Abstract
It is not known at present whether neuronal cell-type diversity—defined by cell-type-specific anatomical, biophysical, functional and molecular signatures—can be reduced to relatively simple molecular descriptors of neuronal identity1. Here we show, through examination of the expression of all of the conserved homeodomain proteins encoded by the Caenorhabditis elegans genome2, that the complete set of 118 neuron classes of C. elegans can be described individually by unique combinations of the expression of homeodomain proteins, thereby providing—to our knowledge—the simplest currently known descriptor of neuronal diversity. Computational and genetic loss-of-function analyses corroborate the notion that homeodomain proteins not only provide unique descriptors of neuron type, but also have a critical role in specifying neuronal identity. We speculate that the pervasive use of homeobox genes in defining unique neuronal identities reflects the evolutionary history of neuronal cell-type specification.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
All newly generated data, including the expression pattern of every homeobox gene, are available in Supplementary Tables 1, 2. Additionally, whole-worm confocal images of all homeobox genes analysed are available in Extended Data Figs. 1–8. Newly generated reporter strains made during this study are available from the Caenorhabditis Genetics Center. The most up-to-date version of the community-curated transgene expression resource used is available in Supplementary Table 5.
Code availability
The R code used to generate the Jaccard distance matrix for the clustering of homeobox genes and neuron classes is available on the GitHub of the O.H. laboratory, at https://github.com/hobertlab/Reilly_2020/tree/master/Jaccard_Distance. The MATLAB code used to create the minimal codebook of homeobox genes is available at https://github.com/hobertlab/Reilly_2020/tree/master/Minimal_Codebook.
References
Zeng, H. & Sanes, J. R. Neuronal cell-type classification: challenges, opportunities and the path forward. Nat. Rev. Neurosci. 18, 530–546 (2017).
Hench, J. et al. The homeobox genes of Caenorhabditis elegans and insights into their spatio-temporal expression dynamics during embryogenesis. PLoS ONE 10, e0126947 (2015).
Sebe-Pedros, A. et al. Cnidarian cell type diversity and regulation revealed by whole-organism single-cell RNA-seq. Cell 173, 1520–1534 (2018).
Zeisel, A. et al. Molecular architecture of the mouse nervous system. Cell 174, 999–1014 (2018).
Tasic, B. et al. Shared and distinct transcriptomic cell types across neocortical areas. Nature 563, 72–78 (2018).
Hodge, R. D. et al. Conserved cell types with divergent features in human versus mouse cortex. Nature 573, 61–68 (2019).
Gehring, W. J. Master Control Genes in Development and Evolution: The Homeobox Story (Yale Univ. Press, 1998).
Way, J. C. & Chalfie, M. mec-3, a homeobox-containing gene that specifies differentiation of the touch receptor neurons in C. elegans. Cell 54, 5–16 (1988).
Finney, M., Ruvkun, G. & Horvitz, H. R. The C. elegans cell lineage and differentiation gene unc-86 encodes a protein with a homeodomain and extended similarity to transcription factors. Cell 55, 757–769 (1988).
White, J. G., Southgate, E. & Thomson, J. N. Mutations in the Caenorhabditis elegans unc-4 gene alter the synaptic input to ventral cord motor neurons. Nature 355, 838–841 (1992).
Jin, Y., Hoskins, R. & Horvitz, H. R. Control of type-D GABAergic neuron differentiation by C. elegans UNC-30 homeodomain protein. Nature 372, 780–783 (1994).
Hobert, O. A map of terminal regulators of neuronal identity in Caenorhabditis elegans. Wiley Interdiscip. Rev. Dev. Biol. 5, 474–498 (2016).
Tsuchida, T. et al. Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes. Cell 79, 957–970 (1994).
Lindtner, S. et al. Genomic resolution of DLX-orchestrated transcriptional circuits driving development of forebrain GABAergic neurons. Cell Rep. 28, 2048–2063 (2019).
Stettler, O. & Moya, K. L. Distinct roles of homeoproteins in brain topographic mapping and in neural circuit formation. Semin. Cell Dev. Biol. 35, 165–172 (2014).
