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First published online 14 July 2004
doi: 10.1242/dev.01264

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Conditional ß1-integrin gene deletion in neural crest cells causes severe developmental alterations of the peripheral nervous system

Thomas Pietri^1,*, Olivier Eder¹, Marie Anne Breau¹, Piotr Topilko², Martine Blanche¹, Cord Brakebusch³, Reinhard Fässler³, Jean-Paul Thiery¹ and Sylvie Dufour^1,

¹ UMR144, CNRS – Institut Curie, 26, rue d'Ulm, 75248 Paris Cedex 05, France
² U 368, INSERM – Ecole Normale Supérieure, 46, rue d'Ulm 75230 Paris Cedex 05, France
³ Max Planck Institute of Biochemistry, Department of Molecular Medicine, Martinsried, 82152, Germany

View larger version (85K): [in a new window]   Fig. 1. Targeted deletion of the ß1-integrin gene in NCC derivatives. (A) The lower panel summarises the NCC lineage and its derivatives (at trunk level), showing that the floxed ß1-integrin gene is recombined in the neurons and SC of the sensory nerves and only in the SC of the motor nerves of the PNS. The upper panel depicts the genomic structure of the floxed ß1-integrin gene allele before and after Cre-dependent DNA recombination. Following recombination, the lacZ reporter gene is expressed under the control of the endogenous ß1-integrin gene promoter (ß1 pr). (B) Section (200 µm) of a control embryo (Ht-PA-Cre;ß1fl/ß1+), showing the distribution of ß-galactosidase activity on E13. The entire PNS is stained, including the spinal nerve and enteric nervous system (white arrowhead). (C,D) ß1-integrin immunolocalisation in sagittal sections of an E10 mutant embryo (C; Ht-PA-Cre;ß1fl/ß1–) and a control embryo (D) at the level of the branchial arches. (E-I) ß1-integrin immunodetection in transverse sections, made at the level of the DRG, of E12.5 (E,F) and E13 (G-I) embryos with mutant (E and G, respectively), control (F and I, respectively) or heterozygous (H; Ht-PA-Cre;ß1–/ß1+) genotype. The white arrowheads and arrows indicate the structures derived and not derived from NCC, respectively. The loss of ß1-integrins in the NCC-derived structure of DRG is achieved at E13. DRG, dorsal root ganglia; SG, sympathetic ganglia; NT, neural tube. Scale bars: 50 µm.

View larger version (79K): [in a new window]   Fig. 2. Disappearance of ß1-integrins in glial and neuronal derivatives of NCC. Hu-D immunolocalisation (A,E), DAPI staining (B,F) and ß1-integrin immunolocalisation (C,G) detected in a transverse section of an E13 mutant embryo (A-C) and a control embryo (E-G), at the level of a DRG. Merge pictures are represented in D for the mutant and in H for the control embryo. White arrows and white arrowheads point some of neuronal and glial derivatives, respectively. Note that the two NCC derivatives of the DRG do not express ß1-integrins in the mutant. Structures positives for ß1-integrins in the DRG of the mutant are the vessels, not targeted by the mutation. (I,J) Distribution of ß-galactosidase activity in a transverse section of an E13 mutant (I) and a control (J) embryo, at the level of a DRG. Scale bar: 50 µm.

View larger version (66K): [in a new window]   Fig. 3. Morphology of the PNS in control and mutant embryos on E10.5 and E11.5. (A,B) Detection of ß-galactosidase activity after targeted deletion of the floxed ß1-integrin locus in whole-mount control (A) and mutant (B) embryos, in the DRG and spinal nerves in the trunk on E10.5. (C,D) Hu-D immunolocalisation in transverse sections of control (C) and mutant (D) thoracic DRG of E10.5 embryos. (E-H) Whole-mount NF-160 immunostaining labels cranial sensory nerves in control (E,G) and mutant (F,H) embryos on E10.5 (E,F) and E11.5 (G,H), respectively. E11.5 embryos were treated with BABB. The position of nerves is indicated in E. The black arrowhead in F illustrates the fusion of nerves IX and X in the mutant on E10.5. The white arrowhead indicates a decrease in the number of nerve X roots at the same stage. On E11.5, structures are similar in the control (G) and mutant (H) embryos, except for certain changes to the fasciculation of nerve X roots in the mutant, which exhibits the strongest alteration (white arrowhead in H).

