Final Cell Division Research Articles

Cell-Cell Signaling in Early Cortical Development

The remarkable process of neuronal migration has long been viewed as the key event of corticogenesis. Before neurons complete their final cell division and begin to migrate out of the proliferative zones, however, many important decisions concerning their ultimate fate have already been made. These critical decisions are, in part, under local environmental control. The signals by which proliferating cortical precursor cells interact with each other and with their environment are still largely unknown, but, as these factors are dis covered, they will alter our view of corticogenesis and most likely provide new insights into causes of cerebral malformations. This review is intended to describe the repertoire of cellular interactions detected thus far among proliferating cortical cells and to discuss possible roles of cell-cell signaling pathways. The Neurosci entist 1:268-276, 1995

New Rods Move before Differentiating in Adult Teleost Retina

To understand the cellular context of neuronal differentiation in the vertebrate retina, we analyzed the behavior of a class of progenitor cells in the outer nuclear layer of the teleost retina which divide throughout the animal's life and generate only rod photoreceptors. We present evidence that these progenitors reside adjacent to the outer limiting membrane of the retina during all phases of cell cycle. After final cell division, postmitotic cells move inward, toward the vitreal boundary of the outer nuclear layer, before they give rise to new rods. This movement is remarkable because it occurs in the mature, functioning retina. We hypothesize that only one of the two progenitor daughter cells moves while the other remains adjacent to the outer limiting membrane to divide again.

Complementary expressions of transcripts encoding GAD67 and GABAA receptor alpha 4, beta 1, and gamma 1 subunits in the proliferative zone of the embryonic rat central nervous system

The developmental stage at which nerve cells initially express specific neurotransmitters and their corresponding receptors remains elusive. In the present study, the distribution patterns of transcripts for the GABA-synthesizing enzyme, glutamate decarboxylase (GAD67), and specific GABAA receptor subunits were examined in the proliferative zone of the rat central nervous system using in situ hybridization. In order to define the DNA synthetic zone of the germinal matrix, tissue sections were taken from embryos whose mothers had been injected with 5-bromo-2'-deoxyuridine (BrdU) and had survived for 1 hr. BrdU immunocytochemistry was used to locate the relative position of BrdU-immunoreactive nuclei within the ventricular zone (VZ). At embryonic day (E) 15 in the alar plate of the lumbar spinal cord, and at E17 and E20 in the dorsomedial sector of the neocortex, densely packed BrdU-immunoreactive nuclei were consistently detected in lateral portions of the inner half of the germinal matrix, indicating that the inner half of the germinal matrix corresponded to the VZ, while the outer half corresponded to the transitional (TZ) or subventricular zone (SV). In situ hybridization in tissue sections adjacent to BrdU-immunoreacted ones showed that the transcripts for GABAA receptor alpha 3, beta 3, and gamma 2 subunits were found exclusively in the mantle zone, while those for alpha 4, beta 1, and gamma 1 subunits were predominantly detected in the inner half of the germinal matrix (i.e., VZ). Furthermore, in the E15 germinal matrix of the lumbar spinal cord, cells exhibiting alpha 4 subunit mRNA were much more abundant in the receding intermediate plate, which contains mostly postmitotic cells, than in the alar plate comprised of many DNA-synthesizing cells, strongly suggesting that only those cells completing final cell division expressed the subunit mRNAs. In clear contrast, GAD67 mRNA was abundant in the outer half of the germinal matrix (i.e., TZ or SV), and in the intermediate zone as well. Immunocytochemical staining of E17 neocortex with anti-GABA antibody revealed a well defined band of GABA-immunoreactive cells and processes in the SV and occasional positive cells in the VZ. It appears that cells in the proliferative zone may express GABA at the migratory stage, whereas cells in the VZ may express mRNAs for GABAA receptor alpha 4, beta 1, and gamma 1 subunits at the premigratory stage, just after completing cell division.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in the reproductive system following acute prenatal exposure to ethanol or methylazoxymethanol in the rat: I. Effects on immunoreactive LHRH cell number

