温度对红瘰疣螈胚胎发育的影响

探讨了不同温度对红瘰疣螈Tylototriton shanjing Nussbaum,Brodie et Yang 1995胚胎发育速度、孵化率及胚长增长速度的影响,并对胚胎发育起点温度和发育有效积温等进行研究。结果表明,在试验温度范围内(10³0℃),随着温度的升高,其胚胎发育速度、胚长增长速度加快,发育时间缩短,孵化率上升。其中孵化率与孵化温度成线性关系(Y=0.0017X+0.95230),发育周期与孵化温度成线性关系(Y=-0.377X+24.154)。但红瘰疣螈有一定适宜温度范围(2030℃),随着温度的升高,其胚胎发育速度、胚长增长速度加快,发育时间缩短,孵化率上升。其中孵化率与孵化温度成线性关系(Y=0.0017X+0.95230),发育周期与孵化温度成线性关系(Y=-0.377X+24.154)。但红瘰疣螈有一定适宜温度范围(20²5℃),超过或低于该温度范围,卵的畸形率和最终死亡率明显增加。应用直线回归法和直接最优化法计算红瘰疣螈胚胎发育的发育起点温度和有效积温分别为0.58℃、68.66日度和0.46℃、69.01日度。     

红瘰疣螈皮肤的显微结构观察

对我国Ⅱ级重点保护野生动物红瘰疣螈(Tylototriton shanjing)成体的皮肤结构进行了显微观察。观察结果表明,其皮肤厚薄、微血管、色素分布,以及腺体的分布和大小等方面在身体的不同部位存在差异:头和背部的皮肤厚于腹部;黑色素细胞多分布于真皮浅层,以腹部及背侧无瘰粒处多见;真皮层富含黏液腺和颗粒腺,但分布不均匀;头嵴棱部及背部瘰粒下方颗粒腺的数量多、体积大,并充满分泌物;表皮下腺体外突,其表皮细胞层数减少,形成分泌物有效通道。同时,将红瘰疣螈皮肤与其他两栖类相比可知,其以上皮肤结构特征均体现了红瘰疣螈对山区阴湿环境中陆栖生活的重要适应。     

红瘰疣螈的体温调节

红瘰疣螈(Tylototriton shanjing)为我国Ⅱ级重点保护野生动物。本实验测定了在不同环境温度条件下红瘰疣螈体温及代谢率变化。结果表明,在10～35℃环境温度范围内红瘰疣螈体温(Tb)与环境温度(Ta)呈正相关,其直线回归方程为:Tb=3.99+0.86Ta(R2=0.99,P0.01);其代谢率(MR)在15～30℃的环境温度范围内随环境温度的升高而升高,在35℃时,其代谢率由于体温过高而急剧降低;在15～35℃之间的6个温度条件下雄性代谢率的回归方程为:MR1=0.374 1-0.355 1Ta+0.113 9T2a-0.010 5T3a(R2=0.47,P0.01,df1=3,df2=46);雌性代谢率的回归方程为:MR2=0.478 8-0.420 3Ta+0.130 4T2a-0.011 8T3a(R2=0.40,P0.01,df1=3,df2=46)。不同于内热源动物的代谢特征,红瘰疣螈的体温调节表现出外热源动物的特点:其体温受环境影响较大,体温生理调节能力较弱。     

红瘰疣螈的繁殖生态

红瘰疣螈(Tylototriton shanjing)为国家Ⅱ级重点保护野生动物,对其繁殖生态尚无系统的研究.2007 ～2010年在云南省新平县哀牢山对红瘰疣螈形态、繁殖栖息地、求偶和交配行为、产卵及孵化等繁殖特征进行了研究.结果表明,红瘰疣螈雌雄两性在广泛的形态学度量特征上存在着差异;繁殖栖息地主要为稻田和潮湿的沟渠;参与繁殖的雌雄成体性比随繁殖时间的推移而不断变化,繁殖前期和后期雄性比例高,中期雌性比例高.求偶和交配主要在陆地潮湿的水沟中完成,也可在静水中进行.成体产卵活动从5月初持续至6月下旬,呈现出波浪式的产卵进程,个体完成产卵平均时间为(22.2 ±2.7)h.繁殖前期雌螈产卵于稻田,中后期产于多杂草的田埂草丛和泥壁,平均产卵数( 126±18)枚(n=17).平均孵化期(17.3 ±0.1)d(n =225),孵化率59.8％ (n =79),孵化时幼体平均体长12.7mm(n =6).     