Tahayato, A. et al. Otd/Crx, a dual regulator for the specification of ommatidia subtypes in the Drosophila retina. Dev. Cell 5, 391–402 (2003).
Blochlinger, K., Bodmer, R., Jack, J., Jan, L. Y. & Jan, Y. N. Primary structure and expression of a product from cut, a locus involved in specifying sensory organ identity in Drosophila. Nature 333, 629–635 (1988).
Sugino, K. et al. Mapping the transcriptional diversity of genetically and anatomically defined cell populations in the mouse brain. eLife 8, e38619 (2019).
Davis, F. P. et al. A genetic, genomic, and computational resource for exploring neural circuit function. eLife 9, e50901 (2020).
Allen, A. M. et al. A single-cell transcriptomic atlas of the adult Drosophila ventral nerve cord. eLife 9, e54074 (2020).
White, J. G., Southgate, E., Thomson, J. N. & Brenner, S. The structure of the nervous system of the nematode Caenorhabditis elegans. Phil. Trans. R. Soc. Lond. B 314, 1–340 (1986).
Hobert, O., Glenwinkel, L. & White, J. Revisiting neuronal cell type classification in Caenorhabditis elegans. Curr. Biol. 26, R1197–R1203 (2016).
Bürglin, T. R. & Affolter, M. Homeodomain proteins: an update. Chromosoma 125, 497–521 (2016).
Bürglin, T. R., Finney, M., Coulson, A. & Ruvkun, G. Caenorhabditis elegans has scores of homoeobox-containing genes. Nature 341, 239–243 (1989).
Lambert, S. A. et al. The human transcription factors. Cell 172, 650–665 (2018).
Fuxman Bass, J. I. et al. A gene-centered C. elegans protein–DNA interaction network provides a framework for functional predictions. Mol. Syst. Biol. 12, 884 (2016).
Murray, J. I. et al. Automated analysis of embryonic gene expression with cellular resolution in C. elegans. Nat. Methods 5, 703–709 (2008).
Yemini, E. et al. NeuroPAL: a neuronal polychromatic atlas of landmarks for whole-brain imaging in C. elegans. Preprint at https://www.biorxiv.org/content/10.1101/676312v1 (2019).
Hobert, O. Terminal selectors of neuronal identity. Curr. Top. Dev. Biol. 116, 455–475 (2016).
Merabet, S. & Mann, R. S. To be specific or not: the critical relationship between Hox and TALE proteins. Trends Genet. 32, 334–347 (2016).
Packer, J. S. et al. A lineage-resolved molecular atlas of C. elegans embryogenesis at single-cell resolution. Science 365, eaax1971 (2019).
Cao, J. et al. Comprehensive single-cell transcriptional profiling of a multicellular organism. Science 357, 661–667 (2017).
Kratsios, P. et al. An intersectional gene regulatory strategy defines subclass diversity of C. elegans motor neurons. eLife 6, e25751 (2017).
Schneider, J. et al. UNC-4 antagonizes Wnt signaling to regulate synaptic choice in the C. elegans motor circuit. Development 139, 2234–2245 (2012).
Hobert, O. Development of left/right asymmetry in the Caenorhabditis elegans nervous system: from zygote to postmitotic neuron. Genesis 52, 528–543 (2014).
Serrano-Saiz, E. et al. Modular control of glutamatergic neuronal identity in C. elegans by distinct homeodomain proteins. Cell 155, 659–673 (2013).
Serrano-Saiz, E., Oren-Suissa, M., Bayer, E. A. & Hobert, O. Sexually dimorphic differentiation of a C. elegans hub neuron is cell autonomously controlled by a conserved transcription factor. Curr. Biol. 27, 199–209 (2017).
Pereira, L. et al. A cellular and regulatory map of the cholinergic nervous system of C. elegans. eLife 4, e12432 (2015).
Lloret-Fernández, C. et al. A transcription factor collective defines the HSN serotonergic neuron regulatory landscape. eLife 7, e32785 (2018).
Doitsidou, M. et al. A combinatorial regulatory signature controls terminal differentiation of the dopaminergic nervous system in C. elegans. Genes Dev. 27, 1391–1405 (2013).
