Injection Of Tritiated Thymidine Research Articles

Differential cell proliferation and differentiation in developing and growing claws of turtles and alligator determine their shape

AbstractDifferential cell proliferation and differentiation in developing and growing claws of turtles and alligator determine their shape. Acta Zoologica (Stockolm). Morphogenesis and cell proliferation in claws of turtle and alligator have been analysed by immunolabelling and thymidine autoradiography. Proliferating cells are randomly distributed in the epidermis and mesenchyme at the tip of forming digits. Developing claws elongate with a dorsal curvature forming the unguis. Numerous proliferating keratinocytes are present in the unguis at 4 hr–1 day after injection of 5BrdU or by PCNA detection. Their size increases before being incorporated into the ungueal corneous layer. Immunolabelling at 3–6 days post‐injection of 5BrdU indicates that proliferating keratinocytes move towards the tip of the claw giving rise to the claw pad. The ventral side of the claw termed sub‐unguis shows a lower proliferation that expands the surface and produces thin corneocytes that are desquamated. Autoradiography and immunolabelling indicates that proliferating cells of growing turtle claws are present along most of the germinal epidermis 7 hr–1 day after injection of tritiated thymidine or 5BrdU. Low labelled corneocytes incorporated into the corneous layer are detected at 6–12 days post‐injection, generally localized towards the tip of the claw, indicating they have migrated distally. The study confirms that claws of reptiles have an extended proliferative zone instead of a localized proximal germinal matrix as in mammalian claws.

Higher environmental temperatures promote acceleration of spermatogenesis in vivo in mice (Mus musculus)

Temperature is considered a crucial modulator of reproductive activity and testis homeostasis. It is well known that elevated temperatures cause several effects on testicular components, particularly on germ cells, which might lead to the impairment of spermatogenesis and loss of male fertility. The present study aimed to evaluate the effects of different environmental temperatures on several morphofunctional testis parameters, with emphasis on duration of spermatogenesis and spermatogenic efficiency. Thirty sexually mature Swiss mice (Mus musculus) were allocated in three different experimental groups, being kept in vivarium for three weeks at 16 °C, 23 °C (control group) and 32 °C. In order to estimate the duration of spermatogenesis, three animals per each group received intraperitoneal injections of tritiated thymidine and the testes were perfused-fixed and routinely processed for histological, morphometrical and immunoperoxidase analyses. Although the lower temperature (16 °C) did not change most of the evaluated testicular parameters, our findings showed that higher environmental temperature (32 °C) is able to alter important testis parameters, resulting for instance in acceleration of spermatogenesis, alterations in the stages frequencies, increased number of germ and Leydig cells apoptosis and reduced Sertoli cell and spermatogenic efficiencies. As in many conditions infertile men exhibit higher mean scrotal temperature, we believe that experimental studies with mice involving temperature might represent an interesting approach to better understand the mechanisms related to human testis function and sperm production.

Spermatogenesis in a neotropical marsupial species, Philander frenatus (Olfers, 1818)

Despite the singular morphology of the male genital system and the different reproductive strategies of marsupials, little emphasis has been given to the testis morphology and spermatogenic kinetics in this mammalian order. The present study aimed to investigate the testis function and the duration of spermatogenesis in the southeastern four-eyed opossum, Philander frenatus. Testes of six adult males were routinely processed for histological and stereological analyses. In order to determine the duration of spermatogenesis, intratesticular injections of tritiated thymidine were performed 1h, 13days and 21days before the sacrifice. Based on the development of the acrosomic system, ten stages of the seminiferous epithelium cycle were characterized. The mean body and testis weights for the P. frenatus were respectively 326±20g and 0.4±0.05g, providing a gonadosomatic index of 0.3±0.02%. The most advanced germ cell types labeled at 1h, 13days and 21days after thymidine injections were, respectively, preleptotene spermatocytes at stage IV, pachytene spermatocytes at stage IV and diplotene spermatocytes at stage IX. Based on the stages frequencies and the most advanced labeled germ cells, each spermatogenic cycle and the entire spermatogenic process lasted respectively 13.5±0.5 and 60.9±2.4days. When compared to the vast majority of eutherian mammals already investigated, these data indicate that the Philander frenatus presents a relatively long duration of spermatogenesis.