降雨决定繁殖期红瘰疣螈的活动性

有尾两栖类的繁殖活动常常受到环境因子,特别是如温度、湿度和降雨等气候因子的限制。红瘰疣螈Tylototriton shanjing是适应云南山地环境的典型有尾两栖类动物。本研究期望通过对繁殖期红瘰疣螈活动个体数量的野外调查和气候因子的分析,弄清影响红瘰疣螈繁殖行为活动的关键气候因子。研究结果显示,繁殖期红瘰疣螈活动个体数量与气温呈显著的负相关关系,与空气相对湿度和降雨量呈显著的正相关关系。然而偏相关和多元回归分析则表明,红瘰疣螈繁殖行为活动与相对湿度和降雨量关系更密切。红瘰疣螈总是在降雨后引起空气湿度增加以及气温下降时进行各种繁殖行为活动,降雨是影响红瘰疣螈繁殖行为活动关键的气候因子。红瘰疣螈的繁殖行为活动与降雨节律保持一致是对云南山地环境的重要适应。     

红瘰疣螈玉龙雪山种群的两性异形

分别测量于2018年6—8月采自云南丽江玉龙雪山的32只(17♂,15♀)红瘰疣螈Tylototriton shanjing成体的全长、头体长、头长等13项形态特征指标,并检验该物种的两性异形。结果表明:雌性平均全长为160.72 mm±3.02 mm(n=15),雄性平均全长为138.58 mm±2.57 mm(n=17),雌性与雄性平均全长比为1.160,两性异形指数为0.138,属雌性大于雄性的两栖类;全长在两性间的差异有统计学意义;除了雌性的头宽、尾宽、尾高和腋至胯距外,其他形态特征与全长均有显著或极显著相关性;以全长为协变量的协方差分析结果显示,红瘰疣螈的头体长、头长、吻长、尾长和前肢长在两性间的差异有统计学意义;雌性的尾长和前肢长随全长的生长速率大于雄性,而头体长、头长和吻长随全长的生长速率小于雄性。生育力选择假说能解释红瘰疣螈玉龙雪山种群的两性异形现象。     

捕食风险对红瘰疣螈幼体生长发育的影响

捕食风险是两栖动物幼体表型和生活史进化重要的选择压力之一。红瘰疣螈Tylototriton shanjing为云南山地环境中典型的有尾两栖类,山地环境水体水量的迅速减少常导致红瘰疣螈在幼体发育阶段捕食风险增加。本研究通过实验,观察红瘰疣螈幼体的体长和体质量在不同捕食风险环境中的变化、变态时体型以及完成变态发育的时间差异,探讨捕食风险对红瘰疣螈幼体个体早期发育的影响。实验设计4个不同的捕食风险处理:无任何捕食者的无捕食组、有同种个体化学信号的同种异体组、有同种尾受伤个体化学信号的断尾组、有入侵物种——克氏原螯虾Procambarus clarkii信息的外来物种组。结果显示,红瘰疣螈幼体在不同捕食风险环境中的生长发育过程不同。在生长发育前期,所有处理组的幼体生长均相似,而在中后期,有捕食风险存在的3个处理组生长发育显著加快,最终变态时的体长和体质量要比无捕食风险组更长和更重,体型更大。幼体在有捕食风险存在的处理组完成变态的时间要比无捕食风险组显著更短。这表明,红瘰疣螈幼体在面对捕食风险增加时,通过在胚后发育后期加快生长和缩短发育时间,尽快完成变态,离开幼体发育水体来适应高捕食风险的水环境。     

