Biomedical subjects

R S Nowakowski

Publications and source records attributed to R S Nowakowski.

At least 37 records · Page 2Linked to original sources

PubMed1999

Cyclin E-p27 opposition and regulation of the G1 phase of the cell cycle in the murine neocortical PVE: a quantitative analysis of mRNA in situ hybridization.

We have analyzed the expression patterns of mRNAs of five cell cycle related proteins in the ventricular zone of the neocortical cerebral wall over the course of the neuronogenetic interval in the mouse. One set of mRNAs (cyclin E and p21) are initially expressed at high levels but expression then falls to a low asymptote. A second set (p27, cyclin B and cdk2) are initially expressed at low levels but ascend to peak levels only to decline again. These patterns divide the overall neuronogenetic interval into three phases. In phase 1 cyclin E and p21 levels of mRNA expression are high, while those of mRNAs of p27, cdk2 and cyclin B are low. In this phase the fraction of cells leaving the cycle after each mitosis, Q, is low and the duration of the G1 phase, TG1, is short. In phase 2 levels of expression of cyclin E and p21 fall to asymptote while levels of expression of mRNA of the other three proteins reach their peaks. Q increases to approach 0.5 and TG1 increases even more rapidly to approach its maximum length. In phase 3 levels of expression of cyclin E and p21 mRNAs remain low and those of the mRNAs of the other three proteins fall. TG1 becomes maximum and Q rapidly increases to 1.0. The character of these phases can be understood in part as consequences of the reciprocal regulatory influence of p27 and cyclin E and of the rate limiting functions of p27 at the restriction point and of cyclin E at the G1 to S transition.

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PubMed1998

Cerebellar microfolia and other abnormalities of neuronal growth, migration, and lamination in the Pit1dw-J homozygote mutant mouse.

The Snell dwarf mouse (Pit1dw-J homozygote) has a mutation in the Pit1 gene that prevents the normal formation of the anterior pituitary. In neonates and adults there is almost complete absence of growth hormone (GH), prolactin (PRL), thyroxin (T4), and thyroid-stimulating hormone (TSH). Since these hormones have been suggested to play a role in normal development of the central nervous system (CNS), we have investigated the effects of the Pit1dw-J mutation on the cerebellum and hippocampal formation. In the cerebellum, there were abnormalities of both foliation and lamination. The major foliation anomalies were 1) changes in the relative size of specific folia and also the proportional sizes of the anterior vs posterior cerebellum; and 2) the presence of between one and three microfolia per half cerebellum. The microfolia were all in the medial portion of the hemisphere in the caudal part of the cerebellum. Each microfolium was just rostral to a normal fissure and interposed between the fissure and a normal gyrus. Lamination abnormalities included an increase in the number of single ectopic granule cells in the molecular layer in both cerebellar vermis (86%) and hemisphere (40%) in comparison with the wild-type mouse. In the hippocampus of the Pit1dw-J homozygote mouse, the number of pyramidal cells was decreased, although the width of the pyramidal cell layer throughout areas CA1-CA3 appeared to be normal, but less densely populated than in the wild-type mouse. Moreover, the number of granule cells that form the granule cell layer was decreased from the wild-type mouse and some ectopic granule cells (occurring both as single cells and as small clusters) were observed in the innermost portion of the molecular layer. The abnormalities observed in the Pit1dw-J homozygote mouse seem to be caused by both direct and indirect effects of the deficiency of TSH (or T4), PRL, or GH rather than by a direct effect of the deletion of Pit1.

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PubMed1998

Postembryonic neurogenesis in the procerebrum of the terrestrial snail, Helix lucorum L.

Neuronogenesis during posthatching development of the procerebrum of the terrestrial snail Helix lucorum was analyzed using bromodeoxyuridine immunohistochemistry to label proliferating cells. Comparison of the distribution of labeled cells in a series of animals which differed in age at the time of incubation with bromodeoxyuridine, in survival time after incubation, and in age at sacrifice reveals a clear pattern and developmental sequence in neuron origin. First, the proliferating cells are located only at the apical portion of the procerebrum. Second, cells which are produced at any particular age remain, for the most part, confined to a single layer in the procerebrum. Third, as development proceeds, each layer of previously produced neurons is displaced toward the basal part of the procerebrum by the production of additional neurons. Our results suggest that the vast majority of the neurons (probably about 70-80%) of the snail procerebrum are produced during the first 1-2 months of posthatching development.

