Characterization of Amphioxus AmphiVent, an evolutionarily conserved marker for chordate ventral mesoderm

Structure and developmental expression are described for amphioxus AmphiVent, a homolog of vertebrate Vent genes. In amphioxus, AmphiVent-expressing ventral mesoderm arises at midneurula by outgrowth from the paraxial mesoderm, but in vertebrates, Vent-expressing ventral mesoderm originates earlier, at the gastrula stage. In other embryonic tissues (nascent paraxial mesoderm, neural plate, endoderm, and tailbud), AmphiVent and its vertebrate homologs are expressed in similar spatiotemporal domains, indicating conservation of many Vent gene functions during chordate evolution. The ventral mesoderm evidently develops precociously in vertebrates because their relatively large embryos probably require an early and extensive deployment of the mesoderm-derived circulatory system. The vertebrate ventral mesoderm, in spite of its strikingly early advent, still resembles the nascent ventral mesoderm of amphioxus in expressing Vent homologs. This coincidence may indicate that Vent homologs in vertebrates and amphioxus play comparable roles in ventral mesoderm specification.     

Regulatory expression of Brachyury and Goosecoid in P19 embryonal carcinoma cells

Mouse P19 embryonal carcinoma cells can differentiate into various cell types depending on culture conditions. Here we show that the expression of the mesodermal genes Brachyury (Bra) and Goosecoid (Gsc) are under regulatory control in P19 cells. When P19 cells were cultured in a tissue culture dish in the presence of serum, Bra and Gsc were unexpectedly expressed. Expression of Bra and Gsc was greatly reduced with culture time, and expression levels at 144 h of culture were below 25% those at 48 h of culture. Members of the Tgf-beta family such as Activin and Nodal have been known to up-regulate expression of mesodermal genes. Treatment with SB431542, an Alk4/5/7 inhibitor, decreased Bra and Gsc in a dose-dependent manner, whereas it induced the expression of the neuroectodermal genes Mash-1 and Pax-6. Quantitative RT-PCR and dsRNAi transfection indicated Nodal as a possible ligand responsible for the regulation of Bra and Gsc. In addition, exogenous Nodal increased expression of Bra and Gsc in a dose-dependent manner. Serum concentration in culture medium positively related to expression of Nodal, Bra, Gsc, and Cripto, which encodes a membrane-tethered protein required for Nodal signaling. Addition of the culture supernatant of P19 cells at 144 h of culture to medium decreased expression of these genes. The present study reveals that stimulation and inhibition of the Nodal pathway increases mesodermal genes and neuroectodermal genes, respectively, indicating the importance of control of Nodal and Cripto expression for mesodermal formation and neurogenesis.     

Dissociation of expression patterns of homeodomain transcription factors in the evolution of developmental mode in the sea urchins Heliocidaris tuberculata and H. erythrogramma

The direct-developing sea urchin species Heliocidaris erythrogramma has a radically modified ontogeny. Along with gains of novel features, its entire ectoderm has been reorganized, resulting in the apparent absence of a differentiated oral ectoderm, a major module present in the pluteus of indirect-developing species, such as H. tuberculata. The restoration of an obvious oral ectoderm in H. erythrogrammaxH. tuberculata hybrids, indicates the action of dominant regulatory factors from the H. tuberculata genome. We sought candidate regulatory genes based on the prediction that they should include genes that govern development of the oral ectoderm in the pluteus, but play different roles in H. erythrogramma. Such genes may have a large effect in the evolution of development. Goosecoid (Gsc), Msx, and the sea urchin Abd-B-like gene (Hox11/13b) are present and expressed in both species and the hybrid embryos. Both Gsc and Msx are oral ectoderm specific in H. tuberculata, and show novel and distinct expression patterns in H. erythrogramma. Gsc assumes a novel ectodermal pattern and Msx shifts to a novel and largely mesodermal pattern. Both Gsc and Msx show a restoration of oral ectoderm expression in hybrids. Hox11/13b is not expressed in oral ectoderm in H. tuberculata, but is conserved in posterior spatial expression among H. tuberculata, H. erythrogramma and hybrids, serving as a control. Competitive RT-PCR shows that Gsc, Msx, and Hox11/13b are under different quantitative and temporal controls in the Heliocidaris species and the hybrids. The implications for the involvement of these genes in the rapid evolution of a direct developing larva are discussed.     

