Cell Lineage of the Ilyanassa Embryo: Evolutionary Acceleration of Regional Differentiation during Early Development

Cell lineage studies in mollusk embryos have documented numerous variations on the lophotrochozoan theme of spiral cleavage. In the experimentally tractable embryo of the mud snail Ilyanassa, cell lineage has previously been described only up to the 29-cell stage. Here I provide a chronology of cell divisions in Ilyanassa to the stage of 84 cells (about 16 hours after first cleavage at 23°C), and show spatial arrangements of identified nuclei at stages ranging from 27 to 84 cells. During this period the spiral cleavage pattern gives way to a bilaterally symmetric, dorsoventrally polarized pattern of mitotic timing and geometry. At the same time, the mesentoblast cell 4d rapidly proliferates to form twelve cells lying deep to the dorsal ectoderm. The onset of epiboly coincides with a period of mitotic quiescence throughout the ectoderm. As in other gastropod embryos, cell cycle lengths vary widely and predictably according to cell identity, and many of the longest cell cycles occur in small daughters of highly asymmetric divisions. While Ilyanassa shares many features of embryonic cell lineage with two other caenogastropod genera, Crepidula and Bithynia, it is distinguished by a general tendency toward earlier and more pronounced diversification of cell division pattern along axes of later differential growth.     

Suspensor Length Determines Developmental Progression of the Embryo in Arabidopsis

The first structure that differentiates during plant embryogenesis is the extra-embryonic suspensor that positions the embryo in the lumen of the seed. A central role in nutrient transport has been ascribed to the suspensor in species with prominent suspensor structures. Little is known, however, about what impact the size of the rather simple Arabidopsis (Arabidopsis thaliana) suspensor has on embryogenesis. Here, we describe mutations in the predicted exo-polygalacturonase gene NIMNA (NMA) that lead to cell elongation defects in the early embryo and markedly reduced suspensor length. Mutant nma embryos develop slower than wild-type embryos, and we could observe a similar developmental delay in another mutant with shorter suspensors. Interestingly, for both genes, the paternal allele has a stronger influence on the embryonic phenotype. We conclude that the length of the suspensor is crucial for fast developmental progression of the embryo in Arabidopsis.Annual, self-pollinating weeds such as Arabidopsis (Arabidopsis thaliana) benefit from a short life cycle in their natural ephemeral habitats (Snell and Aarssen, 2005). A rapid progression through embryogenesis is a prerequisite for early seed maturation, and it is therefore not surprising that Arabidopsis sets up its body plan already after a very limited number of cell divisions (Aarssen, 2000; Lau et al., 2012).Arabidopsis embryogenesis starts with the fertilized egg cell, the zygote, which elongates about 3-fold before it divides asymmetrically. The smaller apical cell is the founder of the embryo proper that contributes to most of the later seedling, while the larger basal cell develops into a support structure called the suspensor (Jeong et al., 2011a). The suspensor is formed by a series of transverse cell divisions followed by longitudinal cell expansion forming a stalk-like structure. Only the upper-most suspensor cell, the hypophysis, will contribute to parts of the root meristem, while the rest of the suspensor remains extraembryonic and will cease its growth at the heart stage of the embryo (Yeung and Meinke, 1993). The suspensor is thought to be important for pushing the embryo into the lumen of the seed, where the embryo is surrounded by the nourishing endosperm. In addition, a key function in nutrient and hormone transport to the embryo is assigned to the suspensor (Kawashima and Goldberg, 2010).The Arabidopsis suspensor achieves its maximum length with a minimum number of cells by having a rod-shaped structure built by a single cell file. Although several mutants with distorted or shorter suspensors have been described in Arabidopsis, little is known about what impact suspensor length has on embryo development (Schwartz et al., 1994; Vernon and Meinke, 1994; Lukowitz et al., 2004; Breuninger et al., 2008; Bayer et al., 2009; Jeong et al., 2011b).As in all plant cells, the size and shape of suspensor cells is primarily determined by the elasticity of the cell wall. While rigid cellulose microfibrils determine the direction of cell expansion, it is the pectin matrix that plays a major role in determining the stiffness of the cell wall (Peaucelle et al., 2012). Several pectin-modifying enzymes have been described that modulate the elasticity of the cell wall. Among these, pectin methyl esterases that determine the degree of homogalacturonan methylation seem to be major players (Palin and Geitmann, 2012). Pectin-degrading enzymes such as polygalacturonases (PGs) have been implicated with cell expansion, but their role in this process is much less clear (Hadfield and Bennett, 1998).The pectin-rich middle lamella serves as cement to aid cell adhesion, and it is therefore not surprising that several PG genes are involved in dehiscence and abscission events (Rhee et al., 2003; González-Carranza et al., 2007, 2012; Ogawa et al., 2009). The expression profile of many PG genes correlates temporally and spatially with cell elongation and therefore implies a role in cell expansion (Sitrit et al., 1999). The exact function of PGs during cell elongation is still unclear, and the lack of elongation defects in loss-of-function mutants further complicates matters.Here, we describe mutations in a PG gene, which we called NIMNA (NMA), that severely affect cell elongation in the embryo and suspensor.We demonstrate that embryos with shorter suspensors show a significant delay in development during embryogenesis, possibly caused by reduced access to nutrients from the endosperm. Interestingly, reciprocal crosses reveal an unequal parental contribution to NMA function in cell elongation of the suspensor.     

