Generation of Pmel-dependent conditional and inducible Cre-driver mouse line for melanocytic-targeted gene manipulation.

Conditional and inducible gene targeting using Cre/loxP-mediated recombination is a powerful reverse genetics approach used to study spatiotemporal gene functions in specified cell types. To enable temporal gene manipulation in the melanocyte lineage, we established a novel inducible Cre-driver mouse line by targeting an all-in-one tetracycline/doxycycline (Dox)-inducible Cre expression cassette into the Pmel locus (PmelP2A-TetON3G-TRE3G-iCre ), a gene locus preferentially expressed in pigment cells. By crossing these Cre-driver mice with a strong Cre-reporter mouse line, Gt(ROSA)26Sortm9(CAG-tdTomato)Hze , we show the effectiveness of the PmelP2A-TetON3G-TRE3G-iCre mouse line in facilitating Dox-inducible Cre/loxP recombination in a wide variety of pigment cell lineages including hair follicle melanocytes and their stem cells. Furthermore, to demonstrate proof of concept, we ablated Notch signaling postnatally in the PmelP2A-TetON3G-TRE3G-iCre mice. In agreement with the previously reported phenotype, induced ablation of Notch signaling in the melanocyte lineage resulted in premature hair graying, demonstrating the utility of the PmelP2A-TetON3G-TRE3G-iCre allele. Therefore, the PmelP2A-TetON3G-TRE3G-iCre mouse line is suitable for assessing gene functions in melanocytes using an in vivo inducible reverse genetics approach. Furthermore, we unexpectedly identified previously unrecognized PMEL-expressing cells in non-pigmentary organs in the mice, suggesting unanticipated functions of PMEL other than melanosome formation.

Role of MEKK1 in the anterior-posterior patterning during planarian regeneration.

Planarians have established a unique body pattern along the anterior-posterior (AP) axis, which consists of at least four distinct body regions arranged in an anterior to posterior sequence: head, prepharyngeal, pharyngeal (containing a pharynx), and tail regions, and possess high regenerative ability. How they reconstruct the regional continuity in a head-to-tail sequence after amputation still remains unknown. We use as a model planarian Dugesia japonica head regeneration from tail fragments, which involves dynamic rearrangement of the body regionality of preexisting tail tissues along the AP axis, and show here that RNA interference of the gene D. japonica mek kinase 1 (Djmekk1) caused a significant anterior shift in the position of pharynx regeneration at the expense of the prepharyngeal region, while keeping the head region relatively constant in size, and accordingly led to development of a relatively longer tail region. Our data suggest that DjMEKK1 regulates anterior extracellular signal-regulated kinase (ERK) and posterior ß-catenin signaling pathways in a positive and negative manner, respectively, to establish a proper balance resulting in the regeneration of planarian's scale-invariant trunk-to-tail patterns across individuals. Furthermore, we demonstrated that DjMEKK1 negatively modulates planarian ß-catenin activity via its serine/threonine kinase domain, but not its PHD/RING finger domain, by testing secondary axis formation in Xenopus embryos. The data suggest that Djmekk1 plays an instructive role in the coordination between the establishment of the prepharyngeal region and posteriorizing of pharynx formation by balancing the two opposing morphogenetic signals along the AP axis during planarian regeneration.

Unusually Large Number of Mutations in Asexually Reproducing Clonal Planarian Dugesia japonica.

We established a laboratory clonal strain of freshwater planarian (Dugesia japonica) that was derived from a single individual and that continued to undergo autotomous asexual reproduction for more than 20 years, and we performed large-scale genome sequencing and transcriptome analysis on it. Despite the fact that a completely clonal strain of the planarian was used, an unusually large number of mutations were detected. To enable quantitative genetic analysis of such a unique organism, we developed a new model called the Reference Gene Model, and used it to conduct large-scale transcriptome analysis. The results revealed large numbers of mutations not only outside but also inside gene-coding regions. Non-synonymous SNPs were detected in 74% of the genes for which valid ORFs were predicted. Interestingly, the high-mutation genes, such as metabolism- and defense-related genes, were correlated with genes that were previously identified as diverse genes among different planarian species. Although a large number of amino acid substitutions were apparently accumulated during asexual reproduction over this long period of time, the planarian maintained normal body-shape, behaviors, and physiological functions. The results of the present study reveal a unique aspect of asexual reproduction.

Comparative transcriptomic analysis of two closely related ground beetle species with marked genital divergence using pyrosequencing.