It has long been recognized that ethanol (EtOH) interferes with the hypothalamo-pituitary-gonad axis in adults of many species, and recent studies have provided evidence for similar effects after prenatal EtOH exposure. Since EtOH is capable of injuring dividing cells, we investigated the possibility that a single acute in utero EtOH exposure during the period of LHRH neuron formation might change the number of immunoreactive LHRH cells in the hypothalamus. Final LHRH cell division in Long-Evans rats was determined by [3H]thymidine autoradiography to take place over a short period between gestation days 12 and 13. Subsequently, pregnant rats were treated acutely with either EtOH or methylazoxymethanol (MAM), a known neuroteratogen, and the numbers of immunoreactive LHRH cells were counted. On gestation day 22, LHRH-positive cell numbers were significantly fewer than control numbers in both EtOH- and MAM-exposed offspring. On postnatal day 60, cell numbers in EtOH-exposed offspring did not differ from control numbers, whereas cells in MAM-exposed offspring remained significantly reduced. In controls, there were 40% fewer LHRH-positive cells on postnatal day 4 than in late gestation or at maturity. We conclude that 1) acute exposure to a high dose of EtOH at a critical time in early gestation can alter the expression of LHRH in late gestation; 2) exposure to MAM in the same period alters LHRH expression before birth and in the adult; and 3) in the early postnatal period, LHRH expression decreases profoundly.

Modulation of CTP: Phosphocholine cytidylyltransferase activity in choline-sensitive and choline-resistant tobacco cells

Microsomes of cultured tobacco cells exhibited a CTP:phosphocholine cytidylyltransferase (CT) activity which was involved in the regulation of phosphatidylcholine (PC) biosynthesis. Some properties of this cellular activity measured in vitro are reported, especially its changes over a 35-day culture period. The changes were modulated by the presence of choline in the culture medium and differed between choline-sensitive (ChS) and choline-resistant (ChR) cell lines. In ChS cells a high CT stimulation, which was partially cycloheximide insensitive, occurred during the lag phase and the first wave of cell divisions, and was followed by an abrupt drop during the final cell divisions. The total activity did not parallel the microsomal protein content of the cells. The early stimulation of CT activity was also found in ChS cells which were cultivated in the presence of choline but was not as high as in cells cultivated in the absence of choline. CT activity, as well as microsomal protein content of the cells, remained high in the stationary growth phase of ChS cells cultivated on a choline-supplemented medium. In marked contrast, in ChR cells, routinely cultivated on choline, CT activity was constant so that the total activity only varied in accordance with microsomal protein content during the exponential and stationary growth phases. A non-specific nucleotidase catalysing the hydrolysis of CDP-choline exhibited an activity opposite to the overall activity of CT. In conclusion, PC biosynthesis appears to be controlled either by de novo synthesis of CT either by a reversible association of this protein to membranes, or by variable levels of nucleotidase and PC precursors. Alternative pathways concerned with ethanolamine methylation should also be considered.

Fetal Development and Neuronal/Glial Cell Specificity of cAMP-Dependent Protein Kinase Subunit mRNAs in Rat Brain

All known actions of cAMP in the brain require cAMP-dependent protein kinase (cAMPdPK), which consists of regulatory (R) and catalytic (C) subunits (R2C2). Using homologous rat cDNAs for all known cAMPdPK subunit isoforms found in the brain (RI alpha, RI beta, RII alpha, RII beta, C alpha, C beta) we observe that, in the fetal rat brain from 12 days of gestation to birth, while alpha subunit (RI alpha, RII alpha, C alpha) mRNA levels are abundant, beta subunit (RI beta, RII beta, C beta) mRNA levels increase from undetectable or very low levels to abundant levels. Furthermore, while alpha subunit mRNA levels are abundant in both primary neuronal and primary glial cultures, beta subunit mRNA levels are very low (C beta) or undetectable (RI beta, RII beta) in primary glial cultures, but are abundant in primary neuronal cultures. Thus, prior to about 12 days of gestation, cAMP in the brain may act only via the alpha cAMPdPK subunits in neuronal and glial precursor cells. After 12 days of gestation, coincident with the onset of final cell division in neurons, beta cAMPdPK subunits may also mediate the effects of cAMP predominantly in neurons.

