Skeletogenesis in sea urchin larvae under modified gravity conditions.

From many points of view, skeletogenesis in sea urchins has been well described. Based on this scientific background and considering practical aspects of sea urchin development (i.e. availability of material, size of larvae, etc.), we wanted to know whether orderly skeletogenesis requires the presence of gravity. The objective has been approached by three experiments successfully performed under genuine microgravity conditions (in the STS-65 IML-2 mission of 1994; in the Photon-10 IBIS mission of 1995 and in the STS-76 S/MM-03 mission of 1996). Larvae of the sea urchin Sphaerechinus granularis were allowed to develop in microgravity conditions for several days from blastula stage onwards (onset of skeletogenesis). At the end of the missions, the recovered skeletal structures were studied with respect to their mineral composition, architecture and size. Live larvae were also recovered for post-flight culture. The results obtained clearly show that the process of mineralisation is independent of gravity: that is, the skeletogenic cells differentiate correctly in microgravity. However, abnormal skeleton architectures were encountered, particularly in the IML-2 mission, indicating that the process of positioning of the skeletogenic cells may be affected, directly or indirectly, by environmental factors, including gravity. Larvae exposed to microgravity from blastula to prism/early pluteus stage for about 2 weeks (IBIS mission), developed on the ground over the next 2 months into normal metamorphosing individuals.

The sea urchin larva, a suitable model for biomineralisation studies in space (IML-2 ESA Biorack experiment '24-F urchin').

By the ESA Biorack 'F-24 urchin' experiment of the IML-2 mission, for the first time the biomineralisation process in developing sea urchin larvae could be studied under real microgravity conditions. The main objectives were to determine whether in microgravity the process of skeleton formation does occur correctly compared to normal gravity conditions and whether larvae with differentiated skeletons do 'de-mineralise'. These objectives have been essentially achieved. Postflight studies on the recovered 'sub-normal' skeletons focused on qualitative, statistical and quantitative aspects. Clear evidence is obtained that the basic biomineralisation process does actually occur normally in microgravity. No significant differences are observed between flight and ground samples. The sub-normal skeleton architectures indicate, however, that the process of positioning of the skeletogenic cells (determining primarily shape and size of the skeleton) is particularly sensitive to modifications of environmental factors, potentially including gravity. The anatomical heterogeneity of the recovered skeletons, interpreted as long term effect of an accidental thermal shock during artificial egg fertilisation (break of climatisation at LSSF), masks possible effects of microgravity. No pronounced demineralisation appears to occur in microgravity; the magnesium component of the skeleton seems yet less stable than the calcium. On the basis of these results, a continuation of biomineralisation studies in space, with the sea urchin larva as model system, appears well justified and desirable.

Preservation of viable biological samples for experiments in space laboratories.

Standard viable preservation methods for biological samples using low temperatures have been investigated concerning their storage capabilities under higher temperature levels than usual. For a representative set of organism classes (plants, mammalian cells, arthropods and aquatic invertebrates), the minimum appropriate storage conditions have been identified by screening storage temperatures at -196 degrees, -80 degrees, -20 degrees, +4 degrees, +20 degrees/25 degrees C for periods from 2 days to 4 weeks. For storage below 0 degree C, as a typical cryopreservative, dimethylsulfoxide (DMSO) was used. For some samples, the addition of trehalose (as cryopreservative) and the use of a nitrogen atmosphere were investigated. After storage, the material was tested for vitality. The findings demonstrated that acceptable preservation can be achieved under higher storage temperatures than are typically applied. Small, dense cultured plant cells survive for 21 d when moderately cooled (+4 degrees to -20 degrees C); addition of trehalose enhances viability at -20 degrees C. For mammalian cells, the results show that human lymphocytes can be preserved for 3 d at 25 degrees C, 7 d at 4 degrees C and 28 d at -80 degrees C. Friend leukaemia virus transformed cells can be stored for 3 d at 25 degrees C, 14 d at 4 degrees C and 28 d at -80 degrees C. Hybridoma cells can be kept 7 d at 4 degrees C and 28 d at -20 degrees C or -80 degrees C. Model arthropod systems are well preserved for 2 weeks if maintained at lower temperatures that vary depending on the species and/or stage of development; e.g., 12 degrees C for Drosophila imagoes and 4-6 degrees C for Artemia nauplii. For aquatic invertebrates such as sea urchins, embryonic and larval stages can be preserved for several weeks at +6 degrees C, whereas sperm and eggs can best be stored at + 4 degrees C for up to 5 d at maximum. These results enhance the range of feasible space experiments with biological systems. Moreover, for typical terrestrial preservation methods, considerable modification potential is identified.

