Kinetics of telencephalic neural cell proliferation during the fetal regeneration period following a single X-irradiation at the late organogenesis stage

Summary A sequential study of the acute effects of prenatal X-irradiation on telencephalic cell population dynamics was performed by combining a pathological evaluation with autoradiographic methods. This study confirmed that a dose of 0,95 Gy affects nearly all neuroblasts in the G₁-, G₂-, and M-stage lethally. Within ventricular cells staying in the S-phase at the time of X-irradiation, lethal effects are in the range between 50% and 75%. The surviving remainder of these neuroblasts is responsible for subsequent attempts for regeneration of the telencephalic roof. Two subpopulations of the surviving S-phase cells were observed: The first subpopulation which seems to be restricted to the rudiments of the ventricular zone shows a S/G₂-blockade for 8–12 h after X-irradiation. Thereafter these cells start again with mitotic activity. The second subpopulation is morphologically manifested by the formation of heterotopic cell nests, i.e., so-called rosettes. These cells continue with an intense DNA-replication after the 12th h post irradiation and proceed to the mitosis stage only after about another 4–8 h, i.e., the 16th to 20th hour following X-irradiation. These findings indicate the existence of two different pools of regenerative cells within the telencephalic roof, giving rise to either orthotopic or heterotopic growth processes after irradiation injury.Dedicated to Prof. Dr. H. Kriegel on occasion of his 60th anniversary     

Prenatal pathogenesis of hydro-micrencephaly induced by X-rays

Summary Experimental results are presented which describe induction and pathogenesis of the hydro-micrencephaly in NMRI-mice after single X-irradiation with 0.9 Gy or 1.9 Gy on gestation day (gd) 12. The ultrastructural alterations in the ventricular walls are sequentially investigated up to gross developmental damage. The ventricular zone is the most sensitive region in the developing brain. Its constituting undifferentiated and proliferating cells lose their palisade like orientation and fail subsequently to differentiate into primitive neurons, glia cells, or ependymal cells. Structurally this results in the thinning of ventricular walls by more than 50% associated with periventricular oedema and a dilation of the brain ventricles by 20–60%. Damage is clearly more pronounced with the higher dose. Repair processes originate from regions with intact Zonulae adherentes which give also rise to typical globular or cylindrical heterotopic structures; these are known as rosettes and made up from undifferentiated proliferating ventricular cells. Perinatally in these rosettes cell replication persists, at a time when cell production in the ventricular zone has ceased. Histological changes are most prominent in and around the telencephalic roof consisting in replacement of the ependymal lining by a felt of glial fibers, faulty myelinisation, and periventricular oedema; postnatally these structural alterations lead to hydro-micrencephaly. Results from this animal model can be translated to the human situation because the fundamental developmental processes in the brain of mammals are similar despite species related differences of the time scale.This paper is dedicated to Prof. Dr. W. Gössner on the occasion of his 72nd birthday     

Ontogeny of Neurochemical Markers for Noradrenergic, GABAergic and Cholinergic Neurons in Neocortex Lesioned with Methylazoxymethanol Acetate

Abstract: Methylazoxymethanol acetate (MAM), a potent, rapidly eliminated nucleic acid alkylating agent, produces microencephaly in rat pups when injected into their dams on day 15 of gestation. In the adult microencephalic rats, neuronal loss is largely confined to telencephalic structures, such as the superficial neocortical laminae, whose neuroepithelial progenitor cells were undergoing vigorous replication during the chemical exposure. Histological examination of the forebrain 2 days after injection revealed early selective damage to the ventricular geminal zone with relative sparing of cortical plate neurons generated on earlier days. The degree of specificity of MAM's action on neurochemically defined neuronal populations was examined by measuring presynaptic markers for GABAergic, noradrenergic and cholinergic neurons in atrophic lateral cortex from 20 days gestation to adulthood. Although treatment reduced GABAergic markers (GABA, its synthetic enzyme and synaptosomal uptake process) in proportion to loss of cortex mass (-67%), the maturational pattern for remaining GABAergic neurons was virtually normal. Although the maturational sequence of noradrenergic markers was similar to control, the concentration of endogenous norepinephrine, [³H]norepinephrine uptake and tyrosine hydroxylase specific activity were two- to fourfold higher than control at each time. However, total noradrenergic markers per cortex section were nearly identical to control throughout development, indicating that development of the noradrenergic axonal arbor in neocortex was insensitive to loss of neurons in the terminal field. Maturation of cholinergic markers (endogenous acetylcholine, its synthetic enzyme and [³H]choline uptake) in the atrophic cortex was biphasic: concentrations were similar to control values for the first 12 postnatal days, but gradually rose to levels twofold higher than control. These results indicate that neurochemical alterations observed in cortex from prenatally MAM-treated rats are primarily the result of early selective elimination of neuronal subpopulations. Fetal MAM exposure appeared to have minimal effects on biochemical differentiation of neurons remaining intact in the atrophic cortex. MAM appears to be a useful toxin for producing selective loss of neuronal groups based on their time of generation in the fetus.     