Ground beetles of the subgenus Ohomopterus (genus Carabus) show marked divergence in species-specific male and female genital morphologies, which contributes to reproductive isolation among species. Characterizing the genetic basis of species-specific genital morphology is essential for understanding their diversification, but genomic information on Ohomopterus is not yet available. We analyzed mRNA extracted from abdominal sections of the last instar larvae and pupae of two sister species, Carabus (Ohomopterus) iwawakianus and C. (O.) uenoi, which show marked differences in genital morphology, to compare transcriptomic profiles using Roche 454 pyrosequencing. We obtained 1,608,572 high-quality reads and assembled them into 176,278 unique sequences, of which 66,049 sequences were combined into 12,662 clusters. Differential expression analyses for sexed pupae suggested that four and five clusters were differentially expressed between species for males and females, respectively. We also identified orthologous sequences of genes involved in genital development in Drosophila, which potentially affect genital development and species-specific genital morphology in Ohomopterus. This study provides the first large transcriptomic data set for a morphologically diversified beetle group, which can facilitate future studies on the genetic basis of species-specific genitalia.

How are plant and fungal communities linked to each other in belowground ecosystems? A massively parallel pyrosequencing analysis of the association specificity of root-associated fungi and their host plants.

In natural forests, hundreds of fungal species colonize plant roots. The preference or specificity for partners in these symbiotic relationships is a key to understanding how the community structures of root-associated fungi and their host plants influence each other. In an oak-dominated forest in Japan, we investigated the root-associated fungal community based on a pyrosequencing analysis of the roots of 33 plant species. Of the 387 fungal taxa observed, 153 (39.5%) were identified on at least two plant species. Although many mycorrhizal and root-endophytic fungi are shared between the plant species, the five most common plant species in the community had specificity in their association with fungal taxa. Likewise, fungi displayed remarkable variation in their association specificity for plants even within the same phylogenetic or ecological groups. For example, some fungi in the ectomycorrhizal family Russulaceae were detected almost exclusively on specific oak (Quercus) species, whereas other Russulaceae fungi were found even on "non-ectomycorrhizal" plants (e.g., Lyonia and Ilex). Putatively endophytic ascomycetes in the orders Helotiales and Chaetothyriales also displayed variation in their association specificity and many of them were shared among plant species as major symbionts. These results suggest that the entire structure of belowground plant-fungal associations is described neither by the random sharing of hosts/symbionts nor by complete compartmentalization by mycorrhizal type. Rather, the colonization of multiple types of mycorrhizal fungi on the same plant species and the prevalence of diverse root-endophytic fungi may be important features of belowground linkage between plant and fungal communities.

The molecular logic for planarian regeneration along the anterior-posterior axis.

The planarian Dugesia japonica can regenerate a complete individual from a head, trunk or tail fragment via activation of somatic pluripotent stem cells. About a century ago, Thomas Hunt Morgan attempted to explain the extraordinary regenerative ability of planarians by positing two opposing morphogenetic gradients of formative "head stuff" and "tail stuff" along the anterior-posterior axis. However, Morgan's hypothesis remains open to debate. Here we show that extracellular signal-related kinase (ERK) and Wnt/ß-catenin signalling pathways establish a solid framework for planarian regeneration. Our data suggest that ERK signalling forms a spatial gradient in the anterior region during regeneration. The fibroblast growth factor receptor-like gene nou-darake (which serves as an output of ERK signalling in the differentiating head) and posteriorly biased ß-catenin activity negatively regulate ERK signalling along the anterior-posterior axis in distinct manners, and thereby posteriorize regenerating tissues outside the head region to reconstruct a complete head-to-tail axis. On the basis of this knowledge about D. japonica, we proposed that ß-catenin signalling is responsible for the lack of head-regenerative ability of tail fragments in the planarian Phagocata kawakatsui, and our confirmation thereof supports the notion that posterior ß-catenin signalling negatively modulates the ERK signalling involved in anteriorization across planarian species. These findings suggest that ERK signalling has a pivotal role in triggering globally dynamic differentiation of stem cells in a head-to-tail sequence through a default program that promotes head tissue specification in the absence of posteriorizing signals. Thus, we have confirmed the broad outline of Morgan's hypothesis, and refined it on the basis of our proposed default property of planarian stem cells.

Transcriptome pyrosequencing of the parasitoid wasp Cotesia vestalis: genes involved in the antennal odorant-sensory system.

