Changes In Cell Height Research Articles

Mechanisms of cell height changes that mediate epithelial invagination.

Epithelial invagination is a morphogenetic process that converts flat cell sheets into tubular structures and contributes to the formation of three-dimensional organs during development. Because the cells in tubular structures have smaller apical than basal surfaces, apical constriction is thought to be critical for the process of epithelial invagination. In addition, the invagination process is also accompanied by cell elongation, followed by cell shortening and basal expansion. While the mechanisms involved in apical constriction have been well-characterized, recent technical advances are just beginning to unravel the mechanisms involved in cell height control, which include cytoskeletal changes, cortical tension generation, cell adhesion, and cytoplasmic flow. Furthermore, cell height changes associated with mitosis and apoptosis have recently been shown to contribute to epithelial invagination. To develop a comprehensive understanding of epithelial invagination, it is important to elucidate the mechanisms that mediate cell shape changes and facilitate their coordination. In this review, we summarize the recent advances in this field, focusing on the mechanisms that control cell height.

Z-scan fluorescence profile deconvolution of cytosolic and membrane-associated protein populations

This study introduces a technique that characterizes the spatial distribution of peripheral membrane proteins that associate reversibly with the plasma membrane. An axial scan through the cell generates a z-scan intensity profile of a fluorescently labeled peripheral membrane protein. This profile is analytically separated into membrane and cytoplasmic components by accounting for both the cell geometry and the point spread function. We experimentally validated the technique and characterized both the resolvability and stability of z-scan measurements. Furthermore, using the cellular brightness of green fluorescent protein, we were able to convert the fluorescence intensities into concentrations at the membrane and in the cytoplasm. We applied the technique to study the translocation of the pleckstrin homology domain of phospholipase C delta 1 labeled with green fluorescent protein on ionomycin treatment. Analysis of the z-scan fluorescence profiles revealed protein-specific cell height changes and allowed for comparison between the observed fluorescence changes and predictions based on the cellular surface area-to-volume ratio. The quantitative capability of z-scan fluorescence profile deconvolution offers opportunities for investigating peripheral membrane proteins in the living cell that were previously not accessible.

A Novel AFM-MEA Platform for Studying the Real Time Mechano-Electrical Behavior of Cardiac Myocytes

AbstractWe present a novel experimental platform based on a combined Atomic Force Microscopy (AFM) and Micro-Electrode Array (MEA) set-up. We have used it to measure minimal changes in the morphological/mechanical properties of electrically active cell cultures as well as to measure the changes in the extracellular electrical activity when a single cell is stimulated by means of the AFM tip. In particular, we studied the dynamical changes in cell elasticity of embryonic rat cardiac myocytes along the contraction-relaxation cycle. Applying high load indentations, we also recorded the effects of mechanical stimulations on the cell electrophysiology. The dynamic elastic modulus of the cell related to the contraction-relaxation cycle reveals a temporal behavior that closely follows the changes in cell height. Observed values of dynamic elastic modulus at a maximum indentation depth of 1500nm varied between 8.93 ± 0.78 kPa during systolic (contraction) phase and 4.26 ± 0.47 kPa during diastolic (relaxation) phase. Induced electrophysiological responses were observed when applying loads in the range 40-150 nN. The probability P of recording an induced electrical response (P = 0.16 for a maximum load of 100nN) increased with the maximum applied load. Pulling-like stimulations due to the tip-cell adhesion could also evocate electrical responses.