Aspartate Aminotransferase Activity during Early Development of Chicken Embryo

Aspartate aminotransferase (AAT) activity is studied, employing two different procedures, during early developmental stages of chicken embryo. AAT activity is steady from pre-streak to the definitive primitive streak stage after which it suddenly increases as growth proceeds. INH or IIH administration in this embryonic system led to almost instantaneous and complete disappearance of AAT activity which could be reversed to 80 percent by treatment with equimolar pyridoxal phosphate. Histochemical studies from the literature support a view that the period of intense differentiation coincides with an increased RNA content. The present study shows more AAT activity per mg wet embryo during the same development stages. Whether this is due to availability of more aspartic acid for pyrimidine is not clear due to possible presence of two AAT activities, the many competing reactions that can use aspartic acid, and In situ conditions during differentiation.     

发育中玉米胚结合蛋白(BiP)的动态变化及定位

Western blot检测表明,在玉米胚发育过程中结合蛋白(BiP)含量与胚可溶性蛋白含量变化一致,在授粉16d后BiP含量随发育而增加;对热激不敏感.组织化学免疫定位表明,在玉米胚发育的不同时期,BiP主要定位在胚芽端、初生维管组织和糊粉层中,提示胚在构建器官的同时,也为其功能执行准备了条件;热激不影响其定位.     

Dynamics of DNA Methylation during Early Development of the Preimplantation Bovine Embryo

There is species divergence in control of DNA methylation during preimplantation development. The exact pattern of methylation in the bovine embryo has not been established nor has its regulation by gender or maternal signals that regulate development such as colony stimulating factor 2 (CSF2). Using immunofluorescent labeling with anti-5-methylcytosine and embryos produced with X-chromosome sorted sperm, it was demonstrated that methylation decreased from the 2-cell stage to the 6–8 cell stage and then increased thereafter up to the blastocyst stage. In a second experiment, embryos of specific genders were produced by fertilization with X- or Y-sorted sperm. The developmental pattern was similar to the first experiment, but there was stage × gender interaction. Methylation was greater for females at the 8-cell stage but greater for males at the blastocyst stage. Treatment with CSF2 had no effect on labeling for DNA methylation in blastocysts. Methylation was lower for inner cell mass cells (i.e., cells that did not label with anti-CDX2) than for trophectoderm (CDX2-positive). The possible role for DNMT3B in developmental changes in methylation was evaluated by determining gene expression and degree of methylation. Steady-state mRNA for DNMT3B decreased from the 2-cell stage to a nadir for D 5 embryos >16 cells and then increased at the blastocyst stage. High resolution melting analysis was used to assess methylation of a CpG rich region in an intronic region of DNMT3B. Methylation percent decreased between the 6–8 cell and the blastocyst stage but there was no difference in methylation between ICM and TE. Results indicate that DNA methylation undergoes dynamic changes during the preimplantation period in a manner that is dependent upon gender and cell lineage. Developmental changes in expression of DNMT3B are indicative of a possible role in changes in methylation. Moreover, DNMT3B itself appears to be under epigenetic control by methylation.     

组蛋白在胚胎早期发育转录调控中的作用

组蛋白是一组等电点大于１０．０的碱性蛋白质,在进化上十分保守。真核生物的染色体上主要含有５种组蛋白,即核心组蛋白Ｈ２ａ、Ｈ２ｂ、Ｈ３、Ｈ４及连接组蛋白Ｈ１。核心组蛋白八聚体、蛋白Ｈ１和２００ｂｐ的ＤＮＡ共同组成了染色体的基本单位——核小体。组蛋白不仅...     

