Nancy L. Hayes

Dynamics of cell proliferation in the adult dentate gyrus of two inbred strains of mice

The output potential of proliferating populations in either the developing or the adult nervous system is critically dependent on the length of the cell cycle (T(c)) and the size of the proliferating population. We developed a new approach for analyzing the cell cycle, the Saturate and Survive Method (SSM), that also reveals the dynamic behaviors in the proliferative population and estimates of the size of the proliferating population. We used this method to analyze the proliferating population of the adult dentate gyrus in 60 day old mice of two inbred strains, C57BL/6J and BALB/cByJ. The results show that the number of cells labeled by exposure to BUdR changes dramatically with time as a function of the number of proliferating cells in the population, the length of the S-phase, cell division, the length of the cell cycle, dilution of the S-phase label, and cell death. The major difference between C57BL/6J and BALB/cByJ mice is the size of the proliferating population, which differs by a factor of two; the lengths of the cell cycle and the S-phase and the probability that a newly produced cell will die within the first 10 days do not differ in these two strains. This indicates that genetic regulation of the size of the proliferating population is independent of the genetic regulation of cell death among those newly produced cells. The dynamic changes in the number of labeled cells as revealed by the SSM protocol also indicate that neither single nor repeated daily injections of BUdR accurately measure proliferation.

Targeted mutagenesis of Lis1 disrupts cortical development and LIS1 homodimerization

Lissencephaly is a severe brain malformation in humans. To study the function of the gene mutated in lissencephaly (LIS1), we deleted the first coding exon from the mouse Lis1 gene. The deletion resulted in a shorter protein (sLIS1) that initiates from the second methionine, a unique situation because most LIS1 mutations result in a null allele. This mutation mimics a mutation described in one lissencephaly patient with a milder phenotype. Homozygotes are early lethal, although heterozygotes are viable and fertile. Most strikingly, the morphology of cortical neurons and radial glia is aberrant in the developing cortex, and the neurons migrate more slowly. This is the first demonstration, to our knowledge, of a cellular abnormality in the migrating neurons after Lis1 mutation. Moreover, cortical plate splitting and thalomocortical innervation are also abnormal. Biochemically, the mutant protein is not capable of dimerization, and enzymatic activity is elevated in the embryos, thus a demonstration of the in vivo role of LIS1 as a subunit of PAF-AH. This mutation allows us to determine a hierarchy of functions that are sensitive to LIS1 dosage, thus promoting our understanding of the role of LIS1 in the developing cortex.

Exploiting the dynamics of S-phase tracers in developing brain: interkinetic nuclear migration for cells entering versus leaving the S-phase

Two S-phase markers for in vivo studies of cell proliferation in the developing central nervous system, tritiated thymidine (3H-TdR) and bromodeoxyuridine (BUdR), were compared using double-labeling techniques in the developing mouse cortex at embryonic day 14 (E14). The labeling efficiencies and detectability of the two tracers were approximately equivalent, and there was no evidence of significant tracer interactions that depend on order of administration. For both tracers, the loading time needed to label an S-phase cell to detectability is estimated at <0.2 h shortly after the injection of the label, but, as the concentration of the label falls, it increases to ∼0.65 h after about 30 min. Thereafter, cells that enter the S-phase continue to become detectably labeled for ∼5–6 h. The approximate equivalence of these two tracers was exploited to observe directly the numbers and positions of nuclei entering (labeled with the second tracer only) and leaving (labeled with the first tracer only) the S-phase. As expected, the numbers of nuclei entering and leaving the S-phase both increased as the interval between the two injections lengthened. Also, nuclei leaving the S-phase rapidly move towards the ventricular surface during G2, but, unexpectedly, the distribution of the entering nuclei does not differ significantly from the distribution of the nuclei in the S-phase. This indicates that: (1) the extent and rate of abventricular nuclear movement during G1 is variable, such that not all nuclei traverse the entire width of the ventricular zone, and (2) interkinetic nuclear movements are minimal during S-phase.

Descending projections from brainstem and sensorimotor cortex to spinal enlargements in the cat. Single and double retrograde tracer studies.

