水螅营养积累对基盘组织更新进程的影响

目的观察大乳头水螅（Hydra magnipapillata ）基盘组织更新进程,探讨水螅营养积累对基盘组织更新进程的影响。方法设定水螅喂食频率梯度（代表不同的营养积累水平）,记录和观察喂食频率对水螅更新基盘组织进程的影响。通过ABTS细胞化学染色法检测水螅基盘分子标志物过氧化物酶的表达,观察水螅老基盘组织脱落后水螅体主体新生基盘组织的再生过程。结果喂食频率对水螅更新基盘组织进程有明显的影响。水螅基盘组织更新的标准过程如下;在一定的喂食频率下培养水螅,水螅体出芽区逐渐有芽体产生,随后在出芽区和基盘之间靠近芽体的位置出现缢痕,最后水螅体在缢痕处断裂为水螅体主体和老基盘组织两部分。缢痕断裂后对水螅体主体保持既定的喂食频率,其伤口能愈合但不能再生出新的基盘组织;对其降低喂食频率直至其伤口上方的芽体全部脱落后伤口处重新启动新生基盘组织的再生进程。另外,脱落的老基盘组织有两种不同的命运,即大部分老基盘组织不能发育成正常水螅体、最终解体;而小部分的老基盘组织能发育成正常的水螅体。结论水螅营养积累可能促进基盘组织更新进程,靠近断裂伤口处的芽体能抑制水螅体主体新生基盘的再生进程。     

水螅的无性生殖

淡水水螅的无性繁殖方式是出芽生殖。水螅胃区的皮肌细胞摄取的营养物质,经细胞间传递方式,转移到芽体的内、外胚层,为芽体的发育提供了能量。胃区的干细胞与增殖的细胞不断迁移到芽体,保障芽体发生时的细胞数。水螅的头部与基盘对芽体的发生,同时存在着激活与抑制2对位置信息素,共同控制并决定了芽体在体柱上的发生位置。芽体先发生垂唇与触手芽,其发生位置必需远离母体头部抑制素的作用。芽体发育后期,发生基盘时,必须远离母体基盘抑制素的作用。     

大乳头水螅基盘再生进程中过氧化物酶的表达

目的观察大乳头水螅(Hydra magnipapillata)基盘再生进程中基盘过氧化物酶的表达情况,探讨水螅基盘过氧化物酶的生理作用。方法通过ABTS细胞化学染色法显示水螅基盘过氧化物酶的表达。结果水螅基盘再生20h后其基盘过氧化物酶开始出现少量表达,其后过氧化物酶表达量逐渐增加;基盘再生52h后该酶表达量趋于稳定。过氧化物酶仅在基盘周边区域外胚层中表达,而在基盘中央区域(反口孔)外胚层中无表达。结论水螅基盘再生进程中过氧化物酶的表达量逐渐增加直接反映了基盘再生时细胞分化过程,基盘表达的过氧化物酶可能在维持基盘结构的稳定上起一定的作用。     

水螅异常极性体轴的形成及矫正进程的观察

目的:如何建立和维持体轴是一个基本的发育生物学问题,而淡水水螅是适合进行形态发生和个体发育调控机制研究的重要模式生物。本文观察了大乳头水螅异常极性体轴的形成及矫正进程,初步探讨水螅极性体轴的维持和调控机制。方法:先切取水螅的整个头部,再获得带二根触手的口区组织。通过ABTS细胞化学染色法检测水螅基盘分子标志物过氧化物酶的表达,判别水螅基盘组织(水螅足区的末端)是否形成。结果:从40块口区组织再生得到的水螅个体中有1例极性体轴发育异常的个体,其身体两端均发育成头区,且两端的头区均具有捕食能力。随后水螅其中一端头区的触手逐渐萎缩、退化,最终该端头区转化成具有吸附能力的基盘组织。结论:水螅组织的再生涉及极性体轴的重建,而一些特殊因素可能造成临时性的水螅极性体轴调控紊乱。本研究表明水螅具备自我矫正异常极性体轴的能力。另外,本研究结果显示水螅触手可以萎缩直至退化,该现象涉及的细胞学过程可能是非常复杂的,有可能涉及到触手细胞的凋亡转化过程,也可能是触手的高度分化细胞仍然具备去分化能力、去分化后再转移到身体其他地方,其具体机制值得进一步探究。     

双头无基盘水螅的初步观察

自然形成的双头无基盘畸形体水螅极为罕见,为探索其形成原因,人工嫁接水螅14例,获得5例相同的双头水螅。对其分别进行连续观察发现:嫁接的双头水螅的双头可同时摄食,食物集聚于1个胃区。随个体生长其体柱延长,逐渐发育为2个胃区,在2个胃区间发生2~6个芽体,所有子代发育正常。出芽位置至触手环的距离同正常水螅。之后分化为2个出芽区,出芽区之间体柱较透明,同茎区组织。第21天开始发生基盘,发生过程异常缓慢。     