Atomic force microscopy study of germination and killing of Bacillusatrophaeus spores

Bacterial spores such as Bacillus atrophaeus are one of the most resistant life forms known and are extremely resistant to chemical and environmental factors in the dormant state. During germination, as bacterial spores progress towards the vegetative state, they become susceptible to anti-sporal agents. B. atrophaeus spores were exposed to the non-nutritive germinant dodecylamine (DDA), a cationic surfactant that can also be used as a killing agent, for up to 60 min, or to the nutrient germinant L-alanine. In kinetic studies, 99% of the spores were killed within 5 min of exposure to DDA. Atomic force microscopy (AFM) can be used as a sensitive tool to assess how the structure of the spore coat changes upon exposure to germinants or killing agents. Changes in cell height and roughness over time of exposure to DDA were examined using AFM. DDA caused the spore height to decrease by >50%, which may have been due to a partial breakdown of the spore coat. Treatment of B. atrophaeus with the nutrient germinant resulted in a decrease in height of spores after 2 h of incubation, from 0.7 +/- 0.1 microm to 0.3 +/- 0.2 microm. However, treatment with L-alanine did not change the surface roughness of the spores, indicating that the changes that occur during germination take place underneath the spore coat. We propose that exposure to DDA at high concentrations causes pores to form in the coat layer, killing B. atrophaeus without the need to fully germinate spores.

Interrogation of living cells using alternating current scanning electrochemical microscopy (AC-SECM)

In this paper we present the application of alternating current scanning electrochemical microscopy (AC-SECM) to the study of living cells. Commercial AFM instrumentation was modified to allow for performing robust AC-SECM measurements. Constant height AC imaging of the Cos-7 cells, performed directly in cell culture medium without the addition of a redox mediator, provided topographical information of the cell. Stationary tip measurements on the AC current were carried out to investigate the cellular activity of a single cell. The dependence of AC current magnitude on tip-to-sample separation distance was used to monitor real time changes in cell height of individual Cos-7 cells. Furthermore, AC-SECM was employed to observe changes in metabolic cellular activity stimulated by ethanol and phorbol-1,2-myristate-acetate-3. The effect of changing cellular activity on constant height AC-SECM imaging was also studied.

Swelling-Activated K+ Efflux and Regulatory Volume Decrease Efficiency in Human Bronchial Epithelial Cells

This study describes the correlation between cell swelling-induced K+ efflux and volume regulation efficiency evaluated with agents known to modulate ion channel activity and/or intracellular signaling processes in a human bronchial epithelial cell line, 16HBE14o(-1). Cells on permeable filter supports, differentiated into polarized monolayers, were monitored continuously at room temperature for changes in cell height (T(c)), as an index of cell volume, whereas (86)Rb efflux was assessed for K+ channel activity. The sudden reduction in osmolality of both the apical and basolateral perfusates (from 290 to 170 mosmol/kg H(2)O) evoked a rapid increase in cell volume by 35%. Subsequently, the regulatory volume decrease (RVD) restored cell volume almost completely (to 94% of the isosmotic value). The basolateral (86)Rb efflux markedly increased during the hyposmotic shock, from 0.50 +/- 0.03 min(-1) to a peak value of 6.32 +/- 0.07 min(-1), while apical (86)Rb efflux was negligible. Channel blockers, such as GdCl(3) (0.5 mM), quinine (0.5 mM) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB, 100 microM), abolished the RVD. The protein tyrosine kinase inhibitors tyrphostin 23 (100 microM) and genistein (150 microM) attenuated the RVD. All agents decreased variably the hyposmosis-induced elevation in (86)Rb efflux, whereas NPPB induced a complete block, suggesting a link between basolateral K(+) and Cl(-1) efflux. Forskolin-mediated activation of adenylyl cyclase stimulated the RVD with a concomitant increase in basolateral (86)Rb efflux. These data suggest that the basolateral extrusion of K+ and Cl(-1) from 16HBE14o(-1) cells in response to cell swelling determines RVD efficiency.

Swelling‐activated K efflux and regulatory volume decrease efficiency in human bronchial epithelial cells

