Development of axonal transport of RNA and its precursors in the optic pathway of the chick embryo

The axonal migration of RNA has been investigated at various developmental stages in the optic pathway of chick embryo after intraocular administration of labeled uridine. The somatofugal movement of soluble RNA precursors has also been studied. An excess of radioactivity in RNA within the optic lobe contralateral to the injected eye relative to that within the ipsilateral lobe was found 3 days after ³H-uridine injection in 13- and 18-day-old embryos. In 9-day-old embryos no asymmetry in radioactive RNA from paired optic lobes developed even 6 days after isotope injection. At all stages no asymmetry was found 6 and 24 hr after isotope administration. These results suggest a slow retinotectal flow of RNA beginning at about Day 13 of incubation. In the newly hatched chick, 3 days after monocular ³H-uridine injection, the asymmetry in label within RNA from paired optic lobes was much greater than was that of embryos. At all stages studied a considerable asymmetry of radioactivity within nucleotides and nucleosides from paired optic lobes also developed 3 days after monocular ³H-uridine injection. However, this was at least as high in 9-day-old embryos as in newly hatched chicks. Intraocular injection of colchicine prior to ³H-uridine blocked the migration of labeled RNA in the newly hatched chick, suggesting such transport to be dependent on the integrity of neuronal microtubules. It is concluded that a significant proximodistal migration of RNA occurs in the optic pathway of chick embryos prior to the onset of electrical activity.     

Rohon-beard cells and other large neurons in Xenopus embryos originate during gastrulation

The time of origin (birthday) of Rohon-Beard cells in Xenopus laevis was studied by ³H-thymidine autoradiography. Rohon-Beard cells were selected because they are a morphologically identifiable population of neurons in which the development of chemical and electrical excitability has been studied. A single injection of a radioactive DNA precursor was given to animals in successive stages of development from blastula to late tail bud (Nieuwkoop and Faber stages 8–33/34). The label was available throughout the stage of injection and longer. The labeling pattern was examined when animals had reached stage 42, when Rohon-Beard cells are easily recognized. All neurons including Rohon-Beard cells were labeled in animals injected with ³H-thymidine before stage 10 1/2 (early gastrula). Unlabeled Rohon-Beard cells were observed in animals injected with ³H-thymidine in and after stage 10 1/2. The percentage of unlabeled Rohon-Beard cells increased as development progressed. About 80% were born by the completion of gastrulation (stage 13). The other ～20% were born during neurulation and early tail bud stages. By stage 27, no Rohon-Beard neuron incorporated ³H-thymidine. In addition to Rohon-Beard neurons, five other neuronal populations begin generation during gastrulation: Mauthner neurons (Vargas-Lizardi and Lyser, '74), trigeminal ganglion cells, large basal plate cells of the medulla, extramedullary neurons, and primary motor neurons. The first birthdays in any of the six populations are temporally close to but appear to be independent of the others.     

Cochlear cytogenesis visualized through pulse labeling of chick embryos in culture

Cytogenesis in the basilar papilla sensory epithelium of the chicken was investigated through pulse labeling of proliferative cells. Tritiated-thymidine was injected intravenously in chick embryos cultured in petri dishes. All embryos received the injection on the seventh day of incubation (E7), when the progenitors of hair cells and supporting cells are replicating deoxyribonucleic acid (DNA). Cells that were in the synthesis phase of the cell cycle, either at the time of the ³H-thymidine pulse or within 2 hours, incorporated detectable levels of the radioactive DNA precursor. Labeled cells were identified in cochleae from embryos fixed at 0.5, 1, 2, 4, 6, 12 hours, 6 and 8 days after the pulse. One hour after the injection the majority of labeled nuclei were in the basal and middle strate of the sensory epithelium. Four to 6 hours after the injection, a greater number of labeled cells appeared in the lumenal stratum. The patterns of labeled cells in embryos fixed immediately after the injection of ³H-thymidine and in others fixed 6 to 8 days after the injection were unchanged, suggesting that the progenitor cells divide and their progeny differentiate in the sensory epithelium without appreciable transverse migration. Mitotic figures were usually observed only in the lumenal stratum. Analysis of DNA content in the population of Feulgen-stained nuclei at three levels of depth through the epithelium also produced results consistent with the conclusion that vertical nuclear migration occurs during development of the cells in this sensory epithelium. © 1993 Wiley-Liss, Inc.     