Dobzhansky, T. Biology, molecular and organismic. Am. Zool. 4, 443–452 (1964).
Dickinson, D. J., Pani, A. M., Heppert, J. K., Higgins, C. D. & Goldstein, B. Streamlined genome engineering with a self-excising drug selection cassette. Genetics 200, 1035–1049 (2015).
Dokshin, G. A., Ghanta, K. S., Piscopo, K. M. & Mello, C. C. Robust genome editing with short single-stranded and long, partially single-stranded DNA donors in Caenorhabditis elegans. Genetics 210, 781–787 (2018).
Sarov, M. et al. A genome-scale resource for in vivo tag-based protein function exploration in C. elegans. Cell 150, 855–866 (2012).
Feng, W. et al. A terminal selector prevents a Hox transcriptional switch to safeguard motor neuron identity throughout life. eLife 9, e50065 (2020).
Patel, T. & Hobert, O. Coordinated control of terminal differentiation and restriction of cellular plasticity. eLife 6, e24100 (2017).
Leyva-Díaz, E. & Hobert, O. Transcription factor autoregulation is required for acquisition and maintenance of neuronal identity. Development 146, dev177378 (2019).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Cassata, G. et al. The LIM homeobox gene ceh-14 confers thermosensory function to the AFD neurons in Caenorhabditis elegans. Neuron 25, 587–597 (2000).
Way, J. C. & Chalfie, M. The mec-3 gene of Caenorhabditis elegans requires its own product for maintained expression and is expressed in three neuronal cell types. Genes Dev. 3 (12A), 1823–1833 (1989).
Miller, D. M., III & Niemeyer, C. J. Expression of the unc-4 homeoprotein in Caenorhabditis elegans motor neurons specifies presynaptic input. Development 121, 2877–2886 (1995).
Kellerer, H., Pferschy, U. & Pisinger, D. Knapsack Problems (Springer, 2004).
Schrijver, A. Theory of Linear and Integer Programming (John Wiley & Sons, 1998).
Mukaka, M. M. Statistics corner: a guide to appropriate use of correlation coefficient in medical research. Malawi Med. J. 24, 69–71 (2012).
Acknowledgements
We thank Q. Chen for expert technical assistance in generating transgenic lines; E. Berghoff for communicating the unpublished expression of unc-42; E. Leyva-Díaz for CRISPR-tagging ceh-44 and ceh-48; R. Dowen for sending a ceh-60 reporter allele; V. Bertrand for communicating unpublished results on ceh-10 and ttx-3 reporter alleles; L. Glenwinkel for updating and sharing the community-curated gene expression resource; J. Booth for creation of select extrachromosomal fosmid reporter strains; Y. Ramadan for help with mutant analysis; and T. Bürglin for comments on the manuscript. This work was funded by a predoctoral fellowship to M.B.R. (F31 NS105398), by NIH R21 NS106843, and by the Howard Hughes Medical Institute. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).
Author information
Authors and Affiliations
Contributions
M.B.R. and O.H. designed the experiments and wrote the manuscript, M.B.R. generated constructs and conducted the expression pattern analysis, C.C. conducted genetic loss-of-function experiments and contributed to writing the paper, E.V. and E.Y. conducted the bioinformatic analysis and contributed to writing the paper.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Peer review information Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 ANTP and HOXL homeodomain protein expression patterns.
Neuronal cell identifications with NeuroPAL are provided for one member of each class and shown as the GFP reporter alone, the NeuroPAL landmark alone and a merged image of the GFP reporter with the NeuroPAL landmark. Neurons are circled in yellow and the identities of those neurons by NeuroPAL identifier are immediately beside them. V indicates the vulva of the worm. Heads, midbodies and tails of each reporter are shown and labelled accordingly. The outer body of the worm and pharynx are outlined in white. All images are of L4 or young adult worms unless otherwise noted as an expression outside of the L4 nervous system. Ten worms were analysed for each reporter strain and characteristic images were chosen.
Extended Data Fig. 2 NKL homeodomain protein expression patterns.