This study describes the correlation between cell swelling-induced K+ efflux and volume regulation efficiency evaluated with agents known to modulate ion channel activity and/or intracellular signaling processes in a human bronchial epithelial cell line, 16HBE14o−1. Cells on permeable filter supports, differentiated into polarized monolayers, were monitored continuously at room temperature for changes in cell height (Tc), as an index of cell volume, whereas 86Rb efflux was assessed for K+ channel activity. The sudden reduction in osmolality of both the apical and basolateral perfusates (from 290 to 170 mosmol/kg H2O) evoked a rapid increase in cell volume by 35%. Subsequently, the regulatory volume decrease (RVD) restored cell volume almost completely (to 94% of the isosmotic value). The basolateral 86Rb efflux markedly increased during the hyposmotic shock, from 0.50 ± 0.03 min−1 to a peak value of 6.32 ± 0.07 min−1, while apical 86Rb efflux was negligible. Channel blockers, such as GdCl3 (0.5 mM), quinine (0.5 mM) and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB, 100 μM), abolished the RVD. The protein tyrosine kinase inhibitors tyrphostin 23 (100 μM) and genistein (150 μM) attenuated the RVD. All agents decreased variably the hyposmosis-induced elevation in 86Rb efflux, whereas NPPB induced a complete block, suggesting a link between basolateral K+ and Cl−1 efflux. Forskolin-mediated activation of adenylyl cyclase stimulated the RVD with a concomitant increase in basolateral 86Rb efflux. These data suggest that the basolateral extrusion of K+ and Cl−1 from 16HBE14o−1 cells in response to cell swelling determines RVD efficiency.

Proliferation and cell shape changes during ciliary body morphogenesis in the mouse

Very little is known about the structure and development of the ciliary processes in the mouse eye. Our scanning electron microscope (SEM) studies reveal that, unlike other mammals, the ciliary processes form an irregular pattern, crossing over and interweaving rather than lying parallel to one another. Histological and SEM studies from embryonic day (E) 14.5 to postnatal day (P) 7 reveal that the first morphological sign of the ciliary zone is an annular bulge; this is then gradually molded to form discrete ciliary processes. The striking similarity between the developing capillary network and the adult ciliary folds suggests that the patterning template for the ciliary processes could be the underlying capillary network. Cell proliferation measurements and cell height assessments indicated that one of the first events occurring during the morphogenesis of ciliary processes is a proliferative surge around P0 in the outer ciliary epithelium. It is likely that this surge together with increasing cell heights leads to a bulging of this layer. After a slight delay, the inner ciliary epithelium responds by proliferating and extending inward toward the lens. Final shaping of the ciliary processes is achieved through cell height reductions in the inner ciliary epithelium. Thus, in the mouse, the temporal correlation between mitotic and cell height changes during ciliary body morphogenesis suggests that these processes play an integral role in the shaping of ciliary processes.

Height Changes Associated with Pigment Aggregation inXenopus laevisMelanophores

Melanophores are pigment cells found in the skin of lower vertebrates. The brownish-black pigment melanin is stored in organelles called melanosomes. In response to different stimuli, the cells can redistribute the melanosomes, and thereby change colour. During melanosome aggregation, a height increase has been observed in fish and frog melanophores across the cell centre. The mechanism by which the cell increases its height is unknown. Changes in cell shape can alter the electrical properties of the cell, and thereby be detected in impedance measurements. We have in earlier studies of Xenopus laevis melanophores shown that pigment aggregation can be revealed as impedance changes, and therefore we were interested in investigating the height changes associated with pigment aggregation further. Accordingly, we quantified the changes in cell height by performing vertical sectioning with confocal microscopy. In analogy with theories explaining the leading edge of migrating cells, we investigated the possibility that the elevation of plasma membrane is caused by local swelling due to influx of water through HgCl2-sensitive aquaporins. We also measured the height of the microtubule structures to assess whether they are involved in the height increase. Our results show that pigment aggregation in X. laevis melanophores resulted in a significant height increase, which was substantially larger when aggregation was induced by latrunculin than with melatonin. Moreover, the elevation of the plasma membrane did not correlate with influx of water through aquaporins or formation of new microtubules, Rather, the accumulation of granules seemed to drive the change in cell height.

Morphological changes and proliferative activity in the oviductal epithelium during hormonally defined stages of the oestrous cycle in the bitch.

