Experimental reconstruction of mouse eggs and embryos: an analysis of mammalian development

The scope of experimental approaches applicable to the study of mammalian eggs and embryos has advanced in recent years to provide unprecedented opportunities for understanding mammalian embryology. Amongst these significant advances has been the ability to alter the genetic constitution of eggs by pronuclear and nuclear transplantation as well as by the introduction of specific cloned genes into eggs and embryos. These techniques can be used in conjunction with the experimental reconstruction of preimplantation embryos to investigate more precisely a number of aspects of mammalian embryology. Recently, a most intriguing aspect of development has been uncovered, one that is apparently unique to mammals; experiments have revealed that the parental genomes are not functionally equivalent during embryogenesis. Hence, the parental origin of chromosomes determines their influence during embryogenesis. The mechanistic aspects responsible for the germ line modifications of homologous chromosomes, their role during development, and the wide-ranging implications of these findings for mammalian development have yet to be fully defined. An understanding of this process will provide the basis for developing genetic and reproductive strategies that can be applied to domestic animals and to humans.     

Obtainment of chimeric blastocysts of mice by methods of laser nanosurgery

The procedure of obtainment of chimeric blastocysts of mice by laser nanosurgery methods with-out using any other techniques is described. To perform the experiments, a special laser micromanipulator was invented. The murine embryonic stem cells (ESC), which were transformed with pEF-GFP vector, encoding the green fluorescent protein, were used in the experiments. ESC were introduced into the perivitelline space of murine embryos at the stage of 8 cells using the laser micromanipulator. The operated embryos were cultured in vitro until the stage of emergence from zona pellucida. The fluorescence and its precise localization were registered using a confocal microscope. It was shown for the first time that the inclusions of ESC introduced with the lased micromanipulator were found not only in the inner cell mass (ICM) but also in the trophectoderm of the chimeric blastocyst. The technology of nanosurgical operations at early stage preimplanted mammalian embryos using laser techniques opens great opportunities not only for solution of fundamental tasks of experimental embryology of mammals but also for obtainment of chimeric and transgenic animals with predetermined genotype.     

Developmental biology in Geneva: a three century-long tradition

It was in the first half of the 18th century when life sciences started to flourish in the independent republic of Geneva. However, it is difficult to identify a genuine school of developmental biologists during that era. Nevertheless, several prominent scientists over the past two and a half centuries have established and maintained a strong tradition of studies in embryological development and reproduction. In this short historical account, we briefly pay tribute to these famous forerunners, by emphasizing both the originality and quality of their work, as well as the many accompanying conceptual and methodological advances. We start with Abraham Trembley (1710-1784) and the discovery of Hydra and of regeneration, and with Charles Bonnet (1720-1793) who, amongst other contributions, first observed parthenogenetic development. In the 19th century, Carl Vogt (1817-1895) and Edouard Claparède (1832-1871) were well-known scientists in this field of research, whereas Hermann Fol (1845-1892) can be considered as one of the pioneers, if not the founder, of causal embryology, through his experiments on lateral asymmetry in manipulated chicken. More recently, Emile Guyénot (1885-1963) and Kitty Ponse (1897-1982) perpetuated this tradition, which is well alive nowadays in the city of Calvin.     

Residues SFQ (173-175) in the large extracellular loop of CD9 are required for gamete fusion

Gamete fusion is the fundamental first step initiating development of a new organism. Female mice with a gene knockout for the tetraspanin CD9 (CD9 KO mice) produce mature eggs that cannot fuse with sperm. However, nothing is known about how egg surface CD9 functions in the membrane fusion process. We found that constructs including CD9's large extracellular loop significantly inhibited gamete fusion when incubated with eggs but not when incubated with sperm, suggesting that CD9 acts by interaction with other proteins in the egg membrane. We also found that injecting developing CD9 KO oocytes with CD9 mRNA restored fusion competence to the resulting CD9 KO eggs. Injecting mRNA for either mouse CD9 or human CD9, whose large extracellular loops differ in 18 residues, rescued fusion ability of the injected CD9 KO eggs. However, when the injected mouse CD9 mRNA contained a point mutation (F174 to A) the gamete fusion level was reduced fourfold, and a change of three residues (173-175, SFQ to AAA) abolished CD9's activity in gamete fusion. These results suggest that SFQ in the CD9 large extracellular loop may be an active site which associates with and regulates the egg fusion machinery.     