Somatostatin immunoreactive structures in the developing rat spinal cord

Somatostatin immunoreactive (SOM-I) perikarya were first observed in the ventral horn at E12, in the presumptive intermediate gray area at E14, and in the alar plate of the rostral spinal cord at E14. In general, after their initial appearance, their density increased and then decreased during development. A moderate density of SOM-I varicosities became obvious in the superficial laminae of the E20 dorsal horn. By E12 a few SOM-I perikarya, interpreted to be dorsal root ganglia, were observed lateral to the spinal cord, and by E13, SOM immunoreactivity was visualized within the central and peripheral processes of dorsal root ganglion axons. In the marginal zone, SOM-I fibers were first demonstrable in the ventral funiculus at E14, and in the lateral funiculus at E15. After their initial appearance, their density increased and then decreased with age, with the exception of the dorsal part of the lateral funiculus where it increased at the early stages of development to an apparently stable level. The early detection of SOM immunoreactivity in specific spinal regions corresponds well with the birth dates of cells in those regions. This indicates that the SOM-I cells are capable of synthesizing the substance at least as early as they have entered their final cell division.

A phenological and cytological study of pollen development in western hemlock (Tsuga heterophylla)

Pollen-cone development, which was divided into nine phenological stages, was compared with pollen cytology in eight field-grown western hemlock (Tsuga heterophylla (Raf.) Sarg.) clones in 1983 and three container-grown clones in 1984. Phenology proved to be an accurate indicator of cytology, independent of collection dates and rate of development. The breaking of dormancy, the resumption of development, and meiosis occurred during stage 1 (quiescent bud). During stage 2 (swollen pollen-cone bud) the tetrad of microspores separated. The exine wall was completely developed during stage 3 (bud burst). The one-celled microspores began to expand as a result of accumulation of reserves during stage 4, when the pollen cones were one-quarter to one-half emerged through the bud scales. The first cell division occurred during stage 5, when the cones were greater than half emerged. The second and third cell divisions occurred during stage 6, after the cones had completely emerged through the bud scales. The fourth and final cell division, resulting in mature five-celled pollen grains occurred during stage 7 (stalk elongation). Mature pollen consisted of two prothallial cells and a stalk, a body, and a tube cell. Pollen shed occurred at stage 8. The empty cones (stage 9) remained on the trees until the following winter. Some practical implications of relating pollen-cone phenology to cytology are discussed.

Genesis of GABA-immunoreactive neurons in the ferret visual cortex

The pattern of neurogenesis of GABA-immunoreactive neurons in the ferret primary visual cortex was determined using immunohistochemical and 3H-thymidine autoradiographic techniques. Neurons in the visual cortex of the ferret undergo their final cell division during a period extending from embryonic day 20 (E20) to postnatal day 14 (P14) and follow an inside-out pattern of neuronal production (Jackson et al., 1984) similar to that observed in other mammals. Earlier-generated neurons are found at deeper cortical positions in the adult than are those generated later. Layer I is an exception to this rule, since neurons destined for this layer are produced at both the beginning and end of neurogenesis. In this study, the pattern of neurogenesis of GABA-immunoreactive neurons is compared to the pattern observed for nonimmunoreactive neurons. The overall pattern of cortical neurogenesis (inside-out pattern) is similar for GABA-immunoreactive neurons and neurons that are not GABA-immunoreactive. However, the GABA-immunoreactive neurons born on a given day of development are more broadly distributed across the radial axis of the adult cortex than are nonimmunoreactive neurons generated on the same day. GABA-immunoreactive neurons generated later in neurogenesis are, on average, slightly smaller than those generated early. If GABA-immunoreactive neurons in the visual cortex are interneurons, then these findings suggest that interneurons follow the same pattern of neurogenesis as do projecting neurons in the visual cortex.

Cell formation in the motor nucleus and mesencephalic nucleus of the trigeminal nerve of rats made hypothyroid by propylthiouracil.