The aim of the present study was to investigate morphological changes and proliferative activities in the epithelium of the canine oviduct with regard to the part of the oviduct - possibly indicating the existence of a locally restricted sperm reservoir - and the stage of the oestrous cycle. Nine healthy adult nulliparous bitches were submitted to ovariohysterectomy at three stages of the cycle: anoestrus (n = 3), late follicular phase (n = 3) and mid-luteal phase (n = 3). The whole oviduct ranging from the utero-tubal junction (UTJ) to the infundibulum (IN) was collected, divided into UTJ, IN plus six segments of equal length, i.e. eight oviductal specimens per animal were studied by light microscopy. Morphological characteristics of ovaries and endometrium were recorded macroscopically and verified histologically. The height of oviduct epithelial cells and percentage of ciliated cells (CC) were assessed and the respective data analysed statistically. Proliferative activity was immunohistochemically visualized by means of Ki-67 antigen detection. Blood was collected and concentrations of oestradiol-17beta and progesterone (P(4)) were measured. Within the IN and five of the six tissue samples collected from the ampulla and isthmus in anoestrous bitches, the oviductal surface epithelium consisted of low cuboidal cells demonstrating a uniform dark staining intensity. Only a very few scattered lighter staining CC could be detected. Under the influence of oestrogens during late follicular phase, the oviductal epithelium was highly differentiated. Lighter stained CC with apically located nuclei were easily distinguishable from basophilic secretory cells with apical cytoplasmic protrusions. Cell height and percentage of CC were significantly higher than in anoestrus (p <or= 0.05). During mid-luteal phase, high levels of P(4) were associated with differentiated and dedifferentiated cells as well as cells in regression seen in the mucosal folds of all samples. The percentage of CC and cell height were significantly lower than during late follicular phase (p <or= 0.05). Further signs of dedifferentiation consisted of a loss of cilia, a pinching off of the apical cytoplasm as well as the presence of debris and macrophages within the oviductal lumen. In the oviductal part of UTJ and the caudal isthmus hormone-dependent variations in cellular morphology were less distinct. Changes in cell height were minimal and did not differ significantly throughout the oestrous cycle. Hypertrophic cells with large nuclei were predominantly present at these sites, but did not consistently demonstrate signs of ciliation or secretion. Sporadic proliferating activity, visualized by means of Ki-67 antigen, was mainly seen in some cells of the late follicular phase samples. Thus, overall proliferative activity is generally very low or may occur within a relatively short period of time. It therefore cannot be excluded, that periods exhibiting higher mitotic rates are not included in the present study. It should, however, be mentioned that cells demonstrating morphological signs of apoptosis can only be seen very sporadically within a few specimens during mid-luteal phase, thus, reflecting low proliferative capacities and minimal cellular turnover found during this study. The results of the present study strongly indicate that oestrogens cause hypertrophy and differentiation, whereas P(4) induces gradual dedifferentiation or regression of the oviductal epithelium. Furthermore, they reveal clearly visible changes in the morphology of the tubal epithelium during the oestrous cycle. Depending on the tubal segment, these are, however, variably expressed. Whether the low degree of cellular variation of the UTJ and caudal isthmus is caused by specific hormone concentrations at these sites or specific regulatory mechanisms and may be associated with specific functional properties such as the formation of a locally restricted sperm reservoir needs further investigations.

Scanning electrochemical microscopy of model neurons: imaging and real-time detection of morphological changes.

Living PC12 cells, a model cell type for studying neuronal function, were imaged using the negative feedback mode of a scanning electrochemical microscope (SECM). Six biocompatible redox mediators were successfully identified from a large pool of candidates and were then used for imaging PC12 cells before and after exposure to nerve growth factor (NGF). When exposed to NGF, cells differentiate into a neuron phenotype by growing narrow neurites (1-2 microm wide) that can extend > 100 microm from the cell proper. We demonstrate that carbon fiber electrodes with reduced tip diameters can be used for imaging both the cell proper and these neurites. Regions of decreased current, possibly resulting from raised features not identifiable by light microscopy, are clearly evident in the SECM images. Changes in the morphology of undifferentiated PC12 cells could be detected in real time with the SECM. After exposure to hypotonic and hypertonic solutions, reversible changes in cell height of <2 microm were measured.