TGF-beta type I receptor Alk5 regulates tooth initiation and mandible patterning in a type II receptor-independent manner

TGF-beta superfamily members signal through a heteromeric receptor complex to regulate craniofacial development. TGF-beta type II receptor appears to bind only TGF-beta, whereas TGF-beta type I receptor (ALK5) also binds to ligands in addition to TGF-beta. Our previous work has shown that conditional inactivation of Tgfbr2 in the neural crest cells of mice leads to severe craniofacial bone defects. In this study, we examine and compare the defects of TGF-beta type II receptor (Wnt1-Cre;Tgfbr2(fl/fl)) and TGF-beta type I receptor/Alk5 (Wnt1-Cre;Alk5(fl)(/fl)) conditional knockout mice. Loss of Alk5 in the neural crest tissue resulted in phenotypes not seen in the Tgfbr2 mutant, including delayed tooth initiation and development, defects in early mandible patterning and altered expression of key patterning genes including Msx1, Bmp4, Bmp2, Pax9, Alx4, Lhx6/7 and Gsc. Alk5 controls the survival of CNC cells by regulating expression of Gsc and other genes in the proximal aboral region of the developing mandible. We conclude that ALK5 regulates tooth initiation and early mandible patterning through a pathway independent of Tgfbr2. There is an intrinsic requirement for Alk5 signal in regulating the fate of CNC cells during tooth and mandible development.     

Early genetic control of craniofacial development is affected by the in vitro exposure of rat embryos to the fungicide triadimefon

BACKGROUND: Previous published experiments reported that in vitro exposure of postimplantation rat embryos to the triazole fungicide triadimefon (FON) resulted in specific abnormalities at the branchial apparatus and that the sensitive period is restricted to the first 24 hr of culture and is associated with the abnormal expression of TGF family genes (some of a large panel of genes regulated by retinoic acid (RA) and involved in branchial arch morphogenesis). The aim of this study is the determination of the sensitive window to FON‐induced abnormalities during in vitro development and the evaluation of the expression of some genes controlled by RA and involved in early branchial arch morphogenesis (Gsc, Msx1, Msx2, Dlx1, Dlx2, Shh, Patched (the main Shh receptor)). METHODS: Rat embryos were exposed in vitro to the FON under condition known to be able to induce 100% of abnormal embryos (250 µ M) at different stages and examined after 48 hr of culture. The sensitive window for FON‐induced abnormalities was during the hours E9 h8.00 PM–E10 h8.00 AM. To evaluate the expression of selected genes, embryos exposed during the sensitive stages were processed to perform quantitative PCR after 18 and 24 hr of culture. RESULTS: FON was able to affect the expression of some genes in a stage‐specific manner: earlier embryos were characterized by the downregulation of Msx2 and Gsc, later embryos showed the downregulation of Gsc, Shh, and Patched. The obtained data suggest that FON‐induced abnormalities are mediated, at least in part, through the imbalance of the expression of RA‐related signals. Birth Defects Res (Part B) 92:77‐81, 2011. © 2011 Wiley‐Liss, Inc.     

Cloning of the Candida albicans homolog of Saccharomyces cerevisiae GSC1/FKS1 and its involvement in beta-1,3-glucan synthesis.

Saccharomyces cerevisiae GSC1 (also called FKS1) and GSC2 (also called FKS2) have been identified as the genes for putative catalytic subunits of beta-1,3-glucan synthase. We have cloned three Candida albicans genes, GSC1, GSL1, and GSL2, that have significant sequence homologies with S. cerevisiae GSC1/FKS1, GSC2/FKS2, and the recently identified FKSA of Aspergillus nidulans at both nucleotide and amino acid levels. Like S. cerevisiae Gsc/Fks proteins, none of the predicted products of C. albicans GSC1, GSL1, or GSL2 displayed obvious signal sequences at their N-terminal ends, but each product possessed 10 to 16 potential transmembrane helices with a relatively long cytoplasmic domain in the middle of the protein. Northern blotting demonstrated that C. albicans GSC1 and GSL1 but not GSL2 mRNAs were expressed in the growing yeast-phase cells. Three copies of GSC1 were found in the diploid genome of C. albicans CAI4. Although we could not establish the null mutation of C. albicans GSC1, disruption of two of the three GSC1 alleles decreased both GSC1 mRNA and cell wall beta-glucan levels by about 50%. The purified C. albicans beta-1,3-glucan synthase was a 210-kDa protein as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and all sequences determined with peptides obtained by lysyl endopeptidase digestion of the 210-kDa protein were found in the deduced amino acid sequence of C. albicans Gsc1p. Furthermore, the monoclonal antibody raised against the purified beta-1,3-glucan synthase specifically reacted with the 210-kDa protein and could immunoprecipitate beta-1,3-glucan synthase activity. These results demonstrate that C. albicans GSC1 is the gene for a subunit of beta-1,3-glucan synthase.     

Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells

Preparation of specific lineages at high purities from embryonic stem (ES) cells requires both selective culture conditions and markers to guide and monitor the differentiation. In this study, we distinguished definitive and visceral endoderm by using a mouse ES cell line that bears the gfp and human IL2R alpha (also known as CD25) marker genes in the goosecoid (Gsc) and Sox17 loci, respectively. This cell line allowed us to monitor the generation of Gsc+ Sox17+ definitive endoderm and Gsc- Sox17+ visceral endoderm and to define culture conditions that differentially induce definitive and visceral endoderm. By comparing the gene expression profiles of definitive and visceral endoderm, we identified seven surface molecules that are expressed differentially in the two populations. One of the seven markers, Cxcr4, to which a monoclonal antibody is available allowed us to monitor and purify the Gsc+ population from genetically unmanipulated ES cells under the condition that selects definitive endoderm.     

Polymerase-dependent DNA synthesis from phosphoramidate-activated nucleotides

Nucleoside triphosphate mimetics, which are substrates for polymerases, can be used in the enzymatic synthesis of nucleic acids. Alternatively, they might also become reversible or irreversible enzyme inhibitors. In order to analyze the effects of 5'-phosphoramidate modification of deoxynucleotide in DNA synthesis, 3-phosphono-L-Ala-dNMP (N = A, T, or G) were evaluated as substrates of HIV-1 RT, Vent (exo(-)), and Therminator polymerase, respectively. The DNA-dependent DNA polymerase activity is significantly higher for Vent exo(-) polymerase than for HIV-1 RT, which is reflected by the capacity of Vent exo(-) polymerase to efficiently synthesize DNA without stalling effects. In addition, Vent (exo(-)) polymerase proved to be more accurate than Therminator polymerase, based on Watson-Crick base-pairing. The optimal yield (88%-97%) of full-length elongation can be obtained in 60 minutes by Vent (exo(-)) polymerase at 0.025 U/μL, with the phosphoramidate analogues as substrates. These data led us to conclude that the optimal pyrophosphate mimetic for the enzyme-catalyzed synthesis of DNA is polymerase dependent.     

The morphogenetic role of midline mesendoderm and ectoderm in the development of the forebrain and the midbrain of the mouse embryo

The anterior midline tissue (AML) of the late gastrula mouse embryo comprises the axial mesendoderm and the ventral neuroectoderm of the prospective forebrain, midbrain and rostral hindbrain. In this study, we have investigated the morphogenetic role of defined segments of the AML by testing their inductive and patterning activity and by assessing the impact of their ablation on the patterning of the neural tube at the early-somite-stage. Both rostral and caudal segments of the AML were found to induce neural gene activity in the host tissue; however, the de novo gene activity did not show any regional characteristic that might be correlated with the segmental origin of the AML. Removal of the rostral AML that contains the prechordal plate resulted in a truncation of the head accompanied by the loss of several forebrain markers. However, the remaining tissues reconstituted Gsc and Shh activity and expressed the ventral forebrain marker Nkx2.1. Furthermore, analysis of Gsc-deficient embryos reveals that the morphogenetic function of the rostral AML requires Gsc activity. Removal of the caudal AML led to a complete loss of midline molecular markers anterior to the 4th somite. In addition, Nkx2.1 expression was not detected in the ventral neural tube. The maintenance and function of the rostral AML therefore require inductive signals emanating from the caudal AML. Our results point to a role for AML in the refinement of the anteroposterior patterning and morphogenesis of the brain.