A functional genomics screen identifies an Importin-α homolog as a regulator of stem cell function and tissue patterning during planarian regeneration

Background

Planarians are renowned for their regenerative capacity and are an attractive model for the study of adult stem cells and tissue regeneration. In an effort to better understand the molecular mechanisms underlying planarian regeneration, we performed a functional genomics screen aimed at identifying genes involved in this process in Schmidtea mediterranea.

Methods

We used microarrays to detect changes in gene expression in regenerating and non-regenerating tissues in planarians regenerating one side of the head and followed this with high-throughput screening by in situ hybridization and RNAi to characterize the expression patterns and function of the differentially expressed genes.

Results

Along with five previously characterized genes (Smed-cycD, Smed-morf41/mrg-1, Smed-pdss2/dlp1, Smed-slbp, and Smed-tph), we identified 20 additional genes necessary for stem cell maintenance (Smed-sart3, Smed-smarcc-1, Smed-espl1, Smed-rrm2b-1, Smed-rrm2b-2, Smed-dkc1, Smed-emg1, Smed-lig1, Smed-prim2, Smed-mcm7, and a novel sequence) or general regenerative capability (Smed-rbap46/48-2, Smed-mcm2, Smed-ptbp1, and Smed-fen-1) or that caused tissue-specific defects upon knockdown (Smed-ddc, Smed-gas8, Smed-pgbd4, and Smed-b9d2). We also found that a homolog of the nuclear transport factor Importin-α plays a role in stem cell function and tissue patterning, suggesting that controlled nuclear import of proteins is important for regeneration.

Conclusions

Through this work, we described the roles of several previously uncharacterized genes in planarian regeneration and implicated nuclear import in this process. We have additionally created an online database to house our in situ and RNAi data to make it accessible to the planarian research community.

     

Molecular cloning and expression analysis of a gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase from <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

Camptotheca acuminata is a Chinese tree that produces the anti-cancer monoterpenoid indole alkaloid camptothecin (CPT). 3-Hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) catalyzes the condensation of acetyl CoA and acetoacetyl CoA to form 3-hydroxy-3-methylglutaryl-CoA as an early step in the CPT biosynthetic pathway. A full-length cDNA encoding HMGS (designated as CaHMGS, GenBank accession no. EU677841) was successfully isolated from young leaves of C. acuminata by rapid amplification of cDNA ends (RACE). The full-length cDNA of CaHMGS was 1801 bp long and contained a 1413-bp open reading frame encoding a polypeptide of 471 amino acids. Comparative and bioinformatic analyses revealed that CaHMGS showed extensive homology with HMGSs from other plant species. Southern hybridization analysis showed that there were at least two HMGS gene members in the C. acuminata genome. CPT content was found to be much higher in cotyledons and hypocotyls as compared to roots. RT-PCR analysis revealed strong expression in hypocotyls and cotyledons, but no expression in roots, indicating good correlation between CaHMGS expression and CPT content in the tested tissues. The expression of CaHMGS could be regulated by exogenous elicitors, including salicylic acid and methyl jasmonate, suggesting that CaHMGS was elicitor-responsive. This work is a first step to acquire a better understanding on the role of HMGS in CPT biosynthesis.     

Specific features of eye regeneration in multiocular planarians <Emphasis Type="Italic">Polycelis tenuis</Emphasis>

Regeneration and negative phototaxis were studied in planarians Polycelis tenuis, in which the anterior body end is fringed with many eyes. Comparative data for the same indices are given for binocular planarians Girardia tigrina. Multiple eyes regenerated gradually with a decrease in the rate of regeneration and independently from the rate of restoration of the anterior body end, where they are located. Negative phototaxis was restored independently from the total amount of regenerated eyes. It was unstable in both planarian species.     

