Otx2 expression in anterior neuroectoderm and forebrain/midbrain is directed by more than six enhancers

Otx2 plays essential roles in each site at each step of head development. We previously identified the AN1 enhancer at 91 kb 5' upstream for the Otx2 expressions in anterior neuroectoderm (AN) at neural plate stage before E8.5, and the FM1 enhancer at 75 kb 5' upstream and the FM2 enhancer at 122 kb 3' downstream for the expression in forebrain/midbrain (FM) at brain vesicle stage after E8.5. The present study identified a second AN enhancer (AN2) at 88 kb 5' upstream; the AN2 enhancer also recapitulates the endogenous Otx2 expression in choroid plexus, cortical hem and choroidal roof. However, the enhancer mutants indicated the presence of another AN enhancer. The study also identified a third FM enhancer (FM3) at 153 kb 5' upstream. Thus, the Otx2 expressions in anterior neuroectoderm and forebrain/midbrain are regulated by more than six enhancers located far from the coding region. The enhancers identified are differentially conserved among vertebrates; none of the AN enhancers has activities in caudal forebrain and midbrain at brain vesicle stage after E8.5, nor do any of the FM enhancers in anterior neuroectoderm at neural plate stage before E8.5.     

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.     

The regulation of Dkk1 expression during embryonic development

During embryogenesis, the Dkk1 mediated Wnt inhibition controls the spatiotemporal dynamics of cell fate determination, cell differentiation and cell death. Furthermore, the Dkk1 dose is critical for the normal Wnt homeostasis, as alteration of the Dkk1 activity is associated with various diseases. We investigated the regulation of Dkk1 expression during embryonic development. We identified nine conserved non-coding elements (CNEs), located 3′ to the Dkk1 locus. Analyses of the regulatory potential revealed that four of these CNEs in combination drive reporter expression very similar to Dkk1 expression in several organs of transgenic embryos. We extended the knowledge of Dkk1 expression during hypophysis, external genitalia and kidney development, suggesting so far to unexplored functions of Dkk1 during the development of these organs. Characterization of the regulatory potential of four individual CNEs revealed that each of these promotes Dkk1 expression in brain and kidney. In combination, two enhancers are responsible for expression in the pituitary and the genital tubercle. Furthermore, individual CNEs mediates craniofacial, optic cup and limb specific Dkk1 regulation. Our study substantially improves the knowledge of Dkk1 regulation during embryonic development and thus might be of high relevance for therapeutic approaches.     

Characterization and expression of cry4Cb1 and cry30Ga1 from Bacillus thuringiensis strain HS18-1

We characterized a novel Bacillus thuringiensis isolate native to China (HS18-1) that shows a spherical crystal harboring two major proteins of about 70 and 130 kDa, and contains three novel cry genes (cry4Cb1, cry30Ga1, cry54-type). Furthermore, the cry4Cb1 and cry30Ga1 genes were expressed in Escherichia coli BL21 (DE3): pLysS. Insecticidal activity tests showed that the cry4Cb1 protein exhibited larvicidal activity against Aedes aegypti (Diptera) and the cry30Ga1 protein was toxic to both A. aegypti and P. xylostella (Lepidoptera).     

Rpi-blb2-mediated late blight resistance in Nicotiana benthamiana requires SGT1 and salicylic acid-mediated signaling but not RAR1 or HSP90

The Rpi-blb2 recognizes the presence of the Phytophthora infestans AVRblb2 and initiates effector-triggered immunity (ETI). We performed gain-of-function and loss-of-function studies in Nicotiana benthamiana to elucidate Rpi-blb2-mediated resistance to P. infestans. Rpi-blb2 triggered a hypersensitive response through SGT1-mediated, but not RAR-mediated or HSP90-mediated, pathways. NbSGT1 was also required for basal and ETI-mediated by Rpi-blb2 in N. benthamiana. Moreover, salicylic acid (SA) affected basal defense and Rpi-blb2-mediated resistance against P. infestans. The increased susceptibility of Rpi-blb2-transgenic plants in the NahG-background correlated with reduced levels of SA. These findings provide evidence for the roles of SGT1- and SA-signaling in Rpi-blb2-mediated resistance against P. infestans.     

Retinoic acid signaling in perioptic mesenchyme represses Wnt signaling via induction of Pitx2 and Dkk2

Morphogenesis during eye development requires retinoic acid (RA) receptors plus RA-synthesizing enzymes, and loss of RA signaling leads to ocular disorders associated with loss of Pitx2 expression in perioptic mesenchyme. Several Wnt signaling components are expressed in ocular tissues during eye development including Dkk2, encoding an inhibitor of Wnt/β-catenin signaling, which was previously shown to be induced by Pitx2 in the perioptic mesenchyme. Here, we investigated potential cross-talk between RA and Wnt signaling during ocular development. Genetic studies using Raldh1/Raldh3 double null mice deficient for ocular RA synthesis demonstrated that Pitx2 and Dkk2 were both down-regulated in perioptic mesenchyme. Chromatin immunoprecipitation and gel mobility shift studies demonstrated the existence of a DR5 RA response element upstream of Pitx2 that binds all three RA receptors in embryonic eye. Axin2, an endogenous readout of Wnt/β-catenin signaling, was up-regulated in cornea and perioptic mesenchyme of RA deficient embryos. Also, expression of Wnt5a was expanded in perioptic mesenchyme of RA deficient eyes. Our findings demonstrate excessive activation of Wnt signaling in the perioptic mesenchyme of RA deficient mice which may be responsible for abnormal development leading to defective optic cup, cornea, and eyelid morphogenesis.     

The ascl1a and dlx genes have a regulatory role in the development of GABAergic interneurons in the zebrafish diencephalon

During development of the mouse forebrain interneurons, the Dlx genes play a key role in a gene regulatory network (GRN) that leads to the GABAergic phenotype. Here, we have examined the regulatory relationships between the ascl1a, dlx, and gad1b genes in the zebrafish forebrain. Expression of ascl1a overlaps with dlx1a in the telencephalon and diencephalon during early forebrain development. The loss of Ascl1a function results in a loss of dlx expression, and subsequent losses of dlx5a and gad1b expression in the diencephalic prethalamus and hypothalamus. Loss of Dlx1a and Dlx2a function, and, to a lesser extent, of Dlx5a and Dlx6a, impairs gad1b expression in the prethalamus and hypothalamus. We conclude that dlx1a/2a act downstream of ascl1a but upstream of dlx5a/dlx6a and gad1b to activate GABAergic specification. This pathway is conserved in the diencephalon, but has diverged between mammals and teleosts in the telencephalon.     

The interplay between SUCLA2, SUCLG2, and mitochondrial DNA depletion

SUCLA2-related mitochondrial DNA (mtDNA) depletion syndrome is a result of mutations in the β subunit of the ADP-dependent isoform of the Krebs cycle succinyl-CoA synthase (SCS). The mechanism of tissue specificity and mtDNA depletion is elusive but complementation by the GDP-dependent isoform encoded by SUCLG2, and the association with mitochondrial nucleoside diphosphate kinase (NDPK), is a plausible link.We have investigated this relationship by studying SUCLA2 deficient fibroblasts derived from patients and detected normal mtDNA content and normal NDPK activity. However, knockdown of SUCLG2 by shRNA in both patient and control fibroblasts resulted in a significant decrease in mtDNA amount, decreased NDPK and cytochrome c oxidase activities, and a marked growth impairment. This suggests that, SUCLG2, to a higher degree than SUCLA2, is crucial for mtDNA maintenance and that mitochondrial NDPK is involved. Although results pertain to a cell culture system, the findings might explain the pathomechanism and tissue specificity in mtDNA depletion caused by defective SUCLA2.     

Isolation and Characterization of OsGSTL1 Promoter from Rice

OsGSTL1 gene was isolated from the rice genomic library. Semi-quantitative RT-PCR analysis demonstrated that the expression of the OsGSTL1 in rice was not induced by chlorsulfuron, ethylene, abscisic acid, salicylic acid, and methyl jasmonate. In order to investigate the cis-elements of OsGSTL1 promoter, the promoter regions with different lengths were fused to the β-glucuronidase (GUS) reporter gene. All constructs were transformed into onion epidermal cells or A. thaliana plants to detect the expression patterns. In onion epidermal cells, the 160 bp fragment and longer ones were functional for directing GUS expression. In transgenic A. thaliana, the 2?155 bp upstream region of OsGSTL1 gene directed the GUS expression only in cotyledon after germination, but not in the root of young seedlings. In the later seedling, the 2?155 bp upstream region of OsGSTL1 gene directed GUS expression in roots, stems, and leaves. However, the GUS gene directed by a 1?224 bp upstream fragment is expressed in all the checked tissues. These results suggest that the spatiotemporal expression response elements of OsGSTL1 existed in the 5′-upstream region between −2?155 and −1?224 bp.     

Genetic interactions between Pax9 and Msx1 regulate lip development and several stages of tooth morphogenesis

Developmental abnormalities of craniofacial structures and teeth often occur sporadically and the underlying genetic defects are not well understood, in part due to unknown gene-gene interactions. Pax9 and Msx1 are co-expressed during craniofacial development, and mice that are single homozygous mutant for either gene exhibit cleft palate and an early arrest of tooth formation. Whereas in vitro assays have demonstrated that protein-protein interactions between Pax9 and Msx1 can occur, it is unclear if Pax9 and Msx1 interact genetically in vivo during development. To address this question, we compounded the Pax9 and Msx1 mutations and observed that double homozygous mutants exhibit an incompletely penetrant cleft lip phenotype. Moreover, in double heterozygous mutants, the lower incisors were consistently missing and we find that transgenic BMP4 expression partly rescues this phenotype. Reduced expression of Shh and Bmp2 indicates that a smaller “incisor field” forms in Pax9^+/−;Msx1^+/− mutants, and dental epithelial growth is substantially reduced after the bud to cap stage transition. This defect is preceded by drastically reduced mesenchymal expression of Fgf3 and Fgf10, two genes that encode known stimulators of epithelial growth during odontogenesis. Consistent with this result, cell proliferation is reduced in both the dental epithelium and mesenchyme of double heterozygous mutants. Furthermore, the developing incisors lack mesenchymal Notch1 expression at the bud stage and exhibit abnormal ameloblast differentiation on both labial and lingual surfaces. Thus, Msx1 and Pax9 interact synergistically throughout lower incisor development and affect multiple signaling pathways that influence incisor size and symmetry. The data also suggest that a combined reduction of PAX9 and MSX1 gene dosage in humans may increase the risk for orofacial clefting and oligodontia.