Anterior dorsal ventricular ridge in the lizard: Embryonic development

In lacertids the telencephalic vesicle starts its development at stage E = 30, at which time it is lined by a homogeneous nucleated zone in which particular ventricular zone territories or sulci cannot be distinguished. At stage E = 32 coinciding with the initial development of the anterior dorsal ventricular ridge (ADVR), one may distinguish the ventricular zone b in the dorsolateral wall of the ventricle adjacent to the sulcus lateralis. The ADVR continues growing by incorporation of cells produced in two proliferative zones (zone b and wall of the sulcus lateralis) and appears fully developed in postnatal lizards. Ultrastructural characteristics of young ADVR neurons between stages E-32 and E-33 are typical of those in immature cells. Beginning at stage E-34, some of these neurons appear to be degenerating (pycnotic). Thereafter, neurons of the ADVR develop abundant cytoplasmic organelles and the neuropile grows quickly. Myelination starts in the ADVR between stages E-38 and E-40, but is not observed in other striatal masses in the same period. Vascularization begins and is well developed at E-40. The first synaptic contacts were observed in embryos of stage E=38; they are chiefly axo-dendritic, although some are axo-somatic. Degenerating neurons were found in the ADVR up to hatching. From stage E-40 onward, the ADVR shows a greater and more rapid differentiation than all other striatal nuclei, including the ventral and amygdaloid complex.     

Developmental-stage-dependent radiosensitivity of neural cells in the ventricular zone of telencephalon in mouse and rat fetuses

Pregnant ICR mice were treated with single whole-body X-radiation at a dose of 0.24 Gy on day 10, 13, or 15 of gestation. Fetuses were obtained from mothers during 1 and 24 hours after irradiation. Pyknotic cells in the ventricular zone of telencephalon were counted in serial histological sections. Incidence of pyknotic cells peaked during 6 and 9 hours after irradiation in each gestation day group. Then, dose-response curves were obtained 6 hours after 0-0.48 Gy of irradiation. All three dose-response curves showed clear linearity in the dose range lower than 0.24 Gy. Ratios of radiosensitivity estimated from the slopes of dose-response curves in day 10, 13, and 15 groups were 1, 1.4, and 0.4, respectively. These demonstrated that ventricular cells in the day 13 fetal telencephalon were the most radiosensitive among the three different age groups. In order to confirm the presence of the highly radiosensitive stage common to mammalian cerebral cortical histogenesis, pregnant F344 rats were treated with single whole-body gamma-irradiation at a dose of 0.48 Gy on day 13, 14, 15, 17, or 19 of gestation. The incidence of pyknotic cells in the ventricular zone of telencephalon was examined microscopically during 1 and 24 hours after irradiation. The peak incidence was shown 6 hours after irradiation in all the treated groups, and the highest peak incidence was shown in day-15-treated group. The developmental stage of telencephalon of day 15 rat fetuses was comparable to that of day 13 mouse fetuses. Thus, the highest radiosensitivity in terms of acute cell death was shown in the same developmental stage of brain development, i.e., the beginning phase of cerebral cortical histogenesis, in both mice and rats.     

