In-house derived hES cells (KIND1) were subjected to directed differentiation into cardiovascular progenitors (D12) and beating cardiomyocytes (D20). Transcriptome profiling of undifferentiated (D0) and differentiated (D12 and 20) cells was undertaken by microarray analysis. ChIP and sequential ChIP were employed to study role of transcription factor NR2F2 during hES cells differentiation. Microarray profiling showed that an alteration of about 1400 and 1900 transcripts occurred on D12 and D20 respectively compared to D0 whereas only 19 genes were altered between D12 and D20. This was found associated with corresponding expression pattern of chromatin remodelers, histone modifiers, miRNAs and lncRNAs marking the formation of progenitors and cardiomyocytes on D12 and D20 respectively. ChIP sequencing and sequential ChIP revealed the binding of NR2F2 with polycomb group member EZH2 and pluripotent factor OCT4 indicating its crucial involvement in cardiac differentiation. The study provides a detailed insight into genetic and epigenetic changes associated with hES cells differentiation into cardiac cells and a role for NR2F2 is deciphered for the first time to down-regulate OCT-4 via EZH2 during cardiac differentiation.Human embryonic (hES) stem cells are widely used as an  Pre-clinical animal studies undertaken over a decade involving transplantation of hES derived cardiomyocytes have also shown considerable promise. Studies describing differentiation and outcome of transplantation using hES cells derived cardiac cells are compiled as Supplementary Table 3. In order to address these questions, it is necessary to understand stage-wise gene expression during early cardiac development and hES cells serve as an excellent model to study this differentiation in vitro.The successful isolation of human embryonic stem (hES) cells in the late nineties5 and plays an important role when pluripotent hES cells become committed. Chromatin modification is one of the fundamental epigenetic mechanisms associated with active or repressed state of gene expression in a cell8. Active states are marked by methyl modifications like H3K4me3 and H3K36me3 brought about by Trithorax group (TrxG) of proteins10 while repression is catalysed by addition of H3K27me3 by Polycomb group (PcG) of proteins11 grouped as Polycomb Repressive Complex 1 (PRC1)  and Polycomb Repressive Complex 2 (PRC2) 13.In addition to various signalling cascades and transcription factors, epigenetics is another player involved in major functional genomics decisionsin vitro15. Dynamics of PcG proteins during differentiation of human ES cells into the three lineages was for the first time reported by our group17. Transcripts of both PRC1 and PRC2 were found up regulated during endodermal differentiation while during ectodermal differentiation only PRC1 transcripts were found to be up regulated. On the other hand, both PRC1 and PRC2 were down regulated during mesoderm lineage formation in vitro. A study by Murry’s group determined the evolution of chromatin modifications like H3K4me3 and H3K27me3 along cardiac lineage formation wherein the dynamic acquisition of H3K4me3 and H3K27me3 by various transcription factors during differentiation at the five different time points was identified18. Bruneau and colleagues19 also revealed a unique epigenetic pattern of H3K27ac, H3K4me1, H3K4me3 and H3K4me3 during the gene expression changes of cardiomyocytes differentiation in vitro.Various efforts are now focussed to characterize the ES cells and their differentiated progeny with respect to epigenetic mechanisms. Importance of PcG proteins for differentiation of ES cells and not in their self-renewal was identified when the ES cells deficient for PRC2 proteins  could still be maintained in their undifferentiated state 23. Though much of the research has focused on providing key conclusions regarding genetic and epigenetic regulatory mechanisms, we are still left with key questions like how are only the right genes expressed or repressed in a given cell type? How does the gene activation or repression machinery gain or keeps its access into the cell? In the present study, we aim to understand the gene expression and histone modification pattern during cardiac differentiation from hES cells using directed differentiation protocol.All these emerging evidences of requirement of unique pattern of epigenetic mechanisms during lineage specification indicate that the disturbed or faulty epigenetic program may contribute to various cardiac disorders. Mutations in a number of histone methyltransferases like JMJD2A, H3K36me3 methyltransferase SETD2, H3K4me3 methyltransferase MLL2, H3K27me3 methyltransferase EZH2 are implicated in congenital heart disordersIn-house derived hES cell line KIND1) was subjected to cardiac differentiation using a directed differentiation protocol , cardiovascular progenitors (D12) and beating cardiomyocytes (D20). A list of genes (expected and predicted) was generated showing a fold change value of >=2.0 and a p-value score < 0.05. Comparisons were made amongst three groups including D0 versus D12, D0 versus D20 and D12 versus D20.About 1400 genes were found to be differentially expressed when KIND1 cells (D0) differentiated into cardiovascular progenitors D12)  on days 0, 12 and 20 of cardiac differentiation protocol was performed by gene ontology study. Genes enriched during the differentiation of undifferentiated cells (D0) into cardiac progenitors (D12) were found to be associated with biological processes related to early mesoderm and cardiac derivatives formation like ‘extracellular region development’, ‘vasculature development’, ‘regulation of cell proliferation’, ‘skeletal system development’, ‘embryonic morphogenesis’  as they represent the key players, the repressors and