Prediction and characterisation of a highly conserved, remote and cAMP responsive enhancer that regulates Msx1 gene expression in cardiac neural crest and outflow tract

Double knockouts of the Msx1 and Msx2 genes in the mouse result in severe cardiac outflow tract malformations similar to those frequently found in newborn infants. Despite the known role of the Msx genes in cardiac formation little is known of the regulatory systems (ligand receptor, signal transduction and protein-DNA interactions) that regulate the tissue-specific expression of the Msx genes in mammals during the formation of the outflow tract. In the present study we have used a combination of multi-species comparative genomics, mouse transgenic analysis and in-situ hybridisation to predict and validate the existence of a remote ultra-conserved enhancer that supports the expression of the Msx1 gene in migrating mouse cardiac neural crest and the outflow tract primordia. Furthermore, culturing of embryonic explants derived from transgenic lines with agonists of the PKC and PKA signal transduction systems demonstrates that this remote enhancer is influenced by PKA but not PKC dependent gene regulatory systems. These studies demonstrate the efficacy of combining comparative genomics and transgenic analyses and provide a platform for the study of the possible roles of Msx gene mis-regulation in the aetiology of congenital heart malformation.     

Initiation of olfactory placode development and neurogenesis is blocked in mice lacking both Six1 and Six4

Mouse olfactory epithelium (OE) originates from ectodermally derived placode, the olfactory placode that arises at the anterior end of the neural plate. Tissue grafting and recombination experiments suggest that the placode is derived from a common preplacodal domain around the neural plate and its development is directed by signals arising from the underlying mesoderm and adjacent neuroectoderm. In mice, loss of Six1 affects OE morphogenesis but not placode formation. We show here that embryos lacking both Six1 and Six4 failed to form the olfactory placode but the preplacodal region appeared to be specified as judged by the expression of Eya2, which marks the common preplacodal domain, suggesting a synergistic requirement of Six1 and Six4 in patterning the preplacodal ectoderm to a morphologic placode. Our results show that Six1 and Six4 are coexpressed in the preplacodal ectoderm from E8.0. In the olfactory pit, Six4 expression was observed in the peripheral precursors that overlap with Mash1-expressing cells, the early committed neuronal lineage. In contrast, Six1 is highly distributed in the peripheral regions where stem cells reside at E10.5 and it overlaps with Sox2 expression. Both genes are expressed in the basal and apical neuronal progenitors in the OE. Analyses of Six1;Six4 double mutant embryos demonstrated that the slightly thickened epithelium observed in the mutant was not induced for neuronal development. In contrast, in Six1^−/− embryos, all neuronal lineage markers were initially expressed but the pattern of their expression was altered. Although very few, the pioneer neurons were initially present in the Six1 mutant OE. However, neurogenesis ceased by E12.5 due to markedly increased cell apoptosis and reduced proliferation, thus defining the cellular defects occurring in Six1^−/− OE that have not been previously observed. Our findings demonstrate that Six1/4 function at the top of early events controlling olfactory placode formation and neuronal development. Our analyses show that the threshold of Six1/4 may be crucial for the expression of olfactory specific genes and that Six1 and Six4 may act synergistically to mediate olfactory placode specification and patterning through Fgf and Bmp signaling pathways.     

Direct activation of a mouse Hoxd11 axial expression enhancer by Gdf11/Smad signalling

A Hoxd11/lacZ reporter, expressed with a Hoxd11-like axial expression pattern in transgenic mouse embryos, is stimulated in tailbud fragments when cultured in presence of Gdf11, a TGF-β growth/differentiation factor. The same construct is also stimulated by Gdf11 when transiently transfected into cultures of HepG2 cells. Stimulation of the reporter in HepG2 cells is enhanced where it contains only the 332 bp Hoxd11 enhancer region VIII upstream or downstream of a luciferase or lacZ reporter. This enhancer contains three elements conserved from fish to mice, one of which has the sequence of a Smad3/4 binding element. Mutation of this motif inhibits the ability of Gdf11 to enhance reporter activity in the HepG2 cell assay. Chromatin immunoprecipitation experiments show direct evidence of Smad2/3 protein binding to the Hoxd11 region VIII enhancer. The action of Gdf11 upon Hoxd11 in HepG2 cells is inhibited, at least in part, by SIS3, a specific inhibitor of Smad3. SIS3 also produces partial inhibition of Hoxd11/lacZ expression in cultured transgenic tailbuds, indicating that Smad3 may play a similar role in the embryonic expression of Hoxd11. Transgenic mouse experiments show that the Smad binding motif is essential for the axial expression of Hoxd11/lacZ reporter in the embryo tailbud, posterior mesoderm and neurectoderm.     