Mouse chimaeras developed from electrofused blastocysts: new evidence for developmental plasticity of the inner cell mass

Blastocysts obtained from mice differing in pigmentation (albino versus pigmented) and the isoforms of glucose phosphate isomerase (GPI 1A versus 1B) were electrofused and those containing a single chimaeric inner cell mass (ICM) were transferred to the uterus of pseudopregnant recipients. The pups were recovered on the 20(th) day by Caesarian section and fostered by females that had littered on the previous night or 24 h earlier. Altogether nine adult animals and two pups, which died soon after delivery, were available for GPI analysis. Between 9 and 13 organs/tissues were examined and the relative contribution of the GPI 1A and 1B isoforms was estimated using an electrophoretic GPI assay. Eight adult animals were overtly chimaeric and one was chimaeric in some internal tissues only. Eight mice were males: seven were fertile, one was infertile. The ninth adult mouse was a hermaphrodite. The fertile animals produced sperm of one genotype only, i.e. derived either from the albino or from the pigmented component. This is the first report showing that adult chimaeras can be produced from two combined blastocysts, provided that fusion of the adhering trophectoderm cells is first induced and the orientation of blastocysts enables the two ICMs to integrate into a single ICM. Our results suggest that in the preimplantation blastocyst, the organisation of the ICM remains labile thus making it possible for the fused blastocysts to establish new embryonic organisation and to develop into a single organism.     

Imaging filopodia dynamics in the mouse blastocyst

During mammalian development, the first cell lineage diversification event occurs in the blastocyst, when the trophectoderm (TE) and the inner cell mass (ICM) become established. Part of the TE (polar) remains in contact with the ICM and differs from the mural TE (mTE) which is separated from the ICM by a cavity known as the blastocoele. The presence of filopodia connecting ICM cells with the distant mural TE cells through the blastocoelic fluid was investigated in this work. We describe two types of actin-based cell projections found in freshly dissected and in vitro cultured expanding blastocysts: abundant short filopodia projecting into the blastocoelic cavity that present a continuous undulating behavior; and long, thin traversing filopodia connecting the mural TE with the ICM. Videomicroscopy analyses revealed the presence of vesicle-like structures moving along traversing filopodia and dynamic cytoskeletal rearrangements. These observations, together with immunolocalization of the FGFR2 and the ErbB3 receptors to these cell extensions, suggest that they display signal transduction activity. We propose that traversing filopodia are employed by mitotic mTE cells to receive the required signals for cell division after they become distant to the ICM.     

Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro

The in vitro viability of polyspermic pig eggs was investigated. Immature oocytes were matured and fertilized in vitro. Approximately 10 h after insemination, the eggs were centrifuged at 12 000 x g for 10 min and individually classified into two (2PN)- and poly-pronuclear (PPN, 3 or 4 pronuclei) eggs. The classified eggs were cultured in vitro or in vivo. Nuclei numbers of inner cell mass (ICM) and trophectoderm (TE) were compared between 2PN- and PPN-derived blastocysts. The frequency of development in vitro of 2PN and PPN eggs to the blastocyst stage was 53.6% and 40.7%, respectively. The mean number (8.2 +/- 0.7, n = 48) of ICM nuclei of 2PN-derived blastocysts was higher than that (4.2 +/- 0.8, n = 37) of PPN-derived blastocysts (p < 0.001), whereas there was no difference (p > 0.05) in mean numbers of total (46.7 +/- 3.4 vs. 39. 9 +/- 3.9) and TE nuclei (38.5 +/- 2.9 vs. 35.7 +/- 3.3) between the two groups. Development of 2PN and PPN eggs cultured in vivo to the blastocyst stage was 33.3% and 27.4%, respectively. The numbers of ICM and TE nuclei of these embryos cultured in vivo showed a pattern similar to that for the in vitro-produced blastocysts. Additionally, fetuses were obtained on Day 21 from both the 2PN and the PPN groups. This suggests that polyspermic pig embryos develop to the blastocyst stage and beyond, although showing a smaller ICM cell number as compared to normal embryos.     