Neurogenesis in the motor and mesencephalic nuclei of the trigeminal nerve was examined using autoradiographic techniques. Two groups of pregnant rats (control and experimental) were injected with two successive daily doses of 3H thymidine in an overlapping series starting from day nine of gestation in order to label in their progeny, the dividing precursor of neurons of the motor nucleus and mesencephalic nucleus of the trigeminal nerve. Control group of rats was raised on a standard diet, while the experimental group was made hypothyroid by propylthiouracil (PTU). At postnatal ages ranging between 20-30 days in the pups of both the control group and experimental group, the percentage of cells labelled and the proportion of cells added during each embryonic day were determined quantitatively throughout the rostro-caudal extent for both motor and mesencephalic nuclei. The neurons of the mesencephalic nucleus undergo their final cell divisions between gestational days 9 and 10 (E9 and E10). More than 80% of the population is generated by E10. The neurons of the motor nucleus undergo their final cell divisions between E9 and E11, and nearly 88% of the cells is generated by E11. In the thyroid deficient rats, in both nuclear centers, only 61% of the cells is generated by E12, and labelled cells are observed even as late as E18 and E19. In the hypothyroid state, there is a significant lengthening of the proliferative period. On the basis of absolute datings and duration of neuron production, it is postulated that in normal development, thyroid hormone determines the duration of the proliferative period, and push cells into the differentiative phase by taking them out of the proliferative phase.

Interrelationship among the proliferating ability, morphological development and acetylcholin-esterase activity of the neural tube cells in early chick embryos.

Using new methods which perform 3H-thymidine autoradiography and AChE histochemistry on the same sections, the interrelationship among loss of proliferating ability, morphological development and increase of AChE activity during the course of differentiation of the neural tube cells were investigated in early chick embryos. The neural tube wall consisted of four kinds of cells: (1) AChE negative spindle shaped cells, (2) weakly positive spindle shaped cells showing AChE activity in the cisternae of the nuclear envelope and in a few short profiles of r-ER, (3) moderately positive spindle shaped cells showing AChE activity in the cisternae of the nuclear envelope and in a moderate number of r-ER profiles and (4) intensely positive large round cells showing AChE activity in the cisternae of the nuclear envelope and in a large number of r-ER profiles. Nuclei of the former three kinds of cells slet located in the ependymal layer, while the AChE intensely positive cells were in the mantle layer. The AChE negative and weakly positive cells were capable of proliferation and were regarded as undifferentiated neuroepithelial cells. The AChE moderately positive and intensely positive cells were, in contrast, no longer capable of proliferation and were regarded as neurons, especially the former as newly formed neurons. These findings indicate that the r-ER increases rapidly in number and in volume in newly formed neurons, soon after their final cell division, and that AChE activity increases in the neurons parallel to the development of the r-ER.

Genesis of morphologically identified neurons in the dorsal lateral geniculate nucleus of the cat.

Neurons in the dorsal lateral geniculate nucleus of the cat can be grouped into five morphological classes based on a variety of structural characteristics. These same structural characteristics can serve as morphological signatures for the three physiological classes (X, Y, W) of neurons found in this nucleus. The purpose of this study was to determine if a relationship exists between the birthdate of neurons within the dorsal lateral geniculate nucleus and the adult morphology of those neurons. Seven cats, each of which had received a single injection of 3H-thymidine, were studied. A total of 2,138 Golgi-impregnated neurons were identified in the dorsal lateral geniculate nuclei of these seven cats; 1,517 of these neurons were successfully resectioned and recovered, of which 385 (25%) were found to contain the 3H label. Neurons from each of the five morphological classes were labeled in each of the six animals that received a 3H-thymidine injection between embryonic day 24 (E24) and E28. Class 3 and class 5 neurons were labeled in a cat injected with 3H-thymidine on E30. These findings demonstrate that the development of the morphological class of a neuron in the dorsal lateral geniculate nucleus is independent of the time of its final cell division. Further, given the relationship that exists in the cat's dorsal lateral geniculate nucleus between neuronal structure and function, the present findings suggest that the different physiological classes of cells found in this nucleus undergo their final cell divisions throughout most of the period of neurogenesis except that the functional role of neurons born late in this period may be more restricted.

Genesis of neurons in the dorsal lateral geniculate nucleus of the cat.

