Additional Cell Divisions Research Articles

The Combination of Stem Cell Factor and Oncostatin M Maintains Cord Blood-Derived NOD/SCID-Repopulating Cells.

We here investigated whether receptor tyrosine kinase signaling (ckit, flt3) was sufficient to maintain hematopoietic stem cells (HSC) in culture, or whether, like in embryonic stem cells, signaling through gp130 is also required. Sorted CD34+ AC133+ (CD33/ CD38/ CD71)- cells from human umbilical cord blood (CB) were cultured in the presence of combinations of ckit-ligand stem cell factor (SCF), flt3-ligand (FL) and different stimulators of gp130 (IL-6, IL-11, LIF, OSM, CT1 and CNTF). Of these cytokines, SCF stimulated cell division by itself. Gp130-stimulating cytokines did not give any additional cell division activity. When SCF was included in the cytokine cocktail, progenitor levels (CFC, CAFCw2, CAFCw6) was very similar for all combinations of cytokines tested after this initital 6-day culture. To study more immature progenitors, cells were harvested after 6 days of serum-free culture and cultured for another 6 weeks on FBMD-1 stromal cells (LTC-CAFC). These experiments showed that in initial 6 day cultures with FL or gp130-stimulators alone, very little hematopoietic activity remained. SCF maintained CFC, and LTC-CAFCw2 and LTC-CAFCw6 at about half the original level. However, combinations of SCF with OSM consistently increased the number of LTC-CAFCw2 and w6 about two-fold. This finding suggests that very immature hematopoietic progenitors were at least maintained in serum-free cultures. To study the most immatore cells, we performed in transplantations into sublethally irradiated NOD/SCID mice. When CB cells were cultured with SCF alone, NOD/SCID-repopulating cell (CRU) frequency (1 in 900) dropped to about 20% in 6 days (1 in 5500). Similarly, cultures without growth factors or with FL or OSM showed a decrease to less than 10% of the original CRU number. In contrast, the combination of SCF and OSM maintained CRU at about half the original level (1 in 1900). This study shows that signals through ckit and gp130 synergize to maintain CRU level. However, to achieve expansion of human NOD/SCID-repopulating cell numbers, additional signaling pathways need to be stimulated.

Transformation fingerprint: induced STAT3-C, v-Src and Ha-Ras cause small initial changes but similar established profiles in mRNA.

Induced transformation of mouse fibroblasts was carried out by releasing tetracycline-repressed expression of an oncogenic mutant of STAT3, STAT3-C, or of v-Src or Ha-Ras. At 15 days after derepression of each oncogene, DNA microarrays showed elevation (>3-fold) of a similar group of approximately 25 mRNAs compared to untransformed cells. RT-PCR confirmed a number of these mRNA elevations. RNA samples were then analysed at intervals during the first 24 h after doxycycline removal to determine the time of early changes. Extensive changes were not observed by array analysis, except in v-Src-expressing cells where about 10 mRNAs were elevated threefold or more. However, RT-PCR did uncover changes in each derepressed cell type that included some of the changes observed after the 15-day transformation period. In addition, STAT3-C target genes such as BclXI and cyclin D1, which were not observed on array analysis, were elevated by RT-PCR analysis. We conclude, therefore, that early after oncogene induction, transcriptional changes, including those initiated by STAT3-C, may occur only in scarce mRNA and/or to a limited extent. However, with additional time and probably additional cell division, a new epigenetic state is established that is mirrored by a changed transcriptional profile emblematic of transformation by each of three oncogenes.

The uniquely human in human nature

features, as manufacturers do when they provide a description of the parts of an assembly-required crib; or by comparing the object with one from a related category, as parents do when they tell their child that a zebra has stripes but a horse does not. Most answers to the question What is human nature? adopt this second strategy when they nominate the features that are either uniquely human or that are quantitative enhancements on the properties of apes. I adopt such a comparative posture here and describe seven psychological features that are either restricted to or enhanced in humans compared with our closest relatives, the chimpanzees. Four are of the 1⁄2rst category; the other three are quantitative enhancements on chimpanzee talents. The distinct psychological qualities of Homo sapiens are traceable to genetic changes that permitted the founder cells that become mature neurons to continue to divide for an extra seventy-two hours. Those additional cell divisions signi1⁄2cantly expanded the size of the human cortex and contributed to the novel cognitive, emotional, and motor skills that emerge in humans over the course of development. However, some scientists remain inexplicably resistant to acknowledging that any human quality is unique. When a linguist claims that only humans have a language with a grammar, a scientist will reply that chimpanzees can be taught to communicate with pieces of plastic. Jane Goodall’s discovery that chimpanzees use tools is celebrated because of its implication that my use of a hammer to hang my granddaughter’s recent artwork on the wall is not fundamentally different from a chimpanzee’s use of a twig to ferret out termites. But the modern syn-

New plant growth-modifying properties of the Agrobacterium T-6b oncogene revealed by the use of a dexamethasone-inducible promoter.

