The complete set of genetic material within a cell is known as a genome. The genomes of human and other animal cells have regions of active genes interspersed with ‘dark’ regions known as heterochromatin, which contain genes and other types of genetic material that have been inactivated.

Heterochromatin commonly contains sections of genetic material known as transposons. When a transposon is active it is able to move around the genome, therefore, inactivating (or ‘silencing’) transposons helps to maintain the integrity of the genetic material in a cell. It is particularly important to silence transposons in the stem cells that produce sperm and egg cells – known as germline stem cells – to ensure genetic information is faithfully passed on to the next generation.

A protein called HP1a plays a major role in directing where heterochromatin forms in the genome. This process requires an enzyme called dLsd1 to remove a small tag from the genetic material but it is not clear how HP1a regulates the activity of dLsd1. To address this question, Yang et al. studied how egg cells form in fruit flies, which are often used as models of animal biology in experiments.

The team screened a population of fruit flies that carried mutations in many different genes to identify genes that affect the fertility of female flies. This revealed a gene named as ovaries absent (or ova for short) is required for egg cells to form. In germline stem cells ova silences transposons and in the surrounding tissue it represses a specific signal that usually maintains stem cells to allow the stem cells to divide to make egg cells. Further experiments using biochemical techniques found that the protein encoded by ova acts as a bridge to bring HP1a and dLsd1 together to silence genes in heterochromatin.

The next step would be to identify the functional counterpart of the ova gene in mammals, including humans, which may help to discover causes of infertility and develop new fertility treatment.

In eukaryotic genomes, heterochromatin is mainly composed of repetitive sequences such as transposons that require active silencing (Slotkin and Martienssen, 2007). Heterochromatin is defined by the presence of repressive epigenetic methylation of histone H3 at lysine 9 (H3K9me) and by heterochromatin protein 1 (HP1), which binds to H3K9me sites (Lachner et al., 2001; Bannister et al., 2001). Heterochromatin formation is mediated by co-transcriptional gene silencing, a process that requires small RNAs and their associated argonaute proteins (Martienssen and Moazed, 2015). In Drosophila, the argonaute protein Piwi and Piwi-interacting RNAs (piRNAs) use base-pairing to target nascent transcripts to the corresponding transposon regions. The Piwi/piRNAs then recruit gene silencing machinery, including HP1a and the H3K9 methyltransferase Egg to form heterochromatin (Yang and Xi, 2017; Czech and Hannon, 2016; Brower-Toland et al., 2007). The formation of heterochromatin also requires erasing of active epigenetic mark by the H3K4 demethylase dLsd1 (Rudolph et al., 2007), but the molecular link between HP1a and local H3k4 demethylation remains elusive.

Piwi, a founding member of the piRNA pathway in Drosophila, was initially identified as a fertility factor; its mutation results in germline degeneration and sterility (Cox et al., 1998; Lin and Spradling, 1997). To identify new genes involved in Piwi/piRNA-mediated gene silencing, we here conducted a female fertility screen by EMS mutagenesis and identified a novel recessive mutation on the second chromosome. The homozygous mutant males are semi-lethal (Supplementary file 1, Table 1), but females are viable but do not lay any eggs; other than sterility, these females do not have other notable defects. Dissection revealed that these females had rudimentary ovaries: rather than a normal ovary, each oviduct in these mutant females was connected to only a tiny mass of cells (Figure 1a,b). Given this nearly ‘ovaryless’ phenotype, we named the gene associated with this mutation as ovaries absent (ova) and named this mutant allele ova¹ .

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Complementation mapping with deficiency lines, followed by sequencing of candidate genes led us to identify a single nucleotide deletion in an exon of CG5694, which results in a truncated protein of 387 amino acids (aa) rather than the predicted 623 aa full length protein (Figure 1—figure supplement 1a,b). CG5694 encodes a protein with no obvious sequence similarity to any existing proteins in the NCBI database, but does have a conserved nuclear respiratory factor-1 (NRF-1)- like domain at its N-terminus (15–105 aa); this DNA-binding domain was initially identified in the mammalian transcription factor NRF-1 and are known to occur in at least one other Drosophila transcription factor, Erect Wing (Ewg) (Figure 1—figure supplement 1c,d). We used CRISPR-Cas9 to generate a knock-out allele in which the entire coding region of CG5694 was deleted (Figure 1—figure supplement 1e). Homozygous knock-out allele females are sterile and exhibit virtually identical ‘ovaryless’ phenotypes as the ova¹ females (Figure 1a). Additionally, transgenic expression of a genomic DNA fragment containing the ova gene region was able to effectively rescue the ovary defect and restore fertility of ova¹ homozygous or CG5694 null females (Figure 1a,b). Therefore, ova is allelic to CG5694.