Neuronal cell identifications with NeuroPAL are provided for one member of each class and shown as the GFP reporter alone, the NeuroPAL landmark alone and a merged image of the GFP reporter with the NeuroPAL landmark. Neurons are circled in yellow and the identities of those neurons by NeuroPAL identifer are immediately beside them. V indicates the vulva of the worm. Heads, midbodies and tails of each reporter are shown and labelled accordingly. The outer body of the worm and pharynx are outlined in white. All images are of L4 or young adult worms unless otherwise noted as an expression outside of the L4 nervous system. Ten worms were analysed for each reporter strain and characteristic images were chosen.
Extended Data Fig. 3 CUT and LIM homeodomain expression patterns.
Neuronal cell identifications with NeuroPAL are provided for one member of each class and shown as the GFP reporter alone, the NeuroPAL landmark alone and a merged image of the GFP reporter with the NeuroPAL landmark. Neurons are circled in yellow and the identities of those neurons by NeuroPAL identifier are immediately beside them. V indicates the vulva of the worm. Heads, midbodies and tails of each reporter are shown and labelled accordingly. The outer body of the worm and pharynx are outlined in white. All images are of L4 or young adult worms unless otherwise noted as an expression outside of the L4 nervous system. Ten worms were analysed for each reporter strain and characteristic images were chosen.
Extended Data Fig. 4 POU, PRD and SIX homeodomain protein expression patterns.
Neuronal identifications with NeuroPAL are provided for one member of each class and shown as the GFP reporter alone, the NeuroPAL landmark alone, and a merged image of the GFP reporter with the NeuroPAL landmark. Neurons are circled in yellow and the identities of those neurons by NeuroPAL identifier are immediately beside them. V indicates the vulva of the worm. Heads, midbodies and tails of each reporter are shown and labelled accordingly. The outer body of the worm and pharynx are outlined in white. All images are of L4 or young adult worms unless otherwise noted as an expression outside of the L4 nervous system. Ten worms were analysed for each reporter strain and characteristic images were chosen.
Extended Data Fig. 5 PRD-like homeodomain protein expression patterns.
Neuronal cell identifications with NeuroPAL are provided for one member of each class and shown as the GFP reporter alone, the NeuroPAL landmark alone and a merged image of the GFP reporter with the NeuroPAL landmark. Neurons are circled in yellow and the identities of those neurons by NeuroPAL identifier are immediately beside them. V indicates the vulva of the worm. Heads, midbodies and tails of each reporter are shown and labelled accordingly. The outer body of the worm and pharynx are outlined in white. All images are of L4 or young adult worms unless otherwise noted as an expression outside of the L4 nervous system. Ten worms were analysed for each reporter strain and characteristic images were chosen.
Extended Data Fig. 6 HNF, PROS, TALE and ZF homeodomain protein expression patterns.
Neuronal cell identifications with NeuroPAL are provided for one member of each class and shown as the GFP reporter alone, the NeuroPAL landmark alone and a merged image of the GFP reporter with the NeuroPAL landmark. Neurons are circled in yellow and the identities of those neurons by NeuroPAL identifier are immediately beside them. V indicates the vulva of the worm. Heads, midbodies and tails of each reporter are shown and labelled accordingly. The outer body of the worm and pharynx are outlined in white. All images are of L4 or young adult worms unless otherwise noted as an expression outside of the L4 nervous system. Ten worms were analysed for each reporter strain and characteristic images were chosen.
Extended Data Fig. 7 Divergent homeodomain protein expression patterns.
Neuronal cell identifications with NeuroPAL are provided for one member of each class and shown as the GFP reporter alone, the NeuroPAL landmark alone and a merged picture of the GFP reporter with the NeuroPAL landmark. Neurons are circled in yellow and the identities of those neurons by NeuroPAL identifier are immediately beside them. V indicates the vulva of the worm. Heads, midbodies and tails of each reporter are shown and labelled accordingly. The outer body of the worm and pharynx are outlined in white. All images are of L4 or young adult worms unless otherwise noted as an expression outside of the L4 nervous system. Ten worms were analysed for each reporter strain and characteristic images were chosen.