View larger version (90K): [in a new window]   Fig. 4. Morphology of the spinal nerves in control and mutant embryos, on E12.5 and E13.5. (A-H) ß-Galactosidase activity was detected in whole-mount preparations. Lateral view of control embryo (A) showing intense labelling of craniofacial structures and PNS. The mutant (B) displays an absence of labelling in the trunk PNS. The 200 µm transverse sections of whole-mount E13.5 control (C) and mutant (D) embryos reveal the loss of reporter gene expression in the distal part of the mutant innervation network (white arrows in D). (E-H) Detail of cutaneous lateral ramus and lateral muscular innervation. The immunolocalisation of NF-160 on similar 200 µm transverse sections from control (E,F) and mutant (G,H) embryos shows the presence of nerves in both cases. However, SC have been specifically lost from the distal part of the mutant nerves (black arrowhead in G,H).

View larger version (139K): [in a new window]   Fig. 5. Subcutaneous and muscular innervation network at E13.5 and E14.5. (A-H) NF-160 immunolocalisation in whole-mount preparation. (A-D) E13.5 embryos. Ventral (A,C) and lateral (B,D) views of the body wall of control (A,B, n=6) and mutant embryos (C,D, n=7). Control (E) and mutant embryonic hindlimbs (F) on E14.5. (G,H) Cutaneous anterior ramus emergence point (asterisk) and its arborisation at the forelimb level in control (G) and mutant (H) E14.5 embryos (E,G, n=4; F,H, n=6). Scale bars: in D, 1 mm for A-D; in E, 500 µm for E,F; in H, 500 µm for G,H.

View larger version (106K): [in a new window]   Fig. 6. Postnatal features of the mutant and control mice. (A) Pictures of the animals on P21 on an inclined surface, showing the strong postural defect of mutants (Ht-PA-Cre; ß1fl/ß1–), particularly of the hindquarters (arrow) with respect to controls (Ht-PA-Cre; ß1fl/ß1+). (B) Body weight of the controls (red bars) and mutants (blue bars) at birth and P21. (C) Survival curve of the mutant animals. n, number of scored animals.

View larger version (129K): [in a new window]   Fig. 7. Morphological analysis of sciatic nerves. Semi-thin sections of sciatic nerves of controls (A,B) and mutants (C,D). ß-galactosidase activity (labelling sensory axons in light blue) was detected in semi-sections, which were also counterstained with Alcian Blue. In B,D, red arrowheads, red arrows and black arrows indicate the unmyelinated sensory axons and the myelinated sensory and motor axons, respectively. The black arrowheads in D indicate the unsegregated sensory axons of small to large diameter. (E) Electron microscopy image of a mutant sciatic nerve transverse section on P21. Scale bar: 0.5 µm.

View larger version (98K): [in a new window]   Fig. 8. Immunohistochemical analysis of sciatic nerves on P1 and P21. Transverse sections of sciatic nerves of control (A,B,E,F,I-M) and mutant (C,D,G,H,N-R) embryos on P1 (A-D,I,K,N,P) and P21 (E-H,J,L,M,O,Q,R). Immunolocalisation of NF-160 (A,C,E,G), the major myelin protein P0 (B,D,F,H), laminin (I,J,N,O), fibronectin (K,L,P,Q) and tenascin (M,R). The levels of labelling for different markers cannot be compared. Scale bars: in H, 25 µm for A-D and 50 µm for E-H; in R, 20 µm for I-R.

View larger version (77K): [in a new window]   Fig. 9. Innervation profile of muscles of controls and mutants. Whole-mount preparation of abdominal muscle of control (A,C,E) and mutant (B,D,F) animals on P1 (A,B) and P21 (C-F). The presynaptic compartment is visualised by immunolocalisation of synaptophysin (Synapto) and of the postsynaptic compartment with -bungarotoxin (-BTX), which reveals clusters of AChR. SC are labelled for S100. Each inset shows a higher magnification of the corresponding panel, making it possible to focus on the structure of NMJ.