Targeted ablation and reorganization of the principal preplate neurons and their neuroblasts identified by golli promoter transgene expression in the neocortex of mice

The present study delineates the cellular responses of dorsal pallium to targeted genetic ablation of the principal preplate neurons of the neocortex. Ganciclovir treatment during prenatal development (E11–E13; where E is embryonic day) of mice selectively killed cells with shared S-phase vulnerability and targeted expression of a GPT [golli promoter transgene, linked to HSV-TK (herpes simplex virus-thymidine kinase), τ-eGFP (τ-enhanced green fluorescent protein) and lacZ (lacZ galactosidase) reporters] localized in preplate neurons. Morphogenetic fates of attacked neurons and neuroblasts, and their successors, were assessed by multiple labelling in time-series comparisons between ablated (HSV-TK+/0) and control (HSV-TK0/0) littermates. During ablation generation, neocortical growth was suppressed, and compensatory reorganization of non-GPT ventricular zone progenitors of dorsal pallium produced replacements for killed GPT neuroblasts. Replacement and surviving GPT neuroblasts then produced replacements for killed GPT neurons. Near-normal restoration of their complement delayed the settlement of GPT neurons into the reconstituted preplate, which curtailed the outgrowth of pioneer corticofugal axons. Based on this evidence, we conclude that specific cell killing in ablated mice can eliminate a major fraction of GPT neurons, with insignificant bystander killing. Also, replacement GPT neurons in ablated mice originate exclusively by proliferation from intermediate progenitor GPT neuroblasts, whose complement is maintained by non-GPT progenitors for inductive regulation of the total complement of GPT neurons. Finally, GPT neurons in both normal and ablated mice meet all morphogenetic criteria, including the ‘outside-in’ vertical gradient of settlement, presently used to identify principal preplate neurons. In ablated mice, delayed organization of these neurons desynchronizes and isolates developing neocortex from the rest of the brain, and permanently impairs its connectivity

Transmission of Mitochondrial DNA Diseases and Ways to Prevent Them

Recent reports of strong selection of mitochondrial DNA (mtDNA) during transmission in animal models of mtDNA disease, and of nuclear transfer in both animal models and humans, have important scientific implications. These are directly applicable to the genetic management of mtDNA disease. The risk that a mitochondrial disorder will be transmitted is difficult to estimate due to heteroplasmy—the existence of normal and mutant mtDNA in the same individual, tissue, or cell. In addition, the mtDNA bottleneck during oogenesis frequently results in dramatic and unpredictable inter-generational fluctuations in the proportions of mutant and wild-type mtDNA. Pre-implantation genetic diagnosis (PGD) for mtDNA disease enables embryos produced by in vitro fertilization (IVF) to be screened for mtDNA mutations. Embryos determined to be at low risk (i.e., those having low mutant mtDNA load) can be preferentially transferred to the uterus with the aim of initiating unaffected pregnancies. New evidence that some types of deleterious mtDNA mutations are eliminated within a few generations suggests that women undergoing PGD have a reasonable chance of generating embryos with a lower mutant load than their own. While nuclear transfer may become an alternative approach in future, there might be more difficulties, ethical as well as technical. This Review outlines the implications of recent advances for genetic management of these potentially devastating disorders

Subtypes of active cell death in the granulosa of ovarian atretic follicles in the quail (Coturnix coturnix japonica)

Follicular atresia in the ovaries of Japanese quail was studied by cytochemistry and electron microscopy. Three different types of cell death coexisted in the granulosa. A large number of cells showed signs of apoptosis. The DNA fragmentation in these cells was demonstrated in a previous study using in situ end-labeling. A second and non-negligible type of cell death consisted of extensive autophagocytosis of the cytoplasm occurring simultaneously with late nuclear alterations. Finally, a few detached cells displayed cytoplasmic disintegration and small irregular clumps of chromatin condensation indicative of primary cell necrosis. Apoptotic versus autophagic cell death revealed a different pattern of acid phosphatase activity (lysosomal versus cytoplasmic). We propose that these observations may be linked to the existence of distinct subpopulations in the granulosa as has been shown by others. This study confirms the biochemical data on granulosa cell death, but demonstrates that apoptosis is not the exclusive mode of active cell death in follicular atresia

