Variability of quantitative morphogenetic parameters during early morphogenesis of the loach, <Emphasis Type="Italic">Misgurnus fossilis</Emphasis> L.

Analysis of normal variation in quantitative morphological characters during the early embryonic development of the loach, based on observations on individual developmental trajectories of living embryos, shows that the dorsoventral differentiation of the blastoderm proceeds in two stages. Initially, at the onset of epiboly, the sagittal (short) and transverse (long) blastoderm meridians are marked off, and only then, upon germ ring (GR) formation, differentiation between the opposite poles of the sagittal meridian takes place. The embryonic shield (ES) usually appears in the segment of the blastoderm where the radius of its external curvature reaches a maximum and, therefore, the active surface tension at the blastoderm boundary with the YSL periblast) and yolk is the highest. In this case, the convergence of inner cells toward the future dorsal segment (leading to ES formation) is a mechanical consequence of surface tension anisotropy. The normal course of epiboly is associated with periodic changes in the curvature of the blastoderm external surface, with new structures (the dorsal segment, GR, and ES) are marked off only when the surface curvature becomes maximally uniform. Although the ES in most embryos appears within the initial dorsal segment, individual developmental trajectories have been traced where the GR starts to form at the dorsal pole of the blastoderm but the ES develops on its opposite site, at the point of GR closure. In both cases, GR formation is initiated at the point of convergence of centrifugal cell migration flows that arise in the marginal zone of the blastoderm upon GR initiation or closure.     

Mitotic cells and their microappendages in the primitive streak of the chick embryo.

Fresh pullet eggs (White Leghorn Strain) were incubated to the primitive streak stage of development. Blastoderms were fixed in situ with isotonic aldehyde fixatives and prepared for scanning electron miscropy by means of post-osmication, critical point drying and gold-palladium coating. Cells judged to be in various stages of mitosis by their surface contours were numerous on the ventral surface of the chick blastoderm. Cells which were in the late preparatory stages for mitosis had rounded up from their surroundings. Microvilli dominated the surface. The degree of separation and number of microvilli increased until late metaphase or anaphase. Mitotic cells did not completely separate themselves from adjacent cells. Ruffles and blebs were not prominent during mitotis and long filopodia were absent. A definite localization of microappendages (microvilli, blebs, ruffles) to the area of cytokinesis was evident in early telophase and persisted through daughter cell formation.     

Scanning electron microscopy of Drosophila embryogenesis: 1. The structure of the egg envelopes and the formation of the cellular blastoderm

As part of a series of detailed observations on embryogenesis in Drosophila, the protective coverings of the egg and surface changes in the embryo prior to gastrulation have been studied with the SEM. Four specializations of the chorion are described: the plastron, micropylar cone, operculum, and the posterior thickening. After removal of the protective coverings the surface changes during development can be observed. During the first eight synchronous nuclear divisions a dense array of thin microprojections covers the whole embryo. After the ninth division between 373 and 408 nuclei reach the surface and become located in cytoplasmic projections. From counts of the number of surface bulges during the syncytial blastema stages, it was established that 13 synchronous divisions take place producing between 5600 and 6500 surface nuclei. During formation of the cellular blastoderm, the location of the prospective cells becomes obscured by a dense pattern of microprojections from each cell. However, with the completion of the blastoderm, the surfaces of the cells become smooth and the cell outlines distinct. The usefulness of the SEM in developmental studies is discussed.     

Morphometrical changes in the apical surface of the colonic absorptive cells in perinatal rats with special reference to the effect of fetal oral administration of milk in utero

Perinatal changes in the apical surface of the colonic absorptive cells in the rat were studied morphometrically. Cell microvilli length increased from day 20 through neonatal day 3, during which a maximum incremental growth rate was noted between fetal day 22 and neonatal day 1. Microvilli width remained almost constant throughout the period. Enlargement of the apical surface of the microvilli showed a similar developmental pattern as was seen from the measurement of length and surface area of any one of the microvilli. Fetal oral administration of milk in utero caused incremental growth in length and surface area, as well as an associated apical surface enlargement. The present study indicates that the function of the colonic absorptive cells, which is acquired later on in utero, is activated by ingestion of maternal milk after birth.     