鹤顶兰胚囊发育过程中Ca~（2＋）分布的超微细胞化学定位（英文）

用焦锑酸盐沉淀法对鹤顶兰（Phaius tankervilliae）胚囊发育过程中的Ca2＋状态进行超微细胞化学定位。观察结果发现：功能大孢子时期,珠孔端的胚囊壁上开始出现小颗粒的Ca2＋沉淀,但功能大孢子细胞内未见明显的Ca2＋标记;四核胚囊时期胚囊壁上的Ca2＋沉淀明显增多,液泡膜上有Ca2＋沉淀出现,珠孔处的Ca2＋沉淀颗粒较大;成熟胚囊时期,胚囊壁上的Ca2＋沉淀进一步增多,且胚囊内Ca2＋分布明显增多,且极性明显,珠孔端助细胞、卵细胞比合点端反足细胞有更多的Ca2＋沉淀。鹤顶兰成熟胚囊内Ca2＋积累的来源有：（1）在胚囊成熟前主要由珠被细胞、珠细胞通过胞间连丝向胚囊运输;（2）以沉淀有大量Ca2＋的小泡形式跨过胚囊壁进入胚囊。     

Histochemical Localization of Several Enzymes in Early Young Embryo of Pinus tabulaeformis

Histochemical localization of adenosine triphosphatase, acid phosphatase and peroxidase in young embryo of Pinus tabulaeformis has been studied. All cells of the embryo proper showed very intense lead phosphate disposite with ATP in the Wachs- tein Meisel medium and with B-glyceropbosphate in Gomori medium. In the suspensor 1–3 suspensor cells closed to embryo proper appear blackening with both the ATP and the B-glycerophosphate, and other cells of the suspensor showed less adenosine triphosphatase and B-glyeerophosphatase. In the young embryo the peroxidase distribution may vary with the developmental stages of the young embryo, for instance cone-shaped embryo proper showed dark blue, and which peroxidase occupies only 1–2 suspensor cells closed to embryo proper. When the young embryo developed further into columnus-shape, the peroxidase was mainly distributed in regions of both the posterior part of embryo proper and just derived suspensor cells. However the anterior part of embryo proper showed slight peroxidase. The experimental results indicate that all of these enzymes mentioned above are mainly present in both the embryo proper and the connecting end of the suspensor, and that these regions show great matabolic activity. From the above mentioned observation, the possible relationship between the young embryo and near surrounding tissue during the development of embryo is discussed.     

Distribution of Cytosolic mRNAs Between Polysomal and Ribonucleoprotein Complex Fractions in Alfalfa Embryos : Stage-Specific Translational Repression of Storage Protein Synthesis during Early Somatic Embryo Development

Cell-free translational and northern blot analyses were used to examine the distribution of storage protein messages in the cytoplasmic polysomal and mRNA-protein complex (mRNP) fractions during development of somatic and zygotic embryos of alfalfa (Medicago sativa cv Rangelander RL-34). No special array of messages was identified in the mRNP fraction; however, some messages were selectively enriched in either the polysome or mRNP fractions, and their distribution pattern varied quantitatively during development of the embryos. During the earliest stages of somatic embryo development, storage protein messages already were present, but there was no detectable accumulation of the proteins. Selective enrichment of messages for the 11S, 7S, and 2S storage proteins occurred in the mRNP fraction during the globular, heart, and torpedo stages of somatic embryogenesis, but the distribution pattern was shifted toward the polysomal fraction at the beginning of cotyledon development. Thus, there was translational repression of storage protein synthesis at the early stage of somatic embryo development that was relieved later. During the cotyledonary development stages in the somatic and zygotic embryos, storage protein synthesis and distribution of the messages were similar in that these specific messages were predominantly in the polysomal fraction.     

Chk2 Regulates Cell Cycle Progression during Mouse Oocyte Maturation and Early Embryo Development

As a tumor suppressor homologue during mitosis, Chk2 is involved in replication checkpoints, DNA repair, and cell cycle arrest, although its functions during mouse oocyte meiosis and early embryo development remain uncertain. We investigated the functions of Chk2 during mouse oocyte maturation and early embryo development. Chk2 exhibited a dynamic localization pattern; Chk2 expression was restricted to germinal vesicles at the germinal vesicle (GV) stage, was associated with centromeres at pro-metaphase I (Pro-MI), and localized to spindle poles at metaphase I (MI). Disrupting Chk2 activity resulted in cell cycle progression defects. First, inhibitor-treated oocytes were arrested at the GV stage and failed to undergo germinal vesicle breakdown (GVBD); this could be rescued after Chk2 inhibition release. Second, Chk2 inhibition after oocyte GVBD caused MI arrest. Third, the first cleavage of early embryo development was disrupted by Chk2 inhibition. Additionally, in inhibitor-treated oocytes, checkpoint protein Bub3 expression was consistently localized at centromeres at the MI stage, which indicated that the spindle assembly checkpoint (SAC) was activated. Moreover, disrupting Chk2 activity in oocytes caused severe chromosome misalignments and spindle disruption. In inhibitor-treated oocytes, centrosome protein γ-tubulin and Polo-like kinase 1 (Plk1) were dissociated from spindle poles. These results indicated that Chk2 regulated cell cycle progression and spindle assembly during mouse oocyte maturation and early embryo development.     