Novel enhancer and promoter elements indispensable for the tissue-specific expression of the sericin-1 gene of the silkworm Bombyx mori

Sericins are glue proteins produced specifically in the middle silk gland (MSG) of the silkworm Bombyx mori, while the silk fiber protein, fibroin, is produced in the posterior silk gland (PSG). These silk proteins are expected to be useful biomaterials in medical technology as well as biotechnology. In this study, we analyzed promoter elements of the sericin-1 gene (ser1) in vivo by introducing reporter constructs into silk glands via gene gun technology. The region from −1602 to +47 was sufficient to induce MSG-specific expression. The 5′ deletion mutants showed a three-step decrease in promoter activity with the key sequences located between −1362 and −1250, −201 and −116, and −115 and −37. We detected a tissue- and stage-specific factor complex (MSG-intermolt-specific complex: MIC) bound to the sequence elements around the −1350, −320, −180, and −70 regions. A mutation in the −70 region, which inhibits MIC-binding, diminished almost all promoter activity, while another mutation that did not inhibit MIC-binding showed no effect on promoter activity. The results suggest that the binding of MIC to the above elements is intrinsic for the spatiotemporal specificity of ser1 in vivo.     

Comparative genomic analysis of Lactobacillus mucosae LM1 identifies potential niche-specific genes and pathways for gastrointestinal adaptation

Lactobacillus mucosae is currently of interest as putative probiotics due to their metabolic capabilities and ability to colonize host mucosal niches. L. mucosae LM1 has been studied in its functions in cell adhesion and pathogen inhibition, etc. It demonstrated unique abilities to use energy from carbohydrate and non-carbohydrate sources. Due to these functions, we report the first complete genome sequence of an L. mucosae strain, L. mucosae LM1. Analysis of the pan-genome in comparison with closely-related Lactobacillus species identified a complete glycogen metabolism pathway, as well as folate biosynthesis, complementing previous proteomic data on the LM1 strain. It also revealed common and unique niche-adaptation genes among the various L. mucosae strains. The aim of this study was to derive genomic information that would reveal the probable mechanisms underlying the probiotic effect of L. mucosae LM1, and provide a better understanding of the nature of L. mucosae sp.     

Key steps in the morphogenesis of a cranial placode in an invertebrate chordate, the tunicate Ciona savignyi

Tunicates and vertebrates share a common ancestor that possessed cranial neurogenic placodes, thickenings in embryonic head epidermis giving rise to sensory structures. Though orthology assignments between vertebrate and tunicate placodes are not entirely resolved, vertebrate otic placodes and tunicate atrial siphon primordia are thought to be homologous based on morphology and position, gene expression, and a common signaling requirement during induction. Here, we probe key points in the morphogenesis of the tunicate atrial siphon. We show that the siphon primordium arises within a non-dividing field of lateral-dorsal epidermis. The initial steps of atrial primordium invagination are similar to otic placode invagination, but a placode-derived vesicle is never observed as for the otic vesicle of vertebrates. Rather, confocal imaging reveals an atrial opening through juvenile stages and beyond. We inject a photoactivatable lineage tracer to show that the early atrial siphon of the metamorphic juvenile, including its aperture and lining, derives from cells of the atrial placode itself. Finally, we perturb the routing of the gut to the left atrium by laser ablation and pharmacology to show that this adaptation to a sessile lifestyle depends on left-right patterning mechanisms present in the free-swimming chordate ancestor.     

The IE2 promoter/enhancer region from mouse CMV provides high levels of therapeutic protein expression in mammalian cells

Protein expression in mammalian cells is key for the production and manufacturing of bio-therapeutics with human-like properties and activities. As a molecular basis for reaching high protein expression levels, efficient promoter/enhancer systems are a prerequisite. Here we identify a novel enhancer from the mouse cytomegalovirus (CMV) immediate early 2 (IE2) region as a strong expression-promoting element. We further demonstrate its activity in bi-directional promoter architecture and apply it to generate production clones for IL-18BP, a protein with therapeutic indications in autoimmune diseases. These data show that the IE region from mouse CMV, and the IE2 enhancer/promoter in particular, have a broad potential for application in novel gene expression systems for research, development, and manufacturing of protein drugs.     

