鸡胚早期神经系统发育中凋亡细胞的分布研究

研究鸡胚18S（Stage）神经系统发育中凋亡细胞的分布及其生物学意义。采用HNK－1和TUNEL免疫组化双染及改变切片方向的方法观察了神经管和神经嵴的凋亡细胞,结果显示：凋亡的细胞在间隔10张横向切片上呈非均匀分布,但在矢状切面凋亡细胞有节段性分布趋势。体节处连续神经嵴没有发现凋亡细胞,而体节与体节之间神经嵴迁离神经管呈游离状并有凋亡细胞,神经管腹侧面体节处间充质细胞呈现细胞凋亡节段性分布。结果表明：鸡胚早期神经系统发育中选择性发生细胞凋亡作用。     

脊椎动物体节形成的分节时钟调控机制

脊椎动物胚胎发育早期中胚层细胞的分节时钟控制着体节的周期性形成。体节是沿身体轴的重复结构,最终发育形成椎骨和肋骨。如果分节时钟受到干扰,体节形成就会出现缺陷,从而导致身体发育异常,最终产生脊柱先天性疾病。参与体节发育的主要模型是时钟和波前模型。中胚层分化由组合梯度系统调节,该系统涉及成纤维细胞生长因子（FGF）、Wnt/β-catenin和视黄酸（RA）信号通路。FGF信号和Wnt/β-catenin信号控制后中胚层处于未分化状态,RA信号则诱导前中胚层细胞分化导致体节成熟。因此相反的信号梯度在特定位点达到平衡。当分子振荡器从尾芽起始表达并以行波模式向前传播至信号平衡临界点时,将启动分节时钟程序,触发Mesp2等分化基因表达,表现为未成熟的前体节中胚层发育形成一对体节。随着细胞二维培养体系和时事报告系统的成熟,研究人员成功在体外将干细胞诱导分化至中胚层并实现了分节时钟的二维可视化振荡。研究表明,细胞通信中的耦合延迟可以保持相邻细胞之间同步振荡,因此导致体节边界和双侧对称形成。此后研究人员在体外重建了诱导多能干细胞的三维培养系统,再现了具有前-后（AP）轴特征的体节样结构的形成。这为解码分节时钟网络调控机理、探索体节双侧对称形成以及不同物种发育速率的代谢调控机制提供了一个宝贵的研究体系。同时为探索病理性体节缺陷发展中的失调机制创造了一个平台。     

鸡胚模型在生物研究中的应用进展

实验动物模型在预防、诊断、治疗疾病和探讨疾病的发生机制等方面起到了至关重要的作用。鸡胚发育过程清楚,利用鸡胚本身的结构特点,可作为研究与胚胎发育相关的生物学实验模型。另外,鸡胚绒毛尿囊膜（CAM）血管丰富,是天然免疫缺陷宿主,可作为血管药理学、肿瘤学等方面研究的一个较为理想的实验模型。本文综述了鸡胚模型在生物实验研究中的应用进展。     

非洲爪蟾体节发生标志基因Mespo的表达调控研究

脊椎动物的脊柱来源于体节,体节是胚胎发生过程中的过渡性结构。体节的形成过程被称为体节发生（somitogenesis）。胚胎发育过程中正确的分节保证了脊椎动物具有正常的体轴,因此体节发生在时间和空间上受到严格的遗传控制,对体节发生的分子机制的研究是发育生物学中的重要课题。     

关于鸡胚孵化中的预热时间

在胚胎学、组织细胞培养的某些实验以及鸡胚的生物标本制作过程中,首先需要获得特定发育时期的鸡胚材料,而往往由于种蛋来源、孵化条件、季节的不同,不易获得准确发育期的材料,尤其是以小时计算胚龄的胚胎学材料,如孵化16小时的原条期,18小时的头突起期,20小时的头褶期,24小时的4体节期,33小时的13对体节期等等。常感到取胚的时间很难掌握,取出的鸡胚发育差别甚大,具有代表性的发育期比例很小,不但造成人力、物力上的浪费,而且直接影响教学和科研的顺利进行。     