SummarySingle and double retrograde tracer techniques were employed in cats to investigate: (1) the topographical relationships between supraspinal neurons projecting to either the brachial or lumbosacral enlargement, (2) the distribution and relative frequency of single supraspinal neurons which project to both enlargements by means of axonal branching.In one group of cats large injections of horseradish peroxidase (HRP) were made throughout either the brachial or lumbosacral enlargement. The results from these experiments support recent observations on the multiplicity of brainstem centers giving origin to descending spinal pathways and provide evidence for a population of corticospinal neurons in area 6.In a second set of experiments, HRP was injected in one enlargement, and 3H-apo-HRP (enzymatically inactive) was injected in the other enlargement. Relatively large numbers of neurons with collateral projections to both enlargements (double-labeled) were observed in the medullary and pontine reticular formation, the medial and inferior vestibular nuclei bilaterally, the ipsilateral lateral vestibular nucleus, Edinger-Westphal nucleus, caudal midline raphe nuclei and nuclear regions surrounding the brachium conjunctivum. By contrast, double-labeled neurons were infrequently observed in the red nucleus and sensorimotor cortex, contralateral to the injections.In the red nucleus, lateral vestibular nucleus and sensorimotor cortex, neurons projecting to the brachial enlargement were largely segregated topographically from neurons projecting to the lumbosacral enlargement. However, there was some overlap, and double-labeled neurons were consistently observed within the region of overlap. In the sensorimotor cortex, the overlap between brachial- and lumbar-projecting neurons was most prominent in areas 4 and 3a, along the cruciate sulcus, but also involved other cytoarchitectonic regions in the medial aspect of the hemisphere.

Population Dynamics During Cell Proliferation and Neuronogenesis in the Developing Murine Neocortex

During the development of the neocortex, cell proliferation occurs in two specialized zones adjacent to the lateral ventricle. One of these zones, the ventricular zone, produces most of the neurons of the neocortex. The proliferating population that resides in the ventricular zone is a pseudostratified ventricular epithelium (PVE) that looks uniform in routine histological preparations, but is, in fact, an active and dynamically changing population. In the mouse, over the course of a 6-day period, the PVE produces approximately 95% of the neurons of the adult neocortex. During this time, the cell cycle of the PVE population lengthens from about 8 h to over 18 h and the progenitor population passes through a total of 11 cell cycles. This 6-day, 11-cell cycle period comprises the neuronogenetic interval (NI). At each passage through the cell cycle, the proportion of daughter cells that exit the cell cycle (Q cells) increases from 0 at the onset of the NI to 1 at the end of the NI. The proportion of daughter cells that re-enter the cell cycle (P cells) changes in a complementary fashion from 1 at the onset of the NI to 0 at the end of the NI. This set of systematic changes in the cell cycle and the output from the proliferative population of the PVE allows a quantitative and mathematical treatment of the expansion of the PVE and the growth of the cortical plate that nicely accounts for the observed expansion and growth of the developing neocortex. In addition, we show that the cells produced during a 2-h window of development during specific cell cycles reside in a specific set of laminae in the adult cortex, but that the distributions of the output from consecutive cell cycles overlap. These dynamic events occur in all areas of the PVE underlying the neocortex, but there is a gradient of maturation that begins in the rostrolateral neocortex near the striatotelencephalic junction and which spreads across the surface of the neocortex over a period of 24-36 h. The presence of the gradient across the hemisphere is a possible source of positional information that could be exploited during development to establish the areal borders that characterize the adult neocortex.

Barrels in somatosensory cortex of normal and reeler mutant mice

The pattern of projection of the ventrobasal thalamic complex (VB) upon the neocortical barrel field of normal and reeler mutant mice was determined by orthograde degeneration experiments and by Timms histochemical method. In both reeler and normal mice the thalamic terminals are concentrated at the midcortical zone dominated by granule cells and the adjacent zone dominated by medium-sized pyramidal cells. The medium-sized pyramids are principally supragranular in normal cortex but infragranular in the reeler. Throughout the barrel fields of reeler and normal mice thalamic terminals are organized as a mosaic of radially oriented columns coextensive in the tangential plane with barrels whose cross-sectional areas and shapes are similar in both genotypes. The radial extent of the columns in reeler is 2-3 times that in normal cortex. These observations suggest that the tangential organization of the thalamocortical projection is normal in reeler and that thalamic terminals are distributed among the same neuronal classes in normal and reeler mice despite cell malposition in the mutant.

Size distribution of retrovirally marked lineages matches prediction from population measurements of cell cycle behavior