Agrobacterium 6b oncogenes induce tumours on Nicotiana glauca and enations and associated modifications in transgenic N. tabacum plants. 2x35S-AB-6b tobacco rootstocks produced a graft-transmissible factor that induced enations in wild-type scions; the nature of this enation factor remains to be identified. Here, we report on the properties of tobacco plants carrying a dexamethasone-inducible T-6b gene (dex-T-6b). Induction with dex led to complex growth modifications, many of which have not been reported previously. Modifications were only found in growing tissues; mature tissues remained unaffected. Growth could be either stimulated or inhibited. Dex induction of young plants led to morphogenetic gradients that included enations, tubular leaves and fragmented leaf primordia. Root elongation was increased or slowed down, while radial root growth was strongly enhanced. Additional cell divisions were found in the root pericycle and vasculature. Enation factor import from mature tissues did not have the same effects on growing tissues as local T-6b synthesis: normal scions grafted on induced dex-T-6b rootstocks formed enations, whereas local dex-T-6b induction at the shoot apex led to numerous dark-green spots on the abaxial side of the leaves. In leaf patch assays, the 23-kDa T-6b protein was found to move through leaves and to enter the vascular system. This and the fact that rootstocks of spontaneous tobacco enation mutants did not modify wild-type scions contrary to 6b plants indicate that the 6b protein might be the enation factor.

Bacterial cell division: a moveable feast.

Recent advances in fluorescence microscopy technologies have revolutionized our views on bacterial cell organization. A prolific area of this revolution in tiny cell visualization has been cell division. Most bacteria divide in their middle, implying that they are aware of at least one particular site of themselves, the midcell division site. This raises the fundamental question: How do bacterial cells identify midcell? It all started with the immunolocalization studies of the FtsZ division protein, which showed that FtsZ assembles at midcell in a ring-like structure (1). Since then, additional cell division proteins have been shown to localize to midcell (2). In a recent issue of the Proceedings , Raskin and de Boer (3) extend this concept of site-specific protein targeting by describing the unique dynamic properties of the localization of the MinD division inhibitor. This discovery adds a new dimension to our exploration of the unexpected complexity in the spatial organization of bacterial cells. One cannot talk about bacterial cell division without considering the FtsZ protein, which shares properties with cytoskeletal molecules. FtsZ, which binds GTP and has a GTPase activity (4–6), plays a central role in cytokinesis as a major component of a contractile ring (1). The assembly of the FtsZ ring at midcell occurs well before the constriction is initiated (1). In addition to ftsZ (7), many more genes are specifically involved in cell division (2, 8). The other cell division proteins are later recruited to the FtsZ ring to form the membrane-associated septal ring that mediates septation. The answer to the problem of midsite selection is still largely unknown, but important insights have come from studies on minicell-producing ( min ) mutants (9). These mutants often divide at the poles (¼ and ¾ sites on the cell), generating small, chromosomeless minicells. Here, we discuss the roles …

