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.     

Construction and characterization of a BAC library from a gynogenetic channel catfish Ictalurus punctatus

A bacterial artificial chromosome (BAC) library was constructed by cloning HindIII-digested high molecular weight DNA from a gynogenetic channel catfish, Ictalurus punctatus, into the vector pBeloBAC11. Approximately 53 500 clones were arrayed in 384-well plates and stored at -80°C (CCBL1), while clones from a smaller insert size fraction were stored at -80°C without arraying (CCBL2). Pulsed-field gel electrophoresis of 100 clones after NotI digestion revealed an average insert size of 165 kb for CCBL1 and 113 kb for CCBL2. Further characterization of CCBL1 demonstrated that 10% of the clones did not contain an insert. CCBL1 provides a 7.2-fold coverage of the channel catfish haploid genome. PCR-based screening demonstrated that 68 out of 74 unique loci were present in the library. This represents a 92% chance to find a unique sequence. These libraries will be useful for physical mapping of the channel catfish genome, and identification of genes controlling major traits in this economically important species.     

Identification of a novel adrenomedullin gene family in teleost fish

Adrenomedullin (AM) is a multifunctional peptide known to form a hormone family with calcitonin gene-related peptide (CGRP) and amylin. We have cloned five distinct AM cDNAs from the pufferfish, Takifugu rubripes, and named them TrAM-1, -2, -3, -4, and -5. Judging from the deduced precursor sequences and processing pattern of the C-terminal mature peptides, TrAMs may be divided into at least two groups; AM-2 and -3, and AM-1, -4, and possibly -5. Phylogenetic analysis of the mature peptides, exon-intron structure of their genes, and tissue distribution of their mRNA also support this classification. TrAM-1 and -4 were ubiquitously expressed in various tissues including the kidney and interrenal (adrenal homolog) as in the case of mammalian AM, while TrAM-2 and -3 were expressed most abundantly in the brain followed by the vascular tissues. Synteny of the genes around AM gene showed that TrAM-1 is the ortholog of mammalian AM. The presence of a PAMP-like sequence in the prosegment of TrAM-1 also supports this notion. Multiple AMs were also detected in another pufferfish, Tetraodon nigroviridis, and in zebrafish, Danio rerio. The present study shows for the first time the presence of a novel AM family in teleost fish that is independent from CGRP and amylin, which further suggests the possible existence of multiple AMs in mammals.     

Otx2 and Otx1 protect diencephalon and mesencephalon from caudalization into metencephalon during early brain regionalization

Otx2 is expressed in each step and site of head development. To dissect each Otx2 function we have identified a series of Otx2 enhancers. The Otx2 expression in the anterior neuroectoderm is regulated by the AN enhancer and the subsequent expression in forebrain and midbrain later than E8.5 by FM1 and FM2 enhancers; the Otx1 expression takes place at E8.0. In telencephalon later than E9.5 Otx1 continues to be expressed in the entire pallium, while the Otx2 expression is confined to the most medial pallium. To determine the Otx functions in forebrain and midbrain development we have generated mouse mutants that lack both FM1 and FM2 enhancers (DKO: Otx2^{ΔFM1ΔFM2/ΔFM1ΔFM2}) and examined the TKO (Otx1^−/−Otx2^{ΔFM1ΔFM2/ΔFM1ΔFM2}) phenotype. The mutants develop normally until E8.0, but subsequently by E9.5 the diencephalon, including thalamic eminence and prethalamus, and the mesencephalon are caudalized into metencephalon consisting of isthmus and rhombomere 1; the caudalization does not extend to rhombomere 2 and more caudal rhombomeres. In rostral forebrain, neopallium, ganglionic eminences and hypothalamus in front of prethalamus develop; we propose that they become insensitive to the caudalization with the switch from the Otx2 expression under the AN enhancer to that under FM1 and FM2 enhancers. In contrast, the medial pallium requires Otx1 and Otx2 for its development later than E9.5, and the Otx2 expression in diencepalon and mesencephalon later than E9.5 is also directed by an enhancer other than FM1 and FM2 enhancers.     

Bm-TFF2, a trefoil factor protein with platelet activation activity from frog Bombina maxima skin secretions

In mammals, trefoil factor family (TFF) proteins are involved in mucosal maintenance and repair, and they are also implicated in tumor suppression and cancer progression. A novel two domain TFF protein from frog Bombina maxima skin secretions (Bm-TFF2) has been purified and cloned. It activated human platelets in a dose-dependent manner and activation of integrin alpha(IIb)beta(3) was involved. Aspirin and apyrase did not largely reduce platelet response to Bm-TFF2 (a 30% inhibition), indicating that the aggregation is not substantially dependent on ADP and thromboxane A2 autocrine feedback. Elimination of external Ca(2+) with EGTA did not influence the platelet aggregation induced by Bm-TFF2, meanwhile a strong calcium signal (cytoplasmic Ca(2+) release) was detected, suggesting that activation of phospholipase C (PLC) is involved. Subsequent immunoblotting revealed that, unlike in platelets activated by stejnulxin (a glycoprotein VI agonist), PLCgamma2 was not phosphorylated in platelets activated by Bm-TFF2. FITC-labeled Bm-TFF2 bound to platelet membranes. Bm-TFF2 is the first TFF protein reported to possess human platelet activation activity.     

Development of an activation tagging system for the basidiomycetous medicinal fungus Antrodia cinnamomea

This study describes the development of an efficient and reliable activation tagging system for the medicinal fungus Antrodia cinnamomea. For successful Agrobacterium tumefaciens-mediated transformation, different parameters were considered. The Agrobacterium concentration of 5 × 10⁸ cfu ml⁻¹, 1 mm acetosyringone, 25-d-old mycelia at 0.2 g ml⁻¹, and co-culture period of 6 d were found to be the most optimal conditions for enhancing the transformation efficiency. The mitotic stability of transferred DNA (T-DNA) was demonstrated by growing eight randomly selected putative transformants in malt extract agar medium for five subcultures. Insertion of T-DNA into the genome of transformants was confirmed by PCR and Southern hybridization. Results showed that 88 % of the mutants contained a single T-DNA insertion. Two of the mutants were observed with different triterpenoid profiles compared with the untransformed cultures. Our results suggest a new functional genomics approach to tag the triterpenoid biosynthesis genes in A. cinnamomea.     

A major bristle QTL from a selected population of Drosophila uncovers the zinc-finger transcription factor poils-au-dos, a repressor of achaete-scute

Traditional screens aiming at identifying genes regulating development have relied on mutagenesis. Here, we describe a new gene involved in bristle development, identified through the use of natural variation and selection. Drosophila melanogaster bears a pattern of 11 macrochaetes per heminotum. From a population initially sampled in Marrakech, a strain was selected for an increased number of thoracic macrochaetes. Using recombination and single nucleotide polymorphisms, the factor responsible was mapped to a single locus on the third chromosome, poils au dos, that encodes a zinc-finger-ZAD protein. The original, as well as new, presumed null, alleles of poils au dos, is associated with ectopic achaete-scute expression that results in the additional bristles. This suggests a possible role for Poils au dos as a repressor of achaete and scute. Ectopic expression appears to be independent of the activity of known cis-regulatory enhancer sequences at the achaete-scute complex that mediate activation at specific sites on the notum. The target sequences for Poils au dos activity were mapped to a 14 kb region around scute. In addition, we show that pad interacts synergistically with the repressor hairy and with Dpp signaling in posterior and anterior regions of the notum, respectively.