Carbohydrate histochemistry of the epidermis of Natrix piscator during its sloughing cycle

Carbohydrate histochemistry of the scale and hinge epidermis of the chequered water snake, Nalrix piscator , throughout the sloughing cycle, has been described. The small amount of mucopolysaccharide present in the Oberhautchen, mesos layer, α-layer and β-Mayer (in its initial stage of differentiation) is comparable with that in amphibian epidermis and the epidermis of certain freshwater fish undergoing keratinization. Moderate amounts of mucopolysaccharide in the lacunar tissue and clear layer may protect against environmental pathogens and retain water to protect the epidermis from desiccation. Mucous cells could not be located in the epidermis throughout the sloughing cycle, contrary to some previous observations that they occur in the hinge region. The general absence of glycogen in the epidermis in most stages of the sloughing cycle suggests that the glycogen metabolized in the epidermis is utilized immediately, in view of the high energy requirements of proliferation and differentiation. Accumulation of glycogen granules in the presumptive α-layer in stage 2 and in the clear layer, presumptive Oberhautchen and presumptive β-Mayer in stage 3 is correlated with low energy requirements, indicating a slowing down of the process of keratinization of cells in these layers.     

(beta)-catenin mediates the specification of endoderm cells in ascidian embryos

In the present study, we addressed the role of (beta)-catenin in the specification of embryonic cells of the ascidians Ciona intestinalis and C. savignyi and obtained the following results: (1) During cleavages, (beta)-catenin accumulated in the nuclei of vegetal blastomeres, suggesting that it plays a role in the specification of endoderm. (2) Mis- and/or overexpression of (beta)-catenin induced the development of an endoderm-specific alkaline phosphatase (AP) in presumptive notochord cells and epidermis cells without affecting differentiation of primary lineage muscle cells. (3) Downregulation of (beta)-catenin induced by the overexpression of cadherin resulted in the suppression of endoderm cell differentiation. This suppression was compensated for by the differentiation of extra epidermis cells. (4) Specification of notochord cells did not take place in the absence of endoderm differentiation. Both the overexpression of (beta)-catenin in presumptive notochord cells and the downregulation of (beta)-catenin in presumptive endoderm cells led to the suppression of Brachyury gene expression, resulting in the failure of notochord specification. These results suggest that the accumulation of (beta)-catenin in the nuclei of endoderm progenitor cells is the first step in the process of ascidian endoderm specification.     

A new triple staining method for double in situ hybridization in combination with cell lineage tracing in whole-mount Xenopus embryos

To analyze cell to cell interaction effects on cell differentiation, we developed a new triple staining method for double in situ hybridization with cell lineage tracing in whole-mount Xenopus embryos. The method provides high color contrast views, and also enabled us to examine inside the embryos. Wild-type embryos whose blastomere(s) had been injected with a cell lineage tracer were cultured, fixed, hemisectioned when necessary, and first served for the double in situ hybridization, with two sequential chromogenic reactions. They were postfixed, and the labeled cells were retraced immunohistochemically. Finally, the pigment of the embryos was bleached to obtain a clear view. We applied this method to a blastomere transplantation experiment to examine whether the spatial gene expression patterns along the anteroposterior axis can be induced by cell to cell interactions. The presumptive organizer of a 32-cell embryo was replaced by the labeled presumptive epidermis of another synchronous embryo. The resultant triple-stained late gastrula showed quite similar anteroposterior expression patterns of gsc and Xbra to those of a normal embryo in the axial mesoderm derived from the transplanted presumptive epidermis, indicating that cell to cell interactions had induced these patterns.     