2 Drosophila Myogenesis and insights into the Role of nautilus

Several aspects of muscle development appear to be conserved between Drosophila and vertebrate organisms. Among these is the conservation of genes that are critical to the myogenic process, including transcription factors such as nautilus. From a simplistic point of view, Drosophila therefore seems to be a useful organism for the identification of molecules that are essential for myogenesis in both Drosophila and in other species. nautilus, the focal point of this review, appears to be involved in the specification and/or differentiation of a specific subset of muscle founder cells. As with several of its vertebrate and invertebrate counterparts, it is capable of inducing a myogenic program of differentiation reminiscent of that of somatic muscle precursors when expressed in other cell types. We therefore favor the model that nautilus implements the specific differentiation program of these founder cells, rather than their specification. Further analyses are necessary to establish the validity of this working hypothesis. Studies have revealed a critical role for Pax-3 in specifying a particular subset of myogenic cells, the progenitors of the limb muscles. These myogenic cells migrate from the somite into the periphery of the organism, where they differentiate. These myoblasts do not express MyoD or myf5 until they have arrived at their destination and begin the morphologic process of myogenesis (Bober et al., 1994; Goulding et al., 1994; Williams and Ordahl, 1994). They then begin to express these genes, possibly to put the myogenic plan into action. Thus, as with nautilus, MyoD and myf5 may be necessary for the manifestation of a muscle-specific commitment that has already occurred. By comparison with vertebrates, it was anticipated that the single Drosophila gene would serve the purpose of all four vertebrate genes. However, its restricted pattern of expression and apparent loss-of-function phenotype are inconsistent with this expectation. It remains to be determined whether nautilus functions in a manner similar to just one of the vertebrate genes. Since the myf5- and MyoD-expressing myoblasts are proliferative, the loss of one cell type appears to be compensated by proliferation of the remaining cell type. This apparent plasticity may obscure differences in mutant phenotype resulting from the loss of particular cells that express each of these genes. In Drosophila, by comparison, nautilus-expressing cells committed to the myogenic program undergo few, if any, additional cell divisions, and thus no other cells are available to compensate for the loss of nautilus. Therefore, the apparent differences between the Drosophila nautilus gene and its vertebrate counterparts may reflect, at least in part, differences in the developmental systems rather than differences in the function of the genes themselves.

Calcium, Calcium Oxalate Crystals, and Leaf Differentiation in the Common Bean (Phaseolus vulgaris L.)*

AbstractYoung plants of Phaseolus vulgaris were grown in nutrient solutions at different levels of calcium concentration. When the calcium concentration was low more palisade parenchyma and less extended bundle sheath was formed at the adaxial side of minor veins of the leaves as compared to leaves of plants grown with higher calcium supply. The number of calcium oxalate crystals in the bundle sheath extensions was positively correlated to the amount of calcium fed to the plants. The ion induces additional cell divisions in the bundle sheath extensions. A high supply of calcium leads to the formation of a second type of crystal in the bundle sheath.

Polyamines and related enzymes in rice seeds differing in germination potential

In ungerminated rice seeds, (Japonica rice variety, CV Tapei 309), the content of free amines (putrescine, spermidine, spermine, tyramine) was higher in seed lots having a low germination frequency compared to those with high germination potential. Conversely, amine conjugates (di-feruloylputrescine, di-feruloylspermidine, diferuloyldiaminopropane and feruloyltyramine) decreased with loss of viability. Thus, these compounds appeared to constitute biochemical markers of seed viability. In seeds with high germination potential, conjugates decreased drastically during germination, with an early and rapid increase in free amines (putrescine, spermidine, tyramine). Arginine decarboxylase (ADC) activity was highest during the germination of high germination potential seeds, its activity gradually declining with loss of viability and being closely correlated with agmatine content. The polyamine biosynthetic inhibitors (α-DL-difluoromethylarginine, DFMA, a specific and irreversible inhibitor of ADC; α-DL-difluoromethylornithine, DFMO, a specific irreversible inhibitor of ornithine decarboxylase (ODC); cyclohexylammonium sulfate, CHA, inhibitor of spermidine synthase) neither depleted putrescine and spermidine levels nor inhibited germination in high germination potential seeds. In low germination potential seeds, the germination process was inhibited by DFMA or CHA. Application of agmatine resulted in a reversal of inhibition. DFMA inhibited ADC activity in both categories of seeds. In low germination potential seeds treated with CHA no ADC activity was found. These results suggest that amines are involved in the germination process of rice seeds. It appears that amine conjugates may serve as a storage form of amines which, upon enzymatic hydrolysis, could supply the cell with an additional amine reserve and influence cell division and/or cell elongation.