Cotesia vestalis is an endoparasitic wasp that attacks larvae of the diamondback moth (Plutella xylostella), a herbivore of cruciferous plants. Females of C. vestalis use herbivore-induced plant odorants released from plants infested by P. xylostella as a host-searching cue. Transcriptome pyrosequencing was used to identify genes in the antennae of C. vestalis adult females coding for odorant receptors (ORs) and odorant binding proteins (OBPs) involved in insect olfactory perception. Quantitative gene expression analyses showed that a few OR and OBP genes were expressed exclusively in the antenna of C. vestalis adult females whereas most other classes of genes were expressed in the antennae of both males and females, indicating their diversity in importance for the olfactory sensory system. Together, transcriptome profiling of C. vestalis genes involved in the antennal odorant-sensory system helps in detecting genes involved in host- and food-search behaviors through infochemically-mediated interactions.

Temperature-dependent, behavioural, and transcriptional variability of a tritrophic interaction consisting of bean, herbivorous mite, and predator.

Different organisms compensate for, and adapt to, environmental changes in different ways. In this way, environmental changes affect animal-plant interactions. In this study, we assessed the effect of temperature on a tritrophic system of the lima bean, the herbivorous spider mite Tetranychus urticae and the predatory mite Phytoseiulus persimilis. In this system, the plant defends itself against T. urticae by emitting volatiles that attract P. persimilis. Over 20-40 °C, the emission of volatiles by infested plants and the subsequent attraction of P. persimilis peaked at 30 °C, but the number of eggs laid by T. urticae adults and the number of eggs consumed by P. persimilis peaked at 35 °C. This indicates that the spider mites and predatory mites performed best at a higher temperature than that at which most volatile attractants were produced. Our data from transcriptome pyrosequencing of the mites found that P. persimilis up-regulated gene families for heat shock proteins (HSPs) and ubiquitin-associated proteins, whereas T. urticae did not. RNA interference-mediated gene suppression in P. persimilis revealed differences in temperature responses. Predation on T. urticae eggs by P. persimilis that had been fed PpHsp70-1 dsRNA was low at 35 °C but not at 25 °C when PpHsp70-1 expression was very high. Overall, our molecular and behavioural approaches revealed that the mode and tolerance of lima bean, T. urticae and P. persimilis are distinctly affected by temperature variability, thereby making their tritrophic interactions temperature dependent.

A LIM-homeobox gene is required for differentiation of Wnt-expressing cells at the posterior end of the planarian body.

Planarians have high regenerative ability, which is dependent on pluripotent adult somatic stem cells called neoblasts. Recently, canonical Wnt/ß-catenin signaling was shown to be required for posterior specification, and Hedgehog signaling was shown to control anterior-posterior polarity via activation of the Djwnt1/P-1 gene at the posterior end of planarians. Thus, various signaling molecules play an important role in planarian stem cell regulation. However, the molecular mechanisms directly involved in stem cell differentiation have remained unclear. Here, we demonstrate that one of the planarian LIM-homeobox genes, Djislet, is required for the differentiation of Djwnt1/P-1-expressing cells from stem cells at the posterior end. RNA interference (RNAi)-treated planarians of Djislet [Djislet(RNAi)] show a tail-less phenotype. Thus, we speculated that Djislet might be involved in activation of the Wnt signaling pathway in the posterior blastema. When we carefully examined the expression pattern of Djwnt1/P-1 by quantitative real-time PCR during posterior regeneration, we found two phases of Djwnt1/P-1 expression: the first phase was detected in the differentiated cells in the old tissue in the early stage of regeneration and then a second phase was observed in the cells derived from stem cells in the posterior blastema. Interestingly, Djislet is expressed in stem cell-derived DjPiwiA- and Djwnt1/P-1-expressing cells, and Djislet(RNAi) only perturbed the second phase. Thus, we propose that Djislet might act to trigger the differentiation of cells expressing Djwnt1/P-1 from stem cells.

Planarian Hedgehog/Patched establishes anterior-posterior polarity by regulating Wnt signaling.