The effect of prenatal procarbazine treatment on brain development in the rat

Groups of pregnant Sprague-Dawley rats were treated orally with procarbazine, an antineoplastic drug, at dose levels of 0, 1.0, 2.5, 5.0, 7.5, and 10.0 mg/kg/day from days 12 through 15 of gestation. Following normal delivery, offspring were raised until day 21 and sacrificed, and their brains removed and weighed. A dose-dependent micrencephaly, characterized by hypoplasia of the cerebral hemispheres, was seen starting at 2.5 mg/kg/day. In a second study, groups of pregnant female rats were given a single dose of 10 mg/kg procarbazine on gestation day 12, 13, 14, or 15. Micrencephaly occurred in 21-day-old offspring from all groups, with the greatest effect induced on days 13, 14 and 15. Analysis of brain region weights revealed a maximum reduction in neocortex weight in offspring from groups treated on days 13 and 14. The hippocampus, cerebellum, and diencephalon-midbrain were also reduced in size, depending on the day of treatment, while the corpus striatum and pons-medulla were spared. In a final study, embryos from females treated on gestation days 12 through 15 were removed, fixed, and sectioned at 24-hour intervals starting on gestation days 13. Necrosis and cellular degeneration were observed with decreasing severity in the telencephalon, diencephalon, mesencephalon, and medulla. The neocortex of 20-day treated fetuses was characterized by a thickening of the ventricular zone and reduced cellularity of the cortical plate.     

Appearance of immunoreactive endocrine cells during the development of the rat pancreas, with special reference to polypeptide-secreting cells

The chronological appearance of PP cells in fetal pancreatic islets was studied using specific anti-PP serum and the direct peroxidase method. The presence of A and B cells was also studied, using the same immunocytochemical technique, as a reference pattern related to data previously reported. Our data confirm that the A cell is the earliest endocrine cell type, appearing on the 12th day of gestation, followed by B cells (14th day) and later on by PP cells (19th day). Primitive islets were identified in the pancreas after the 15th day. However, the spatial cell disposition observed in the adult islet was only recognized at the 20th day of gestation. The data reported provide the necessary information to establish the complete chronology in the rat fetus. Consequently, the development of pancreatic islets in the rat fetus could be employed as a useful model to study the existence of factors that control the sequential appearance of endocrine cells and the possible changes occurring in the islets of animals with genetic diabetes during the fetal period.     

Differentiation and innervation of the atrioventricular bundle and ventricular Purkinje system in sheep heart

The development of the atrioventricular bundle (AVB) and ventricular Purkinje system and their innervation have been studied in fetal sheep from 27 to 140 days gestation (term is 147 days). The AVB initially consisted of a primordium, which lacked innervation and was composed of small, relatively undifferentiated myocytes. Differentiation of Purkinje-like cells within the AVB began near its distal end and extended towards the atrioventricular node (AVN). Differentiation of the ventricular Purkinje system extended distally from the region of bifurcation of the AVB from cells that were indistinguishable from the working myocardium and continuous with the AVB primordium. Differentiation of Purkinje-like AVB cells was complete by 46 days gestation but Purkinje fibres were still differentiating within the ventricular wall at 60 days gestation. The main morphological changes included a large increase in cell size and organization into strands, development of characteristic glycogen-filled regions containing many intermediate filaments and early development of myofibrillar M lines compared to the working myocardium. The differentiation of AVB cells and the ventricular Purkinje system preceded their innervation. The AVB became innervated earlier than ventricular Purkinje fibres, intimate contacts between proximal AVB cells and nerve axons being present at 60 days gestation. Nerve fibres were present in the septomarginal band at this time, however, en passant associations with ventricular Purkinje fibres were rarely observed until 140 days gestation and intimate contacts were not present at any stage. Although the AVB and ventricular Purkinje system of adult sheep are composed of morphologically similar cells, the present study demonstrates that they differ in origin and their mode of differentiation as well as timing and intimacy of innervation. Innervation is not part of the developmental mechanism leading to the differentiation of Purkinje fibres. No primordium of the ventricular Purkinje system could be identified, suggesting that the mechanism of differentiation of ventricular Purkinje fibres involves recruitment from early working myocardium.     

