Distribution of diaphragmatic lymphatic lacunae.

The morphology of the submesothelial lymphatic lacunae on the pleural and peritoneal surface over the tendinous and muscular portion of the diaphragm was studied in 10 anesthetized rabbits. The lymphatic network was evidenced by injecting 1 ml of colloidal carbon solution in the pleural (n = 5) or the peritoneal (n = 5) space. After 1 h of spontaneous breathing, the animal was killed and the diaphragm was fixed in situ by injection of approximately 5 ml of fixative in pleural and peritoneal spaces. Then both cavities were opened and the diaphragm was excised and pinned to a support. According to which cavity had received the injection, the peritoneal or the pleural side of the diaphragm was scanned by sequential imaging of the whole surface by use of a video camera connected to a stereomicroscope and to a video monitor. The anatomic design appeared as a network of lacunae running either parallel or perpendicular to the major axis of the tendinous or muscular fibers. The lacunae were more densely distributed on the tendinous peritoneal area than on the pleural one. Scanty lacunae were seen on the muscular regions of both diaphragmatic sides, characterized by large areas without lacunae. The average density of lacunae on tendinous and muscular regions was 6 and 1.7/cm2 for the pleural side and 25 and 3.4/cm2 for the peritoneal side, respectively. The average width of lacunae was 137.9 +/- 1.6 and 108.8 +/- 1.7 microns on the tendinous pleural and the peritoneal side, respectively, and 163 +/- 1.8 microns on the muscular portion of the pleural and peritoneal surfaces.     

Light and electron microscope observations of the lymphatic drainage units of the peritoneal cavity of rodents

Fluid, particles, and cells are taken up from the peritoneal cavity by lymphatic drainage units, which, in the mouse and rat, are located along the peritoneal surface of the muscular portion of the diaphragm. The drainage units are composed of three specifically differentiated components: a lymphatic lacuna, a covering of lacunar mesothelium, and intervening submesothelial connective tissue. The units are drained by connecting lymphatic vessels that cross the diaphragm to empty into collecting lymphatic vessels running along the pleural surface of the diaphragm. The collecting lymphatics empty into parasternal lymphatic trunks. In this report, we briefly review critical features of the drainage apparatus and describe new observations, summarized below, about their structure. Around the rim of stomata, the mesothelial openings that lead into the lymphatic lacunae, plasma membranes of lacunar mesothelial cells and of lacunar endothelial cells abut but are not linked to one another by recognizable junctional specializations. Lacunar endothelial cells often extend valve-like processes that bridge the distal end of the channel beneath the stoma. The configuration of the endothelial processes may be complex. Occasionally, processes from fibroblasts in the submesothelial connective tissue adjacent to stomata make contact with the interstitial surface of lacunar endothelial cells. A discontinuous elastic layer in the submesothelial connective tissue spans the roof of each lacuna. Connecting and collecting lymphatics, which drain lymphatic lacunae, possess endothelial valves. Possible functions for each of these newly described structural features are discussed.     

Study on the ultrastructure of the peritoneal stomata in humans.

In 16 human specimens the topography and organization of stomata and mesothelial cells of the diaphragmatic, pelvic wall and anterior abdominal wall peritoneum were studied by transmission electron microscopy, scanning electron microscopy and the image processing technique. The mesothelial cells were organized into two discrete populations, cuboidal cells and flattened cells. The stomata were found only among cuboidal cells, either on the muscular portion or on the tendinous portion of the diaphragm. The size and shape of stomata, which were arranged in a cluster or a strip, were often irregular. The average area of a stoma on the muscular portion was 10.43 +/- 1.61 microns2, on the tendinous portion 7.93 +/- 1.67 microns2. Stomata opened to submesothelial connective tissue, under which numerous lymphatics were observed. Stomata were not discovered in the pelvic and anterior abdominal wall peritoneum. In animal experiments intraperitoneally injected trypan blue particles were rapidly removed from the peritoneal cavity through stomata of the diaphragmatic peritoneum in rabbits. It is suggested that stomata may be the main pathway for draining matter from the peritoneal cavity and that the diaphragmatic peritoneum shows the strongest absorption in all parts of the peritoneum.     

