Bovine fetal microchimerism in normal and embryo transfer pregnancies and its implications for biotechnology applications in cattle

Fetal cells and DNA have been detected in the maternal circulation during and after pregnancy in a few mammalian species. The incidence of similar microchimerism in cattle could have repercussion for the application of modern biotechnologies such as the transfer of transgenic embryos. To determine if feto-maternal leakage can occur in pregnant cows, we have analyzed maternal blood samples for the presence of fetal DNA during gestation and post-partum periods. Y chromosome-specific DNA was detected in up to 73% of blood samples from naturally mated heifers carrying conventional bull calves and a transgene-specific sequence in up to 50% of recipient cows carrying transgenic fetuses. These findings document for the first time that transplacental leakage of fetal DNA into the maternal circulation can occur in cattle despite the epitheliochorial placenta of ruminants, with potential implications for the utilization of recipient cows in the food chain.     

OPGmRNA在牙齿萌出过程中牙台方组织内的表达

目的：探讨骨保护素（osteoprotegerin OPG）mRNA在牙齿萌出过程中胎方组织内的表达。方法：运用原位杂交方法检测大鼠下颌第一磨牙胎方组织内OPGmRNA的表达。结果：大鼠下颌第一磨牙牙台方组织内牙囊成纤维细胞中OPGmRNA在牙齿骨内萌出阶段中期阳性表达,与萌出早、晚期比较差异有统计学意义（P〈0．01）,其中早期比晚期差异显著（P〈0．01）;成釉细胞中OPGmRNA在牙齿骨内萌出阶段中期阳性表达,与萌出早、晚期比较差异亦有统计学意义（P〈0.01）,早期与晚期没有显著性差异（P〉0．05）。结论：OPGmRNA在大鼠出生后下颌第一磨牙牙囊成纤维细胞、成釉细胞骨内萌出阶段中期表达最强,牙齿萌出过程中可能通过OPGmRNA表达量的变化来调节牙齿的萌出。     

刀鲚胚胎及胚后发育早期脂肪酸组成变化

为掌握刀鲚(Coilia nasus)胚胎及胚后发育早期的脂肪酸变化规律,采用生化分析手段对刀鲚的胚胎(原肠期,受精后7～9 h)、0日龄仔鱼(初孵仔鱼)、3日龄仔鱼和5日龄仔鱼(开口前)的脂肪酸组成和含量进行了检测分析。结果显示,刀鲚发育早期的总脂占干物质的相对含量均较高(53.10%～60.97%),干物质的总脂相对含量随个体发育显著降低,单个个体的总脂含量随个体发育呈现剧烈下降趋势,数值从胚胎的43.62μg/ind剧烈下降到5日龄仔鱼的16.27μg/ind;水分含量随个体发育而升高。刀鲚发育早期上述4个时期的干样中检出6种饱和脂肪酸(SFA)、4种单不饱和脂肪酸(MUFA)和8种多不饱和脂肪酸(PUFA)。4个发育时期脂肪酸相对含量,C18:1n9c占绝对优势(50.39%～57.00%),C16:1丰富且稳定(13.77%～14.24%),C16:0也较丰富(7.45%～9.15%)。单不饱和脂肪酸(MUFA)比例占绝对优势(65.14%～72.26%),n-3与n-6系列多不饱和脂肪酸含量的比值(∑n3PUFA/∑n6PUFA)较小(1.78～2.38)。刀鲚胚胎孵化出膜期间,单个个体单不饱和脂肪酸(MUFA)实际减少程度较高,尤其是C18:1n9c(减少量为13.21μg/ind,减少比例达到55.49%)和C16:1(减少量和比例分别为3.30μg/ind和53.12%),而二十碳五烯酸(EPA)加二十二碳六烯酸(DHA)的减少程度较低(1.44μg/ind和38.41%),尤其是DHA(0.95μg/ind和36.52%)。然而,出膜后,仔鱼对单不饱和脂肪酸(MUFA)的利用相对较低(1.94μg/ind和14.17%),尤其是C18:1n9c(13.21μg/ind和12.41%)和C16:1(0.63μg/ind和21.81%);而对EPA+DHA利用相对较高(1.04μg/ind和45.10%),尤其是DHA(0.71μg/ind和42.61%)。研究表明,刀鲚胚胎优先蓄留EPA和DHA,仔鱼在摄食前大量利用EPA+DHA(特别是DHA),呈现出海水鱼类脂肪酸的利用特点。因此,建议在刀鲚亲本强化培育及产后培育中,增加投喂富含单不饱和脂肪酸(MUFA)(特别是C18:1和C16:1)的饵料,以加强刀鲚亲本的营养积累和产卵后亲本生理机能的恢复;在刀鲚育苗前期,要及时补充富含DHA和EPA的饵料,如,单胞藻、蛋黄等,以提高刀鲚仔鱼开口期间的成活率。     