Brain Barriers and a Subpopulation of Astroglial Progenitors of Developing Human Forebrain Are Immunostained for the Glycoprotein YKL-40

YKL-40, a glycoprotein involved in cell differentiation, has been associated with neurodevelopmental disorders, angiogenesis, neuroinflammation and glioblastomas. We evaluated YKL-40 protein distribution in the early human forebrain using double-labeling immunofluorescence and immunohistochemistry. Immunoreactivity was detected in neuroepithelial cells, radial glial end feet, leptomeningeal cells and choroid plexus epithelial cells. The subpial marginal zone was YKL-40-positive, particularly in the hippocampus, from an early beginning stage in its development. Blood vessels in the intermediate and subventricular zones showed specific YKL-40 reactivity confined to pericytes. Furthermore, a population of YKL-40-positive, small, rounded cells was identified in the ventricular and subventricular zones. Real-time quantitative RT-PCR analysis showed strong YKL-40 mRNA expression in the leptomeninges and the choroid plexuses, and weaker expression in the telencephalic wall. Immunohistochemistry revealed a differential distribution of YKL-40 across the zones of the developing telencephalic wall. We show that YKL-40 is associated with sites of the brain barrier systems and propose that it is involved in controlling local angiogenesis and access of peripheral cells to the forebrain via secretion from leptomeningeal cells, choroid plexus epithelium and pericytes. Furthermore, we suggest that the small, rounded, YKL-40-positive cells represent a subpopulation of astroglial progenitors, and that YKL-40 could be involved in the differentiation of a particular astrocytic lineage.     

Relationships between regional myocardial wall stress and bioenergetics in hearts with left ventricular hypertrophy

This study utilized porcine models of postinfarction left ventricular (LV) remodeling [myocardial infarction (MI); n = 8] and concentric LV hypertrophy secondary to aortic banding (AoB; n = 8) to examine the relationships between regional myocardial contractile function (tagged MRI), wall stress (MRI and LV pressure), and bioenergetics ((31)P-magnetic resonance spectroscopy). Physiological assessments were conducted at a 4-wk time point after MI or AoB surgery. Comparisons were made with size-matched normal animals (normal; n = 8). Both MI and AoB instigated significant LV hypertrophy. Ejection fraction was not significantly altered in the AoB group, but significantly decreased in the MI group (P < 0.01 vs. normal and AoB). Systolic and diastolic wall stresses were approximately two times greater than normal in the infarct region and border zone. Wall stress in the AoB group was not significantly different from that in normal hearts. The infarct border zone demonstrated profound bioenergetic abnormalities, especially in the subendocardium, where the ratio of PCr/ATP decreased from 1.98 +/- 0.16 (normal) to 1.06 +/- 0.30 (MI; P < 0.01). The systolic radial thickening fraction and the circumferential shortening fraction in the anterior wall were severely reduced (MI, P < 0.01 vs. normal). The radial thickening fraction and circumferential shortening fraction in the AoB group were not significantly different from normal. The severely elevated wall stress in the infarct border zone was associated with a significant increase in chemical energy demand and abnormal myocardial energy metabolism. Such severe metabolic perturbations cannot support normal cardiac function, which may explain the observed regional contractile abnormalities in the infarct border zone.     

Studies concerning the effects of low level prenatal X-irradiation on postnatal growth and adult behaviour in the Wistar rat

Fifty-nine pregnant Wistar strain rats were sham irradiated or subjected to a 0.1 or 0.2 Gy exposure of X-radiation on the 9th or 17th day of gestation. Twenty-seven of the females were killed at term for teratologic analysis. The remaining mothers raised their young. At 60 days of age the 252 offsprings were randomly assigned three of six tests: open field, swimming, hanging, activity wheel, water T-maze, or conditioned avoidance response. Male offspring exposed at the 0.2 Gy level exhibited retarded growth only during the first few weeks of postnatal life. Female offspring exposed on the 17th day to 0.2 Gy X-radiation were growth retarded throughout the test period. Postnatal growth rates, however, were not significantly different between the irradiated and control groups. There were no significant alterations in adult behaviour due to prenatal X-irradiation. There were sex differences in activity wheel and forelimb hanging performance, unrelated to radiation exposure. These results indicate that prenatal low level X-irradiation on the 9th or 17th day of gestation does not result in significant alterations in adult behavioural performance in the rat, but prenatal growth retardation persists postnatally. Growth may be a more sensitive indicator of the effects of prenatal exposure to X-radiation than postnatal behaviour.     