Regulation of Islet β-Cell Proliferation by Prolactin in Rat Islets

This study examined the effects of prolactin on beta-cell proliferation in pancreatic islet of Langerhans. Insulin secretion and beta-cell proliferation were significantly increased from neonatal rat islets cultured for 4 days in the presence of either 500 ng/ml ovine prolactin (oPRL) or rat prolactin (rPRL). These effects could be prevented by including anti-oPRL serum in the culture media. Although insulin secretion and beta-cell proliferation were slightly higher during the first 24 h of exposure to rPRL, maximal response was observed after 4 days for insulin secretion and 6-10 days for beta-cell proliferation. The initial mitogenic response of beta-cell to rPRL occurred by the limited recruitment of nondividing beta-cells into the cell cycle and by most daughter cells proceeding directly into additional cell division cycles. Subsequently, the maximal effect of rPRL on beta-cell proliferation was maintained by a higher rate of recruitment of previously nondividing beta-cells into cell cycle with only one fourth of the daughter cells continuing to divide. These observations are difficult to reconcile with the proposal that a limited pool of beta-cells capable of undergoing cell division exists in islets. Instead, these observations suggest that individual beta-cells are transiently re-entering the cell cycle and dividing infrequently in response to rPRL. In this case, the majority of the beta-cells would not be expected to be in an irreversible Go phase. We also demonstrated that the effects of rPRL on beta-cell proliferation occur at normal serum glucose concentrations and are affected by inhibitors of polyamine metabolism. Additional studies on the effects of rPRL on beta-cells should provide important information on the regulation of beta-cell proliferation during conditions of increased insulin demand.

A late role for a subset of neurogenic genes to limit sensory precursor recruitments in Drosophila embryos.

In Drosophila, mutations in a class of genes, the neurogenic genes, produce an excess of neurons. This neural hyperplasia has been attributed to the formation of more than the normal number of neuronal precursor cells at the expense of epidermal cells. In order to find out whether the neurogenic genes only act at this intial step of neurogenesis, we studied the replication pattern of the sensory organ precursor cells by monitoring BrdU incorporation in embryos mutant for Notch (N), Delta (Dl), mastermind (mam), almondex (amx), neuralized (neu), big brain (bib) and the Enhancer of split-Complex (E(spl)-C). Using temperature sensitive alleles of two of the neurogenic genes, DI and N, we also induced an acute increase of replicating sensory precursors by shifting briefly to the restricted temperature. We have found that the loss of function of all the seven neurogenic loci that were tested causes an increase in replicating sensory precursor cells, consistent with the model that these neurogenic genes normally participate in the process of restricting the number of neuronal precursors. Whereas the temporal pattern of replication appeared normal in mutants of five of the seven neurogenic loci, in N and mam embryos replicating PNS cells are present beyond the time when they normally undergo replication. Experiments with colchicine suggest that many of these late replicating cells may be newly emerging precursors and probably not additional cell divisions of already recruited precursors. Thus, different neurogenic genes may be required over different periods of time for the specification of sensory precursor cells.

The patterns of cell division and chloroplast reproduction in young leaflets ofSphagnum

The pattern of cell division in Sphagnum leaflets is reviewed. The review is supplemented by data on additional cell divisions, symmetry, and the pattern of chloroplast reproduction. The results are compared with Neuburg's (1960) results about Protosphagnum nervatum from the Upper Permian. The regular shift of the spindle orientation between subsequent mitoses (exceptions occur), the temporal pattern of asymmetrical and symmetrical cell divisions, and the handedness of cell triads are described and discussed. The pattern could hardly be more regular. Additional cell divisions modify the pattern but do not really change it.

Relationship between glial reaction to a stab wound and tumor development after receiving transplacental ethylnitrosourea in the rat

Fisher 344 rats born from mothers treated with ethylnitrosourea (ENU) 50 mg/kg intravenously were injured at the 1st and 2nd month of extrauterine life by a transcranial stab. The wound affected cerebral cortex, white matter and basal ganglia. The animals were killed 15 and 45 days and 5 months after injury and cell reaction was studied histologically and immunohistochemically. Bromodeoxyuridine (BrdUrd) was administered 1 h before sacrifice and the labeled cells were evaluated. In ENU-treated rats injured at 1 month of age only minor differences were found in comparison with injured controls. In ENU rats injured at 2 months of age and killed 15 days later, a higher number of BrdUrd-labeled cells was found in comparison with controls; 45 days after injury the cell reaction acquired the aspect of a microtumor, however, no microtumor unrelated with the needle track was present. In ENU rats killed 5 months after the injury, there was no difference between injured and not injured ENU-treated rats, as far as the aspect and the number of tumors were concerned. The tumor phenotype was, thus, anticipated by the cell response to trauma in ENU rats. The interpretation is that the additional cell division, in response to trauma, anticipate not only the phenotypic, but also the cell kinetics changes, as indicated by BrdUrd labeling.