Features of expression of the <Emphasis Type="Italic">PsSst1</Emphasis> and <Emphasis Type="Italic">PsIgn1</Emphasis> genes in nodules of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) symbiotic mutants

The sequences of the PsSst1 and PsIgn1 genes of pea (Pisum sativum L.) homologous to the symbiotic LjSST1 and LjIGN1 genes of Lotus japonicus (Regel.) K. Larsen are determined. The expression level of PsSst1 and PsIgn1 genes is determined by real-time PCR in nodules of several symbiotic mutants and original lines of pea. Lines with increased (Sprint-2Fix^– (Pssym31)) and decreased (P61 (Pssym25)) expression level of both genes are revealed along with the lines characterized by changes in the expression level of only one of these genes. The revealed features of the PsSst1 and PsIgn1 expression allow us to expand the phenotypic characterization of pea symbiotic mutants. In addition, PsSst1 and PsIgn1 cDNA is sequenced in selected mutant lines, characterized by a decreased expression level of these genes in nodules, but no mutations are found.     

Molecular cloning,characterization, and functional analysis of a gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A synthase from <Emphasis Type="Italic">Matricaria chamomilla</Emphasis>

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA as the first committed enzyme in the mevalonate (MVA) pathway. HMGS plays an important role in the biosynthesis of the sesquiterpene, which is the main constituent of essential oil in Matricaria chamomilla. In this paper, a HMGS gene designated as McHMGS (GenBank Accession No. KU529970) was successfully cloned from M. chamomilla. The full-length cDNA of McHMGS was 1495-bp and contained a 1374-bp open reading frame. It encoded a 458-amino-acid protein with a calculated molecular weight of about 50.7 kDa and isoelectric point of 5.69. Sequence comparison revealed that McHMGS showed extensive homology with HMGSs from other plant species. Phylogenetic tree analysis indicated that McHMGS is clustered with the HMGS of Asteraceae in the dicotyledoneae clade. Further functional complementation of McHMGS in hmgs-deficient mutant yeast strain YSC6274 demonstrated that cloned McHMGS cDNA encodes a functional HMGS and mediates the MVA biosynthesis in yeasts. The tissue expression pattern analysis revealed that McHMGS expression level is highest in the flowers and lowest in the stems. Quantitative real-time PCR analysis showed that the expression of McHMGS was induced by MeJA, and the expression level is highest 24 h after induction. The characterization and expression of McHMGS can help in further studying the role of McHMGS gene in the biosynthesis of sesquiterpene in M. chamomilla.     

Cloning of gonadal aromatase gene <Emphasis Type="Italic">Cyp19a</Emphasis> in an endemic fish (<Emphasis Type="Italic">Schizothorax kozlovi</Emphasis>) of the Upper Yangtze River,and temperature effects on its expression

In order to elucidate the role of Cyp19a in sex differentiation of Schizothorax kozlovi, the full length cDNA of Cyp19a was cloned from the mature ovary of S. kozlovi by using rapid amplification of cDNA ends method, and then its relative mRNA expression levels among tissues and temperature groups were determined by using quantitative real-time PCR. The complete Cyp19a cDNA of 1795 bp of S. kozlovi was obtained, which encoded 517 amino acids and belonged to gonadal aromatase. Its deduced amino acid sequence had the above 70 % identity compared with gonadal aromatase genes of teleost fishes, but only 62–67 % when compared with brain aromatase genes of fishes. It was expressed only in heart and gonad, but no expression in other tissues, presenting relatively high tissue specificity. It also exhibited sex-specific expression pattern in gonads, but no sex differences in heart. Comparing with the Cyp19a expression levels at 12 days post hatching (dph), significant temperature effects were revealed in low temperature group (10 °C) at 18 dph, and in high temperature group (26 °C) at 40 dph. It suggested that gonadal aromatase Cyp19a gene may play important roles on the feminization or masculinization of S. kozlovi affected by temperature during the early developmental stage.     

Natural genetic variation in <Emphasis Type="Italic">Brassica</Emphasis> homologs of <Emphasis Type="Italic">FLOWERING LOCUS T</Emphasis> and characterization of its expression domains