Spermatogenic efficiency in the spiny rat, Trinomys moojeni (Rodentia: Echimyidae)

The spiny rat ( Trinomys moojeni) is a rodent found in the Atlantic Forest, which is considered one of the most diverse and threatened biomes in the world. Knowledge on reproductive biology and physiology is critical to conservation and species management, allowing the prevention of extinction and the use of males in natural and artificial reproduction programs. The main objectives of the present study were to investigate the testis structure as well as spermatogenic and Sertoli cell efficiency in the spiny rat captured in the Caraça Natural Reserve, a fragment of the Atlantic Forest located in the State of Minas Gerais, Brazil. Ten sexually mature spiny rats were analyzed. Intraperitoneal injections of tritiated thymidine were administered to estimate duration of spermatogenesis. The testes were perfused-fixed in buffered glutaraldehyde and routinely processed for histological and morphometric analyses as well as the characterization of the stages of seminiferous epithelium cycle. Volume density (%) of seminiferous tubules and Leydig cells were 97 ± 0.3 and 0.3 ± 0.02, respectively. The duration of one spermatogenic cycle and total duration of spermatogenesis were 8.6 ± 0.1 and 38.5 ± 0.5 days, respectively. Due to the very great volume density of the seminiferous tubules, short duration of spermatogenesis, tubule length per gram of testis (∼40 m), great Sertoli cell efficiency (∼15 spermatids per Sertoli cell) and large number of Sertoli cells per testis gram (53 million), spermatogenic efficiency in the spiny rat (82 million) is by far the greatest of the mammalian species investigated thus far.

The seminiferous epithelium cycle and its duration in different breeds of dog (Canis familiaris)

Testis structure and function in dogs are relatively poorly investigated. The aim of the present study was to carry out a comparative investigation of the stages of the seminiferous epithelium cycle and its duration in different breeds of dog. Fifty-six sexually mature dogs (mongrel, n = 12; pinscher, n = 12; beagle, n = 5; American pit bull, n = 9; poodle, n = 12; and Labrador retriever, n = 6) were analysed. Intratesticular injections of tritiated thymidine were given to determine the duration of spermatogenesis. Orchiectomy was performed at different time periods following injection (1 h, 2 and 4 weeks). Testis fragments were embedded in plastic and routinely prepared for histological and autoradiographic evaluations. Eight stages were characterized based on the acrosome system. Significant (P < 0.05) differences were found for the frequencies of the different stages characterized (except Stages V, VI and VIII), particularly for the mongrel. Stage IV (when spermiation occurs) was the most frequent in all six breeds (~25%), whereas Stages II and VIII were the least frequent (< 8%). Each spermatogenic cycle and the total duration of spermatogenesis lasted 13.73 +/- 0.03 and 61.9 +/- 0.14 days, respectively, for the mongrel, poodle, pinscher, beagle, and Labrador retriever. These values were approximately 10% lower (P < 0.03) for the American pit bull (12.55 +/- 0.26 and 56.5 +/- 1.17 days, respectively). To our knowledge, this is the first comprehensive study to perform a careful investigation of stage frequencies and seminiferous epithelium cycle duration in this very important domestic species.

Keratinocyte stem cells, label-retaining cells and possible genome protection mechanisms.

Stem cells are the crucial cells upon which the entire tissue is dependent. Here we define and discuss what is meant by and known about keratinocyte stem cells. One way in which these cells have been studied is by their ability to retain radioactivity labelled thymidine for long periods of time (label retaining cells, LRCs). The underlying mechanism has been assumed in the past to be slow cycling but a more likely explanation is the selective segregation of old and new DNA strands (Cairns's hypothesis). Experiments in the small intestine indicate that the stem cells here are selectively sorting their DNA and becoming LRCs. A possible role for p53 in stem cell biology is presented.