August Rauber (1841-1917): from the primitive streak to Cellularmechanik

In the early 19th century Karl Ernst von Baer initiated a new research program searching for the mechanisms by which an egg transforms itself into an embryo. August Rauber (1841-1917) took up this challenge. He considered the phylogenetic principle as the right tool to explain the similitude of embryogenetic processes. In extending Baer's approach, he combined comparative embryology and histology in his studies of avian and mammalian embryos. His earlier work demonstrated that the two-layered chick embryo is a modified gastrula and not a "disc" as Wilhelm His had claimed. From the 1880s onwards, he concentrated on the issue of how the development of germ layers is related to tissue differentiation. To address this, he studied the blastopore, epiblast, primitive streak, teratology and the relative importance of nucleus and cytoplasm in heredity. This paper reconstructs some of Rauber's work and concludes that his observations and reflections constituted a new approach combining embryology and histology with "phylogenetic" reasoning.     

IGF-2 receptors are first expressed at the 2-cell stage of mouse development.

A specific IGF-2 receptor antiserum was used to reveal the presence of IGF-2 receptors during preimplantation development of mice. Receptors were present on 2-, 4- and 8-cell embryos, morulae, blastocysts, and on ICMs isolated prior to staining. There was no evidence for receptors on fertilized eggs. These observations confirm reports of the expression of IGF-2 receptor mRNA as early as the 2-cell stage and refine similar observations in blastocysts to confirm expression in both the TE and ICM. A potential auto/paracrine loop is thus one of the first products of activation of the embryonic genome and is expressed constitutively through preimplantation development.     

An amphioxus homeobox gene: sequence conservation, spatial expression during development and insights into vertebrate evolution.

The embryology of amphioxus has much in common with vertebrate embryology, reflecting a close phylogenetic relationship between the two groups. Amphioxus embryology is simpler in several key respects, however, including a lack of pronounced craniofacial morphogenesis. To gain an insight into the molecular changes that accompanied the evolution of vertebrate embryology, and into the relationship between the amphioxus and vertebrate body plans, we have undertaken the first molecular level investigation of amphioxus embryonic development. We report the cloning, complete DNA sequence determination, sequence analysis and expression analysis of an amphioxus homeobox gene, AmphiHox3, evolutionarily homologous to the third-most 3' paralogous group of mammalian Hox genes. Sequence comparison to a mammalian homologue, mouse Hox-2.7 (HoxB3), reveals several stretches of amino acid conservation within the deduced protein sequences. Whole mount in situ hybridization reveals localized expression of AmphiHox3 in the posterior mesoderm (but not in the somites), and region-specific expression in the dorsal nerve cord, of amphioxus neurulae, later embryos and larvae. The anterior limit to expression in the nerve cord is at the level of the four/five somite boundary at the neurula stage, and stabilises to just anterior to the first nerve cord pigment spot to form. Comparison to the anterior expression boundary of mouse Hox-2.7 (HoxB3) and related genes suggests that the vertebrate brain is homologous to an extensive region of the amphioxus nerve cord that contains the cerebral vesicle (a region at the extreme rostral tip) and extends posterior to somite four.(ABSTRACT TRUNCATED AT 250 WORDS)     

The history of algology, algotherapy, and the role of inhibition

Cephalalgia (1st century AD), nostalgia (1678), neuralgia (18th century), causalgia (1872) were terms followed in the 1950's by Bonica's 'algology... a disease state of its own', addressed by ever-growing numbers of pain clinics, strongly foreshadowed by Leriche's douleur maladie in the 1930's. (Hence also 'algotherapy'). Philosophers first, then early academic physiologists began to exhibit interest in pain, that all too common phenomenon, only too often unyielding to theoretical as well as practical efforts. Was it, after all, an instance of built-in self-preservation, a reflex? Identification of the nervous energy and its anatomical pathways in the 19th century, endless arguments as to their 'specificity', led to new surgical attempts to control and interpret pain, by now supported by general, then local anesthesia. Early in this century Henry Head's much-discussed notion of 'epicritic' sensation exerting some control over 'protopathic' pain was soon followed by Otfried Foerster's insistence on a central role of inhibition providing pain relief. Almost forgotten, Foerster's idea found expression in Melzack and Wall's 'gate control theory' of 1965. Gasser and Erlanger's classification of sensory nerve fibers began to dominate research in the 1930's thanks to the cathode ray oscillograph invented in 1897. The pain inhibition concept was given another boost in the seventies when the role of the midline mesencephalic and oblongata nuclei was established as both opium receptors and producers of opioids. Finally, inhibition may also be seen as the principle underlying the age-old therapeutic effect of 'counter-irritation', mostly in the form of electrical stimulation.     

