A study of diurnal proliferative activity in tumour and small intestine of C3H mice bearing a transplanted mammary carcinoma

Diurnal changes in proliferative activity were investigated in tumour and small intestinal epithelium of mice bearing a transplanted mammary carcinoma. In addition to mitotic and labelling index studies, the metaphase-arrest technique with vincristine (VCR) was employed. In the tumour there was no clear evidence of a significant diurnal rhythm in proliferative activity but in the small intestinal epithelium such a rhythm was clearly demonstrated. A higher cell production rate (kB) measured by metaphase-arrest and higher labelling and mitotic indices were seen in the mid to late part of the dark period. The peak mitotic index was seen 3 to 6 h after the labelling peak in the small intestine. The basal third of the crypt which is believed to include the stem cell compartment of this tissue showed larger diurnal fluctuations in both labelling index and kB than the rest of the proliferative compartment.     

The cell kinetics of the epidermis and follicular epithelium of the rat: variations with age and body site

The skin from rats of differing age was used to quantify variations in the cell kinetics of the epidermis and the follicular epithelium of different body sites. Four parameters were assessed, namely the basal cell density (BCD), the labelling index (LI), the duration of DNA synthesis (ts) and the basal cell turnover time (tT). The BCDs of the epidermis of the dorsum and the upper surface of the foot were similar in rats of 7, 14 and 52 weeks of age, but there was an indication of a progressive decline with increasing age in the BCD of the epidermis of the ear and tail. There were no age-related changes in the length of ts in any of the four body regions. The rate of cell proliferation, as indicated by the values of the LI and tT, was relatively rapid in the epidermis of the dorsum, foot and tail of rats aged 7 weeks (LI greater than 12%; tT less than 80 h). In rats aged 14 weeks this rate of proliferation was maintained in the epidermis of the dorsum. However, in the foot and tail the rate of cell proliferation was decreased (LI less than 10%; tT greater than 85 h). A fall in the rate of proliferation of the epidermis of the dorsum was only seen in 52-week-old animals. In these animals the rates of proliferation in the foot and tail were similar to those at the age of 14 weeks. In the epidermis of the ear there was no appreciable change in the rate of cell proliferation with age. The values of the cell kinetic parameters varied in the different body sites. For example, in 52-week-old animals values for tT were relatively short in the epidermis of the tail and foot and appreciably longer in the epidermis of the dorsum and ear. Considered overall, values for the cell kinetic parameters of the epidermis were comparable with those for the follicular epithelium. The only major differences between the epidermis and the follicular epithelium were in the upper surface of the foot at 7 weeks of age, and in the tail at 7 and 14 weeks of age, where the LI was higher and the tT shorter in the epidermis than in the follicular epithelium. The relevance of the observed age- and body-site-related variations in the cell kinetics of the epidermis are discussed in relation to previously described differential changes in the radiosensitivity of the skin in this strain of rat.     

CIRCADIAN RHYTHMS OF PRESUMPTIVE STEM CELLS IN THREE DIFFERENT EPITHELIA OF THE MOUSE

Variation in the percentage of labelled cells (LI), mitoses (MI) and apoptosis (AI: i.e. shrinkage necrosis) have been studied throughout a 24 hr period (40 min after labelling with ³H-TdR) for tongue epithelium, epidermis and intestinal epithelium in the mouse. A room with reversed light cycle was used to obtain data for half of the 24 hr period. All three tissues showed marked variations in LI with peak values between 24.00 and 03.00 hours. In the intestine a maximum value for MI was observed 3-6 hr after that for LI and with a maximum value for AI slightly later. In all three epithelia the circadian rhythm was most striking in cells at positions which can be correlated with presumptive stem cell activity; e.g. in the crypts the labelling and mitotic peaks reflecting a circadian rhythm were most clearly distinguishable at the basal part of the crypts. These observations are discussed in relation to the validity of various proliferative models.     

