Double-band sarcomeric SHG pattern induced by adult skeletal muscles alteration during myofibrils preparation.

To understand the reported difference between double band, sarcomeric second harmonic generation pattern of isolated myofibril and predominant single band pattern found in thick muscle tissues, we studied the effect of myofibril preparation on the second harmonic generation pattern. We found that double band sarcomeric second harmonic generation pattern usually observed in myofibrils (prepared from fresh tissue) is due to muscle alteration during the mixing and triton treatment processes. Single band sarcomeric second harmonic generation pattern could be observed in isolated myofibrils when this alteration is previously prevented using paraformaldehyd fixed tissue. We conclude that single band sarcomeric second harmonic generation pattern is a signature of adult muscle myofibrils in normal physiological condition, suggesting that sarcomeric second harmonic generation patterns could be used as a valuable diagnosis tool of muscle health.

Morphogenetic domains in the yolk syncytial layer of axiating zebrafish embryos.

The yolk syncytial layer (YSL) of the teleostean yolk cell is known to play important roles in the induction of cellular mesendoderm, as well as the patterning of dorsal tissues. To determine how this extraembryonic endodermal compartment is subdivided and morphologically transformed during early development, we have examined collective movements of vitally stained YSL nuclei in axiating zebrafish embryos by using four-dimensional confocal microscopy. During blastulation, gastrulation, and early segmentation, zebrafish YSL nuclei display several highly patterned movements, which are organized into spatially distinct morphogenetic domains along the anterior-posterior and dorsal-ventral axes. During the late blastula period, with the onset of epiboly, nuclei throughout the YSL initiate longitudinal movements that are directed along the animal-vegetal axis. As epiboly progresses, nuclei progressively recede from the advancing margin of the epibolic YSL. However, a small group of nuclei is retained at the YSL margin to form a constricting blastoporal ring. During mid-gastrulation, YSL nuclei undergo convergent-extension behavior toward the dorsal midline, with a subset of nuclei forming an axial domain that underlies the notochord. These highly patterned movements of YSL nuclei share remarkable similarities to the morphogenetic movements of deep cells in the overlying zebrafish blastoderm. The macroscopic shape changes of the zebrafish yolk cell, as well as the morphogenetic movements of its YSL nuclei, are homologous to several morphogenetic behaviors that are regionally expressed within the vegetal endodermal cell mass of gastrulating Xenopus embryos. In contrast to the cellular endoderm of Xenopus, the dynamics of zebrafish YSL show that a syncytial endodermal germ layer can express a temporal sequence of morphogenetic domains without undergoing progressive steps of cell fate restriction.

Confocal microscopic analysis of morphogenetic movements.

Confocal microscopy is an excellent means of imaging cellular dynamics within living zebrafish embryos because it provides a means of optically sectioning tissues that have been labeled with specific fluorescent probe molecules. In order to study genetically encoded patterns of cell behavior that are involved in the formation of germ layers and various organ primordia, it is possible to vitally stain an entire zebrafish embryo with one or more fluorescent probe molecules and then examine morphogenetic behaviors within specific cell populations of interest using time-lapse confocal microscopy. There are two major advantages to this "bulk-labeling" approach: (1) the applied fluorescent probe (a contrast-enhancing agent) allows all of the cells within an intact zebrafish embryo to be rapidly stained; (2) the morphogenetic movements and shape changes of hundreds of cells can then be examined simultaneously in vivo using time-lapse confocal microscopy. The neutral fluorophore Bodipy 505/515 and its sphingolipid-derivative Bodipy-C5-ceramide are particularly useful, nonteratogenic vital stains for imaging cellular dynamics in living zebrafish embryos. These photostable fluorescent probes (when applied with 2% DMSO) percolate through the enveloping layer epithelium of the embryo, and localize in yolk-containing cytoplasm and interstitial space, respectively, owing to their different physiochemical characteristics. Bodipy-ceramide, for instance, remains highly localized to interstitial fluid once it accumulates within a zebrafish embryo, allowing the boundaries of deep cells to be clearly discerned throughout the entire embryo. Through the use of either of these fluorescent vital stains, it is possible to rapidly convert a developing zebrafish embryo into a strongly fluorescent specimen that is ideally suited for time-lapse confocal imaging. For zebrafish embryos whose deep cells have been intentionally "scatter-labeled" with fluorescent lineage tracers (e.g., fluorescent dextrans), sequential confocal z-series (i.e., focus-throughs) of the embryo can be rendered into uniquely informative 3D time-lapse movies using readily available image-processing programs. Similar time-lapse imaging, combined with rapidly advancing computer-assisted visualization techniques, may soon be applied to study the dynamics of GFP-fusion proteins in vivo, as well as other types of synthetic probe molecules designed to reveal the cytological processes associated with the patterning and morphological transformations of the zebrafish's embryonic tissues.