Succinic dehydrogenase activity in the epidermis ofNatrix piscator during its sloughing cycle

Succinic dehydrogenase activity, in the epidermis of Nairix piscutor in different stages of sloughing cycle, has been localized using a nitro-BT technique with appropriate controls. The staining properties of different layers in scale epidermis are similar to the corresponding layers in hinge epidermis.
In the stratum germinativum, the layers of undifferentiated epidermal cells in all stages of the sloughing cycle, and in the lacunar tissue of Stages 3,4 and 5, a positive though weak reaction for SDH activity reflects the active metabolic state of the cells in these layers. Loss of SDH activity in Stage 6 indicates an inactive metabolic state of the lacunar tissue cells, corresponding with their disintegration owing to the cessation of nutrients as a result of keratinization of cells in the underlying layers.
The Oberhautchen, mesos and alpha layers in all Stages, and the clear layer cells in Stages 5 and 6 (outer epidermal generation), the presumptive Oberhautchen, presumptive mesos layer and presumptive alpha layer in all stages of their differentiation, and the presumptive beta layer in Stages 3 and 4 (inner epidermal generation) all stain purple with nitro-BT technique even in sections incubated in the medium without the substrate-succinate. The reaction is inhibited by prior treatment with 0.1 M N-ethyl maleimide blocking protein-bound -SH groups. This suggests that the reaction is due to the presence of protein-bound -SH groups in these sites. The reduced intensity of reaction in the mature beta layer of the outer epidermal generation, and in the presumptive beta layer in Stages 5 and 6 of the inner epidermal generation, is due to simultaneous loss of their content of -SH groups with maturation and keratinization.     

In vitro pancreas formation from Xenopus ectoderm treated with activin and retinoic acid

In the present study, isolated presumptive ectoderm from Xenopus blastula was treated with activin and retinoic acid to induce differentiation into pancreas. The presumptive ectoderm region of the blastula consists of undifferentiated cells and is fated to become epidermis and neural tissue in normal development. When the region is isolated and cultured in vitro, it develops into atypical epidermis. Isolated presumptive ectoderm was treated with activin and retinoic acid. The ectoderm frequently differentiated into pancreas-like structures accompanied by an intestinal epithelium-like structure. Sections of the explants viewed using light and electron microscopy showed some cells clustered and forming an acinus-like structure, including secretory granules. The pancreas-specific molecular markers insulin and XIHbox8 were also expressed in the treated explants. The pancreatic hormones, insulin and glucagon, were detected in the explants using immunohistochemistry. Therefore, sequential treatment with activin and retinoic acid can induce presumptive ectoderm to differentiate into a morphological and functional pancreas in vitro. When ectoderm was immediately treated with retinoic acid after treatment with activin, well-differentiated pronephric tubules were seen in a few of the differentiated pancreases. Treatment with retinoic acid 3-5 h after activin treatment induced frequent pancreatic differentiation. When the time lag was longer than 15h, the explants developed into axial mesoderm and pharynx. The present study provides an effective system for analyzing pancreas differentiation in vertebrate development.     

INDUCTION OF THE FROG HATCHING GLAND CELL FROM EXPLANTED PRESUMPTIVE ECTODERMAL TISSUE BY LICL

When cells of the superficial layer explanted from the presumptive ectoderm of a Rana japonica early gastrula embryo at stage 10 were cultured in standard salt solution for 4–7 days, they differentiated into cement gland cells (CGCs), cilia cells (CCs) and common epidermal cells (CECs). When, however, these explants were treated with LiCl and transferred to Barth's solution, hatching gland cells (HGCs) and pigment cells were induced.
The optimum condition for inducing differentiation of HGC was treatment with 70 mM LiCl for 6–8 hr at 18°C. The best ability to react to the HGC-inducing stimuli resided in the superficial layer of the dorsal presumptive epidermis of the embryo at stage 10. Upon repeated stimulation, explants from stage 8 embryos underwent differentiation into nerve and pigment cells, whereas those from stage 11 embryos differentiated into CCs and CECs. Under optimum conditions, the total volume of HGCs induced amounted to about 70% of the explanted tissue. The culture media from LiCl-induced HGCs showed an apparent jelly-digesting activity, strongly indicating that the cells were functionally identical with those differentiated in situ .     