云南新平哀牢山红瘰疣螈的陆地求偶交配行为模式

红瘰疣螈Tylototriton shanjing为云南山地环境中典型的有尾两栖类,其求偶交配行为模式尚未被系统研究。2013—2014年对云南省新平县哀牢山繁殖期红瘰疣螈在陆地环境中的求偶交配模式进行了观察。研究结果显示,红瘰疣螈的求偶交配行为是按照一定时序进行的,整个过程由定位、静止展示、劝导展示和精子转移4个阶段组成。在求偶交配中雄性个体行为包括:警戒、追逐、阻拦、蹑行、嗅探、轻推、诱导、扇尾、侧行和产精包等类型。雌性个体的行为包括离开、静止、跟随、纳精包等类型。雄性行为较雌性复杂,雄性在求偶交配行为过程中占据主动。雌雄的识别主要以视觉为主,整个求偶交配过程中无抱握行为,雄性尾部的运动也比较单一,这些行为的变化可能是对在陆地求偶交配的适应。     

氧化乐果对红瘰疣螈胚胎及蝌蚪生长发育的影响

本文研究了氧化乐果对红瘰疣螈Tylototriton shanjing胚胎及蝌蚪生长发育的影响。结果表明,氧化乐果对红瘰疣螈胚胎发育过程中的出膜期和开口期毒性较大,其余各期毒性较小。随着氧化乐果浓度的增大,红瘰疣螈的畸形率和死亡率逐渐增大。gosner22期蝌蚪的安全浓度是0.05 mg·L~(-1)。在安全浓度内,0.03 mg·L~(-1)氧化乐果溶液对蝌蚪的生长具有明显的促进作用,浓度高于0.03 mg·L~(-1)则对蝌蚪的生长发育有明显的滞育作用,对其生长的影响主要表现在10 d以后,这与处理时间延长,毒物在蝌蚪体内富集有关。氧化乐果对红瘰疣螈胚胎及蝌蚪生长发育有显著的影响和损伤,该研究为农业生产科学合理使用农药提供了依据。     

文县疣螈早期胚胎发育

对湖南省怀化市黄岩地区的文县疣螈(Tylototriton wenxianensis)早期胚胎发育进行了观察。整个胚胎发育分为21个时期,在实验室19～21℃下,受精卵完成胚胎发育历时(513.30±7.93)h(n=8);本文描述了各时期的形态特征,并讨论了与其他有尾类胚胎发育的异同,以及怀化产文县疣螈与甘肃产文县疣螈的主要区别。     

Early fetal hematopoietic development from in vitro differentiated embryonic stem cells.

In this report we describe the efficient hematopoietic differentiation of embryonic stem (ES) cells in vitro. When cultured in semisolid medium two of five ES cell lines efficiently generated embryoid bodies (EBs) containing blood islands in which hematopoietic cells from all six myeloid lineages could be detected. Among a variety of growth factors tested, only erythropoietin significantly increased blood island formation. We directly demonstrate the presence of hematopoietic progenitors in the EBs by employing an in vitro precursor assay. Colony-forming cells (CFC) of all myeloid lineages as well as bi- and multipotent (CFC-MIX) progenitors were readily identified, and a detailed time-course analysis of their appearance was performed. Despite a high frequency of CFC-MIX in vitro, we did not observe any spleen colony-forming cells (CFU-S) in vivo. We conclude that hematopoietic differentiation of ES cells under these conditions reflects formation of the complete range of blood cells found in the yolk sac of the early fetus. Therefore this system provides a unique model in which to study the earliest events of hematopoietic development in vitro.     

CCN5 Expression in mammals. III. Early embryonic mouse development

CCN proteins play crucial roles in development, angiogenesis, cell motility, matrix turnover, proliferation, and other fundamental cell processes. Early embryonic lethality in CCN5 knockout and over-expressing mice led us to characterize CCN5 distribution in early development. Previous papers in this series showed that CCN5 is expressed widely in mice from E9.5 to adult; however, its distribution before E9.5 has not been studied. To fill this gap in our knowledge of CCN5 expression in mammals, RT-PCR was performed on preimplantation murine embryos: 1 cell, 2 cell, 4 cell, early morula, late morula, and blastocyst. CCN5 mRNA was not detected in 1, 2, or 4 cell embryos. It was first detected at the early morula stage and persisted to the preimplantation blastocyst stage. Immunohistochemical staining showed widespread CCN5 expression in post-implantation blastocysts (E4.5), E5.5, E6.5, and E7.5 stage embryos. Consistent with our previous study on E9.5 embryos, this expression was not limited to a particular germ layer or cell type. The widespread distribution of CCN5 in early embryos suggests a crucial role in development.     