The cell kinetics of the epidermis and follicular epithelium of the rat: variations with age and body site

Abstract The skin from rats of differing age was used to quantify variations in the cell kinetics of the epidermis and the follicular epithelium of different body sites. Four parameters were assessed, namely the basal cell density (BCD), the labelling index (LI), the duration of DNA synthesis (t_s) and the basal cell turnover time (t^T). The BCDs of the epidermis of the dorsum and the upper surface of the foot were similar in rats of 7, 14 and 52 weeks of age, but there was an indication of a progressive decline with increasing age in the BCD of the epidermis of the ear and tail. There were no age-related changes in the length of t_s in any of the four body regions. The rate of cell proliferation, as indicated by the values of the LI and t_T, was relatively rapid in the epidermis of the dorsum, foot and tail of rats aged 7 weeks (LI > 12%; t_T < 80 h). In rats aged 14 weeks this rate of proliferation was maintaned in the epidermis of the dorsum. However, in the foot and tail the rate of cell proliferation was decreased (LI < 10%; t^T > 85 h). A fall in the rate of proliferation of the epidermis of the dorsum was only seen in 52-week-old animals. In these animals the rates of proliferation in the foot and tail were similar to those at the age of 14 weeks. In the epidermis of the ear there was no appreciable change in the rate of cell proliferation with age. The values of the cell kinetic parameters varied in the different body sites. For example, in 52-week-old animals values for t_T were relatively short in the epidermis of the tail and foot and appreciably longer in the epidermis of the dorsum and ear. Considered overall, values for the cell kinetic parameters of the epidermis were comparable with those for the follicular epithelium. The only major differences between the epidermis and the follicular epithelium were in the upper surface of the foot at 7 weeks of age, and in the tail at 7 and 14 weeks of age, where the LI was higher and the t_T shorter in the epidermis than in the follicular epithelium. The relevance of the observed age- and body-site-related variations in the cell kinetics of the epidermis are discussed in relation to previously described differential changes in the radiosensitivity of the skin in this strain of rat.     

Pig epidermis: a cell kinetic study

The basal cell density (BCD), labelling index (LI), duration of DNA synthesis (TS) and cell cycle time (TC) have been calculated for the epidermis of pigs in the age range 4-27 months. The BCD declined progressively from 143.4 +/- 6.5 cells/mm at 4 months to 128.8 +/- 8.3 cells/mm at 15 months, whereafter the values showed little change. There was a small decrease in LI with increasing age, from 7.9 +/- 1.5% at 4 months to 5.9 +/- 1.0% at 27 months. However, the change to housing animals outdoors as compared with indoors had a greater effect on the LI (approximately 10%). Severe weathering in the skin of animals housed outdoors resulted in a very high LI (approximately 20%). Neither TS or TC varied significantly with age. TS was within the range 8.8-9.2 hr and TC 127-161 hr. In animals housed outdoors TC was reduced relative to animals housed indoors. The BCD and TS were not affected by housing conditions. The kinetic parameters investigated in the pig were similar to those reported for man.     

Circadian variations in influx and efflux of the S phase in a partially synchronized cell system Double-labelling with [3H]thymidine in the epithelium of the hamster cheek pouch

Abstract. By means of a double-labelling experiment, circadian variations in the kinetic parameters of the S phase of the hamster cheek pouch epithelium were studied. The evaluation of the experiment included a recently developed correction for deviations from the strict pulse interpretation of the labelling technique.
Pronounced circadian variations were found in S phase influx and efflux; the diurnal mean of both was estimated as 0.5%/hr, when based on measurements of all nucleated epithelial cells. Variations in S phase influx seem mainly responsuble for the diurnal variation in cell proliferation, although diurnal variation in DNA synthesis rate, and thus in mean transit time, was also found. The increases in LI and influx were closely correlated and related to the beginning of the dark period.
A circadian variation in cell number was also observed.     

