Arterial Blood Supply of Liver Segment IV and Its Possible Surgical Consequences.

The risk of ischemia of segment IV after split liver resection is high. This anatomical study was done to identify the arterial blood supply and the intrahepatic distribution of liver segment IV. The anatomy of segment IV was studied in 29 livers from adult cadavers. To identify the arterial blood supply of segment IV, water and ink were injected into the various branches of the hepatic artery and the outflow through segment IV and discoloration of the liver parenchyma were observed. In 23 of the 29 livers (79.3%), the arterial perfusion of segment IV was separated by a line drawn from the left side of the inferior vena cava at the top of and lateral to the falciform ligament to the medial point of the gallbladder bed. The area lateral to this line was supplied mainly by the right hepatic artery, and the area medial to it was supplied mainly by the left hepatic artery. In addition to the classification system of Couinaud, we describe here a new division of liver segment IV based on arterial blood supply. These anatomical findings may be useful in defining the resection line for split liver to prevent necrosis of segment IV.

Primordial germ cell development in the marmoset monkey as revealed by pluripotency factor expression: suggestion of a novel model of embryonic germ cell translocation.

Primordial germ cells (PGCs) are the embryonic progenitors of sperm and egg cells. Mammalian PGCs are thought to actively migrate from the yolk sac endoderm over long distances across the embryo to reach the somatic genital ridges. The general principles of mammalian PGC development were discovered in mice. In contrast, little is known about PGC development in primates due to extremely limited access to primate embryos. Here, we analyzed 12 well preserved marmoset monkey (Callithrix jacchus) embryos covering the phase from PGC emergence in the endoderm to the formation of the sexually differentiated gonad (embryonic day (E) 50 to E95). We show using immunohistochemistry that the pluripotency factors OCT4A and NANOG specifically mark PGCs throughout the period studied. In contrast, SALL4 and LIN28 were first expressed ubiquitously and only later down-regulated in somatic tissues. We further show, for the first time, that PGCs are located in the endoderm in E50 embryos in close spatial proximity to the prospective genital ridge, making a long-range migration of PGCs dispensable. At E65, PGCs are already present in the primitive gonad, while significantly later embryonic stages still exhibit PGCs at their original endodermal site, revealing a wide spatio-temporal window of PGC distribution. Our findings challenge the 'dogma' of active long-range PGC migration from the endoderm to the gonads. We therefore favor an alternative model based primarily on passive translocation of PGCs from the mesenchyme that surrounds the gut to the prospective gonad through the intercalar expansion of mesenchymal tissue which contains the PGCs. In summary, we (i) show differential pluripotency factor expression during primate embryo development and (ii) provide a schematic model for embryonic PGC translocation.

The canine hepatic progenitor cell niche: molecular characterisation in health and disease.

Hepatic progenitor cells (HPCs) are an adult stem cell compartment in the liver that contributes to liver regeneration when replication of mature hepatocytes is insufficient. In this study, laser microdissection was used to isolate HPC niches from the livers of healthy dogs and dogs with lobular dissecting hepatitis (LDH), in which HPCs are massively activated. Gene expression of HPC, hepatocyte and biliary markers was determined by quantitative reverse transcriptase PCR. Expression and localisation of selected markers were further studied at the protein level by immunohistochemistry and immunofluorescent double staining in samples of normal liver and liver from dogs with LDH, acute and chronic hepatitis, and extrahepatic cholestasis. Activated HPC niches had higher gene expression of the hepatic progenitor markers OPN, FN14, CD29, CD44, CD133, LIF, LIFR and BMI1 compared to HPCs from normal liver. There was lower expression of albumin, but activated HPC niches were positive for the biliary markers SOX9, HNF1ß and keratin 19 by immunohistochemistry and immunofluorescence. Laminin, activated stellate cells and macrophages are abundant extracellular matrix and cellular components of the canine HPC niche. This study demonstrates that the molecular and cellular characteristics of canine HPCs are similar to rodent and human HPCs, and that canine HPCs are distinctively activated in different types of liver disease.

Subcellular redistribution of the mitochondrial PG2 epitope during development from cleavage to primordial germ cell formation in the rabbit embryo.