水螅摄食中的特殊行为

取经中性红活染后的杆吻虫喂水螅,仔细观察了水螅的摄食行为.结果表明,水螅垂唇端部能够相互识别出同类;水螅的垂唇与胃区能协力把杆吻虫吞入胃腔,触手经常随食物进入胃腔;吞食时经常出现头部内翻或外翻;个体间及其成体与芽体间常出现争夺食物的现象,当垂唇端部互不相触时,体型较大的个体常常把较小个体或芽体连同食物一起吞食;垂唇内胚层腺细胞对食物有消化作用,对同类无伤害;水螅的神经系统已有初步的整合功能.     

水螅摄食中的特殊行为

取经中性红活染后的杆吻虫喂水螅,仔细观察了水螅的摄食行为。结果表明,水螅垂唇端部能够相互识别出同类;水螅的垂唇与胃区能协力把杆吻虫吞入胃腔,触手经常随食物进入胃腔;吞食时经常出现头部内翻或外翻;个体间及其成体与芽体间常出现争夺食物的现象,当垂唇端部互不相触时,体型较大的个体常常把较小个体或芽体连同食物一起吞食;垂唇内胚层腺细胞对食物有消化作用,对同类无伤害;水螅的神经系统已有初步的整合功能。     

水螅畸形触手转分化过程的观察

水螅胃区诱导出的触手芽，１４天后在茎区诱导体柱组织分化头结构。胃区诱导出的畸形触手芽，８天后在出芽区脱落，在母体上留下一个基盘，脱落的畸形触手呈２触手水螅个体，其直径相当于头部触手直径的２／３。在人工帮助下能吞食１个未分化的蚤状卵。第１２天能自行捕食幼。最后对２种额外触手芽的不同演化结果做了简单比较。     

水螅AChE和NPY类似物的定位

乙酰胆碱脂酶(AChE)组织化学定位方法常用于研究动物的神经系统,但迄今未见水螅AChE分布定位的相关报道。本文使用免疫细胞化学、组织化学和亚甲基蓝活染方法,探索AChE、NPY类似物在水螅上的连续分布状态,对阳性标本进行内胚层、外胚层人工分离手术,并与亚甲基蓝染色结果进行比较。本文首次成功地在水螅上定位出AChE,展示了AChE、NPY类似物在水螅上的分布特点。AChE定位在水螅体柱和触手基部外胚层的近外表,呈网状结构,正常水螅上没有发现明显的阳性神经细胞,外胚层表面受损部位内有阳性神经细胞,我们认为是一种临时的生理现象。成熟刺丝囊的刺针内有AChE 产物。NPY免疫组织化学反应(NPYIR)在水螅外胚层近外表处上均有分布,呈网状结构。垂唇和近基盘处NPYIR最强,NPYIR神经细胞仅存在于垂唇和近基盘处的外胚层基部。刺丝囊分化、发育初期NPYIR较强。随刺丝囊发育成熟而逐渐减弱。最后,进行了分析和比较,认为NPY、AChE来源于上皮细胞。水螅刺丝囊的分化和发育与NPY的作用密切相关;刺丝囊发射刺丝的机理与胆碱能的作用可能有联系     

大乳头水螅增殖细胞核抗原基因的克隆及再生进程中的表达分析

研究以增殖细胞标记物-增殖细胞核抗原(Proliferating cell nuclear antigen, PCNA)蛋白为切入点, 采用RACE(Rapid amplification of cDNA ends)方法克隆了大乳头水螅(Hydra magnipapillata)PCNA基因的cDNA序列。该cDNA序列包含1240 bp, 其中837 bp为编码区, 编码的蛋白质预测分子量为30.93 kD。把PCNA基因ORF中的部分序列克隆到原核表达质粒pET-28b(+)中, 重组质粒转化E. coli BL21 (DE3)菌株, IPTG诱导后成功表达重组PCNA蛋白, 再用该重组蛋白免疫新西兰兔制备多克隆抗体用于PCNA蛋白的免疫印迹分析(Western blotting assay, WB)。采用实时定量PCR (quantitative Real-time PCR, qPCR)及WB方法检测水螅头部再生进程中其PCNA表达水平的动态变化, 结果表明在再生进程的中后期阶段水螅PCNA表达量呈现一定程度的上调。结果暗示水螅头部再生进程的中后期阶段其伤口及附近区域可能存在细胞分裂活动。     