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PubMed1997

Patterns of connectivity in a Drosophila nerve.

We investigated the spatial patterns of synaptic profiles in en passant synapses between the premotor axon of a peripherally synapsing interneuron (PAPSI) and a set of individually identifiable motoneuron axons in Drosophila melanogaster. These synaptic profiles are distributed as the axons travel parallel to each other in a bundle; the synapses begin as the axons leave the thoracic ganglion and continue peripherally for 45-65 microm. We found that the number of synaptic profiles per micron length of the motoneuron axons was greatest close to the ganglion; the cumulative distribution of profiles could be fitted to curves of the form f(x) = alpha(1 - e(-beta x)), where x = the distance from the thoracic ganglion, and alpha and beta are constants. The distribution of synaptic profiles was also examined in a mutant strain, Passover (Pas), known to affect connectivity in a pathway that includes the PAPSI. The synaptic profiles between the PAPSI and the motoneuron axons appeared ultrastructurally unremarkable in Pas. Also, the total number of synaptic profiles between the PAPSI and the motoneuron axons did not differ between Pas and wild type flies. However, the distribution of synaptic profiles among the individual motoneuron axons did differ significantly from wild type flies, as did the area of contiguity between the motoneuron axons and the PAPSI, which was much greater in Pas than in wild type flies.

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PubMed1997

Local homogeneity of cell cycle length in developing mouse cortex.

We have measured the amount of variation in the length of the cell cycle for cells in the pseudostratified ventricular epithelium (PVE) of the developing cortex of mice on embryonic day 14. Our measurements were made in three cortical regions (i.e., the neocortex, archicortex, and periarchicortex) using three different methods: the cumulative labeling method (CLM), the percent labeled mitoses (PLM) method, and a comparison of the time needed for the PLM to ascend from 0 to 100% with the time needed for the PLM to descend from 100 to 0%. These 3 different techniques provide different perspectives on the cytokinetic parameters. Theoretically, CLM gives an estimate for a maximum value of the total length of the cell cycle (TC), whereas PLM gives an estimate of a minimum value of TC. The difference between these two estimates indicates that the range for TC is +/-1% of the mean TC for periarchicortex, +/-7% for neocortex, and +/-8% for archicortex. This was confirmed by a lengthening of the PLM descent time in comparison with its ascent time. The sharpness of the transitions and the flatness of the plateau of the PLM curves indicate that 99% of the proliferating cells are within this narrow estimated range for TC; hence, only approximately 1% deviate outside of a relatively restricted range from the average TC of the population. In the context of the possible existence within the cortical PVE of two populations with markedly dissimilar cell cycle kinetics from the mean, one such population must comprise approximately 99% of the total population, and the other, if it exists, is only approximately 1% of the total. This seems to be true for all three cortical regions. The narrow range of TC indicates a homogeneity in the cell cycle length for proliferating cells in three different cortical regions, despite the fact that progenitor cells of different lineages may be present. It further predicts the existence of almost synchronous interkinetic nuclear movements of the proliferating cells in the ventricular zone during early development of the cerebral cortex.

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PubMed1997

Synchrony of clonal cell proliferation and contiguity of clonally related cells: production of mosaicism in the ventricular zone of developing mouse neocortex.