Mitochondria are central players in diverse cellular functions and their efficient functioning has dramatic impact on embryonic development. Apparently, proliferation and transmission of well functioning mitochondria to the next generation require ingeniously adapted mechanisms, one of which, the 'mitochondrial bottleneck', is thought to occur early in mammalian development during primordial germ cell (PGC) specification. We used an antibody directed against the mitochondrial PG2 epitope, a reliable marker of primordial and adult female germ cells to monitor mitochondrial differentiation in the early rabbit embryo. Early development shows the PG2 epitope either tightly mitochondria-associated (zygote) or diffusely distributed throughout cytoplasm (cleavage stages). Mitochondrial colocalization of the PG2 epitope is regained in the early blastocyst but expression is then retained by the hypoblast and epiblast only, with the epiblast, although the forerunner of PGCs, showing weak and diffuse labeling only. At gastrulation, hypoblast cells lose PG2 expression but intensive PG2 labeling is found again on all mitochondria in the first PGCs and reveals the number of mitochondria to be in the range of 50 to 100 per PGC at this stage. The results highlight the dynamics of PG2 expression during early development and the usefulness of the epitope for testing the bottleneck theory.

O-GlcNAc expression in developing and ageing mouse brain.

In order to understand whether there is a specific role for the posttranslational N-acetylglucosamine modification linked O-glycosidically (O-GlcNAc) to serine and threonine residues of proteins during development and/or ageing of the brain, we investigated the O-GlcNAc expression of early postnatal cerebellar neurons as well as of mouse brain of different ages. In all cells either in culture or of cryosections mainly the nuclei and nuclear membranes were stained with an O-GlcNAc specific monoclonal antibody. In cerebellar neurons in culture the level of expression could be manipulated by directly interfering with either the biosynthesis of GlcNAc or the removal of O-GlcNAc from proteins confirming the dynamic nature of this protein modification. O-GlcNAc was ubiquitously expressed in mouse brains from embryonic day 10 until late adulthood with some variations in expression strength from cell to cell. In addition, no significant difference in O-GlcNAc expression of subcellular fractions from brains of mice which age at an accelerated rate could be detected compared to normal mice. Taken together these observations support the view that the O-GlcNAc modification has important functional roles for physiological processes of neural cell throughout development, in adulthood and ageing.

Brachyury is expressed in gastrulating bovine embryos well ahead of implantation.

Brachyury is a T-box-containing transcription factor involved in mesoderm formation during vertebrate gastrulation. To analyse whether the regulation of gastrulation varies as much as the timing of gastrulation does with respect to implantation, we isolated a bovine brachyury cDNA fragment. The amino acid sequence shows high similarity to mouse and human Brachyury and clear differences to other T-box genes. Whole-mount in situ hybridisation reveals a normal expression pattern except for a transiently reduced expression in the anterior part of the primitive streak. According to these results, gastrulation in mammals is implemented and regulated irrespective of implantation.

Proteolipid protein 2 mRNA is expressed in the rabbit embryo during gastrulation.

Differential display technology applied to rabbit blastocysts identified an mRNA that encodes a motif similar to that of the proteolipid protein PLP2/A4 of man, mouse and sheep. The open reading frame (456bp) has 88% amino acid identity to human PLP2/A4. The gene is maximally expressed at the beginning of gastrulation: in situ hybridizations exhibited a sickle-shaped area of labelling at the posterior pole of day 7 post-coitum embryos, which appeared at day 6.5 and decreased in size up to day 8. Weaker labelling was found in the extraembryonic mesoderm, in the anterior part of the primitive streak and in the trophoblast. Time and site of gene expression coincide with emerging morphogenetic activities at the posterior pole of the embryo at the beginning of gastrulation.

Hensen's node.

With the 125th anniversary of the original description of Hensen's node in the rabbit coming up and with current research focused on the function of this pivotal embryonic structure, this article proposes a simplified use of terms for the gastrulation organizer in amniote embryos and reemphasizes the achievements of Victor Hensen (1835-1924) in embryology. A partial translation of Hensen's paper (originally published in German) is accompanied by a short historical introduction that concentrates on the framework of embryological research at Hensen's time and on the subsequent reception of his classic paper.

Mitotic arrest of female germ cells during prenatal oogenesis. A colcemid-like, non-apoptotic cell death.