Rapid, sequential changes in surface morphology of PC12 pheochromocytoma cells in response to nerve growth factor

The effect of nerve growth factor (NGF), a substance that promotes the differentiation and maintenance of certain neurons, was studied via scanning electron microscopy utilizing the PC12 clonal NGF-responsive pheochromocytoma cell line. After 2-4 d of exposure to NGF, these cells acquire many of the properties of normal sympathic neurons. However, by phase microscopy, no changes are discernible within the first 12-18 h. Since the primary NGF receptor appears to be a membrane receptor, it seemed likely that some of the initial responses to the factor may be surface related. PC12 cells maintained without NGF are round to ovoid and have numerous microvilli and small blebs. After the addition of NGF, there is a rapidly initiated sequential change in the cell surface. Ruffles appear over the dorsal surface of the cells with 1 min, become prominent by 3 min, and almost disappear by 7 min. Microvilli, conversely, disappear as the dorsal ruffles become prominent. Ruffles are seen at the the periphery of cell at 3 min, are prominent on most of the cells by 7 min and are gone by 15 min. The surface remains smooth from 15 min until 45 min when large blebs appear. The large blebs are present on most cells at 2 h and are gone by 4 h. The surface remains relatively smooth until 6-7 h of NGF treatment, when microvilli reappear as small knobs. These microvilli increase in both number and length to cover the cell surface by 10 h. These changes were not observed with other basic proteins, with α-bungarotoxin (which binds specifically to PC12 membranes), and were not affected by an RNA synthesis inhibitor that blocks initiation of neurite outgrowth. Changes in the cell surface architecture appear to be among the earlist NGF responses yet detected and may represent or reflect primary events in the mechanism of the factor’s action.     

Early development of the kelp fly,Coelopa frigida (Diptera). II. Morphology of cleavage and blastoderm formation

Cleavage and blastoderm formation in Coelopa frigida are extremely rapid developmental processes. In short (6–7 minutes) successive cell cycles, nuclei multiply and spread out through the egg. The movement seems to be aided by endoplasmic vesicles and cisternae which are in direct contact with the nuclear membrane. The first cells to separate from the egg plasmodium in early superficial cleavage stages are the pole cells. Precursor material from multivesicular bodies forms the pole cell membranes. The primary nuclei from the posterior pole region are removed from the blastoderm by the pole cell segregation. Blastoderm nuclei from the regions adjacent to the posterior pole migrate into the residual periplasm after pole cell segregation has been completed and constitute the blastoderm nuclei in that region of the egg. Nucleoli are not revealed during internal cleavage. They appear in pole cells shortly after their segregation. The generation time of the blastoderm nuclei increases after the twelfth cleavage. Concurrently, nucleoli form in the blastoderm nuclei and permanent cell membranes separate individual blastoderm cells. After blastoderm cells have been separated from each other, they remain in contact with the interior yolk sac by means of cytoplasmic canals. This contact is maintained at least during the early phases of blastokinesis. Observations on nuclear migration and rapid membrane formation are discussed as examples of protein assembly from subunits as an alternative to de novo protein synthesis in early stages of development.     

The corpus luteum of the pig. Scanning electron microscopic study of surface features at different times of incubation

The surface ultrastructure of porcine early corpus luteum cells (days 1-3 of the luteal phase) was studied in SEM and correlated with progesterone secretion. Luteal cells were divided into 2 groups: small cells (10-20 microns) and large cells (20-30 microns) and their surface features were observed after 1, 3, and 5 h of incubation in the control medium and in a medium supplemented with prolactin (PRL). The surface morphology of control cells was characterized by numerous smooth blebs and the presence or absence of thin microvilli. Small and large cells showed a tendency to adhere to the glass during the experiment, but on the large cells the number of thin adhesive filopodia was greater. After the 1st and 3rd h of incubation with PRL the number of microvilli and numerous filopodia on the small cells increased substantially. Nodular blebs were scattered and appeared to protrude from the cell surface. Many small cells adhered to the glass by thick, layered and thin thread-like cytoplasmic processes. After the 5th h distinct smoothing of the surface of the small cells was seen. The number of microvilli seen on the PRL stimulated surface of the large cells was smaller and in some cases even entirely absent. After the 1st and 3rd h of the experiment the large cell surface was ruffled with minute folds. Numerous nodular blebs protruded from the cell surface. The number of adhesive filopodia attaching the cells to the glass decreased or vanished during the experiment. After the 5 h of incubation most of the cells had smooth surface with smooth blebs. Progesterone secretion was measured by radioimmunoassay. The cells in the medium without exogenous hormone (control) secreted relatively low levels of progesterone throughout 1-5 h of the incubation period. After addition of PRL to the medium the amount of secreted progesterone increased.     

