Whole-mount RNA Research Articles

Determination of gene expression patterns using in situ hybridization to Drosophila testes.

We describe a whole-mount RNA in situ hybridization (ISH) method optimized for detection of the cellular and subcellular distributions of specific mRNA within Drosophila testes and male genital tract. Digoxygenin (dig)-labeled antisense RNA probes are in vitro transcribed from a template synthesized by (RT)-PCR; the probe length is reduced by hydrolysis. Testes and male genital tracts are dissected from adult flies, fixed and processed for hybridization. Both probe and fixed testes can be stored before use. Extensive post-hybridization washing reduces the background. Detection is through alkaline phosphatase-conjugated anti-dig antibodies followed by a color reaction. This protocol is suitable for low-medium throughput applications with parallel processing of 2-48 samples, and takes 4-5 d to complete. We have used this protocol, which is similar to other RNA ISH protocols, but optimized for whole-mount Drosophila testes, to document the expression of about 1,000 genes in Drosophila melanogaster male genital tract.

Znrg, a novel gene expressed mainly in the developing notochord of zebrafish

The notochord, a defining characteristic of the chordate embryo is a critical midline structure required for axial skeletal formation in vertebrates, and acts as a signaling center throughout embryonic development. We utilized the digital differential display program of the National Center for Biotechnology Information, and identified a contig of expressed sequence tags (no. Dr. 83747) from the zebrafish ovary library in Genbank. Full-length cDNA of the identified gene was cloned by 5'- and 3'- RACE, and the resulting sequence was confirmed by polymerase chain reaction and sequencing. The cDNA clone contains 2,505 base pairs and encodes a novel protein of 707 amino acids that shares no significant homology with any known proteins. This gene was expressed in mature oocytes and at the one-cell stage, and persisted until the 5th day of development, as determined by RT-PCR. Transcripts were detected by whole-mount RNA in situ hybridization from the two-cell stage to 72 h of embryonic development. This gene was uniformly distributed from the cleavage stage up to the blastula stage. During early gastrulation, it was present in the dorsal region, and became restricted to the notochord and pectoral fin at 48 and 72 h of embryonic development. Based on its abundance in the notochord, we hypothesized that the novel gene may play an important role in notochord development in zebrafish; we named this gene, zebrafish notochord-related gene, or znrg.

Androgen receptor gene expression in the developing and adult zebrafish brain

Androgens play a central role in the regulation of male sexual differentiation and behavior in many vertebrates, including zebrafish. Their signaling is mediated by activation of the androgen receptor. A single androgen receptor (ar) gene was recently identified in zebrafish, which encodes a protein that binds androgens in vitro. However, the tissue-specific expression pattern of this receptor in vivo has not been described. Using whole-mount RNA in situ hybridization, we characterized expression of the ar gene in developing zebrafish and in the adult brain. In embryos, transcripts were found in the presumptive pronephros and in olfactory placodes. By 3-5 days postfertilization, ar transcripts were also detected in the pineal organ anlage and the retina. In the adult brain, ar was expressed in discrete regions of the telencephalon, in the preoptic area, and throughout the periventricular hypothalamus, regions previously implicated in the regulation of sexually dimorphic behaviors in mammals.

Whole Mount RNA In Situ Hybridization on Zebrafish Embryos: Probe Synthesis

INTRODUCTIONThis protocol describes methods for synthesizing digoxigenin- and fluorescein-labeled RNA probes from linearized plasmids or PCR products for use in whole-mount RNA in situ hybridization on zebrafish embryos.

Cloning and characterization of the sympathetic nervous system mutant, Sym3, in zebrafish

