Use of the Coelomic Grafting Technique for Prolonged ex utero Cultivation of Late Preprimitive Streak-Stage Rabbit Embryos.

Due to its morphological similarity with the early human embryo, the pregastrulation-stage rabbit may represent an appropriate mammalian model for studying processes involved in early human development. The usability of mammalian embryos for experimental studies depends on the availability of whole embryo culture methods facilitating prolonged ex utero development. While currently used culture methods yield high success rates for embryos from primitive streak stages onward, the success rate of extended cultivation of preprimitive streak-stage mammalian embryos is low for all previously established methods and for all studied species. This limits the usability of preprimitive streak-stage rabbit embryos in experimental embryology. We have tested whether the extraembryonic coelom of 4-day-old chick embryos may be used for prolonged ex utero culture of preprimitive streak-stage rabbit embryos (stage 2, 6.2 days post coitum). We found that, within this environment, stage 2 rabbit blastocysts can be cultured at decreasing success rates (55% after 1 day, 35% after 2 days, 15% after 3 days) up to a maximum of 72 h. Grafted blastocysts can continue development from the onset of gastrulation to early organogenesis and thereby form all structures characterizing age-matched controls (e.g. neural tube, somites, beating heart). Compared to normal controls, successfully cultured embryos developed at a slower rate and finally showed some structural and gross morphological anomalies. The method presented here was originally developed for whole embryo culture of mouse embryos by Gluecksohn-Schoenheimer in 1941. It is a simple and inexpensive method that may represent a useful extension to presently available ex utero culture systems for rabbit embryos.

Whole-mount in situ hybridization to assess advancement of development and embryo morphology.

Whole-mount in situ hybridization (WISH) is widely used to visualize the site and dynamics of gene expression during embryonic development. Various methods of probe labeling and hybridization detection are available nowadays. Meanwhile the technique was adapted to be used on many different species and has evolved from a manual to a larger scale and automated procedure. Standardized automated protocols improve the chance to compare different experimental settings reliably. The high resolution of this method is ideally suited for examination of manipulated (e.g., cloned) embryos often displaying subtle changes only. Embedding and sectioning of in situ hybridized specimen further enhance the detailed examination of their gene expression and morphology.

Diagnosis of stapedial footplate fixation in archaeological human remains.

This study analyses changes in the region of the oval window suggestive of stapedial footplate fixation in archaeological human skeletal remains. We endoscopically investigated 621 temporal bones of 385 individuals from five medieval sites in Germany to identify fixations of the stapedial footplate. For differential diagnosis, four cases suspicious of representing stapes fixation or remnants of the fixed footplate were further investigated using microscopic techniques (brightfield and darkfield imaging, phase-contrast microscopy, fluorescence microscopy, CLSM, SEM-BSE imaging), and EDX-analysis, either alone or in combination. Our findings suggest that only two of the four cases represented an intravital fixation of the stapedial footplate. The first case was diagnosed as caused by sclerosis of the annular ligament, the second cases as representing an example of congenital footplate fixation. In a third case, structures that were initially diagnosed as remnants of the footplate were shown to be soil particles. In the fourth case the structures attached to the oval window were identified as apatitic deposits formed by diagenetic agents. Our findings highlight the need for microscopic analyses to distinguish intravital from postmortem changes in the region of the oval window and the differential diagnosis of intravital footplate fixations.

Gastrulation in rabbit blastocysts depends on insulin and insulin-like-growth-factor 1.