Effet synchroniseur du rythme circadien de la corticostérone sur la vitesse de synthèse du DNA dans le foie du jeune rat

Post-natal liver proliferation is synchronized by corticosterone circadian variation from the 21st day of life in rats. After tritiated thymidine injection, we compare the labelling index, the specific radioactivity of DNA, the DNA synthesis rate, the plasma corticosterone in un-rhythmed rats at day 15 and in rhythmed rats at day 25. No variation of these parameters is found during the 15th nycthemeron. During the 25th nycthemeron, the corticosterone circadian wave peaking at 18h is followed by the circadian variations of the 3 proliferation parameters peaking at 24 h.

Undifferentiated cells in the snail myocardium are capable of DNA synthesis and myodifferentiation.

Cellular mechanisms of heart-muscle growth in the snail Achatina fulica have been studied using cytophotometry and electron microscopic autoradiography. Cytophotometric DNA measurements showed that the snail cardiomyocytes are mononucleated cells with diploid nuclei. Ultrastructural analysis of the snail myocardium revealed that, in addition to mature myocytes, it contains small roundish undifferentiated cells (UCs) and poorly differentiated muscle cells. EM autoradiography detected silver grains over the nuclei of UCs 2 h after injection of tritiated thymidine ([(3)H]Tdr), while the nuclei of both mature and poorly differentiated myocytes remained unlabeled. In EM autographs of the myocardial tissue fixed 14 days after [(3)H]Tdr administration, labeled myonuclei were evident, which may suggest some myodifferentiation of prelabeled UCs. Many labeled UCs persist for 14 days after a single [(3)H]Tdr injection, suggesting that not all UCs undergo myodifferentiation after passing through the cell cycle, and that those that do not can enter the next cycle. UCs in the snail myocardium presumably provide not only reserve but also stem cells for myocytes. Thus, the heart muscle of the adult snail consists of mononucleated diploid myocytes with blocked proliferative activity and a renewable population of precursor myogenic cells. The results obtained suggest that the growth of this muscle involves a myoblastic mechanism of myogenesis; this mechanism differs from that of vertebrate cardiac muscle growth, which is non-myoblastic-that is, based on proliferation or polyploidization of cardiomyocytes. Evolutionary aspects of cellular mechanisms of the heart-muscle growth are discussed.

Cell kinetic studies in murine ventral tongue epithelium: cell cycle progression studies using double labelling techniques.

The dorsal and ventral epithelia on the murine tongue exhibit very pronounced circadian rhythms in terms of the cell cycle. These rhythms are such that three injections of tritiated thymidine 3 h apart spanning the circadian peak in S phase cells labelled between 40 and 50% of the basal cells. Injection of bromodeoxyuridine generally gave slightly lower labelling indices. Approximately the same proportion (54% of the basal cells) could be accumulated in metaphase over a 24-h period using vincristine as a stathmokinetic agent. The experiments reported here using mouse ventral tongue epithelium use double-labelling approaches to address the question: what proportion of the approximately 50% of the basal cells that are proliferating have a 24-h cell cycle and can therefore be labelled by a similar labelling protocol the following day? The results suggest a heterogeneity amongst the proliferating basal cells, similar to the heterogeneity proposed for the dorsal tongue epithelium. Although not all the basal component has been accounted for, the data presented here suggest that about 20% of the basal cells may have a cell cycle time of 24 h, about 30% appear to have a longer cell cycle time (48 or 72 h), while about 20% of the basal cells appear to be postmitotic maturing G1 cells, awaiting the appropriate signals for migration into the suprabasal layer.

Effect of myenteric denervation on intestinal epithelium proliferation and migration of suckling and weanling rats.

