The autonomous cell fate specification of basal cell lineage: the initial round of cell fate specification occurs at the two‐celled proembryo stage

In angiosperms, the first zygotic division usually gives rise to two daughter cells with distinct morphologies and developmental fates, which is critical for embryo pattern formation; however, it is still unclear when and how these distinct cell fates are specified, and whether the cell specification is related to cytoplasmic localization or polarity. Here, we demonstrated that when isolated from both maternal tissues and the apical cell, a single basal cell could only develop into a typical suspensor, but never into an embryo in vitro. Morphological, cytological and gene expression analyses confirmed that the resulting suspensor in vitro is highly similar to its undisturbed in vivo counterpart. We also demonstrated that the isolated apical cell could develop into a small globular embryo, both in vivo and in vitro, after artificial dysfunction of the basal cell; however, these growing apical cell lineages could never generate a new suspensor. These findings suggest that the initial round of cell fate specification occurs at the two‐celled proembryo stage, and that the basal cell lineage is autonomously specified towards the suspensor, implying a polar distribution of cytoplasmic contents in the zygote. The cell fate transition of the basal cell lineage to the embryo in vivo is actually a conditional cell specification process, depending on the developmental signals from both the apical cell lineage and maternal tissues connected to the basal cell lineage.     

Tobacco zygotic embryogenesis in vitro: the original cell wall of the zygote is essential for maintenance of cell polarity, the apical-basal axis and typical suspensor formation

We have developed a reliable in vitro zygotic embryogenesis system in tobacco. A single zygote of a dicotyledonous plant was able to develop into a fertile plant via direct embryogenesis with the aid of a co-culture system in which fertilized ovules were employed as feeders. The results confirmed that a tobacco zygote could divide in vitro following the basic embryogenic pattern of the Solanad type. The zygote cell wall and directional expansion are two critical points in maintaining apical-basal polarity and determining the developmental fate of the zygote. Only those isolated zygotes with an almost intact original cell wall could continue limited directional expansion in vitro, and only these directionally expanded zygotes could divide into typical apical and basal cells and finally develop into a typical embryo with a suspensor. In contrast, isolated zygote protoplasts deprived of cell walls could enlarge but could not directionally elongate, as in vivo zygotes do before cell division, even when the cell wall was regenerated during in vitro culture. The zygote protoplasts could also undergo asymmetrical division to form one smaller and one larger daughter cell, which could develop into an embryonic callus or a globular embryo without a suspensor. Even cell walls that hung loosely around the protoplasts appeared to function, and were closely correlated with the orientation of the first zygotic division and the apical-basal axis, further indicating the essential role of the original zygotic cell wall in maintaining apical-basal polarity and cell-division orientation, as well as subsequent cell differentiation during early embryo development in vitro.     

Embryology ofCymbidium sinense:the Microtubule Organization of Early Embryos

InCymbidium sinense, the pattern of embryo development is unusualin that oblique cell divisions result in the formation of severalsuspensor cells prior to the development of the embryo proper.Characteristic changes in microtubular distribution can be foundwithin the zygote and the proembryo during their development.After fertilization, the ellipsoid-shaped zygote has randomlydistributed microtubules within its cytoplasm. As the zygotetakes on a more rounded appearance, microtubules organize intoa dense meshwork. Furthermore, microtubule bundles appear atthe chalazal region of the cell prior to the first mitotic divisionof the zygote. At the preprophase stage of mitosis, a preprophaseband of microtubules appears in the cytoplasm of the zygote.The zygote divides obliquely and unequally and gives rise toan apical cell and a slightly larger basal cell. Many randomly-alignedmicrotubules can be found in the cortex of the basal cell. Theincrease in the abundance of microtubules coincides with theisotropic expansion of the basal cell. The early division ofthe basal cell and subsequent division of the apical cell resultsin the formation of a four-celled embryo, of which three cellsnear the micropylar pole develop as suspensor cells. In thesuspensor cells, the microtubules tend to orient in the samedirection as the long axis of the cell. In addition, prominentmicrotubules can also be found near the adjoining cell wallsof the four-celled embryo. The terminal cell is highly cytoplasmicwith abundant microtubules within the cell. Subsequent divisionsof the terminal cell give rise to additional suspensor cellsand the embryo proper. In the mature embryo, five suspensorcells are usually present; one eventually grows through themicropyle of the inner integument and four grow towards thechalazal pole. The cortical microtubules of suspensor cellsredistribute from a longitudinal to a transverse direction asthey grow towards their respective poles.Copyright 1998 Annalsof Botany Company Embryogenesis, endosperm, microtubules, preprophase band, suspensor cells,Cymbidium sinense(Andr.) Willd.     