Astrocyte-endothelial cell calcium signals conveyed by two signalling pathways

Astrocytes and endothelial cells are in close contact with each other at the blood-brain barrier, where important molecular transports take place. Despite these key morphological and functional properties, little is known regarding the dynamic signalling processes that occur between these two cell types. We investigated astrocyte-endothelial cell calcium signalling mechanisms in a coculture model prepared from primary rat cortical astrocytes and ECV304 cells. We used flash photolysis of caged inositol-trisphosphate (IP3) and gentle mechanical stimulation to trigger astrocyte-endothelial cell calcium signals and to investigate the underlying propagation mechanisms. Photolytically releasing IP3 in a single cell triggered increases in cytoplasmic calcium concentration that propagated between astrocytes and endothelial cells in either direction. These propagating calcium signals did not cross cell-free zones and were not affected by fast superfusion or by the purinergic inhibitors apyrase and suramin, indicating that they are communicated through an intracellular pathway in conjunction with gap junctions. Electrophysiological experiments confirmed a tow degree of astrocyte-endothelial cell electrical cell-to-cell coupling, Mechanical stimulation of a single cell also triggered astrocyte-endothelial cell calcium signals but, in contrast to the former triggering mode, these signals crossed cell-free zones and were significantly inhibited by apyrase, thus indicating the involvement of an extracellular and purinergic messenger. Astrocyte-endothelial cell calcium signalling also occurred in cocultures prepared with astrocytes and primary rat brain capillary endothelial cells. We conclude that astrocytes and endothelial cells can exchange fast-acting calcium signals (time scale of seconds) that can be communicated through an intracellular/gap junctional pathway and an extracellular purinergic pathway

Post-mortem Reperfusion of a Pig: a First Step to a New Surgical Training Model?

The purpose of this experimental study was to establish a short-term post-mortem circulation in a pig model using liquid paraffin. This study also investigated the quality of vascular perfusion in the peripheral tissues. This is the first step in the development of a new revascularized human surgical training model. This first experience was performed on the hind leg of a pig. Initial cannulation of the external iliac artery and vein was followed by connection of the arterial inflow to a heart-lung machine and using the venous outflow to flush post-mortem clots and blood. Subsequently, after connecting the venous outflow to the heart-lung machine, circulation was initiated. Circulation was established during 27 min, during which the flow was constantly 130 mL/min. A steady increase in inlet pressure was observed during the experiment, which finally reached a minimum value of 124 mmHg. Perfusion was interrupted early due to an uncontrollable fluid leak. Afterwards, the distal hind leg was incised showing an equal distribution of paraffin. A short-term revascularization was successfully re-established under excellent conditions. Although the results are promising, further experiments are necessary to eventually perform a wide range of surgical procedures on revascularized human cadavers

Homology between mitochondriogenesis in the avian and amphibian oocyte

Cytochrome oxidase cytochemistry was used to unequivocally identify the spread of mitochondria during oogenesis in the adult Japanese quail. This enabled us to compare their distribution with the distribution in the Xenopus laevis oocyte (Tourte ef al. 1984). In the quail the paranuclear mitochondrial cloud initially disperses homogeneously but afterwards segregates into 2 populations: (i) a population localized in the basophilic cortical layer (surrounding the vegetal pole); and (ii) clusters of mitochondria distributed geometrically around the germinal vesicle in the animal pole. The mitochondria in these clusters have a high cytochrome oxidase activity, which reflects their functionality. This perinuclear crown of mitochondrial clusters actively replicates mtDNA in both animal species and builds up most of the stock of the mitochondria in the full-grown oocyte. Our study suggests that the perinuclear group of mitochondria will segregate in the somatic cells of the future embryo, whilst the original subcortical group will become localized in the primordial germ cells