Cell population kinetics in pig epidermis: further studies

The cell population kinetics of the epidermis were studied in 4-month-old pigs. Mitotic figures were confined to the basal cell (L1) and the first suprabasal cell layer (L2). The mitotic index (MI) was 0.17 +/- 0.04% for L1 and 0.08 +/- 0.03% for L2. Labelled nuclei were distributed throughout the viable epidermis, the majority (79.1 +/- 1.1%) were in L1 with 19.5 +/- 1.2% in L2. The labelling indices (LI) in layers L1 and L2 were 7.1 +/- 0.4% and 3.4 +/- 0.1%, respectively. After labelling with two injections of tritiated thymidine [3H]TdR separated by 90 min, the LI increased to 8.2 +/- 0.3% in L1 and to 4.0 +/- 0.2% in L2. This increased labelling confirmed that cell proliferation occurs in both layers, L1 and L2, of the epidermis. The cell production rate (K) in L1 and L2 had an upper limit of 10.7 +/- 1.0 and 6.2 +/- 1.8 cells per 1000 cells per hour respectively. The cell flow rate per hour (cell flux), into and out of the DNA synthesis phase (S), and the duration of DNA synthesis were determined from double-labelling studies with [3H]TdR and [14C]TdR. The cell flux into and out of S was identical and was calculated as 0.6 +/- 0.1%/hr (L1) and 0.5 +/- 0.1%/hr (L2). Values for tS varied from 8 to 10 hr. The cell turnover times (tT) were in the range 89-129 hr and 180-261 hr for L1 and L2, respectively. Log normal curves were fitted to the fraction labelled mitoses data for L1 and L2. Values for tS for cells in L1 and L2 were 9.8 hr and 11.9 hr, respectively. tG2 + 1/2tM was 7.2 hr in L1 and 9.1 hr in L2.     

Cell population kinetics in the epithelium of the colon of the male rat.

The present study was undertaken in order to try to define some of the kinetic parameters in the colonic mucosa of normal Wistar rats. Preliminary observations showed considerable morphological differences in the mucosa from site to site along the length of the colon. In particular the height of the crypts (measured in cells) was variable. In addition labelling index studies demonstrated dramatic variations in the distribution of labelling along the length of the crypts from site to site in the bowel. A single site in the descending colon was selected for more detailed study using a stathmokinetic agent, vincristine, and the continous labelling technique with tritiated thymidine. The results of these investigations suggest that there exists at the base of the crypt a subpopulation of cells cycling more slowly than the cells in the rest of the proliferative compartment. Growth fraction appears to fall with rising cell positions within the crypt.     

Evidence for discrete cell kinetic subpopulations in mouse epidermis based on mathematical analysis

Abstract. Continuous (repeated) labelling studies in mouse epidermis indicate that nearly all cells are labelled after about 100 hr. Percentage labelled mitoses studies ([³H]TdR at 15.00 and 03.00 hours) have a first peak that does not reach 100% and has a half-width of about 10 hr. Small second and third peaks can be detected at about 90 and 180 hr, respectively. The changes with time in the number of labelled cells show a difference dependent on the time of day of [³H]TdR administration. Both curves show an early doubling in labelled cells which then decline, forming a peak of labelled cells. A second peak occurs at about 120 hr. This is followed by a progressive decline with no further peaks until values of about 1% labelling are obtained at 340 hr.
These experiments have been investigated mathematically. A computer programme has been devized that permits all three types of experiments to be analysed simultaneously. More importantly, it can analyse situations with a heterogeneity in cell cycle parameters in all proliferative subpopulations.
Various models for epidermal cell replacement have been considered. The data as a whole can best be explained if the basal layer contains at least two distinct subpopulations of cells and an exponentially decaying post-mitotic population with a half-life of about 30 hr. The proliferative sub-populations must be characterized by near integer differences in the length of cycle, the precursor (stem) compartment having the longer cycle. An inverse relationship is required for the length of S, i.e. the shortest time for the stem cells.
A full range of cell kinetic parameters can be calculated and are tabulated for the most appropriate model system which is one involving three transit proliferating subpopulations.     