Early embryonic development in vitro by coculture with oviductal epithelial cells in pigs

This experiment was designed to evaluate the ability of three different somatic cell cultures to promote development of early cleavage stage pig embryos. A total of 245 2-cell, 4-cell, 8-cell, and 16-cell pig embryos were cocultured for 5 days with porcine oviductal epithelial cells (POEC), porcine fetal fibroblast monolayer (PEF), a combined POEC and PEF coculture system (PEF-POEC), or Dulbecco's Modified Eagle Medium alone (DMEM). Embryos were collected at slaughter from the reproductive tracts of superovulated prepubertal gilts. Embryos were recovered, evaluated, and randomly placed in one of the four treatment groups. POEC were recovered from oviductal flushes, washed, and placed in 24-well plates. PEF were obtained from 30-day to 60-day fetuses and established in culture. Finally, PEF-POEC consisted of a confluent monolayer of PEF in the bottom of 24-well plates also containing a Costar semipermeable membrane chamber with POEC in it. Embryos were evaluated every 24 h to determine stage of development. More (p less than 0.05) embryos developed to blastocysts in POEC (70% and 54%, respectively) and PEF-POEC (67% and 61%, respectively), than in either DMEM (16% and 2%, respectively) or PEF (27% and 23%, respectively). However, development of embryos did not differ (p less than 0.05) for POEC and PEF-POEC. These data indicate the presence of a primary culture of POEC promotes in vitro development of early cleavage stage pig embryos.     

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.     

Reconstructing embryonic development

Novel approaches to bio-imaging and automated computational image processing allow the design of truly quantitative studies in developmental biology. Cell behavior, cell fate decisions, cell interactions during tissue morphogenesis, and gene expression dynamics can be analyzed in vivo for entire complex organisms and throughout embryonic development. We review state-of-the-art technology for live imaging, focusing on fluorescence light microscopy techniques for system-level investigations of animal development, and discuss computational approaches to image segmentation, cell tracking, automated data annotation, and biophysical modeling. We argue that the substantial increase in data complexity and size requires sophisticated new strategies to data analysis to exploit the enormous potential of these new resources.     

Early embryonic development of the dipteran insect Heteropeza pygmaea in the presence of cytoskeleton-affecting drugs

Summary Embryos of the paedogenetically reproducing gall midge Heteropeza pygmaea develop floating in the haemocoel of a so-called mother larva. The egg membranes remain permeable and the embryos increase in size during embryonic development by taking up nutrients from the haemolymph. Such embryos can be cultured in vitro, i.e. in haemolymph drops obtained from mother larvae. We tested the effects of several drugs known to interact with cytoskeletal elements on different stages of embryonic development, including cleavage and gastrulation. The drugs were added to the in vitro cultures and the effects were studied with time-lapse cine-micrography. Colchicine and vinblastine blocked cleaving eggs in metaphase stage and arrested yolk globule oscillation. In spite of such a block blastoderms once formed continued development through germ band formation and extension and also increased in size. Cytochalasin B did not affect the stage of cleavage; however, it inhibited gastrulation and subsequent morphogenetic processes and also prevented size increase. We conclude that (1) the functioning of microtubules is needed for yolk globule oscillation during cleavage interphases but not for the gastrulation processes subsequent to blastoderm formation and (2) microfilaments do not play an important role in cleavage, at least not for the orderly succession of the cleavage divisions, but are essential for the morphogenetic movements associated with gastrulation. We suggest that during cleavage a limited stock of microtubules and their precursors is responsible for both transport of chromosomes during mitoses and translocation of organelles during interphase. Yolk oscillation seems to be a secondary effect and of minor or no importance for the normal course of embryonic development.Dedicated to Professor Gerhard Krause on the occasion of his 80th birthday