Adult mammalian hepatocyte as target cell for retroviral gene transfer: a model for gene therapy.

Gene therapy models have recently been developed based on the repopulation of a major organ, such as mammalian bone marrow, with cells genetically modified in vitro by infection with retroviral vectors (1-4). Another important target organ for therapeutically useful gene transfer is the liver. Unfortunately, the murine leukemia viruses (MLV) used to construct most retroviral vectors do not infect newborn, adult, or regenerating liver cells in vivo (5-8), although fetal liver can be infected and continues to express retrovitally transduced genes postnatally (7, 8). We now show that adult primary rat hepatocyte cultures (9, 10) are in fact susceptible to infection with MLV-based retroviral vectors during a period of transient dedifferentiation in vitro. Primary hepatocyte cultures exhibit many adult liver-specific functions--including gluconeogenesis and the expression of albumin, alcohol dehydrogenase, and type I pyruvate kinase--during their early lag (days 0-2) and late stationary (after day 11) phases of culture (10 16). During the intervening phase of logarithmic growth, these mature liver-specific functions are transiently but markedly decreased, and fetal hepatocyte functions such as alphal-fetoprotein and type III pyruvate kinase are expressed (12). Hepatocyte cultures were exposed at varying times during their growth cycle to a retroviral vector expressing a mutagenized but fully functional human hypoxanthine guanine phosphoribosyl transferase (HPRT) (Fig. 1, LSAPALM) (J.-K. Yee et al., in preparation). Human HPRT enzymic activity was detected in isoelectric focusing polyacrylamide gels (17) under conditions that distinguish it from the endogenous rat enzyme by its altered isoelectric point. Twoto three-dayold cultures infected with 5 x 1 0 6 infectious units of the HPRT vector exhibited human HPRT enzymic activity on day 14 postplating, as indicated by the arrows in Fig. 2A (lane a). The transduced human HPRT activity was at least 10-25% of the endogenous rat HPRT enzymic activity, equivalent to the high levels achieved in primary rat fibroblasts (Fig. 2, lane c) and human lymphoblasts (18). Since the upper bands in Fig. 2A (lanes a and c) may also contain human HPRT multimers (17), the actual transduced levels of human HPRT enzymic activity may be even higher. Figure 2B shows that human HPRT activity was present only when the hepatocyte cultures were infected during the early proliferative, dedifferentiated period of growth (days 2-3). Very low levels of human HPRT expression, not evident in Fig. 2B, were also detected on days 1 and 5, but not at any other times of infection. In addition, quiescent hepatocytes on day 1 1 can also be stimulated to reinitiate DNA synthesis and undergo additional cell division by exposure to fresh medium containing rat serum or polypeptide mitogens, mimicking the regenerating adult liver following partial hepatectomy (9, 19).

Iron deficiency stress induced morphological and physiological changes in root tips of sunflower

Typical morphological and physiological changes were observed in iron deficient sunflower (Helianthus annuus L. cv. Sobrid) roots. These changes, or so‐called iron stress reactions, are exclusively confined to the root tips. Typical morphological changes included additional cell division in the rhizodermis layer and enhanced formation of root hairs, leading to an increase in root diameter (“swollen root tips”). These morphological changes were correlated with physiological changes such as increased release of protons, accumulation of phenols in the rhizodermis, and an increased ability of the roots to reduce iron‐III compounds (“reducing capacity”). A marked increase in ability of the root tips to take up and translocate iron occurred simultaneously with these changes. There is good evidence that these morphological and physiological changes are reflections of an effective regulatory mechanism for enhanced mobilization of sparingly soluble iron‐III compounds in the rhizosphere and for iron uptake by sunflower plants.

