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.

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.

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.

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.

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.

A modified anti-roll plate as a remedy for the ill-effects of electrical charge during cryosectioning.

This report describes problems commonly encountered in cryosectioning of fragile tissues, such as jamming and scrambling of sections between knife and anti-roll plate. The electrostatic nature of the factors causing these derangements is demonstrated here using simple methods, and several modifications to the anti-roll plate surface are tested for their help in obviating these difficulties. The anti-roll plate material found to be most effective consists of a metal oxide-coated glass which is commercially available at low cost.

Intermediate filaments in Sertoli cells.

Using immunohistochemical techniques both at light and electron microscopic levels, the arrangement and distribution of intermediate filaments in Sertoli cells of normal testis (in rat and human), during pre- and postnatal development (in rabbit, rat, and mouse) and under experimental and pathological conditions (human, rat), have been studied and related to the pertinent literature. Intermediate filaments are centered around the nucleus, where they apparently terminate in the nuclear envelope providing a perinuclear stable core area. From this area they radiate to the plasma membranes; apically often a close association with microtubules is seen. Basally, direct contacts of the filaments with focal adhesions occur, while the relationship to the different junctions of Sertoli cells is only incompletely elucidated. In the rat (not in human) a group of filaments is closely associated with the ectoplasmic specializations surrounding the head of elongating spermatids. Both in rat and human, changes in cell shape during the spermatogenic cycle are associated with a redistribution of intermediate filaments. As inferred from in vitro studies reported in the literature, these changes are at least partly hormone-dependent (vimentin phosphorylation subsequent to FSH stimulation) and influenced by local factors (basal lamina, germ cells). Intermediate filaments, therefore, are suggested to be involved in the hormone-dependent mechanical integration of exogenous and endogenous cell shaping forces. They permit a cycle-dependent compartmentation of the Sertoli cell into a perinuclear stable zone and a peripheral trafficking zone with fluctuating shape. The latter is important with respect to the germ cell-supporting surface of the cell which seems to limit the spermatogenetic potential of the male gonad.

Cytokeratin expression and early lens development.

Immunohistochemical analysis of cytokeratins and vimentin in human, rabbit and rat lens epithelium during development revealed transient coexpression of both types of intermediate filaments. Cytokeratins were still detectable after the closure of the lens vesicle (rat and rabbit embryos 13 days post conception) and in the epithelial cells located at the anterior side of the lens in 7-week-old human embryos. Different monoclonal antibodies against cytokeratin 8 reacted differently in lens cells but not in other embryonic tissues. In addition, early human and rabbit specimens exhibited cytokeratin immunostaining in the neuroectodermal cells of the eye cup as well as in the surrounding mesenchyme, and in the hyaloid artery. Possible explanations for the loss of cytokeratins during the differentiation of ectodermal and neuroectodermal cells are discussed.

Dendrite bundles in lamina II/III of the rabbit neocortex.

The present investigation systematically analyzes the course and arrangement of dendrites in lamina II/III of the visual and the motor cortex of the rabbit on the basis of Klüver-PAS stained 10 micron paraffin sections, 1 micron plastic-embedded semithin sections and ultrathin sections. In both areas the dendritic pattern of lamina II/III is characterized by vertical bundles reminiscent of the pattern in lamina IV/V. The bundles form in the upper half of lamina II/III. They consist mainly of apical dendrites from lamina II/III pyramidal cells and receive branches from dendrite bundles in lamina IV/V, i.e., branches from apical dendrites arising from lamina V pyramidal cells. Besides these features in common, the lamina II/III bundles in the visual cortex on the one hand and in the motor cortex on the other differ with regards to the size and shape of individual bundles as well as to the extent of connections with bundles in lamina IV/V.

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.

Signs of the principle body axes prior to primitive streak formation in the rabbit embryo.