The history of the discovery of the nerve growth factor

The Nerve Growth Factor (NGF) is the progenitor of a family of growth factors which is still expanding. The history of its discovery is very colorful; it is a rare combination of scientific reasoning, intuition, fortuities, and good luck. In addition, I believe that the collaboration of three scientists with very different backgrounds contributed to the success: I had grown up in a laboratory of experimental embryology, Dr. Levi-Montalcini came from neurology, and Dr. Stanley Cohen was from biochemistry. The decision where to begin the history of a discovery is always arbitrary. I shall give my reasons why I begin this story with my wing bud extirpations on chick embryos and the analysis of the effects of the operation on the development of spinal nerve centers, published in 1934. Of course, I am aware of the fact that the analysis of neurogenesis had been pioneered by Dr. R. G. Harrison and his students at Yale University since the beginning of this century. It should be mentioned that their experiments had been done on amphibian embryos. My own interest in problems of neurogenesis dates back to my Ph.D. thesis in the Zoology Department of Professor H. Spemann at the University of Freiburg in (the Federal Republic of) Germany; it dealt with the influence of the nervous system on the development of limbs in frog embryos. After I had obtained some inconclusive results I did the crucial experiment of producing nerveless legs. I removed the lumbar part of the spinal cord and the spinal ganglia before the outgrowth of nerve fibers. The nerveless legs developed normally in every respect, but the muscles atrophied eventually.     

Trophoblast-specific DNA methylation occurs after the segregation of the trophectoderm and inner cell mass in the mouse periimplantation embryo

The first cell differentiation in the mammalian development separates the trophoblast and embryonic cell lineages, resulting in the formation of the trophectoderm (TE) and inner cell mass (ICM) in blastocysts. Although a lower level of global DNA methylation in the genome of the TE compared with ICM has been suggested, the dynamics of the DNA methylation profile during TE/ICM differentiation has not been elucidated. To address this issue, first we identified tissue-dependent and differentially methylated regions (T-DMRs) between trophoblast stem (TS) and embryonic stem (ES) cells. Most of these TS–ES T-DMRs were also methylated differentially between trophoblast and embryonic tissues of embryonic day (E) 6.5 mouse embryos. Furthermore, we found that the human genomic regions homologous to mouse TS–ES T-DMRs were methylated differentially between human placental tissues and ES cells. Collectively, we defined them as cell-lineage-based T-DMRs between trophoblast and embryonic cell lineages (T–E T-DMRs). Then, we examined TE and ICM cells isolated from mouse E3.5 blastocysts. Interestingly, all T-DMRs examined, including the Elf5, Pou5f1 and Nanog loci, were in the nearly unmethylated status in both TE and ICM and exhibited no differences. The present results suggest that the establishment of DNA methylation profiles specific to each cell lineage follows the first morphological specification. Together with previous reports on asymmetry of histone modifications between TE and ICM, the results of the current study imply that histone modifications function as landmarks for setting up cell-lineage-specific differential DNA methylation profiles.     

Incorporation of mammalian metabolism into mutagenicity testing

In the 1950's and 1960's it became obvious that many chemicals in daily use were mutagenic or carcinogenic, but there seemed to be little relation between the two activities. As scientists were debating the cause of this discrepancy, it was hypothesized that mammalian metabolism could form highly reactive intermediates from rather innocuous chemicals and that these intermediates could react with DNA and were mutagenic. This commentary presents the historical development of metabolic activation in mutagenicity tests, beginning with Udenfriend's hydroxylation system, which mimics aspects of mammalian metabolism in a purely chemical mixture, and extending through procedures that moved closer and closer to incorporating actual mammalian metabolism into the test systems. The stages include microsomal activation systems, host-mediated assays, incorporation of human P450 genes into the target cells or organisms, and detecting mutations in single cells in vivo. A recent development in this progression is the insertion of recoverable vectors containing mutational targets into the mammalian genome. Since the target genes of transgenic assays are in the genome, they are not only exposed to active metabolites, but they also undergo the same repair processes as endogenous genes of the mammalian genome.     