Simultaneous Measurement of Ca 2+ and Cellular Dynamics: Combined Scanning Ion Conductance and Optical Microscopy to Study Contracting Cardiac Myocytes

We have developed a distance modulated protocol for scanning ion conductance microscopy to provide a robust and reliable distance control mechanism for imaging contracting cells. The technique can measure rapid changes in cell height from 10 nm to several micrometers, with millisecond time resolution. This has been demonstrated on the extreme case of a contracting cardiac myocyte. By combining this method with laser confocal microscopy, it was possible to simultaneously measure the nanometric motion of the cardiac myocyte, and the local calcium concentration just under the cell membrane. Despite large cellular movement, simultaneous tracking of the changes in cell height and measurement of the intracellular Ca 2+ near the cell surface is possible while retaining the cell functionality.

Rapid flow-induced responses in endothelial cells.

Endothelial cells alter their morphology, growth rate, and metabolism in response to fluid shear stress. To study rapid flow-induced responses in the 3D endothelial cell morphology and calcium distribution, coupled fluorescence microscopy with optical sectioning, digital imaging, and numerical deconvolution techniques have been utilized. Results demonstrate that within the first minutes of flow application nuclear calcium is increasing. In the same time frame whole cell height and nuclear height are reduced by about 1 microm. Whole cell height changes may facilitate reduction of shear stress gradients on the luminal surface, whereas nuclear structural changes may be important for modulating endothelial growth rate and metabolism. To study the role of the cytoskeleton in these responses, endothelial cells have been treated with specific disrupters (acrylamide, cytochalasin D, and colchicine) of each of the cytoskeleton elements (intermediate filaments, microfilaments, and microtubules, respectively). None of these compounds had any effect on the shear-induced calcium response. Cytochalasin D and acrylamide did not affect the shear-induced nuclear morphology changes. Colchicine, however, completely abrogated the response, indicating that microtubules may be implicated in force transmission from the plasma membrane to the nucleus. A pedagogical model based on tensegrity theory principles is presented that is consistent with the results on the 3D endothelial morphology.

Changes in secretory status, cell height and percentage ciliation of epithelial lining of sheep fimbria oviduct during early pregnancy.

Studies have shown that the biosynthetic activity of the fimbria oviduct in the sheep changes during the first few weeks of pregnancy. The objective of the present study was to determine whether the secretory epithelial lining of the fimbria undergoes pregnancy-associated morphological alterations in a concordant manner. Oviducts obtained from ovariectomized, oestrous (day 0) and pregnant (day 1.5, 2, 3, 4, 6 and 16) ewes were analysed quantitatively and qualitatively using light and electron microscopy. The epithelium was low cuboidal in fimbria from ovariectomized sheep, significantly increased in height at day 1.5 of pregnancy, immediately decreased at day 2, and underwent an additional slight reduction at day 6 and day 16 of pregnancy. Ciliated and nonciliated cells were present in the epithelium at all reproductive stages. The proportions of ciliated and nonciliated cells varied during pregnancy and ciliated cells were always the major cell type. Consistent with previous data, the secretory organelles of nonciliated epithelial cells obtained from ovariectomized ewes were poorly developed. Maturation of secretory organelles in nonciliated cells occurred at oestrus and was maintained until days 2-3 of pregnancy; regressive changes were then observed, characterized by atrophy of the Golgi apparatus and rough endoplasmic reticulum, and increases in heterochromatin and pronounced nuclear folds. Large, electron-dense lipid droplets were present throughout the cytoplasm of ciliated and nonciliated epithelial cells, beginning at day 1.5 of pregnancy and increasing at later stages of pregnancy. Apical protrusions and microvilli were observed at the luminal domains of nonciliated cells and were reduced in extent after day 3 of pregnancy. Cells were extruded from the epithelial lining at day 16 of pregnancy. These data show that the epithelial lining of the fimbria oviduct in the sheep undergoes distinct changes in cell height, percentage ciliation and secretory organelle development during the first few weeks of pregnancy.