Neurons that will ultimately form the dorsal and ventral lateral geniculate nuclei, the medial interlaminar nucleus, the perigeniculate nucleus, and the nucleus reticularis of the cat undergo their final cell division beginning on, or slightly before, embryonic day 22 (E22) and ending on, or before, E32. Early in this period, neurogenesis proceeds for all of these geniculate nuclei, whereas only in the dorsal lateral geniculate nucleus does cell birth continue until E32. Distinct spatiotemporal gradients of cell birth are not obvious within any of the individual geniculate nuclei. For the dorsal lateral geniculate nucleus in particular, and for the other geniculate nuclei in general, neurons born early in this period exhibit a full range of adult soma sizes, including large and small neurons. Neurons born late in this period exhibit only small adult somas. The location and size of a neuron within the dorsal lateral geniculate nucleus provide clues to that cell's functional properties. On the basis of presently available information regarding the relationship between structure and function of neurons in the cat's dorsal lateral geniculate nucleus, the findings described here suggest that all functional classes of neurons in the dorsal lateral geniculate nucleus are born at the same time throughout most of this period.

Appearance of acetylcholine receptors in the myotome cells of the early chick embryo.

Appearance and distribution of acetylcholine receptor (AChR) in the myotome cells of the chick embryo were investigated by autoradiography using 3H-α-bungarotoxin (α-BGT). Acetylcholinesterase (AChE) histochemistry was also performed on the same sections.In the 1, 5 day-old embryo (H-H stage 11), the myotome was not formed and the specific labeling of the toxin was not found on any tissue components. In the 2 day-old embryo (stage 13), when the myotome first formed in the dorsomedial angle of the dermatome, some silver grains were demonstrated on the AChE-positive cells constituting the myotome, and thereafter, the grains on the myotome increased in number as well as in density, as development progressed. On the other hand, the specific labeling of the toxin was not observed on the neural tube, sclerotome (mesenchyme), dermatome or spinal nerves.These findings indicate that since the AChE-positive myotome cells are no longer capable of proliferation (26), AChR appears in the myotome cells shortly after their final cell division.

Stimulation of adenylate cyclase activity by catecholamines and prostaglandins E during differentiation of rabbit bone marrow erythroid cells

After fractionation of rabbit bone marrow into erythroid cells at different developmental stages adenylate cyclase activity of membrane ghosts was assayed in the presence of sodium fluoride, catecholamines or prostaglandins E. Both basal and fluoride-stimulated adenylate cyclase decreased continuously during differentiation. Only catecholamines having β 2-adrenergic activity stimulated adenylate cyclase and their effect was restricted to the most immature cells, the proerythroblasts and, to a lesser extent, the basophilic erythroblasts. Thus, uncoupling of β-adrenergic receptors occurs early in erythroblast development and hormone responsiveness is lost before the final cell division. Prostaglandin E receptors and adenylate cyclase remain coupled throughout erythroid cell development.

A review of the neuroembryology of monoamine systems

The technique of monoamine histofluorescence has been used successfully for neuroembryologic studies in a number of species ranging from amphibia to the human. Monoamine systems can be visualized early, often before final cell division and migration have taken place. Neuronal cell bodies are seen before axon terminals. Unlike the adult animal, preterminal axons can often be visualized, even in the untreated animal. Anatomical studies have shown major analogies in most of the species studied. CA-containing neuronal cell bodies are restricted to the brainstem and hypothalamus. Those neurons containing 5-HT are largely restricted to the brainstem raphe, although other cell groups may take up 5-HT, or related compounds, under experimental conditions. Monoamine nerve terminals are found throughout the entire nervous system, with some of the regions of highest density being the hypothalamus and striatum. Dynamic studies have indicated that biochemical differentiation precedes morphologic maturity, often by a long period of time. Although attempts have been made to determine a specific role of monoamines in the complex programs of neurogenesis, there is little specific data currently available.

MIT-23: A mitochondrial marker for terminal neuronal differentiation defined by a monoclonal antibody

We describe a monoclonal antibody directed against a neuron-specific mitochondrial protein from rat brain. On protein blots the antibody recognizes a single polypeptide of apparent molecular weight 23,000. By solid-phase immunoassay the antigen was detected in all brain regions tested but was not detected in nonneural tissues. Within neurons, the antibody stains cytoplasmic granules that immunoelectron microscopy shows are mitochondria, hence the designation MIT-23. Immunocytochemical staining of the cerebellar cortex showed that MIT-23 occurs in all the neuronal types but is absent from glial and other nonneuronal cells. During neonatal development of the cerebellum, MIT-23 appears in neurons after their final cell division or migration is completed, suggesting that specific proteins associated with mitochondria participate in neuronal maturation.