Cloning of Homo sapiens? No!

Animal cloning by nuclear transplantation was first developed in the northern leopard frog, Rana pipiens. It was soon extended to other amphibian species and within time, to various mammalian species. The production of a cloned sheep (Dolly) from an adult nuclear donor reawakened interest in human cloning. Nuclear transfer for the production of animal clones has served experimental biology well. Nonetheless, the potential burden of developmental hazards, scientists and funds diverted from more needy causes, as well as the potential assault on the concept of family has led the author to oppose human cloning.     

CDX2 in the formation of the trophectoderm lineage in primate embryos

The first lineage decision during mammalian development is the establishment of the trophectoderm (TE) and the inner cell mass (ICM). The caudal-type homeodomain protein Cdx2 is implicated in the formation and maintenance of the TE in the mouse. However, the role of CDX2 during early embryonic development in primates is unknown. Here, we demonstrated that CDX2 mRNA levels were detectable in rhesus monkey oocytes, significantly upregulated in pronuclear stage zygotes, diminished in early cleaving embryos but restored again in compact morula and blastocyst stages. CDX2 protein was localized to the nucleus of TE cells but absent altogether in the ICM. Knockdown of CDX2 in monkey oocytes resulted in formation of early blastocyst-like embryos that failed to expand and ceased development. However, the ICM lineage of CDX2-deficient embryos supported the isolation of functional embryonic stem cells. These results provide evidence that CDX2 plays an essential role in functional TE formation during primate embryonic development.     

Three thousand years of questioning sex determination

Of all the inborn differences that distinguish individual humans, as well as other animals, sex exerts the most far-reaching effects, and the question, what determines it, has been debated throughout history. A discriminating reading of Biblical and Ancient Greek sources reveals surprising insights that are relevant to present-day biology. The material basis of generation was inaccessible until, following the invention of the microscope and the discovery of "spermatic animalcules" in the 17th century, the 19th century witnessed the discovery of the mammalian egg, the nature of sperm, and the process of fertilization. Sex was thought to be determined by external conditions. The 20th century developed the genetics of sex determination. The search for the mammalian testis-determining gene during the last quarter century culminated in the discovery of SRY, soon to be accompanied by non-Y chromosome sex- determining genes. During the same period, data accumulated that testicular differentiation was accompanied by accelerated gonadal growth; subsequently, differences in growth were shown to distinguish early XX from early XY embryos. Other research showed that temperature-dependent sex determination was widely distributed among reptiles, thus illustrating that the mammalian system of sex determination is of recent evolutionary origin, adopted in response to homoiothermy and placentation. The recent discovery that Sry induces cell proliferation in the gonads of fetal mice suggests that the task for the 21st century will be to aim beyond simple genotype/phenotype correlations by unraveling the relationship between genes and epigenetic factors acting on cell growth during development and affecting the phenotype in later life.     

Johann Friedrich Meckel, Jr. as founder of scientific teratology

n the occasion of the 150th anniversary of his death, the scientific work of the famous German anatomist Johann Friedrich Meckel (1781 to 1833) in Halle is appreciated. The Younger Meckel is counted to the most outstanding figures in the history of anatomy and medicine in the first third of 19th century. According to his founded knowledges in the normal, comparative, and pathologic anatomy and embryology he was able to give a scientific argument of malformations first of all in the history of medicine and biology. The edition of Meckel's Handbook of Pathologic Anatomy (in German language; 1st vol. 1812) is the birth of scientific teratology. Through his contributions to teratology Meckel directly participated in the raising of general pathology and pathologic anatomy to scientific disciplines. Meckel's interceding for C. F. Wolff's theory of epigenesis, not at last by translation of Wolff's paper "De formatione intestinorum" (1768 to 1769) into the German language, accelerated the development of the general and special embryology during the 19th century. In the contemporary medicine the succeeding eponyms are reminding of the imposing German physician and anatomist: the Meckel's diverticulum of ileum (1809), the Meckel's cartilage of the mandibular arch (1820) and the so-called Meckel syndrome (1822).