We have analyzed clonal cell proliferation in the ventricular zone (VZ) of the early developing mouse neocortex with a replication-incompetent retrovirus encoding human placental alkaline phosphatase (AP). The retrovirus was injected into the lateral ventricles on embryonic day 11 (E11), i.e., at the onset of neuronogenesis. Three days postinjection, on E14, a total of 259 AP-labeled clones of various sizes were found in 7 fetal brains. There are approximately 7 cell cycles between E11 and E14 (), and there is a 1-2 cell cycle delay between retroviral injection and the production of a retrovirally labeled "founder" cell; thus, we estimate that the "age" of the clones was about 5-6 cell cycles. Almost one-half of the clones (48.3%) identified were pure proliferating clones containing cells only in the VZ. Another 18.5% contained both proliferating and postproliferative cells, and 33.2% contained only postproliferative cells. It was striking that over 90% of the clonally related proliferating cells occurred in clusters of two or more apparently contiguous cells, and about 73% of the proliferating cells occurred in clusters of three or more cells. Regardless of the number of cells in the clone, these clusters were tightly packed and confined to a single level of the VZ. This clustering of proliferating cells indicates that clonally related cells maintain neighbor-neighbor relationships as they undergo interkinetic nuclear migration and progress through several cell cycles, and, as a result, the ventricular zone is a mosaic of small clusters of clonally related and synchronously cycling cells. In addition, cells in the intermediate zone and the cortical plate were also frequently clustered, indicating that they became postproliferative at a similar time and that the output of the VZ is influenced by its mosaic structure.

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PubMed1997

The mathematics of neocortical neuronogenesis.

Neocortical neuronogenesis occurs in the pseudostratified ventricular epithelium (PVE) which forms the margin of the ventricular system of the embryonic cerebral wall. We have determined that in mouse the neuronogenetic interval continues 6 days and is divisible into 11 integer cycles. The fraction of daughter cells which leaves the cycle (Q) following a curvilinear path from 0 to 1.0 over the neuronogenetic interval. Q reaches 0.5 in the course of cell cycle 8 at which point the number of daughter cells which leaves the PVE is equal to the number that remains to renew the proliferative process. Over the course of the neuronogenetic interval in mouse, the founder population is amplified 140 fold. If, theoretically, the operation of the process were altered so that the total number of integer cycles were kept constant but that Q varied so that it reached 0.5 in the course of cell cycles 7, 9 and 10, the total neuronal production from the same founder population would be 60%, 160% and 550%, respectively, that observed in mouse. If, on the other hand, the number of integer cycles was varied but the relative rate of progression of Q with respect to the neuronogenetic interval was kept the same as in mouse, the total neuronal production would be 50% with 9 cycles, 2 fold with 13 cycles and 60 fold with 22 cycles with respect to the 140 fold amplification of the founder population seen in mouse. These large scale amplifications in neuronal output from the PVE illustrate the predominant effect of the number of integer cycles, but also the substantial effect of variations in Q as potential control parameters in the regulation of neuronogenesis.

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PubMed1996

The leaving or Q fraction of the murine cerebral proliferative epithelium: a general model of neocortical neuronogenesis.

Neurons of neocortical layers II-VI in the dorsomedial cortex of the mouse arise in the pseudostratified ventricular epithelium (PVE) through 11 cell cycles over the six embryonic days 11-17 (E11-E17). The present experiments measure the proportion of daughter cells that leave the cycle (quiescent or Q fraction or Q) during a single cell cycle and the complementary proportion that continues to proliferate (proliferative or P fraction or P; P = 1 - Q). Q and P for the PVE become 0.5 in the course of the eighth cycle, occurring on E14, and Q rises to approximately 0.8 (and P falls to approximately 0.2) in the course of the 10th cycle occurring on E16. This indicates that early in neuronogenesis, neurons are produced relatively slowly and the PVE expands rapidly but that the reverse happens in the final phase of neuronogenesis. The present analysis completes a cycle of analyses that have determined the four fundamental parameters of cell proliferation: growth fraction, lengths of cell cycle, and phases Q and P. These parameters are the basis of a coherent neuronogenetic model that characterizes patterns of growth of the PVE and mathematically relates the size of the initial proliferative population to the neuronal population of the adult neocortex.

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PubMed1996

Interkinetic and migratory behavior of a cohort of neocortical neurons arising in the early embryonic murine cerebral wall.