Altered placental morphology associated with murine trisomy 16 and murine trisomy 19

The morphology of placentas from trisomy 16 and trisomy 19 mouse conceptuses aged 12 to 18 gestational days was studied at the light microscopic level. Comparisons were made with placentas from normal littermate animals. Trisomy 16 placentas showed marked changes from normal: 1) the junctional zone showed little indication of normal morphologic differentiation throughout gestation; 2) clusters of germinal trophoblast cells persisted in the labyrinth throughout gestation, whereas these cells disappeared by gestational day 16 in the normal littermate placentas; 3) the labyrinth was reduced in size in the trisomic placentas, and the differentiation of the interhemal membranes was delayed. The size of the labyrinths from trisomy 19 placentas appeared to be decreased, but otherwise the placentas appeared to have normal morphology. These observations and others from the literature show that placental development is affected by the presence of a trisomic genome, and that different trisomies influence the development of the placenta differently. For trisomy 16, we propose that the striking changes of the junctional zone may be associated with the trisomy 16-related gene dosage effect for alpha- and beta-interferon cell surface receptors. Because of the homology for this and other genes on mouse chromosome 16 with genes on human chromosome 21, findings related to the altered development of the trisomy 16 mouse may be relevant to understanding some of the phenotypic variations associated with human trisomy 21, the Down syndrome.     

Expression of the protein tyrosine phosphatase-like protein IA-2 during pancreatic islet development.

A tyrosine phosphatase-like protein, IA-2, is a major autoantigen in Type 1 diabetes but its role in islet function is unclear. Tyrosine phosphorylation mediates regulation of cellular processes such as exocytosis, cell growth, and cell differentiation. To investigate the potential involvement of IA-2 in islet differentiation and insulin secretion, we analyzed by immunohistochemistry expression of IA-2 during islet development in fetal rats and during the maturation of insulin secretory responses after birth. In the fetus, IA-2 immunoreactivity was detected in primitive islets positive for insulin and glucagon at 12 days' gestation. Subsequently, IA-2 was only weakly detectable in the fetal pancreas. In neonatal rat, a progressive increase in IA-2 immunoreactivity was observed in islets from very low levels at 1 day of age to moderate labeling at 10 days. In the adult, relatively high levels of IA-2 were detected in islets, with heterogeneous expression in individual cells within each islet. IA-2 marks a population of endocrine cells that transiently appear early in pancreatic ontogeny. Islet IA-2 expression reappears after birth concomitant with the development of mature insulin secretory responses, consistent with a role for this protein in regulated hormone secretion.     

The progenitor cells of the embryonic telencephalon and the neonatal anterior subventricular zone differentially regulate their cell cycle

For the last 10 years our laboratory has been studying the proliferation, migration and differentiation of neuronal progenitor cells located in the anterior part of the postnatal forebrain subventricular zone (SVZa). SVZa-derived cells possess a number of proliferative characteristics that distinguish them from the other progenitor cells in the central nervous system. This review summarizes our recent findings, in which we compared the pattern of cell cycle inhibitory proteins expressed by the neonatal SVZa to that of telencephalic ventricular zone cells.     