被子植物胚柄研究进展

胚柄在胚胎发育过程中是一个暂时性的结构, 但却起着为胚体提供营养和生长调节因子的作用。相对胚体而言, 胚柄具有细胞类型单一、结构相对独立及发育时间短等特点, 其在胚胎发育的基因调控和细胞命运决定研究中具有独特的优势。该文从胚柄的形成及特点、胚柄在胚胎发育中的作用、信号传递系统对胚柄的影响以及与胚柄相关基因的功能等方面进行综述, 以期为研究胚柄在胚胎发育过程中所起的作用以及胚柄的命运决定提供参考信息。     

Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts

The extra-embryonic endoderm lineage plays a major role in the nutritive support of the embryo and is required for several inductive events, such as anterior patterning and blood island formation. Blastocyst-derived embryonic stem (ES) and trophoblast stem (TS) cell lines provide good models with which to study the development of the epiblast and trophoblast lineages, respectively. We describe the derivation and characterization of cell lines that are representative of the third lineage of the blastocyst -extra-embryonic endoderm. Extra-embryonic endoderm (XEN) cell lines can be reproducibly derived from mouse blastocysts and passaged without any evidence of senescence. XEN cells express markers typical of extra-embryonic endoderm derivatives, but not those of the epiblast or trophoblast. Chimeras generated by injection of XEN cells into blastocysts showed exclusive contribution to extra-embryonic endoderm cell types. We used female XEN cells to investigate the mechanism of X chromosome inactivation in this lineage. We observed paternally imprinted X-inactivation, consistent with observations in vivo. Based on gene expression analysis, chimera studies and imprinted X-inactivation, XEN cell lines are representative of extra-embryonic endoderm and provide a new cell culture model of an early mammalian lineage.     

The embryonic cell lineage of the nematode Caenorhabditis elegans

The embryonic cell lineage of Caenorhabditis elegans has been traced from zygote to newly hatched larva, with the result that the entire cell lineage of this organism is now known. During embryogenesis 671 cells are generated; in the hermaphrodite 113 of these (in the male 111) undergo programmed death and the remainder either differentiate terminally or become postembryonic blast cells. The embryonic lineage is highly invariant, as are the fates of the cells to which it gives rise. In spite of the fixed relationship between cell ancestry and cell fate, the correlation between them lacks much obvious pattern. Thus, although most neurons arise from the embryonic ectoderm, some are produced by the mesoderm and a few are sisters to muscles; again, lineal boundaries do not necessarily coincide with functional boundaries. Nevertheless, cell ablation experiments (as well as previous cell isolation experiments) demonstrate substantial cell autonomy in at least some sections of embryogenesis. We conclude that the cell lineage itself, complex as it is, plays an important role in determining cell fate. We discuss the origin of the repeat units (partial segments) in the body wall, the generation of the various orders of symmetry, the analysis of the lineage in terms of sublineages, and evolutionary implications.     