Pig Epidermis: A Cell Kinetic Study

The basal cell density (BCD), labelling index (LI), duration of DNA synthesis (T_s) and cell cycle time (T_c) have been calculated for the epidermis of pigs in the age range 4–27 months. the BCD declined progressively from 143.4 ± 6.5 cells/mm at 4 months to 128.8 ± 8.3 cells/mm at 15 months, whereafter the values showed little change. There was a small decrease in LI with increasing age, from 7.9 ± 1.5% at 4 months to 5.9 ± 1.0% at 27 months. However, the change to housing animals outdoors as compared with indoors had a greater effect on the LI (～10%). Severe weathering in the skin of animals housed outdoors resulted in a very high LI (～20%). Neither T_s or T_c varied significantly with age. T_s was within the range 8.8–9.2 hr and T_c 127–161 hr. In animals housed outdoors T_c was reduced relative to animals housed indoors. the BCD and T_s were not affected by housing conditions. the kinetic parameters investigated in the pig were similar to those reported for man.     

Proliferation in murine epidermis after minor mechanical stimulation. Part 2. Alterations in keratinocyte cell cycle fluxes

We have recently shown that a mild mechanical irritation (tape strip) of the epidermis on the back skin of adult mice induces a strong and long lasting increase in proliferative activity and cell production. This was revealed by following the fate of 3HTdR-pulse labelled cells within the basal and suprabasal layers. To obtain further insight into the dynamics of cell kinetic changes we also performed double labelling experiments with 3HTdR and BrdUrd at various times after tape stripping. The technique for analysing the data had to account for a non stationary cell flux. A novel biometrical technique was developed which provides parameter estimates on the S-phase duration, the cell cycle duration and a parameter characterizing the degree of nonstationarity. When applied to the mechanically irritated epidermis we observed that the cell flux through the S-phase in the basal layer was accelerated by a factor of 10 between 18 and 36 h post tape strip. This activation declined slightly in the subsequent days and remained 4-6 fold higher than in the normal steady state for over 7 days post tape strip. The duration of the S-phase was 3-5 h and showed little variation. We conclude that mild mechanical irritation only affecting the stratum corneum has major stimulatory effects on the cell kinetics of proliferative keratinocytes in the basal layer of the epidermis indicating the existence of a powerful regulatory mechanism.     

Proliferation in murine epidermis after minor mechanical stimulation. Part 1. Sustained increase in keratinocyte production and migration

It was our objective to obtain an insight into the details and dynamics of the cell proliferative changes following minor barrier disruption, the mechanisms of recovery, and their regulation. Hair of the dorsal area of DBA2-mice was removed and the epidermis was tape stripped. Tritiated thymidine was injected into groups of mice at daily intervals thereafter. Labelling and nuclear densities were measured at several time intervals later in the various epidermal strata to characterize cell production and cell fluxes through the tissue. A dramatic proliferative response was observed at 24 h when the labelling density increased more than sixfold in the basal layer. Labelled cells rapidly appeared in suprabasal layers within a few hours in large quantities while this process took over 2 days in normal skin. Some cycling cells were also found in the suprabasal layer (pulse labelling at 24 h) in contrast with the controls. The cellular flux through the suprabasal layers was drastically (20-fold) increased and the transit time was shortened. Although the nuclear density in the basal layer showed only moderate changes it increased four-fold in the suprabasal layer within 5 days. A kinetic model analysis suggested that the cell cycle time of proliferative cells dropped from a normal value of about 200 h to less than 12 h post tape strip. After 7 days, the proliferative activation still persisted, even though at 3 days post tape strip the stratum corneum had been re-established. Hence, a mild mechanical alteration with removal of some parts of the cornified layer in mouse backskin epidermis triggers a huge proliferative response with massive overproduction of cells that lasts at least 7 days. Our findings suggest that the re-establishment of the cornified layer does not immediately shut down cell proliferation and that more complex, slower (long-term) regulatory processes are involved.     