Effects of reserpine on fast, intermediate and slow axonal transport of proteins in rat locus coeruleus neurons

The effects of a single injection of reserpine (5 mg/kg, i.p.) on protein turnover and axonal transport (AT) in locus coeruleus (LC) noradrenergic neurons was investigated in the rat. Reserpine pretreatment, at intervals of 1–21 days prior to [³H]-fucose or leucine injection into the LC, resulted in marked alterations in the turnover of [³H]glycoproteins and proteins in the LC and hypothalamus which were present for up to 14 days and varied according to the time after reserpine pretreatment. Reserpine produced an intermittent blockade, of variable degree, in rapidly and intermediately transported proteins for up to 2 weeks following injection. Slow AT was uniformly decreased over the first 10 post-treatment days to 2–42% of controls. Blockade and not a change in the rate or time of onset of transport appeared to be responsible for the observed changes. The suggested mechanism for these alterations is a re-ordering of metabolic priorities in the synthesis and transport of proteins in these noradrenergic cells secondary to a reserpine-induced depletion of norepinephrine in the nerve terminals.     

Genesis of resting microglia in the gray matter of mouse hippocampus

The genesis of resting microglia in the gray matter of mouse hippocampus was studied by ³H-thymidine autoradiography in combination with electron microscopy. Newborn mice were injected with ³H-thymidine singly or repeatedly at different postnatal stages, and killed shortly after the injection or after various intervals. Tissue specimens of the hippocampus at CA1 and CA2 were processed for light and electron microscopic autoradiography. The results showed that at least 91% of glial cells in the stratum radiatum of the hippocampus are produced after birth. About three-fourths of astroglia in this area are produced before the sixth postnatal day, and a larger part of resting microglia are formed after the ninth postnatal day. Morphological transition can be traced from either proliferating cells in the stratum radiatum at late postnatal days to resting microglia or from those in early postnatal days to astroglia. A continuous morphological transition was observed between the proliferating cells at the late postnatal days (microglial production period) and those at the early postnatal days (astroglial production period). The latter retain some fine structural characteristics similar to small glioblasts in the subependymal layer. These findings strongly suggest that resting microglia, as well as astroglia, are derived from glioblasts, and are of neurectodermal origin.     

Maturation of cerebellar granule cells is delayed in cultures derived from ethanol-treated chick embryos: Survival and proliferation studies

Previous studies from this laboratory have shown that ethanol administration to chick embryos during embryonic days 1–3, a critical period of neuroembryogenesis, differentially affects primordial CNS structures. In this study, chick embryos were treated in ovo with ethanol (10 mg/50 μl/day) at E1 to E3. At 14 days of embryonic age cerebellar (E14CE) granule cell cultures were prepared from both control and ethanol-treated embryos. Growth patterns were evaluated morphologically and the neuronal nature of these cultures was evaluated immunocytochemically. E14CE granule cell cultures exhibited neurofilament immunoreactivity demonstrating the neuronal-nature of these cultures. In addition E14CE granule cultures contained numerous glutamateric neurons as assessed by positive glutamate immunoreactivity and also some GABAergic neurons as assessed by positive GABA immunoreactivity. Cultures derived from both control and ethanol-treated embryos were labeled with ³H-thymidine and assessed for effects on survival and proliferation in culture. Cultures derived from ethanol-treated embryos showed a higher rate of proliferation and survival during the first 3 days in culture as compared to those derived from controls. However, after 3 days in culture, survival was lower in the cultures from ethanol-treated embryos as compared to those derived from controls. We interpret these findings to mean that (a) ethanol arrested cerebellar granule cell development at an immature state; (b) immature neurons have a higher survival capacity than differentiated neurons; and (c) ethanol accelerates normal neuronal cell death as previously reported.     