The sequence of events and a possible reason for germ cell death during oogenesis in the prenatal ovary were studied in rat and mouse embryos. ED 14-22 rat and ED 14-16 mouse embryos were studied using semithin sections for light microscopy and serial ultrathin sections for electron microscopy. In addition, the rat material was 3H-thymidine labelled for historadioautography and cytospin preparations of freshly obtained gonads were immunohistochemically analysed. During the transition from the proliferating oogonial stage to the meiotic prophase about 16% of the postmitotic oocytes do not pass the initial meiotic checkpoint on ED 18/19 in the rat (ED 15/16 in the mouse). These germ cells first show structural signs of mitosis; the diploid number of 'super-condensed' chromosomes are globally formed and are concentrated in the center of the cell. Although the germ cells show all morphological signs of living cells they never have mitotic spindles; the micro-tubulus-organisation-centres (MTOCs) are found peripherally and become concentrated, forming a single centrosomal body (acentriolar MTOC) as detected by immunohistochemistry for the centrosomal protein MPM2 and gamma-tubulin. EM studies show 25 nm tubule-like profiles within the MTOC bodies. The centrioles frequently lie separate from the MTOC material or are not present at all; the germ cells are apparently arrested in a prophase- or metaphase-like stage when they have reached the postmitotic G2/preleptotenal transition and are unable to enter meiosis. Forty-eight to 72 h after the first mitotically arrested germ cells are found, degeneration is seen in these germ cells. This second event, the germ cell death proper, shows neither criteria of apoptosis (cell shrinkage, marginal condensation of chromatin, DNA fragmentation) nor signs of necrosis (cell swelling, pycnosis, inflammation). Both arrested pro- and metaphase-like stages are found with signs of cell death and phagocytosis. The morphological signs of phagocytosis are found in neighbouring pregranulosa cells. The final heterocytotic bodies contain the remnants of the centrosomal (MTOC) material and DAPI-positive DNA material. The pregranulosa cells are mitotically silent during the phase when mitotic arrest and germ cell degeneration is found. The results suggest the presence of a hypothetical 'anti-spindle' factor, which under normal conditions is necessary for induction of meiotic prophase. The structural events of 'arrested mitosis' is reminiscent of those induced by the antimitotic, tubule-degrading drug colcemid. This type of arrest may inhibit meiosis of more than 33% prenatal germ cells and induce their cell death.

Anterior neural induction by nodes from rabbits and mice.

The organizer of vertebrate embryos represents the major regulatory center for the formation of the embryonic axis during gastrulation. The early blastopore lip of amphibia and Hensen's node of the chick at the full-length primitive streak stage possess both a head- and a trunk-inducing potential. In mice, a head-inducing activity was identified in the extraembryonic, anterior visceral endoderm (AVE) by tissue ablation and genetic experiments. Evidence for a similar activity in the AVE from the rabbit was obtained by transplanting below the avian epiblast. However, it was still unclear whether the AVE is the exclusive origin of anterior neural induction or if this activity is recapitulated by the node and/or its derivatives. We report here that nodes from both rabbit and mouse embryos can induce a complete neural axis including forebrain structures upon grafting to chick hosts. Thus, in rabbits and mice not only the AVE, but also the node, possesses a potential for the induction of anterior neural tissue.

The term cell epitope PG-2 is expressed in primordial germ cells and in hypoblast cells of the gastrulating rabbit embryo.

Rapid progress in the functional analysis of germline segregation has been made recently using the mouse as an experimental and molecular model. However, comparative vertebrate embryology suggests that the time point and mode of germline segregation may vary between mammalian species to a greater extent than hitherto suspected. Therefore, we started to make use of the monoclonal antibody PG-2 specific for primordial germ cells (PGCs) of the rabbit as an opportunity to investigate the early phases of germ cell formation in a mammalian species other than the mouse. Using immunohistochemistry on whole mount preparations and frozen sections we describe the typical mitochondrial labelling of PGCs in the posterior part of the primitive streak at 7.0 days post conception (d.p.c.) and the subsequent distribution of labelled PGCs at early somite stages (8 d.p.c.) within a bilobed area that flanks the posterior margin of the embryo. At these later stages, PGCs were found close to, and within, the yolk sac epithelium but they were still within the confines of the embryo as defined by the peripheral margin in the epiblast/ectoderm layer. Interestingly, cells expressing the PG-2 epitope in an atypical, finely granulated intracellular pattern were found in the hypoblast layer, but not in the epiblast, at the primitive streak stage. This atypical expression pattern may be interpreted as a sign of cells gradually losing the PG-2 epitope and this, in turn, may indicate that PGC progenitors are allocated to the hypoblast layer before appearing in the mesoderm compartment of the primitive streak. These results raise the question as to whether the germline in the rabbit is separated during early blastocyst stages, i.e. rather earlier than in the mouse.

The anterior margin of the mammalian gastrula: comparative and phylogenetic aspects of its role in axis formation and head induction.

Recent findings on morphology and gene expression in several mammalian embryos suggest that there is a new landmark and possibly a center with organizer activity in the anterior margin of the embryo at the onset of gastrulation. This review compiles morphological variations and similarities found among mammals during gastrulation stages and, at the same time, stresses the common aspects, at the morphological and the molecular level, of setting up the body plan with regard to axis formation and head induction. Both morphological and functional aspects are then used to draw comparisons with equivalent developmental stages in lower vertebrate species, such as birds, amphibia, and bony fish. Finally, a suggestion is made as to how gastrulation may have evolved in the vertebrate phylum.

