Spermatogenesis in animals as revealed by electron microscopy

Summary The early spermatid nuclei of the grasshopper, Acrida lata, have been observed electron microscopically. The irregularly compact chromatin mass appears closely attached to the nuclear envelope. This mass migrates subsequently into a more central portion. It seems to participate in the formation of the nucleolus as a nucleolar organizer. At the time when the chromatin mass and frequently the nucleolus undergo involution, clusters of peculiar granular bodies 130 m in average diameter and 200 A wide filamentous elements among the bodies make their appearance in the nucleoplasm. The particles constituting the granular bodies are composed of DNA, but their matrix consists of RNA. The term microkaryosome is proposed for such granular body, because it is similar in chemical components to karyosome, but the former is smaller in size than the latter. It is suggested that the microkaryosome may be related with the paracrystalline formation of nucleoprotein.     

核仁组成区相关嗜银蛋白染色革新法

核仁组成区相关嗜银蛋白（ＡｇＮＯＲ）银染技术已广泛用以研究细胞生长活性,根据ＡｇＮＯＲ数目多少来判定肿瘤的良恶性和对肿瘤进行鉴别诊断。然而ＡｇＮＯＲ技术至今还存在背景非特异性银颗粒沉积问题而影响结果判定。本研究发现影响背景染色结果的主要因素是明胶的质量,采用Ｆａｒｍｅｒ’ｓ液可以清除背景染色,运用微波炉染色不仅可以缩短染色时间而且可以减少银用量。     

Cytosine methylation and nucleolar dominance in cereal hybrids

In wheat-rye hybrids the nucleolus organizer regions (NORs), the sites of ribosomal RNA genes, from rye are suppressed. Wheat and wheat-rye hybrid genetic stocks containing different numbers of wheat and rye nucleolus organizers, as well as addition lines and rye-barley hybrids, were used in Southern hybridization experiments to determine the cause of nucleolar dominance and suppression in cereal hybrids. Based on the use of restriction endonucleases that cleave near the ends of the spacer unit and an additional, methylation-sensitive enzyme, HpaII, which does not recognize the CCGG restriction site if the internal C is methylated, an indirect method of assaying NOR expression was established. The results indicated that cleavage by the HpaII enzyme of the rye NOR sequences, is reduced when major NORs from other cereals were present. The reduction in the number of rye rRNA genes containing an unmethylated CCGG site in the promoter was associated with the suppression of the rye nucleolus. These results are consistent with a model in which promoter and upstream regulatory repeats of ribosomal RNA genes compete for limited concentrations of regulatory proteins, and genes that are methylated at key binding sites fail to engage these regulatory proteins and thus remain inactive. Received: 15 November 1996 / Accepted: 19 March 1997     

Nucleolar Organizer Regions In Pituitary Gland Adenomas

Nucleolar organizer regions are DNA segments concerned with the synthesis of ribosomal RNA (rRNA). Some specific proteins related to NORs can be demonstrated by a silver technique and are then referred to as Ag-NORs. The mean number of Ag-NORs per cell has been used as a marker of cell proliferation in recent years and there is some evidence to suggest that Ag-NOR counts can be used to differentiate between normal and neoplastic cells. Paraffin block sections of 42 pituitary gland adenomas and 24 normal adult pituitary glands have been examined using the Ag-NOR staining technique. Touch imprints of nine adenomas and 12 normal adult pituitary glands were also examined using the same Ag-NOR staining technique. Using light microscopy at x2000 magnification, the Ag-NORs were located as black dots in the cell nuclei and the mean number of Ag-NORs per cell was determined. No significant difference was observed between blocked sections and imprints of adenomas or between imprints and paraffin sections of normal glands. However, a significant difference was observed between the mean number of Ag-NORs in pituitary gland adenomas and normal glands in paraffin block sections and touch preparations (P less than 0.001). The modified Ag-NOR technique can be a useful aid in the differential diagnosis between normal pituitary gland tissue and pituitary adenomas, especially during surgical operations.     