Proliferation and turnover of glial cells in the forebrain of young adult mice as studied by repeated injections of 3 H-thymidine over a prolonged period of time

The magnitude of glial cell renewal was studied on young adult mice using repeated intraperitoneal injections of ³H-thymidine every eight hours over a period of 30 days. Mean labeling indices one hour after the last injection were as follows: Glial cells of the subependymal layer of the lateral ventricle, 61.5%; oligodendrocytes (various sites), 24 to 36.2%; astrocytes (various sites), 14.3 to 30.8%, and satellites in the cerebral cortex, 32.7%. Since DNA synthesis time of the proliferating, immature glial cells is unknown and may be shorter than the time interval of eight hours chosen for repeated injections of ³H-thymidine, these results are interpreted as representing minimum values for turnover, during 30 days, of the various cell types in different areas of the forebrain. The significance of a marked proliferative activity of the glial cells as related to differentiation and possible migration of subependymal cells, is discussed.     

Cell dynamics in the adult mouse olfactory epithelium: a quantitative autoradiographic study

The dynamics of cell genesis in the olfactory epithelium of the adult mouse were investigated using 3H-thymidine autoradiography. Mice were injected once with 3H-thymidine, and their olfactory epithelia were examined 7, 14, 30, 60, and 90 d later. The number of silver grains over each nucleus was counted, and the relative distance from the basement membrane was measured for each labeled nucleus. At 7 and 14 d, the average number of labeled cells in each section was about 20 per mm. By 30 d, and for the following 60 d, the average number of labeled cells was only about 6 per mm. Thus, most cells labeled by the injection died 2-4 weeks after injection. When the labeled cells were compared by nuclear grain density, time after injection (the "survival period"), and distance of the nucleus from the basement membrane (the "migration distance"), it was apparent that there was a small population of "nonmigrating" cells that remained close to the basement membrane. These cells, at first heavily labeled, divided a second time about 60 d after the 3H-thymidine injection, indicated by a significant decrease in nuclear grain density. This nonmigrating, slowly dividing basal cell is probably the neural stem cell, which gives rise to another stem cell and an olfactory neuron precursor by an asymmetric division. When the relative numbers of nonmigrating and migrating cells were compared, the results indicated that, after the asymmetric division, there are at least 2 or 3 rapid, symmetric divisions of the precursor cells, producing many immature receptor cells. Most of these die within 4 weeks of the 3H-thymidine injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Passive avoidance training is correlated with decreased cell proliferation in the chick hippocampus

One-trial passive avoidance learning (PAL), where the aversive stimulus is the bitter-tasting substance methylanthranilate (MeA), affects neuronal and synaptic plasticity in learning-related areas of day-old domestic chicks (Gallus domesticus). Here, cell proliferation was examined in the chick forebrain by using 5-bromo-2-deoxyuridine (BrdU) at 24 h and 9 days after PAL. At 24 h post-BrdU injection, there was a significant reduction in labelling in MeA-trained chicks in both the dorsal hippocampus and area parahippocampalis, in comparison to controls. Moreover, double-immunofluorescence labelling for BrdU and the nuclear neuronal marker (NeuN) showed a reduction of neuronal cells in the dorsal hippocampus of the MeA-trained group compared with controls (35 and 49%, respectively). There was no difference in BrdU labelling in hippocampal regions between trained and control groups of chicks at 9 days post-BrdU injection; however, the number of BrdU-labelled cells was considerably lower than at 24 h post-BrdU injection, possibly due to migration of cells within the telencephalon rather than cell loss as apoptotic analyses at 24 h and 9 days post-BrdU injection did not demonstrate differences in cell death between treatment groups. Cortisol levels increased in the chick hippocampus of MeA-trained birds 20 min after PAL, suggesting the possibility of a stress-related mechanism of cell proliferation reduction in the hippocampus. In contrast to hippocampal areas, the olfactory bulb, an area strongly stimulated by the strong-smelling MeA, showed increased cell genesis in comparison to controls at both 24 h and 9 days post-training.     