粘皮鲻虎鱼胚胎及仔鱼的发育

粘皮鲻鰕虎鱼(Mugilogobius myxodermus)的卵产出时为球型,直径0.40~0.60mm,具黏性,卵颜色淡黄,半透明,油球多且数量不固定。受精5~10min后吸水膨胀成椭圆形。水温16~18℃的条件下,胚胎发育共需138h。初孵仔鱼体长2.30~3.10mm,体高0.35~0.40mm,总肌节29~30。出膜30min~24h内鳔充气,从孵出到各鳍分化完全约需要40d。     

Prospects for transgenesis in the chick

Research to develop a useful method for genetic modification of the chick has been on-going since the first demonstrations in the mouse in the 1980s that genetic modification is an invaluable tool for the study of gene function. Manipulation of the chick zygote is possible but inefficient. Considerable progress has been made in developing potentially pluripotent embryo stem cells and their contribution to somatic chimeric birds well-established. Germ line transmission of gametes derived from genetically modified embryo cells has not been described. Transfer of primordial germ cells from a donor embryo to a recipient and production of functional gametes from the donor-derived cells is possible. Genetic modification of primordial germ cells before transfer and their recovery through the germ line has not been achieved. The first transgenic birds described were generated using retroviral vectors. The use of lentiviral vectors may make this approach a feasible method for transgenic production, although there are limitations to the applications of these vectors. It is likely that a method will be developed in the next few years that will enable the use of transgenesis as a tool in the study of development in the chick and for many other applications in basic research and biotechnology.     

Systematic analysis of hematopoietic, vasculogenetic, and angiogenetic phases in the developing embryonic avian lung, Gallus gallus variant domesticus

In the embryonic lung of the domestic fowl, Gallus gallus variant domesticus, hematogenetic and vasculogenetic cells become ultrastructurally clear from day 4 of development. In the former group of cells, filopodial extensions coalesce, cytoplasm thickens, and accumulating hemoglobin displaces the nucleus peripherally while in the latter, conspicuous filopodial extensions and large nuclei develop as the cells assume a rather stellate appearance. From day 5, erythrocytes and granular leukocytes begin forming from cytoarchitecturally cognate hematogenetic cells. The cells become distinguishable when hemoglobin starts to accumulate in the erythroblasts and electron dense bodies form in the leukoblasts. Vasculogenesis begins from day 7 in different areas of the developing lung: erthrocytes (but not granular leukocytes) appear to attract committed vasculogenetic cells (angioblasts) that form an endothelial lining and vessel wall. Arrangement of angioblasts around forming blood vessels sets the direction along which the vessels sprout (angiogenesis). In some areas of the developing lung, through what seems like an inductive erythropoietic process, arcades of erythrocytes organize. Once endothelial cells surround such continuities, discrete vascular units organize. By day 10, the major parts of the in-built (intrinsic) pulmonary vasculature are assembled. Complete pulmonary circulation (i.e., through the exchange tissue) is not established until after day 18 when the blood capillaries start to develop. Since the precursory erythrocytes do not have a respiratory role, it is imperative that de novo erythropoiesis is essential for vasculogenesis. Diffuse (fragmentary) development and subsequent piecemeal assembly of the pulmonary vascular system may explicate the fabrication of a complex circulatory architecture that grants cross-current, counter-current, and multicapillary serial arterialization designs in the exchange tissue of the avian lung. The exceptional respiratory efficiency of the avian lung is largely attributable to the geometries (physical interfacing) between the bronchial and vascular elements at different levels of morphological organization.     