9541 Background: Neuroblastoma (NB) is a neural crest tumor manifesting in the adrenal gland and the sympathetic ganglia. The goal of this project was to use the zebrafish model to identify genes involved in the development of the peripheral sympathetic nervous system (PSNS). We used the expression of tyrosine hydroxylase (TH) in the cervical sympathetic ganglion (cervical complex, CC), as a marker for PSNS development. TH expression can be observed by whole-mount RNA in situ hybridization in 2–3-day zebrafish embryos in the CC. Methods: In an ethyl nitrosourea mutagenesis screen for mutations that affect PSNS development, we identified a putative mutant, Sym3, showing a change in modeling of the TH staining cells in the region of the CC. Results and Conclusion: The TH-positive cells were scattered rather than conglomerated together as is typical of cells in the normal CC. The mutant was also characterized by a “curly-tail” phenotype. After the mutation was recovered from the F3 generation, heterozygous pairs harboring the mutation were identified. Using genome wide scanning PCR assay, linkage of the Sym3 mutation to microsatellite markers z9704 and z1351, located on Linkage Group 1, was established. Complementation assays were performed with other known “curly tail” mutants in the genetic interval, which revealed that Sym3 and a previously described vic mutant were in the same complementation group. The vic mutant is characterized by curved body axis, left to right asymmetry and kidney cysts and results from a mutation in the ARL13b gene, a protein of the Ras superfamily involved in intracellular trafficking and cilial motility. The vic mutation is a C to A transition at position 104 in the second exon of the open reading frame (T35L). The ARL13b gene in Sym3 has a four aa insertion at the 5’ end of the 4th exon of the coding region.The mutant Sym3 phenotype can be rescued by overexpression of the normal ARL13b gene. The vic mutant showed defective cilia formation in the kidney. However, immunocytochemistry showed normal cilia in the Sym3 mutant, suggesting that the Sym3 allele of ARL13b affects the development of alternative tissues during development, such as the aggregation of sympathetic neurons into discrete ganglia. Further functional characterization of the Sym3 mutation is ongoing. No significant financial relationships to disclose.

NXT2 is required for embryonic heart development in zebrafish

BackgroundNXT2 is a member of NXT family proteins that are generally involved in exporting nuclear RNA in eukaryotic cells. It is not known if NXT2 has any function in specific biological processes.ResultsA zebrafish mutant exhibiting specific heart defects during embryogenesis was generated by animal cloning-mediated retroviral insertions. Molecular analysis indicated that the mutant phenotype was caused by a disruption of NXT2. Whole-mount RNA in situ hybridization showed that NXT2 transcripts were clearly detectable in embryonic heart as well as other tissues. Further analysis revealed that expression level of one form of alternative splicing NXT2 mRNA transcripts was significantly reduced, resulting in deficient myocardial cell differentiation and the malformation of cardiac valve at the atrioventricular boundary. The defects could be reproduced by morpholino anti-sense oligo knockdown of NXT2.ConclusionNXT2 has a critical role in maintaining morphogenetic integrity of embryonic heart in vertebrate species.

Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo.

We have used the Tol2 transposable element to design and perform effective enhancer trapping in zebrafish. Modified transposon DNA and transposase RNA were delivered into zebrafish embryos by microinjection to produce heritable insertions in the zebrafish genome. The enhancer trap construct carries the EGFP gene controlled by a partial epithelial promoter from the keratin8 gene. Enhanced green fluorescent protein (EGFP) is used as a marker to select F1 transgenic fish and as a reporter to trap enhancers. We have isolated 28 transgenic lines that were derived from the 37 GFP-positive F0 founders and displayed various specific EGFP expression patterns in addition to basal expression from the modified keratin 8 promoter. Analyses of expression by whole-mount RNA in situ hybridization demonstrated that these patterns could recapitulate the expression of the tagged genes to a variable extent and, therefore, confirmed that our construct worked effectively as an enhancer trap. Transgenic offspring from the 37 F0 EGFP-positive founders have been genetically analyzed up to the F2 generation. Flanking sequences from 65 separate transposon insertion sites were identified by thermal asymmetric interlaced polymerase chain reaction. Injection of the transposase RNA into transgenic embryos induced remobilization of genomic Tol2 copies producing novel insertions including some in the germ line. The approach has great potential for developmental and anatomical studies of teleosts.

The expression of the imprinted gene Ipl is restricted to extra-embryonic tissues and embryonic lateral mesoderm during early mouse development.