Normally, oogenesis initiates in the germarium, an anterior part of the ovariole where germline stem cells (GSCs) reside. Each germarium normally harbors 2–3 GSCs that can be distinguished by spherically-shaped spectrosome and by their direct contact with the cap cell niche (Figure 1c). The decedents of GSCs move posteriorly as they differentiate into germline cyst, and then bud off from the germarium to form egg chambers (Xie, 2013; Spradling, 1993). Immunostaining of ova¹ homozygous and ova^1/4 trans-heterozygous ovaries revealed that the mutant ovaries completely lacked vitellaria, and the germaria were either full of GSC-like cells [77% (n = 117) of ova¹ germaria] (Figure 1c,d,g) or entirely germless (lacking Vasa expression (Figure 1c,d,g), suggesting that ova is required for GSC differentiation and for germline survival.

To determine whether ova functions cell-autonomously in the germline and/or non-cell-autonomously in somatic supporting cells to regulate GSCs, we conducted mosaic analysis by inducing mitotic clones using a FLP-FRT system (Xu and Rubin, 1993). Similar to wild-type control clones, ova¹ mutant GSC clones behaved normally: the mutant GSCs were properly maintained in the niche, and their descendant cells were properly differentiated into germline cysts and egg chambers with properly specified oocytes (Figure 1—figure supplement 2a–d), although germline mutant eggs failed to hatch (Figure 1b). Similarly, germline-specific knocking down ova by UAS-Dcr2; nos-GAL4 (thereafter referred as ova GLKD) also did not cause any obvious defects in ovary morphology, and the number of GSCs and their immediate daughter cystoblasts (collectively referred to as GSC-like cells) per germarium remained largely normal (Figure 1—figure supplement 2g,h). Collectively, these data demonstrate that ova is not cell-autonomously required for the early stages of GSC differentiation. We next used a temperature sensitive GAL4/UAS system (Brand and Perrimon, 1993; McGuire et al., 2004) to specifically deplete ova in somatic escort cells with c587-GAL4 (c587 >ova RNAi) (Song et al., 2004). The somatic escort cells, which usually send out long protrusions that encapsulate the germline, are known to provide the niche environment required for germline cyst differentiation (Kirilly et al., 2011; Morris and Spradling, 2011). After treatment at the restrictive temperature, c587 >ova RNAi germaria began to exhibit a significantly increased number of spectrosome-containing GSC-like cells in a time-dependent manner (Figure 1e,g). The mutant escort cells were still able to send protrusions to the encapsulate the germline cells (Figure 1e), indicating that the GSC differentiation defects is likely not caused by defects in escort cell morphology. The requirement for ova i in somatic escort cells for proper GSC differentiation was further supported by the observation that escort cell-specific expression of an ova transgene was sufficient to rescue the ovary defects of ova mutant females (hereafter referred to as ova germline mutants) (Figure 1f). Therefore, ova functions in somatic escort cells and regulates germline differentiation in the germarium in a non-cell-autonomous manner.

