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First published online 30 January 2008
doi: 10.1242/dev.006742

Development 135, 899-908 (2008)
Published by The Company of Biologists 2008
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Oscillatory lunatic fringe activity is crucial for segmentation of the anterior but not posterior skeleton

Emily T. Shifley, Kellie M. VanHorn, Ariadna Perez-Balaguer*, John D. Franklin{dagger}, Michael Weinstein and Susan E. Cole{ddagger}

Department of Molecular Genetics, The Ohio State University, 984 Biological Sciences Building, 484 West 12th Avenue, Columbus, OH 43210-1292, USA.


Figure 1
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  		Fig. 1. Deletion of FCE1 from the endogenous locus alters Lfng expression in the posterior PSM. (A) The Lfng endogenous locus (boxes signify coding exons and FCE1), the targeting vector replacing the 110 bp FCE1 sequence with an EcoRV site and the structure of the targeted locus are shown. The floxed Neo/Testis-CRE cassette is excised upon passage through the male germline (Bunting et al., 1999Go). Locations of probes (solid lines) and primers (numbered arrows) used for genotyping are indicated. (B) After electroporation into TC1 cells (Deng et al., 1996Go), G418 resistant colonies were screened by Southern blot. A representative colony containing the Lfng{Delta}FCE1 allele and a mouse genotyping PCR are shown. Arrows, endogenous band; arrowheads, targeted bands. (C) RNA in situ analysis demonstrates cyclic Lfng expression in wild-type embryos at 10.5 dpc (c-e, n=4/14 Phase 1, 5/14 Phase 2, 5/14 Phase 3). In homozygous mutant embryos, expression is seen only in the anterior PSM (g, n=11). PSM expression patterns are summarized (f,h). Lfng RNA expression at other sites is unaffected (a,b).

 

Figure 2
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  		Fig. 2. The Lfng{Delta}FCE1 allele interferes with normal skeletal development during primary body formation. (A) Representative phenotypes of Lfng+/-, Lfng{Delta}FCE1/{Delta}FCE1 and Lfng-/- mice. The Lfng{Delta}FCE1/{Delta}FCE1 mouse has a shortened body and kinked tail. (B) Skeletal preparations of wild-type (a,b,g), Lfng{Delta}FCE1/{Delta}FCE1 (c,d,h) and Lfng-/- (e,f,i) mice. Ventral (a,c,e) and dorsal (b,d,f) views of the ribs and dorsal views of the lumbar and sacral spine (g-i) are shown. The thoracic regions of Lfng{Delta}FCE1/{Delta}FCE1 (c,d) and Lfng-/- (e,f) mice exhibit rib fusions (arrows) and disorganized vertebrae. In Lfng{Delta}FCE1/{Delta}FCE1 skeletons, vertebral disorganization extends through the lumbar region (bar, h), but normal vertebral condensations are seen in the sacral spine (*). By contrast, vertebral disorganization extends throughout the lumbar (bar) and sacral (*) regions of Lfng-/- skeletons (i), and the tail appears severely truncated. (C) Rib abnormalities were quantified in Lfng wild-type (n=17), Lfng{Delta}FCE1/{Delta}FCE1 (n=11) and Lfng-/- (n=8) neonates. Results are shown as bar and whisker graphs (solid horizontal line indicates the mean), with the number of rib abnormalities indicated on the y-axis. The number of rib abnormalities is similar in Lfng-/- and Lfng{Delta}FCE1/{Delta}FCE1 animals (P=0.236, the null hypothesis is accepted). (D) Tail anomalies were quantified in adult animals. The proportion of animals with 0-1 kinks, 2-5 kinks or truncated tails are shown. Forty percent of Lfng{Delta}FCE1/{Delta}FCE1 animals exhibit mild tail defects (0-1 kinks), while the remaining animals had between 2 and 5 kinks. By contrast, Lfng-/- animals exhibit truncation in the tail region.

