Proper function of the nervous system relies on the formation of precise connections between nerve cells that span complex substrates and long distances. Axon guidance is a key process in establishing this architecture, in which cells must appropriately integrate and respond to multiple signals presented in the extracellular environment 1
. These cues come from a wide range of signaling systems, including “canonical” guidance molecules (e.g.
Netrins, Slits, Semaphorins, and Ephrins), morphogens (e.g.
Hedgehog, BMP, and Wnt families), growth factors (e.g.
NGF, BDNF, HGF, and FGFs), and cell adhesion proteins (e.g.
N-cadherin, NCAM, L1-CAM). Extensive cross-talk between these pathways has been demonstrated, introducing additional layer of complexity to the mechanisms controlling axonal navigation. Moreover, guidance molecules that are often associated with extracellular matrix components are received and interpreted by nerve cells in a highly localized manner within the growth cones found at the tips of growing axons. Axon guidance is thus an intricate process occurring on a subcellular scale, requiring highly refined experimental techniques to study and manipulate.
Contemporary microfabrication methods have much promise for enhancing the tools that are used to study how localized signaling drives cell function. For example, Campenot chambers, which consist of millimeter-scale barriers that isolate axons and cell bodies into separate compartments, have been widely used to investigate local signaling by restricting the activity of pharmacological agents to distinct parts of the neuron 2
. This allows localized study of receptors on the cell surface and limits the effects of detrimental or toxic drugs to the rest of the cell. Microfabricated versions of the Campenot chamber have been more recently developed that provide higher reproducibility and better compatibility with a broad range of nerve cells by eliminating the need for a sealant compound between the chamber and substrate through which neurons must extend their axons 3-6
. The classic stripe assay method, in which matrices are patterned with two different guidance molecules to study axonal substrate preference, has similarly been updated by microfabrication. Specifically, the use of microchannel and microcontact printing methods allow patterning of finer features as well as the combination and alignment of more than two molecular cues 7, 8
In this report, we combine the benefits of the microfabricated chamber with those of the microcontact printing to study local signaling in motor axons guided along multicomponent protein patterns 8
. Our chamber system is modified from previously described systems to accommodate aggregate cultures, such as explants or embryoid bodies containing embryonic stem cell-derived neurons, both being more suitable for the study of axon guidance than dissociated cells. As a first demonstration of this system, we investigate guidance of embryonic stem cell-derived motor neuron axons by N-cadherin, a well-known type I cadherin that mediates homophilic, calcium-dependent cell-cell interactions 9
and plays multiple roles in the vertebrate nervous system development, including stimulation of axonal extension 10-14
. These actions are mediated in large part through classic cadherin/catenins interactions. Crosstalk of N-cadherin with FGF receptor (FGFR) and downstream PLCγ/DAG lipase/CAM kinase/Ca2+ pathway has been posed as an additional mechanism 15, 16
, global inhibition of FGFR reduces neurite outgrowth on N-cadherin, with no effect on axon outgrowth on other proteins such as laminin 17, 18
. However, the precise mechanism of this interaction remains unclear. In this study we utilize the new microfabricated system to examine localization of N-cadherin / FGFR interaction in axon guidance.