Genes with restricted expression within the developing embryo represent valuable tools as they allow distinct tissue types to be distinguished and studied. In order to identify genes that are expressed within a particular germ layer, a differential screen was performed using germ layer-specific cDNA libraries derived from gastrulation stage mouse embryos. The gene expression profiles of the germ layers were compared following the hybridisation of some 20,000 cDNA clones with probes derived from germ layer-specific Ectoderm, Mesoderm and Endoderm libraries. A cDNA clone (50c15) was identified that hybridised with the Mesoderm-derived probe but not Ectoderm or Endoderm. 50c15 derives from Ipl/Tssc3/BWR1C, an imprinted gene which in human maps to chromosome 11p15.5. This region has been associated with Beckwith-Weidemann Syndrome, Wilms' tumour and ovarian, breast and lung cancer. In the gastrulating mouse embryo, wholemount RNA in situ hybridisation revealed that Ipl expression is restricted not only to the mesodermal germ layer, but specifically to lateral mesoderm and the most posterior extent of the primitive streak from which lateral and extra-embryonic mesoderm is derived. Moreover, Ipl is expressed in extra-embryonic tissues prior to gastrulation and afterwards in extra-embryonic mesoderm, ectoderm and endoderm. This expression profile indicates that Ipl is a good molecular marker for embryonic mesoderm and extra-embryonic tissues. In addition heterotopic grafting studies indicate that nascent mesoderm, which expresses Ipl, is restricted in its potential and therefore may be committed to its fate.

The MADS box gene ZmMADS2 is specifically expressed in maize pollen and during maize pollen tube growth

We screened a cDNA library of mature maize pollen for clones encoding transcription factors. Two novel cDNAs (ZmMADS1 and ZmMADS2) encoding MADS box proteins were isolated. To our knowledge, ZmMADS1 and ZmMADS2 represent the first transcription factors isolated from pollen of a monocot plant species. ZmMADS1 can be classified as a member of the TM3 subfamily of MADS box proteins whereas ZmMADS2, which is described here in more detail, belongs to the AGL17 subfamily. Temporal and spatial expression analyses showed that ZmMADS2 expression is restricted to pollen and roots. During pollen development ZmMADS2 is detectable at all stages of pollen development analyzed by RT-PCR but is most abundant in mature pollen after dehiscence. The establishment of a whole-mount RNA in situ hybridization protocol with in-vitro germinated pollen enabled the localization of ZmMADS2 transcripts in pollen tubes. ZmMADS2 transcripts are translocated into pollen tubes immediately upon germination and display a gradient with increasing concentration towards the tube tip. The vegetative nucleus and the sperm cells, putative targets of ZmMADS2, enter the pollen tube about 2 h later.

Expression and imprinting of MAGEL2 suggest a role in Prader-willi syndrome and the homologous murine imprinting phenotype.

Prader-Willi syndrome (PWS) is caused by the loss of expression of imprinted genes in chromosome 15q11-q13. Affected individuals exhibit neonatal hypotonia, developmental delay and childhood-onset obesity. Necdin, a protein implicated in the terminal differentiation of neurons, is the only PWS candidate gene to reduce viability when disrupted in a mouse model. In this study, we have characterized MAGEL2 (also known as NDNL1), a gene with 51% amino acid sequence similarity to necdin and located 41 kb distal to NDN in the PWS deletion region. MAGEL2 is expressed predominantly in brain, the primary tissue affected in PWS and in several fetal tissues as shown by northern blot analysis. MAGEL2 is imprinted with monoallelic expression in control brain, and paternal-only expression in the central nervous system as demonstrated by its lack of expression in brain from a PWS-affected individual. The orthologous mouse gene (Magel2) is located within 150 kb of NDN:, is imprinted with paternal-only expression and is expressed predominantly in late developmental stages and adult brain as shown by northern blotting, RT-PCR and whole-mount RNA in situ hybridization. Magel2 distribution partially overlaps that of NDN:, with strong expression being detected in the central nervous system in mid-gestation mouse embryos by in situ hybridization. We hypothesize that, although loss of necdin expression may be important in the neonatal presentation of PWS, loss of MAGEL2 may be critical to abnormalities in brain development and dysmorphic features in individuals with PWS.