Functional arrangement of rat diaphragmatic initial lymphatic network

Fluid and solute flux between the pleural and peritoneal cavities, although never documented under physiological conditions, might play a relevant role in pathological conditions associated with the development of ascitis and pleural effusion and/or in the processes of tumor dissemination. To verify whether a pleuroperitoneal flux might take place through the diaphragmatic lymphatic network, the transdiaphragmatic pressure gradient (Delta P(TD)) was measured in five spontaneously breathing anesthetized rats. Delta P(TD) was -1.93 cmH2O (SD 0.59) and -3.1 cmH2O (SD 0.82) at end expiration and at end inspiration, respectively, indicating the existence of a pressure gradient directed from the abdominal to the pleural cavity. Morphometrical analysis of the diaphragmatic lymphatic network was performed in the excised diaphragm of three additional rats euthanized with an anesthesia overdose. Optical and electron microscopy revealed that lymphatic submesothelial lacunae and lymphatic capillaries among the skeletal muscles fibers show the ultrastructural features of the so-called initial lymphatic vessels, namely, a discontinuous basal lamina and anchoring filaments linking the outer surface of the endothelial cells to connective tissue or to muscle fibers. Primary unidirectional valves in the wall of the initial lymphatics allow entrance of serosal fluid into the lymphatic network preventing fluid backflow, while unidirectional intraluminar valves in the transverse vessels convey lymph centripetally toward central collecting ducts. The complexity and anatomical arrangement of the two valves system suggests that, despite the existence of a favorable Delta P(TD), in the physiological condition no fluid bulk flow takes place between the pleural and peritoneal cavity through the diaphragmatic lymphatic network.     

The ultrastructure of the peritoneal membrane in chronically dialysed rats with spontaneous peritonitis: preliminary observations

In this paper we describe ultrastructure of the peritoneal membrane from single peritoneal biopsies collected from chronically dialysed rats with spontaneous peritonitis. The results were compared with those obtained in chronically dialysed animals without peritonitis. In rats with peritonitis, peritoneum was much thicker than in peritonitis-free animals. The increased thickness of the peritoneum during peritonitis was due to infiltration of the submesothelial tissue with oedematous fluid and to the presence of huge amount of cells in the stroma. The connective tissue cells were accumulated just underneath the peritoneal surface. In deeper parts of the interstitium, infiltrating acute inflammatory cells were present (lymphocytes, polymorphonuclear cells: neutrophils and eosinophils). Inversely, the increased thickness of the peritoneum in peritonitis-free animals was mainly due to enhanced amounts of collagen. Additionally, in rats with peritonitis, the surface was often denuded of mesothelial cells. The damaged mesothelial cells that detached from the peritoneal surface were also found. In conclusion, the morphological changes observed in rats with peritonitis are similar to those reported in humans, thus the model of peritonitis in dialysed rats can be used for the study of peritoneal remodeling during peritoneal dialysis complicated by peritonitis.     

The microencapsulation of cells within alginate poly-L-lysine microcapsules prepared with the standard single step drop technique: histologically identified membrane imperfections and the associated graft rejection.

Standard alginate-polylysine microcapsules containing isolated rat hepatocytes were prepared. These capsules were intraperitoneally implanted into mice, and retrieved after seven days. Histological sections of the recovered microcapsules showed peritoneal lymphocyte and macrophage infiltration. Additional microscopic observations at various stages of the microencapsulation procedure, and histological observations of control non-implanted microcapsules; illustrate that encapsulated cells became embedded within the microcapsular membrane matrix. The microcapsular membrane at these sites appeared thin and often poorly formed. The cellular infiltration into the implanted microcapsules can occur through holes developed in these thin and poorly formed areas found in the microcapsular membrane. Similar observations were seen in microcapsules prepared with 20 x 10(6) and at a lower cell concentration of 10 x 10(6) suspended cells per millilitre of sodium alginate.     

Effect of cytokines on polymorphonuclear neutrophil infiltration in the mouse. Prostaglandin- and leukotriene-independent induction of infiltration by IL-1 and tumor necrosis factor

