaired cyclic Lfng expression. Whole-mount in situ hybridisation with myogenin antisense riboprobes revealed a reduced number of segmental units anterior to the forelimb bud in LfngHA3, LfnglacZ/lacZ and Dll1lacZ/lacZ 10.5 day embryos. In wild-type (A) and homozygous msd::Dll1dn embryos (B) seven myotomes were detected anterior to the fore limb bud. Transgenic msd::LfngHA embryo (C) with six segments anterior to the forelimb bud, and homozygous mutant LfnglacZ/lacZ (D) and Dll1lacZ/lacZ (E) embryos with five segments anterior to the forelimb bud, respectively. (G,G') In situ hybridisation on a day 12.5 LfnglacZ/lacZ embryo section showing anterior-most expression of Hoxb6 between pv4 and pv5 in contrast to pv7 in wild type (F,F'). Brackets in F,G indicate the extent of the first five pv.

View larger version (81K): [in a new window]   Fig. 4. Homeotic transformations in the cervical vertebral column. Isolated individual vertebrae and cervical vertebral columns after Alcian blue/alizarin red staining. Ventral processes (anterior tuberculi) present at wild-type vertebrae C6 (A,A') were unilaterally or bilaterally missing (black arrowheads) in hemizygous (B,B'') and homozygous (C,C') msd::Dll1dn and most double heterozygous msd::Dll1dn; Dll1lacZ/+ (D,D') mice. In hemi- or homozygous animals this was accompanied by the presence of ventral structures (white arrowheads, B,B',C,C') and/or transverse foraminae (arrows, B,C) on C7 and reduction of ribs at T1 (grey arrowheads, C). In msd::Dll1dn; Dll1lacZ/+ mice aberrant ventral processes were found on C5 or C7 (white arrowheads, D,D'). In some heterozygous Dll1lacZ mutant mice rudimentary ribs were attached to C7 (asterisks, E,E'). Arrows in (A) point to the foramina at C3-C6.

View larger version (38K): [in a new window]   Fig. 5. Mesp2::Dll1dn transgene expression and phenotype. Schematic representation of transgenic constructs (A) directing heterologous gene expression into the anterior region of the psm corresponding approximately to somitomere S-1 as indicated by whole-mount in situ hybridisation of a day 9.5 Mesp2::lacZ transgenic embryo (B). Expression of Dll1dn detected by RT-PCR in transgenic lines #4 and #5 (C). Hes5 expression (asterisks) in tailbuds of day 9.5 (dorsal views) and 11.5 (lateral views) wild-type (left) and transgenic embryos (right) (D). Note the reduction of Hes5 in the psm of transgenic embryos. Skeletal preparation of a homozygous Mesp2::Dll1dn line 4 mouse showing 14 ribs (E), with 8 ribs attached to the sternum (F).

View larger version (98K): [in a new window]   Fig. 6. Alterations in Hox gene expression in msd::Dll1dn transgenic mice. In situ hybridisation on sections of paraffin-embedded day 12.5 embryos using Digoxigenin-labeled antisense riboprobes. Hoxb6 and Hoxc5 transcripts were detected in prevertebra (pv) 7 and subsequent posterior prevertebrae in wild type (A,A',D,D'), whereas anterior-most expression in transgenic embryos was limited to pv8 (B,B',E,E'). In Dll1lacZ embryos Hoxb6 expression was detected in pv6 (C,C'). Arrowheads indicate the anterior-most pv with detected Hox gene expression.

View larger version (53K): [in a new window]   Fig. 7. Transformations in msd::LfngHA3 and LfnglacZ/lacZ mice. (A) Skeletal preparations of cervical vertebral columns and sterna of transgenic msd::LfngHA3 and mutant LfnglacZ/lacZ mice. Anterior tuberculi (asterisks) typical for C6 and the anterior-wards directed dorsal spine typical for T2 in the wild type (a) were present in msd::LfngHA3 (c,d) and LfnglacZ/lacZ (g,h) mice, but the number of cervical vertebrae was frequently reduced in msd::LfngHA3 and LfnglacZ/lacZ mice. In addition, the number of ribs attached to the sternum (seven in wild type, b) varied from six to eight in msd::LfngHA3 mice (e,f) and was often reduced to five or six in Lfnglacz/lacZ mice (i,j). (B) Schematic overview of changes in numbers and identities of cervical vertebrae in msd::LfngHA and LfnglacZ/lacZ mice. Vertebrae carrying ventral processes (anterior tuberculi) and dorsal spines used as landmarks are indicated in black, the first rib-bearing vertebra is indicated in grey. In one case rudimentary ribs were found on the seventh cervical vertebra of a msd::LfngHA skeleton. (C) Summary of numbers of cervical vertebrae, ribs, and ribs attached to the sternum in msd::LfngHA3 transgenic and Lfng mutant animals. Normal numbers of skeletal elements in wild-type mice are highlighted.

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