非洲爪蟾（Xenopus laevis）囊胚,神经胚两个cDNA文库的建立

随着分子发育生物学的发展,非洲爪蟾(Xenopus laevis)作为发育生物学,尤其是胚胎诱导、分化机制研究的重要实验材料,受到人们的重视。作者建立的非洲爪蟾囊胚(分期8)和神经胚(分期14)两个不同发育时期胚胎的cDNA文库,为进一步筛选有关的发育基因创造了条件。     

邻近组织对爪蟾脊索决定和分化的影响

影响爪蟾脊索决定和分化的因素,过去未见报道。本文采用体外共同培养的方法检测了肌节和神经上皮在脊索决定和分化中的作用。结果指出,脊索与两侧肌节共同培养,肌节的量为脊索的决定和分化提供了足够的条件,一侧肌节的量满足不了使所有脊索进行决定和分化的条件,在少数例子里甚至分化不出脊索。在神经上皮的包裹中,部份脊索细胞向肌细胞分化,单独与脊索接触也不利于脊索的正常分化。讨论了中轴器官之间在决定和分化中错综复杂的关系。     

邻近组织对爪蟾脊索决定和分化的影响

影响爪蟾脊索决定和分化的因素，过去未见报道。本文采用体外共同培养的方法检测了肌节和神经上皮在脊索决定和分化中的作用。结果指出，脊索与两侧肌节共同培养，肌节的量为脊索的决定和分化提供了足够的条件；一侧肌节的量满足不了使所有脊索进行决定和分化的条件，在少数例子甚至分化不出脊索。在神经上皮的包囊中，部分脊索细胞向肌细胞分化，单独与脊索接触也不利于脊索的正常分化。讨论了中轴器官之间的决定和分化中错综复杂的     

非洲爪蟾在神经系统疾病研究中的应用

在生物医学研究领域,使用合适的动物模型研究大脑运行机制和疾病机理至关重要。作为经典的脊椎动物模型,非洲爪蟾在研究神经环路构建和功能以及神经疾病的分子机制中具有一定优势。因为非洲爪蟾的胚胎发育过程与人类器官形成过程相似,同时,神经系统疾病往往与神经系统功能异常和发育缺陷有关,所以一些人类疾病在基因和形态上的功能机制可以通过非洲爪蟾的胚胎发育模型进行在体研究,且神经前体细胞的增殖分化以及视觉刺激依赖的神经环路稳态和功能研究已经相对成熟。目前,该模型已经在癫痫和自闭症及表观遗传调控等相关疾病的研究上得到了应用。该文将分别介绍非洲爪蟾蝌蚪模型在环境毒物诱发疾病、神经发育障碍以及表观遗传疾病机制研究领域的最新进展。     

非洲爪蟾肌肉决定基因Xmyf—5表达调控的研究

Ｘｍｙｆ－５是爪蟾胚胎肌细胞决定的关键基因之一,研究Ｘｍｙｆ－５的表达调控有助于揭示肌肉原基的形成和肌肉发育的分子机制。从爪蟾部分基因组文库筛选到Ｘｍｙｆ－５５‘上游４．９ｋｂ片段。该片段指导报告基因在爪蟾胚胎内的表达以及其缺失片段指导的报告基因活性分析结果显示,Ｘｍ６ｓｆ－５５’上游４．９ｋｂ片段内含有指导Ｘｎｙｆ－５在胚胎内的表达以及其缺失片段指导的报告基因活性分析结果显示,Ｘｍｙｆ－５５‘     

爪蟾(Xenopus laevis)的四倍体及其子一代

在爪蟾受精卵第一次有丝分裂中期进行静液压处理,抑制细胞质分裂从而得到四倍体。实验中获得三只存活了三年以上的四倍体雌性爪蟾。其中一只雌性与二倍体雄性爪蟾作人工催青得到受精卵。现已产卵三批,发育的胚胎400余,经检查子代染色体为三倍体。     

p21活化蛋白激酶2在卵母细胞成熟中的作用

研究p21活化蛋白激酶2（p21-activated kinase 2,PAK2）在爪蟾卵母细胞成熟中的作用。利用特异性抑制PAK2活性的PAK2-N端（PAK2-N terminal,PAK2-NT）片段显微注射爪蟾卵母细胞。荧光显微镜下比较PAK2-NT mRNA注射组和未注射对照组卵母细胞胚泡破裂发生。共聚焦显微镜下,时间延迟摄影法观察两组卵母细胞胞质分裂过程中肌动蛋白和纺锤体的变化。与未注射PAK2-N端mRNA的对照组卵母细胞相比,注射组卵母细胞胚泡破裂发生无异常,但未见胞质分裂发生和极体形成。结果提示PAK2可能参与爪蟾卵母细胞胞质分裂过程。     