Cell population kinetics in pig epidermis: further studies

Abstract. The cell population kinetics of the epidermis were studied in 4-month-old pigs. Mitotic figures were confined to the basal cell (L1) and the first suprabasal cell layer (L2). The mitotic index (MI) was 0.17 ± 0.04% for L1 and 0.08 ± 0.03% for L2. Labelled nuclei were distributed throughout the viable epidermis, the majority (79.1 ± 1.1%) were in L1 with 19.5 ± 1.2% in L2. The labelling indices (LI) in layers L1 and L2 were 7.1 ± 0.4% and 3.4 ± 0.1%, respectively. After labelling with two injections of tritiated thymidine [³H]TdR separated by 90 min, the LI increased to 8.2 ± 0.3% in L1 and to 4.0 ± 0.2% in L2. This increased labelling confirmed that cell proliferation occurs in both layers, L1 and L2, of the epidermis.
The cell production rate ( K ) in L1 and L2 had an upper limit of 10.7 ± 1.0 and 6.2 + 1.8 cells per 1000 cells per hour respectively. The cell flow rate per hour (cell flux), into and out of the DNA synthesis phase (S), and the duration of DNA synthesis were determined from double-labelling studies with [³H]TdR and [¹⁴C]TdR. The cell flux into and out of S was identical and was calculated as 0.6 ± 0.1%/hr (L1) and 0.5 ± 0.1%/hr (L2). Values for t _S varied from 8 to 10 hr. The cell turnover times ( t _T) were in the range 89–129 hr and 180–261 hr for L1 and L2, respectively.
Log normal curves were fitted to the fraction labelled mitoses data for L1 and L2. Values for t _S for cells in L1 and L2 were 9.8 hr and 11.9 hr, respectively. t _G2+ 1/2 t _M was 7.2 hr in L1 and 9.1 hr in L2.     

Heterogeneity in the mouse epidermal cell cycle analysed by computer simulations

Abstract. Different sets of cell kinetic data obtained over many years from hairless mouse epidermis have been simulated by a mathematical model including circadian variations. Simulating several independent sets of data with the same mathematical model strengthens the validity of the results obtained. The data simulated in this investigation were all obtained with the experimental system in a state of natural synchrony. The data include cell cycle phase distributions measured by DNA flow cytometry of isolated epidermal basal cells, fractions of tritiated thymidine ([³H]TdR) labelled cells within the cell cycle phases measured by cell sorting at intervals after [³H]TdR pulse labelling, bivariate bromodeoxyuridine (BrdUrd)/DNA data from epidermal basal cells isolated at intervals after pulse labelling with BrdUrd, mitotic rate and per cent labelled mitosis (PLM) data from histologic sections. The following main new findings were made from the simulations: the second PLM peak observed at about 35 h after pulse labelling is hardly influenced by circadian variations; the peak is mainly determined by persisting synchrony of a rapidly cycling population with a G₁-duration (T_G1) of 20 h to 30 h; and there is a highly significant population of slowly cycling G₁-cells (G_1σ). However, no significant circadian variations were found in the number of these cells.     

Trophic effects of essential fatty acids on pig skin

The daily oral administration of 3 ml of two oils (So-5407 and So-1129) containing essential fatty acids (EFAs) for 16 weeks resulted in a transient increase in cell proliferative activity in the skin of female Large White pigs. The So-5407 oil contained 7% gamma-linolenic acid (GLA) whereas So-1129 was an oil of similar composition, but with no GLA. Hyperplasia of the epidermis was observed after the administration of both oils, and this was characterized by an increase in the size of the rete pegs. The maximum effect occurred at 4 weeks after the start of oil administration, at which time the number of viable cell layers had increased by a factor of approximately 1.5, and mean epidermal thickness (excluding the stratum corneum) was approximately 40% greater than that of the epidermis prior to oil administration. There was a marked increase in the labelling index (LI) of the basal cell layer of the epidermis in pigs receiving So-5407. Maximum LIs were quantified at 4 weeks after the start of administration and were 18.8 ± 1.3% and 13.1 ± 1.7% for pigs receiving So-5407 and So-1129, respectively. After this time the LI declined prgressively and had returned to values within normal limits (P>0.1) by 8 weeks after the start of administration of both oils. A similar pattern of change in the LI was seen in the follicular epithelium, although the peak values at 4 weeks after the start of oil administration of 12.2 ± 1.8% and 10.8 ± 0.9 for the groups receiving So-5407 and So-1129, respectively, were lower than in the epidermis. Labelled cells were also counted in the papillary dermis and maximum values were again seen at 4 weeks after the start of oil administration. Of the two oils, So-1129 had the greatest effect, with the number of labelled cells in the papillary dermis being a factor of three to four-fold higher than in skin prior to oil administration, between 2 and 12 weeks after the start of administration.     