Extended Data Fig. 8 Homeobox gene expression ordered by lineage and neurotransmitter identity.
Representation of homeobox gene expression pattern with neurons ordered by their lineage and genes ordered by similarity of expression by the Jaccard index (as in Fig. 3). Neurons are further coloured by their neurotransmitter identity: acetylcholine, red; glutamate, yellow; GABA, blue; amine, green; unknown, grey.
Extended Data Fig. 9 Features of the homeobox gene code.
a, The 70 conserved homeobox genes alone are sufficient to codify all neuron classes. Neuron classes are coloured by neuron type (sensory, blue; motor, pink; interneuron, yellow; and pharyngeal, grey) and ordered by similarity between neuron classes defined by the Jaccard index as in Fig. 2b. Homeobox genes are coloured by subfamily, and ordered by similarity of neuron class expression and sparsity. b, Theoretical minimal code of conserved homeobox genes required to distinguish every neuron class (determined mathematically, as described in Methods). Colours are as in a.
Extended Data Fig. 10 Homeobox codes define neuronal subclasses.
a, Homeobox codes subdivide neuron classes in the head. Images of head neuron classes are provided from http://www.WormAtlas.org. Homeobox genes expressed in each neuron are listed. Yellow colour indicates the homeobox gene is expressed in all members of that neuron class. Orange and green colours indicate that the homeobox gene is expressed in only a subset of neuron class members. b, Tabular representation of homeobox gene expression subdividing members of the ventral nerve cord motor neurons. Red and blue boxes correspond to cholinergic and GABAergic neurotransmitter identity, respectively. Green boxes indicate the gene is a member of the HOX subfamily; grey boxes indicate that a gene is expressed in some neurons of the VNC; black boxes indicate that a gene is expressed in all neurons of the VNC.
Extended Data Fig. 11 Homeobox genes affecting neuronal marker gene expression.
a, Correlation between predicted gene expression patterns and actual expression patterns in each neuron class. Actual expression patterns for each neuron class were determined by a community-curated list of 1,132 fluorescent reporter expression patterns in all 118 neuron classes (Supplementary Table 5). Predicted gene expression was found by multivariate linear regression of known reporter expression patterns using homeobox gene expression atlas. Correlation coefficient was calculated by Pearson, in which a coefficient of 0.5–0.7 is moderate, 0.7–0.9 is a strong and 0.9–1.0 is a very strong correlation, consistent with ref. 54. b–e, Additional characteristic images for the quantification shown in Fig. 4a–c. We analysed about 15 independent worms per genotype, acquired over at least two separate imaging sessions with an equal mix of both genotypes. Characteristic images were chosen. b, Expression of dop-2, a dopamine receptor, is lost in RIA in the ceh-8-mutant worm. c, Expression of flp-10, a neuropeptide, is lost or becomes dimmer in PVR in the ceh-31-mutant worm. d, e, Expression of glr-3, a glutamate receptor, is lost in RIA in both the ceh-8- (c) and the ceh-32- (d) mutant worms. f, Summary of effects of the loss of homeobox gene on neuronal identity throughout entire nervous system of C. elegans, based on previous studies (specified in Supplementary Table 2) and the mutant analysis conducted here. Dark grey boxes indicate gene activator function; black boxes indicate repressor function.
Supplementary information
41586_2020_2618_MOESM2_ESM.xlsx
Supplementary Table 1 Previously reported homeobox gene expression patterns. Summary of previously reported expression patterns of neuronally expressed homeobox genes, ordered by homeobox gene class and color-coded as in other supplemental tables. Available reagent previously analyzed and the status of reported expression in identified neurons is provided. All information is extracted from Wormbase where relevant references can be found. “locus reporter” = exon/introns + upstream region, but not always with 3’ UTR or all intergenic region (in contrast, fosmid reporters usually contain several genes up and downstream of relevant gene). “transcriptional, incomplete reporter” = 5’ upstream sometimes with exons/introns but never with all exons and introns. If our analysis of fosmid-based reporters and/or CRISPR/Cas9-engineered reporter alleles has revealed differences from this original identification it is noted as “yes” and if it has not changed it is noted as “no”. Differences may not solely be due to novel sites of expression compared to previous reports, but our improved ability for cell identification using the NeuroPAL landmark strain.