The homeobox gene Pitx2: mediator of asymmetric left-right signaling in vertebrate heart and gut looping.

Left-right asymmetry in vertebrates is controlled by activities emanating from the left lateral plate. How these signals get transmitted to the forming organs is not known. A candidate mediator in mouse, frog and zebrafish embryos is the homeobox gene Pitx2. It is asymmetrically expressed in the left lateral plate mesoderm, tubular heart and early gut tube. Localized Pitx2 expression continues when these organs undergo asymmetric looping morphogenesis. Ectopic expression of Xnr1 in the right lateral plate induces Pitx2 transcription in Xenopus. Misexpression of Pitx2 affects situs and morphology of organs. These experiments suggest a role for Pitx2 in promoting looping of the linear heart and gut.

Head induction in the chick by primitive endoderm of mammalian, but not avian origin.

Different types of endoderm, including primitive, definitive and mesendoderm, play a role in the induction and patterning of the vertebrate head. We have studied the formation of the anterior neural plate in chick embryos using the homeobox gene GANF as a marker. GANF is first expressed after mesendoderm ingression from Hensen's node. We found that, after transplantation, neither the avian hypoblast nor the anterior definitive endoderm is capable of GANF induction, whereas the mesendoderm (young head process, prechordal plate) exhibits a strong inductive potential. GANF induction cannot be separated from the formation of a proper neural plate, which requires an intact lower layer and the presence of the prechordal mesendoderm. It is inhibited by BMP4 and promoted by the presence of the BMP antagonist Noggin. In order to investigate the inductive potential of the mammalian visceral endoderm, we used rabbit embryos which, in contrast to mouse embryos, allow the morphological recognition of the prospective anterior pole in the living, pre-primitive-streak embryo. The anterior visceral endoderm from such rabbit embryos induced neuralization and independent, ectopic GANF expression domains in the area pellucida or the area opaca of chick hosts. Thus, the signals for head induction reside in the anterior visceral endoderm of mammals whereas, in birds and amphibia, they reside in the prechordal mesendoderm, indicating a heterochronic shift of the head inductive capacity during the evolution of mammalia.

Expression patterns of gap junctional proteins connexin 32 and 43 suggest new communication compartments in the gastrulating rabbit embryo.

A central problem in embryological research is the identification of mechanisms by which control over the development of a viable individual is maintained. An important role in this process is attributed to intercellular communication the preconditions of which were examined in the present study. Using a range of monoclonal antibodies, the expression patterns of the gap junctional proteins connexin 32 (Cx32) and connexin 43 (Cx43) were examined in whole-mount preparations and cryosections of gastrulating rabbit embryos between 6.0 and 7.5 days post conception. Distinct distribution patterns for Cx32 and Cx43, respectively, were found: Cx32 was exclusively expressed in the hypoblast and yolk sac epithelium (the lower layer of the embryo) whereas Cx43-expression was limited to the epiblast (in the upper layer) and its derivatives. Moreover, the dynamics of the Cx32 and Cx43 expression patterns indicate the existence of smaller tissue compartments within the three embryonic cell layers present at the beginning of gastrulation (epiblast, mesoderm and hypoblast). The most striking one of these smaller compartments is a belt-like area within the lower layer which straddles the epiblast-trophoblast border seen in the upper layer of the embryonic disc. The significance of these compartments for initiating and maintaining the gastrulation process is discussed.

Neurulation in the rabbit embryo.

Among a broad range of factors and mechanisms involved in the complex process of neurulation a relationship between the curvature of the craniocaudal body axis and rate of neural tube closure has been proposed, but more examples and models are needed to further substantiate the existence of this relationship. This is particularly true for mammals, where marked differences in embryonic body curvature between species exist. The rabbit embryo has virtually no curvature during the main phase of neurulation and is therefore a suitable model, but neurulation is hardly documented in this species. In the present study, therefore, neural tube closure in the rabbit embryo is presented in detail by morphological and morphometrical parameters, as well as from scanning electron microscopic investigations. At the stages of 6-8 somites, the flat neural plate transforms into a V-shaped neural groove, beginning at the rhombo-cervical level. Between the stages of 8 and 9 somites, multiple closure sites occur simultaneously at three levels: at the incipient pros-mesencephalic transition, at the incipient mes-rhombencephalic transition, and at the level of the first pairs of somites. This results in four transient neuropores. The anterior and rhombencephalic neuropores close between the stages of 9-11 somites. The mesencephalic neuropore is very briefly present. The posterior neuropore is the largest and remains longest. Its tapered (cranial) portion closes fast within somite stages 9-10. Subsequently its wide (caudal) portion closes up to a narrow slit, but further closure slows down till full closure is achieved at the 22-somite stage. In comparing rabbit neurulation with that of chick and mouse, the sequence of multiple site closure resembles that of the mouse embryo, but other important aspects of neurulation resemble those of the chick embryo. In contrast to mouse and chick, no time lag between closure at the three closure sites in the rabbit was seen.

