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First published online 15 November 2006
doi: 10.1242/dev.02662

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Eph/ephrins and N-cadherin coordinate to control the pattern of sympathetic ganglia

¹ Stowers Institute for Medical Research, 1000 E. 50th St., Kansas City, MO 64110, USA.
² Cell Biology and Neuroscience Department, Montana State University, Bozeman, MT 59717, USA.
³ Developmental Neurobiology Department, The Burnham Institute, La Jolla, CA 92037, USA.

View larger version (159K): [in this window] [in a new window]   Fig. 1. Expression patterns of ephrinB1, EphB2 and N-cadherin. Changes in Eph/ephrin and N-cadherin expression correlate in space and time with neural crest cell segregation from inter-ganglionic regions and sorting into sympathetic ganglia. Immunohistochemistry performed on HH St. 17 and HH St. 20 sagittal cryostat sections, 15 µm. (A-C) HH St. 17 sections. hnk-1-labeled (green) neural crest cells dispersed adjacent to the dorsal aorta. (D-F) HH St. 20 sections. hnk-1-labeled (green) neural crest cells formed sympathetic ganglia adjacent to the rostral somite. (A) ephrinB1 (red) expressed in caudal somite. Expression ends at ventral edge of caudal somite, arrowheads. (D) ephrinB1 (red) expressed in inter-ganglionic regions between sympathetic ganglia anlagen, arrowheads. (B) EphB2 (red) coexpressed on hnk-1-labeled neural crest cells in the rostral somite and dispersed adjacent to the dorsal aorta. Inset shows separate green channel (hnk-1) and red channel (EphB2). (E) EphB2 (red) coexpressed on neural crest cells that formed sympathetic ganglia. (C) NCD2 (red) expressed on neural crest cells dispersed adjacent to dorsal aorta. (F) NCD2 (red) expressed in sympathetic ganglia. Arrowheads indicate inter-ganglionic region, dashed circles indicate sympathetic ganglia. Scale bars: in C, 20 µm for A-C,F; in E, 20 µm for D,E. c, caudal somite; r, rostral somite; SG, sympathetic ganglia.

View larger version (53K): [in this window] [in a new window]   Fig. 2. EphB2 and ephrinB1 fusion proteins disrupt proper sympathetic ganglia formation. (A) E3 embryo schematic with EGFP-labeled trunk neural crest cells migrating through the rostral somite and dispersed adjacent to the dorsal aorta. The red oval indicates axial level and size of the fusion protein injection. (B) Magnified trunk region of the embryo where the inhibitor was injected, showing focal delivery the length of one somite (red oval). (C) IgG-Fc-control injected embryo (n=8) with normal sympathetic ganglia formed adjacent to the rostral somite. The horizontal grid indicates the rows where mean fluorescence intensity was calculated, and plotted onto the graph to the right. (D) EphB2-Fc injected embryos (n=6). Neural crest cells found along the anteroposterior axis of the dorsal aorta in a random orientation. Circles indicate the location of presumptive sympathetic ganglia. (E) ephrinB1-Fc injected embryo (n=6). Neural crest cells evenly distributed along the anteroposterior axis of the dorsal aorta. The red oval indicates the inhibitor injection site. Scale bar: 20 µm. c, caudal somite; r, rostral somite; sg, sympathetic ganglion.

View larger version (53K): [in this window] [in a new window]   Fig. 3. Time-lapse analysis of Eph/ephrin Fc-fusion protein injected embryos reveals dynamic neural crest cell behavior. (A-D) Static images taken from time-lapse movie. Premigratory neural crest cells injected with EGFP. ephrinB1-Fc fusion protein injected in ovo at E3 when neural crest cells were dispersed adjacent to the dorsal aorta. Asterisk indicates location of injection. Sagittal explants cultured at E3.5 and imaged with time-lapse confocal microscopy. At A, t=0; at D, t=5 hours. Cells extend and retract numerous filopodia but fail to significantly translocate. Sympathetic ganglia at axial level of fusion protein injection do not sort into ganglia but stay continuous along the dorsal aorta. The dashed circle indicates a region of presumptive sympathetic ganglia. The arrowhead indicates a presumptive inter-ganglionic region. The black arrow indicates time increasing from left to right. (E) An EphB2-Fc injected embryo after 5 hours of incubation. Sympathetic ganglia at level of fusion protein injection (asterisk) do not segregate, but sympathetic ganglia caudal to the injection site segregate normally (brackets), indicating that the inhibitor is only acting at the axial level of the injection. (F) A graph representing the average number of filopodia on neural crest cells in control IgG-Fc injected embryos, and ephrinB1-Fc injected embryos. Control embryos extended 3.6±0.79 (s.d.) filopodia. ephrinB1-Fc injected embryos extended 6.0±0.6 (*P<0.001) filopodia with 5.5±1.0 (*P<0.001) further extensions off primary filopodia (secondary) extended from the cell body. (G) A graph representing the total distance migrated (blue bars) compared to net displacement (purple bars) for individual control and ephrinB1-Fc-inhibited neural crest cells. Scale bars: 40 µm in A-D; 20 µm in E. A, anterior; c, caudal somite; P, posterior; r, rostral somite.