Mechanisms that regulate neuron production in the developing mouse neocortex were examined by using a retroviral lineage marking method to determine the sizes of the lineages remaining in the proliferating population of the ventricular zone during the period of neuron production. The distribution of clade sizes obtained experimentally in four different injection–survival paradigms (E11–E13, E11–E14, E11–E15, and E12–E15) from a total of over 500 labeled lineages was compared with that obtained from three models in which the average behavior of the proliferating population [i.e., the proportion of cells remaining in the proliferative population (P) vs. that exiting the proliferative population (Q)] was quantitatively related to lineage size distribution. In model 1, different proportions of asymmetric, symmetric terminal, and symmetric nonterminal cell divisions coexisted during the entire developmental period. In model 2, the developmental period was divided into two epochs: During the first, asymmetric and symmetric nonterminal cell divisions occurred, but, during the second, asymmetric and symmetric terminal cell divisions occurred. In model 3, the shifts in P and Q are accounted for by changes in the proportions of the two types of symmetric cell divisions without the inclusion of any asymmetric cell divisions. The results obtained from the retroviral experiments were well accounted for by model 1 but not by model 2 or 3. These findings demonstrate that: 1) asymmetric and both types of symmetric cell divisions coexist during the entire period of neuronogenesis in the mouse, 2) neuron production is regulated in the proliferative population by the independent decisions of the two daughter cells to reenter S phase, and 3) neurons are produced by both asymmetric and symmetric terminal cell divisions. In addition, the findings mean that cell death and/or tangential movements of cells in the proliferative population occur at only a low rate and that there are no proliferating lineages “reserved” to make particular laminae or cell types.

Spinothalamic and spinomedullary neurons in macaques: A single and double retrograde tracer study

Abstract The location and perikaryal size of spinothalamic cells and of spinal neurons projecting to the dorsal column nuclei has been compared in adult monkeys ( Macaco fascicularis ) in which horseradish peroxidase has been injected in either the ventrobasal complex or the dorsal medulla. Spinothalamic neurons are found mainly in lamina I and in the lateral portions of laminae IV-VI throughout the side of the cord contralateral to the thalamic injection. Spinomedullary neurons are most ipsilateral to the medullary injection, in medial portions of laminae IV-VI throughout cervical levels but laterally, in the same laminae, in lumbar segments. At these levels, spinothalamic and spinomedullary neurons are also found along the lateral border of the ventral horn; neurons in this location, labelled by either injection, are large (40–65 μm in diameter), and are indistinguishable from ‘spinal border cells’ at the origin of the ventral spinocerebellar tract. Computer-assisted measurement of perikaryal size reveals that the mean values for dorsal horn neurons of the two systems differ significantly, although a sizeable fraction of both neuronal populations have identical cytological characteristics, including perikaryal size. A double-labelling strategy employing horseradish peroxidase and [ 3 H]-apo-horseradish peroxidase has been applied (a) to identify simultaneously cells of origin of the two ascending systems and to appreciate better their topographical relationships, and (b) to answer the question of whether at least some of these spinal neurons have axons which, by way of collateral branching, reach both targets. The distribution of neurons at the origin of the two ascending tracts in animals with injection of the two tracers is identical to that revealed in animals in which only horseradish peroxidase has been injected. While primarily single-labelled neurons are found throughout the cord, some large dorsal horn neurons in both brachial and lumbosacral enlargements are labelled by both cytoplasmic horseradish peroxidasepositive granules and by reduced silver grains on the overlying emulsion. These double-labelled cells are interpreted as having an axon which gives origin, presumably at spinal cord levels, to collaterals ascending to both the ipsilateral medulla and the contralateral thalamus. Systematic evaluation of the extent to which ascending pathways to various supraspinal targets originate from common spinal neurons may provide new insights into the functional mechanisms of ascending spinal systems.

CNS development: an overview

The basic principles of the development of the central nervous system (CNS) are reviewed, and their implications for both normal and abnormal development of the brain are discussed. The goals of this review are (a) to provide a set of concepts to aid in understanding the variety of complex processes that occur during CNS development, (b) to illustrate how these concepts contribute to our knowledge of the normal anatomy of the adult brain, and (c) to provide a basis for understanding how modifications of normal developmental processes by traumatic injury, by environmental or experiential influences, or by genetic variations may lead to modifications in the resultant structure and function of the adult CNS.

Competitive interactions during dendritic growth: a simple stochastic growth algorithm

A simple growth algorithm is presented that deals with one feature of dendritic growth, the distance between branches. The fundamental assumption of our growth algorithm is that the lengths of dendritic segments are determined by the branching characteristics of the growing neurite. Realistic-appearing dendritic trees are produced by computer simulations in which it is assumed that: (1) growth of individual neurons occurs only at the tips of each growing neurite; (2) the growing neurite can either branch (as a bifurcation) or continue to elongate; (3) events at any one growing tip do not affect the events at any other growing tip; and (4) the probability of branching is a function only of the distance grown either from the cell body (if branching has not occurred) or from the previous branch point. An analytic solution of a differential equation based on these same assumptions produces a distribution of dendritic segment lengths that accurately fits an experimentally determined distribution of dendritic segment lengths of reconstructed neurons, accounting for about 89% of the sample variance. Our analysis indicates that, immediately following branching, the temporary suppression of further branching during dendritic growth may be an important mechanism for regulating the distance between branches.