Despite long-standing interest, the molecular mechanisms underlying the establishment of anterior-posterior (AP) polarity remain among the unsolved mysteries in metazoans. In the planarians (a family of flatworms), canonical Wnt/beta-catenin signaling is required for posterior specification, as it is in many animals. However, the molecular mechanisms regulating the posterior-specific induction of Wnt genes according to the AP polarity have remained unclear. Here, we demonstrate that Hedgehog (Hh) signaling is responsible for the establishment of AP polarity via its regulation of the transcription of Wnt family genes during planarian regeneration. We found that RNAi gene knockdown of Dugesia japonica patched (Djptc) caused ectopic tail formation in the anterior blastema of body fragments, resulting in bipolar-tails regeneration. In contrast, RNAi of hedgehog (Djhh) and gli (Djgli) caused bipolar-heads regeneration. We show that Patched-mediated Hh signaling was crucial for posterior specification, which is established by regulating the transcription of Wnt genes via downstream Gli activity. Moreover, differentiated cells were responsible for the posterior specification of undifferentiated stem cells through Wnt/beta-catenin signaling. Surprisingly, Djhh was expressed in neural cells all along the ventral nerve cords (along the AP axis), but not in the posterior blastema of body fragments, where the expression of Wnt genes was induced for posteriorization. We therefore propose that Hh signals direct head or tail regeneration according to the AP polarity, which is established by Hh signaling activity along the body's preexisting nervous system.

A wide-range phylogenetic analysis of Zic proteins: implications for correlations between protein structure conservation and body plan complexity.

We compared Zic homologues from a wide range of animals. Striking conservation was found in the zinc finger domains, in which an exon-intron boundary has been kept in all bilateralians but not cnidarians, suggesting that all of the bilateralian Zic genes are derived from a single gene in a bilateralian ancestor. There were additional conserved amino acid sequences, ZOC and ZF-NC. Combined analysis of the zinc finger, ZOC, and ZF-NC revealed the presence of two classes of Zic, based on the degree of protein structure conservation. The "conserved" class includes Zic proteins from the Arthropoda, Mollusca, Annelida, Echinodermata, and Chordata (vertebrates and cephalochordates), whereas the "diverged" class contains those from the Platyhelminthes, Cnidaria, Nematoda, and Chordata (urochordates). The result indicates that the ancestral bilateralian Zic protein had already acquired an entire set of conserved domains, but that this was lost and diverged in the platyhelminthes, nematodes, and urochordates.

Gene expression of FK506-binding proteins 12.6 and 12 during chicken development.

FKBPs are cytosolic receptors for the immunosuppressive drug FK506. FKBP12.6 and FKBP12 associate with cardiac and skeletal muscle isoforms of ryanodine receptors and thereby regulate intracellular Ca(2+) release. The amino acid sequences of FKBP12.6 and FKBP12 are highly conserved among mammals and chicken. The expression profiles of FKBP12.6 and FKBP12 genes during chicken development were compared by Northern blot and in situ hybridization analyses. Transcripts of the FKBP12 gene were abundant throughout the embryo at early stages of development but subsequently underwent gradual down-regulation. Expression of the FKBP12.6 gene was mostly restricted to the heart during early embryogenesis and also underwent subsequent down-regulation, becoming localized to the atrium after hatching. Treatment of early embryos with FK506 had no apparent effect on embryogenesis. Retinoic acid induced marked abnormalities in cardiogenesis, but it did not affect FKBP gene expression. These results suggest that, even though FKBP12.6 and FKBP12 genes are expressed in chick embryos, FK506-sensitive functions of the encoded proteins do not appear to contribute to early embryogenesis or cardiogenesis.

Heart-selective expression of the chicken FK506-binding protein (FKBP) 12.6 gene during embryonic development.

FKBP12.6, a member of the family of FK506-binding proteins, selectively associates with the cardiac isoform of the ryanodine receptor and thereby stabilizes this Ca(2+) release channel. A chicken FKBP12.6 (chFKBP12.6) cDNA was cloned and shown to encode a protein of 108 amino acids. The deduced amino acid sequence of chFKBP12.6 is 91-92% identical to those of mammalian FKBP12.6 proteins. Northern blot analysis revealed that chFKBP12.6 mRNA is largely restricted to the heart during embryonic development and that the abundance of this mRNA in the heart decreases, and it becomes restricted to the atrium during cardiogenesis. In situ hybridization revealed that chFKBP12.6 mRNA is localized to the precardiac mesoderm before formation of the primitive heart tube. Expression of the chFKBP12.6 gene was initially apparent throughout the developing multichambered heart but became restricted to the atria before hatching. Reverse transcription and polymerase chain reaction analysis demonstrated that chFKBP12.6 mRNA is present in the embryo from early gastrulation and is most abundant immediately after the onset of the heartbeat. These observations suggest that the chFKBP12.6 gene is expressed before heart morphogenesis to play a role in excitation-contraction coupling in cardiomyocytes and that the function of the encoded protein becomes increasingly restricted to the atrium during embryonic development.