碱性磷酸酶在文昌鱼胚胎和幼体中表达图式的研究(英文)

研究组织特异性酶在胚胎发育中的表达图式对于研究细胞、组织和器官的分化具有重要意义。本文以 ＢＣＩＰ为底物研究了碱性磷酸酶在文昌鱼胚胎和幼体中的表达图式。在囊胚、原肠胚和神经胚早期（１２～１３小时）未检测到碱性磷酸酶的特异性表达;到神经胚中期（１５小时,约６个体节）,碱性磷酸酶在胚胎每一体节的中部开始表达,在胚胎中形成３～６个条纹（Ｆｉｇ．１）;在１８小时神经胚（９～１０体节）中,碱性磷酸酶在肌节中表达（Ｆｉｇ．２）;到２４小时在后部内胚层中也开始表达（Ｆｉｇ．３）;在３６～４８小时幼体中,碱性磷酸酶在消化道中强烈表达,但在咽鳃区不表达,同时在肌节中仍有弱的表达（Ｆｉｇ．４）。研究表明碱性磷酸酶可能在肌节的发育过程中具有一定作用。碱性磷酸酶在消化道中的表达可能与这一时期消化道开始行使功能有关。另外,Ｌ－苯丙氨酸只抑制３６、４８小时文昌鱼肠碱性磷酸酶活性,不抑制其体节肠碱性磷酸酶活性（Ｆｉｇ．５）、表明文昌鱼至少存在两种碱性磷酸酶：在消化道中表达的是一种肠型的,与脊椎动物碱性磷酸酶可能为同一类型;另一种主要在肌节中表达,可能是组织非特异性的破性磷酸酶。以上结果说明文昌鱼早期发育中碱性磷酸酶的表达与脊椎动物相似,具     

鸡Vezf1基因RCAS干扰载体构建及鉴定

为研究Vezf1基因在鸡胚早期发育过程中的作用,构建了针对鸡Vezf1基因的RCAS病毒RNA干扰载体,分别在鸡胚细胞和胚胎水平对Vezf1基因实施沉默,通过Real-time PCR和原位杂交法检测目的基因m RNA表达水平。结果表明,基于RCAS病毒的干扰载体可以在鸡胚成纤维细胞和活体鸡胚中成功沉默Vezf1基因的表达。此研究为利用鸡胚模型深入了解Vezf1基因在早期发育过程中的作用提供了素材。     

鸡胚早期发育过程中细胞迁移的基因调控

鸡胚是发育生物学研究的经典动物模型,通过基因导入技术调节胚胎发育的基因功能,研究鸡胚早期发育过程中的细胞迁移,有助于更好地诠释相关先天性疾病的发生发展过程。在早期胚胎发育的过程中,原肠胚期三胚层的形成、心管的发生及神经嵴的发育都伴随着显著的细胞迁移过程。该文将结合近年来国内外对该过程的研究进展,介绍这三个不同时期细胞的迁移及相关基因调控。     

Scribble在原肠期鸡胚表达的研究

旨在研究极性脚手架蛋白Scibble(Scrib)在原肠期的表达及意义,明确Scrib在早期鸡胚发育中的作用.以含有全长人Scrib的质粒pEGFP-N2-Scrib作为模板克隆出N端一段770 bp左右的片段,从而构建一个新的质粒pSPT18-Scrib;以pSPT18-Scrib为模板进行体外转录制备cRNA探针;并采用原位杂交的方法用此探针检测鸡原肠胚各个时期Scrib的表达情况.结果显示,Scrib在鸡胚四期开始逐渐在原条顶端及两侧的外胚层表达,并随着发育过程向外胚层两侧蔓延扩散,并且在发展到十期时呈现包括神经管和体节在内的广泛的弥散性表达.Scrib的表达规律提示在胚胎发育早期Scrib对外胚层细胞迁移和分化以及而后的器官发生起到重要的作用,为进一步研究Scrib在鸡胚早期发育中的作用提供参考.     