The ova mutant phenotype is reminiscent of the piwi mutant phenotypes: piwi mutant flies also have rudimentary ovaries that contain an abnormal number of differentiation-blocked GSC-like cells, and piwi also functions primarily in somatic escort cells to regulate GSC differentiation (Jin et al., 2013; Ma et al., 2014). Loss of piwi in escort cells causes de-repression of decapentaplegic (dpp), a major self-renewal signal for GSCs, leading to GSC-like cell accumulation in the germarium (Jin et al., 2013; Ma et al., 2014). Interestingly, we found that the ova phenotype was also associated with increased dpp signaling. The extra GSC-like cells in c587 >ova RNAi germaria had dramatically increased expression of Dad-lacZ and phosphorylated Mad (pMad) (Figure 1h,i), both of which are reporters of BMP pathway activity, and decreased expression of bam (Figure 1h), a gene whose expression is normally suppressed by BMP signaling (Song et al., 2004; Chen and McKearin, 2003). Collectively, these data suggest that loss of ova in escort cells leads to ectopic dpp signaling that blocks further GSC differentiation, leading to GSC-like cell accumulation in the germarium.

The phenotypic and molecular similarities between the ova and piwi mutants led us to further test whether Ova and Piwi act via the same genetic pathway to regulate GSCs. As expected, we observed that homozygous piwi mutant females had rudimentary ovaries. As a positive control, escort cell-specific expression of a piwi transgene was sufficient to rescue the piwi mutant ovary phenotype (Figure 2a,b). On the one hand, transgenic expression of ova in escort cells of piwi mutants also partially rescued the ovary morphology phenotype with the frequent appearance of developing germline cysts, including late stages of egg chambers, although oogenesis was still abnormal, GSC-like tumor still remained, and no mature eggs were produced (Figure 2a,b,c). On the other hand, transgenic expression of piwi in escort cells of ova mutants could not rescue any ovary phenotypes (Figure 2a,b). Consistent with previous reports of piwi phenotypes, escort cell-specific knocking down of other Piwi/piRNA pathway effectors, such as panx, also showed a similar GSC-l accumulation phenotype (Figure 2d,e), further supporting the idea that Ova may participate in the same Piwi/piRNA pathway. Next, we tested whether overexpression of ova could rescue the germline TE upregulation phenotype caused by piwi mutation. As a control, ubiquitous expression of piwi, but not soma-only expression of piwi was able to effectively rescue the TE upregulation phenotype. However, neither ubiquitous nor soma-specific expression of ova could rescue the TE upregulation phenotype in piwi mutants (Figure 2f). These observations suggest that, genetically, ova acts downstream of piwi, but there must be additional factors downstream of piwi that cooperatively function with ova to regulate GSC differentiation and transposon silencing.

Figure 2

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Given that Piwi is associated with a number of chromatin factors that are known to regulate heterochromatin formation and germline transposon silencing, and considering that dpp silencing in escort cells can be attributed to Piwi-dependent gene silencing, we asked whether Ova is also associated with these silencing machinery components and somehow participates in these processes. We performed a yeast two-hybrid (Y2H) screen for potential physical interactions among Ova and other known essential components of the heterochromatin silencing machinery (Yu et al., 2015; Sienski et al., 2015; Sienski et al., 2012), including: Panoramix (Panx), Arx, and Mael, which participates in Piwi/piRNA-mediated gene silencing (Yu et al., 2015; Sienski et al., 2015; Sienski et al., 2012; Muerdter et al., 2013; Dönertas et al., 2013; Ohtani et al., 2013); HP1a, the H3K9me3 methyltransferase Eggless (Egg), and the Egg cofactor Windei (Wde) (Seum et al., 2007; Tzeng et al., 2007; Koch et al., 2009); the H3K4me2 demethylase dLsd1 and its cofactor CoREST (Rudolph et al., 2007); and Piwi. The Y2H screen identified two positive interactions: Ova and HP1a, and Ova and dLsd1 (Figure 3a and Figure 3—figure supplement 1). Notably, the previously reported interaction between HP1a and Piwi was not observed in our screen here (Figure 3—figure supplement 1) (Brower-Toland et al., 2007), possibly due to different expression systems used in the studies. Co-immunoprecipitation experiments also showed positive interactions between Ova and HP1a and between Ova and dLsd1 in ovary lysates (Figure 3b,c). Collectively, these results indicate that Ova is physically associated with the co-transcriptional silencing machinery and directly interacts with HP1a and with dLsd1. Previous studies have reported that HP1a and dLsd1 function in the escort cell niche to restrict dpp signaling and to facilitate GSC differentiation (Wang et al., 2011; Eliazer et al., 2011). These reports, considered alongside the known role of Piwi-dependent gene silencing of the dpp gene locus in normal escort cells, further supporting the notion that these three Piwi-associated factors (Ova, HP1a, dLsd1) function in a shared pathway in escort cells to establish a repressive chromatin state for the dpp gene locus.