The effect of increased crypt cell proliferation on the activity and subcellular localization of esterases and alkaline phosphatase in the rat small intestine

Synopsis The activity and ultrastructural localization of alkaline phosphatase and esterase has been studied in normal rat intestine and after the increased crypt cell proliferation that occurs during recovery after 400 rad X-irradiation. Alkaline phosphatase activity is not present in crypt cells of normal intestine, but becomes apparent after the cell has migrated on to the villus. The enzyme is localized in the microvilli, along the lateral cell membranes and in dense bodies. Its activity increases 10 to 15-fold from the base to the tip of the villus. Morphometric analysis of the cell structureswhere this enzyme is localized reveals no marked changes in their relative proportions during crypt cell development.The expansion of the proliferative cell compartment along the whole length of the crypt which occurs during recovery after irradiation (72 hr after 400 rad X-irradiation) results in a marked reduction of alkaline phosphatase activity in the lower 10–15 cell positions at the base of the villus. During subsequent migration of these cells, the activity increases with cell age but normal values are not attained. From a morphometric analysis it was found that the ultrastructural development is similar to that in controls. These results suggest that during cell maturation, normal values for alkaline phosphatase activity are only attained after a 10–12 hr period of maturation in a non-proliferative state and only after the cell has migrated on to the functional villus compartment.In normal intestine, esterase activity shows a 3-fold increase from the bottom to the tip of the crypt and a 3 to 4-fold increase during migration up to the middle of the villus. Enzyme activity is localized in the endoplasmic reticulum, the dense bodies and the perinuclear space. Morphometric analyses reveal a 2 to 3-fold increase in the absolute size of these subcellular compartments during crypt cell differentiation and a 2-fold increase at the crypt-villus junction. The relative sizes increase 1.5-fold during crypt cell differentiation and at the time of transition of the cells on to the villus.Increased crypt cell proliferation after irradiation leads to a marked decrease in esterase activity both in crypts and villi. Morphometric analyses of electron micrographs indicate that these changes in activity are not related to any changes in the subcellular structures in which the enzyme is localized. It appears that the normal development of esterase activity depends both on the functional state of the cell and its localization in the crypt or villus.     

Rac1 deficiency in the forebrain results in neural progenitor reduction and microcephaly

The Rho family of small GTPases has been implicated in many neurological disorders including mental retardation, but whether they are involved in primary microcephaly (microcephalia vera) is unknown. Here, we examine the role of Rac1 in mammalian neural progenitors and forebrain development by a conditional gene-targeting strategy using the Foxg1-Cre line to delete floxed-Rac1 alleles in the telencephalic ventricular zone (VZ) of mouse embryos. We found that Rac1 deletion in the telencephalic VZ progenitors resulted in reduced sizes of both the striatum and cerebral cortex. Analyses further indicated that this abnormality was caused by accelerated cell-cycle exit and increased apoptosis during early corticogenesis (approximately E14.5), leading to a decrease of the neural progenitor pool in mid-to-late telencephalic development (E16.5 to E18.5). Moreover, the formation of patch-matrix compartments in the striatum was impaired by Rac1-deficiency. Together, these results suggest that Rac1 regulates self-renewal, survival, and differentiation of telencephalic neural progenitors, and that dysfunctions of Rac1 may lead to primary microcephaly.     

Analysis of developing ependymal and choroidal surfaces in rat brains using scanning electron microscopy

The ultrastructural surface features of normal differentiating ventricles in prenatal rat brains, days 13–21 of pregnancy, were examined with a Cambridge “Stereoscan” scanning electron microscope (SEM) (40-30,000 ×). All tissues were fixed in 3% buffered glutaraldehyde, glycerinated (16%), dehydrated in graded ethanol, infiltrated with amyl acetate, and critical-point dried in CO₂. Specimens were plated with approximately 50–100 Å of gold and palladium prior to viewing with the SEM (5–20 kV).Ependyma and choroid plexuses showed early, consistent differences in their respective morphology and relative rates of differentiation. Within the telencephalic and myelencephalic ventricles distinct regional variations in cellular projections were observed along the ependyma as early as day 13 of gestation. Choroid plexuses in day 14 embryos were densely covered with microvilli and short cilia. By day 16 of pregnancy, embryonic ventricular surfaces revealed tufts of cilia and numerous, varied microvilli, reminiscent of the adult picture. The results demonstrate the early development of a variety of surface features previously thought to arise later in organogenesis. Based on information received from the SEM, it appears early rat embryos definitely possess the morphological differentiation commensurate with early cerebrospinal fluid (CSF) functioning, e.g., fluid movement, removal of debris, and secretion and absorption of CSF.