Circadian variations in the DNA synthesis of the rat corneal epithelium. A study using double labelling with tritiated thymidine

A prominent circadian rhythm was found in the labelling indices (LI) of the peripheral rat corneal epithelium and of the adjacent conjunctival epithelium, while almost no diurnal variation was found in the central area. Application of a double labelling technique indicated that there are rhythmic pulses of high and low influx of cells into the S phase and similar pulses of efflux of cells from the S phase. Results of the study indicate that there are different cohorts of cycling cells all over the rat corneal epithelium. Cells belonging to a rapidly proliferating cohort are observed in the peripheral cornea. There is a gradual reduction in the fraction of labelled DNA-synthesizing cells towards the centre. The considerably lower fraction of cells taking up tritiated thymidine (3H)TdR in the central cornea may be due to a higher fraction of basal cells having reached higher levels of differentiation. This may result in a shift from the salvage to the de novo pathway. The slowly proliferating cohort seems to have a prolonged S phase duration and displays practically no diurnal variation in the LI. The DNA-synthesizing cells belonging to this latter cohort probably use the salvage pathway for DNA synthesis resulting in uptake of (3H)TdR all over the cornea. The LI is thus not a reliable indicator of cell proliferation in the corneal epithelium, due both to the heterogeneity of the cell proliferation, and in particular due to the lack of labelling of the centrally located DNA-synthesizing cells. To what extent these properties may also be present in other proliferating tissues with different levels of differentiations, may be questioned.     

Basal Keratinocytes Contribute to All Strata of the Adult Zebrafish Epidermis

The epidermis of terrestrial vertebrates is a stratified epithelium and forms an essential protective barrier. It is continually renewed, with dead corneocytes shed from the surface and replaced from a basal keratinocyte stem cell population. Whilst mouse is the prime model system used for epidermal studies, there is increasing employment of the zebrafish to analyse epidermis development and homeostasis, however the architecture and ontogeny of the epidermis in this system are incompletely described. In particular, it is unclear if adult zebrafish epidermis is derived entirely from the basal epidermal stem cell layer, as in the mouse, or if the most superficial keratinocyte layer is a remnant of the embryonic periderm. Furthermore, a relative paucity of cellular markers and genetic reagents to label and manipulate the basal epidermal stem cell compartment has hampered research. Here we show that the type I keratin, krtt1c19e, is a suitable marker of the basal epidermal layer and identify a krtt1c19e promoter fragment able to drive strong and specific expression in this cell type. Use of this promoter to express an inducible Cre recombinase allowed permanent labelling of basal cells during embryogenesis, and demonstrated that these cells do indeed generate keratinocytes of all strata in the adult epidermis. Further deployment of the Cre-Lox system highlighted the transient nature of the embryonic periderm. We thus show that the epidermis of adult zebrafish, as in the mouse, derives from basal stem cells, further expanding the similarities of epidermal ontogeny across vertebrates. Future use of this promoter will assist genetic analysis of basal keratinocyte biology in zebrafish.     

