Cloning and expression analysis of piRNA-like RNAs: adult testis-specific small RNAs in chicken

Many small RNAs have been cloned from animal gonads, for example, endogenous small interfering RNAs (endo-siRNAs) were found in oocytes and piwi-interacting RNAs (piRNAs) were found in testis. Gallus gallus (chicken) is an important model organism, but few small RNAs have been identified from its gonads. In this study, we isolated and cloned 156 small RNAs from adult chicken testes. Since there is a reasonably even distribution from 22 to 33 nt, these small RNAs are slightly longer than miRNAs and endo-siRNAs. Genome mapping indicated that these small RNAs were derived from intergenic regions, exons, introns, and repetitive elements including chicken repeat 1, long terminal repeats, and simple repeats. Since they are similar with piRNAs, we named them piRNA-like RNAs (pil-RNAs). Northern blotting of 16 selected sequences showed that nine are specifically expressed in the adult testis. The vast majority of these pil-RNAs are poorly conserved between species, suggesting that they are unique to the adult chicken testis. Further analysis of the cloned pil-RNAs will improve our understanding of the function of small RNAs in animal gonad development.     

Cloning, characterization and widespread expression analysis of testicular piRNA-like chicken RNAs

Piwi-interacting RNAs (piRNAs) are small RNAs abundant in the germline that have been implicated in germline development and maintenance of genomic integrity across several animal species including human, mouse, rat, zebrafish and drosophila. Tens of thousands of piRNAs have been discovered, yet abundant piRNAs have still not been detected in various eukaryotic organisms. This is a report on the characterization, cloning and expression profiling of piRNA-like chicken RNAs. Here, we identified 19 piRNAs, each 23–39 nucleotides long, from chicken testis using a small RNA cDNA library and T-A cloning methods. Three different pilRNAs were selected according to size, homology and secondary structure for temporal and spatial expression by Q-PCR technology in different tissues at five growth and four development stages of Chinese indigenous Rugao chickens (RG) and introduced recessive white feather chickens (RW). We found that, consistent to other organisms, pilRNA-encoding sequences within the chicken genome were asymmetrically distributed on the chromosomes while displaying a preference for intergenic regions across the genome. Interestingly, unlike miRNAs with unique stem-loop structures (mature miRNAs form stem section and the rest form loop section), distinct secondary structures of pilRNAs were predicted. In addition, chicken pilRNAs were not only abundant in the germline but also existed in somatic tissues, where, expression levels were influenced mainly by different pilRNAs, breed and gender. Taken together, our results suggest that two distinct secondary structures exist between pilRNAs and miRNAs, which may clarify the splicing and processing mechanisms of the two small RNAs are possible different. Moreover, our results suggest that pilRNAs may not only be confined to development and maintenance of the germline but may also play important roles in somatic tissues. Additionally, different pilRNAs may be involved in the unique regulatory machinery of complex biological processes.     

Cell proliferation study at various stages of gastrulation in axolotl embryos

A study of cell proliferation in different regions of axolotl embryos has shown a rather uniform distribution of the S phase and mitotic indices in the animal half of the early and midgastrulae. The dorsal blastoporal lip is characterized by a very low S phase index as compared with the other regions of the embryo.     