Apc1 is required for maintenance of local brain organizers and dorsal midbrain survival

The tumor suppressor Apc1 is an intracellular antagonist of the Wnt/β-catenin pathway, which is vital for induction and patterning of the early vertebrate brain. However, its role in later brain development is less clear. Here, we examined the mechanisms underlying effects of an Apc1 zygotic-effect mutation on late brain development in zebrafish. Apc1 is required for maintenance of established brain subdivisions and control of local organizers such as the isthmic organizer (IsO). Caudal expansion of Fgf8 from IsO into the cerebellum is accompanied by hyperproliferation and abnormal cerebellar morphogenesis. Loss of apc1 results in reduced proliferation and apoptosis in the dorsal midbrain. Mosaic analysis shows that Apc is required cell-autonomously for maintenance of dorsal midbrain cell fate. The tectal phenotype occurs independently of Fgf8-mediated IsO function and is predominantly caused by stabilization of β-catenin and subsequent hyperactivation of Wnt/β-catenin signalling, which is mainly mediated through LEF1 activity. Chemical activation of the Wnt/β-catenin in wild-type embryos during late brain maintenance stages phenocopies the IsO and tectal phenotypes of the apc mutants. These data demonstrate that Apc1-mediated restriction of Wnt/β-catenin signalling is required for maintenance of local organizers and tectal integrity.     

Conserved and divergent genes in apex and axis development of cnidarians

Despite their radial organization and their sister group position in the life tree, cnidarian species express during morphogenesis a large number of genes that are related to bilaterian developmental genes. Among those, homologs to forkhead, emx, aristaless, goosecoid, brachyury, wnt and nanos genes are regulated during apical patterning in cnidarians, suggesting that key components of early organizer activity were conserved across evolution and recruited for either anterior, axial, or dorso-ventral patterning in bilaterians. In contrast, the expression patterns of the cnidarian Hox-related genes suggest that the apical-basal axis of the cnidarian polyp and the anterior-posterior axis of bilaterians do not differentiate following homologous processes.     

Convergent Wnt and FGF signaling at the gastrula stage induce the formation of the isthmic organizer

The development of the vertebrate brain depends on the formation of local organizing centres within the neural tube that express secreted signals that refine local neural progenitor identity. The isthmic organizer (IsO) forms at the isthmic constriction and is required for the growth and ordered development of mesencephalic and metencephalic structures. The formation of the IsO, which is characterized by the generation of a complex pattern of cells at the midbrain-hindbrain boundary, has been described in detail. However, when neural plate cells are initially instructed to form the IsO, the molecular nature of the inductive signals remain poorly defined. We now provide evidence that convergent Wnt and FGF signaling at the gastrula stage are required to generate the complex polarized pattern of cells characteristic of the IsO, and that Wnt and FGF signals in combination are sufficient to reconstruct, in na?ve forebrain cells, an IsO-like structure that exhibits an organizing activity that mimics the endogenous IsO when transplanted into the diencephalon of chick embryos.     

High Ag-NOR-site variation associated to a secondary contact in brown trout from the Iberian Peninsula

The analysis of nucleolar organizer regions (NORs) using silver (Ag-) staining and in situ hybridization (ISH) in brown trout (Salmo trutta) from various river basins in the Iberian Peninsula revealed high variation in the number and location of NORs. A total of 17 different Ag-NOR sites were revealed in 10 different chromosome pairs. Three different Ag-NOR patterns clustered by river basins and strongly associated to the internal transcribed spacer 1 (ITS1) variation were detected. The main variability in NOR-sites was found in a secondary contact between two divergent lineages of brown trouts at Duero basin. Our results confirmed the abrupt break in the spatial distribution of genetic variation of brown trout populations previously reported at Duero basin. We hypothesize that NOR-site variation might be a consequence of hybridization between divergent lineages of brown trouts and that NORs could play a major role in the maintenance of a hybrid zone in Duero basin via post-zygotic isolation mechanisms.     

β-catenin plays a central role in setting up the head organizer in hydra

In an adult hydra the head organizer, located in the hypostome, is constantly active in maintaining the structure of the animal in the context of its steady state tissue dynamics. Several Wnt genes, TCF, and elevated levels of β-catenin are expressed in the hypostome as well as during the formation of a new organizer region in developing buds suggesting they play a role in the organizer. Transgenic hydra were generated in which a modified hydra β-catenin gene driven by an actin promoter is continuously expressed at a high level throughout the animal. These animals formed heads and secondary axes in multiple locations along the body column. Transplantation experiments indicate they have a high and stable level of head organizer activity throughout the body columns. However, none of the Wnt genes are expressed in the body columns of these transgenic animals. Further, in alsterpaullone-treated animals, which results in a transient rise in head organizer activity throughout the body column, the time of expression of the Wnt genes is much shorter than the time of the elevated level of head inducing activity. These results for the first time provide direct functional evidence that β-catenin plays a crucial role in the maintenance and activity of the head organizer and suggest that Wnt ligands may be required only for the initiation but not in maintenance of the organizer in Hydra.