The effects of myenteric denervation on the cell kinetics of the intestinal epithelium of suckling and weanling rats were investigated. The myenteric plexus of an ileal segment was partially ablated by serosal application of benzalkonium chloride (BAC) in three groups of rats: those that underwent surgery at 13 days and were killed 15 (13/28-day-old) or 23 (13/36-day-old) days after treatment, and those that were operated at 21 days (21/36-day-old) and were killed 15 days after treatment. The extent of denervation was assessed in whole-mount preparations. The cell bodies of myenteric neurones were stained by NADH-diaphorase histochemical technique. Cell proliferation was estimated by the mitotic index (MI) and morphometric analysis of villus and crypt lengths using an image analysis system. Thickness of the muscle layers was also assessed by morphometry. Cell migration on the villi was estimated by the position of the leading labelled cell 24 h after tritiated thymidine injection. The number of neurones was reduced by around 80% in rats operated at 13 days, and reduced by 98% in those operated at 21 days. The thickness of the muscle layers was increased in all groups of treated animals. MI was significantly higher 15 days after BAC-treatment in the 13/28 group. Morphological changes in the intestinal mucosa were observed 15 days after BAC-treatment, when there was an increase in villus height (13/28 group) and crypt depth (13/28 and 21/36 groups). Cell migration rate was accelerated in the 21/36 group. No differences where found in the 13/36 group. These results show the strong effect of myenteric ablation on cell proliferation and migration in the ileal epithelium in the first 15 days of treatment in suckling and in weanling rats, and the subsequent recovery of intestinal mucosa homeostasis later on.

Neuron loss in the mouse hippocampus following prenatal injection of tritiated thymidine or saline

To investigate possible effects of injections of tritiated thymidine ([3H]dThd) into pregnant mice or the injection procedure itself on the proliferation of neuronal precursor cells in the fetuses, pregnant mice received intraperitoneal injections of either [3H]dThd or saline on embryonic days 12, 14, and 19, while their offspring remained untreated. A second group of dams was not injected but their male offspring received a subcutaneous injection of again either [3H]dThd or saline on postnatal day 10. Then total numbers of hippocampal pyramidal cells (areas CA1 to CA3) and granular cells (dentate gyrus) were determined stereologically for 20-day-old as well as for 80-day-old male pups. No significant differences were found for the mean total number of pyramidal cells between the investigated groups of pups. However, the mean total number of granular cells was significantly reduced in those groups in which the dams had received an intraperitoneal injection, irrespective of whether [3H]dThd or saline was injected. This revives the repeated warning in the literature to consider the effect of the injection procedure on the developing brain when interpreting possible effects of agents administered during pregnancy.

The length of the cycle of seminiferous epithelium in goats (Capra hircus)

This is the first report in literature showing the length of the seminiferous epithelium cycle in goats. In the present study, the duration of spermatogenesis was estimated using intratesticular injections of tritiated thymidine. Animals were castrated at 4 h, 7 days, and 11 days after injections. The duration of each spermatogenic cycle in goats is 10.6 ± 0.5 days (SEM). Considering that the total duration of spermatogenesis takes about 4.5 cycles of seminiferous epithelium, spermatogenesis was estimated to last 47.7 days. The approximate primary spermatocytes life span is 14.1 days, while spermiogenesis in goats lasts 14.9 days. Staging in goats was based on the tubular morphology, where 8 stages of the cycle are yielded for all species. The relative stage frequencies in goats, based on 400 seminiferous tubule cross sections for each animal were as follows: stage 1: 15.8 ± 1.0%; stage 2: 12.8 ± 0.5%; stage 3: 20.5 ± 0.9%; stage 4: 10.7 ± 0.7%; stage 5: 11.6 ± 0.6%; stage 6: 9.3 ± 1.1%; stage 7: 7.6 ± 0.4%; stage 8: 11.7 ± 0.6%. The pre-meiotic, meiotic and post-meiotic phases’ relative frequencies were 49.1%, 10.7% and 40.2%, respectively. The duration of spermatogenesis in goats is very similar to that found in rams.

Seminiferous epithelium cycle and its duration in capybaras (Hydrochoerus hydrochaeris)

