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First published online 29 March 2006
doi: 10.1242/dev.02346

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Lack of ß1 integrins in enteric neural crest cells leads to a Hirschsprung-like phenotype

Marie A. Breau¹, Thomas Pietri^1,*, Olivier Eder¹, Martine Blanche¹, Cord Brakebusch², Reinhardt Fässler², Jean P. Thiery¹ and Sylvie Dufour^1,

¹ UMR144, CNRS-Institut Curie, 26, rue d'Ulm, 75248 Paris cedex 05, France.
² Max Planck Institute of Biochemistry, Department of Molecular Medicine, Martinsried, Germany.

View larger version (82K): [in a new window]   Fig. 1. Loss of ß1 integrin in mutant ENCCs and expression of other ß integrins. ENCCs, or neurons and their precursors, are detected with p75NTR and HuD, respectively. (A-H) Frozen sections of control and mutant guts stained with anti-ß1 integrin (green) and anti-p75NTR (red) antibodies. (A-D) At E9.5, the ß1 integrin loss is mostly complete in mutant p75NTR+ cells, even though a few of them still express small amounts of ß1 at their surface (D, arrowheads). (E-H) At E11.5, mutant p75NTR+ cells have completely lost the ß1 integrin subunit. (I-P) Frozen sections of E13.5 control and mutant midguts stained for ß5 integrin (green) and HuD (red) (I-L), or for ß3 integrin (green) and p75NTR+ (red) (M-P). Control and mutant ENCCs express high levels of ß5 integrin and small amounts of ß3 integrin. Scale bar: 50 µm.

View larger version (85K): [in a new window]   Fig. 2. ENS defects in ß1 integrin conditional mutants. (A-F) Whole-mount X-Gal staining. (A,B) E12.5 guts. Black arrowheads indicate the position of the ENCC migratory front. The control hindgut is being invaded, whereas the mutant ENCC front is still located in the proximal caecum. Insets show higher magnification of distal midguts. ENCCs have a scattered distribution in the control but form clusters in the mutant. (C,D) E16.5 guts. The mutant ENCCs have stopped in the middle of the hindgut, whereas control ENCCs have reached the rectum (black arrowheads). (E,F) Descending colon at P14, with higher magnification in insets. Aganglionosis occurs in the mutant, with abnormal extrinsic innervation, compared with the control regular network. (G,H) Freshly dissected P4 guts. The mutant ascending colon and caecum are distended (megacolon) compared with control. (J-O) Whole-mount X-Gal staining of small intestines at E16.5 (J,K), P1 (L,M) and P14 (N,O). Mutant ENCCs form abnormal aggregates surrounded by ENCC-free spaces, compared with the regular ganglia network in the control. (P,Q) Confocal images of a whole-mount immunostaining on P14 small intestines with anti-NF160 (red) and anti-S100 (green) antibodies, recognising neuronal processes and glial cells, respectively. Green and red arrows in P indicate the circular and longitudinal orientations of the samples, respectively. Green and red arrowheads show control ganglia and neuronal processes following the circular and longitudinal directions, respectively. In the mutant, ganglia and neuronal processes do not follow these directions. ac, ascending colon; caec, caecum; dc, descending colon; dsi, distal small intestine; hg, hindgut; mg, midgut. Scale bar: 500 µm in B,D,K; 2 mm in F; 1 mm in M,O.

View larger version (75K): [in a new window]   Fig. 3. Expression pattern of intercellular adhesion proteins in ENCCs. Immunolocalisation of intercellular adhesion proteins (in green) on frozen sections of E11.5 distal midgut (A,C,F,H,J) and paraffin sections of E15.5 small intestine (B,D,G,I,K). ENCCs, or neurons and their precursors, are detected with p75NTR (in red, A,B,J,K) and HuD (in red, C,D,F-I), respectively. (B) N-cad is expressed at the same level by all the neurons and glial cells of the E15.5 myenteric ganglia (p75NTR+ cells). (D) At E15.5, Cad6 is expressed at high levels by a population of myenteric neurons (HuD+ cells, white arrowheads), and at lower levels by other neurons and HuD- cells, closely associated to neurons, very likely to be glial cells (white arrow). (F-I) Cad11 and NCAM are expressed by both neurons and glial cells (white arrows), and at lower level by the smooth muscles and the submucosal zone. (K) L1-CAM is detected in fibre-like structures in the E15.5 p75NTR+ myenteric ganglia, likely to be neuronal processes. All these proteins, except L1-CAM, are already expressed by isolated ENCCs located near the migratory front at E11.5 (lines A,C,F,H,J). (E) Deconvolution analysis shows a cell surface localisation for N-cad (in green) and intracytoplasmic accumulation of Cad6 (in red) in E13.5 ENCCs. m, smooth muscles; sz, submucosal zone. Scale bar: 5 µm for A,C,E,F,H,J; 50 µm for B,D,G,I,K.