Neocortical neuronogenesis occurs in the pseudostratified ventricular epithelium (PVE) where nuclei of proliferative cells undergo interkinetic nuclear movement. A fraction of daughter cells exits the cell cycle as neurons (the quiescent, or Q, fraction), whereas a complementary fraction remains in the cell cycle (the proliferative, or P, fraction). By means of sequential thymidine and bromodeoxyuridine injections in mouse on embryonic day 14, we have monitored the proliferative and post-mitotic migratory behaviors of 1 and 2 hr cohorts of PVE cells defined by the injection protocols. Soon after mitosis, the Q fraction partitions into a rapidly exiting (up to 50 microns/hr) subpopulation (Qr) and a more slowly exiting (6 microns/hr) subpopulation (Qs). Qr and Qs are separated as two distributions on exit from the ventricular zone with an interpeak distance of approximately 40 microns. Cells in Qr and Qs migrate through the intermediate zone with no significant change in the interpeak distance, suggesting that they migrate at approximately the same velocities. The rate of migration increases with ascent through the intermediate zone (average 2-6.4 microns/hr) slowing only transiently on entry into the developing cortex. Within the cortex, Qr and Qs merge to form a single distribution most concentrated over layer V.

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PubMed1996

The abnormal distribution of mossy fiber bundles and morphological abnormalities in hippocampal formation of dreher(J) (dr(J)/dr(J))mouse.

The organization of pyramidal cells and mossy fibers in the hippocampal formation of homozygous dreher(J) mutant mice was investigated using Timm's and Golgi methods. Five clear abnormalities were found: (1) some pyramidal cells were located below the infrapyramidal mossy fiber layer, (2) mossy fibers emerged in diffuse fashion from between the suprapyramidal and infrapyramidal mossy fiber layers, and their fibers invaded within the pyramidal cell layer, where they traveled as 3-6 small, usually quite short, bundles, (3) some normally situated pyramidal cells had unusual contacts with mossy fibers at two or three places on their apical and/or basal dendrites, (4) some normally situated pyramidal cells had abnormal dendritic trees typified by the occurrence of fine-caliber dendritic branches extending out of the apical dendrite or the apical portion of the soma, and (5) a few Timm positive fibers extending from the dentate hilus to the dentate molecular layer in both dreher(J) and control mice were observed. These abnormalities indicate that in the hippocampal formation a variety of cell populations and neuronal circuits can be indirectly modified by the dreher mutation.

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PubMed1996

Regulation of normal proliferation in the developing cerebrum potential actions of trophic factors.

We review here a computational model of neocortical histogenesis based upon experiments in the developing cerebral wall of the mouse. Though based upon experiments in mouse, commonalities of developmental history and structure of neocortex across mammalian species suggest that the principles which support this model will be generally applicable to neocortical evolution and development across species. In its scope the model spans the successive histogenetic events: cell proliferation, cell migration, and the positioning of cell somata in neocortical layers following migration. Neurons are produced in a pseudostratified epithelium (PVE) which lines the ventricular cavaties of the embryonic cerebrum. The parameters which determine the rate and total number of neurons produced in the PVE are (1) the size of the founder population, (2) the number of integer cell cycles executed by the founder population and its progeny in the course of the neuronogenetic interval, (3) the growth fraction, and (4) the fraction of cells which exits the cycle (Q fraction) with each integer cycle. There is a systematic relationship between the integer cycle of origin and the sequence of cell migration, position in the cortex, and the extent to which a set of postmigratory neurons will be diluted in the cortex by the combined effects of tissue growth and cell death. Variation across species in the number of integer cell cycles as a function of the rate of progression of Q may be expected to modulate profoundly the total numbers of neurons that are produced but not the relative proportions of neurons assigned to the major neocortical layers.

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PubMed1995

Morphological abnormalities in the hippocampus of the weaver mutant mouse.