Cytogenesis in the monkey retina

Time of cell origin in the retina of the rhesus monkey (Macaca mulatta) was studied by plotting the number of heavily radiolabeled nuclei in autoradiograms prepared from 2- to 6-month-old animals, each of which was exposed to a pulse of 3H-thymidine (3H-TdR) on a single embryonic (E) or postnatal (P) day. Cell birth in the monkey retina begins just after E27, and approximately 96% of cells are generated by E120. The remaining cells are produced during the last (approximately 45) prenatal days and into the first several weeks after birth. Cell genesis begins near the fovea, and proceeds towards the periphery. Cell division largely ceases in the foveal and perifoveal regions by E56. Despite extensive overlap, a class-specific sequence of cell birth was observed. Ganglion and horizontal cells, which are born first, have largely congruent periods of cell genesis with the peak between E38 and E43, and termination around E70. The first labeled cones were apparent by E33, and their highest density was achieved between E43 and E56, tapering to low values at E70, although some cones are generated in the far periphery as late as E110. Amacrine cells are next in the cell birth sequence and begin genesis at E43, reach a peak production between E56 and E85, and cease by E110. Bipolar cell birth begins at the same time as amacrines, but appears to be separate from them temporally since their production reaches a peak between E56 and E102, and persists beyond the day of birth. Müller cells and rod photoreceptors, which begin to be generated at E45, achieve a peak, and decrease in density at the same time as bipolar cells, but continue genesis at low density on the day of birth. Thus, bipolar, Müller, and rod cells have a similar time of origin. The maximal temporal separation of cell birth is between cones and amacrine cells so that cell generation exhibits two relatively distinct phases: the first phase gives rise to ganglion, horizontal, and cone cells, and the second phase to amacrine, bipolar, rod, and Müller cells. In addition, cells of the first phase are generated faster than the second phase cells, and there are differences in the topography of spread of labeled cells between the two phases. Each cell class displays a central-to-peripheral gradient in genesis, although the spatiotemporal characteristics of the gradients differ between the classes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characteristics of norepinephrine uptake in developing peripheral nerve terminals

In the chick the various characteristics of [³H]norepinephrine (NE) uptake do not appear simultaneously during the development of the iris. Na⁺-dependence and ouabain sensitivity are both first seen at a significant level at 10 days of incubation (d.i.) Na⁺-dependence achieves its peak value at 14 d.i. and is unchanged at 3 months after hatching (a.h.). Ouabain sensitivity increases after its initial appearance to 14 days a.h. and then slowly declines. Temperature sensitivity develops only at hatching and is modified subsequently. Both desmethylimipramine (DMI) and cocaine inhibition develop at 10 d.i. and follow the same developmental pattern; however, DMI appears to be a more effective inhibitor than cocaine. Metanephrine, an inhibitor of extraneuronal uptake, first inhibits NE uptake to a significant degree at hatching. These changes may be related to changes in membrane structure and metabolic function in the developing neuronal membrane.No evidence of [³H]NE uptake is seen at 7 d.i. After this age the K_m values tend to fall into two groups, the first 1.3−1.9 × 10⁻⁵ M, up to one month a.h., the second, 3.5–5.0 × 10⁻⁶ M, after one month a.h. There is a large increase in V_max from 10 to 14 d.i., a period of rapid growth of sympathetic innervation of the iris.Our study suggests that Na⁺-dependent uptake is a more reliable and constant index of innervation in development than temperature-dependent uptake. It also indicates that in longitudinal developmental studies it is necessary to determine the characteristics of the transport process of each stage, as characteristics such as Na⁺-, drug- and temperature-sensitivity may all have unique developmental profiles.     

3H-Thymidine autoradiographic analysis of telencephalic histogenesis in the chick embryo: I. Neuronal birthdates of telencephalic compartments in situ

The birthdates of neuronal populations comprising the chick telencephalon were determined by ³H-thymidine labeling and were mapped with respect to their terminal positions in the 16-day embryo. Essentially all neurons were generated between four and nine days of embryonic development. Each telencephalic structure (based on terminology used by Karten and Hodos, '67) was characterized by a specific range of birthdates: some regions such as the core of the ectostriatum or the paleostriatum primitivum, were generated within a single day, while others, such as the hyperstriatum accessorium, required up to five days for generation of the complete population. Spatial-temporal gradients of neuronal birthdates, lateromedial and ventrodorsal, were seen in the telencephalon as a whole and within individual subcompartments as well. An “outside-in” pattern of histogenesis predominated throughout the entire telencephalon, including the dorsolateral cortex. However, notable exceptions pertaining to the paleostriatum augmentatum, hyperstriatum intercalatus and field “L” were observed. Glial cells, generated for the most part after day ten, were found to be distributed homogeneously throughout all areas of the telencephalon. These data provide the first birthdating data for an avian telencephalon and bring greater resolution to previous analyses of the histogenesis of this brain region. Further, the compartmentalization of the proliferative neuroepithelium is revealed by these data, and the possibility of a common time of origin in the neuroepithelium for neurons of related function is discussed.     