An expanded role for the TWN1 gene in embryogenesis: defects in cotyledon pattern and morphology in the twn1 mutant of Arabidopsis (Brassicaceae)

The suspensor is a specialized basal structure that differentiates early in plant embryogenesis to support development of the embryo proper. Suspensor differentiation in Arabidopsis is maintained in part by the TWIN1 (TWN1) gene, which suppresses embryogenic development in suspensor cells: twn1 mutants produce supernumerary embryos via suspensor transformation. To better understand mechanisms of suspensor development and further investigate the function of TWN1, we have characterized late-embryo and post-embryonic development in the twn1 mutant, using seedling culture, microscopy, and genetics. We report here that the twn1 mutation disrupts cotyledon number, arrangement, and morphology and occasionally causes partial conversion of cotyledons into leaves. These defects are not a consequence of suspensor transformation. Thus, in addition to its basal role in suspensor differentiation, TWN1 influences apical pattern and morphology in the embryo proper. To determine whether other genes can similarly affect both suspensor and cotyledon development, we looked for twinning in Arabidopsis mutants previously identified by their abnormal cotyledon phenotypes. One such mutant, amp1, produced a low frequency of twin embryos by suspensor transformation. Our results suggest that mechanisms that maintain suspensor identity also function later in development to influence organ formation at the embryonic shoot apex. We propose that TWN1 functions in cell communication pathways that convey local positional information in both the apical and basal regions of the Arabidopsis embryo.     

The Arabidopsis Receptor Kinase ZAR1 Is Required for Zygote Asymmetric Division and Its Daughter Cell Fate

Asymmetric division of zygote is critical for pattern formation during early embryogenesis in plants and animals. It requires integration of the intrinsic and extrinsic cues prior to and/or after fertilization. How these cues are translated into developmental signals is poorly understood. Here through genetic screen for mutations affecting early embryogenesis, we identified an Arabidopsis mutant, zygotic arrest 1 (zar1), in which zygote asymmetric division and the cell fate of its daughter cells were impaired. ZAR1 encodes a member of the RLK/Pelle kinase family. We demonstrated that ZAR1 physically interacts with Calmodulin and the heterotrimeric G protein Gβ, and ZAR1 kinase is activated by their binding as well. ZAR1 is specifically expressed micropylarly in the embryo sac at eight-nucleate stage and then in central cell, egg cell and synergids in the mature embryo sac. After fertilization, ZAR1 is accumulated in zygote and endosperm. The disruption of ZAR1 and AGB1 results in short basal cell and an apical cell with basal cell fate. These data suggest that ZAR1 functions as a membrane integrator for extrinsic cues, Ca²⁺ signal and G protein signaling to regulate the division of zygote and the cell fate of its daughter cells in Arabidopsis.     

CAPSELLA EMBRYOGENESIS: THE EGG,ZYGOTE, AND YOUNG EMBRYO

The ultrastructure and composition of the egg, zygote, and young embryo of Capsella bursa-pastoris were examined. The egg is a highly polarized cell; one-half to one-third of the micropylar end is filled with a large vacuole while the chalazal end contains the nucleus and much of the cytoplasm of the cell. The wall which surrounds the cell is incomplete at the chalazal end. Ribosomes fill the cytoplasm and show little or no aggregation into polysomes. The structure of the nucleolus suggests that ribosomes are not being produced. Following fertilization and the formation of the zygote, the cell decreases slightly in volume as the large central vacuole becomes smaller. The zygote soon increases in size as the small chalazal vacuoles present before fertilization begin to enlarge. The dictyosomes become active and a continuous wall forms around the zygote. Aggregation of the ribosomes begins and numerous polysomes are formed. Before division of the zygote all plasmodesmata between the zygote and the surrounding cells are lost. The first division of the zygote is unequal as a result of its marked polarity. A large basal cell and a small terminal cell are produced. The basal cell appears to contain more protein, RNA, carbohydrate, and cell organelles than the terminal cell. Ribosomal aggregation is even more pronounced at this stage. Starch accumulates in the plastids. Numerous plasmodesmata are present between the terminal and basal cells but there are no connections between the endosperm or other cells. The basal cell divides next to give rise to a three-celled linear embryo consisting of the basal cell, the suspensor cell, and the terminal cell. The terminal cell stains more intensely for protein and RNA as a result of increased numbers of ribosomes. Starch in all the cells is about equal and reaches a maximum in the embryo at this stage.     