An early common element during anterior-posterior axis formation amongst amniotes is the primitive streak, running longitudinally in the two-layered embryonic disc. In mammals the primordium of this transient structure is the first definite morphological sign of the anterior-posterior axis, while in avian embryos the axis is visible and apparently defined earlier. Here we scrutinize suggestions that in mammals also there are earlier signs of axis formation by using correlative low- and high-resolution light microscopy on tissues from rabbit embryos at 6.3 and 6.5 days post-conception, i.e. immediately before and after primitive streak formation. A series of semithin sections were cut from resin-embedded embryonic discs that had been photographed previously at low power. In embryos at 6.5-days post-conception the primitive streak is as long as up to half the diameter of the embryonic disc, extending anteriorly from a thickening, here called the posterior node, at the posterior margin, which contains the first mesoderm cells ingressing from the epiblast. On both sides of the primitive streak there is a triangular area that appears light in surface views of fixed embryos and correlates with stretches of low-columnar simple epithelium in an otherwise high-columnar pseudostratified epiblast. Within the anterior margin, which has a sharper contour than the rest of the circumference of the embryonic disc, there is a narrow, crescent-shaped dark zone caused by increased cellular height and number in both epiblast and hypoblast. These characteristics of the anterior margin are also found at 6.3 days post-conception, at which stage there is no sign of a primitive streak or a posterior node. The posterior margin, in contrast, is ill-defined in these earlier embryos, or there is a light crescent within the posterior margin, which has the same histological characteristics as the bilateral posterior triangular areas of primitive streak stages. Because the anterior differentiation occurs prior to primitive streak formation and is a sign of both the anterior-posterior and the transverse axes of the embryonic disc, and because some of its histological characteristics are found in primate and human embryos, we propose to name this structure the 'anterior marginal crescent' and to add it to the list of transient structures that gradually establish the principal body axes in mammals. The anterior manifestation of body axes in mammals is thus essentially different from axis development in the avian embryo, where differentiation of these axes is first manifest at the posterior margin.

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.

Expression and organization of muscle specific proteins during the early developmental stages of the rabbit heart.

The expression and intracellular distribution patterns of muscle-specific proteins were studied during rabbit embryo development (7-13 dpc) using monoclonal antibodies against titin, myosin, tropomyosin and actin, as well as the intermediate filament proteins desmin, keratin and vimentin. From our panel, titin appeared to be the first muscle-specific protein to be exclusively expressed in the embryonic rabbit heart. Upon differentiation (myocyte and myotube formation), titin reorganizes from dot-like aggregates into a cross-striated pattern (in 9- to 30-somite embryos) via a transiently filamentous distribution. When the expression and organization of the other muscle proteins was studied in relation to titin, it became apparent that tropomyosin followed upon titin with respect to its exclusive expression in the heart anlagen and its organization into a striated pattern. Myosin and desmin were organized into cross-striated patterns after titin and tropomyosin, but this arrangement had not reached its final form in 13-dpc embryos. Actin, keratin and vimentin were distributed in cytoplasmic filaments in the embryonic stages we investigated. Since the first pulsations are already detected in 3-somite embryos, we conclude that the organization of titin, tropomyosin, myosin and desmin into a striated pattern does not seem to be essential for the initiation of muscle cell contraction in the heart anlagen. Furthermore, this study shows that, in comparison with studies on mouse, chick and rat, the sequence of expression of muscle-specific and intermediate filament proteins during cardiomyogenesis is species-dependent, and that their expression and organization varies in time in different regions of the developing heart.

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.

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.

Correlation between differences in the structure of dendrite bundles and cytoarchitectonic patterns in the cerebral cortex of the rabbit.

The bundling patterns of apical dendrites are investigated in the neocortex of adult rabbits using 10-15 microns paraffin serial sections. In order to extend observations to 12 neocortical areas, four vertical planes of sectioning differing with regard to their angle with the frontal plane are chosen. In alternate sections, stained with either Klüver-PAS or a modified Gomori-method, the shape and structure of dendrite bundles are investigated in relation to the cytoarchitectonic pattern. In all 12 areas, bundling of apical dendrites is found both in lamina IV and II/III. Differences in bundle structure are found to be more pronounced amongst bundles in lamina IV than in lamina II/III. The principal difference between the bundling patterns in the precentral (motor) and occipital (visual) areas known from previous studies is confirmed, i.e. in the precentral areas short and stout bundles with a complex arrangement of apical dendrites are encountered, whereas in the occipital areas long and slender bundles with a simpler alignment of dendrites are found. Bundling patterns in the temporal cortex resemble the ones found in the occipital areas, whereas in the postcentral areas bundles are seen which show characteristics common to bundles of both the precentral and occipital areas.