Imaging the Cell and Molecular Dynamics of Craniofacial Development

Abstract

The development of the vertebrate head requires cell–cell and tissue–tissue interactions between derivatives of the three germ layers to coordinate morphogenetic movements in four dimensions (4D: x, y, z, t). The high spatial and temporal resolution offered by optical microscopy has made it the main imaging modularity for capturing the molecular and cellular dynamics of developmental processes. In this chapter, we highlight the challenges and new opportunities provided by emerging technologies that enable dynamic, high-information-content imaging of craniofacial development. We discuss the challenges of varying spatial and temporal scales encountered from the biological and technological perspectives. We identify molecular and fluorescence imaging technology that can provide solutions to some of the challenges. Application of the techniques described within this chapter combined with considerations of the biological and technical challenges will aid in formulating the best image-based studies to extend our understanding of the genetic and environmental influences underlying craniofacial anomalies.

Introduction

Embryonic processes are driven by the action of gene regulatory networks (GRNs) that control cell–cell interactions, differentiation, divisions, and coordinated movements. These concerted outputs of the GRN execute the patterning and the morphogenetic changes that generate the features of the head and face or any organ within the embryo. Thus, understanding how the GRN translates into the cellular behaviors that coordinate the cell and tissue rearrangements giving rise to a functioning organ has been both fascinating and a major challenge to scientists for generations. The ability to directly observe morphogenetic processes and cell behaviors that contribute to coordinated tissue rearrangements has been a long-standing goal for developmental biologists. However, the dynamic imaging of key events in embryogenesis has been hampered by the inherent nature of developmental processes, as they take place over multiple spatial and temporal scales. To resolve cell behaviors in their in vivo context, it is necessary to image at cellular resolution (~ 1–10 μm scale), as the cells are migrating, dividing, or undergoing differentiation, within tissues that are patterning at a much larger scale (~ 1 mm scale). In addition to this mismatch in scale, there is a similar mismatch in timing: cellular processes such as rearrangements, division, and migration can occur in seconds to minutes; in contrast, the entire morphogenetic event can take place over days to weeks, depending on the animal model system being studied. The ability to capture these broad spatial and temporal scales is further complicated by the need to understand the multicomponent molecular networks that contribute to the dynamic cell and tissue behavior.

Imaging studies of craniofacial development encounter these same multiple, opposing needs that have provided long-standing challenges to dynamic imaging of developmental events. The entire process of craniofacial development, from the initial specification and migration of neural crest cells to the eventual formation of the bony skeleton structures, ranges from several days in animal models such as zebrafish to over a week in mouse and avian embryos (Neuhauss et al., 1996, Rossant and Tam, 2002, Wilkie and Morriss-Kay, 2001, Yelick et al., 1996, Yelick and Schilling, 2002). Depending on the developmental stages and particular model organisms studied, there are distinct challenges to achieving both cellular and tissue resolution due to the spatial and temporal scales over which the cellular processes occur, the morphological structures that can obscure the imaging window, and the composition of the tissue. These challenges and their potential solutions are the major issues that will be presented in the following materials.

Adding to challenges of dynamic imaging is the growing recognition that the formation of structures like the face is not the result of a single mechanism acting in isolation; instead such structures derive from interactions among many distinct components, at several levels of regulation—genes, regulatory networks, and cells—in varying contexts. Such interacting systems exhibit properties of nonlinear dynamics and emergent behavior that cannot easily be inferred from studies of the individual components in isolation. Thus, researchers requires the means to follow multiple cellular and molecular events in the same preparation. We require multiplex and multimodal imaging tools, able to detect multiple gene expression patterns in a single specimen, and to perform quantitative studies of the molecular components within the live embryo so that the interplay between gene networks can be addressed.

In this chapter, we discuss the challenges mentioned above and highlight some of the emerging technological solutions to these challenges. We discuss the issues related to imaging craniofacial development in terms of both the biological and the instrumental temporal and spatial scales. We highlight current imaging approaches and consider new opportunities in imaging to provide higher spatial and molecular content to imaging data. Finally, we reflect on the potential for these emerging technologies to provide high-resolution spatial, temporal, and molecular content that will provide new insights into normal and abnormal craniofacial development.