中国早熟禾属植物部分种叶下表皮微形态的研究

通过光学显微镜及扫描电子显微镜对中国早熟禾属20种、6亚种及2变种植物的叶下表皮微形态特征进行了观察, 以明确叶表皮微形态特征在早熟禾属植物中的分类学意义。结果显示：(1)早熟禾属植物的叶下表皮长细胞多为长筒状或纺锤形,少数短筒状,细胞壁波状弯曲或近平直;脉间具有短细胞或缺如;气孔器多数常见,副卫细胞平行形至低圆屋顶形;脉上具有刺细胞或缺如,脉间刺细胞多为缺如;脉上硅细胞单生或对生,椭圆形、肾形、新月形、近方形、长方形边缘波状弯曲或结节形;部分早熟禾属植物叶下表皮存在冠细胞。(2)早熟禾属植物叶下表皮微形态特征在长细胞的形状及其细胞壁的弯曲与否、短细胞的有无及其形状、气孔器的分布与副卫细胞的形状、刺细胞的分布、脉上硅细胞的形状、冠细胞的有无这些方面存在着一定的差异,可为该属植物种间分类提供参考依据。叶表皮微形态证据支持高原早熟禾、细叶早熟禾作为草地早熟禾亚种的处理意见。     

Embryonic development of Xenopus studied in a cell culture system with tissue-specific monoclonal antibodies

An in vitro microculture system of early gastrula cells of Xenopus laevis has been developed; deep layer cells from the lateral marginal zone (prospective somite region) or ventral ectoderm (prospective epidermis region) were fully dissociated, and the desired number of each (1-100) was distributed into a microculture well and cultured under appropriate conditions. When examined with the tissue-specific Mabs (Mu1 for muscle and E3 for epidermis, respectively), a substantial portion of the deep layer cells from the two regions followed their respective normal embryonic fates. It was found that reproducible cellular differentiation was dependent on the intimate reaggregation of dissociated cells and on the size of the resultant aggregate. About 20 lateral marginal zone cells were found to be sufficient, when put into a culture well, for supporting successful muscle differentiation, whereas about 100 ventral ectoderm cells were necessary for epidermal differentiation.     

Solute sorting in grass leaves: the transpiration stream

Solutes distribute differentially between leaf tissues and cells. The present study tested the hypothesis that certain solutes are supplied preferentially to the epidermis in the transpiration stream, by-passing mesophyll cells along bundle sheath extensions. Using energy dispersive X-ray analysis of extracted cell sap, the distribution of solutes was studied in the emerged zone (transpiring) and the elongation zone (non-transpiring) of the developing leaf three of barley (Hordeum vulgare L.). The basic distribution of Cl, K, P and Ca between epidermis and bulk tissue, and between cells within the epidermis, was similar in the two leaf regions. However, in the emerged zone differences in solute concentrations between tissues and cells were greater. A local reduction in transpiration rate along the emerged portion of the blade specifically prevented Ca from accumulating to high levels in epidermal cells close to stomata. It is concluded that differences in solute concentrations between epidermal cells and other leaf tissues can be established in the absence of transpiration, but that they require transpiration for their full expression. Peristomatal transpiration appears to be responsible for high Ca in interstomatal cells.Abbreviations EDX-analysis Energy-dispersive X-ray analysis - IS-cell Interstomatal cell - R-cell Ridge cell - TR-cell Trough cell     

pag gene-like protein (ABP-25) of the Cynops embryo: regional distribution and gene expression during early embryogenesis

A maternal protein showing a unique distribution during early Cynops embryogenesis was screened by monoclonal antibody. The antigen protein, designated as ABP-25 (animal blastomere protein, molecular weight 25,000), was distributed uniformly in the uncleaved egg and concentrated into blastomeres of the animal half during cleavage. At the blastula stage, ABP-25 was definitely localized in cells of the animal half and a polarized distribution was observed within the cytoplasm. During gastrulation, immunohistochemical analysis indicated that the reactivity of the marginal zone (presumptive mesoderm) to the monoclonal antibody ABP-25 decreased after involution. At the end of gastrulation, a polarized distribution was still clearly observed in the ventral epidermis, but not in the neuroectoderm. Both Western and Northern blots indicated that the amount of antigen protein and the intensity of gene expresion were almost constant until the neurula stage. The deduced amino acid sequence of the ABP-25 cDNA showed a strong homology (84%) with that of the pag gene associated with cell proliferation.The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases with the accession number D37808     