Fatty acid esterification in the yolk sac membrane of the avian embryo

The transfer of lipid from the yolk to the avian embryo is mediated by the yolk sac membrane (YSM). Some, but not all, of the published morphological evidence supports the view that the lipid undergoes a cycle of hydrolysis and re-esterification during translocation across the YSM. The present study aims to test this view by investigating the capacity of the YSM to perform esterification of free fatty acids to form acyl-lipids. YSM pieces (area vasculosa), obtained from the chicken embryo at day 10 of development, were incubated in vitro in medium containing [¹⁴C]-palmitic acid. Radioactivity was rapidly incorporated into the tissue lipid indicating a high capacity for esterification. The incorporation was linear with time during the 1-h incubation. Approximately 84% of the incorporated label was recovered in triacylglycerol, 12% was incorporated into phospholipid and less than 1% was detected in cholesteryl ester. [¹⁴C]-palmitic acid was incorporated primarily at the sn-1/3 positions in the triacylglycerol molecule and at the sn-1 position of phospholipid. The incorporation of label into tissue pieces obtained from the non-vascularized peripheral region of the YSM (area vitellina) was much more limited than that observed for the area vasculosa. The results support the hypothesis that yolk lipid is hydrolyzed and re-esterified during transfer across the YSM.Abbreviations YSM yolk sac membrane - VLDL very-low density lipoprotein Communicated by G. Heldmaier     

Drapc1 expression during mouse embryonic development

We identified the mouse homolog of human DRAPC1 (APCDD1) gene, shown to be a target of Wnt/beta-catenin signaling pathway in cancer cell lines. Analysis of its spatiotemporal expression in mouse embryos from E7.5 to E14 showed that Drapc1 is expressed during development of the extraembryonic structures, nervous system, vascular system and inner ear. In addition, Drapc1 is expressed in the mesenchyme of several developing organs at sites of epithelio-mesenchymal interactions. Drapc1 expression was also found in the hair follicles of the adult mouse skin. Similarity of Drapc1 expression pattern to location of active beta-catenin in developing mouse embryo further suggests that mouse Drapc1 is a novel in vivo target gene of Wnt/beta-catenin signaling pathway.     

Interaction of FIE,a Polycomb protein,with pRb: a possible mechanism regulating endosperm development

Inactivation of the Arabidopsis protein FERTILIZATION INDEPENDENT ENDOSPERM (FIE) induces division of the central cell of the embryo sac, leading to endosperm development in the absence of fertilization. The mechanism whereby FIE regulates this process is unknown. We postulated that activation of central cell division in fie mutant plants might involve the retinoblastoma protein (pRb), a cell cycle regulatory element. Pull-down and surface plasmon resonance assays demonstrated that FIE interacts in-vitro with the pRb homologues from Arabidopsis (AtRb), maize (ZmRb) and human (HuRb). The interaction of FIE with ZmRB and HuRb in the yeast two-hybrid system supports the possibility that a FIE-pRb interaction may occur also in planta. Mutational analysis showed that this interaction does not occur via the LxCxE motif of the FIE protein nor via the pocket B domain of pRb. These results suggest that FIE may inhibit premature division of the central cell of the embryo sac, at least partly, through interaction with pRb, and suppression of pRb-regulated genes.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. G. Herrmann     

Dissecting Drosophila embryonic brain development using photoactivated gene expression

The Drosophila brain is generated by a complex series of morphogenetic movements. To better understand brain development and to provide a guide for experimental manipulation of brain progenitors, we created a fate map using photoactivated gene expression to mark cells originating within specific mitotic domains and time-lapse microscopy to dynamically monitor their progeny. We show that mitotic domains 1, 5, and 9 give rise to discrete cell populations within specific regions of the brain. Two novel observations were that the antennal sensory system, composed of four disparate cell clusters, arose from mitotic domain 5 and that mitotic domain B produced glial cells, while neurons were produced from mitotic domains 1, 5, and 9. Time-lapse analysis of marked cells showed complex mitotic and migratory patterns for cells derived from these mitotic domains. Photoactivated gene expression was also used either to kill, to induce ectopic divisions, or to alter cell fate. This revealed that deficits were not repopulated, while ectopic cells were removed and extra glia were tolerated.