The lamination of the hippocampus in the homozygous B6CBA weaver mouse (wv/wv) was compared with that in normal B6CBA littermates (+/+) and C57BL/6J mice using Nissl and Timm's staining. In Nissl-stained preparations, the normal littermates exhibit a compact, regular arrangement of pyramidal cells in area CA3 of the hippocampus. In contrast, in homozygous weaver mutant mice, the pyramidal cell layer of area CA3 frequently appears to be thicker than normal with an apparent increase of neuropil, as evidenced by the presence of cell-free spaces within the layer. Also, small ectopic clusters of pyramidal cells and sometimes the subdivision of the pyramidal cell layer into 2 or 3 layers were found throughout the dorsoventral extent of the hippocampus. In Timm's stained preparations of the normal mouse hippocampus, two clearly separated bundles of axons were seen emerging from the hilus: one bundle running above the pyramidal cell layer of area CA3 (i.e., the suprapyramidal mossy fiber layer, SPMFL), and the second bundle running below the pyramidal cell layer (i.e., the infrapyramidal mossy fiber layer, IPMFL). In contrast, in some homozygous weaver mice, the origin of the mossy fiber bundles is clearly different from normal; specifically, mossy fibers emerge in a diffuse fashion from the area between suprapyramidal and infrapyramidal mossy fiber layers. In other weaver mice, short, discontinuous bundles diverge from the infrapyramidal mossy fiber layer and invade the thickened pyramidal cell layer. In addition, ectopic pyramidal cells are situated below the IPMFL in area CA3. The morphological changes observed in hippocampus of weaver mutants are likely to be secondary to a more basic genetic defect.

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PubMed1995

Numbers, time and neocortical neuronogenesis: a general developmental and evolutionary model.

The number of neurons in the neocortex is the product of the size of the preneuronogenetic founder population, that is, the number of proliferative cells that are present at the onset of neuronogenesis, and neuronogenetic amplification occurring as neurons are being produced. The amount of neuronogenetic amplification is determined by changes in the output fraction, Q, from 0 to 1, over a fixed number of cell cycles. Greater neuronogenetic amplification would occur across species if the number of cell cycles during which Q < 0.5 increased. Since neither the length of the cell cycle nor the length of the neuronogenetic interval, that is, time per se, influence neuron number directly, it is speculated that changes in these parameters are essential to neuronal diversity.

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PubMed1995

An immortalized mouse neuroepithelial cell line with neuronal and glial phenotypes.

Evidence from retroviral marking techniques and immortalized cell lines indicates that multipotential stem cells exist in many areas of the developing central nervous system. However, the factors that influence the commitment of these stem cells into distinct neuronal or glial lineages are not known. We have created an immortalized hypothalamic cell line derived from embryonic day 14 hypothalamic cells with a replication-defective retroviral construct containing a temperature-sensitive allele (tsA58) of the large T antigen of the simian virus 40. The clonality of this cell line, which we have named V1, was established by single cell cloning and by Southern blot analysis. V1 cells exhibit two different morphologies: the vast majority of cells are flat and stellate, and a smaller number are phase-bright round cells with processes. V1 cells express nestin and neural-cell adhesion molecule, typical of proliferating neuroepithelial cells. They also express glial fibrillary acidic protein and S100 as well as the low molecular weight neurofilament protein. In addition, the phase-bright, process-bearing V1 cells stain intensely for many typical neuronal proteins, such as low, medium and high molecular weight neurofilament proteins, tau protein, microtubule-associated protein-2, and neuron-specific enolase. The phase-bright cells also have condensed chromatin and display mitotic spindles, indicating that they are in mitosis. When V1 cells are transferred from the permissive temperature (33 degrees C) to the restrictive temperature (39 degrees C), there is a decrease in expression of NF-L and an increase in expression of NF-H and glial fibrillary acidic protein in the flat V1 cells. The enhanced expression of neuronal antigens in mitotically active V1 cells is novel and may represent a more general property of the differentiation process. We suggest that V1 cells arise from a mixed neural/glial neuroepithelial progenitor cell that expresses both neuronal- and glial-specific proteins in the developing hypothalamus.

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PubMed1995

The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall.