Although capybara is the largest rodent in the world and largely distributed in Central and South America, there is no report in the literature concerning the cycle of seminiferous epithelium in this species. In the present study, the length of spermatogenic cycle was estimated using intratesticular injections of tritiated thymidine. Animals were sacrificed at 1 h, 8 days, and 17 days after injections. The duration of one spermatogenic cycle in capybaras is 11.9 ± 0.1 days (SEM). Spermatogenesis was estimated to last 53.6 days, when considering that the total duration of spermatogenesis takes about 4.5 cycles of seminiferous epithelium. The approximate life span of primary spermatocytes is 19.1 days, while spermiogenesis lasts 16.7 days. Staging in capybaras was based on the spermatid nuclei shape and location of spermatids, named tubular morphology method, which consists of 8 stages in all species. The relative stage frequencies in capybaras, based on the analysis of approximately 200 cross sections of seminiferous tubule for each of the ten animals were as follow: stage 1: 14.0 ± 1.5%; stage 2: 15.1 ± 1.0%; stage 3: 15.7 ± 1.1%; stage 4: 14.6 ± 1.1%; stage 5: 8.7 ± 0.7%; stage 6: 7.0 ± 0.7%; stage 7: 9.4 ± 0.9%; stage 8: 15.5 ± 1.0%. The pre-meiotic, meiotic and post-meiotic phases relative frequencies were 44.8%, 14.6% and 40.6%, respectively. Compared to most rodents investigated so far, the duration of spermatogenesis in capybaras is relatively long.

Progenitor cell cycling during hair cell regeneration in the vestibular and auditory epithelia of the chick.

We investigated nucleotide-labeling patterns during ongoing hair cell regeneration in the avian vestibular epithelium and during drug-induced regeneration in the avian auditory epithelium. For utricle experiments, post-hatch chicks received an injection of bromodeoxyuridine (BrdU) and were allowed to survive from 2 hours to 110 days after the injection. Utricles were fixed and immunoreacted to detect BrdU. The number of BrdU-labeled nuclei in the hair cell and support cell layers of the utricular sensory epithelium changes significantly between 2 hours and 110 days post-BrdU. At 2 hours, most labeled cells are isolated, while by 5-10 days, the majority of labeled cells are organized in pairs that are most frequently composed of a hair cell and a support cell. Pairs of labeled cells are seen as late as 110 days. Clusters of more than 3 labeled cells are uncommon at all time-points. The total number of labeled cells increases approximately 1.5-fold between 5 and 60 days post-BrdU. This increase is due primarily to a rise in the number of labeled support cells, and it is likely that it represents additional rounds of division by a subset of cells that were labeled at the time of the BrdU injection. There is a significant decrease in labeled nuclei in the hair cell layer between 60 and 110 days post-BrdU, suggesting that hair cells die during this period. To investigate support cell recycling in the drug-damaged auditory epithelium, we examined nucleotide double labeling after separate injections of BrdU and tritiated thymidine. A small number of support cells that incorporate BrdU administered at 3 days post-gentamicin treatment also label with tritiated thymidine administered between 17 and 38 hours later. We conclude that a small population of support cells recycles during regeneration in both the normal utricle and the drug-damaged basilar papilla.

Neurogenesis and Commitment of Corticospinal Neurons inreeler

In the homozygous (but not the heterozygous) reeler mutant, disruption of neuron migration leads to a major perturbation of the cortical environment that in turn could modify (1) the specification of neuronal fate and (2) the proliferation dynamics of cortical precursors. To investigate these issues, tritiated thymidine injections during cortical neurogenesis were coupled with postnatal injections of a retrograde tracer in the spinal cord to accurately measure the neurogenesis of corticospinal neurons in the heterozygous and homozygous mutant. The homozygous reeler shows (1) strict conservation of area-specific timetables of corticospinal neuron generation; (2) neurons with the appropriate birthdates show an enhanced probability of projecting to the spinal cord; (3) during early stages of corticogenesis, there is a reduced rate of neuron production followed at later stages by an increased rate of neuron production; and (4) these changes in the rate of neuron production were shown to be at least partially attributable to changes in the proportions of differentiative divisions. Taken together, our results show that in the developing cortex, the neurogenesis and specification of a given neuronal phenotype are partially controlled by the postmigratory compartment. On the other hand, neither areal identity nor the chronology of production of layer-specific neuronal phenotype seems to depend on the integrity of the cellular environment.