Axial differentiation and early gastrulation stages of the pig embryo

Differentiation of the principal body axes in the early vertebrate embryo is based on a specific blueprint of gene expression and a series of transient axial structures such as Hensen's node and the notochord of the late gastrulation phase. Prior to gastrulation, the anterior visceral endoderm (AVE) of the mouse egg-cylinder or the anterior marginal crescent (AMC) of the rabbit embryonic disc marks the anterior pole of the embryo. For phylogenetic and functional reasons both these entities are addressed here as the mammalian anterior pregastrulation differentiation (APD). However, mouse and rabbit show distinct structural differences in APD and the molecular blueprint, making the search of general rules for axial differentiation in mammals difficult. Therefore, the pig was analysed here as a further species with a mammotypical flat embryonic disc. Using light and electron microscopy and in situ hybridisation for three key genes involved in early development (sox17, nodal and brachyury), two axial structures of early gastrulation in the pig were identified: (1) the anterior hypoblast (AHB) characterised by increased cellular height and density and by sox17 expression, and (2) the early primitive streak characterised by a high pseudostratified epithelium with an almost continuous but unusually thick basement membrane, by localised epithelial–mesenchymal transition, and by brachyury expression in the epiblast. The stepwise appearance of these two axial structures was used to define three stages typical for mammals at the start of gastrulation. Intriguingly, the round shape and gradual posterior displacement of the APD in the pig appear to be species-specific (differing from all other mammals studied in detail to date) but correlate with ensuing specific primitive streak and extraembryonic mesoderm development. APD and, hence, the earliest axial structure presently known in the mammalian embryo may thus be functionally involved in shaping extraembryonic membranes and, possibly, the specific adult body form.     

Global alteration of microRNAs and transposon-derived small RNAs in cotton (Gossypium hirsutum) during Cotton leafroll dwarf polerovirus (CLRDV) infection

Small RNAs (sRNAs) are a class of non-coding RNAs ranging from 20- to 40-nucleotides (nts) that are present in most eukaryotic organisms. In plants, sRNAs are involved in the regulation of development, the maintenance of genome stability and the antiviral response. Viruses, however, can interfere with and exploit the silencing-based regulatory networks, causing the deregulation of sRNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs). To understand the impact of viral infection on the plant sRNA pathway, we deep sequenced the sRNAs in cotton leaves infected with Cotton leafroll dwarf virus (CLRDV), which is a member of the economically important virus family Luteoviridae. A total of 60 putative conserved cotton miRNAs were identified, including 19 new miRNA families that had not been previously described in cotton. Some of these miRNAs were clearly misregulated during viral infection, and their possible role in symptom development and disease progression is discussed. Furthermore, we found that the 24-nt heterochromatin-associated siRNAs were quantitatively and qualitatively altered in the infected plant, leading to the reactivation of at least one cotton transposable element. This is the first study to explore the global alterations of sRNAs in virus-infected cotton plants. Our results indicate that some CLRDV-induced symptoms may be correlated with the deregulation of miRNA and/or epigenetic networks.     

Characterization of a maternal type VI collagen in Xenopus embryos suggests a role for collagen in gastrulation

We characterized a novel extracellular matrix element that is present in the earliest developmental stages of Xenopus laevis, and is recognized by an mAb 3D7. Based on amino acid composition, breakdown patterns by bacterial collagenases, and the molecular weights of the components of the antigen (240, 200, and 140 kD), we found it very similar to mammalian collagen type VI. The antigen is evenly distributed in unfertilized eggs. Shortly after fertilization, it becomes localized intracellularly in the periphery of the cleaving embryo as well as in the extracellular spaces. During gastrulation, the antigen was localized in the cells lining the blastopore and in the extracellular space between the two cell layers, in the presumptive archenteron. When Fab elements of the 3D7 antibody were added to the culture medium, gastrulation was blocked, suggesting a role for the antigen in gastrulation movements.     

Spatial-temporal characteristics of intercellular junctions in early zebrafish and loach embryos before and during gastrulation

