Volume 130, Issue 4, Pages 1221-1232 (April 2006) The Cell Adhesion Molecule L1 Is Required for Chain Migration of Neural Crest Cells in the Developing Mouse Gut  Richard B. Anderson, Kirsty N. Turner, Alexander G. Nikonenko, John Hemperly, Melitta Schachner, Heather M. Young  Gastroenterology  Volume 130, Issue 4, Pages 1221-1232 (April 2006) DOI: 10.1053/j.gastro.2006.01.002 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Confocal micrographs of whole-mount preparations of gut from (A–C) E10.5 and (D–F) and E11.5 mice processed for immunohistochemistry. (A–C) In the E10.5 caudal midgut, Phox2b-positive cells also expressed L1. (D–F) At the E11.5 migratory wavefront, all Phox2b-positive cells were also L1 positive (scale bar = 20 μm). Gastroenterology 2006 130, 1221-1232DOI: (10.1053/j.gastro.2006.01.002) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 Diagrams and micrographs of catenary cultures of gut from RetTGM/+ mice. (A) E11.5 midgut and hindgut were grown in culture for 24 hours. At the beginning of the culture period, GFP-positive neural crest cells were located at the cecal region. (B) During the culture period, neural crest cells continued their caudal migration. The distance migrated was determined by measuring the distance from the ileocecal junction (dotted line) to the most caudal neural crest cell. Gastroenterology 2006 130, 1221-1232DOI: (10.1053/j.gastro.2006.01.002) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 Effect of blocking L1 activity on neural crest migration. (A–C) Explants of E11.5 RetTGM/+ gut cultured in the presence of (A) control media or media containing (B) control immunoglobulins or (C) L1 function-blocking antibodies. (D and E) Quantification of (D) RetTGM/+ and (E) DβH-nlacZ neural crest location when L1 activity was inhibited. The most caudal neural crest cells were significantly closer to the ileocecal junction when cultured in the presence of L1 function-blocking antibodies. (F and G) Percentage of neural crest cells undergoing (F) cell proliferation or (G) cell death in E11.5 gut cultured in the presence of either control immunoglobulins or L1 function-blocking antibodies. No significant difference was detected. (H–J) Explants of E11.5 RetTGM/+ gut cultured in media containing (H) control protein or (I) exogenous soluble L1 protein. (J) Quantification of RetTGM/+ neural crest location when L1 activity was disrupted. The most caudal neural crest cells were significantly closer to the ileocecal junction when cultured in the presence of exogenous soluble L1 protein. (K–M) Higher-magnification images of hindgut explants cultured in the presence of (K) control immunoglobulins, (L) L1 function-blocking antibodies, or (M) exogenous soluble L1 protein. Solitary GFP-positive cells (arrows) are common at or close to the migratory wavefront in explants cultured in the presence of L1 function-blocking antibodies or exogenous soluble L1 protein. Solitary cells tend to be round in shape. (N) Density of solitary neural crest cells at the migratory wavefront when L1 activity was disrupted. There are significantly more solitary neural crest cells at the migratory wavefront in explants exposed to L1 function-blocking antibodies or exogenous soluble L1 protein (scale bar = 100 μm). Gastroenterology 2006 130, 1221-1232DOI: (10.1053/j.gastro.2006.01.002) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 Effect of blocking L1 activity on the behavior of living neural crest cells within the gut. (A1–A5) Selected frames of an explant of hindgut from an E11.5 RetTGM/+ mouse that had been cultured in the presence of control immunoglobulins for 14 hours before imaging. GFP-positive neural crest cells migrated caudally as chains of cells. At the beginning of filming, there were 2 chains of cells (arrows), which united to form a single chain that followed an unpredictable trajectory caudally along the gut and gave off branches (scale bar = 50 μm). (B1–B5) A solitary cell (asterisk) in an explant of gut that had been exposed to L1 function-blocking antibodies for 14 hours. Although the solitary cell sent out processes in a variety of directions, the cell body did not change position. In contrast, chains of cells in the field of view (arrows) progressed caudally. (C1–C5) Explant of gut that had been exposed to L1 function-blocking antibodies for 14 hours. A cell (asterisk) broke free from a chain of cells (arrow). The cell migrated a short distance, rounded up, and then ceased movement (B and C: scale bars = 20 μm). Note that the levels of GFP shown by neural crest cells in these preparations of living gut from RetTGM/+ mice are low compared with fixed preparations where the GFP staining was amplified with an anti-GFP antibody. Gastroenterology 2006 130, 1221-1232DOI: (10.1053/j.gastro.2006.01.002) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 Effect of blocking metalloprotease activity on neural crest migration. (A–C) E11.5 RetTGM/+ gut cultured in the presence of (A) dimethyl sulfoxide, (B) N-t-butoxycarbonyl-l-leucyl-l-tryptophan methylamide, or (C) the metalloprotease inhibitor GM6001. (D) Quantification of neural crest cell migration when metalloprotease activity was inhibited. The rate of migration of neural crest cells was significantly reduced when cultured in the presence of GM6001 (scale bar = 100 μm). Gastroenterology 2006 130, 1221-1232DOI: (10.1053/j.gastro.2006.01.002) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 E11.5 gut from (A) wild-type, (B) L1 heterozygous, and (C) L1-deficient mice immunostained for p75. Arrow indicates the position of the most caudal neural crest cell. (D–F) Quantification of neural crest cell migration from (D) E11.5, (E) E12.5, and (F) E13.5 gut of wild-type, L1 heterozygous, and L1-deficient mice. The distance from the ileocecal junction to the most caudal p75-positive cell was significantly lower in E11.5 L1-deficient gut compared with wild-type gut. No significant difference was detected in the location of p75-positive cells in E12.5 and E13.5 gut (scale bar = 100 μm). Gastroenterology 2006 130, 1221-1232DOI: (10.1053/j.gastro.2006.01.002) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions