Identification of a distant cis‐regulatory element controlling pharyngeal arch‐specific expression of zebrafish gdf6a/radar

Skeletal formation is an essential and intricately regulated part of vertebrate development. Humans and mice deficient in growth and differentiation factor 6 (Gdf6) have numerous skeletal abnormalities, including joint fusions and cartilage reductions. The expression of Gdf6 is dynamic and in part regulated by distant evolutionarily conserved cis‐regulatory elements. radar/gdf6a is a zebrafish ortholog of Gdf6 and has an essential role in embryonic patterning. Here, we show that radar is transcribed in the cells surrounding and between the developing cartilages of the ventral pharyngeal arches, similar to mouse Gdf6. A 312 bp evolutionarily conserved region (ECR5), 122 kilobases downstream, drives expression in a pharyngeal arch‐specific manner similar to endogenous radar/gdf6a. Deletion analysis identified a 78 bp region within ECR5 that is essential for transgene activity. This work illustrates that radar is regulated in the pharyngeal arches by a distant conserved element and suggests radar has similar functions in skeletal development in fish and mammals. Developmental Dynamics 239:1047–1060, 2010. © 2010 Wiley‐Liss, Inc.     

Expression profiling of the mouse early embryo: reflections and perspectives.

The laboratory mouse model plays important roles in our understanding of early mammalian development and provides an invaluable model for human early embryos, which are difficult to study for ethical and technical reasons. A comprehensive collection of cDNA clones, their sequences, and complete genome sequence information, which have been accumulated over the past two decades, reveal even further the value of the mouse models. Here, the progress in global gene expression profiling in early mouse embryos and, to some extent, stem cells is reviewed and future directions and challenges are discussed. The discussions include the restatement of global gene expression profiles as a snapshot of cellular status, and subsequent distinction between the differentiation state and physiological state of the cells. The discussions then extend to the biological problems that can be addressed only through global expression profiling, including a bird's-eye view of global gene expression changes, molecular index for developmental potency, cell lineage trajectory, microarray-guided cell manipulation, and the possibility of delineating gene regulatory cascades and networks.     

Mutants in a conserved region near the carboxy-terminus of HIV-1 Rev identify functionally important residues and exhibit a dominant negative phenotype

The rev protein (Rev) of human immunodeficiency virus increases the cytoplasmic expression of viral structural gene mRNAs. We had previously reported the existence of a region (residues 73-98) near the carboxy-terminus in HIV-1 Rev essential for its function. To further define the structural elements in this region, we examined the effects of substitution mutations in highly conserved residues in this region, between amino acids 75-81, on Rev function. Mutations in Pro76-77 and Arg80 retained Rev function, whereas those in Leu75 and Leu81 abolished Rev activity and exhibited trans-dominant suppression of wt Rev function. The Leu81 mutation, in particular, exhibited an efficient dominant negative phenotype. Leu75 and Leu81 thus appear to define residues essential to the Rev "effector" function.     

Expression microarray and mouse virulence analysis of four conserved two-component gene regulatory systems in group a streptococcus

Group A streptococcus (GAS) is a gram-positive human bacterial pathogen that causes diseases ranging from relatively mild epithelial cell surface infections to life-threatening invasive episodes. Much is known about the extracellular molecules that contribute to host-pathogen interactions, but in contrast, far less information is available about regulatory genes that control the expression of individual or multiple GAS virulence factors. The eight GAS genomes that have been sequenced have 12 conserved two-component gene regulatory systems (TCSs), but only 3 of these 12 have been studied in detail. Using an allelic replacement strategy with a nonpolar cassette, we inactivated the response regulator of four TCSs that have only weak homology with TCS genes of known or inferred function in other bacteria. The mutant strains were analyzed by expression microarray analysis at four time points and tested in two mouse infection models. Each TCS influenced expression (directly or indirectly) of 12 to 41% of all chromosomal genes, as assessed by growth in Todd-Hewitt broth and a custom Affymetrix GeneChip. None of the isogenic mutant strains was significantly altered for mouse virulence based on intraperitoneal inoculation. Similarly, compared to the wild-type strain, there was no significant difference in skin lesion size for three of the four mutants. In contrast, the DeltaM5005_Spy_0680 mutant strain produced significantly larger abscesses after subcutaneous inoculation into mice, consistent with a hypervirulence phenotype. The mutant strain had significantly higher in vitro expression of several proven and putative virulence genes, including scpA, encoding a peptidase that inactivates complement protein C5a. Together, the data provide new information about previously uncharacterized GAS TCSs.     

Organization and expression analysis of the zebrafish hepcidin gene, an antimicrobial peptide gene conserved among vertebrates

Hepcidin is an antimicrobial peptide and iron-regulatory molecule that is conserved among vertebrates. Mutations or over-expression of the human hepcidin gene have been found in patients with hemochromatosis and refractory anemia. To further understand the function and regulation of hepcidin, animal models are needed. We sequenced cDNA, genes and upstream regions of zebrafish hepcidin and analyzed gene expression by kinetic PCR. Zebrafish hepcidin genes consist of two introns and three exons that encode a prepropeptide (91 amino acids). The amino acid sequences and gene organization were remarkably conserved between zebrafish and other species. Elevated gene expression was observed in abdominal organs, skin, and heart in fish that developed signs of infection following bacterial injection. Zebrafish may be a suitable model organism for further study of hepcidin gene regulation.     

Development and maturation of the immune system in zebrafish,Danio rerio: a gene expression profiling,in situ hybridization and immunological study

The development and maturation of the immune system in zebrafish was investigated using immune-related gene expression profiling by quantitative real-time polymerase chain reaction, in situ hybridization (ISH), immunoglobulin (Ig) detection by immuno-affinity purification and Western blotting as well as immersion immunization experiments. Ikaros expression was first detected at 1 day post-fertilization (dpf) and thereafter increased gradually to more than two-fold between 28 and 42dpf before decreasing to less than the initial 1dpf expression level in adult fish (aged 105dpf). Recombination activating gene-1 (Rag-1) expression levels increased rapidly (by 10-fold) between 3 and 17dpf, reaching a maximum between 21 and 28dpf before decreasing gradually. However, in adult fish aged 105dpf, the expression level of Rag-1 had dropped markedly, and was equivalent to the expression level at 3dpf. T-cell receptor alpha constant region and immunoglobulin light chain constant region (IgLC) isotype-1, 2 and 3 mRNAs were detected at low levels by 3dpf and their expression levels increased steadily to the adult range between 4 and 6 weeks post-fertilization (wpf). Using tissue-section ISH, Rag-1 expression was detected in head kidney by 2wpf while IgLC-1, 2 and 3 were detected in the head kidney and the thymus by 3wpf onwards. Secreted Ig was only detectable using immuno-affinity purification and Western blotting by 4wpf. Humoral response to T-independent antigen (formalin-killed Aeromonas hydrophila) and T-dependent antigen (human gamma globulin) was observed in zebrafish immunized at 4 and 6wpf, respectively, indicating that immunocompetence was achieved. The findings reveal that the zebrafish immune system is morphologically and functionally mature by 4-6wpf.     