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We next tested whether Ova, similar to HP1a and dLsd1 (Wang and Elgin, 2011; Czech et al., 2013), is required for heterochromatin formation and germline transposon silencing. The white locus of In(l)w^m4h chromosomal reversion flies is relocated to a position next to a heterochromatin region, and this relocation often causes heterochromatin-based silencing of this gene, resulting from a genomic phenomenon referred to as position effect variegation (PEV), these flies typically display mosaic eyes with red and white facets as a result of this relocation based silencing (Wallrath and Elgin, 1995; Schotta et al., 2003) (Figure 3—figure supplement 2a). Interestingly, removing one functional copy of ova from the In(l)w^m4h background was sufficient cause fully-pigmented eyes (Figure 3—figure supplement 2a). Analysis using several additional PEV reporter fly lines (118E-10, 118E-15, 39 C-72, and 6 M-193), each of which has its white gene locus relocated (inserted) into the heterochromatin rich fourth chromosome, showed that ova acts as a suppressor of PEV: the ova transheterozygous flies had fully-pigmented eyes with increased pigment level whereas the ova heterozygous flies from all three of the reporter lines had mosaic eyes (P values by two-tailed Student t-test, Figure 3—figure supplement 2b,c). It thus appears that ova has a functional role in heterochromatic gene silencing.

To test whether or not ova functions in germline transposon silencing, we performed germline-specific knock-down of ova using the UAS-Dcr2; nos-GAL4 driver (ova GLKD), followed by RNA-seq analysis. Interestingly, nos > ova RNAi ovaries had dramatically up-regulated transcripts of a subset of transposons that included the LTR element 412 and the telomeric non-LTR repeats Het-A, TAHRE, Tart (Figure 3d). By comparison, the expression of protein-coding genes and piRNAs was largely un-altered (Figure 3—figure supplement 3a,b,e). Somatic cell-specific knock-down of ova (tj-GAL4 >ova RNAi) only caused mild, if any, TE upregulation (Figure 3—figure supplement 3f). Consistent with a role in germline transposon silencing, a previously reported genetic screen for genes involved in germline transposon silencing identified ova (CG5694) as one of the top hits (Muerdter et al., 2013; Czech et al., 2013). Notably, germline-specifc knock-down of either ova (ova GLKD) or dLsd1 (dLsd1 GLKD) exhibited de-repression of a similar subset of transposons (R = 0.9463 by Pearson’s correlation coefficient, Figure 3e); this subset is distinguished by enrichment for bivalent histone marks (both H3K9me3 and H3K4me2) (Czech et al., 2013; Klenov et al., 2014). In mutants deficient in piRNA biogenesis, the inability to form Piwi/piRNA complexes typically results in retention of Piwi in the cytoplasm (Wang and Elgin, 2011; Malone et al., 2009; Olivieri et al., 2010). The fact that nuclear Piwi localization was largely unaffected in the ova mutant germline (Figure 3—figure supplement 3c) further supports our conclusion that ova is not required for piRNA biogenesis, but may function at the chromatin to mediate Piwi/piRNAs- induced transcriptional gene silencing, a phase that has been referred to as ‘effector step’ (Czech et al., 2013).