ANATOMY OF MACROZAMIA COMMUNIS LATERAL ROOTS AND ROOT NODULES FORMED IN VITRO STUDIED WITH LIGHT AND SCANNING ELECTRON MICROSCOPY

The anatomy of Macrozamia communis L. Johnson lateral roots and nodules was studied following axenic culture in light and darkness. Pointed lateral roots from dark cultures had an open apical organization similar to that of other cycads and gymnosperms. A distinct protoderm-derived epidermis was not observed. At the apex, the dermis was formed by the outer root capcortical cell layer. Subapically, the outer cortex formed the dermis. No evidence of an algal zone was observed in these roots. The stele was bounded by a distinct endodermis and contained an exarch, diarch xylem. Apogeotropic nodules which developed at the root-shoot junction in darkness, branched dichotomously and had rounded tips covered by tangentially-enlarged root cap cells. The root cap was reduced to a few cell layers and was confined to the extreme nodule apex. The central region of the apical meristem was enlarged, and meristematic cells contained differentiated amyloplasts. A presumptive algal zone was present in some but not all nodules and divided the cortex into inner and outer regions. Stelar anatomy was similar to that observed in pointed, dark-grown lateral roots, except that there was greater xylem differentiation. Nodules which developed in the light were similar to dark-formed nodules, except that root cap cells were radially enlarged and extended over the flanks of the nodule forming a persistent root cap. The heteromorphic lateral roots of M. communis formed a developmental continuum not a heterorhizic root system.     

Generation and migration of cells in the developing striatum.

The development of the rat striatum was investigated using a combination of two histochemically distinguishable retrovirus vectors. Using this method, it was possible to identify clonal boundaries within the embryonic striatum and thus determine patterns of proliferation, migration, and some lineal relationships. Several novel aspects of striatal histogenesis were discovered. Striatal progenitor cells do not exhibit a stem cell pattern of division between embryonic day 15 (E15) and E19; a progenitor-progeny relationship appears to exist for ventricular zone and subventricular zone (SVZ) cells; striatal progenitors produce a variety of clone types; some SVZ cells migrate radially, and some migrate tangentially within the SVZ; and radial glia and presumptive neurons can occur in the same clone.     

Localization of a nervous system-specific class II beta-tubulin gene in Xenopus laevis embryos by whole-mount in situ hybridization.

A neural-specific beta-tubulin mRNA is expressed in the developing central nervous system shown by whole-mount in situ hybridization experiments. Of special interest is the fact that from the late blastula (stage 9; Nieuwkoop and Faber, 1967; Hausen and Ribesell, 1991) until the early neurula (stage 13) the signal can be found not only in the presumptive neural plate but also in the presumptive epidermis. Later in development (from stage 13) the specific mRNA becomes restricted to the presumptive brain and spinal cord area. The results are discussed in the context of predisposition and (pre)determination.     

中国看麦娘属植物叶表皮微形态研究

看麦娘属是禾本科早熟禾亚科的重要类群,中国分布有8种。该研究采用光学显微镜和电子扫描电镜,对中国看麦娘属7种植物的叶下表皮(含叶鞘下表皮)微形态特征进行观察并探讨其分类学意义。结果表明：(1)看麦娘属叶鞘下表皮长细胞的形状呈长筒状或短筒状,深波状弯曲的细胞壁,脉间短细胞密布,单生或对生,形状多为新月形或椭圆形,脉区硅细胞为椭圆形,平行形或低圆屋顶形的气孔器,看麦娘属叶鞘下表皮特征高度一致,分类价值不大。(2)看麦娘属植物叶下表皮和叶鞘下表皮的显微结构存在很大不同,看麦娘属植物叶下表皮长细胞多为长筒状或短筒状或长菱形,平周壁平直或不明显弯曲,脉间区短细胞分布或无,脉区刺毛密布,脉间区有或无刺细胞的分布,平行形的气孔器,脉区分布的硅细胞多为长方形边缘呈波状弯曲。看麦娘属植物叶下表皮的不同主要体现在脉间区长细胞的形状及平周壁的弯曲与否,脉间区短细胞出现或无,脉间区刺细胞的分布数量以及气孔器的列数。7种植物的叶表皮微形态的分类检索表据此列出。依据叶表皮微形态划分的组与根据外部特征划分的组并不完全一致。但是,由于该文仅研究了7种,叶表皮微形态对于看麦娘属的分类意义还需要更深入的研究。     

