METHODS Migration AssaysMigration assays were performed as described (3)(4)(5). Briefly, 16 h before the assay, 80% confluent 75 cm 2 flasks (Corning Costar) of human microvessel endothelial cells (HMVEC; Cambrex, Walkersville, MD), human coronary artery endothelial cells (HCAEC; Cambrex), human umbilical artery endothelial cells (HUAEC; Promocell, Heidelburg, Germany), or human umbilical vein endothelial cells (HUVEC; Promocell), were washed with Hank's Balanced Salt Solution (HBSS, Invitrogen) and serum-starved overnight in endothelial basal media (EBM-2, Cambrex) with 0.1% fatty-acid-free BSA (Sigma) and 0.5% fetal calf serum (FCS, Hyclone). The following day cells were lifted with Trypsin/EDTA solution (Promocell), mixed with an equal volume Trypsin Neutralization Solution (Promocell), and washed 3 times in migration media (EBM-2 with 0.1% fatty-acid-free BSA and 0.2% FCS). Cells were resuspended at a density of 1.5×10 6 cells/ml and were allowed to recover for 1 h at 37°C (5% CO 2 ). 3.75 × 10 4 cells were plated into each well of a 48-well Boyden chamber apparatus (NeuroProbe, Cabin John, MD), and the wells were overlayed with an 8 μm pore polycarbonate membrane (NeuroProbe) that had been previously coated with 50 μg/ml human fibronectin (Biomedical Technologies, Inc., Stoughton, MA). Experiments performed with membranes coated with acetylated 1% gelatin from porcine skin (Sigma, St. Louis, MO) gave similar results. The apparatus was assembled and stored inverted at 37°C (5% CO 2 ) for 2 h. The apparatus was then re-inverted and 52 μl of purified chemoattractants [murine netrin-1 (R&D Systems, Minneapolis, MN), chicken netrin-2 (R&D Systems), murine netrin-4 (R&D Systems), murine netrin-G1a (R&D Systems), human VEGF 165 (R&D Systems), or control/ migration media (EBM-2 with 0.1% fatty-acid-free BSA and 0.2% FCS) were added to the upper chambers, and the migration was allowed to proceed for 2 h at 37°C (5% CO 2 ). The membranes were then removed, fixed in methanol, stained with a Hema 3 stain set (Fisher Scientific, Pittsburgh, PA), and placed (migrated-side down) onto 50 × 75 mm glass slides. Before 90% mounting medium (in xylenes) and coverslips were applied, the non-migrated cells were removed from the exposed (non-migrated) side of the membrane with a moistened swab. Cells present on the migrated side of the membrane were manually counted (three random 200× fields per well), and data points for each experiment represent the average number of migrated cells from six separate wells (three 200× fields counted per well).Another method was employed in a separate laboratory to evaluate the effects of the netrins on mouse (MS1) endothelial cells (ATCC, Manassas, VA) using a modified Boyden chamber assay as described previously (6). Briefly, a 5 μm-polycarbonate filter (Poretics) was placed between upper and lower chamber. Cell suspensions (5×10 4 cells/well) were placed in the upper chamber, and the lower chamber was filled with serum-free medium containing

The lateral line sensory system, found in fish and amphibians, is used in prey detection, predator avoidance and schooling behavior. This system includes cell clusters, called superficial neuromasts, located on the surface of head and trunk of developing larvae. Mechanosensory hair cells in the center of each neuromast respond to disturbances in the water and convey information to the brain via the lateral line ganglia. The convenient location of mechanosensory hair cells on the body surface has made the lateral line a valuable system in which to study hair cell damage and regeneration. One way to measure hair cell survival and recovery is to assay behaviors that depend on their function. We built a system in which orientation against constant water flow, positive rheotaxis, can be quantitatively assessed. We found that zebrafish larvae perform positive rheotaxis and that, similar to adult fish, larvae use both visual and lateral line input to perform this behavior. Disruption or damage of hair cells in the absence of vision leads to a marked decrease in rheotaxis that recovers upon hair cell repair or regeneration.

There is emerging evidence that the canonical neural guidance factor netrin can also direct the growth of blood vessels. We deleted the gene encoding UNC5B, a receptor for the netrin family of guidance molecules, specifically within the embryonic endothelium of mice. The result is a profound structural and functional deficiency in the arterioles of the placental labyrinth, which leads first to flow reversal in the umbilical artery and ultimately to embryonic death. As this is the only detectable site of vascular abnormality in the mutant embryos, and because the phenotype cannot be rescued by a wild-type trophectoderm, we propose that UNC5B-mediated signaling is a specific and autonomous component of fetal-placental angiogenesis. Disruption of UNC5B represents a unique example of a mutation that acts solely within the fetal-placental vasculature and one that faithfully recapitulates the structural and physiological characteristics of clinical uteroplacental insufficiency. This pro-angiogenic, but spatially restricted requirement for UNC5B is not unique to murine development, as the knock-down of the Unc5b ortholog in zebrafish similarly results in the specific and highly penetrant absence of the parachordal vessel, the precursor to the lymphatic system.

Whether neurons can restore their original connectivity patterns during circuit repair is unclear. Taking advantage of the regenerative capacity of zebrafish retina, we show here the remarkable specificity by which surviving neurons reassemble their connectivity upon regeneration of their major input. H3 horizontal cells (HCs) normally avoid red and green cones, and prefer ultraviolet over blue cones. Upon ablation of the major (ultraviolet) input, H3 HCs do not immediately increase connectivity with other cone types. Instead, H3 dendrites retract and re-extend to contact new ultraviolet cones. But, if regeneration is delayed or absent, blue-cone synaptogenesis increases and ectopic synapses are made with red and green cones. Thus, cues directing synapse specificity can be maintained following input loss, but only within a limited time period. Further, we postulate that signals from the major input that shape the H3 HC's wiring pattern during development persist to restrict miswiring after damage.