Apical membrane sodium and chloride entry during osmotic swelling of renal (A6) epithelial cells

To assess the role of chloride in cell volume and sodium transport regulation, we measured cell height changes (CH), transepithelial chloride and sodium fluxes, and intracellular chloride content during challenge with hyposmotic solutions under open circuit (OC) conditions. CH maximally increased following hyposmotic challenge within approximately 5 minutes. The change in CH was smaller under short circuit (SC) conditions or following replacement of chloride in the mucosal solution by gluconate or cyclamate (Cl(-)-freem). When corrected for the osmotically inactive cell volume (30 +/- 2%), delta CH for controls (OC) were greater than predicted for an ideal osmometer. In contrast, delta CH for Cl(-)-freem or SC conditions were similar to that predicted for an ideal osmometer. Na+ and Cl- mucosa-to-serosa fluxes increased following hyposmotic challenge. Chloride fluxes increased maximally within 5 min, then decreased. In contrast, the Na+ flux increased slowly and reached a steady state after approximately 25 min. Under isosmotic conditions, exposure to Cl(-)-freem solutions led to decreases in the transepithelial conductance, Na+ flux, and CH. Chloride permeabilities in the apical and basolateral membranes were detected using the fluorescent intracellular chloride indicator MQAE. The results indicate that during osmotic swelling, the entry of both sodium and chloride is increased. The time courses of these increases differ, suggesting distinct mechanisms for the osmotic regulation of these apical membrane transport processes.

An automatic monitoring system for epithelial cell height

This paper describes an automatic method to measure cell height (h) of epithelia grown as monolayers on transparent filter supports. Tissues are mounted in an Ussing-type chamber enabling solution exchange on both sides. The apical and basal side of the epithelial cells are marked with fluorescent beads. The image of the fluospheres is captured with a video camera and processed by a computer-based video imaging system. One basal reference bead in a gelatin layer on the filter support and up to three beads attached at the apical surface are used to monitor changes in cell height of three cells simultaneously. The focusing of the microbeads is done automatically by moving the objective with a piezoelectric device mounted on the nosepiece of the microscope. The algorithm for locating the bead is based on the changes in fluorescent light intensity emitted by the fluospheres. The method has an accuracy higher than 0.1 micron and a time resolution as low as 6 s if measurements are restricted to one bead at the apical side. The method was tested on artificial model systems and used to measure volume changes in renal cultured epithelia (A6) after exposing the serosal surface to hypotonic solutions and replacing cell-impermeable sucrose by an organic compound (glycerol) with a smaller reflection coefficient. Serosal hypotonicity elicited a rapid volume increase followed by regulatory volume decrease, whereas the organic compound replacement caused a steady increase in cell volume.

A simple method for monitoring changes in cell height using fluorescent microbeads and an Ussing-type chamber for the inverted microscope

In this study, we report two developments for studies of ion transport in cultured epithelial cells. First, a convenient method is presented for measuring apparent cell height using fluorescent microbeads as high-contrast landmarks of the apical and basal cell surfaces. The apparent cell height is then used as an indicator to monitor the time course of changes in cell volume in response to osmotic perturbations. Second, an Ussing-type chamber design for the inverted fluorescence microscope is presented, which allows determination of transepithelial electrical properties. Using these two methods, we obtained simultaneous measurements of cell height and transepithelial electrical parameters for cultured renal (A6) epithelium. Cell height was measured by alternately focusing the microscope between microbeads marking the apical and basal surfaces. The distance between these two surfaces was measured electrically from the voltage output of a potentiometer that was mechanically coupled to the fine-focusing knob of the microscope. Following decreases in the bathing solution osmolality, the cell height and transepithelial Na+ transport rate (measured as short-circuit current, ISC) increased. The increase in cell height preceded changes in ISC by several minutes, suggesting a lack of direct linkage between changes in cell volume and transepithelial Na+ transport. Both the fluorescent microbead cell height method and the Ussing-type chamber can be used in conjunction with patch-clamp techniques, intracellular microelectrode impalements, or fluorescent probes of intracellular composition. Therefore, this system may be advantageous for studies of epithelial cell volume and channel regulation.