非洲爪蟾GATA-1转录因子与爪蟾发育

GATA－1是GATA结合蛋白（GATA－binding protein）家族的成员之一,正向调节红细胞特异性基因的表达,是红系终末分化所必需的因子。与其他物种不同的是,非洲爪蟾GATA－1转录因子具有两业型,两者结构极为相似,但存在功能上的差异,非洲爪蟾GATA－1转录因子在爪蟾发育过程中起着重要的调节作用。     

人胚与鼠胚神经干细胞体外培养的差异

为比较人胚与鼠胚神经干细胞体外培养的差异。实验采用具有丝裂原作用的细胞生长因子。结合无血清细胞培养技术从人胚和鼠胚皮层分离神经干细胞。在连续传代过程中观察其体外培养特性,免疫荧光染色检测Nestin抗原和分化后特异性成熟神经细胞抗原的表达,并用流式细胞仪检测神经干细胞分化情况。结果表明：（1）使用单一生长因子即可从鼠胚皮层分离神经干细胞,但在人胚却需同时使用多种生长因子,协同使用bFGF,EGF和LIF是人胚神经干细胞体外培养的较佳条件;（2）鼠胚皮层神经干细胞在连续传代过程中增殖速度快于人胚,其Nestin阳性率和BrdU标记的阳性率亦高于人胚,表明其增殖能力明显高于人胚,（3）人胚神经干细胞较鼠胚更易分化为神经元。     

爪蟾胚胎原肠中期背方中胚层的组织分化

原肠中期内卷的背方中胚层出现了分别控制脊索和肌肉发育的专一分子的区域化表达。为了研究这个时期的背方中胚层是否已经能够在脱离体内信号的情况下向预定命运分化,我们进行了预定脊索和预定肌肉组织的体外培养,以及两者的共培养,并检测了细胞表达组织专一性分子的情况。原肠中期的预定脊索区域和预定体节区域都能在体外分化成相应的组织——空泡化的脊索和肌细胞,但脊索只能微弱表达其功能分子Shh,肌细胞不能形成肌节。预定脊索区域和预定肌肉区域的共培养也无法增强脊索表达Shh和促进肌细胞形成肌节。我们的结论是,原肠中期内卷的中胚层细胞已经具有了朝预定命运独立分化的能力,但进一步形成功能和结构都完整的相应组织可能还需要周围组织的作用。     

The effect of a phorbol ester on amphibian myogenesis

The effect of a phorbol ester (TPA) on myogenesis of urodelian embryo has been investigated. Presumptive somites were extirpated from mid-neurula, the stage when they have already attained certain degree of determination. The explants were treated with TPA (10 ng/ml in Steinberg solution with the addition of 10% Leibovitz's L-15) for 4 days, the period during which the frequency and size of gap junctions in normal myogenic cells are at the maximum. After the treatment many cells became dispersed and isolated while others spread out and remained connected in the form of a sheet. During further culture for 6 days in the solution without TPA myogenesis was blocked. Both the dispersed cells and the cells in a sheet remained at the earliest stage of muscle differentiation with irregular shape or in the form of myoblast, while cells of the control series formed myotubes containing bundles of myofibrils. The result suggests that the blockage of myogenesis is the consequence of interruption of junctional communication evoked by TPA treatment.     

