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Ectopic expression of Gcm1 induces congenital spinal cord abnormalities

Brahim Nait-Oumesmar1, Barbara Stecca1, Girish Fatterpekar2, Thomas Naidich2, Joshua Corbin1,* and Robert A. Lazzarini1,{dagger}

1 Department of Molecular, Cellular and Developmental Biology and
2 Department of Radiology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA
* Present address: Skirball Institute, New York University, New York, NY, USA



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  		Fig. 1. Expression pattern of the Hoxa7-Gcm1 transgene. (A) A diagrammatic representation of the two transgenes used in this work (see Materials and Methods for details). A cDNA fragment containing the entire coding sequence of Gcm1 (1.6 kb) is under the control of the 470-bp enhancer element of the mouse Hoxa7 gene and thymidine kinase minimal promoter (TK). SV40 polyadenylation signal sequences terminate the transgene transcript. In a separate transgene, the E. coli lacZ gene replaces the Gcm1 gene. (B) Northern blot of poly(A)-selected RNA from E9.5 wild-type and double transgenic embryos were probed for Gcm1. Expression of the Gcm1 transgene was confirmed by the detection of 1.8 kb band corresponding to the predicted size of the transgene transcript. (C-E) Lateral views of a E9.5 wild-type embryo (C), a E9.5 transgenic embryo (D) and a E10.5 transgenic embryo (E) stained for ß-galactosidase activity. The expression domain of the transgene is restricted to the caudal part of the embryo with an anterior boundary at the level of somite 18-20. At E9.5, note the enlargement of the posterior neuropore in the transgenic embryo as compared to the small oval shape of the posterior neuropore in wild type (arrow in C,D).

 


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  		Fig. 2. The gross morphology of Hoxa7-Gcm1 transgenic mice. (A,B) Lateral views of E12.5 wild-type (A) and transgenic (B) embryos. The arrow in B indicates spina bifida in the transgenic embryo. (C,D) Lateral and posterior aspect of a transgenic E16.5 embryo revealing a subcutaneous neural plate-like structure (arrow). (E,F) Posterior aspects of 1-week-old wild-type and transgenic littermates. Scarring and necrosis is evident in the transgenic pup (arrow).

 


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  		Fig. 3. Histologic analyses of the spina bifida and ectopic neural tubes in transgenic mice. (A,B) Transverse sections through the lumbo-sacral level of the spinal cord showing the histological features of the spina bifida (A) and an ectopic neural tube (B) in E12.5 transgenic embryos. Dorsal root ganglia are indicated by the arrows. (C,D) View of the filum terminale (ft) in 1-monthold wild-type (C) and transgenic (D) spinal cord. Note the presence of a lipoma (lp) in the transgenic spinal cord. (E) Transverse section of the adult transgenic spinal cord illustrating the attached lipoma and the split cord. cc, central canal; A,B,E, Hematoxylin and Eosin staining. Bars: 100 µm.

 


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  		Fig. 4. Magnetic resonance images of fixed, whole-mount transgenic embryos. The serial MRI transverse sections of an E16.5 transgenic embryo are arranged in a rostral (A) to caudal (D) series. The ectopic neural tubes (arrows) emerge ventrally from the spinal cord in the vicinity of the posterior neuropore (D) and proceed rostrally with progressively diminishing caliber to the level of the kidneys (A-C). The arrowheads in C and D indicate the area of spina bifida with a lesion in the surface ectoderm.

 


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  		Fig. 5. Free-floating neural tubes in the transgenic tail buds. Transverse sections of E12.5 wild-type (A) and transgenic tail buds (B,C). In the transgenic tail bud, free-floating tubular structures (B, arrowheads) are formed in place of mesodermal tissues. Immunostaining for tubulin ß 3 confirms the neural nature of the ectopic tubes in the transgenic tail bud (C). (A-C) Dorsal is at the top and ventral is at the bottom. (A,B) Hematoxylin and Eosin staining, (C) Immuno-peroxidase labeling. Bars: 100 µm.

 


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  		Fig. 6. Brightfield photomicrographs of Shh and Pax3 expression in wild-type and transgenic neural tubes. 35S-in situ hybridization for Shh (A,C) and Pax3 (B,D) at E12.5. (A) Shh is expressed in the floor plate (fp) and notocord (nc) in the wild-type embryo. (C) In the transgenic embryos a single notocord is visible and each ectopic neural tubes (arrowheads) has a floor plate. (B,D) Pax-3 mRNA is normally expressed in the dorsal neural tube in wild-type (B) and transgenic (D) embryos. Ventrally located neural tubes (arrowheads) show no Pax3 signal in the transgenic conceptuses. Bar: 50 µm.

 


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  		Fig. 7. Immunostaining reveals cellular differentiation within the ectopic neural tubes. Immunostaining for tubulin ß3 (A) and MAP2 (B) confirms the neural identity of the ectopic tubes in E12.5 transgenic embryos. (C,D) Differentiating motoneurons and dorsal root ganglia are identified by Islet-1 immunostaining in wild-type (C) and transgenic (D) neural tubes at E12.5. Ectopic neural tubes are indicated by arrowheads and dorsal root ganglia by arrows. Immunoperoxidase labeling was used in all sections. The section in A was counterstained with Methyl Green. Bar: 50 µm.

 


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  		Fig. 8. Gcm1 restricts expression of genes encoding mesodermal differentiation factors. Transverse sections through the tail bud of E9.5 wild-type (A,C,E,G) and transgenic (B,D,F,H) embryos were hybridized with Gcm1 (A,B), Fgfr1 (C,D), Notch1 (E,F) and Tbx6 (G,H) 35S-labeled riboprobes, and then counterstained with Hematoxylin. In the transgenic tail bud, Gcm1-expressing cells form secondary neural tube-like structures (arrowheads, B). In contrast, no expression of Gcm1 is detected in the wild-type tail bud (A). Expression levels of Fgfr1, Notch1 and Tbx6 are reduced in the secondary neural tubes of the transgenic tail buds (D,F,H). (H) The small rosettes of cells with no Tbx6 expression are those that tend to form secondary neural tubes (arrowheads). Bar: 50 µm.

 




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