View larger version (44K): [in this window] [in a new window]   Fig. 4. Disrupting N-cadherin adhesion in neural crest cells alters sympathetic ganglion formation. (A,B) EGFP-labeled trunk neural crest cells in anlagen of sympathetic ganglia (dashed circle), E3.5 embryo. (A) IgG control injected embryo with discrete sympathetic ganglion formation. (B) NCD2 in ovo injected embryo analyzed at E3.5. Discrete ganglion formation observed with increase in length (anteroposterior direction), compared to control injected embryos. (C) Schematic showing length comparison of sympathetic ganglia (Lsg) compared to length of somites (Ls) at the same axial level in injected embryos. (D) Area comparison of EGFP control (n=9), FL-cadherin (n=11; *P<0.001) and DN-cadherin (n=14; *P<0.004) electroporated embryos imaged at E3.5 after sympathetic ganglion formation is complete. (E) Length comparison of sympathetic ganglia in control EGFP, 48.6±4.1% (s.d.; n=10), IgG control inhibitor, 50.0±2.5% (n=8), FL-cadherin-pMES, 52.0±2.9% (n=14), NCD2 inhibitor, 70.5±4.9% (n=18) and DN-cadherin-pMES, 75.8±4.6% (n=11) electroporated embryos at E3, incubated and imaged at E3.5. Yellow bar, EGFP control electroporated neural crest cells; light blue, IgG control injected blocking antibody into EGFP control labeled embryo; dark blue bar, FL-cadherin electroporated neural crest cells; pink bar, NCD2 blocking antibody injected into EGFP labeled embryos; red bar, DN-cadherin electroporated neural crest cells.*P<0.0001. Scale bar: 40 µm in A,B. c, caudal somite; drg, dorsal root ganglia; nt, neural tube; r, rostral somite; sg, sympathetic ganglia.

View larger version (44K): [in this window] [in a new window]   Fig. 5. Cell shape and velocity comparisons in neural crest cells transfected with EGFP, DN-cadherin or FL-cadherin. Cells imaged at E3-3.5 during sympathetic ganglion formation adjacent to the dorsal aorta. (A) Control EGFP-expressing neural crest cells. (B) FL-cadherin-expressing neural crest cell. (C) DN-cadherin-expressing neural crest cells. (D) Graph of number of filopodia on cells expressing EGFP control, 3.6±0.79 (s.d.), FL-cadherin, 6.1±0.91 and DN-cadherin, 1.9±0.6. (E) Graph showing average cell velocities during ventral migration and after reaching dorsal aorta. The schematic shows the region within the embryo that cells were migrating through when the velocity was measured. Ventral migration through the rostral somite for EGFP control, 50±12.1 µm/hour, FL-cadherin, 19.3±5.14 µm/hour, and DN-cadherin, 24.7±4.63 µm/hour. Cell velocities after reaching dorsal aorta, EGFP control 62.1±15.3 µm/hour, FL-cadherin, 16.2±2.4 µm/hour, and DN-cadherin, 29.2±3.5 µm/hour. Asterisk and caret (D,E) indicate significance, P<0.01, P<0.001, respectively. Scale bars: 10 µm in A-C; 20 µm in D. c, caudal somite; r, rostral somite.

View larger version (64K): [in this window] [in a new window]   Fig. 6. Model of events needed for proper sympathetic ganglion formation. Schematic model showing important events in a spatiotemporal pattern for discrete sympathetic ganglion formation adjacent to the ventral edge of a rostral somite. 1. Neural crest cells migrate from the neural tube through the rostral somites. 2. Neural crest cells disperse adjacent to the dorsal aorta. ephrinB1 expression is established in inter-ganglionic regions between developing ganglia. 3. N-cadherin adhesion and inhibitory ephrinB1 expression induce formation of discrete ganglia. A, anterior; c, caudal somite; da, dorsal aorta; nc, notochord; nt, neural tube; P, posterior; r, rostral somite.

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