佛波酯对蝾螈肌细胞分化的影响(英文)

佛波酯(TPA)是一种有效的皮肤癌促变剂。有不少实验事实指出,它的原始作用部位是质膜,而且对间隙连接的影响也已从各方面得到了证实。本文利用这种药物研究细胞通讯在肌细胞分化中的重要性。割取蝾螈神经胚中期躯干部体节中胚层,用组织培养的方法培养。实验组在培液(Steinberg+Leibovitz's L-15,9∶1)中加适量TPA,共试用了4种浓度,5、10、20和40ng/ml,以10ng/ml为适当,本文报道用这一浓度得到的结果。处理时间为4天,天天换新鲜配制的TPA溶液,连续三天,以后在正常培液中培养。对照组和实验组一样,在20℃下培养10—16天。比较对照组和实验组的结果,看到TPA的处理使预定体节细胞分化为肌细胞的过程受到了明显的阻抑。对照组在10天左右的培养中,可以观察到从预定体节细胞转变为成肌细胞,再融合为肌管。但是,实验组培养早期有一定量的脱落细胞,它们大多为不规则形,分散在外植块周围;还有一些不规则形的细胞,成片地自外植块铺展出来。不论是前者还是后者,在10天左右的培养过程中都未能表现进一步分化。除了看到一些梭形或长形的细胞夹杂在分散或成片的不规则细胞之间,没有找到过多核的肌管细胞。我们的实验结果结合过去形态的观察,说明细胞间通讯的时间性在肌细胞分化中是十分重要的。TPA处理的时间正相当于正常肌细胞分化中间隙连接大量存在并发挥作用的期间,很可能,TPA使间隙连接失去作用,处理之后再恢复正常培液,错过了时间,间隙连接的作用以后也不再能得到补偿。     

Characterization and localization of tropomyosin proteins in Xenopus embryos with specific antibodies

In the process of monoclonal antibody (mAb) production against the 38kDa protein which is lacking in the gastrula arrested mutant embryos in Xenopus we incidentally obtained two kinds of mAb (designated as B11 and 2D10 antibodies, respectively) recognizing tropomyosin (TM) proteins in Xenopus embryos. The characterization of the corresponding antigens to those mAb was performed by immunoblotting and silver staining for two-dimensional (2-D) gels in the present study. The localization of the antigens in Xenopus embryos was also investigated by fluorescent microscopy.
By 2-D immunoblots with those mAb, three distinct protein spots or TM isoforms were recognized in Xenopus embryos; a 38 kDa spot with a pl of approximately 4.8 reacted with both antibodies in embryos at stages later than the mid-tailbud (stage 28) and two 30 kDa spots, which are probably isomers, with a pl of approximately 4.8 were detected with 2D10 antibody in embryos at stages extending from the fertilized to the mid-neurula (stage 20). By immunofluorescent microscopy, B11 antibody was shown to react mainly with muscle cells and their precursor cells. In contrast, 2D10 antibody stained the cytoplasm of almost all cells in embryos at stages from the fertilized to the tadpole.
Judging from the results obtained with immunoblotting and fluorescent microscopy, it is likely that the 38 kDa spot is a skeletal muscle TM isoform and the two 30 kDa spots are non-muscle TM isoforms.     

Multinucleation during myogenesis of the myotome of Xenopus laevis: a qualitative study

Ultrastructural studies of myogenesis in the myotome of Xenopus laevis reveal that the myotubes developed by stage 33/34 have peripheral myofibrils but are still uninucleate with a single large nucleus. By stage 45, the cytoplasm of the muscle cells is filled with myofibrils and there are many small peripheral nuclei, resulting in multinucleate muscle fibres. With the electron microscope, we have examined myotomes from stages 33/34 to 59 of development and some stages were also investigated by autoradiography. There was no evidence from autoradiographic studies for DNA synthesis in muscle cells, and the increase in the number of myonuclei was accompanied by a decrease in their size. Satellite cells were not seen at the myotube stage but were first seen after the cells had become multinucleate, with many small nuclei close together forming rows. Constrictions were frequently observed in the large single nuclei. It is concluded that division of the myonuclei by amitosis is mainly responsible for the multinucleation that occurs during development of the myotome muscle in Xenopus laevis.     

Development of smooth and skeletal muscle cells in the iris of the domestic duck,chick and quail

Summary Embryonic development of the avian iris muscle was studied by light and electron microscopy in order to clarify the origin of the iridial skeletal muscle cells. In normal development of the domestic duck, chick, and quail, the muscle bundles appearing in the iris at stage 35 consisted solely of smooth muscle cells. Undifferentiated cells appeared at stage 36, and finally skeletal muscle cells were observed at stage 37. This sequence suggests that stromal mesenchymal cells migrate into the muscle bundles to become skeletal muscle cells.Tissue culture of whole indes removed from duck embryos at stages 30 through 34 produced skeletal muscle cells while culture of isolated iridial epithelia removed at stages 31 and 32 did not. Removal of the midbrain region of duck embryos at stage 10 frequently produced severe disorganization of the eye concomitant with craniofacial deformities typical of a neural crest mesenchymal defect. These severely disorganized eyes were devoid of iridial skeletal muscle cells. These results also suggest mesenchymal origin of iridial skeletal muscle cells.     