Spatially distinct domains of cell behavior in the zebrafish organizer region.

To determine the sequence of cell behaviors that is involved in the morphogenesis of the zebrafish organizer region, we have examined the dorsal marginal zone of vitally stained zebrafish embryos using time-lapse confocal microscopy. During the late-blastula stage, the zebrafish dorsal marginal zone segregates into several cellular domains, including a group of noninvoluting, highly endocytic marginal (NEM) cells. The NEM cell cluster, which lies in a superficial location of the dorsal marginal zone, is composed of both enveloping layer cells and one or two layers of underlying deep cells. The longitudinal position of this cellular domain accurately predicts the site of embryonic shield formation and occupies a homologous location to the organizer epithelium in Xenopus laevis. At the onset of gastrulation, deep cells underneath the superficial NEM cell domain undergo involution to form the nascent hypoblast of the embryonic shield. Deep cells within the NEM cell cluster, however, do not involute during early shield formation, but instead move in front of the blastoderm margin to form a loose mass of cells called forerunner cells. Forerunner cells coalesce into a wedge-shaped mass during late gastrulation and eventually become overlapped by the converging lateral lips of the germ ring. During early zebrafish tail elongation, most forerunner cells are incorporated into the epithelial lining of Kupffer's vesicle, a transient teleostean organ rudiment long thought to be an evolutionary vestige of the neurenteric canal. Owing to the location of NEM cells at the dorsal margin of blastula-stage embryos, as well as their early segregation from other deep cells, we hypothesized that NEM cells are specified by an early-acting dorsalizing signal. To test this possibility, we briefly treated early-blastula stage embryos with LiCl, an agent known to produce hyperdorsalized zebrafish embryos with varying degrees of expanded organizer tissue. In Li(+)-treated embryos, NEM cells appear either within expanded spatial domains or in ectopic locations, primarily within the marginal zone of the blastoderm. These results suggest that NEM cells represent a specific cell type that is specified by an early dorsal patterning pathway.

A cluster of noninvoluting endocytic cells at the margin of the zebrafish blastoderm marks the site of embryonic shield formation.

In zebrafish embryos, the nascent embryonic shield first appears as a thickening in the germ ring of the mid-epiboly blastoderm. This site defines the dorsal side of the developing embryo. In this paper, we report that the site of embryonic axis formation is marked earlier at the late-blastula stage by the appearance of a cluster of cells with unique endocytic activities. This cluster of cells is composed of enveloping layer epithelial cells and one to two layers of underlying deep cells. Unlike other marginal blastomeres, cells in this cluster do not participate in involution as the blastoderm undergoes epiboly. These noninvoluting endocytic marginal (NEM) cells can be selectively labeled by applying membrane impermeant fluorescent probes to pre-epiboly and mid-epiboly embryos. During embryonic shield formation, deep cells in the NEM cell cluster rearrange and are displaced forward to the leading edge of the blastoderm. As deep NEM cells move into this location, they become a group of cells known as "forerunner cells." Between 60%- and 80%-epiboly, the forerunner cells coalesce into a coherent cell cluster that forms a wedge-shaped cap at the leading edge of the blastoderm. During embryonic axis formation, deep cells migrate and converge toward the embryonic midline, which is defined by the center of the forerunner cell cluster. At approximately 90% epiboly, the forerunner cell cluster becomes overlapped by the constricting germ ring. At tailbud stage, forerunner cells form the dorsal roof of Kupffer's vesicle, which is located ventral to the nascent chordoneural hinge. On the basis of previous grafting studies and known dorsal gene expression patterns, we discuss possible roles that the NEM/forerunner cell cluster may play in teleost axis formation.