喜树各器官解剖学及其喜树碱含量测定的研究

运用植物解剖学方法研究了喜树各器官的结构,对喜树不同器官中喜树碱含量进行了HPLC法测定。结果表明:(1)喜树根尖纵切面的结构由根冠、分生区、伸长区和根毛区4个部分组成。根和茎的初生结构都由表皮、皮层和维管柱3部分组成;在根和茎初生结构的皮层和髓部具有被锇酸染成黑色的嗜锇细胞;在次生生长中,根的木栓形成层起源于中柱鞘,茎的木栓形成层起源于皮层细胞;随着次生维管组织的增加,茎的中央形成髓腔。(2)叶片由表皮、叶肉和叶脉组成,叶肉细胞中分布有嗜锇细胞;光学显微镜和扫描电子显微镜观察显示,喜树叶的上表皮没有气孔器分布;下表皮气孔器的保卫细胞呈肾形,没有副卫细胞,被几个表皮细胞不规则的围绕,属于无规则型;在上下表皮均分布着单细胞非腺毛和2种类型的腺毛,其中下表皮的分布相对稠密;上表皮腺毛呈球形,下表皮腺毛大部分为长卵形,其中间杂着一些球形腺毛;观察发现,越幼嫩的叶中,分布的腺毛和非腺毛越多。(3)喜树不同器官中喜树碱含量以幼叶和种子中较高,木质部中较低,髓中含量最少;随着叶的发育成熟,叶中喜树碱的含量逐渐降低,且同一叶的不同部位,喜树碱含量也有差异;喜树幼苗发育过程中喜树碱含量也在不断变化。     

EFFECT OF ACTINOMYCIN D ON THE DIFFERENTIATION OF HATCHING GLAND CELL AND CILIA CELL IN THE FROG EMBRYO

The forehead epidermis of the stage 18–20 R. japonica embryo includes the hatching gland cell (HGC) which contains cell-specific secretory granules. The cilia cell (CC) and common epidermal cell (CEC) constitute the epidermis of the entire body surface, in addition to the forehead region.
Culture of superficial epidermal explants from various embryonic portions at various developmental stages revealed that HGCs are derived from cells localized on the neural crest in the stage 13a (early neural plate) embryo. When explants from the presumptive HGC area were treated with 1 ug/ml actinomycin D (AMD), the formation of secretory granules in HGCs was inhibited either by continuous treatment from stage 13 or by an 8-hr treatment at stage 13b. Similarly, the ciliogenesis in CCs was inhibited. The differentiation of CECs was entirely unaffected by any of the AMD treatment. After release from AMD, mucous vesicles, characteristic of the CEC, were formed in cells whose differentiation into HGC and CC had been suppressed by the antibiotic. Thread complexes and clumps of coiled strings were found in the nuclei of AMD-affected cells.
It is concluded that the DNA-dependent RNA syntheses which direct secretory granule formation in the HGC and ciliogenesis in the CC occur during a limited period at stage 13b, viz. , 20 hr before their cytodifferentiation becomes appreciable.     