Fertilization of sea urchin eggs in space and subsequent development under normal conditions.

Sea urchin eggs are generally considered as most suitable animal models for studying fertilization processes and embryonic development. In the present study, they are used for determining a possible role of gravity in fertilization and the establishment of egg polarity and the embryonic axis. For this purpose, eggs of the particularly well known and suitable species Paracentrotus lividus have been automatically fertilized under microgravity conditions during the Swedish sounding rocket flights MASER IV and MASER V. It turns out, that fertilization "in Space" occurs normally and that subsequent embryonic and larval development of such eggs, continued on the ground, is normal, leading to advanced pluteus stages.

Primary effects of singlet oxygen sensitizers on eggs and embryos of sea urchins.

Photodynamic effects of rose bengal, a well-known singlet oxygen sensitizer, and of haematoporphyrin derivative, the most widely used sensitizer in photodynamic therapy of tumours, could be visualized using sea urchin eggs and embryos. This biological material is a valuable model for the analysis of mechanisms and/or sites of the photodynamic action occurring in any living tissue. Depending on the sensitizer used, singlet oxygen may be identified as the main mediator of the cytotoxic effects observed. Besides observations made on the living, in particular within the context of fertilization ability of the egg cell, gross damages of the cells are morphologically analysed by scanning electron microscopy. The results support the working hypothesis explaining the different susceptibility of healthy and tumour cells for photosensitization as a cell cycle phenomenon.

Individual migration of mesentodermal cells in the early embryo of the squid Loligo vulgaris: in vivo recordings combined with observations with TEM and SEM.

In the translucent preorganogenetic embryo of the squid Loligo vulgaris a population of single cells between the ectodermal layer and the yolk syncytium can be studied continuously in vivo during migration to the vegetal hemisphere of the egg. The results from 2 different preparations are reported: 1. An intact embryo served to view locomotive cell behavior through the translucent ectoderm with undisturbed cell-substrate interactions. 2. In an embryo a patch of ectoderm was microsurgically removed thereby exposing migrating cells to direct observation and experimental manipulation. In vivo time lapse microcinematographic recordings for 22 h (in 1.) and 10 h (in 2.) revealed the following: cell migration is neither directional nor dependent on the presence of the ectodermal layer (in 2.). Although the migrating cells primarily use the syncytial surface as a substrate for locomotion, under natural conditions they also adhere to the basal ectodermal surface as revealed by TEM and SEM. Migration rates were 18.3 +/- 12.6 mu/h in 1. Locally directed cell migration was observed in a group of cells in 1. which were involved in a process of aggregation, the latter being probably related to precocious formation of organ primordia. A preliminary note has appeared previously (Segmüller and Marthy, 1984).

Organogenesis in Cephalopoda: further evidence of blastodisc-bound developmental information.

The basic mechanisms of organ differentiation in the Cephalopod embryo (telolecithal egg, discoidal cleavage) are studied. The results of ligation experiments, performed in early cleavage stages, confirm earlier conclusions of the author, drawn from transplantation/explantation and heat-shock experiments. The developmental information for cellular differentiation is shown to reside in the blastodisc; the yolk syncytium, in which a large part of the original egg cortex is incorporated, acts as as nutritive substrate for the cellular material involved in organogenesis. On the basis of these results, Arnold's induction model supposing an undisplaceable determining informational pattern laid down in the uncleaved egg cortex must be rejected.

[Establishment of a primary culture of cells from embryonic tissue of Loliyo vulgaris L.(Cephalopod)]. / Mise au point d'une culture primaire de cellules à partir du tissu embryonnaire de Loligo vulgaris L. (Céphalopode)

The author studies the behaviour in vitro of organ fragments of Loligo vulgaris embryos at hatching stage. These fragments are maintained in three different media. The time of survival of cells is 20 to 25 days in the medium I(sea water), 40 days in the medium II(perivitellin fluid) and 70 to 80 days in the medium II(perivetellin fluid + horse serum). It is this medium III that the original explant is transformed into a typical primary cell culture.