Homeostatic and developmental control of cell size and shape in an insect epithelium, the epidermis ofManduca sexta

In contrast to previously described transformations of insect epidermal cells, morphological changes in the larval epidermis of Manduca sexta involve large changes in cell volume: a threefold increase at the beginning and a twofold decrease at the end of the last feeding phase. The volume changes are determined, in part, or entirely, by changes in cell height (cell elongation and cell shortening). Initial cell elongation occurs in a region-specific manner, whereas subsequent cell shortening affects all of the epidermis equally. As shown by ligation experiments and hormone treatments in tissue culture, larval changes in cell height, unlike cell elongation and differentiation in prepupae, are not regulated by developmental hormones (juvenile hormones, ecdysteroids). Instead, the maintenance of a normal columnar epithelium in fifth instars depends on continuous growth. Lack of food, especially protein, results in reversible cell shortening at any time during the feeding phase. Intake of water does not mitigate this cellular response; cell shortening is also insensitive to ouabain but inhibited by cold treatments. We propose that during larval growth epidermal cell height is under specific homeostatic control, independent of mechanisms regulating cell width, ploidy levels, or mitotic activity.

Cell enlargement during leaf development in Catharanthus roseus

Cell enlargement and tissue differentiation have been examined using the leaf plastochron index (LPI) as a measure of developmental stage in Catharanthus roseus (L.) G. Don. Observations were made from plastic-embedded, 2-μm serial sections and measurements were made using a microprojector. At the earliest stages examined, six cell layers are present; periclinal divisions in the third abaxial layer give rise to seven cell layers at maturity. The initial six layers are similar in size, shape, and staining characteristics but palisade cells are slightly elongated. Relative rates of change in leaf length are high during early stages before LPI 1.0 whereas relative rates of change in leaf thickness increase after LPI 1.0 largely because of continuing high rates of change in palisade cell height. The predominant plane of enlargement in palisade mesophyll is perpendicular to the predominant plane of enlargement in spongy mesophyll. Relative rates of change in cell height in upper epidermis are higher than in lower epidermis throughout most of the period of development and absolute rates of change in cell size in upper epidermis are always higher than in lower epidermis. Formation of intercellular space in mesophyll seems well correlated with rates of change in cell width in developing dermal layers. These observations show how the rate, duration, and direction of cell expansion in each tissue type results in the histology of the mature leaf.

Histometrical estimation of the corticotrophin secreting cells in the adenohypophysis of bilaterally adrenalectomized rats.

A histological and histometrical estimation was made on the adenohypophyseal cells of bilaterally adrenalectomized adult rats, which were sacrificed at various time-intervals from 6 hours to 32 days. The thyroid weight decreased, then rose again, being stabilized below the normal level after 3 days. The pituitary weight dropped slightly at 3 days, but soon returned to normal. The change in cell-height of the central follicles in thyroids was variable and exhibited no definite tendency. The height of epithelium of the ventral prostate tended to decrease gradually in a straight line. The cell-count resulted in a conclusion that the ranges of acidophils and chromophobes have an early and late peak. A slight decreasing shift was found in gonadotrophs. The curve of thyrotrophs represented a decreasing shift following an initial temporary rise. The population of intermediate cells between the chromophobes and chromophils was so intricate that its significance was hard to determine. The curve, including two peaks, of pituitary ACTH content in rats after adrenalectomy (Gemzell et al., 1951 and Fortier, 1959) was compatible with the population curve of chromophobes and acidophils, but inconsistent with that of the other cell elements. An early degranulation and subsequent accumulation of the α-granules in acidophils might be signs of discharge and secondary storage of the hormonal precursor in acidophils. It was suggested that the axis of the cellular transmigration from a chromophobe into an acidophil is likely to be concerned with ACTH secretion, because the population of chromophobes usually precedes that of the acidophils.