 

Figure 3
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  		Fig. 3. Early somitogenesis is perturbed in Lfng{Delta}FCE1/{Delta}FCE1 embryos. Parasagittal sections of Lfng+/{Delta}FCE1 and Lfng{Delta}FCE1/{Delta}FCE1 embryos at 9.5 dpc (A,B) and 10.5 dpc (C-F). At 9.5 dpc, recently formed somites are irregularly sized and shaped in Lfng{Delta}FCE1/{Delta}FCE1 embryos (bracket, B). At 10.5 dpc, mature somites in the thoracic region remain irregular in Lfng{Delta}FCE1/{Delta}FCE1 embryos with fused (arrow), small (*) and large (bracket) somites seen (D). At this stage, however, the recently formed somites appear relatively normal in wild-type and Lfng{Delta}FCE1/{Delta}FCE1 embryos (E,F; lines represent intersomitic boundaries). Anterior is towards the left.

 

Figure 4
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  		Fig. 4. R/C patterning in Lfng{Delta}FCE1/{Delta}FCE1 embryos. (A) Whole-mount in situ hybridization for Mesp2, defining the presumptive rostral compartment of the pre-somite. In wild-type (a,c) and Lfng{Delta}FCE1/{Delta}FCE1 (b,d) embryos, a single clear band of Mesp2 expression is seen at both 9.0 (a,b) and 10.5 (c,d). The anterior border of this band is sometimes less defined in Lfng{Delta}FCE1/{Delta}FCE1 embryos (arrows). (B) Whole-mount in situ hybridization with a probe against Uncx4.1, which demarcates the caudal half of the somites. At 9.5 and 10.5 dpc, wild-type somites have clear rostral and caudal compartments (a-d). During primary body formation, Lfng{Delta}FCE1/{Delta}FCE1 embryos exhibit some compartmentalization with stronger staining in the caudal region of the somite (e,f, arrowheads), although compartments are frequently irregular (e, bracket). At this stage, little compartmentalization in seen in Lfng-/- embryos (i,j). The mature derivatives of these somites are patterned; clear rostral compartments are observed in Lfng{Delta}FCE1/{Delta}FCE1 embryos in the sclerotome of mature somites in the thoracic region, but compartments may be misshapen or irregularly spaced (arrow, g). Somites in the thoracic region of Lfng-/- embryos exhibit no compartmentalization at this stage (k). During secondary body formation, somites in Lfng{Delta}FCE1/{Delta}FCE1 embryos are of regular size and are correctly patterned (h), while in Lfng-/- embryos at this stage, little to no compartmentalization is observed (bar, l). (C) Whole-mount in situ analysis of Mox1 mRNA demonstrates a regular pattern of mature somitic derivatives in the thoracic region of wild-type embryos at 10.5 dpc. (a) In Lfng{Delta}FCE1/{Delta}FCE1 embryos, somitic derivatives in this region are distinct but irregularly spaced (b, arrow, bar). (D) Staining with 2H3 reveals the regular pattern of axon projections in the trunk region of wild-type embryos at 10.5 dpc (a). In Lfng{Delta}FCE1/{Delta}FCE1 embryos, these projections are spaced irregularly (b, arrow). Anterior is towards the left in all panels. *Hindlimb bud.

 

Figure 5
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  		Fig. 5. Notch1 signaling is altered in Lfng{Delta}FCE1/{Delta}FCE1 embryos. Whole-mount immunohistochemistry using an antibody specific for activated Notch1 was performed. (A) At 8.5 dpc, dynamic domains of Notch activation are seen in wild-type embryos, with anterior bands and a posterior band of varying width (a,b, n=29). In both Lfng{Delta}FCE1/{Delta}FCE1 (c, n=12) and Lfng-/- (d, n=7) embryos, Notch1 activation is seen ubiquitously throughout the PSM. (B) At 10.5 dpc, dynamic Notch1 activation is observed in wild-type embryos, with four distinct phases observed [a-d n=9/38 Phase 1, 8/38 Phase 2, 11/38 Phase 3 and 10/38 Phase 4, as defined in Morimoto et al. (Morimoto et al., 2005Go)]. By contrast, Lfng{Delta}FCE1/{Delta}FCE1 (e, n=17) and Lfng-/- (f, n=8) embryos exhibit a gradient of Notch1 activation throughout the PSM. Yellow bars indicate the extent of the stained regions.