Surface features of Entamoeba histolytica and rabbit kidney (RK13) cell surface changes after trophozoite contact--observations by scanning electron microscopy.

Trophozoites of Entamoeba histolytica grown on glass, fixed in situ, and examined by scanning electron microscopy (SEM) were seen to possess microfilopodia on their lower sides and under surfaces, a hitherto undescribed feature. No surface lysosomes were seen. After trophozoites had been brought into contact with a host monolayer of cultured RK13 cells, immediate surface changes were observed. First the RK13 cell surface microvilli elongated, then decreased in number and finally disappeared. These changes were accompanied by erosion of the cell surface and cellular swelling.     

Characteristics of the upper cell surface in cultures of normal and SV-40 virus-transformed epithelium of mouse kidney. Study with the aid of scanning electron microscopy]

Central cells of the normal epithelial sheet are sparsely covered by microvilli. Numerous microvilli were seen in the regions of intercellular contacts. Marginal cells of sheets had a finely developed lamellar cytoplasm (lameloplasm) with smooth upper surface at their free margins. A transformed cell line (MPTR) resembled normal parent cells by its ability to form monolayered sheets in cultures. More microvilli of increased length appeared on the upper surface of central MPTR cells. The normal structure of lamelloplasm was changed at the free edge of the MPTR sheets. It is suggested that abnormal cell attachment to the substratum may be responsible for the altered cell surface morphology (increased length of microvilli, defective, structure of lamelloplasm) in the MPTR cultures.     

Functional changes in the relief of the apical surface of epithelial endometrial cells in the rat

The surface topography of the uterine born epithelial lining was studied in adult rats using scanning electron microscopy. Cyclic changes in the apical surface relief were observed that involved the formation of numerous microvilli with the increase in blood estrogen content. with the decrease in estrogen content and with the increase in that of progesterone the microvilli reduced in number and the epithelium folding diminished. Similar changes were seen in ovariectomised rats following the injection of corresponding hormones. In neonatal androgenized animals hypersecretion of estrogens and deficit of progesterone provided the persistence of microvilli and caused an intense migration of lymphoid system cells to the uterine born cavity.     

Blastoderm degeneration, an early embryonic failure in dwarf Single Comb White Leghorn chickens.

Blastoderm degeneration is an early embryonic lethal condition observed in selected paired matings within a line of dwarf Single Comb White Leghorn chickens that results in a 25% reduction of the hatch of fertilized eggs. The disorder is macroscopically evident at 32 h of incubation by the presence of a small localized indentation on the outer periphery of the expanding blastoderm. The affected blastoderms undergo a series of rapid macroscopic degenerative changes that conclude at about 120 h characterized by the presence of dispersed blastoderm fragments on the surface of the egg's yolk. Microscopically, this embryonic failure appears to manifest itself between Hamburger-Hamilton stages 8 and 9 of development and is characterized by a series of retarded developmental processes: closure of the anterior neuropore, brain vesicle differentiation, somite formation, and cardiac development. The disorder is inherited as an autosomal recessive trait. Attempts to identify factors that influence the disorder have thus far been unsuccessful. The symbol bld is proposed for this recessive gene.     

Studies on the surface of chick blastoderm cells. I. Electrophoretic mobility and pH-mobility relationships

The electrophoretic mobilities (E.P.M.) and pH-mobility relationships of chick blastoderm cells at successive developmental stages were determined. Mobilities in 0.145 M NaCl, pH 7.2, showed a wide spread and decreased from a mean value of 1.63 μ/sec/V/cm in the prestreak blastoderm, to 1.49 in the intermediate streak and 1.34 in the head process stages. Cell surface charge density changes as detected by E.P.M. were observed in all regions of the embryo. pH-mobility relationship studies showed that two cell populations differing in their approximate isoelectric point appeared at the intermediate streak and head process stages. Neuraminidase treatment indicated that the contribution of sialic acid residues to the negatively charged groups at the cell surfaces is very small at the stages studied.     