3H-Thymidine autoradiadiographic analysis of telencephalic histogenesis in the chick embryo: II. Dynamics of neuronal migration,displacement, and aggregation

The movements of cells from their sites of origin in the proliferative neuroepithelium to their final positions in the chick telencephalon were traced by autoradiographic analysis of ³H-thymidine-labeled brains. A series of chick embryos were labeled on successive days of development between days 5 and 9 and fixed for autoradiography between days 6 and 10. Isochrone maps visualizing neuronal positions on each day of development between days 6 and 10 provided direct information concerning cell migrations, displacements, and aggregations leading to compartmentalization of the telencephalic wall and the generation of the “outside-in” pattern of histogenesis characteristic of the avian telencephalon. The topological divisions of the telencephalic wall appear to result from two factors: (1) the specification of neuronal precursors within the neuroepithelium and (2) the intrinsic associative and migratory properties of postmitotic neuronal populations expressed within the mantle layers. There was no evidence that glial cell barriers mediated the initial compartmentalization of neuronal populations.     

Development of hypothalamic opioid neurons: A combined immunocytochemical and [H]thymidine autoradiographic study

Using a combined technique of immunocytochemistry and [³H]thymidine autoradiography, we have determined the “birth-date” of opioid peptide containing neurons in three hypothalamic nuclei. These include proopiomelanocortin neurons (indicated by ACTH immunoreactivity) in the arcuate nucleus, dynorphin a neurons in the supraoptic nucleus, and [Leu]enkephalin neurons in the periventricular nucleus. Arcuate proopiomelanocortin neurons were born very early in embryonic development, with peak heavy [³H]thymidine nuclear labelling occurring on embryonic day E12. Supraoptic dynorphin A neurons were also labelled relatively early (peak at E13). By contrast, [Leu]enkephalin neurons in the periventricular nucleus exhibited peak heavy nuclear labelling on day E14. The results indicate a differential genesis of these three opioid peptide containing neuronal groups in three different hypothalamic nuclei.     

Intracerebral injection of interferon-gamma inhibits the astrocyte proliferation following the brain injury in the 6-day-old rat

The present study examines the influence of interferon-gamma (IFN-γ) on the astrocyte proliferation in the rat brain injured within the early period of postnatal development. Six-day-old male rats received a lesion in the left cerebral hemisphere and a single injection of recombinant rat IFN-γ into the lesion cavity. One or 2 days after the injury the rats were injected with ³H-thymidine. Brain sections were immunostained for glial fibrillary acidic protein (GFAP), subjected to autoradiography, and examined microscopically to record proliferating GFAP-immunopositive astrocytes labeled with ³H-thymidine. In the IFN-γ-injected rats, a statistically significant decrease in the intensity of reactive astrocyte proliferation was revealed. On day 1 after injury the intensity of astrocyte proliferation showed dose-dependent changes. Relations between the astrocyte reactivity and multiple factors existing in the injured and IFN-γ-injected brain are discussed. The results represent the first in vivo evidence of a dose-dependent action of IFN-γ on the astrocyte proliferation in response to injury. J. Neurosci. Res. 50:1018–1022, 1997. © 1997 Wiley-Liss, Inc.     

Neurogenesis of subpopulations of rat lumbar dorsal root ganglion neurons including neurons projecting to the dorsal column nuclei

The time of birth of subpopulations of dorsal root ganglion (DRG) neurons was studied with immunohistochemistry for 5-bromodeoxyuridine (BrdU). Pregnant rats were injected with BrdU i.p. to label the neurons on one of the embryonic days (E) E11-E16. When they were adults, the rats were given injections of Fluoro-Gold (FG) into the gracile nucleus to identify DRG neurons projecting to this structure. Following a 5 day survival period, the animals were perfused with aldehyde fixative. Sections from the L3-L5 DRGs were processed for BrdU immunohistochemistry followed by either immunostaining for the antineurofilament antibody RT97, as marker of the light neuronal subpopulation, or histochemical staining for the B4 isolectin from Griffonia simplicifolia I, as marker of the small dark subpopulation. The results indicated that the DRG neurons were generated between E12 and E16. The RT97⁺ neurons were generated on E12–E15, with a peak at E13. FG⁺ neurons, the majority of which were RT97⁺, were also generated on E12–E15. The B4⁺ neurons were generated on E13–E16, with a peak around E14. The overall pattern of neurogenesis of the DRG neurons showed that the RT97⁺ neurons were produced prior to the B4⁺ neurons. These findings are in agreement with earlier observations that the large DRG neurons are generated earlier than the small dark neurons. Our findings also suggest the existence of a third neuronal subpopulation that might be produced at the latest period of DRG neurogenesis at E15–E16. © 1996 Wiley-Liss, Inc.     