Primordial germ cells of the rabbit are specifically recognized by a monoclonal antibody labelling the perimitochondrial cytoplasm.

Instrumental for studies investigating the development of germ cells, and especially the separation of the germline in the early embryo, are molecular markers which reliably label germ cells and with which regulative factors of germ cell development may be analyzed. Here, we describe the monoclonal antibody PG-2, which is highly specific for the germ cells of the rabbit embryo and labels the perimitochondrial cytoplasm, as demonstrated by immunogold-silver staining. Identical expression patterns are found in germ cells of either sex from early organogenesis at 10 days post-conception (d.p.c.), when the germ cells leave the hindgut epithelium and settle in the gonadal anlage as primordial germ cells (PGCs), until the time immediately prior to birth (30 d.p.c.), when germ cells are either in their oogonial or prospermatogonial state. The antibody is the first to recognize specifically a cytoplasmic epitope in germ cells of a higher vertebrate and may well recognize the mammalian equivalent of the germ plasm found in invertebrates and lower vertebrates. The antibody can be used for early identification of PGCs and may be of help in the elucidation of mammalian germ cell development towards the gonial stages of spermatogenesis and oogenesis.

Cytoskeleton gradients in three dimensions during neurulation in the rabbit.

Morphogenetic movements leading to the formation of the neural tube and cellular differentiation leading to neuronal and glial cell lineages are both part of early development of the vertebrate nervous system. In order to analyze the degree of overlap between these processes, cellular differentiation during the shaping of the neural plate is investigated immunohistochemically by using monoclonal intermediate filament protein antibodies and the 7.5-8.0-day-old rabbit embryo as a model. Western blotting is used to confirm the specificity of the antibodies, which include a new monoclonal vimentin antibody suitable for double-labeling in combination with monoclonal cytokeratin (and fibronectin) antibodies. Starting in the early somite embryo and concomitant with neural plate folding, a gradual loss of cytokeratin 8 (and 18) expression in the neuroepithelium is mirrored by a gain in vimentin expression with partial coexpression of both proteins. At the prospective rhombencephalic and spino-caudal levels, vimentin expression, in particular, changes (i.e., increases) along gradients in three dimensions: along the longitudinal axis of each neuroepithelial cell from basal to apical, in the transverse plane of the embryo from dorsolateral to ventromedial and along the craniocaudal axis from prospective rhombencephalic toward spino-caudal levels of the neural plate. At the prospective mes- and prosencephalic levels, the expression change also proceeds from basal to apical within each neuroepithelial cell, but along the other axes described here, the progress in expression change is more complex. Although the functional meaning of these highly ordered expression changes is at present unclear, the gradients suggest a novel pattern of neuroepithelial differentiation which may be functionally related to the process of interkinetic nuclear migration (Sauer [1935] J. Comp. Neurol. 62:377-402) and which partially coincides with the morphogenetic movements involved in the shaping of the neural plate.

Rearrangement of intercellular junctions and cytoskeletal proteins during rabbit myocardium development.

A direct and close association between desmosomes and intermediate-sized filaments of the keratin type exists in embryonic and in adult epithelial tissues. Cardiomyocytes are interconnected by spot-desmosomes, which are found in the intercalated disks and can be immunocytochemically detected by antibodies to desmoplakins. In this study, at the light microscopical level, we describe an interaction of keratin filaments with desmoplakins during rabbit myocardiogenesis. In the early stages (0-1 somites), desmoplakins are more abundant in the heart anlagen than in the adjacent intra- and extraembryonic mesoderm. During development of the myocardium, desmoplakin expression gradually rearranges from an apicolateral into an intercalated disk localization in later states. Keratin expression in the developing myocardium of the rabbit heart decreases with the age of the embryo. Keratin filaments are gradually lost via dot-like aggregates which colocalize with desmoplakin-positive clusters. Our results suggest a role for keratins in the developmental rearrangement of desmoplakins into the intercalated disks. A direct relation of desmin and titin reorganization to desmoplakin rearrangement, which was examined because of the dominant role of these proteins in cardiogenesis, was not found.