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.     

Cell surface changes during mitosis and cytokinesis of epithelial cells

Summary PtK2 cells were studied with scanning electron microscopy to record changes on the cell surface during mitosis and cytokinesis. During prophase, prometaphase and metaphase, the cells remain very flat with few microvilli on their surfaces. In anaphase cells, there is a marked increase in the number of microvilli, most of which are clumped over the separating chromosomes and polar regions of the mitotic spindle leaving the surface of the interzonal spindle region relatively smooth. Microvilli appear over the interzonal spindle region in telophase and the cells also increase in height. At the beginning of cleavage, the distribution of microvilli is roughly uniform over the surface but it becomes asymmetric at the completion of cleav-age when the daughter cells begin to spread. At this time most microvilli are over the daughter nuclei and the surfaces that border the former cleavage furrow. The regions of the daughter cells distal to the furrow are the first to spread and their surfaces have very few microvilli. When chromosome movement is inhibited by either Nocodazole or Taxol, microvilli formation is inhibited on the arrested cells. Nevertheless cell rounding still takes place in the normal time period. It is concluded from these observations that the signal for the onset of chromosome movement in anaphase is accompanied by a signal for the formation of microvilli. It is suggested that there is also a separate signal for the cell-rounding event in mitosis and that microvilli do not play a role in this contractile process.     

Blastoderm development in honey bee embryogenesis as seen in the scanning electron microscope

Summary

Based on observations in the scanning electron microscope, we describe the successive changes at the cell surface of fertilized honey bee eggs which, over a period of 20 h, lead to blastoderm formation. Each change starts in the differentiation center located in the anterior egg half and from there spreads as a wave towards both poles. However, one of the waves is heterogeneous: the protrusions or caps of oolemma which start enveloping the preblastoderm nuclei arise in different ways in the anterior third and in the more posterior regions of the egg cell. After the formation of these protrusions, three mitotic waves pass over the peripheral nuclei. The last of these blastemal mitoses results in a two-layered arrangement of peripheral nuclei, each surrounded by an outpocketing of the egg cell basally confluent with the central yolky part. This two-layered state after some time transforms into a single layer of columnar outpocketings which increase in height by extending their borders centripetally into the secondary periplasm (inner ‘Keimhautblastem’). The outpocketings disconnect from the central yolk mass as separate blastoderm cells only a short time before gastrulation. The events described here consume nearly 40% of the time period between oviposition and hatching; thus, and by comparison with most other insects, blastoderm formation in the honey bee is a very lengthy process.     

Developmental analysis of fs(1)gastrulation defective,a dorsal-group gene of Drosophila melanogaster

Summary The gastrulation defective (gd) locus is a maternally expressed gene in Drosophila required for normal differentiation of structures along the embryonic dorso-ventral axis. Cuticular defects of the offspring from females with different combinations of gd alleles comprised a phenotypic continuum. Complementation among several alleles produced normal offspring while progressively more severe mutations produced a graded loss of structures from ventral, and then lateral, blastoderm cells. The most severely affected embryos consisted entirely of structures derived from dorsal blastoderm cells. Histological examination of staged siblings from selected allelic combinations showed that internal tissues were similarly affected. The tissues observed in amorphic embryos support new, more dorsal, assignments of fate map positions for blastoderm precursors of the cephalopharyngeal apparatus, hindgut and ventral nerve cord. The loss of ventral and lateral structures did not occur through cell death and appeared to involve a change in blastoderm cell fate. A direct effect of the mutations on blastoderm cell determination, however, was insufficient to explain the development of the dorsalized embryos. Intermediate phenotypes suggested that cell interactions or movements associated with morphogenesis are required for the determination of some cell fates in the dorsoventral axis. Thus, the developmental fate of all blastoderm cells may not be fixed at the time of blastoderm formation.     

