Mark S. Cooper

Visualizing "green oil" in live algal cells.

We report here that BODIPY 505/515, a green lipophilic fluorescent dye, serves as an excellent vital stain for the oil-containing lipid bodies of live algal cells. BODIPY 505/515 vital staining can be used in combination with fluorescent activated cell sorting to detect and isolate algal cells possessing high lipid content.

Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP.

Green fluorescent protein (GFP) technology is rapidly advancing the study of morphogenesis, by allowing researchers to specifically focus on a subset of labeled cells within the living embryo. However, when imaging GFP‐labeled cells using confocal microscopy, it is often essential to simultaneously visualize all of the cells in the embryo using dual‐channel fluorescence to provide an embryological context for the cells expressing GFP. Although various counterstains are available, part of their fluorescence overlaps with the GFP emission spectra, making it difficult to clearly identify the cells expressing GFP. In this study, we report that a new fluorophore, BODIPY TR methyl ester dye, serves as a versatile vital counterstain for visualizing the cellular dynamics of morphogenesis within living GFP transgenic zebrafish embryos. The fluorescence of this photostable synthetic dye is spectrally separate from GFP fluorescence, allowing dual‐channel, three‐dimensional (3D) and four‐dimensional (4D) confocal image data sets of living specimens to be easily acquired. These image data sets can be rendered subsequently into uniquely informative 3D and 4D visualizations using computer‐assisted visualization software. We discuss a variety of immediate and potential applications of BODIPY TR methyl ester dye as a vital visualization counterstain for GFP in transgenic zebrafish embryos. Developmental Dynamics 232:359–368, 2005.

Somites in zebrafish doubly mutant for knypek and trilobite form without internal mesenchymal cells or compaction.

In vertebrates, paraxial mesoderm is partitioned into repeating units called somites. It is thought that the mechanical forces arising from compaction of the presumptive internal cells of prospective somites cause them to detach from the unsegmented presomitic mesoderm [1-3]. To determine how prospective somites physically segregate from each other, we used time-lapse microscopy to analyze the mechanics underlying early somitogenesis in wild-type zebrafish and in the mutants trilobite(m209) (tri), knypek(m119) (kny), and kny;tri, which are defective in convergent extension during gastrulation. Formation of somite boundaries in all of these embryos involved segregation, local alignment, and cell-shape changes of presumptive epitheloid border cells along nascent intersomitic boundaries. Although kny;tri somites formed without convergence of the presomitic mesoderm and were composed of only two cells in their anteroposterior (AP) dimension, they still exhibited AP intrasegmental polarity. Furthermore, morphogenesis of somite boundaries in these embryos proceeded in a manner similar to that in wild-type embryos. Thus, intersomitic boundary formation in zebrafish involves short-range movements of presumptive border cells that do not require mechanical forces generated by internal cells or compaction of the presomitic mesoderm.

Sustained Bmp signaling is essential for cloaca development in zebrafish

Bone morphogenetic protein (Bmp) signaling has long been known to be important for the early development of the ventral mesoderm, including blood, vasculature and kidney cells. Although Bmp genes are continually expressed in the ventral cells throughout gastrulation and somitogenesis, previous studies in zebrafish have not addressed how the role of Bmp signaling changes over time to regulate ventral mesoderm development. Here, we describe the use of a transgenic inducible dominant-negative Bmp receptor line to examine the temporal roles of Bmp signaling in ventral mesoderm patterning. Surprisingly, we find that Bmp signaling from the mid-gastrula stage through early somitogenesis is important for excluding blood and vascular precursors from the extreme ventral mesoderm, and we show that this domain is normally required for development of the cloaca (the common gut and urogenital opening). Using a novel assay for cloacal function, we find that larvae with reduced mid-gastrula Bmp signaling cannot properly excrete waste. We show that the cloacal defects result from alterations in the morphogenesis of the cloaca and from changes in the expression of genes marking the excretory system. Finally, we show that HrT, a T-box transcription factor, is a Bmp-regulated gene that has an essential function in cloacal development. We conclude that sustained Bmp signaling plays an important role in specification of the zebrafish cloaca by maintaining the fate of extreme ventral cells during the course of gastrulation and early somitogenesis. Furthermore, our data suggest that alterations in Bmp signaling are one possible cause of anorectal malformations during human embryogenesis.

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.

Intercellular signaling in neuronal-glial networks.

Glial cells have recently been found to exhibit electrophysiological and metabolic responses to many neurotransmitters and neuromodulators. These findings have focused attention on the possibility that active signaling between neurons and glia could represent an important form of intercellular communication within the brain. Since glial and neuronal networks are both physically and metabolically interlinked, such intercellular signaling may represent a mechanism for inducing collective changes in the cellular physiology of neuronal and glial cell populations. Within the nervous tissue of both vertebrate and invertebrate organisms, glial cells are known to secrete extracellular signal molecules, modulate carbohydrate metabolism, and control the volume and ionic composition of extracellular space. In this paper, the roles that cytoplasmic [Ca2+] transients may play in regulating these glial cell functions are reviewed. Mechanisms by which intracellular Ca oscillations and intercellular Ca waves may be generated in neurotransmitter-stimulated glial cells are also discussed. In addition, it is proposed that rhythmic glial cell contractions and shape changes, which have been observed for many decades, are linked to Ca-induced secretion of ions, water, and neuroactive compounds. These activities represent mechanisms by which Ca-induced changes in glial cell physiology could potentially alter the excitability of neuronal networks.