To explore the biochemical mechanisms underlying Ova function in greater detail, we used Y2H assays to identify the Ova domains required for its interactions with HP1a and/or dLsd1. We constructed multiple truncated forms of Ova (Figure 4a), and found that the Ova 250–486 fragment and the Ova 388–623 fragment were both able to interact with the chromo shadow domain (CSD) of HP1a (Figure 4b); neither of these Ova fragments could interact with the chromodomain (CD) of HP1a (Figure 4b). We next constructed an Ova fragment composed of the overlapped 388–486 region and confirmed that this fragment was sufficient for interaction with the CSD domain of HP1a (Figure 4b). Mapping the interaction domains of Ova with dLsd1 revealed that both Ova 1–388 and Ova 250–486 fragments, but not Ova 388–486 fragment, could interact with dLsd1 (Figure 4c). Interestingly, transgene expression of the Ova 250–486 fragment, which is able to interact with both HP1a and dLsd1, was sufficient to rescue both the ovary development defect and transposon silencing defect of ova mutant females, similar to the effect produced by transgene expression of a full length ova (Figure 4d,f). In contrast, no rescue effect was observed with the transgenic expression of the Ova 388–623 fragment, which interacts with HP1a only, or with expression of the Ova 1–388 fragment, which interacts with dLsd1 only (Figure 4d). Therefore, the domain that is sufficient to interact with both HP1a and dLsd1 is sufficient for Ova function in ovary development and transposon silencing. These biochemical and genetic experiments indicate that Ova may serve as a protein adaptor that links HP1a and dLsd1. To functionally test this putative adaptor function in vivo, we generated a transgene expressing HP1a::dLsd1 fusion protein. If Ova merely functions as an adapter that bridges the two proteins, the HP1a::dLsd1 transgene should render Ova dispensable and therefore should be able to rescue the ova mutant phenotypes. Strikingly, transgenic expression of HP1a::dLsd1 in escort cells was sufficient to rescue the rudimentary ovary phenotype of ova mutants (Figure 4e). Eighty percent of the HP1a::dLsd1 rescued germaria contained 2–5 GSC-l (n = 41) and all the germaria had properly differentiating cysts. Moreover, ubiquitous expression of HP1a::dLsd1 also significantly rescued the transposon silencing defects of ova mutants and partially restored female fertility (Figure 4f). Given that the genomic fragment transgene of ova (ova-g), which includes the cis-elements of ova, could fully restore fertility (Figure 1a), the incomplete rescue of fertility by the HP1a::dLsd1 fusion could be due to non-physiological levels of the transgene expression. Alternatively, ova could have additional roles beyond the adaptor role that are important for female fertility. In addition to increased expression of transposons, ova germline mutant ovaries also showed moderate upregulation of many protein-coding genes (Figure 4—figure supplement 1). Interestingly, this transgene expression also effectively brought the expression of many protein-coding genes back to wild-type levels (Figure 4—figure supplement 1). These observations indicate that HP1a and Ova may participate in transcriptional silencing of many regular protein-coding genes, in addition to transposons. We conclude that Ova acts as a protein adaptor to link HP1a and dLsd1 to promote HP1a-mediated gene silencing.

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Since dLsd1 catalyzes H3K4me2 demethylation, Ova may function to link dLsd1 and HP1a for local H3K4 demethylation during heterochromatic gene silencing. Indeed, ChIP-seq analysis revealed that the H3K4me2 density was specifically increased at Het-A and TAHRE loci but not other TE loci (Figure 5—figure supplement 1). Further analysis revealed that there was a significant increase in H3K4me2 levels and in RNA Pol II occupancy at the 3’UTR of the Het-A and TAHRE transposons in ova GLKD ovarian germline cells (Figure 5a,b). Note that these telomeric transposons are arranged in a head-to-tail fashion; therefore, the 3’ UTR of one element likely directs the transcription of its downstream neighbor (Danilevskaya et al., 1997). To further test this potential role of Ova in linking H3K4 demethylation during HP1a-mediated gene silencing in vivo, we used a clean lacI/lacO reporter system to tether lacI-HP1a to the promoter of a lacO-GFP reporter (Sienski et al., 2015). We found that 26% of ovarioles examined (n = 131) had reduced GFP signal in their germline upon lacI-HP1a induction (Figure 5c,d), although there was no significant reduced in the overall level of GFP mRNA (P value by two-tailed Student t-test, Figure 5e). Importantly, co-expression of ova in the germline caused a significant increase in the number of ovarioles with reduced or abolished GFP signal [86% (n = 138)], and the overall GFP mRNA level was also significantly reduced in these samples (P value by two-tailed Student t-test, Figure 5c–e). ChIP-seq analysis showed that this reduction in reporter expression was accompanied by significantly reduced H3K4m2 levels near the promoter region of the GFP gene reporter (Figure 5f,h).