Mode of expression of muscle-type enolase isozyme in the developing limb bud of human embryos

The appearance of beta-enolase, a glycolytic enzyme, was studied immunohistochemically using the upper limb bud of human embryos at Carnegie stages from 13 to 21. beta-Enolase-immunoreactive cells first appeared at stage 15 in the proximal portion of the upper limb bud. It was evidenced that glycogen granules first appear at the same stage. These results may suggest that changes in energy metabolism might be one of the earliest events in the differentiating steps of the skeletal muscles because this stage is earlier than the stages of cell fusion, myofilament formation and innervation of the muscle cells.     

Development of the myotomal neuromuscular junction in Xenopus laevis: An electrophysiological and fine-structural study

The normal development of the myotomal neuromuscular junction in Xenopus embryos and tadpoles was investigated electrophysiologically as well as electron microscopically. Spontaneous potentials, considered to be miniature end-plate potentials (MEPPs), were detected by intracellular recording as early as stage 21 and by stage 24 they were observed in every embryo tested. Like MEPPS at later stages they were blocked by curare but not by tetrodotoxin. End-plate potentials (EPPs), subject to block by tetrodotoxin, were evoked by electrical stimulation of the spinal cord in embryos as young as stage 24 and occurred spontaneously as early as stage 22. The durations of MEPPs and EPPs were initially relatively long. Focal external recordings revealed an eightfold decrease in duration during the course of development. Nerve processes emerged from the spinal cord and contacted developing muscle cells as early as stage 21, but junctional specializations were not apparent and vesicles were rare even in stage 24 embryos. During the next 24 hr, between stages 25 and 36, vesicles increased in number and became localized toward the junctional surface of the nerve ending. Basement lamina developed in the cleft and postjunctional ridges and densities were observed. Individual muscle cells also became contacted by several nerve processes. By stages 48–52 there were fewer contacts on individual muscle cells and Schwann cell processes partially covered the nerve endings. Gap junctions were observed between the muscle cells throughout development but occurred less frequently at the later stages. It is concluded that by the time they reach the muscle cells, or very shortly thereafter, at least some of the growing nerve processes can release transmitter, and some of the muscle cells are sufficiently sensitive to acetylcholine in the region of contact to respond with millivolt depolarizations. These earliest functional contacts, however, are morphologically undifferentiated.     

Expression of RhoB in the developing Xenopus laevis embryo

Rho GTPases are signaling components that participate to the control of cell morphology, adhesion and motility through the regulation of F-actin cytoskeleton dynamics. In this paper, we report the identification of RhoB in Xenopus laevis (XRhoB) and its expression pattern during early development. Whole-mount in situ hybridization analysis indicated that XrhoB is expressed at high levels in the dorsal marginal zone early in gastrula and in the dorsal midline at later stages. At mid-neurula stages, XrhoB expression extends to the central nervous system, presomitic mesoderm and somites. Later during development, rhoB mRNA is detected in the eyes, the migrating neural crest cells as well as the dorso-lateral part of the somites.     

The use of fluorescent antimyosin and DNA labeling for the estimation of the myoblast and myocyte population of primary rat heart cell cultures

Cell division in heart muscle cells progressively ceases during the development of the rat heart, leading to an adult stage with muscle cells incapable of cell division. We have quantitatively determined the number of dividing and nondividing heart muscle cells in cultures derived from different stages of the developing rat heart with the use of ³HTdR continuous labeling and fluorescent antimyosin staining. The cultures were derived from 14 and 17 day postcoital (dPC) rat embryos and from 1 and 4 day postnatal (dPN) rats. The percent nondividing cells increased with development and the age of the postnatal rat. The percent nondividing cells in 14 dPC equalled 21%, 17 dPC equalled 25%, 1 dPN equalled 44%, and 4 dPN equalled 60%. This method for the quantitative determination of dividing and nondividing cells in the developing rat heart provides a model that is useful for the study of the mechanism of the loss of cell division capacity.