Cell cycle-dependent surface changes in Chinese hamster cells grown in suspension culture

The cell surface appears to play an important part in the control of cell replication. It has been demonstrated that the cell membrane undergoes cyclic changes in appearance which bear a relation to the cell cycle phase, irrespective of close intercellular contact. The surface of Chinese hamster (CHO) cells was investigated using the scanning electron microscope (SEM). The cells were synchronized in suspension culture and were sampled at frequent intervals during the cell cycle. During mitosis, the cells showed microvilli and few blebs. In early G 1 phase, profuse microvilli were seen. In late G 1 phase, blebs appeared and persisted in great numbers. During the synthesis of DNA in the S phase, blebs were observed in the early stages and then declined in number; in G 2 phase, the blebs appeared to be larger (1–2 μm) and more sparsely distributed than in late S phase. Some of these blebs were pedunculated and, in some instances, the diameter of the pedicles approximated the diameter of microvilli. Since the reasons for these changes are not understood, our long-range goal is to correlate the observed surface changes with internal biochemical events during the cell cycle.     

A fine-structure gynandromorph fate map of the Drosophila head

A gynandromorph fate map of the head of D. melanogaster was produced using 28 landmarks derived from one imaginal disc. An examination of the meaning of fine-structure mapping discloses that the sturt value observed between one pair of landmarks within a disc may approximate the relative physical distance of their progenitor cells at blastoderm, but for another pair of landmarks (assuming no directed cell movements), the sturt value may simply reflect their close geographic location at the time the cells are specified for their particular differentiation, a time much later in development when most cell division within the disc has come to an end. The formation of early developmental compartments has little effect on fate-map distances. Our analysis of the data suggests there are approximately ten cells present at the blastoderm stage that are head progenitors. Each blastoderm cell is likely to be the progenitor of a particular array of landmarks, but there is overlap between arrays from different blastoderm cells.     

Changes in cell surface and cortical cytoplasmic organization during early embryogenesis in the preimplantation mouse embryo

Membrane topography and organization of cortical cytoskeletal elements and organelles during early embryogenesis of the mouse have been studied by transmission and scanning electron microscopy with improved cellular preservation. At the four- and early eight-cell stages, blastomeres are round, and scanning electron microscopy shows a uniform distribution of microvilli over the cell surface. At the onset of morphogenesis, a reorganization of the blastomere surface is observed in which microvilli becomes restricted to an apical region and the basal zone of intercellular contact. As the blastomeres spread on each other during compaction, many microvilli remain in the basal region of imminent cell-cell contacts, but few are present where the cells have completed spreading on each other. Microvilli on the surface of these embryos contain linear arrays of microfilaments with lateral cross bridges. Microtubules and mitochondria become localized beneath the apposed cell membranes during compaction. Arrays of cortical microtubules are aligned parallel to regions of apposed membranes. During cytokinesis, microtubules become redistributed in the region of the mitotic spindle, and fewer microvilli are present on most of the cell surface. The cell surface and cortical changes initiated during compaction are the first manifestations of cell polarity in embryogenesis. These and previous findings are interpreted as evidence that cell surface changes associated with trophoblast development appear as early as the eight-cell stage. Our observations suggest that morphogenesis involves the activation of a developmental program which coordinately controls cortical cytoplasmic and cell surface organization.     

Early embryonic development and diapause stage in the band-legged ground cricket Dianemobius nigrofasciatus

The band-legged ground cricket Dianemobius nigrofasciatus enters diapause at an early embryonic stage when adults are reared under short-day conditions or the eggs are exposed to a low temperature. We examined the morphological features of the embryo during early development and determined the exact stage of entry into diapause. In non-diapause eggs, no periplasmic space was observed in the surface region and a small number of nuclei surrounded by cytoplasm (energids) were found among the yolk granules and lipid droplets 12 h after egg laying (AEL) at 25°C. The energids sparsely but evenly populated the surface region at 40 h AEL, but there were some gaps between these energids. A continuous thin layer of nuclei with cytoplasm had completely covered the egg surface at 56 h AEL, suggesting that the blastoderm is formed between 40 and 56 h AEL. At 72 h AEL, we found a germ band at the posterior pole. Electron microscopy revealed clear cell membranes at 40 h AEL. Staining with rhodamine-dextran dye demonstrated that the cell membrane is formed when the nuclei appear on the egg surface at 12–24 h AEL. These results indicate that cellularization occurs before blastoderm formation. In diapause eggs, neither the embryonic rudiment nor germ band was formed, but a continuous layer of cells covered the egg surface. It is concluded that D. nigrofasciatus enters diapause at the cellular blastoderm.