Duration of spermatogenesis and sperm transit time through the epididymis in the Piau boar

The Piau boar is a rustic breed of economical importance in Brazil. The duration of spermatogenesis and sperm transit through the epididymis in Piau boars was estimated using intratesticular injections of tritiated thymidine. Animals were sacrificed 1 h, 7 days, 14 days, 21 days, 34 days, and 36 days after injections. Each cycle of spermatogenesis in Piau boars lasts 9 ± 0.2 days. At least 9 days are necessary for spermatozoa to traverse the entire epididymis. Considering that the total duration of spermatogenesis takes about 4.5 seminiferous epithelium cycles, spermatogenesis was estimated to take 40.6 days. The primary spermatocytes life span is 13.5 days, while spermiogenesis in Piau boars lasts 14.5 days. Staging in Piau boars was based on tubular morphology system. The relative stage frequencies in these boars, based on approximately 1200 seminiferous tubule cross-sections for each animal, were as follows: stage 1, 11.7 ± 0.7%; stage 2, 14.3 ± 0.3%; stage 3, 5.4 ± 0.1%; stage 4, 12.1 ± 0.6%; stage 5, 9.6 ± 0.4%; stage 6, 17.2 ± 0.4%; stage 7, 15.4 ± 0.8%; and stage 8, 14.3 ± 0.9%. The duration of spermatogenic events and the relative stage frequencies in Piau boars differ slightly from those observed in improved swine breeds.

Using GADlacZ transgenic mice as a marker system for homotopic transplantation

Olfactory bulb (OB) transplantation is a well characterized model that has been widely used for studying neuronal plasticity and regeneration [G. Sekerková, Z. Katarova, E. Mugnaini, F. Joó, J.R. Wolff, S. Prodan, G. Szabó, Intrinsically labeled relay neurons of homotopic olfactory bulb transplants establish proper afferent and afferent synaptic connections with host neurons, Neuroscience, 80 (1997) 973–979 [10]; G. Sekerková, Z. Malatová, J. Orendáčová, T. Žigová, Transplantation of dorsal root ganglion into the olfactory bulb of neonatal rats: a histochemical study, Restor. Neurol. Neurosci., 6 (1993) 1–8 [11]; E. Račeková, I. Vanický, T. Žigová, Correlation of functional alteration with lesion extent after olfactory bulbectomy in rats, Int. J. Neurosci., 79 (1994) 13–20 [12]; T. Žigová, P.P.C. Graziadei, A.G. Monti Graziadei, Olfactory bulb transplantation into the olfactory bulb of neonatal rats: an autoradiographic study, Brain Res., 539 (1991) 51–58 [13]]. In previous studies, the OB grafts have been routinely labeled by tritiated thymidine [S.M. Onifer, L.A. White, S.R. Whittemore, V.R. Holets, In vitro labelling strategies for identifying primary neural tissue and neuronal cell line after transplantation in the CNS, Cell Transplant., 2 (1993) 131–149 [7]; [13]] allowing distinction of graft from the surrounding tissue by the presence of silver grains over the cell nuclei of the transplant. However, this approach has some disadvantages, namely: partial or insufficient labeling of a defined neuronal subclasses due to the length of the period of their generation, variation in the number of labeled cells due to differences in the gestation stage between individual embryos at the time of i.p. injection of tritiated thymidine, inability to follow the dendritic arborization and axonal outgrowth of the transplanted neurons or to detect directly their actual synaptic contacts, and finally, the need to work with radioactive isotopes. In this paper, we describe an alternative approach, in which the donor OBs in a homotopic OB transplantation were derived from transgenic mice carrying the bacterial gene lacZ under control from the regulatory region of GAD67 gene. In these mice, β-galactosidase (β-gal), encoded by lacZ is stably, ectopically expressed in the vast majority of mitral/tufted (M/T) cells of the OB and served as their intrinsic cellular marker in the OB transplant. By using a simple histochemical reaction for β-gal or immunocytochemistry with anti-β-gal antibody, we could detect the cell bodies and processes of the donor M/T cells and their synaptic contacts with host neurons after long-term survival using both light and electron microscopy. Given the great number of existing transgenic mouse lines that express in the nervous system, this approach may have an even wider application in neural transplantation. Themes: Development and regeneration Topics: Transplantation

Cell proliferation and migration in the jejunum of suckling rats submitted to progressive fasting.