Neurons destined for the cerebral neocortex are formed in the pseudostratified ventricular epithelium (PVE) lining the ventricular cavity of the developing cerebral wall. The present study, based upon cumulative S-phase labeling with bromodeoxyuridine, is an analysis of cell cycle parameters of the PVE. It is undertaken in the dorsomedial cerebral wall of mouse embryos from the eleventh to the seventeenth gestational day (E11-E17, day of conception = E0) corresponding to the complete period of neuronogenesis. The growth fraction (fraction of cells in the population which is proliferating) is virtually 1.0 from E11 through E16. The length of the cell cycle increases from 8.1 to 18.4 hr, which corresponds to a sequence of 11 integer cell cycles over the course of neuronal cytogenesis in mice. The increase in the length of the cell cycle is due essentially to a fourfold increase in the length of G1 phase which is the only phase of the cell cycle which varies systematically. Thus, the G1 phase is most likely to be the phase of the cell cycle which is modulated by extrinsically and intrinsically acting mechanisms involved in the regulation of neuronal cytogenesis.

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PubMed1995

Early ontogeny of the secondary proliferative population of the embryonic murine cerebral wall.

The present report is an analysis of the proliferative behavior of the secondary proliferative population (SPP) of the dorsomedial region of the embryonic mouse cerebral wall. It is based upon experiments undertaken on embryonic days 14-16 (E14-E16) and exploits methods in which proliferative cells are labeled in S phase with either or both bromodeoxyuridine and tritiated thymidine. The SPP, which arises from the PVE by E13, is principally the progenitor population to the neuroglial population of the mature neocortex and subjacent cerebral wall. By the end of E14 the SPP comes to be distributed diffusely from the outer margin of the ventricular zone throughout subventricular zone and intermediate zone. The length of the cell cycle of the SPP is constant at approximately 15 hr throughout this interval; thus, this population undergoes 1.6 cell cycles/24 hr or 3.2 cycles in the course of the 48 hr period, E14-E16. Over this 48 hr period, the SPP increases from 11% to 35% of the total proliferative population of the dorsomedial cerebral wall. The absolute size of the SPP increases nearly sixfold. With these values taken together it may be estimated that approximately 87% of postmitotic cells of the SPP reenter S phase after each cell division in this interval which means that only approximately 13% of the proliferative population exits the cycle. These findings illustrate the massive expansion of the SPP antecedent to the explosive diffusion of glial cells through the neocortex and subjacent cerebral wall as neuronal migration comes to completion and neocortical growth and differentiation accelerate.

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PubMed1994

Disruption of neuronal migration in the neocortex of the dreher mutant mouse.

To analyze developmental abnormalities related to neuronal migration in the dreher mutant mouse, the neocortical cytoarchitecture of dreher and control mice were examined in Nissl-stained serial sections by light microscopy. In general, the dreher neocortex has six layers which are similar in size and thickness to those observed in normal mouse neocortex. However, in dreher neocortex, three types of abnormalities were found: (1) an increase in the number of diffusely distributed neurons in layer I, (2) small, ectopic collections of neurons in layer I, and (3) isolated disturbances of local cytoarchitecture characterized by neuron-free space distributed in areas between layer II to IV. The occurrence of small, punctate deficits in the dreher neocortex may be secondary to disruptions of the radial glial fiber system and neuronal migration. The fact that cytoarchitectonic abnormalities of several types were found in the dreher neocortex may be useful in analyzing the relationship between radial glial fibers and migrating young neurons, the synaptic connections which are formed by ectopically situated neurons, and the mechanism of formation of sporadically distributed neocortical abnormalities.

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PubMed1994

Mode of cell proliferation in the developing mouse neocortex.

There are two proliferative populations in the developing cerebral wall: the pseudostratified ventricular epithelium (PVE) and the secondary proliferative population (SPP). The present experiments provide on embryonic day 14 (E14) in the mouse direct measures of the values for the proportions of daughter cells that continue to proliferate (proliferative, P fraction, or P) and for those that leave the cell cycle (quiescent, Q fraction, or Q; Q = 1 - P) for both of these populations. The range of values of P for the PVE, 0.62-0.66, would provide for the relatively low rate of neuronal output and for an expansion of the proliferative population appropriate for E14. The even higher values of P for the SPP, 0.73-1.0, would expand this population rapidly in preparation for the high glial cell output to occur later in cerebral histogenesis.

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