 

Figure 6
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  		Fig. 6. Hes7 transcription is affected in Lfng{Delta}FCE1/{Delta}FCE1 embryos. (A) Hes7 expression in 8.5 dpc embryos, using a probe specific for intronic sequences. In wild-type embryos, several distinct patterns of expression are seen with a Hes7 intron probe (a-c, n=20), reflecting cyclic Hes7 transcription. In Lfng{Delta}FCE1/{Delta}FCE1 (d, n=6) or Lfng-/- (e, n=4) embryos, Hes7 mRNA is transcribed ubiquitously throughout the PSM, suggesting that, at this stage, Lfng activity is required for Hes7 oscillation. (B) Hes7 expression, as detected with a probe specific for intronic sequences in 10.5 dpc embryos. At 10.5 dpc, Hes7 mRNA expression levels and transcription oscillate in wild-type (a-c, n=13/51 phase 1, 18/51 phase 2, 20/51 phase 3), Lfng{Delta}FCE1/{Delta}FCE1 (d-f, n=13/32 phase 1, 8/32 phase 2, 11/32 phase 3) and Lfng-/- (g-i, n=10/22 phase 1, 5/22 phase 2, 7/22 phase 3) embryos. (C) Hes7 RNA expression was examined using a cDNA probe that reveals the steady-state levels of mature Hes7 mRNA. In wild-type 8.5 dpc embryos, several distinct patterns of expression are seen (a-c, n=9), while in Lfng{Delta}FCE1/{Delta}FCE1 (d, n=7) embryos, Hes7 mRNA is found ubiquitously throughout the PSM. At 10.5 dpc, oscillatory expression is seen in both wild-type (e, n=11; f, n=9) and Lfng{Delta}FCE1/{Delta}FCE1 (g, n=8, h, n=9) embryos. (D) 10.5 dpc embryos were bisected along the neural tube, and one half was fixed (a,c,e), while the other half was cultured for 1 hour prior to fixation (b,d,f). The Hes7 expression pattern is altered between the fixed and cultured halves of wild-type (a,b), Lfng{Delta}FCE1/{Delta}FCE1 (c,d) and Lfng-/- (e,f) embryos, confirming that Hes7 RNA levels can oscillate in the absence of LFNG activity.

 

Figure 7
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  		Fig. 7. Oscillatory gene expression is variously perturbed in Lfng{Delta}FCE1/{Delta}FCE1 embryos. (A) Nrarp expression oscillates in wild-type embryos at 8.5 dpc (a,b, n=10), but is stably expressed in Lfng{Delta}FCE1/{Delta}FCE1 embryos (c,d, n=6). At 10.5 dpc, wild-type embryos exhibit two distinct patterns of expression (e, n=7; f, n=8). Both these phases are seen in Lfng{Delta}FCE1/{Delta}FCE1 embryos, but the pattern is more diffuse than that observed in wild-type embryos (g, n=5; h, n=2). (B) Cyclic expression of the Dll1 intron probe is observed in both wild-type (a, n=6; b, n=7) and Lfng{Delta}FCE1/{Delta}FCE1 (c, n=6; d, n=4) embryos at 10.5 dpc. (C) In wild-type embryos, two phases of Hesr1 expression are seen, with a narrow band in the anterior PSM and either a broad (a, n=4) or narrow (b, n=2) band in the posterior PSM. Hesr1 expression is perturbed in Lfng{Delta}FCE1/{Delta}FCE1 embryos, with a diffuse band of staining extending from the posterior into the anterior PSM that overlies a narrow band of expression in the anterior PSM (c, n=6).

 

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© The Company of Biologists Ltd 2008