Building comparative gene expression databases for the mouse preimplantation embryo using a pipeline approach to UniGene

To understand early mammalian development there is a need to compare profiles of gene expression from different stages of the preimplantation mouse embryo. We describe here a method that uses gene expression data held in the UniGene database of the National Institutes of Health (NIH). The full mouse UniGene database (build #151) contains 43,104 gene clusters generated from approximately 4.1 million sequences. The Expressed Sequence Tags (EST) used to build UniGene are derived from cDNA libraries that are archived separately in the database of Expressed Sequence Tags (dbEST) database, with their own catalogue numbers. The mouse dbEST database contains 32 non-normalized dbEST libraries constructed from preimplantation stages (unfertilized oocyte, fertilized oocyte, 2-, 4-, 8- and 16-cell embryo and blastocyst). These libraries contain 219,852 EST sequences mapping to 15,731 UniGene clusters. We have developed a computational pipeline approach that imports and aggregates inventories of gene expression contained in these dbEST libraries. It uses these data to build an annotated web-based database of preimplantation gene expression with an in-built capacity for comparison of expression profiles. Comparison of gene expression profiles obtained for each developmental stage show statistically significant changes in gene expression during preimplantation development. These in silico-generated profiles were validated using RT-PCR.     

Genetic polymorphism of immunoglobulin G in the mink. VI. A regulatory gene controlling the expression of the gamma-chain constant region allotype of mink immunoglobulin

We describe here the inheritance of H6, one of the six known allotypes of the gamma-chain constant region of mink immunoglobulin (IgG). H6 is not present in minor concentrations in the serum, and its phenotypic expression is stable. However, in offspring of some of (H6-)X(H6-) crosses. H6 appeared unexpectedly and, by contrast, it disappeared in some H6+/H6+ homozygote offspring. Based on pedigree analysis, a transregulation of H6 expression in the serum by an autosomal recessive gene not linked to the structural allotype gene is postulated.     

Identification of a hypervariable region containing new Legionella pneumophila Icm/Dot translocated substrates by using the conserved icmQ regulatory signature

Legionella pneumophila is an intracellular pathogen that has been shown to utilize the Icm/Dot type IV secretion system for pathogenesis. This system was shown to be composed of Icm/Dot complex components, accessory proteins, and a large number of translocated substrates. In this study, comparison of the icmQ regulatory regions from many Legionella species revealed a conserved regulatory sequence that includes the icmQ −10 promoter element. Mutagenesis of this conserved regulatory element indicated that each of the nucleotides in it affects the level of expression of the icmQ gene but not in a uniform fashion. A genomic analysis discovered that four additional genes in L. pneumophila contain this conserved regulatory sequence, which was found to function similarly in these genes as well. Examination of these four genes indicated that they are dispensable for intracellular growth, but two of them were found to encode new Icm/Dot translocated substrates (IDTS). Comparison of the genomic regions encoding these two IDTS among the four available L. pneumophila genomic sequences indicated that one of these genes is located in a hypervariable genomic region, which was shown before to contain an IDTS-encoding gene. Translocation analysis that was performed for nine proteins encoded from this hypervariable genomic region indicated that six of them are new IDTS which are translocated into host cells in an Icm/Dot-dependent manner. Furthermore, a bioinformatic analysis indicated that additional L. pneumophila genomic regions that contain several neighboring IDTS-encoding genes are hypervariable in gene content.     

Expression of zTlxA, a Hox11-like gene, in early differentiating embryonic neurons and cranial sensory ganglia of the zebrafish embryo.

We have isolated a cDNA encoding a member of the Tlx/Hox11 family of homeodomain factors from the zebrafish, most closely related to the vertebrate Tlx-1/Hox11 and Tlx-3/Hox11L2 proteins. The gene is expressed in a set of early differentiating neurons that project to a common tract, the lateral longitudinal fascicle. We show that the gene is specifically expressed in spinal cord Rohon Beard neurons, in nucleus of the posterior commissure neurons of the midbrain, in a set of hindbrain neurons that include RoL3 reticulospinal interneurons, and in the trigeminal, statoacoustic, anterior lateral line, glossopharyngeal, and vagal cranial sensory ganglia. Timing of expression of the gene in these neurons correlates with the phase of axonal outgrowth and target innervation. Expression of the gene is also observed in several non-neural tissues, including the pharyngeal arches, budding gill filaments, outgrowing semicircular protrusions in the otic vesicle, and in the pectoral fin buds.