Heterogenous Macromolecular Contributions to Early Mouse Embryo Development

Mouse embryos have been cultured for more than one half of its gestation period by providing sera from definite species of animals as the inducer at the proper stage of development. The processes of normal development at four different but discrete phases have been described.
(1) Fertilized ovum (stage 1) is able to grow up to denuded blastocyst stage and attaches to the culture dish (stage 7) in balanced salt solution with bovine serum albumin as the sole macromolecule. (2) Embryoblast or inner cell mass (ICM) of denuded blastocyst (stage 7) is able to develop in fetal calf serum (FCS), human placenta cord serum (FCS), or sera from mouse (MS), rat (RS) or rabbit (RbS) to the early cylinder stage (stage 11). (3) The early egg cylinder stage (stage 11) of mouse embryo is able to grow in HCS and RS, but not in FCS nor RbS, to the stage of early somite stage (stage 15). (4) Beyond early somite stage (stage 15), mouse embryo is able to develop neural tissues in rat serum. The macromolecular nature of these growth factors in serum has been described (Hsu, 1980).
It indicates that the differential biological activity which induces the early mouse embryogenesis among the sera from different animal species is due to the various degree of sequence homology between the growth factor family among the different species of animals.     

Appearance and Distribution of RNA-Rich Cytoplasms in the Embryo of Xenopus laevis during Early Development

The occurrence of the dorsal yolk-free cytoplasm in the fertilized egg of Xenopus was re-examined, and the appearance and the distribution of RNA-rich cytoplasms in Xenopus embryos during early development were examined with their paraffin sections. The results show that the dorsal yolk-free cytoplasm does not occur solely in the dorsal part of the embryo but is continuous to similar cytoplasmic mass in the central and the ventral part. The whole mass of this continuous cytoplasm is denoted here as the mesoplasm. The locations of the mesoplasm in the embryo can be traced by its high RNA content during cleavage and blastulation. The cells endowed with the mesoplasm constitute a broad band about the equator of the blastula. At the lower edge of this band, the blastopore lip is formed during gastrulation. Another mass of yolk-poor and RNA-rich cytoplasm becomes distinct around every nucleus in the stage 4 embryo and is denoted here as the nucleophilic plasm. This plasm is diminished at every nuclear division and disappears in the stage 10 embryo. Origins and roles of the mesoplasm and the nucleophilic plasm were discussed and a mechanism of blastulation was suggested.     

高浓度葡萄糖对昆明小鼠早期胚胎发育的影响

建立昆明小鼠受孕模型,分离并体外培养胚胎细胞.检测了各培养浓度下的细胞增殖、分化与凋亡.胚胎细胞在0.2mmol/L和5.56mmol/L葡萄糖浓度的KSOM培养基中能正常发育和孵化;而在浓度为15.56mmol/L和25.56mmol/L葡萄糖培养基中胚胎发育和孵化均受到损害(P<0.005),且总细胞数和内细胞团细胞数也明显减少(P<0.01),但其细胞凋亡率与0.2mmol/L和5.56mmol/L葡萄糖浓度下胚胎细胞凋亡率无显著性差异(P>0.05).随着葡萄糖浓度的增高,胚泡总的表面积无明显变化,但胚胎细胞密度呈增加趋势.高血糖对早期胚胎的发育具有毒性作用,提示高糖可能导致妊娠合并糖尿病患者的流产和胎儿畸形率升高.     

胚胎发育早期转基因整合的研究

转基因动物的建立是一项复杂而艰苦的工作,在转基因胚移植受体前对其进行检测,无疑对转基因动物建立具有重要意义。使用小鼠乳清酸蛋白（WAP）控制下的人粒细胞激落刺激因子（G-CSF）基因为显微注射片段,采用PCR方法检测了转基因胚,在1、2和8细胞期的阳性率为100％、77．7％和44．4％。为消除PCR扩增中的假阳性结果,构建了两个具有部分同源性的亚克隆片段进行共注射。PCR扩增片段跨越这一同源区域,转基因的非整合胚不能扩增出特异性片段。结果表明,1、2和8细胞期的阳性率分别为11．1％、55．5％和44．4％,较常规PCR检测获得更为明确的结论,为在大动物转基因的检测提供了新依据。     

Rapid Increase in UDP-Glucose during Early Wheat Embryo Germination

The cellular content of UDP-glucose in isolated wheat (Triticum aestivum L.) embryo increases 8-fold during the first 40 minutes of imbibition. An additional 3-fold increase in the amount of UDP-glucose was observed in the next 5 hours of germination. This communication also describes a unique, quantitative method to achieve a high sensitivity in a direct determination of UDP-glucose with Na [³²P]pyrophosphate and UDP-glucose pyrophosphorylase. The sensitivity of the assay for UDP-glucose is 10 picomoles.