Chemiluminescent determination of H2O2 using 4-(1,2,4-triazol-1-yl)phenol as an enhancer based on the immobilization of horseradish peroxidase onto magnetic beads

This article describes the employment of a novel p-phenol derivative, 4-(1,2,4-triazol-1-yl)phenol (TRP), as a highly potent signal enhancer of the luminol-hydrogen peroxide (H₂O₂)-horseradish peroxidase (HRP) chemiluminescence (CL) system. The CL reaction conditions were optimized, and the enhancement characteristics of TRP were compared with those of p-iodophenol (PIP). TRP produced a strong enhancement of the CL with the effect of prolonging the light emission. The developed system was then applied to the determination of H₂O₂ with immobilized HRP using magnetic beads as a solid support. The linear range for H₂O₂ was 2.0 × 10⁻⁶ to 1.0 × 10⁻³ M. The detection limit for H₂O₂ was 2.0 × 10⁻⁶ M. The proposed sensor was applied successfully to the determination of H₂O₂ in rainwater.     

Comparative analysis of methylthioalkylmalate synthase (MAM) gene family and flanking DNA sequences in Brassica oleracea and Arabidopsis thaliana

Gene BoGSL-PRO is associated with presence of 3-carbon side-chain glucosinolates (GSL). This gene is a member of the methylthioalkylmalate synthase (MAM) gene family. A BAC clone of Brassica oleracea, B21F5, containing this gene, was sequenced, annotated and compared to its corresponding region in Arabidopsis thaliana. Twelve protein-coding genes and 10 transposable elements were found in this clone. The corresponding region in A. thaliana chromosome I has 14 genes and no transposable elements. Analysis of MAM gene family in both species, which also include genes controlling 4-carbon side-chain GSL, separated the genes in two groups based on exon numbers and function. Phylogenetic analysis of the amino acid sequences encoded by these genes suggest that these two groups were produced by a duplication that must have occurred before the divergence of the Rosid and Asterid lineages of angiosperms. Comparison with putative orthologs from several prokaryotes further suggest that the members of the gene family with 10 exons, which encode proteins involved in 4-carbon side-chain GSL biosynthesis, were derived via truncation of the 3′ end from ancestral genes more similar in length to those with 12 exons, which encode proteins involved in 3-carbon side-chain GSL biosynthesis. Lower gene density in B. oleracea compared to A. thaliana is due in part to presence of transposable elements (TE) mostly in inter-genic regions.     

Identification of presomitic mesoderm (PSM)-specific Mesp1 enhancer and generation of a PSM-specific Mesp1/Mesp2-null mouse using BAC-based rescue technology

Bacterial artificial chromosome (BAC) modification technology is a powerful method for the identification of enhancer sequences and genetic modifications. Using this method, we have analyzed the Mesp1 and/or Mesp2 enhancers and identified P1-PSME, a PSM-specific enhancer of Mesp1, which contains a T-box binding site similar to the previously identified P2-PSME. Hence, Mesp1 and Mesp2 use different enhancers for their PSM-specific expression. In addition, we find that these two genes also use distinct enhancers for their early mesodermal expression. Based on these results, we generated a PSM-specific Mesp1/Mesp2-null mouse by introducing a BAC clone, from which only early mesodermal Mesp1 expression is possible, into the Mesp1/Mesp2 double knockout (dKO) genetic background. This successfully rescued gastrulation defects due to the lack of the early mesoderm in the dKO mouse and we thereby obtained a PSM-specific Mesp1/Mesp2-null mouse showing a lack of segmented somites.     