FLOWERING LOCUS T (FT), a major effect gene, regulates flowering time in Arabidopsis. We analyzed evolutionary changes distinguishing two FT homeologous loci in B. rapa, described genetic variation in homologs isolated and reported expression pattern of FT in B. juncea. Synteny analysis confirmed presence of two FT genomic copies in B. rapa ssp. pekinensis and resolved pre-existing anomalies regarding copy number in “AA” genome. Synteny analysis of B. rapa homeologous regions CR1 (129 kb) and CR2 (232 kb) revealed differential gene fractionation and wide-spread re-arrangements. Seven genomic DNA (gDNA) variants (2.1–2.2 kb) and 10 complementary DNA (cDNA) variants (528 bp) were isolated from 6 Brassica species. The gDNA variants shared 72–99 % similarity within Brassica and 58–60 % between Arabidopsis and Brassica. FT cDNA variants shared 92–100 % similarity within Brassica and 87 % between Arabidopsis and Brassica. Phylogenetic analysis of FT gDNA, cDNA and protein sequences revealed two major clades, differentiating homologs derived from species containing shared “BB” and “CC” genomes. Phylogram based on Brassica FT gDNA differentiated homeologs derived from AA-LF (Least fractioned) and AA-MF1 (Moderately fractioned) sub-genomes. Analysis of FT expression pattern in B. juncea revealed increasing levels correlating with attainment of physiological maturity; highest levels were detected in older leaves implying conservation in spatio-temporal expression pattern vis-à-vis Arabidopsis. In conclusion, our study reveals that polyploidy in Brassicas resulted in expansion of FT gene copies with homologs charting independent evolutionary course through accumulation of mutations. However, expression domains of FT remained conserved across Brassicaceae to preserve the critical function of FT in controlling flowering time.     

Connection between proliferation rate and temozolomide sensitivity of primary glioblastoma cell culture and expression of <Emphasis Type="Italic">YB-1</Emphasis> and <Emphasis Type="Italic">LRP/MVP</Emphasis>

Glioblastomas (GBL) are the most common and aggressive brain tumors. They are distinguished by high resistance to radiation and chemotherapy. To find novel approaches for GBL classification, we obtained 16 primary GBL cell cultures and tested them with real-time PCR for mRNA expression of several genes (YB-1, MGMT, MELK, MVP, MDR1, BCRP) involved in controlling cell proliferation and drug resistance. The primary GBL cultures differed in terms of proliferation rate, wherein a group of GBL cell cultures with low proliferation rate demonstrated higher resistance to temozolomide. We found that GBL primary cell cultures characterized by high proliferation rate and lower resistance to temozolomide expressed higher mRNA level of the YB-1 and MDR1 genes, whereas upregulated expression of MVP/LRP mRNA was a marker in the group of GBL with low proliferation rate and high resistance. A moderate correlation between expression of YB-1 and MELK as well as YB-1 and MDR1 was found. In the case of YB-1 and MGMT expression, no correlation was found. A significant negative correlation was revealed between mRNA expression of MVP/LRP and MELK, MDR1, and BCRP. No correlation in expression of YB-1 and MVP/LRP genes was observed. It seems that mRNA expression of YB-1 and MVP/LRP may serve as a marker for GBL cell cultures belonging to distinct groups, each of which is characterized by a unique pattern of gene activity.     

Cloning,characterization and functional analysis of a flavonol synthase from <Emphasis Type="Italic">Vaccinium corymbosum</Emphasis>

Key message

VcFLS from Vaccinium corymbosum promoted myricetin biosynthesis in Arabidopsis thaliana and VcFLS expression was induced by salicylic acid.

Abstract

Flavonoids are polyphenols with important functions in pigmentation, UV filtration, and symbiotic nitrogen fixation. Flavonols are a class of flavonoids that are produced by the desaturation of dihydroflavanols in a reaction that is catalyzed by flavonol synthase (FLS). In the study reported here, we cloned the full-length cDNA of FLS (designated as VcFLS) from Vaccinium corymbosum (blueberry) using rapid amplification of cDNA ends (RACE). The cDNA contained a 1005-bp open reading frame that encoded a 334-amino acid protein. Phylogenetic analysis showed that VcFLS was closely related to FaFLS, a flavonol synthase that catalyzed the formation of kaempferol and had little effect on the formation of quercetin. Quantitative RT-PCR analysis demonstrated that VcFLS was expressed in all of the tissues tested, with particularly high expression in the petals and young leaves (both green and red). The flavanols myricetin and quercetin also occurred in all of these tested tissues, with the highest levels detected in mature leaves. The expression of VcFLS was not consistent with the accumulation of quercetin and myricetin in different tissues, nor were the expressions of VcFLS, VcPAL, VcCHS, VcF3H, and VcF3′5′H consistent with the accumulation of the quercetin during fruit development. However, the change in the trend of VcCHS and VcF3H expression was similar with myricetin accumulation during fruit development. Expression profiling analysis revealed that VcFLS expression was induced by salicylic acid, a phytohormone involved in plant defense against pathogens, and was suppressed by gibberellic acid, a phytohormone involved in various aspects of plant development. Heterologous expression of VcFLS in Arabidopsis thaliana increased the content of myricetin, but did not affect quercetin content. Thus, we conclude that VcFLS is a key enzyme in the flavonol biosynthetic pathway and would appear to be involved in the plant defense response.

