Looking Deeper into Development Applications of High resolution MRI in Developmental Biology

Russell E. Jacobs, Seth Ruffins, and Cyrus Papain
Beckman Institute
California Institute of Technology
Pasadena, CA 91125

Abstract

The exquisite sensitivity of MRI to the local physical and chemical environment provides a wide range of mechanisms giving rise to intrinsic contrast in the MR experiment, thus providing images with dramatic differences between different tissue types (e.g. white versus gray matter, myelinated versus unmyelinated fibers, and brain parenchyma versus ventricles). In this talk we look at two applications of high resolution MRI in developmental biology.

Three-dimensional digital atlases of normal development in the primate, rodent, and avian systems: Real brains and real embryos in the real world exist in 3 dimensions with complex relationships among the various anatomic parts. Print and 2 dimensional web based atlases are extremely useful, but limited tools that describe the various anatomical parts of the animal of interest. We are constructing multidimensional atlases of development of the mouse and quail using high resolution MR imaging. The atlases will not only serve as pedagogical tools, but also present an ideal graphical interface to databases containing non-anatomical information (e.g. gene sequences, gene expression domains, literature citations). In this fashion the atlases will serve as the interface for exploring more detailed anatomical findings such as the organization of axonal tracks, maps of cell lineages and regional fate in the developing brain, and patterns of naturally occurring cell death. Gene expression patterns, receptor domains, arrays of innervation in the developing nervous system, cell lineage patterns, and a host of other types of biological processes in embryonic and adult animals take place in three spatial and one temporal dimensions. They occur within the context of anatomy of the specific sample being examined. Digital atlases provide a means to put such specific data within the context of normal specimen anatomy, analyze the information in three (or more) dimensions, and examine relationships between different types of information. We present preliminary information on the mouse and avian systems, but concentrate on the mouse. The atlas discussed here is composed of three different modules: unprocessed µMR images of fixed embryos aged 6.5 to 15.5 days post conception (dpc); an annotated atlas of the anterior portion of a 13.5dpc mouse where anatomical structures in transverse sections of the embryo have been delineated and linked to descriptive files; a three-dimensional model of the 13.5dpc embryo (Figure 1). As an example of how other types of information can be incorporated into this model, we have painted in the gene expression pattern of Dlx5/Dlx6 genes that are involved in the regulation of forebrain development.

Morphometric Cell Movements: The African clawed frog (Xenopus laevis) is an extremely well studied model system in developmental biology. Even so, the fatty nature of the embryo makes it virtually impossible to examine the complexities of cell movements during crucial events in early embryogenesis with optical microscopy. High resolution MRI coupled with single cell contrast agent labeling (Figure 2) have allowed us to follow morphometric movements during gastrulation in vivo within individual animals. We find that these movements often do not follow the schematics found in most textbooks. In particular, interactions between mesoderm and neurectoderm in the late blastula and through gastrulation are qualitatively different than current dogma suggests.

In Vivo Tract Tracing: We demonstrate that small focal injections of Mn2+ deep within the mouse brain combined with in vivo high resolution Magnetic Resonance Imaging (MRI) can delineate active neuronal tracts originating at the sight of injection. Previous work has shown that manganese ion (Mn2+) is taken up through voltage activated Ca2+ channels, transported along axons, and across synapses. Moreover, Mn2+ ion is a paramagnetic MRI contrast agent, causing positive contrast enhancement in tissues where it has accumulated. These combined properties allow for its use as an effective MRI detectable neuronal tract tracer. Nanoliter injections of 5 mM MnCl2 into both the striatum and the amygdala showed significant contrast enhancement along the appropriate neuronal circuitry.

Figure 1. µMRI allows internal anatomy of opaque specimens to be imaged nondestructively. µMRI is being used to produce an atlas of mouse development at tissue resolution with high 3D fidelity. The image in the left panel shows a surface rendering of a TS24 mouse embryo; in the middle panel the opacity has been decreased to show internal structures; left panel shows a volume rendering of delineated anatomical structures.

Figure 2.Microscopic MR-images of a live stage 8 Xenopus laevis embryo. A single cell (C1 blastomere) was labeled with MR contrast agent at 32 cell stage. A) Single slice from the middle of the sample. The different tissue regions can be distinguished on the basis of their pixel intensities. ac: animal cap; bc: blastocoel; veg: vegetal cell mass. The approximate border between the animal and the vegetal cell mass is highlighted with yellow dots. B) Half the image volume with the front half cut away at the level of the slice in (A) to reveal internal structures. The arrow in A and B points to a bright crescent, which is the liquid-filled cleft between the embryo proper and the vitelline membrane. The magnetite added to the medium does not cross the vitelline membrane and thus cannot eliminate the proton signal from this aqueous moiety. C) Whole image volume. Labeled clone cells (marked with an asterisk) situated in the dorsal marginal zone of the embryo appear as a bright patch. D) Whole image volume in which features of interest have been identified and the opacity lowered to reveal the internal structures. The vegetal cell mass appears dark gray; the animal cap cell is light gray. The blastocoel surface is rendered in lilac. The labeled cells appear greenish-orange. The clone border on the surface and visible in (C) is outlined with white dots.

Return to the Symposium 2003 Main Page