Commitment to erythroid differentiation by friend erythroleukemia cells: a stochastic analysis

A method for the clonal analysis of murine erythroleukemia cells has been developed which allows the precise characterization of the number of progeny produced by each cell and the degree of differentiation of each progeny cell. The potential of almost every cell in the culture can be monitored because a plating efficiency close to 100% has been achieved. The effects of treatment with an inducer of differentiation (DMSO) on the proliferative capacity of the treated cells have been studied with this technique. Cells from a mass culture treated with inducer give rise to colonies of differentiated progeny when subsequently cloned in the absence of inducer. Colonies exhibiting this phenotype represent the progeny of cells committed to the differentiation pathway by treatment with inducer. We observe that the commitment decision limits the subsequent proliferative capacity of the cell to four additional cell divisions. A quantitative analysis suggests that the commitment decision for each cell is made in a stochastic manner. Irreversible commitment to the expression of differentiated functions occurs with discrete probability per cell generation for many cell generations. The value for this probability is a function of the concentration of inducer (DMSO). A correlative biochemical study suggests that an irreversible commitment decision by a significant proportion of the population precedes or accompanies increases in cytoplasmic globin mRNA levels, one of the earliest detectable biochemical markers for erythroid differentiation in this system. A specific kinetic model based on these considerations has been developed to predict clonal phenotypes as a function of time and probability of commitment. Quantitative predictions based on this model are in excellent agreement with experimental observations. The effectiveness of a stochastic model in predicting the behavior of this system is discussed in relation to the stochastic behavior of normal hematopoiesis and the biochemical mechanisms which control these differentiation programs.

Expression of homeotic mutations in duplicated and regenerated antennae ofDrosophila melanogaster

Three homeotic mutants, aristapedia (ssa and ssa-UCl) and Nasobemia (Ns) which involve antenna-leg transformations were analyzed with respect to their time of expression. In particular we studied the question of whether these mutations are expressed when the mutant cells pass through additional cell divisions in culture. Mutant antennal discs were cultured in vivo and allowed to duplicate the antennal anlage. Furthermore, regeneration of the mutant antennal anlage was obtained by culturing eye discs and a particular fragment of the eye disc. Both duplicated and regenerated antennae showed at least a partial transformation into leg structures which indicates that the mutant gene is expressed during proliferation in culture.

Embryo Sac Formation in Wheat

Megagametogenesis of common wheat was examined embryologically paying attentions not only to the egg apparatus and polar nuclei but also to the antipodals whose activities were thought to contribute to the early development of the endosperm. The results of daily observations were summarised as follows: During 10∼8 days before flowering (for short "b. f." in the rest), embryo sac gave rise to 8 nucleate stage through 3 times of nuclear division comprising a micropylar and central quartet (Figs. 1-5). Then of 8 nuclei, 3 at the micropylar end gave rise to the egg apparatus; 3 at the chalazal side of the central quartet gave rise to antipodal cells; and remaining 2, one from each quartet migrated into the micropylar vacuole without cell membrane formation, and gathered to form the polar nuclei. 2 synergid cells started protrution in their charazal sides next day (7 days b. f.), and maturated by 2 days b. f., thus they became slightly senile on flowering day. The egg cell entered into growing at 6 days b. f. and continued its growth till flowering day. 2 polar nuclei getting closer mutually were located near the egg apparatus by 3∼2 days b.f. In mature embryo sac they were in close contact with each other, however, never fused prior to fertilization. Very interesting observations were obtained in the development of antipodal cells. Immediately after the formation of 3 antipodal cells, they entered into cell division to fill up the chalazal vacuole (Figs. 7∼10). These additional cell divisions proceeded 4 times and resulted in about 40 cells which occupied the chalazal half space of the embryo sac by 4∼3 days b. f. (Fig. 14). After the 2nd division, one of the antipodal cells which was located at the chalazal pole showed a singularity and divided to form a haustrium composed of some special shaped cells at 6∼5 days b. f. (Figs. 10∼14). The antipodal haustrium was short-lived and degenerated after the 4th or the last division of other antipodal cells and the contiguous antipodal cells also begun to degenerate continuously. However, antipodal cells in micropylar side increased in their size accompanying with rapid enlargement of the embryo sac that occurred from 2∼1 days b. f. (Fig. 16). on the flowering day, antipodal complex was situated in somewhat lateral side of the embryo sac, and consisted of 12∼17 large, mature cells (Fig. 17). The number of the antipodal cells had little relation with the varietal and environmental differences but varied with the age of the embryo sac remarkably (Table 2). Based upon these observations, the authors thin that the antipodal cells contribute to the growing of the embryo sac during before flowering. By 2 days b. f. the embryo sac came to obtain the ability to fertilize, however, its subsequent development was somewhat incomplete. On the day before flowering the embryo sac was able to give rise to normal seed if artificially pollinated, although its anthers were not yet mature.