Proliferating ability, morphological development and acetylcholinesterase activity of the neural tube cells in early chick embryos. An electron microscopic study.

Using 3H-thymidine autoradiography and AChE histochemistry at the electron microscopic level on the same sections, the interrelationships between loss of proliferating ability, morphological development and increase of AChE activity during the course of differentiation of the neural tube cells were investigated in early chick embryos. The neural tube wall consisted of spindle-shaped cells with no AChE activity, weakly positive spindle-shaped cells showing AChE activity in the cisternae of the nuclear envelope and in a few short profiles of r-ER, moderately positive spindle-shaped cells showing AChE activity in the nuclear envelope and in a moderate number of r-ER profiles and intensely positive large round cells showing AChE activity in the nuclear envelope and in a large number of r-ER profiles. Nuclei of the AChE-negative, weakly positive and moderately positive cells were located in the ependymal layer (matrix). The AChE-intensely positive cells were in the mantle layer. The AChE-negative and weakly positive cells were capable of proliferation and were regarded as undifferentiated neuroepithelial cells. In contrast, the moderately positive and intensely positive cells were no longer capable of proliferation and were considered to be neurons. These findings indicate that the r-ER increases rapidly in amount and volume in newly formed neurons soon after their final cell division, and that AChE increases in the neurons in parallel to the development of the r-ER.

Regional differences in the developing foreleg of Drosophila melanogaster

We transplanted imaginal disks of Drosophila melanogaster from larvae of the second half of the third larval instar into prepupae. Disks from the youngest donors differentiated bristles of only the distal segments of the leg. These disks also produced unusually large areas of cuticle that had no bristles. Disks from older donors differentiated bristles of more proximal segments and the area of cuticle with no bristles was reduced. To account for the regional variation in these results, there must be regional differences among the prospective leg cells at some time during the period from the second half of the third larval instar to the end of adult bristle differentiation. We asked whether prospective distal cells were more advanced than prospective proximal cells during bristle differentiation. We estimated when bristle precursor cells undergo their final cell divisions by heavily irradiating prepupae and pupae. We assumed that cells that were insensitive to the radiation had completed their cell divisions. The distal segments were the first to have insensitive bristles. Most leg bristles became insensitive between 12 and 18 hr after pupariation. The tarsus had a larger proportion of its bristles insensitive than the femur at 15 hr after pupariation. We also investigated when bristle-forming cells begin elongating their bristle shafts. We used the length of bristle rudiments as an indicator of when elongation is initiated. At 35 hr after pupariation, bristle rudiments of distal segments were two to three times longer than bristle rudiments of proximal segments. We discuss how these intersegmental differences observed during bristle differentiation can account for the regional variation in response of discs transplanted into older hosts. However, we do not exclude the possibility that regional differences among cells of the leg tissue exist at stages earlier than the time of bristle differentiation.

Cyclic AMP-binding and cyclic AMP-dependent protein kinase activities in the cytosol of differentiating bone marrow erythroblasts.

Cytosolic cyclic AMP-binding capacity and cyclic AMP-dependent protein kinase activity have been studied in relation to differentiation and maturation of rabbit bone marrow erythroblasts. Using cells fractionated by velocity sedimentation at unit gravity, it was found that both activities decreased in dividing cells when calculated in terms of cell number but remained constant per cell volume. After the final cell division, cyclic AMP-dependent protein kinase activity did not change further, whereas cyclic AMP-binding capacity declined. There were no qualitative, but only quantitative, changes in the cyclic AMP-binding proteins that are present in the cytosol of developing erythroblasts. In the immature cells, the apparent KD for the interaction of binding proteins with cyclic AMP was 4 X 10(-8) M. The data suggest that changes in cyclic AMP-binding activity during differentiation of erythroid cells are due both to changes in the amount of binding proteins and in their affinity for cyclic AMP. Plasma membranes of erythroblasts were also able to bind cyclic AMP but only in dividing cells.