Cell proliferation and migration in the intestinal crypts, and cell migration in the villus are controlled by different mechanisms in adult rats. In the present study, weanling rats and fasting rats were used to quantitatively study the correlation of cell cycle parameters and epithelial cell migration in crypts and intestinal villi. Eighteen-day-old rats received a single injection of tritiated thymidine [3H]TdR (23:00 h); half of the pups were submitted to fasting 5 h earlier. Cell proliferation was determined in radioautographs of jejunal crypts, on the basis of the labeling indices (LI) taken 1, 8, 13 and 19 h after [3H]TdR. The results showed that the labeling index did not differ 1 h or 19 h after [3H]TdR between the fed (38.7% or 48%) and fasting groups (34.6% or 50.4%). The modified method of grain count halving indicated that cell cycle time did not differ between fed (16.5 h) and fasting rats (17.8 h); the growth fraction, however, had lower values in fasting (59%) than in fed rats (77%). Cell migration in the crypt, estimated by the LI obtained for each cell position, did not change with treatment. As for the villi, the cell migration rate was significantly retarded by 3 cell positions (8%). These results suggest that the cell migration in the villi of weanling pups does not depend directly on the cell proliferation and migration in the intestinal crypt, but is directly affected by the absence of food in the lumen.

Neurogenesis in the olfactory bulb of the frogXenopus Laevis shows unique patterns during embryonic development and metamorphosis

We determined the time of origin of neurons in the olfactory bulb of the South African clawed frog,Xenopus laevis. Tritiated thymidine injections were administered to frog embryos and tadpoles from gastrulation (stage 11/12) through metamorphosis (stage 65), paraffin sections were processed for autoradiography, and the distribution of heavily and lightly labeled cells was examined. In the ventral olfactory bulb, we observed that the mitral cells were born as early as stage 11/12 and continued to be generated through the end of metamorphosis. Interneurons (periglomerular and granule cells) were not born in the ventral bulb until stage 41, and birth of these cells also continued through metamorphosis. Labeled cells were observed in the accessory olfactory bulb, beginning at stage 41. In contrast, the cells of the dorsal olfactory bulb were not born until the onset of metamorphosis (stage 54); at this stage in the dorsal bulb, the genesis of mitral cells, interneurons, and glial cells completely overlapped. The results indicate that olfactory axon innervation is not necessary to induce early stages of neurogenesis in the ventral olfactory bulb. On the other hand, the results on the dorsal olfactory bulb are consistent with the hypothesis that innervation from new or transformed sensory neurons in the principal cavity induces neurogenesis in the dorsal bulb.

Correlations between Terminal Mitosis and Differentiated Fate of Retinal Precursor Cellsin Vivoandin Vitro:Analysis with the “Window-Labeling” Technique

We have investigated with high resolution the timing of retinal precursor cell commitment to specific differentiated fates, using anin ovomodification of thein vitro“window-labeling” technique (A. M. Repka and R. Adler,J. Histochem. Cytochem.40, 947–953, 1992a). The method involves an initial injection of tritiated thymidine into chick embryos, followed a specified number of hours later by an injection of bromodeoxyuridine (BrDU); cells born during this period are identified by being labeled with thymidine but not with BrDU. We used this method to determine, in a narrow region adjacent to the choroid fissure, the fate of cells born during defined 5-hr intervals between Embryonic Days (ED) 4–8. All the cohorts gave rise to heterogenous differentiated populations, indicating that time of cell birth is not a major cell fate determinant. A progressive restriction in the developmental potential of precursor cells, however, was suggested by the observed decrease in the number of different populations generated during each 5-hr period from ED 4 to 8, and supported also by dissociated cell culture experiments investigating the fate of cells born at different developmental stages. Microenvironmental influences were testedin vitrousing cells windowed-labeledin ovofor 5 hr on ED 5. After spending at least 72 hr within the retina before their isolation for culture, these cells mimicked theirin vivofate, giving rise predominantly to nonphotoreceptor neurons; a completely different behavior was observed when the cells were isolated after shorter exposures to the retinal microenvironment, when they gave rise predominantly to photoreceptors. Together with data demonstrating that differential cell death cannot account for these results, our results are consistent with the hypothesis that cell fate determination occurs after the time of terminal mitosis.