Quantification of morphologic, cytologic, and kinetic parameters of unirradiated swine skin: a histologic model

The gross changes of erythema and desquamation produced by irradiation indicate population and functional variations occurring in the epidermis, microvasculature, and dermis. However, the parameters do not distinguish the individual population kinetics. This study determines multiple histologic and cell kinetic parameters of unirradiated swine skin. The prickle cell layer exists as a shell three to six cells thick. The proliferative basal layer exists as a confluent monolayer with 2031 +/- 48 cells/cm; it has a growth fraction of 1, an average cell generation time of 12.3 +/- 2.4 days, a TS of 9.4 +/- 2.9 hr, and a TS + T1/2M of 17 to 19 hr. The labeling index is 3.9 +/- 0.1% with a diurnal variation having a 5% peak at 1800 hr and a 2.5% nadir at 0900-1100 hr. The mitotic index varies from 1.7 to 3.2% and has no clear-cut diurnal variation. These values are similar to those available for man. These data and those published previously are utilized to define a histologic model of the irradiated epidermal cell renewal system. These measured values are compared with those derived from analysis of available time-dose isoeffect data using a single-hit multitarget and a linear-quadratic model. The derived dose-survival curve is steeper with a D0 between 97 and 255 rad compared to the measured value of 337 rad.     

THE KINETICS OF CELL PROLIFERATION IN THE TRACHEOBRONCHIAL EPITHELIA OF RATS WITH AND WITHOUT CHRONIC RESPIRATORY DISEASE

Labelling indices of the tracheobronchial epithelia of conventionally-derived rats with chronic respiratory disease (CRD) and minimal-disease rats without CRD have been determined. The duration of the DNA synthesis phase (t_s) computed from the percentage of mitoses labelled at various intervals of time after injection of tritiated thymidine was 7 hr: t_G2 was 3.5 hr. Using the measured value of t_s and the labelling indices, the mean turnover times of the tracheobronchial epithelia in three groups of six 5-week-old conventionally-derived rats were calculated to be 11.2, 14.6 and 22.4 days, while in similar groups of 5-week-old minimal-disease rats the turnover times were found to be 24.3, 36.5 and 41.6 days. The majority of cell divisions in the tracheobronchial epithelium of these minimal disease rats were probably required for growth rather than renewal. The mean turnover time of this tissue in 5-week-old Syrian hamsters was 73 days. The cells of the rat tracheobronchial epithelium have been classed as basal or superficial, depending on their shape and proximity to the basement membrane. The mean turnover time of the basal cells in 5-week-old minimal-disease rats was 11.7 days calculated from labelling indices. The migration method of Brown & Oliver (1968) gave a similar value for the basal cells in minimal-disease rats, and a value of 9.5 days for the basal cells in a group of conventionally-derived rats. The mean turnover time in the latter was only 5.4 days if two rats with tracheobronchitis were included. Consideration of the slow rate of fall in mean grain count over labelled cells at intervals of time after labelling and the calculated turnover times suggests that the proliferative fraction of the basal cell population is close to unity. Well-labelled cells were still present in both basal and superficial populations in the minimal-disease rats at 10 days after labelling. The marked effects of CRD on cell proliferation in this epithelium are emphasized and the significance of this in relation to published work is discussed.     

The spatial relationship between stem cells and their progeny in the basal layer of human epidermis: a new view based on whole-mount labelling and lineage analysis

In order to examine the spatial organisation of stem cells and their progeny in human epidermis, we developed a method for whole-mount epidermal immunofluorescence labelling using high surface beta1 integrin expression as a stem cell marker. We confirmed that there are clusters of high beta1 integrin-expressing cells at the tips of the dermal papillae in epidermis from several body sites, whereas alpha6 integrin expression is more uniform. The majority of actively cycling cells detected by Ki67 or bromodeoxyuridine labelling were found in the beta1 integrin-dull, transit amplifying population and integrin-negative, keratin 10-positive cells left the basal layer exclusively from this compartment. When we examined p53-positive clones in sun-exposed epidermis, we found two types of clone that differed in size and position in a way that was consistent with the founder cell being a stem or transit amplifying cell. The patterning of the basal layer implies that transit amplifying cells migrate over the basement membrane away from the stem cell clusters. In support of this, isolated beta1 integrin-dull keratinocytes were more motile on type IV collagen than beta1 integrin-bright keratinocytes and EGFP-labelled stem cell clones in confluent cultured sheets were compact, whereas transit amplifying clones were dispersed. The combination of whole-mount labelling and lineage marking thus reveals features of epidermal organisation that were previously unrecognised.