P21(Cip1/WAF1) expression in the mouse testis before and after X irradiation

During spermatogenesis, the radiosensitivity of testicular cells changes considerably. To investigate the molecular mechanism underlying these radiosensitivity differences, p21^(Cip1/WAF1) expression was studied before and after irradiation in the adult mouse testis. P21^(Cip1/WAF1) is a cyclin-dependent kinase inhibitor (CDI) and has a role in the G1/S checkpoint and differentiation. P21^(Cip1/WAF1) expression was observed in the normal testis, using Western blotting analysis. After a dose of 4 Gy, but not after 0.3 Gy, an increase in p21^(Cip1/WAF1) expression could be determined in whole testis lysates. To investigate which germ cells are involved in p21^(Cip1/WAF1) protein expression, immunohistochemical analysis was performed on irradiated testis. In the normal testis a weak staining for p21^(Cip1/WAF1) was found in pachytene spermatocytes in epithelial stage V up to step 5 spermatids. A dose of 4 Gy of X-irradiation resulted in a transient increase of p21^(Cip1/WAF1) staining in these cells with a maximum at 6 hr post irradiation, despite the fact that the irradiation did not induce an increase in the number of apoptotic spermatocytes. When a dose of 0.3 Gy was given, no increase in p21^(Cip1/WAF1) staining was observed. Using the TUNEL technique, a 10-fold increase in apoptotic spermatogonia was found after a dose of 4 Gy. However, no staining for p21^(Cip1/WAF1) was observed in spermatogonia, suggesting that these cells do not undergo a p21^(Cip1/WAF1)-induced G1 arrest prior to DNA repair or apoptosis. These data imply that p21^(Cip1/WAF1) is a factor which could be important during the meiotic prophase in spermatocytes and repair mechanisms in these cells, but not in spermatogonial cell cycle delay or apoptosis induction. Mol. Reprod. Dev. 47:240–247, 1997. © 1997 Wiley-Liss, Inc.     

Evolutionary origin of the Otx2 enhancer for its expression in visceral endoderm

In the mouse, the Otx2 gene has been shown to play essential roles in the visceral endoderm during anterior-posterior axis formation and head induction. While these are primary processes in vertebrate embryogenesis, the visceral endoderm is a tissue unique to mammals. Two enhancers (VE and CM) have been previously found to direct Otx2 expression during early embryogenesis. This study demonstrates that in anterior visceral endoderm the CM enhancer does not have an activity by itself, but enhances the activity of the VE enhancer. These two enhancers also cooperate for the activities in anterior mesendoderm and cephalic mesenchyme. Comparative studies suggest that VE enhancer function was most likely established before the divergence of sarcopterygians into Actinistia, Dipnoi and tetrapods, while the nucleotide sequence corresponding to the VE enhancer was already present in the last common ancestor of bony fishes. The CM enhancer sequence and function would have been also established in ancestral sarcopterygians. The VE/CM enhancers and their gene cascades in the ancestral sarcopterygian head organizer would then have been co-opted by amphibian deep endoderm cells and mammalian visceral endoderm cells for the head development.     

Genomic analyses of the Formosan harvest mouse (Micromys minutus) and comparisons to the brown Norway rat (Rattus norvegicus) and the house mouse (Mus musculus)

The harvest mouse, Micromys minutus (MMIN), has a very wide range of distribution (from the British Isles across the Euroasian continent to Japan and Taiwan). We studied an isolated population of MMIN in Taiwan, which is at the southeastern margin of the species’ geographic distribution, and compared its genetic complement with those of the same species previously reported from other geographic locations and with two model rodent species, the house mouse (Mus musculus) and the brown Norway rat (Rattus norvegicus). The diploid number (2N) of MMIN was 68, consistent with that reported for other populations. However, variations were noted in the fundamental number (FN) and the shape and banding patterns of the individual chromosomes among populations. The FN of MMIN was estimated to be 72, including 2 bi-armed autosomes, 31 one-armed autosomes, and one pair of one-armed sex chromosomes. Here, we propose the first ideogram for MMIN. C-banding, Ag-NOR, and the locations of 18S rRNA gene sequences (MMIN chromosomes no. 10, 14, 19, 29, 31, 33, and X) mapped by fluorescence in situ hybridization (FISH) are also reported. Additionally, we compared the 18S rDNA sequences and performed cross-species X chromosome painting (FISH) for M. minutus, M. musculus, and R. norvegicus. The results indicate that both genetic elements are rather conserved across species. Thus, implications for the phylogenetic position of Micromys were limited.     

Sal-like 4 (SALL4) suppresses CDH1 expression and maintains cell dispersion in basal-like breast cancer

In cell cultures, the dispersed phenotype is indicative of the migratory ability. Here we characterized Sal-like 4 (SALL4) as a dispersion factor in basal-like breast cancer. Our shRNA-mediated SALL4 knockdown system and SALL4 overexpression system revealed that SALL4 suppresses the expression of adhesion gene CDH1, and positively regulates the CDH1 suppressor ZEB1. Cell behavior analyses showed that SALL4 suppresses intercellular adhesion and maintains cell motility after cell–cell interaction and cell division, which results in the dispersed phenotype. Our findings indicate that SALL4 functions to suppress CDH1 expression and to maintain cell dispersion in basal-like breast cancer.