Introduction

Characterization of Neurovascular Interactions during Quail Development

Andy Freund, Frances LefcortDepartment of Cell Biology & Neuroscience, Montana State University

August, 2010

Acknowledgments

Works Cited

Results & Discussion

Bead Insertion & Analysis

I would like to thank Dr. Frances Lefcort and Haley Dunkel for all of their assistance in this project. Without them it would not have been possible. This work was supported by the National Institutes of Health, and the Howard Hughes Medical Institute through Montana State University’s Hughes Scholars Program.

Methods Conclusions & Current ResearchBlood vessels and nerves are vital channels to and

from tissues. They use common signals and mechanisms to differentiate, grow and navigate toward their target (1). Also, the vascular and nervous systems cross-talk which, when proper function is lost, contributes to medically important diseases. The realization that both systems use common genetic pathways links vascular biology and neuroscience and also generates many questions as to how they affect one another (2).

During development, cell-fate decision making in the neural crest presents a remarkable illustration of the relationship between the nervous and vascular systems (Figure 1). Endothelial cells (ECs) have more functions than simply providing a channel for blood and may play a role in neural crest cell (NCC) migration, proliferation and differentiation (3-5). One of the mechanisms by which endothelial cells and cells of the nervous system cross talk is through the signal protein vascular endothelial growth factor (VEGF). VEGF acts upon both systems and is necessary for each system to properly develop (Figure 2) (6). Based on this information, we hypothesize that NCCs and ECs communicate directly with one another during migration and development.

In order to study the role of ECs in NCC activity, a bead containing a selective inhibitor of the VEGF receptor was placed in the neural tube of stage 12 quail embryos. This allowed for a constant source of the VEGF inhibiting drug to be released throughout development until stage 22. The quail embryo has proved to be a remarkably important model system in developmental biology. In this research, chickens provided great advantages into investigating loss of function. This model allows for molecular analysis of tissue interactions, cell differentiation and pattern formation within the embryo. Furthermore, the experimental design and methods used provide exciting processes for investigating nervous system development and disease in vivo.

Quail eggs were incubated and gently rocked at 39˚ C for approximately 3 days until they reached Hamburger & Hamilton (HH) stage 12-13 (Figure 3A). After 3 days, 1 mL of albumen was removed and a circular window 1.5 cm in diameter was cut in each egg. Using a tungsten needle, a small incision was made in the embryo and either a bead soaked in Su5416, a VEGF inhibiting drug, or a control bead soaked in DMSO was placed next to the dorsal root ganglion (DRG). The incision was made approximately 3 somites below a large vascular structure. The experimental bead was used at a drug concentration of 10mM and allowed to soak in the drug overnight. A single bead was placed inside each embryo. After placement of the bead, the eggs were resealed with tape, parafilmed and returned to 37˚ C until the embryo reached stage 22 (Figure 3B).

Figure 3. Quail embryos (A) stage 12, (B) stage 21

Figure 4. Stage 20 Quail displaying bead insertion location

Once the embryos with beads reached stage 20-22 (Figure 4), they were dissected and embedded in OCT compound for cryosectioning. In situ cryosections (16 µm) were slide mounted and blocked with NGS for 1 hour at room temperature. Analysis of the samples was then accomplished using immunocytochemistry. Slides from both control and experimental embryos were incubated at 4˚ C overnight with a primary antibody. The slides received a mouse anti-QH1 primary antibody, which is an endothelial cell marker, as well as a mouse anti-HNK antibody, a neural crest cell marker. After the overnight stain, the slides were rinsed 4 times at 10 minutes per wash with NGS and then incubated for 1 hour at room temperature with their secondary antibodies. All slides received a green (488 nm) goat anti-mouse IgG, a red (568 nm) goat anti-mouse IgM, and DAPI. Following the incubation with the secondary antibody, the sections were washed 5 times at 10 minutes per wash with a 3:1 NGS/TBS solution and mounted in Prolong Antifade.

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Figure 2. VEGF attracts the filopodia, driving the endothelial cell or axon to move in the direction of the VEGF gradient.

Figure 1. Schematic of NCC development.

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Endothelial cells were not eliminated using the Su5416 VEGF blocker at 10 mM. Therefore it is not yet possible to say that the absence of ECs has an effect on the activity of NCCs. It is possible that the concentration of the drug that the embryos received was not ideal. Only one concentration was used so it could be plausible that the concentration has a drastic effect. Future work will consist of similar methods and techniques but with different concentrations of the drug to see if that has any effect. Also, the placement of the bead within the embryos may have a crucial effect on the ECs. Overall, the drug concentration as well as the placement of the bead in the embryo need to be tested in order to determine if ECs have an effect on the activity of NCCs.

This work and the methods described promote a pathway for studying the relationship not only between ECs and NCCs but between any cell types that share a common signal protein. It is possible to use the bead injection system to restrict the function of any signal protein and see how the loss of communication affects the communicating cell types.

Current work done in our lab continues to address the relationship between NCCs and ECs. Figures 7 displays static images that present the location of quail NCCs and ECs at stage 15. Work done in our lab will continue to examine this relationship and how NCC activity is determined.

1. LeDouarin NM & Kalcheim C. (1999). The Neural Crest, 2nd Ed. Cambridge: Cambridge University Press.2. Greenberg DA & Jin K. (2005) From angiogenesis to neuropathy. Nature 438, 954-959.3. Carmeliet P & Tessier M. (2005).Common mechanisms of nerve and blood vessel wiring. Nature 436, 193-200.4. McCarty JH. (2009). Cell adhesion and signaling networks in brain neurovascular units. Current Opinion in Hematology 16, 209-214.5. Zacchigna S, Lambrechts D, Carmeliet P. Neurovascular signaling defects in neurodegeneration. Nature Reviews Neuroscience 9:3, 169-181.6. Travazoie M, Van der Veken L, Silva-Vargas V, et al. (2008) A specialized vascular niche for adult neural stem cells. Cell Stem Cell 3, 279-288.

Su5416 drug and bead proved to be ineffective in killing endothelial cells when used at 10 mM.

Beads soaked in either a VEGF-blocker or DMSO (control) were placed next to a DRG of stage 12 quail. When analysis using immunocytochemistry was completed, it was found that the drug was ineffective in killing endothelial cells. Figure 5A displays a cross-section of a spinal cord from a control embryo. Figure 5B shows a cross-section of a spinal cord from a Su5416 experimental embryo. In both figures, endothelial cells are labeled green and neural crest cells red.

Figure 5. (A) DRG of a control embryo with ECs labeled green (QH1) and NCCs labeled red (HNK), (B) DRG of experimental embryo with ECs labeled green (QH1) and NCCs labeled red (HNK)

The purpose of the experimental VEGF drug was to knock out endothelial cells in order to examine their effect on NCC activity. These results do not show any significant difference in the number of endothelial cells between the experimental and the control. Due to this fact, this experiment does not allow for the absence of epithelial cells effect to be examined on neural crest cells. Figure 6 shows a summary of how NCCs and ECs are integrated in the process of NCC activity. If the Su5416 drug were to have killed ECs, it would have been possible to see if NCCs and ECs communicate directly with one another during migration and development or if NCCs and ECs migrate through the same environment independently of each other.

Figure 7. 3D reconstruction of the mid-trunk region of a stage 15 quail stained with QH1 (green) and HNK1 (red)

Figure 6. Diagram displaying how VEGF facilitates the activity of NCCs and ECs

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