41586_2020_2618_MOESM3_ESM.xlsx
Supplementary Table 2 Summary of homeobox genes analyzed including gene subfamily, protein domains, conservation, reporter strains, neuron expression and known function. Homeobox genes ordered by class. Protein domains and gene conservation as reported in26. Strain names and type of reagents analyzed in this study are listed. Expression in the L4 nervous system based on colocalization with NeuroPAL strain as in28 (characteristic images in Extended Data Fig S1-S7). Function of gene in the nervous system is noted and references for previously characterized function listed as PMIDs. Tandem gene duplications and expression profiles highlighted as in text. For easier viewing and manipulation of gene expression, lists of neuron classes where each gene is expressed are provided as well as a binary table of genes by neuron class. The same tables are also provided for expression in single neurons.
41586_2020_2618_MOESM4_ESM.xlsx
Supplementary Table 3 Summary of homeobox gene expression by individual neuron and neuron classes. To easily view the gene expression data, human-readable lists of genes expressed in each neuron class are provided excluding panneuronal genes. Computer-readable binary tables, including panneuronal genes, are also provided. Included in these tables are the neurotransmitter identity and neuron type of each neuron class. The same tables are also provided for expression in individual neurons.
41586_2020_2618_MOESM5_ESM.xlsx
Supplementary Table 4 Comparison of homeodomain protein expression and available single-cell RNA sequencing data in neuron classes. To analyze the similarity between available scRNA sequencing data and our reported homeodomain expression, we used the provided bootstrap median data (averaging resampled RNA levels 1000 times) from31,32 and applied no cutoff (i.e. any TPM>0 counted as real expression). We then directly compared the binary expression profiles of the homeobox gene mRNA in isolated neuron classes with our reported homeodomain protein expression (colored in legend in figure). We found that the scRNA-seq expression data from the 42 identified L2 neuron classes recapitulated only 38% of our homeodomain protein expression. We calculated this percentage by taking the agreed expression (blue) and dividing it by the agreed expression plus the expression seen only in the homeodomain protein analysis (blue+ red). We next asked if scRNA-seq was able to detect mRNA of our homedomain proteins at any point. So, we added the scRNA-seq embryo data available for those 42 neuron classes and found that this increased the coverage to 55%. This percentage was calculated as above with the agreed expression divided by the agreed plus the expression seen only in the homeodomain protein analysis.
41586_2020_2618_MOESM6_ESM.xlsx
Supplementary Table 5 Community-curated reporter expression patterns in the C. elegans nervous system. The most updated version of the community-curated reporter expression patterns in 21. We listed this as a binary table of genes by neuron, where 1 represents reporter expression in that neuron and 0 represents no expression in a neuron or no data. This expression atlas is also made available with gene names by each individual neuron. Additionally, we replaced previously described homeobox gene expression patterns, which were largely inaccurate, with the homeobox gene expression patterns described in this paper.
Rights and permissions
About this article
Cite this article
Reilly, M.B., Cros, C., Varol, E. et al. Unique homeobox codes delineate all the neuron classes of C. elegans. Nature 584, 595–601 (2020). https://doi.org/10.1038/s41586-020-2618-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41586-020-2618-9
This article is cited by
-
A full-body transcription factor expression atlas with completely resolved cell identities in C. elegans
Nature Communications (2024)
-
A lineage-resolved cartography of microRNA promoter activity in C. elegans empowers multidimensional developmental analysis
Nature Communications (2024)
-
The homeodomain transcriptional regulator DVE-1 directs a program for synapse elimination during circuit remodeling
Nature Communications (2023)
-
The homeodomain transcription factor CEH-37 regulates PMK-1/p38 MAPK pathway to protect against intestinal infection via the phosphatase VHP-1
Cellular and Molecular Life Sciences (2023)
-
A global timing mechanism regulates cell-type-specific wiring programmes
Nature (2022)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.