鸡胚电转染-RNA干扰技术在体模型的建立

目的建立在体鸡胚电转染-RNA干扰技术(RNAi)模型。方法通过SOE-PCR方法,利用shRNA中的Loop环作为交叠区序列成功的建立了一种方便的shRNA表达序列构建方法,将Isl-1特异性的shRNA序列插入到pEGFP-H1-shRNA质粒中,通过注射后电转染,利用免疫组织化学方法检测Isl-1在鸡胚神经管和背根神经节(dorsal root ganglia,DRG)中的表达。结果鸡胚神经管和DRG中Isl-1的表达受到明显抑制。结论成功建立了在体鸡胚电转染-RNAi模型,为以鸡胚为模式动物研究神经管和DRG发育相关基因的功能提供了有力的工具。     

Segmentation in vertebrates: a molecular clock linked to periodic somite formation]

In the vertebrate embryo, somites constitute the basis of the segmental body pattern. They give rise to the axial skeleton, the dermis of the back and all striated muscles of the body. In the chick embryo, a pair of somites buds off, in a highly coordinated fashion, every 90 minutes, from the cranial end of the presomitic mesoderm (PSM) while new mesenchymal cells enter the paraxial mesoderm as a consequence of gastrulation. The processes leading to the segmentation of the somite are not yet understood. We have identified and characterised c-hairy1, an avian homologue of the Drosophila segmentation gene, hairy. c-hairy1 is strongly expressed in the presomitic mesoderm where its mRNA exhibits a cyclic posterior-to-anterior wave of expression whose periodicity corresponds to the formation time of one somite (90 min). Fate mapping of the rostral half of the PSM using the quail-chick chimera technique supports a model of cryptic segmentation within the presomitic mesoderm, and indicates that c-hairy1 expression dynamics are not due to massive cell displacement. Analysis of in vitro cultures of isolated presomitic mesoderm demonstrates that rhythmic c-hairy1 mRNA production and degradation is an autonomous property of the paraxial mesoderm. Rather than resulting from the caudal-to-rostral propagation of an activating signal, it arises from pulses of c-hairy1 expression that are coordinated in time and space. Blocking protein synthesis does not alter the propagation of c-hairy1 expression, indicating that negative autoregulation of c-hairy1 expression is unlikely to control its periodic expression. Most of the segmentation models proposed for somite formation rely on the existence of an internal clock coordinating the cells to segment together to form a somite. These results provide the first molecular evidence of a developmental clock linked to segmentation and somitogenesis of the paraxial mesoderm, and support the possibility that segmentation mechanisms used by invertebrates and vertebrates have been conserved.     

A clock-work somite

Somites are transient structures which represent the most overt segmental feature of the vertebrate embryo. The strict temporal regulation of somitogenesis is of critical developmental importance since many segmental structures adopt a periodicity based on that of the somites. Until recently, the mechanisms underlying the periodicity of somitogenesis were largely unknown. Based on the oscillations of c-hairy1 and lunatic fringe RNA, we now have evidence for an intrinsic segmentation clock in presomitic cells. Translation of this temporal periodicity into a spatial periodicity, through somite formation, requires Notch signaling. While the Hox genes are certainly involved, it remains unknown how the metameric vertebrate axis becomes regionalized along the antero-posterior (AP) dimension into the occipital, cervical, thoracic, lumbar, and sacral domains. We discuss the implications of cell division as a clock mechanism underlying the regionalization of somites and their derivatives along the AP axis. Possible links between the segmentation clock and axial regionalization are also discussed. BioEssays 22:72-83, 2000.     

A dual fate of the hindlimb muscle mass: cloacal/perineal musculature develops from leg muscle cells