The i.p. injection of mice with highly purified recombinant human rIL-1 alpha or beta resulted in the rapid influx of a large number of polymorphonuclear neutrophils (PMN) into the peritoneal cavity. Significant increases in the number of PMN were induced by doses of IL-1 which ranged from 0.005 to 5 ng/injection. Interestingly the dose response for PMN influx was bell-shaped because 50 ng of IL-1 did not result in a significant increase in peritoneal PMN. IL-1 induced PMN infiltration was detectable by 1 h with peak levels of PMN obtained by about 2 h, followed by a subsequent decline by 24 h. Other cytokines, IL-2, IFN-gamma, IFN alpha beta, granulocyte-CSF, granulocyte-macrophage-CSF, IL-3, TNF-alpha, and TNF-beta were compared to IL-1 for their ability to induce a PMN influx into the peritoneum. Only TNF-alpha or TNF-beta (lymphotoxin) were able to induce a significant influx of PMN within 2 h. However, based on total protein administered, about 100 times more TNF than IL-1 was required to produce a comparable PMN infiltration. Intraperitoneal injection of inhibitors of the cyclooxygenase or lipoxygenase pathways did not inhibit the IL-1-induced influx of PMN. Also, neither IL-1 nor TNF triggered an increase in PG or leukotriene release from peritoneal cells in vitro. Furthermore, direct peritoneal injection of leukotriene B4, a potent PMN chemoattractant in vitro, did not induce any significant increase in PMN in the peritoneal cavity indicating that chemotactic activity alone is insufficient for inducing peritoneal infiltration. These results suggest that the local production of very low levels of IL-1 in vivo would be sufficient to initiate a sequence of events that results in a rapid accumulation of PMN. Because IL-1 was not chemotactic for PMN in vitro, our data suggest that IL-1 induces production of factors that are chemotactic for PMN. Alternatively, IL-1 may act on other stages of the complex sequence of events that regulates the emigration of PMN into tissue sites in vivo. The synergy apparent in PMN influx when suboptimal concentrations of IL-1 and TNF were injected suggests that the local production of very low concentrations of these cytokines in situ could play a critical role in the emigration of PMN during infection.     

An overview of phrenic nerve and diaphragm muscle development in the perinatal rat.

In this overview, we outline what is known regarding the key developmental stages of phrenic nerve and diaphragm formation in perinatal rats. These developmental events include the following. Cervical axons emerge from the spinal cord during embryonic (E) day 11. At approximately E12.5, phrenic and brachial axons from the cervical segments merge at the brachial plexi. Subsequently, the two populations diverge as phrenic axons continue to grow ventrally toward the diaphragmatic primordium and brachial axons turn laterally to grow into the limb bud. A few pioneer axons extend ahead of the majority of the phrenic axonal population and migrate along a well-defined track toward the primordial diaphragm, which they reach by E13.5. The primordial diaphragmatic muscle arises from the pleuroperitoneal fold, a triangular protrusion of the body wall composed of the fusion of the primordial pleuroperitoneal and pleuropericardial tissues. The phrenic nerve initiates branching within the diaphragm at approximately E14, when myoblasts in the region of contact with the phrenic nerve begin to fuse and form distinct primary myotubes. As the nerve migrates through the various sectors of the diaphragm, myoblasts along the nerve's path begin to fuse and form additional myotubes. The phrenic nerve intramuscular branching and concomitant diaphragmatic myotube formation continue to progress up until E17, at which time the mature pattern of innervation and muscle architecture are approximated. E17 is also the time of the commencement of inspiratory drive transmission to phrenic motoneurons (PMNs) and the arrival of phrenic afferents to the motoneuron pool. During the period spanning from E17 to birth (gestation period of approximately 21 days), there is dramatic change in PMN morphology as the dendritic branching is rearranged into the rostrocaudal bundling characteristic of mature PMNs. This period is also a time of significant changes in PMN passive membrane properties, action-potential characteristics, and firing properties.     

A peritoneal mesothelioma in a captive aardwolf (Proteles cristatus)

A 10-year-old male aardwolf (Proteles cristatus) was presented abdominal distention and emaciation for 3 months. Physical examination revealed firm abdominal masses with effusions. Cytologic assessment of the effusion showed uniform round tumor cells with a surface brush border. Necropsy showed white velvety masses covering the peritoneal surface of the liver, gall bladder, stomach, omentum, mesentery, spleen, intestine, abdominal wall and diaphragm. Histologic examination demonstrated papillary projections, lined with cuboidal tumor cells supported by fibrous connective tissue cores, arising from the serosa of visceral organs. Cytoplasmic vacuolation and a surface brush border were evident on some cells under light microscopy. Tumor cells stained positive for both cytokeratin (AE1/AE3) and vimentin. Electron microscopy showed prominent surface microvilli, rough endoplasmic reticulum, mitochondria and desmosomes in tumor cells. This may be the first reported case of peritoneal mesothelioma in a captive wild aardwolf.     