Overexpression of mSim2 gene in the zona limitans of the diencephalon of segmental trisomy 16 Ts1Cje fetuses, a mouse model for trisomy 21: a novel whole-mount based RNA hybridization study

Trisomy 21 (Down syndrome) is the most common chromosomal abnormality associated with mental retardation in humans. Sim2, a human homologue of Drosophila sim gene, which acts as a master regulator of the early development of the fly central nervous system midline, is located on chromosome 21, in the Down syndrome critical region, and might therefore be involved in the pathogenesis of some of the morphological features and brain anomalies observed in Down syndrome. We report here the detailed expression pattern of murine mSim2 gene in Ts1Cje mice fetuses, a segmental trisomy 16 mouse model for trisomy 21, and its overexpression in the zona limitans of the diencephalon using a new quantitative method based on the whole-mount RNA hybridization technique.

Characterization and developmentally regulated expression of four annexins in the killifish medaka.

Annexins are Ca2+-regulated membrane binding proteins implicated in a wide range of membrane-related and signal transduction events, including the endocytosis of membrane receptors and Ca2+-regulated as well as constitutive secretion. To date, 10 unique members of this multigene family have been identified in a variety of cell types and tissues of higher vertebrates, with different members showing distinct tissue distributions in the adult organisms. To establish whether annexins also function in embryonic development, we analyzed the expression pattern during vertebrate morphogenesis using the medaka fish Oryzias latipes as a model system. From a larval medaka cDNA library, we isolated four types of clones, which were shown by sequence analysis to encode four different annexins (herein referred to as max 1-4). A comparison with known annexin sequences in the databases revealed that two medaka annexins (max 1 and 2) are highly similar in sequence to mammalian annexins V and IV, respectively, whereas the other two medaka annexins (max 3 and 4) are probably novel members of the family most closely related to mammalian annexins I and XI. Using whole-mount RNA in situ hybridization, we showed that the expression of the different medaka annexins during embryogenesis was strictly regulated at both the spatial and the temporal level. High levels of max 1, 2, and 3 transcripts were present in the developing stomach, gut, liver, air-bladder, and rectum during somitogenesis, thus identifying the digestive tract as the prime region of annexin expression. Interestingly, two structures playing crucial roles in neuronal patterning showed a distinct expression of annexins. The mesendoderm of the anterior prechordal plate of neurula-stage embryos was a site of max 4 transcription, and the floor plate of somitogenesis-stage embryos showed expression of max 2 and 3 to differing rostrocaudal extends along the brain and spinal cord. These results suggest specific functions of different annexins during vertebrate morphogenesis.

Fork head domain genes in zebrafish.

Nine members of the fork head domain gene family (fkd1-fkd9) were isolated from early cDNA libraries in the zebrafish. They show unique expression patterns in whole-mount RNA in situ hybridization during the first 24 h of embryonic development. These fkd genes fall into three of ten classes, based on sequence similarities within the DNA-binding domain, whereas members for the other seven classes described in other vertebrates were not found. In addition to conserved residues at certain positions in the fork head domain, characteristic transcription activation domains as well as similarities in expression patterns were found for members of the different classes. Members of class I (fkd1/axial, fkd2/Zffkh1, fkd4 and fkd7) are differentially transcribed in unsegmented dorsal axial structures such as the floor plate, the notochord, the hypochord and, in addition, the endoderm. Transcripts of fkd3 and fkd5 (class II) are mainly detected in the cells of the ectoderm which form neural tissues, as is the case for genes of this class in other species. RNAs of the three members of class V (fkd6, fkd8 and fkd9) are expressed in the paraxial mesoderm and transiently in the neuroectoderm. fkd6 is strongly expressed in neural crest cells from early stages on, whereas fkd2 and fkd7 are transcribed in individual neural crest cells in the pharyngula period.

Hybridizationin situ of whole-mount messenger RNA in plants

A procedure is presented for the detection of mRNA in whole-mount preparations of youngArabidopsis seedlings using digoxigenin (DIG)-labeled RNA probes. It includes tissue preservation with formaldehyde, permeabilization with polyoxyethy lenesorbitan (Tween 20), DMSO, and heptane. Hybridization signal is detected using colloidal gold or alkaline-phosphatase-conjugated anti-DIG antibodies.