Insulin and insulin-like-growth-factor 1 (IGF1) are components of the uterine secretions. As potent growth factors they influence early embryo development. The underlying molecular mechanisms are largely unknown. Here we report on the effects of insulin and IGF1 on early gastrulation in rabbit blastocysts. We have studied blastocysts grown in vivo in metabolically healthy rabbits, in rabbits with type 1 diabetes and in vitro in the presence or absence of insulin or IGF1. Embryonic disc morphology and expression of Brachyury, Wnt3a and Wnt4 were analysed by qPCR and IHC. Pre-gastrulated blastocysts (stage 0/1) cultured with insulin or IGF1 showed a significantly higher capacity to form the posterior mesoderm and primitive streak (stage 2 and 3) than blastocysts cultured without growth factors. In gastrulating blastocysts the levels of the mesoderm-specific transcription factor Brachyury and the Wnt signalling molecules Wnt3a and Wnt4 showed a stage-specific expression pattern with Brachyury transcripts increasing from stage 0/1 to 3. Wnt4 protein was found spread over the whole embryoblast. Insulin induced Wnt3a, Wnt4 and Brachyury expression in a temporal- and stage-specific pattern. Only blastocysts cultured with insulin reached the Wnt3a, Wnt4 and Brachyury expression levels of stage 2 in vivo blastocysts, indicating that insulin is required for Wnt3a, Wnt4 and Brachyury expression during gastrulation. Insulin-induced Wnt3a and Wnt4 expression preceded Brachyury. Wnt3a-induced expression could be depleted by MEK1 inhibition (PD98059). Involvement of insulin in embryonic Wnt3a expression was further shown in vivo with Wnt3a expression being notably down regulated in stage 2 blastocysts from rabbits with type 1 diabetes. Blastocysts grown in diabetic rabbits are retarded in development, a finding which supports our current results that insulin is highly likely required for early mesoderm formation in rabbit blastocysts by inducing a distinct spatiotemporal expression profile of Wnt3a, Wnt4 and Brachyury.

BMP signals and the transcriptional repressor BLIMP1 during germline segregation in the mammalian embryo.

Molecular factors and tissue compartments involved in the foundation of the mammalian germline have been mainly described in the mouse so far. To find mechanisms applicable to mammals in general, we analyzed temporal and spatial expression patterns of the transcriptional repressor BLIMP1 (also known as PRDM1) and the signaling molecules BMP2 and BMP4 in perigastrulation and early neurulation embryos of the rabbit using whole-mount in situ hybridization and high-resolution light microscopy. Both BMP2 and BMP4 are expressed in annular domains at the boundary of the embryonic disc, which--in contrast to the situation in the mouse--partly belong to intraembryonic tissues. While BMP2 expression begins at (pregastrulation) stage 1 in the hypoblast, BMP4 expression commences--distinctly delayed compared to the mouse--diffusely at (pregastrulation) stage 2; from stage 3 onwards, BMP4 is expressed peripherally in hypoblast and epiblast and in the mesoderm at the posterior pole of the embryonic disc. BLIMP1 expression begins throughout the hypoblast at stage 1 and emerges in single primordial germ cell (PGC) precursors in the posterior epiblast at stage 2 and then in single mesoderm cells at positions identical to those identified by PGC-specific antibodies. These expression patterns suggest that function and chronology of factors involved in germline segregation are similar in mouse and rabbit, but higher temporal and spatial resolution offered by the rabbit demonstrates a variable role of bone morphogenetic proteins and makes "blimping" a candidate case for lateral inhibition without the need for an allantoic germ cell niche.

Planar cell movements and oriented cell division during early primitive streak formation in the mammalian embryo.

Formation of the mammalian primitive streak appears to rely on cell proliferation to a minor extent only, but compensating cell movements have not yet been directly observed. This study analyses individual cell migration and proliferation simultaneously, using multiphoton and differential interference contrast time-lapse microscopy of late pregastrulation rabbit blastocysts. Epiblast cells in the posterior gastrula extension area accumulated medially and displayed complex planar movements including U-turns and a novel type of processional cell movement. In the same area metaphase plates tended to be aligned parallel to the anterior-posterior axis, and statistical analysis showed that rotations of metaphase plates causing preferred orientation were near-complete 8 min before anaphase onset; in some cases, rotations were strikingly rapid, achieving up to 45° per min. The mammalian primitive streak appears to be formed initially with its typically minimal anteroposterior elongation by a combination of oriented cell divisions with dedicated planar cell movements.

Sox17 expression patterns during gastrulation and early neurulation in the rabbit suggest two sources of endoderm formation.