Wnt signaling in hydroid development: ectopic heads and giant buds induced by GSK-3beta inhibitors

In Hydractinia, a colonial marine hydroid representing the basal phylum Cnidaria, Wnt signaling plays a major role in the specification of the primary body axis in embryogenesis and in the establishment of the oral pole during metamorphosis. Here we report supplementing investigations on head regeneration and bud formation in post-metamorphic development. Head and bud formation were accompanied by the expression of Wnt, frizzled and Tcf. Activation of Wnt signaling by blocking GSK-3beta affected regeneration, the patterning of growing polyps and the asexual formation of new polyps in the colony. In the presence of lithium ions or paullones, gastric segments excised from adult polyps showed reversal of tissue polarity as they frequently regenerated heads at both ends. Phorbol myristate acetate, a known activator of protein kinase C increased this effect. Global activation of the Wnt pathway caused growing polyps to form ectopic tentacles and additional heads along their body column. Repeated treatment of colonies evoked the emergence of many and dramatically oversized bud fields along the circumference of the colony. These giant fields fell apart into smaller sub-fields, which gave rise to arrays of multi-headed polyps. We interpret the morphogenetic effects of blocking GSK-3beta as reflecting increase in positional value in terms of positional information and activation of Wnt target genes in molecular terms.     

Low-molecular-weight factors from colonial hydroids affect pattern formation

Summary Two morphogenetic factors have been isolated from tissue of colonial hydroids. Both exert strong effects on pattern formation during metamorphosis, regeneration and colony development. Polyp-inhibiting factor (PIF) is a bivalent inhibitor which strongly affects head and bud formation but acts weakly on stolon branching. Proportion-altering factor (PAF) is a distalizing factor. It counteracts the formation of stolon and promotes the formation of head structures during metamorphosis and regeneration. PIF and PAF antagonistically influence the spatial arrangement of polyps within a colony. They are capable of dislocating structures and thus appear to interfere with or are even part of the pattern-controlling mechanism. Both factors are of low molecular size (about 500 daltons), hydrophilic and probably not peptides.     

Control of head morphogenesis in an invertebrate asexually produced larva-like bud (<Emphasis Type="Italic">Cassiopea andromeda</Emphasis>; Cnidaria: Scyphozoa)

Scyphopolyps of Cassiopea andromeda propagate asexually by forming larva-like buds which separate from the parent in a developmentally quiescent state. These buds metamorphose into sessile polyps when exposed to specific biogenic, chemical inducers. Morphogenesis of transversely dissected buds indicates the presence of pattern-determining signals; whereas the basal bud fragments may still form a complete scyphistoma the apical bud fragments develop spontaneously in the absence of an inducer into a polyp head without stalk and foot. Based on these findings Neumann (dissertation, Cologne University, 1980) postulated a head-inhibiting signal which is released at the basal pole and inhibits head formation at the apical end. Contrary to this hypothesis dissection itself might induce the development of head structures. The present study deals with the control of polyp head formation in C. andromeda. It concentrates on two points, namely the postulated head inhibitor and the involvement of compounds known to act during metamorphosis (the enzyme protein kinase C and the specific metamorphosis inducer Z-GPGGPA). We found that compared to intact buds and apical bud fragments transversely incised buds reached an intermediate stage of head development. This confirms Neumann's hypothesis. Consequently we focused on the mode of action and the chemical nature of the head-inhibiting signal in C. andromeda. Our results indicate that the head inhibitor may be included in one of six pooled fractions isolated from bud homogenate via gel filtration on a Sephadex G-50 column. The inhibitor is supposed to be water-soluble and to have a molecular weight of 850-1,500 Da. Furthermore we prove that head formation is not promoted by the metamorphosis-inducer Z-GPGGPA but is prevented by the inhibitors psychosine, chelerythrine and RO-32-0432 showing the involvement of protein kinase C in this process.     

Analysis of spacing in a periodic pattern

Colonies of hydroids exhibit periodic biological patterns. Polyps form on stolons at fixed distances, obeying distinct rules. The spacing mechanism is based on inhibition emanating from existing polyps, predominantly from the head of the polyp. Removal of polyps from young colonies reduces the distance between initiating polyps and newly formed polyps to 50% of the normal values. Head removal suffices to obtain an almost identical reduction. Polyp enlargement which increases the distance between the inhibition-emitting head and the stolon tissue reduces the intrastolonal range of inhibition. In the stolon tissue, decrease of inhibitory activity occurs. An increase in the stolon/polyp ratio of a colony reduces bud distances. The decay is, in part, due to loss of inhibitory activity into the external medium: if colonies are incubated in conditioned culture medium derived from crowded colonies having normally large interpolyp distances, bud distances increase in test colonies. The effectiveness of transfer of inhibitory activity from tissue into the medium depends on culture conditions. If convection is increased by agitation of the culture medium, the distances between polyp and bud decreases; viscosity enhancement of the culture medium reduces convection and bud distances become larger. This effect is compensated for by additional agitation of the viscosity enhanced culture medium. Our results support the idea that a lateral inhibition mechanism controls polyp spacing in the stolon and that inhibition is based on diffusible inhibitory compounds.     