无距虾脊兰胚珠发育及种子形成研究

采用石蜡切片、半薄切片、扫描电镜技术对无距虾脊兰不同时期的子房(蒴果)进行研究.结果表明:(1)无距虾脊兰授粉后19d,胎座上分化出上万个胚珠原基,这些胚珠原基由1列细胞外包1层表皮细胞构成,其中胚珠原基内部顶端的细胞分化为孢原细胞,授粉后45 d,孢原细胞发育分化为大孢子母细胞.(2)无距虾脊兰成熟胚珠为倒生胚珠,双珠被,薄珠心,胚囊发育为蓼型,且胚珠的发育即便在同一个果实内也是不同步的.(3)受精后合子经过一次不均衡横裂形成基细胞和顶细胞;基细胞不参与胚体构成,分化为单细胞的胚柄,最后退化消失;顶细胞经多次分裂形成原球胚,胚胎发育类型为石竹型.(4)成熟种子呈纺锤形,由球形胚和内外双层种皮构成,双层种皮分别由内外珠被发育而来.     

毛竹实时荧光定量PCR内参基因的筛选及成花基因PheTFL1表达分析

通过qRT-PCR对毛竹相关成花基因PheTFL1的表达进行研究,为毛竹开花机理的研究提供理论依据.从毛竹UBC18、PP2A和EF1α等9个候选内参基因中筛选出在叶、幼嫩花序、花序轴、枝、竹青等11个组织器官中都稳定表达的PP2A用于毛竹PheTFL1基因qRT-PCR结果的校正.结果显示:PheTFL1基因在开花竹叶、枝和竹青中低丰度表达,与未开花竹差异不显著,但在花和花序轴中高丰度表达;在实生苗叶和根中高丰度表达,在实生苗茎中低丰度表达.PheTFL1基因在具有分生能力的幼嫩组织中高丰度表达,说明其不仅参与花发育的调控,还参与了分生组织生长的调控.     

FINE STRUCTURE OF THE ZYGOTE AND EARLY EMBRYO IN QUERCUS GAMBELII

This investigation begins with the late zygote and traces ultrastructural development to the late globular stage of the embryo. Two nucleoli and satellite nucleoli sometimes occur in the zygote nucleus. Mitochondria, dictyosomes, cytoplasmic ribosomes, rough ER, and lipid bodies are numerous in the zygote. Microbodies are occasionally seen. The cell wall becomes well developed before the first division. No plasmodesmata occur in the zygote wall. The basal cell of the proembryo and the suspensor cells of the later embryo have very dense cytoplasm with a high concentration of cytoplasmic ribosomes. The nuclei are very electron opaque. The terminal cell and the cells of the embryo proper have a fine structure similar to that of the zygote. Plastids increase in number, size, starch content, and amount of thylakoid lamellae as the embryo develops. Mitochondria are numerous and appear active at all stages. Dictyosome activity, ribosomal aggregation, and the amount of ER are highest during the late globular stage. Lipid bodies are present up to the early globular stage, then disappear. The inner cell walls of the embryo are thin and have many plasmodesmata. These walls begin to thicken at the late globular stage, and at this time the size of the embryo begins to show an increase over that of the zygote. The results show a corresponding increase in the amount and activity of the metabolic machinery as the development of the embryo progresses. Lipids are probably more important as a nutrient source in the zygote and early embryo; starch becomes more important in the late stages. Absorption of nutrient material into the embryo sac and developing embryo appears to be from the chalazal end.     