The cloaca serves as a common opening to the urinary and digestive systems. In most mammals, the cloaca is present only during embryogenesis, after which it undergoes a series of septation events leading to the formation of the anal canal and parts of the urogenital tract. During embryogenesis it is surrounded by skeletal muscle. The origin and the mechanisms regulating the development of these muscles have never been determined. Here, we show that the cloacal muscles of the chick originate from somites 30-34, which overlap the domain that gives rise to leg muscles (somites 26-33). Using molecular and cell labelling protocols, we have determined the aetiology of cloacal muscles. Surprisingly, we found that chick cloacal myoblasts first migrate into the developing leg bud and then extend out of the ventral muscle mass towards the cloacal tubercle. The development of homologous cloacal/perineal muscles was also examined in the mouse. Concordant with the results in birds, we found that perineal muscles in mammals also develop from the ventral muscle mass of the hindlimb. We provide genetic evidence that the perineal muscles are migratory, like limb muscles, by showing that they are absent in metd/d mutants. Using experimental embryological procedures (in chick) and genetic models (in chick and mouse), we show that the development of the cloacal musculature is dependent on proximal leg field formation. Thus, we have discovered a novel developmental mechanism in vertebrates whereby muscle cells first migrate from axially located somites to the pelvic limb, then extend towards the midline and only then differentiate into the single cloacal/perineal muscles.     

Notch signalling is required for cyclic expression of the hairy-like gene HES1 in the presomitic mesoderm

Somitic segmentation provides the framework on which the segmental pattern of the vertebrae, some muscles and the peripheral nervous system is established. Recent evidence indicates that a molecular oscillator, the 'segmentation clock', operates in the presomitic mesoderm (PSM) to direct periodic expression of c-hairy1 and lunatic fringe (l-fng). Here, we report the identification and characterisation of a second avian hairy-related gene, c-hairy2, which also cycles in the PSM and whose sequence is closely related to the mammalian HES1 gene, a downstream target of Notch signalling in vertebrates. We show that HES1 mRNA is also expressed in a cyclic fashion in the mouse PSM, similar to that observed for c-hairy1 and c-hairy2 in the chick. In HES1 mutant mouse embryos, the periodic expression of l-fng is maintained, suggesting that HES1 is not a critical component of the oscillator mechanism. In contrast, dynamic HES1 expression is lost in mice mutant for Delta1, which are defective for Notch signalling. These results suggest that Notch signalling is required for hairy-like genes cyclic expression in the PSM.     

Resegmentation in the mexican axolotl,Ambystoma mexicanum

The segmental series of somites in the vertebrate embryo gives rise to the axial skeleton. In amniote models, single vertebrae are derived from the sclerotome of two adjacent somites. This process, known as resegmentation, is well‐studied using the quail–chick chimeric system, but the presumed generality of resegmentation across vertebrates remains poorly evaluated. Resegmentation has been questioned in anamniotes, given that the sclerotome is much smaller and lacks obvious differentiation between cranial and caudal portions. Here, we provide the first experimental evidence that resegmentation does occur in a species of amphibian. Fate mapping of individual somites in the Mexican axolotl (Ambystoma mexicanum) revealed that individual vertebrae receive cells from two adjacent somites as in the chicken. These findings suggest that large size and segmentation of the sclerotome into distinct cranial and caudal portions are not requirements for resegmentation. Our results, in addition to those for zebrafish, indicate that resegmentation is a general process in building the vertebral column in vertebrates, although it may be achieved in different ways in different groups. J. Morphol. 275:141–152, 2014. © 2013 Wiley Periodicals, Inc.     

Periodic segmental anomalies induced by heat shock in the chick embryo are associated with the cell cycle

This study provides evidence that cells destined to segment together into somites have a degree of cell division synchrony. We have measured the duration of the cell division cycle in somite and segmental plate cells of the chick embryo as 9.5 h using [3H]thymidine pulse- and-chase. Treatment of embryos with any of a variety of inhibitors known to affect the cell division cycle causes discrete periodic segmental anomalies: these anomalies appear about 6-7 somites after treatment and, in some cases, a second anomaly is observed 6 to 7 somites after the first. Since somites take 1.5 h to form, the 6- to 7- somite interval corresponds to about 9-10 h, which is the duration of the cell cycle as determined in these experiments. The anomalies are similar to those seen after heat shock of 2-day chick embryos. Heat shock and some of the other treatments induce the expression of heat-shock proteins (hsp); however, since neither the expression nor the distribution of these proteins relate to the presence or distribution of anomalies seen, we conclude that hsps are not responsible for the pattern of segmental anomalies observed. The production of periodic segmental anomalies appears to be linked to the cell cycle. A simple model is proposed, in which we suggest that the cell division cycle is involved directly in gating cells that will segment together.     