小鼠腹腔内腺病毒介导的基因表达和分布

本文报道了重组腺病毒－带有报道基因ｌａｃＺ的Ａｄ／ＲＳＶ－β－ｇａｌ注入小鼠腹腔后,在胃、小肠、大肠、等均出现被Ｘ－ｇａｌ染色蓝色的细胞,腹膜下面未见染色细胞。结果表明重组腺病毒可广泛感染腹腔内的组织并使外源基因表达,但不扩散出体腔。     

Motility of IL-2-stimulated lymphocytes in neutral and acidified extracellular matrix.

The migration of lymphocytes through extracellular matrix (ECM) is an essential feature of the infiltration process. In the course of their extravasation into poorly perfused neoplastic lesions, lymphocytes often encounter regions of acidified ECM. This study was designed to determine whether lymphocyte adherence and motility in ECM are influenced by ambient pH. Murine splenic lymphocytes, activated by culture with high-titer IL-2, were allowed to migrate into three-dimensional gels of Type I collagen, a major component of interstitial stroma, or into Matrigel, a basement membrane model. After 18 hr at pH 7.1, the leading cell front traveled a mean distance of approx 475 microM into Type I collagen gel. Approx 50% of the cells remained nonadherent, 25% adhered to the gel surface, and 25% were motile (penetrated beneath the surface). At pH 6.7, the leading-front distance increased significantly, by a factor of 1.4X, but there was little change in the proportion of cells exhibiting nonadherence, surface adherence, or motility. The relative motilities of CD3+ and AsGM1+ subsets were also unaltered. It therefore appears that acidification of collagen matrix increases the locomotory activity of motile lymphocytes, but causes little recruitment of nonmotile lymphocytes into the motile pool. Similar results were obtained in experiments with Matrigel. The increased motility observed at pH 6.7 did not reflect breakdown or relaxation of matrix lattices, as measured by the passive diffusion of latex beads of defined diameter. Preincubation of lymphocytes at pH 6.7 did not alter their subsequent motility in pH 7.1 gels. The findings establish ambient pH as a microenvironmental variable which can influence lymphocyte migration through ECM. The weak acidity characteristic of certain tumor microenvironments may be a factor which encourages lymphocyte infiltration through tissue matrix. Treatments which alter intratumor pH could potentially be used to manipulate the infiltration process for immunotherapeutic benefit.     

The ultrastructure of the peritoneal membrane in chronically dialysed rats

The model to estimate peritoneal function in chronically dialysed rats was previously presented by us. The aim of the paper is to report the findings obtained in electron microscopy of peritoneal biopsies from Wistar rats dialysed for 1 month with high glucose dialysis solution. In control animals, thin mesothelial cells were covered with microvilli. The submesothelial tissue was composed of sparse bundles of parallelly oriented collagen fibers with a few resting cells. In chronically dialysed rats, mesothelial cell layer was thicker and cells were fully packed with intracellular structures, mainly secretory granules with a homogeneous content. The submesothelial tissue was expanded due to the increased amount of collagen fibers, oedema and increased amount of submesothelial cells which were activated. The use of electron microscopy to study the peritoneum in dialysed rats is an excellent supplement to the chronic functional model of peritoneal dialysis in rats.     

Origin of the gonad in the juvenile of a solitary ascidian, Ciona intestinalis

In the just-metamorphosed juveniles of Ciona intestinalis, a round mass of tissue debris derived from the resorbed tadpole tail is situated in the broad space enclosed by the peritoneal membrane and the epidermis around the ventral side of the esophagus. In living juveni es, the origin of the gonad rudiment was traced back to the mass of tissue debris. Electron microscopically, the round mass was a clump of irregular-shaped phagocytotic cells engulfing degenerated cell fragments. On the surface of the cell clump, a small number of singly occurring round cells were found and identified as primordial germ cells on the basis of morphological continuity to obvious germ cells in later stages. Presence of nuage around the nucleus characterized the germ cells. In a few days the germ cells assembled to form a solid slender body (gonad rudiment) together with smaller somatic cells. The gonad rudiment left the space around the esophagus, moving into the narrow mesenteric space connecting the stomach and intestine on the fourth day after metamorphosis. It gradually increased in size by proliferation of the germ cells and somatic cells. The solid gonad rudiment changed into an oval vesicle with an eccentrically located cavity on about the seventh day after metamorphosis. The vesicle comprised a thinner wall made of a simple epithelium without germ cells and a thicker wall containing germ cells and somatic cells.     