Cell lineage and determination of cell fate in ascidian embryos

A detailed cell lineage of ascidian embryos has been available since the turn of the century. This cell lineage was deduced from the segregation of pigmented egg cytoplasmic regions into particular blastomeres during embryogenesis. The invariant nature of the cell lineage, the segregation of specific egg cytoplasmic regions into particular blastomeres, and the autonomous development of most embryonic cells suggests that cell fate is determined primarily by cytoplasmic determinants. Modern studies have provided strong evidence for the existence of cytoplasmic determinants, especially in the primary muscle cells, yet the molecular identity, localization, and mode of action of these factors are still a mystery. Recent revisions of the classic cell lineage and demonstrations of the lack of developmental autonomy in certain embryonic cells suggest that induction may also be an important mechanism for the determination of cell fate in ascidians. There is strong evidence for the induction of neural tissue and indirect evidence for inductive interactions in the development of the secondary muscle cells. In contrast to the long-accepted dogma, specification of cell fate in ascidians appears to be established by a combination of cytoplasmic determinants and inductive cell interactions.     

高精度时空转录组揭示小鼠早期胚胎三胚层细胞谱系发生过程

从受精卵发育成具有不同细胞类型个体的过程中,细胞命运受到多个层次的调控。在哺乳动物的早期胚胎发育过程中,原肠运动是外、中、内三个胚层的建立过程,为后续的器官发生和形态建成提供了发育蓝图。然而目前对于三胚层命运建立的分子机制认识并不清晰。该文通过对小鼠早期胚胎的时空转录组分析,从分子层面揭示了外、中、内三胚层谱系发生的整个过程。     

Early Cell Fate Decisions of Human Embryonic Stem Cells and Mouse Epiblast Stem Cells Are Controlled by the Same Signalling Pathways

Human embryonic stem cells have unique value for regenerative medicine, as they are capable of differentiating into a broad variety of cell types. Therefore, defining the signalling pathways that control early cell fate decisions of pluripotent stem cells represents a major task. Moreover, modelling the early steps of embryonic development in vitro may provide the best approach to produce cell types with native properties. Here, we analysed the function of key developmental growth factors such as Activin, FGF and BMP in the control of early cell fate decisions of human pluripotent stem cells. This analysis resulted in the development and validation of chemically defined culture conditions for achieving specification of human embryonic stem cells into neuroectoderm, mesendoderm and into extra-embryonic tissues. Importantly, these defined culture conditions are devoid of factors that could obscure analysis of developmental mechanisms or render the resulting tissues incompatible with future clinical applications. Importantly, the growth factor roles defined using these culture conditions similarly drove differentiation of mouse epiblast stem cells derived from post implantation embryos, thereby reinforcing the hypothesis that epiblast stem cells share a common embryonic identity with human pluripotent stem cells. Therefore the defined growth factor conditions described here represent an essential step toward the production of mature cell types from pluripotent stem cells in conditions fully compatible with clinical use ant also provide a general approach for modelling the early steps of mammalian embryonic development.     

Capsella embryogenesis: The suspensor and the basal cell

Summary The suspensor and basal cell ofCapsella were examined with the electron microscope and analyzed by histochemical procedures. The suspensor cells are more vacuolate and contain more ER and dictyosomes, but fewer ribosomes and stain less intensely for protein and nucleic acids than the cells of the embryo. The end walls of the suspensor cells contain numerous plasmodesmata but there are no plasmodesmata in the walls separating the suspensor from the embryo sac. The lower suspensor cells fuse with the embryo sac wall and the lateral walls of the lower and middle suspensor cells produce finger-like projections into the endosperm. At the heart stage the suspensor cells begin to degenerate and gradually lose their ability to stain for protein and nucleic acids.The basal cell is highly vacuolate and enlarges to a size of 150 X 70. An extensive network of wall projections develops on the micropylar end wall and adjacent lateral wall. The nucleus becomes deeply lobed and suspended in a strand of cytoplasm traversing the large vacuole. The cytoplasmic matrix darkens at the late globular stage and histochemical staining for protein becomes very intense. The basal cell remains active after the suspensor cytoplasm has degenerated. It is proposed that the suspensor and basal cell function as an embryonic root in the absorption and translocation of nutriments from the integuments to the developing embryo.Research supported by NSF grant GB 3460 and NIH grant 5-RO 1-CA-03656-09.