Pattern regulation properties of a Hydra strain which produces additional heads along the body axis

The multiheaded one (mh-1) strain, isolated from inbred crossings of wild type Hydra magnipapillata, develops additional heads along the body axis. This strain reproduces asexually by budding like the wild type (wt) does. We found that young polyps have a wt-like shape and display wt-like properties. When they grow in size and before they produce extra heads along the body axis, the tissue between the head and the budding zone changes its property: in this region, where later on the extra heads preferentially form, foot regeneration is significantly delayed while head regeneration remains unaffected. Further, following various transplantations additional heads form under conditions under which the wild type did not. The observed changes in pattern control and regulation indicate a two-step process of pattern formation. Morphogenetic signalling is suggested to cause the positional value to increase slowly in the form of patches and preferentially in the region between the head and the budding zone. This increase causes an altered morphogenetic signalling, which is eventually responsible for additional head formation.     

Ammonia induces metamorphosis of the oral half of buds into polyp heads in the scyphozoan Cassiopea

Summary The scyphozoan polyp Cassiopea forms vegetative free swimming buds that metamorphose into sessile polyps. In sterile sea water metamorphosis does not take place. Buds keep swimming for weeks. Application of millimolar quantities of NH ₄ ⁺ causes the buds to metamorphose within one day. The resulting animals bear hypostome and tentacles, however, only occasionally peduncle and foot. Almost all transform either completely into solitary polyp head or only the oral half of the bud developes into a head while the aboral half remains bud tissue which becomes constricted off. Under suited conditions this small bud is able to transform into a normal shaped polyp.     

HyAlx, an aristaless-related gene, is involved in tentacle formation in hydra

Developmental gradients are known to play important roles in axial patterning in hydra. Current efforts are directed toward elucidating the molecular basis of these gradients. We report the isolation and characterization of HyAlx, an aristaless-related gene in hydra. The expression patterns of the gene in adult hydra, as well as during bud formation, head regeneration and the formation of ectopic head structures along the body column, indicate the gene plays a role in the specification of tissue for tentacle formation. The use of RNAi provides more direct evidence for this conclusion. The different patterns of HyAlx expression during head regeneration and bud formation also provide support for a recent version of a reaction-diffusion model for axial patterning in hydra.     

Preformation and neoformation in shoots of Nothofagus antarctica (G. Forster) Oerst. (Nothofagaceae) shrubs from northern Patagonia

The size (length and diameter) and number of leaf primordia of winter buds of Nothofagus antarctica (G. Forster) Oerst. shrubs were compared with the size and number of leaves of shoots derived from buds in equivalent positions. Buds developed in two successive years were compared in terms of size and number of leaf primordia. Bud size and the number of leaf primordia per bud were greater for distal than for proximally positioned buds. Shoots that developed in the five positions closest to the distal end of their parent shoots had significantly more leaves than more proximally positioned shoots of the same parent shoots. The positive relationship between the size of a shoot and that of its parent shoot was stronger for proximal than for distal positions on the parent shoots. For each bud position on the parent shoots there were differences in the number of leaf primordia per bud between consecutive years. The correlations between the number of leaf primordia per bud and bud size, bud position and parent shoot size varied between years. Only shoots produced close to the distal end of a parent shoot developed neoformed leaves; more proximal sibling shoots consisted entirely of preformed leaves. Leaf neoformation, a process usually linked with high shoot vigour in woody plants, seems to be widespread among the relatively small shoots developed in N. antarctica shrubs, which may relate to the species' opportunistic response to disturbance.     

Selective inhibition of protein kinases blocks the formation of a new axis,the beginning of budding,in Hydra

In Hydra, head regeneration and bud formation appear to be very similar processes. The fact that there are genes whose expression is specific for one of the two processes suggests that they do not have identical molecular bases. We analyzed the signal transduction pathways regulating bud development using inhibitors of protein kinase C, Src, PI3K and ERK. The four inhibitors reversibly blocked bud formation in Hydra when applied before stage 1. Once the bud reached stage 3, three of them had no effect and the bud developed normally. The inhibitors blocked the expression of Budhead, an early head marker, and of CnOtx which are specific for bud formation. The results are in agreement with the central role of a signaling pathway mediated by Src on bud development.