Temporospatial patterns of apoptosis in chick embryos during the morphogenetic period of development

We examined the temporospatial pattern of naturally occurring apoptosis in chick embryos to five days of incubation (H.H. stages 1-25; Hamburger and Hamilton, 1951) using TUNEL labeling. The initial TUNEL-positive structure was the embryonic shield at stage 1. Apoptotic cells became ubiquitously present within embryos by stage 3, which is early in gastrulation. Until stage 6, TUNEL-positive cells were restricted to the headfold region. In embryos of stages 7-8, most cell death was localized at the most anterior neural plate. TUNEL-positive neural plate, notochord and somites appeared at stage 9. Otic and optic regions became TUNEL-positive at stage 11. The aggregation of cells from which the tail bud arises contains apoptotic cells from stage 11 onwards. At stage 16, scattered TUNEL-positive cells appeared in the branchial arches. Three streams of apoptotic neural crest cells in the cranial region became most clearly visible at stage 18. The secondary neural tube from which caudal structures develop contains apoptotic cells at stage 14. Apoptotic cells are present in the branchial arches and lateral body wall for extended periods, stages 16-25 and 25 respectively. At stages 24-25, intense positive regions of cell death were confined to the caudal regions of the arches, to limb and tail buds and to the lateral body wall, the latter in relation to body wall closure. The new findings in this study are discussed along with past studies to provide the temporospatial pattern of cell death during early chick development.     

A scanning electron microscope study of mouse peritoneal mesothelium.

As seen in the scanning electron microscope, peritoneal mesothelial cells of the mouse diaphragm, anterior abdominal wall and intestinal serosa carry numerous microvilli. These microvilli are absent over certain areas of the cell surface and are sometimes, interlocked in meshwork patterns or coronal formation. The apical cell membranes of the mesothelium at the base of the microvilli, are invaginated by many plasmalemmal vesicles and vacuoles and carry a number of protruding spherical structures. Deep circular craters, giving the impression of stomata, are also visible.     

Tumor angiogenesis: MMP-mediated induction of intravasation- and metastasis-sustaining neovasculature

Metastasis is a distinct stage of cancer progression that requires the development of angiogenic blood vessels serving as conduits for tumor cell dissemination. An accumulated body of evidence indicates that metastasis-supporting neovasculature should possess certain structural characteristics allowing for the process of tumor cell intravasation, an active entry of cancer cells into the vessel interior. It appears that the development of tumor vessels with lumens of a distinctive size and support of these vessels by a discontinuous pericyte coverage constitute critical microarchitectural requirements to: (a) provide accessible points for vessel wall penetration by primary tumor cells; (b) provide enough lumen space for a tumor cell or cell aggregate upon intravasation; and (c) allow for sufficient rate of blood flow to carry away intravasated cells from the primary tumor to the next, proximal or distal site. This review will primarily focus on the functional roles of matrix metalloproteinases (MMPs), which catalytically trigger the development of an intravasation-sustaining neovasculature at the early stages of tumor growth and are also required for the maintenance of a metastasis-supporting state of blood vessels at later stages of cancer progression.     

Physiological characterization of human ovarian cancer cells in a rat model of intraperitoneal antineoplastic therapy.

Destruction of cancer cells by therapies directed against new molecular targets requires their effective delivery to the tumor. To study diffusion and convection of intraperitoneal (ip) therapy to ip tumors, we established a new athymic rat (RNU) model with ovarian tumor cells (SKOV3 and OVCAR3) implanted in the abdominal wall. The model simulates metastatic tumor and facilitates the measurement of physiological parameters that govern transport forces. CD31 immunohistochemistry revealed unique patterns of angiogenesis, with a tissue-averaged vascular volume of approximately 0.01 ml/g for each tumor. The extracellular volume (SKOV3: 0.54 +/- 0.11 ml/g, n=5; OVCAR3: 0.61 +/- 0.03, n=5) was over twice that of the adjacent normal muscle (0.22 +/- 0.06 ml/g, n=5). Intravenous-injected antibody tumor clearance was two to three times that of muscle. Interstitial pressures were higher than normal tissue with a median of 10-15 mmHg. Quantitative autoradiography of frozen tissue slices from rats exposed to ip solutions containing [14C]mannitol or 125I-immunoglobulin G (trastuzumab) was performed to determine transport of small and large molecules. With ip pressure of 0-6 mmHg, both mannitol and immunoglobulin G displayed steep concentration profiles close to the tumor surface with limited penetration deeper within the tumor tissue; antibody penetration was significantly affected by ip pressure. These results demonstrated effects of molecular size, ip pressure, the limited but highly permeable tumor vasculature, and the expanded interstitium on drug penetration from the peritoneal cavity. In conclusion, we have characterized physical and chemical parameters that determine transport of therapeutic agents in our unique tumor-bearing rat model.     