Developmental expression of nitric oxide synthase isoform I and III in chick retina

During our studies on the multiple possible functions of nitric oxide (NO) in chick retinal development and physiology, we have demonstrated the presence and the activity of NO synthase (NOS-I and III) in certain neuronal populations (photoreceptors, amacrine cells in the inner nuclear and ganglion cells) and also in synaptic-rich regions in the developing chick retina. Both enzymes, detected by nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, immunohistochemistry and Western blotting, appeared between embryonic days 6 and 12, and followed a spatial and temporal pattern of expression which correlated with the differentiation of the neuronal layers. Evaluation of the conversion of [³H]-labeled arginine to [³H]-citrulline, confirmed the presence of a calcium-dependent NOS activity in the cytosolic and particulate retinal extracts during the development. This pattern of NOS expression suggests that the regulated release of NO during key phases of development might be one mechanism involved in the regulation of retinal differentiation. J. Neurosci. Res. 50:104–113, 1997. © 1997 Wiley-Liss, Inc.     

Age dependent changes in choline uptake of the chick iris

The kinetics of the uptake of choline, the rate-limiting substrate in the in vivo synthesis of acetylcholine, were studied during the period 1–7 years in the iris of the chick. These changes were correlated to the endogenous levels of acetylcholine and choline in the same organ. V_max values per iris decrease significantly at 5 years and continue to decrease at 7 years, to 64% and 37% of the one year value, respectively. If the variation in V_max is calculated per protein the decline is 34% between 1 and 5 years.K_m does not change significantly during the period 1–7 years. Total acetylcholine and choline levels follow a similar trend, decreasing progressively from 1 to 7 years.Sensitivity to hemicholinium (5.5 × 10⁻⁵M) decrease significantly between 5 days of incubation (d.i.) and 5 years. Inhibition by ouabain (10⁻⁴ M) shows an opposite trend, increasing significantly from 20% at 5 d.i. to 52% at 3 months. At 5 years sensitivity to ouabain is the same as at 3 months. Na⁺ dependence decreases significantly between 5 d.i. and 5 years but no significant changes are seen between 5 d.i. and 3 months. Uptake at 27 °C decreases from 59% of control at 5 d.i. to 45% at 3 months, and is not changed at 5 years.Our observations suggest that the effect of aging on peripheral cholinergic neurons is not generalized, but is specifically directed toward the neuronal perophery (terminals) as opposed to cell bodies.     

Fine structure of reactive cells in injured nervous tissue labeled with 3H-thymidine injected before injury

To examine the fine structure of blood mononuclear cells in injured nervous tissue, mice were given repeated injections of ³H-thymidine with the last injection at least 16 hours before injury. Under ether anesthesia the animals either were given a stab wound to the spinal cord or had their left hypoglossal nerve transected. The animals were killed at 2, 4, 8, or 16 days after injury. Tissue sections containing the spinal cord wound or both hypoglossal nuclei were prepared for electron microscopic radioautography, and all labeled cells were photographed. About half the labeled cells in the injured hypoglossal nerves had nuclei with dark staining peripheral heterochromatin, dark cytoplasm with long cisternae of granular endoplasmic reticulum, and other ultrastructural features characteristic of the cells usually identified as microglia. The remaining labeled cells in the injured spinal cords were macrophages, fibroblasts, cells with pale nuclei, some of which contained cytoplasmic filaments, and vascular cells. Since uninjured nervous tissue has extremely few labeled cells and since ³H-thymidine should be available for only a short time following injection, most of the labeled cells in this experiment should be derived from blood mononuclear cells. However, the possibility is discussed that some or all of the labeled cells proliferating in response to the injury and labeled through reutilization of labeled DNA precursor material.