ИСТИНА

The cnidarian Hydractinia echinata is a marine colony forming species and the first organism in which stem cells, now known as interstitial cells (i-cells), were descried (Weismann, 1883; Plickert et al., 2012). Dr. Frank's lab uses this model to address fundamental questions in stem cell and developmental biology. In Hydractinia, stem cells are generated in the embryo during gastrulation. At the same time, Hydractinia embryo acquires germ layers and establishes oral-aboral axis. This means that differentiation of stem cells is inseparable from the establishment of the primary body plan. Unfortunately, gastrulation and axis formation in Hydractinia was poorly characterized. This lack of information has been problematic for our research, as we have to consider our data in the context of an entire developing system. The main goal of my research visit funded by SFI Incoming Short-Term Fellowship (ISTTF) was a thorough study of Hydractinia normal development. Working on the project, we were answering the following questions: what is the pattern of early cleavage? when does the Hydractinia embryo acquire the epithelial blastoderm (future ectoderm)? do mitotic divisions of ectoblast cells serve as a long-term source of new endoderm cells? when does the outer cell layer segregates from the inner cell mass? what is the first morphological sign allowing the identification of the future posterior and anterior halves of an embryo? when does this identification becomes possible? To answer these questions we have used a number of research techniques: in vivo observations, histology, electron microscopy, immunocytochemistry and molecular biology.

The main results of our research project are as follows: 1) We have staged and created the time-table of Hydractinia embryogenesis for +21C and +15C. All developmental stages have been characterized using in vivo observations, histology (semi-thin sections), scanning and transmission electron microscopy. 2) We characterized the pattern of early cleavage. In this context, we found that early cleavage in Hydractinia resembles the pattern of spiral and radial cleavage of higher Metazoa. Active cell movements and the rate of cytokinesis are the main factors responsible for the high variability of Hydractinia cleavage. 3) It is well known that Hydractinia embryo becomes morula (consisting of tightly packed cells) at 16 cell stage due to the 3D cleavage pattern. We revealed that epithelization of the outer cell layer (the future ectoderm) occurs very early in development, at the 32-64 cell stage (4-5 h of development). 4) Studying the orientation of mitotic spindles, we have detected the time point of segregation between the ectoderm and the endoderm cell populations. This is 128-256 cell stage (6-7 h of development). From this time point, there is no ectoblast contribution to the population of endoderm cells. Using the formal criterion, gastrulation is completed at this stage. 5) We described embryonic morphology and ultrastructure at the set of developmental stages which have never been characterized. These are the stages between early morula and preplanula (7 – 16 h of development). At these stages we have detected high level of morphological variability that does not affect further development. 6) Gastrulation in Hydractinia is uncoupled from axis patterning, which is a very unusual situation in animals in general. Despite gastrulating in an apolar fashion, the Hydractinia embryo transforms into a properly patterned, anterior-posterior polarized larva. We have found absolutely unique morphological landmarks of the anterior and posterior regions. Identification of the posterior half becomes possible at the early preplanula stage (17 h of development). The future posterior half at this stage is the most morphologically disordered region of an embryo. Moreover, in situ hybridization shows that this disordered region expresses Wnt3, the molecular marker of Hydractinia larva posterior pole. It seems that an exact position of the posterior pole becomes strictly determined later in development. 7) We have described succession of stages leading to the formation of planula larva. The main morphological events at these stages are: elongation of anterior-posterior axis and ordering of the shape of the posterior half. Both processes are based on the active intercalating movements of ectoderm cells.