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To further confirm that the alteration of H3K4 deposition is a consequence of Ova recruitment, rather than a secondary effect following altered gene transcription, we performed a similar set of experiments, but with a modified lacO-terminator-GFP reporter that has a transcriptional terminator immediately following the promoter (Figure 5g). This should result in blocked transcription no matter whether a transcriptional activator/repressor is present or not. As expected, this reporter showed a significant reduction of baseline transcription (down to approximately 3.8%) (P value by two-tailed Student t-test, Figure 5i). We found that tethering lacI-HP1a to the promoter failed to alter the H3K4me2 level proximal to the tethering site. Co-expression of Ova, however, almost erased entirely the H3K4me2 marks in the proximal region (Figure 5g,h). These observations further support the notion that Ova links HP1a and dLsd1 for local erasing of H3K4me2 marks.

We also tested whether Ova itself can induce gene silencing by tethering Ova directly to DNA and to mRNA using in vivo reporter systems in the ovarian germline. We used a lacI-Ova and lacO-GFP binary system to tether Ova to genomic DNA (Sienski et al., 2015) and found that such tethering did not have any obvious effect on GFP expression (Figure 5—figure supplement 2a–c). Similarly, tethering Ova to mRNA using a λN-Ova and GFP-boxB binary system did not cause any obvious effect on GFP expression (Figure 5—figure supplement 2d–f). These results are consistent with the idea that Ova acts downstream of HP1a in heterochromatic gene silencing.

Similar to other ‘effector step’ mutations, the loss of ova or dlsd1 only causes de-repression of a subset of transposons; this is in contrast with the widespread transposon de-repression that is common in mutations affecting piRNA biogenesis (Czech et al., 2013). This disparity can possibly be explained by the existence of different silencing mechanisms for particular subsets of transposons. Illustrating this idea, our work suggests that transposons with bivalent histone marks may be preferential targets for Ova and dLsd1. A bivalent pattern of histone methylation may help to regulate the expression of transposons that require a delicate On/Off balance, for example with the expression of telomeric repeats known to be required for normal telomere function (e.g., Het-A, TAHRE, and Tart). The results of our study establishes that Ova has an indispensable role in facilitating dLsd1’s H3K4 demethylation activity during HP1a-induced heterochromatic gene silencing and demonstrates that this Ova function is essential for germline development, heterochromatin formation, and Piwi/piRNA-mediated co-transcriptional gene silencing. Our study suggests that the Piwi/piRNA pathway may adapt a similar effector machinery to repress regular genes, such as the dpp gene in escort cells, in addition to TEs. A study in S. pombe reported a mechanism in which a RNAi protein complex links the activity of the H3K9 methyltransferase Clr4 with H3K4 demethylation by the H3K4 demethylase Lid2 (Li et al., 2008), indicating an evolutionarily conserved interplay of epigenetic marks during transcriptional gene silencing. Given that the mechanisms underlying heterochromatic gene silencing are known to be strongly conserved from Drosophila to mammals, an equivalent functional module that links HP1a with H3K4 demethylation likely exists in mammals as well.

High-throughput sequence data GEO link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE104925

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Author details

1. Fu Yang

   National Institute of Biological Sciences, Beijing, China

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   yangfu@nibs.ac.cn

   Competing interests

   No competing interests declared

2. Zhenghui Quan

   National Institute of Biological Sciences, Beijing, China

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Writing—original draft

   Competing interests

   No competing interests declared

3. Huanwei Huang

   National Institute of Biological Sciences, Beijing, China

   Contribution

   Data curation, Software, Formal analysis, Methodology

   Competing interests

   No competing interests declared

4. Minghui He

   National Institute of Biological Sciences, Beijing, China

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

5. Xicheng Liu

   National Institute of Biological Sciences, Beijing, China

   Contribution

   Data curation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7257-1396

6. Tao Cai

   National Institute of Biological Sciences, Beijing, China

   Contribution

   Software, Formal analysis, Supervision

   Competing interests

   No competing interests declared

7. Rongwen Xi

   1. National Institute of Biological Sciences, Beijing, China
   2. Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, Beijing, China

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   xirongwen@nibs.ac.cn

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5543-1236

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