John B. Morrill

Patterns of cells and extracellular material of the sea urchin Lytechinus variegatus (Echinodermata; Echinoidea) embryo, from hatched blastula to late gastrula

Scanning electron microscopy of six stages of Lytechinus variegatus embryos from hatching through gastrulation reveals changes in the shapes of the ectodermal cells and morphological changes in the extracellular material (ECM) in relation to the locations and migratory activities of mesenchyme cells. The classical optical patterns in the blastular wall (Okazaki patterns) are due to differential orientations of the cells, which bend and extend sheet‐like lamellipodia over adjoining cells toward the eventual location of the primary mesenchymal ring. The blastocoelic surfaces of the blastomeres become covered with a thin basal lamina (BL) composed of fibers and nonfibrous material. During primary mesenchyme cell (PMC) ingression, a web‐like ECM is located in the blastocoel overlying the amassed PMCs. This ECM becomes sparse in migratory mesenchyme blastulae, and is confined to the animal hemisphere. Localized regions of intertwining basal cell processes in the blastular wall are also present during PMC migration. While a distinct BL is present during early and midgastrulation, blastocoelic ECM is absent. Late gastrulae, on the other hand, have an abundance of blastocoelic ECM concentrated near secondary mesenchyme cell protrusive activity. ECM appearing at both the early mesenchyme and late gastrula stages are probably remnants of degraded BL and intercellular matrix preserved by fixation for SEM. Thus, early mesenchyme ECM is formed of BL material whose degradation is necessary for entry of PMCs into the blastocoel. Late gastrula ECM is apparently a degradation product of BL and intercellular material whose destruction is required for fusion of the gut with oral ectoderm in formation of the mouth.

CELLS ARE ADDED TO THE ARCHENTERON DURING AND FOLLOWING SECONDARY INVAGINATION IN THE SEA URCHIN LYTECHINUS VARIEGATUS

In the present investigation, nuclei of endodermal cells, primary and secondary mesenchyme cells (PMCs and SMCs), and small micromere descendants (SMDs) of the sea urchin Lytechinus variegatus were counted and mapped at five developmental stages, ranging from primary invagination to pluteus larva. The archenteron and its derivatives were measured three dimensionally with STERECON analytical software. For the first time SMC production is included in the kinetic analysis of archenteron formation. While the archenteron lumen doubled in length during secondary invagination, the number of archenteron cells increased by at least 38% (over 50% when SMCs that emigrated from the tip of the archenteron were included). The volume of the archenteron epithelial wall plus the volume of 17 new SMCs increased by 40% over the equivalent volumes at the end of primary invagination. Because secondary invagination involves the addition of archenteron cells and an increase in volume of the archenteron epithelium, we conclude that secondary invagination is not accomplished simply by the rearrangement and reshaping of the primary archenteron cells. Both archenteron cell number and wall volume continued to increase at the same rates from the end of secondary invagination until the 27-h prism stage, although the lumen lengthened more slowly. SMCs were also produced at a constant rate from primary invagination until the prism stage. Because the production of both endodermal and mesodermal cells continues until the late prism stage, we conclude that gastrulation (defined as the establishment of the germ layers) also extends into the late prism stage.

A Stereometric Analysis of Karyokinesis, Cytokinesis and Cell Arrangements during and following Fourth Cleavage Period in the Sea Urchin, Lytechinus variegatus

Fourth cleavage of the sea urchin embryo produces 16 blastomeres that are the starting point for analyses of cell lineages and bilateral symmetry. We used optical sectioning, scanning electron microscopy and analytical 3‐D reconstructions to obtain stereo images of patterns of karyokinesis and cell arrangements between 4th and 6th cleavage. At 4th cleavage, 8 mesomeres result from a variant, oblique cleavage of the animal quartet with the mesomeres arranged in a staggered, offset pattern and not a planar ring. This oblique, non‐radial cleavage pattern and polygonal packing of cells persists in the animal hemisphere throughout the cleavage period. Contrarily, at 4th cleavage, the 4 vegetal quartet nuclei migrate toward the vegetal pole during interphase; mitosis and cytokinesis are latitudinal and subequatorial. The 4 macromeres and 4 micromeres form before the animal quartet divides to produce a 12‐cell stage. Subsequently, macromeres and their derivatives divide synchronously and radially through 8th cleavage according to the Sachs‐Hertwig rule. At 5th cleavage, mesomeres and macromeres divide first; then the micromeres divide latitudinally and unequally to form the small and large micromeres. This temporal sequence produces 28‐and 32‐cell stages. At 6th cleavage, macromere and mesomere descendants divide synchronously before the 4 large micromeres divide parasynchronously to produce 56‐ and 60‐cell stages.