     

LjCOCH interplays with LjAPP1 to maintain the nodule development in <Emphasis Type="Italic">Lotus japonicus</Emphasis>

Legume plants develop nodules during their symbiotic interaction with rhizobia, and much progress has been made towards understanding Nod factor perception and downstream signaling pathways, while our knowledge about the maintenance of nodule organogenesis was limited. We report here the knockdown mutants of LjCOCH, an ortholog of COCHLEATA in Pisum sativum, cause severe defects in nodule organogenesis in Lotus japonicus. The mature nodule number was drastically decreased accompanied with abnormal lenticel and vascular bundle developmental defects, but not produce roots from nodules in both Ljcoch mutants and LjCOCH-RNAi transgenic hairy roots. LjAPP1, a membrane-associated soluble aminopeptidase P1, was identified to interact with LjCOCH through yeast two-hybrid screening. Unlike that of Ljcoch mutants, insertion mutants of LjAPP1 and LjAPP1-RNAi transgenic hairy roots showed increased nodule number, while the lenticel and vascular development were not affected. Gene expression analysis indicated that LjCOCH and LjAPP1 were differentially upregulated by rhizobia inoculation, and LjNF-YA1 was the major downstream target of LjCOCH and LjAPP1. Our findings suggested that LjCOCH acts as a key factor involved in determinate nodule development through direct interaction with LjAPP1 to regulate the expression of LjNF-YA1, opposite effects of LjCOCH and LjAPP1 provide a dynamic regulation of nodule development in L. japonicus.     

In vitro regeneration, <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation,and genetic assay of chalcone synthase in the medicinal plant <Emphasis Type="Italic">Echinacea pallida</Emphasis>

In vitro plant regeneration was established in Echinacea pallida, a plant that is commonly used as a folk medicine to treat the common cold, fevers, inflammation and so on. Conditions for callus induction, lateral root and shoot regeneration were determined. Subsequently, two vectors pCHS and pOSAG78, carrying different selection marker genes resistant to kanamycin and hygromycin, respectively, were independently used to transform leaf explants of E. pallida using an Agrobacterium-mediated method. Genomic PCR analysis confirmed the presence of the transgene and selection marker gene in obtained transgenic lines. Southern hybridization indicated that the T-DNA insertion in some transgenic E. pallida was single copy. Among them, transformants carrying Petunia chalcone synthase (CHS) were selected for further study. CHS is a key enzyme in the biosynthesis of diverse flavonoids including anthocyanin pigmentation. Here, we analyzed the roles and compared the gene expression of two clusters of CHSs, EpaCHS-A and EpaCHS-B (EpaCHS-B1 and EpaCHS-B2), isolated from E. pallida. Two of the genes, EpaCHS-A and EpaCHS-B1, were abundantly expressed in petals, whereas EpaCHS-B2 was expressed at high levels in leaves. The expression of EpaCHSs remained constant in leaves and roots of Petunia CHS transformants, while EpaCHS-B2 expression was changed in flowers of transgenic plants. The biosynthesis of caffeic acid derivatives, cichoric acid and caftaric acid, was increased in leaves and roots of CHS transformants, respectively, while the amount of echinacoside in roots of transgenic plants was decreased. This is the first report on genetic engineering of E. pallida. The information contained herein can be used as a tool for further study of the biological pathways and secondary metabolism of specific compounds from medicinal Echinacea species.