Determination of somite cells: independence of cell differentiation and morphogenesis

Somites are mesodermal structures which appear transiently in vertebrates in the course of their development. Cells situated ventromedially in a somite differentiate into the sclerotome, which gives rise to cartilage, while the other part of the somite differentiates into dermomyotome which gives rise to muscle and dermis. The sclerotome is further divided into a rostral half, where neural crest cells settle and motor nerves grow, and a caudal half. To find out when these axes are determined and how they rule later development, especially the morphogenesis of cartilage derived from the somites, we transplanted the newly formed three caudal somites of 2.5-day-old quail embryos into chick embryos of about the same age, with reversal of some axes. The results were summarized as follows. (1) When transplantation reversed only the dorsoventral axis, one day after the operation the two caudal somites gave rise to normal dermomyotomes and sclerotomes, while the most rostral somite gave rise to a sclerotome abnormally situated just beneath ectoderm. These results suggest that the dorsoventral axis was not determined when the somites were formed, but began to be determined about three hours after their formation. (2) When the transplantation reversed only the rostrocaudal axis, two days after the operation the rudiments of dorsal root ganglia were formed at the caudal (originally rostral) halves of the transplanted sclerotomes. The rostrocaudal axis of the somites had therefore been determined when the somites were formed. (3) When the transplantation reversed both the dorsoventral and the rostrocaudal axes, two days after the operation, sclerotomes derived from the prospective dermomyotomal region of the somites were shown to keep their original rostrocaudal axis, judging from the position of the rudiments of ganglia. Combined with results 1 and 2, this suggested that the fate of the sclerotomal cells along the rostrocaudal axis was determined previously and independently of the determination of somite cell differentiation into dermomyotome and sclerotome. (4) In the 9.5-day-old chimeric embryos with rostrocaudally reversed somites, the morphology of vertebrae and ribs derived from the explanted somites were reversed along the rostrocaudal axis. The morphology of cartilage derived from the somites was shown to be determined intrinsically in the somites by the time these were formed from the segmental plate. The rostrocaudal pattern of the vertebral column is therefore controlled by factors intrinsic to the somitic mesoderm, and not by interactions between this mesoderm and the notochord and/or neural tube, arising after segmentation.     

Developmental Fate of Artificially Disordered Somite Cells after Heteroplastic Transplantation

A disordered somite pattern could be produced artificially when the segmental lateral plate of chickembryo was replaced by dissociated cells of quail segmental pate.The artificially disordered somitepattern formed at either place was used in our work as a model to analyze the mechanism of thedevelopment and differentiation of somite on chick embryo.Our conclusions include the following:1.Although the formation of somites from the dissociated segmental plate cells does not requirespecial environment,the development and differentiation of the somltes require a special environmentwhich is related to the neural tube and notochord.The effect of this special environmental factor maydecrease gradually with the increase of the distance from neural tube to lateral plate.2.The somites located on paraxial area at different distances to the axis have different fates indevelopment.3.The formation of epithelial vesicles is the property of somite cells and the epithelial vesicle is thestructural basis of somite differentiation.If and factor interferes with the differentiation of thesomite,the epithelial vesicle of the somite will be degenerated within certain period of time.4.During resegmentation of the somite,the number,size and arrangement of sclerotome in situ donot depend on the somite from which they are derived.5.Somite cells do not transdifferentiate into kidney tubule directly from their original epithelialvesicles,but are reorganized from the free cells dispersed from the disrupted somites.6.The establishment of cell commitment may involve several steps.Before commitment isestablished the of cell commitment is labile.7.The differentiation of sclerotome starts with the rupture of epithelial wall of somites and thedirection of its movement depends not only on the notochord but also on their position with respectto the neural tube and notochord.8.The disordered somite pattern doesn't influence the segmentation of dorsal root ganglia in situ,but causes the formation of the ectopic dorsal root ganglia.Key Words:Somite differentiation;Artificial disordered somite pattern;Chimeral somite;Resegmentation of sclerotome;Distribution of dorsal root ganglia