A multi-dimensional microfluidic platform recapitulating chemotactic and morphogenic chemical gradients

Abstract

The requirement that individual cells be able to communicate with one another over a range of length scales is a fundamental prerequisite for the evolution of multicellular organisms. Often diffusible chemical molecules originate from a source and span the distance between cells in order to establish a line of communication - where the meaning of the signal is a function of both spatial and temporal chemical concentrations. In the case of chemotaxis, cells respond to concentration gradients to establish directionality. In the case of morphogenesis, cells respond to the magnitude of the local concentration field to regulate gene expression. Presented here is an in vitro platform, applicable in the contexts of chemotaxis and morphogenesis, where cells may be exposed to dynamic chemical concentration fields while cultured in a 3-dimensional macromolecular matrix. In the first generation system, cells are exposed to a one-dimensional gradient - constant along the two orthogonal axes. The second-generation system produces two orthogonally oriented gradients intersecting in a 2-dimensional field. These platforms were able to stimulate chemotaxis - both of cultured mammalian cells and emanating from murine skeletal muscle explants. Further, as a developmental tool, we were able to probe the role of Wnt signaling during Sonic Hedgehog based patterning of the vertebrate ventral neural tube. Using the presumptive enhancer for the p3 progenitor domain gene Nkx2.2, our findings indicate that such an enhancer would both negatively and positively regulate Nkx2.2 expression in response to Wnt signaling. However we found that the net effect of positive Wnt signaling - in the context of the cross-repressive interactions between various neural tube transcription factors (Nkx2.2, Olig2, and Pax6) - is inhibition of Nkx2.2 expression and p3 progenitor domain specification. On the basis of our new model, we postulate that the two opposing influences of Wnt on Sonic hedgehog signaling have distinct but dependent functions: first to inhibit Sonic Hedgehog signaling in the dorsal neural tube and secondly to prevent oscillatory behavior at the dorsal p3 boundary.