Imaging brain development and organogenesis in zebrafish using immobilized embryonic explants

Owing to its optical clarity and rapid rate of development, the zebrafish embryo is an ideal model system for studying the cellular mechanics of organogenesis. Unfortunately, extended time‐lapse recordings of zebrafish embryos are often disrupted by the extension and straightening of the embryonic axis, as well as movement artifacts associated with developing musculature. In addition, the embryos massive yolk cell often prevents optical access to tissues of interest. To circumvent these imaging problems, we have developed a procedure to deflate and mechanically remove the yolk cell. A “paralyzing” agent, AMP‐PNP (a membrane‐impermeant nonhydrolyzable analog of ATP), is first injected into the embryos contractile yolk cell. The yolk cell is then removed using sharpened tungsten needles. Deyolked embryos, or organ rudiments explanted from them, are then immobilized on a microscope coverslip using a thin plasma clot. This plasma clot immobilization allows novel mountings of the explants so that ventral, lateral, and even cross‐sectional fields of views are possible using high numerical aperture objectives. We show that isolated head rudiments undergo normal morphogenesis and gene expression for at least 1 day after being explanted into organotypic culture. These procedures can be used to study the cellular mechanics of organogenesis in “deyolked” embryos, as well as in tissues explanted from green fluorescent protein transgenic animals. Developmental Dynamics 2003.

Neuroinflammation, Neuroautoimmunity, and the Co-Morbidities of Complex Regional Pain Syndrome

Complex Regional Pain Syndrome (CRPS) is associated with non-dermatomal patterns of pain, unusual movement disorders, and somatovisceral dysfunctions. These symptoms are viewed by some neurologists and psychiatrists as being psychogenic in origin. Recent evidence, however, suggests that an autoimmune attack on self-antigens found in the peripheral and central nervous system may underlie a number of CRPS symptoms. From both animal and human studies, evidence is accumulating that neuroinflammation can spread, either anterograde or retrograde, via axonal projections in the CNS, thereby establishing neuroinflammatory tracks and secondary neuroinflammatory foci within the neuraxis. These findings suggest that neuroinflammatory lesions, as well as their associated functional consequences, should be evaluated during the differential diagnosis of non-dermatomal pain presentations, atypical movement disorders, as well as other “medically unexplained symptoms”, which are often attributed to psychogenic illness.

Cellular and Genetic Mechanisms of Convergence and Extension

The zebrafish blastula consists of a mound of blastomeres that lies atop a large syncytial yolk cell (Fig. 1). Around the mid-blastula stage, the initial dorsoventral polarity of the embryo has already been established (see Sakaguchi et al.; Hibi et al., this Vol.) and the induction of mesendoderm has begun (see Kimelman and Schier, this Vol.; Warga and Stainier, this Vol.). During the course of the next 10h of development, cells within the blastoderm will acquire specific fates (see Hammerschmidt and Mullins, this Vol.), as well as engage in morphogenetic movements to secure appropriate positions in the nascent embryo. These cell migrations and rearrangements transform the mound of blastomeres into an embryo that features a typical vertebrate body plan. During gastrulation, epibolic movements spread the blastoderm and the yolk syncytial layer towards the vegetal pole. Germ layer formation begins when mesendodermal cells move beneath ectoderm (see Kane and Adams, this Vol.). Epiboly and mesendoderm internalization take place in a relatively uniform manner along the entire dorsoventral axis, except for a small region in the gastrula organizer. This chapter focuses on the third gastrulation movement, convergence and extension, through which dorsoventral and anteroposterior embryonic polarity become morphologically distinct. The process of convergence and extension fashions the dorsoventral axis of the nascent embryo by narrowing its tissues from back to belly.

Treatment of Complex Regional Pain Syndrome (CRPS) Using Low Dose Naltrexone (LDN)

Complex Regional Pain Syndrome (CRPS) is a neuropathic pain syndrome, which involves glial activation and central sensitization in the central nervous system. Here, we describe positive outcomes of two CRPS patients, after they were treated with low-dose naltrexone (a glial attenuator), in combination with other CRPS therapies. Prominent CRPS symptoms remitted in these two patients, including dystonic spasms and fixed dystonia (respectively), following treatment with low-dose naltrexone (LDN). LDN, which is known to antagonize the Toll-like Receptor 4 pathway and attenuate activated microglia, was utilized in these patients after conventional CRPS pharmacotherapy failed to suppress their recalcitrant CRPS symptoms.