Characterization of Involution during Sea Urchin Gastrulation Using Two-Photon Excited Photorelease and Confocal Microscopy.

: Sea urchin embryos have served as a model system for the investigation of many concepts in developmental biology. Their gastrulation consists of two stages; primary invagination, where part of the epithelium invaginates into the blastocoel, and secondary invagination, where the archenteron elongates to completely traverse the blastocoel. Primary invagination involves proliferation of cells within the vegetal plate during primary invagination, but until recently, it was assumed that the larval gastrointestinal (GI) tract developed without further involution of epithelial cells. To investigate rigorously the contribution of epithelial cell involution during archenteron and GI tract development in the sea urchin, Lytechinus variegatus, we developed a new method for cell tracking based on two-photon excited photorelease of caged fluorophores. Single-cell embryos were injected with caged dye and two-photon excitation uncaging was employed to mark small groups of cells throughout gastrulation. Two-photon excitation allowed for noninvasive, three-dimensionally resolved uncaging inside living cells with minimal biological damage. Cellular involution into the archenteron was observed throughout primary and secondary invagination, and the larval intestine was formed by further involution of cells following secondary invagination, which is inconsistent with the traditional model of sea urchin gastrulation. Further, as two-photon excitation microscopy becomes accessible to many researchers, the novel techniques described here will be broadly applicable to development of other invertebrate and vertebrate embryos.

Scanning electron microscopy of embryos.

Publisher Summary This chapter provides descriptions of wet chemical, alcohol dehydration and critical point-drying methods that developed for routine preparation of sea urchin embryos for scanning electron microscopy (SEM) analysis in the secondary electron mode. Therefore, artifacts of specimen preparation and operational parameters of the SEM scope are reviewed. After the basic procedures of preparing specimens and the morphological information derived from SEM secondary electron images are obtained, then is the time to use SEM instrumentation and techniques for further bridging the gap between light microscopy (LM) and transmission electron microscopy (TEM) analyses. In particular, secondary electron images of the labeling of cell surfaces through markers such as latex spheres and colloidal gold coupled with improved techniques for preserving the integrity of hydrated molecules can vastly improve the detection and localization of a number of developmentally interesting macromolecules. Finally, backscatter electron imaging of structures differentially “stained” with various heavy metals in addition to colloidal gold markers offer unexplored opportunities. The main limits of these opportunities are inertia, time, and imagination.

The effects of gymnodinium breve lysate on the larval development of the sea urchin lytechinus variegatus

Abstract Gymnodinium breve Davis, the Florida red tide organism, was obtained in unialgal culture and lysed by adjusting the pH to 4.0 and readjusting it to 8.3. The lysate was crude filtered and its effects upon the larval development of the sea urchin Lytechinus variegatus investigated. Sperm motility, fertilizability of the egg, and morphological development through blastula stage were unaffected. High mortality and various abnormalities were noted for gastrula and pluteus stages. Results suggest that the toxins may interfere with normal potassium flux and/or respiration in the embryos.

Cellular arrangements and surface topography during early development in embryos of Ilyanassa obsoleta

Summary Surface architecture and selected ultrastructural features of normal embryos of the marine gastropod Ilyanassa obsoleta have been investigated using scanning and transmission electron microscopy. A re-examination of the classic account of Anthony Clement on temporal and division patterns in normal and lobeless embryos confirms most of his observations; a few differences, involving the size and positioning of micromere 4d and its derivatives, are noted. Several hypotheses are presented to account for zonular distribution of longer microvilli on some blastomere surfaces and progressive diminution of microvilli on micromere surfaces, especially the leading edge micromeres (LEMs), during postulated epibolic spreading movements. Microfilaments, discovered in the cortical regions of the LEMs, are described, and a role for these structures in gastrulation is proposed.