 Injections of lucifer yellow and fluorescein dyes into loach (Misgurnus fossilis) and zebrafish (Danio rerio) embryos were used to analyse the intercellular communication via gap junctions (GJs) and their role in morphogenetic processes during the period from early blastula to late gastrula. It is shown that the efficiency of dye transfer between the superficial blastomeres increases by the late blastula stage. Blastomeres of the basal layer, on the other hand, become gradually uncoupled from the yolk cell (YC). This process is spatially uneven and finishes by the late gastrula stage. Prior to it, at the early epiboly stage, a local increase in dye transfer is observed in the circular zone of the blastoderm margin. During gastrulation, GJ communication between blastomeres and the YC in this zone and also in the newly-formed germ ring region (the prospective mesoderm domain) persists for a longer period of time (up to the stage of 60–70% epiboly) than in the remaining part of the basal layer (the prospective ectoderm domain). Taking into account the data on changes in the adhesive properties of blastomeres during normal development and observations on embryos with retarded epiboly, we hypothesize that changes in GJ communication between superficial blastomeres, on one hand, and between basal blastomeres and the YC, on the other, are the consequences of the same, more general morphogenetic process of compaction occurring within the blastoderm, which supports epiboly and is probably responsible for the distinction between mesodermal and ectodermal fates of cells differently located within the forming epithelioid sheet. Received: 18 October 1996 / Accepted: 4 April 1997     

Mouse hepatoblasts at distinct developmental stages are characterized by expression of EpCAM and DLK1: Drastic change of EpCAM expression during liver development

Hepatoblasts are hepatic progenitor cells that expand and give rise to either hepatocyte or cholangiocytes during liver development. We previously reported that delta-like 1 homolog (DLK1) is expressed in the mouse liver primordium at embryonic day (E) 10.5 and that DLK1⁺ cells in E14.5 liver contain high proliferative and bipotential hepatoblasts. While the expression of epithelial cell adhesion molecule (EpCAM) in hepatic stem/progenitor cells has been reported, its expression profile at an early stage of liver development remains unknown. In this study, we show that EpCAM is expressed in mouse liver bud at E9.5 and that EpCAM⁺DLK1⁺ hepatoblasts form hepatic cords at the early stage of hepatogenesis. DLK1⁺ cells of E11.5 liver were fractionated into EpCAM⁺ and EpCAM⁻ cells; one forth of EpCAM⁺DLK1⁺ cells formed a colony in vitro whereas EpCAM⁻DLK1⁺ cells rarely did it. Moreover, EpCAM⁺DLK1⁺ cells contained cells capable of forming a large colony, indicating that EpCAM⁺DLK1⁺ cells in E11.5 liver contain early hepatoblasts with high proliferation potential. Interestingly, EpCAM expression in hepatoblasts was dramatically reduced along with liver development and the colony-forming capacities of both EpCAM⁺DLK1⁺ and EpCAM⁻DLK1⁺ cells were comparable in E14.5 liver. It strongly suggested that most of mouse hepatoblasts are losing EpCAM expression at this stage. Moreover, we provide evidence that EpCAM⁺DLK1⁺ cells in E11.5 liver contain extrahepatic bile duct cells as well as hepatoblasts, while EpCAM⁻DLK1⁺ cells contain mesothelial cell precursors. Thus, the expression of EpCAM and DLK1 suggests the developmental pathways of mouse liver progenitors.     

BMP type II receptor is required for gastrulation and early development of mouse embryos

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta superfamily, play a variety of roles during mouse development. BMP type II receptor (BMPR-II) is a type II serine/threonine kinase receptor, which transduces signals for BMPs through heteromeric complexes with type I receptors, including activin receptor-like kinase 2 (ALK2), ALK3/BMPR-IA, and ALK6/BMPR-IB. To elucidate the function of BMPR-II in mammalian development, we generated BMPR-II mutant mice by gene targeting. Homozygous mutant embryos were arrested at the egg cylinder stage and could not be recovered at 9.5 days postcoitum. Histological analysis revealed that homozygous mutant embryos failed to form organized structure and lacked mesoderm. The BMPR-II mutant embryos are morphologically very similar to the ALK3/BMPR-IA mutant embryos, suggesting that BMPR-II is important for transducing BMP signals during early mouse development. Moreover, the epiblast of the BMPR-II mutant embryo exhibited an undifferentiated character, although the expression of tissue-specific genes for the visceral endoderm was essentially normal. Our results suggest that the function of BMPR-II is essential for epiblast differentiation and mesoderm induction during early mouse development.