Most gastrointestinal tract and associated gland epithelia originate from the endoderm germ layer discovered by Pander in 1817. The recent surge in stem cell concepts revived interest in the findings of 30 years ago that the endoderm layer itself originates from the epiblast (which since Pander's time had been held to be the forerunner of the ectoderm and mesoderm germ layers only). However, the question as to which parts of the mammalian gastrulation-stage embryonic disc generate endoderm cells is still unresolved. Therefore, the expression of the gene coding for the transcription factor Sox17, a key transcription factor involved in endoderm formation in mouse, chick, frog, and zebrafish, was analyzed in the rabbit, a model organism for mammalian gastrulation morphology, using whole-mount in situ hybridization and high-resolution histological analysis of embryos at gastrulation and early neurulation stages. Sox17 mRNA in the mesoderm and lower layer (hypoblast) compartments within and adjacent to Hensen's node and the anterior segment of the primitive streak confirmed the validity of this approach, as this region had previously been shown to form endoderm in mouse and chick. However, Sox17 expression in central and posterior epiblast at pregastrulation stages together with a transient expression at the posterior extremity of the primitive streak suggest that endoderm (possibly hindgut) may be formed close to the emerging cloacal membrane, as well.

Tracing and ablation of single cells in the mammalian blastocyst using fluorescent DNA staining and multi-photon laser microscopy.

Comparing the vital DNA dyes Hoechst33342 and DAPI in their ability to visualise cell nuclei of the late rabbit blastocyst, both dyes were found to be equally suited despite differences in staining intensity in embryonic versus extraembryonic tissues and in nuclear versus cytoplasmic domains at the subcellular level: Both dyes stain all nuclei of a given cell layer (e.g. epiblast or hypoblast) evenly and provide satisfactory fluorescence contrast throughout the blastocyst, while not interfering with normal development up to 10 h in vitro. Using short period (60-300 min) irradiation experiments with either dye, single-photon (405 nm) and multi-photon (800 nm) laser excitation was compared in different areas of the same embryo and parameters of multi-photon microscopy were defined for gentle live imaging and tracing of single cells deep to the surface of the embryo. In addition, individual cells were ablated by reducing the "area of interest" to sub-nucleus-size, thus maximally increasing the density of laser energy brought into the tissue. Thickening of epiblast and hypoblast and increased numbers of dense cytoplasmic inclusions within the limits of irradiated areas were found in semithin histological sections in a dose-dependent manner. Ablated cells were found in a necrotic state while neighbouring cells remained apparently unscathed.

Subcellular characterization of the primordial germ cell antigen PG2 in adult oocytes.

Primordial germ cells represent the founder population for establishing the germ line providing the continuity of life between generations. PG2, a germ cell-specific antigen, is one of the few continuously detectable epitopes in mammalian primordial germ cells and it is dynamically expressed during early post-fertilization development and during postnatal germ cell maturation. Immunoelectron microscopy shows a localization of PG2 in the peri-mitochondrial cytoplasm but its further subcellular or biochemical nature remains elusive. For further characterization of the PG2 epitope we used regular and semi-thin cryosection of ovulated and isolated follicular rabbit oocytes and localized all mitochondria with the help of the constitutive mitochondrial antigen MTC02 in double immunofluorescence stainings. Semi-thin cryosections of ovulated oocytes revealed a general close co-localization of both antibody reactions at the level of single mitochondria. In centrifuged follicular oocytes both antigens co-sedimented almost completely indicating a topographical association of the epitopes on the basis of a strong interaction of PG2 with mitochondria. To begin to characterize the germ cell epitope biochemically we treated oocyte cryosections either with acetone to reduce lipids or with N-glycosidase F to remove N-linked glycosylations before the immuoreaction. Neither treatment affected the antibody characteristics, which suggests that the PG2 epitope is most probably a protein. Because of the close interaction of PG2 with the mitochondria we speculate that PG2 is involved in the change of the mitochondrial morphology typically observed during differentiation of germ cells.