Self-organization in the determination of the size of the axial structures in the embryogenesis of the clawed toad

Experiments were performed using X. laevis embryos during gastrulation and neurulation (stages 10, 11 1/2, 12 1/2, 13 1/2, 15 and 18). Part of presumptive epidermis and lateral plate mesoderm was removed, and embryos raised until stage 25. The size of axial structures (notochord, somite mesoderm, central nervous system) was determined using serial histological sections and compared with that of control embryos. In experimental embryos, the size of axial structures was decreased. Until a specific stage of development, close correlation was found between the volume of embryonic compartment corresponding to a particular, structure and the volume of presumptive epidermis and lateral plate mesoderm. This stage is individual for each axial organ: middle gastrula (stage 11 1/2) for notochord, late gastrula (stage 12 1/2) for somite mesoderm, and late neurula (stage 18) for central nervous system. This data suggest that differentiation pattern of ecto-mesodermal rudiment is subject to regulation during gastrulation-neurulation, and subdivision of ectoderm and mesoderm into axial and non-axial tissues is a self-organizing process.     

Studies on the Formation and State of Determination of the Trunk Organizer in the Newt, Cynops Pyrrhogaster III. Tangential Induction in the Dorsal Marginal Zone

Analysis of the course of differentiation of combinants between the presumptive prechordal plate (PcP) and presumptive ectoderm (PE) by time-lapse filming showed that the PcP of early gastrulae has the capacity to induce mesoderm (notochord, muscle cells and migrating cells) in the PE. The mesoderm-inducing capacity of the PcP decreases sharply during gastrulation. Following invagination in the mid-gastrula, the PcP completely loses its mesoderm-inducing capacity. This change also occurred when the PcP of the earliest gastrula was aged in vitro for 18 hr. This shows that the mesoderm-inducing capacity of the PcP decreases autonomously with aging.
PE transplanted into the presumptive trunk organizer region of the dorsal marginal zone of the earlist gastrula, became mesodermized within 12 hr. It is clear that this mesodermization of the transplanted PE is due to "tangential induction" from the PcP. The stepwise formation of the trunk organizer in Cynops pyrrhogaster is discussed in consideration of these results.     

Pattern in the Root Epidermis: An Interplay of Diffusible Signals and Cellular Geometry

The epidermis of roots is composed of hair and non-hair cells. Patterning of this epidermis results from spatially regulated differentiation of these cell types. Root epidermal development in vascular plants may be divided into three broad groups based on the mode of hair development; Type 1: any cell in the epidermis can form a root hair; Type 2: the smaller product of an asymmetric cell division forms a root hair; Type 3: the epidermis is organized into discrete files of hair and non-hair cells. TheArabidopsisroot epidermis is composed of discrete files of hair and non-hair cells (Type 3). Genetic and physiological evidence indicates that ethylene is a positive regulator of hair cell development. Genes with opposite roles in the development of hair cells in the shoot (trichomes) and hair cells in the root have been identified. Plants with presumptive loss of function alleles in theTRANSPARENT TESTA GLABRA (TTG)orGLABRA2(GL2) genes are devoid of trichomes indicating that these genes are positive regulators of trichome development. The development of supernumerary root hair cells in these mutant backgrounds illustrates that these genes are also negative regulators of root hair cell development. A model that explains the spatial pattern of epidermal cell differentiation implicates ethylene or its precursor 1-amino-1-cyclopropane carboxylate as a diffusible signal. Possible roles for theTTGandGL2genes in relation to the ethylene signal are discussed.     

States of determination of single cells transplanted between 512-cell Xenopus embryos

Single cells from 512-cell Xenopus embryos, totally labeled with intracellular horseradish peroxidase, were transplanted orthotopically (from either dorsal or lateral marginal zone) or heterotopically (between these two marginal zones) to unlabeled host embryos at the 512-cell stage. At tailbud stage 23 the locations, numbers, and histotypes of labeled cells were recorded. The transplanted cell had divided many times, giving rise to labeled progeny that expressed a wide range of cell types and were located in several different organs. Locations and cell types of progeny derived from orthotopic grafts to the dorsal marginal zone were different from those derived from grafts to the lateral marginal zone. Single cells grafted heterotopically to either dorsal or lateral positions expressed fates that were appropriate to their final grafted positions, and not to their original positions. We conclude that individual cells of the marginal zone at the 512-cell stage have multiple presumptive fates and have not been committed to any single fate.