View larger version (74K): [in a new window]   Fig. 4. Apoptosis and proliferation of ENCCs. (A,B) Activated caspase 3 (red) and HuD (green) double immunostaining on E13.5 midgut sections. No apoptotic neuron is detected in mutant and control. White arrowheads indicate autofluorescent red blood cells in the submucosal zone. The red arrowhead shows an activated-caspase-3+ HuD- cell in the mesentery. (C,D) PCNA immunostaining on E12.5 X-Gal-stained gut sections. (F,G) BrdU (red) and Phox2b (green) double immunostaining on E12.5 gut sections. (E,H) Percentages of ß-gal+ cells that were PCNA+ (E) and percentages of Phox2b+ cells that were BrdU+ (H), in control and mutant midgut and caecum (cell counts from several preparations were pooled; n, total number of ENCCs examined; 2 test, P>0.05). Scale bar: 100 µm in A,B; 20 µm in C,D,F,G.

View larger version (81K): [in a new window]   Fig. 5. Neuronal differentiation of ENCCs. (A,B) Schematic representation of the E12.5 X-Gal stained guts which were immunostained with anti-NF160 and anti-HuD antibodies, recognising neuronal processes and neuron cell bodies, respectively. The regions which are colonised by ENCCs are stained in blue. The red boxes indicate the regions where pictures C-F were taken. (C,D) Distal midgut. (C) Control neurons and non-labelled ß-gal+ ENCCs form a scattered network occupying all the available space. (D) Mutant neurons and non-labelled ß-gal+ ENCCs form bundles around fasciculated neuronal processes, surrounded by ENCC-free areas indicated by asterisks. (E,F) Migratory front. Black arrowheads indicate NF160+ processes associated with the leading ENCCs, in control and mutant.

View larger version (57K): [in a new window]   Fig. 6. The mutant ENCCs show a migration defect in a 3D tissue environment. (A) Schematic representation of the graft experiment protocol. Segments of control or mutant distal midgut were grafted onto segments of wild type hindguts at E12.5. (B,C) X-Gal staining of the explants after 3 days in culture. The black line represents the limit between the wild-type hindguts and the control or mutant fragments which are grafted onto them. Each red arrowhead indicates the position of the most-caudal ß-gal+ cell in the wild-type hindgut. Mutant ENCCs migrated less far than control ENCCs in wild-type hindguts. (D,E) Quantification of the migration defect. (D) For each of the five independent experiments, noted e1 to e5, the control explant in which ENCCs had migrated the furthest was chosen as the reference (100). The distances travelled by ENCCs in the other explants of the same litter were expressed as percentages of this maximal distance. (E) Histogram showing the average distances covered in wild-type hindguts. The average distance covered by mutant ENCCs is significantly reduced compared with the control (*P<0.002).

View larger version (121K): [in a new window]   Fig. 7. The mutant ENCCs form aggregates on a 2D substrate. Rings of E13.5 control and mutant distal midgut were plated on a mixture of ECM gel and fibronectin. After 2 days in culture, neuron cell bodies were detected with HuD (red), neuronal processes with NF160 (red), glial cells with B-FABP (green) and nuclei with DAPI. (A-G) Control culture, at low (A) and high (B-G) magnification. Neurons and glial cells form scattered networks on SMC (B,C) or directly on ECM (D-G). (H-N) Mutant culture, at low (H) and high (I-N) magnification. Green arrows indicate aggregates of neurons and glial cells that adhere directly to ECM. White arrows show spheroid aggregates that do not adhere to SMC or ECM. (I,J) Aggregates on the SMC layer. (K,L) An aggregate adhering to ECM. (M,N) High magnifications of spheroid aggregates stained for HuD and NF160 (red) and B-FABP (green) (M) and for X-Gal (N). Red arrows indicate neuronal processes linking aggregates to the explants.

View larger version (53K): [in a new window]   Fig. 8. The ENCC migratory response to GDNF is ß1 integrin dependent. Segments of E13.5 proximal midgut were embedded in 3D collagen matrix and cultured in the presence or absence of GDNF. After 2 days of culture, neuron cell bodies were detected with HuD (red), neuronal processes with NF160 (red), glial cells with B-FABP (green) and nuclei with DAPI. (A,B,E,F) Without GDNF, no ENCCs migrated out of the explants. (C,D) In the presence of GDNF, a large number of neurons (red arrows) and glial cells (green arrows) emigrated from control explants. Neurons and glial cells were the only cell types found out of the explants, as shown by the DAPI staining. (G,H) In the mutant, no neuron cell bodies or glial cell entered the collagen matrix in the presence of GDNF, but neuronal processes did (white arrows).

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