Lipid vesicles penetrate into intact skin owing to the transdermal osmotic gradients and hydration force.

Gradients across the outer skin layers may result in fields enforcing a lipid flow into or through the intact skin surface provided that lipids are applied in the form of special vesicles. The osmotic gradient, for example, which is created by the difference in the total water concentrations between the skin surface and the skin interior, provides one possible source of such driving force. It is sufficiently strong to push at least 0.5 mg of lipids per hour and cm2 through the skin permeability barrier in the region of stratum corneum. The lipid concentration gradient, on the contrary, does not contribute much to the lipid penetration into dermis. Occlusion, therefore, is detrimental for the vesicle penetration into intact skin.     

Anatomical Sequence and Morphometric Analysis during Somatic Embryogenesis on Cultured Cotyledon Explants of Camellia japonica L.

This article describes the origin and anatomical developmentof somatic embryos differentiated on Camellia japonica L. cotyledonscultured on Murashige and Skoog's medium containing 1 mg l^-1of 6-benzylaminopurine. Only the abaxial surface of the cotyledonexplants was morphogenetically competent. Embryos developedin abaxial parenchymatic protuberances or nodules arising bydedifferentiation and active cell division in the epidermisand subepidermis. After 12-15 d in culture, successive divisionsat the surface of the nodules led to the formation of embryogenicprecursor cells which dedifferentiated into embryogenic cells;most somatic embryos apparently had a multicellular origin frommulticellular proembryonal complexes, though a number of few-celledproembryos within a thick common wall seemed to have originatedunicellularly. Between days 24 and 27, somatic embryos at theheart-shaped, torpedo-shaped and cotyledonary stages were apparent.Computer-aided image analysis of the histological events showeda progressive increase in the nucleus-to-cell area ratio. Duringthe first 7 d culture the explants exhibited a rapid declinein protein body content, which was high in the initial cotyledon,and an increase in starch content. Developing nodules stronglyPAS-positive, but starch content subsequently declined in thetissues underlaying embryogenic areas and reached a minimumwhen somatic embryos developed.Copyright 1993, 1999 AcademicPress Camellia japonica, camellia, cellular changes, somatic embryogenesis, histology, image analysis     

Myxosoma funduli Kudo (Myxosporida) in Fundulus kansae: Ultrastructure of the Plasmodium Wall and of Sporogenesis*

SYNOPSIS Ultrastructure of the plasmodium wall and of sporogenesis were studied in Myxosoma funduli Kudo infecting the gills of Fundulus kansae (Garman). Plasmodia were located within the lamellar tissues adjacent to sinuses and capillaries. The plasmodium wall consisted of a single unit membrane which was continuous with numerous pinocytic canals extending into the parasite ectoplasm. The plasmodium membrane was covered by a surface coat of almost uniform thickness which prevented direct parasite-host cell contact. Numerous generative cells and cell aggregates, representing early stages of spore development, were seen in immature plasmodia. Later stages of spore development, including mature spores, were observed in older plasmodia. Sporogenesis was initiated by envelopment of one generative cell, the sporont, by a 2nd, nondividing cell, the envelope cell. The sporont and its progeny proceeded through a series of divisions until there were 10 cells, all compartmentalized within the envelope cell. Subsequently, the 10 cells became structurally differentiated and arranged into two 5-celled spore-producing units, each consisting of 1 binucleate sporoplasm and 2 capsulogenic cells, all surrounded by 2 valvogenic cells. Observations of later developmental stages revealed the major events of capsulogenesis, valvogenesis, and sporoplasm maturation, which occurred concomitantly during spore construction.