activators of gene expression respectively. Various studies till date have reported the importance of PcG proteins and that the abolishment of PcG proteins like EZH2, EED or SUZ12 disrupts the stem cell identity and their developmental potential. ES cells in their undifferentiated state maintain the bivalent marks in order to keep the developmental genes poised for further activation. In consistence with all the reports, the core proteins EZH2 and MLL2 forming the bivalent marks (H3K27me3 and H3K4me3 respectively) were found expressed in undifferentiated hES cells. As the differentiation proceeded to progenitors and cardiomyocytes, expression of EZH2 began to down-regulate. Expression of MLL2 core component ASH2L was expressed in both cardiac progenitors and cardiomyocytes. Another PcG group proteins like RING1, BMI1, PHC1, that represent the Polycomb repressive Complex 1 (PRC1) group, were up-regulated while the PRC2 group like SUZ12 and EED were significantly down-regulated in progenitor and cardiomyocyte stages as compared to their status in the undifferentiated state hES cells. Histone methyltransferase expressed during the differentiation is the SET domain containing 2 or SETD2 that is involved in the activation of gene transcription and is reported to be crucial for the cardiac formation21 Fig. . BRG1 an21 Fig. .Figure 4miRNAs and lncRNAs represent a valuable tool to understand the role of cellular proteins. As important regulators of gene expression at the epigenetic level, they activate or repress the transcription. Cardiac formation machinery also includes the number of non-coding RNAs as regulators of gene activation and repression. Among the number of miRNAs identified to be differentially regulated, the miRNAs significantly changed were miR21, miR208a, miR423 and miR27b that have been shown to regulate endothelial and myogenic differentiation. Expression was found increased with the differentiation of hES cells in cardiomyocytes. Similarly, the long non coding RNA represents non-coding ability and act as regulators of development and pathophysiology. ANRIL, CDKN2A/2B and MIAT were among the many lncRNAs identified in our study that might be involved in lineage specific gene expression Fig. .Differential regulation of specific gene transcripts was studied by qRT-PCR to validate the microarray results. This included specific transcripts for pluripotent genes , ectodermal (MAP2), mesodermal into cardiac lineage , LTB, along with transcripts representing epigenetic machinery . The results were found consistent with the microarray data Fig.  and CTNTThe extensive genetic and epigenetic characterization of KIND1 cells during differentiation into cardiac lineage, by microarray along with the qRT-PCR validation depicts the dynamic expression of various transcripts during cardiac differentiation. Though the array and the qRT-PCR validation involved differences in the expression levels, the technique reveals the global view of the genes along with illuminating the differences in their expression within the cells and their differentiated counterparts. Transcription factor NR2F2 was further studied as it showed increased expression during cardiac both by microarray and qRT-PCR.31, transcriptional regulation of OCT4 in mouse embryonal carcinoma cells as well as in hES cells undergoing neuroectoderm development33. NR2F2 is also crucial during mice heart development for angiogenesis and coronary vessel formation35. We thus interrogated the expression of NR2F2 in our differentiation system to evaluate whether it acts as regulator of early cardiac differentiation. The expression levels of NR2F2  were found to be inversely related to that of OCT4  and were comparable to EZH2, a transcriptional repressor as observed both by microarray analysis and qRT-PCR validation  justifying the formation of mature counterparts on D20 compared to D12. The only down-regulated gene LTB between D12 vs D20 (of the 19 genes) was not included in the down-regulated gene list between D0 and D20. All the 18 genes that were up-regulated on D20 compared to D12 were not expressed on D0. Thus, we believe our results show dramatic maturation of cardiac cells on D20 compared to D12 associated with a change in only 19 genes. Of all the genes being altered during ES cells differentiation, transcription factor NR2F2 reported to repress OCT-4 during neural differentiation33, has not yet been reported during cardiac differentiation but its mutation is known to cause atrial septal defects31. NR2F2 showed up regulation as cells differentiate into cardiac progenitors and cardiomyocytes by both microarray and qRT-PCR . A simultaneous down regulation of EZH2 was noted. Further ChIP experiments were undertaken to study whether a cross-talk exists between NR2F2 and EZH2 for repressing OCT-4 during cardiac differentiation. We first observed that the repressive mark H3K27me3 was occupied on NR2F2 promoter on D0 while not in D12 and D20; this explains the increased expression of NR2F2 as differentiation proceeded. The results clearly showed the occupancy of H3K27me3 on OCT4 promoter in progenitor cells on D12 (and not on D0) resulting in repressed OCT4 expression during differentiation. Since this repressive mark is brought about by histone methyltransferase EZH2, we studied its binding to NR2F2 and OCT4 by ChIP and sequential ChIP. QPCR following ChIP displayed the EZH2 binding on both OCT4 and NR2F2 while sequential ChIP further confirmed that NR2F2 was bound to both EZH2 and OCT4. Based on these results, we propose that NR2F2 recruits EZH2 at the OCT4 further leading to its repression during cardiac differentiation from hES cells.Present study provides novel information on the detailed genetic and epigenetic gene expression pattern underlying cardiac differentiation 41; OCT4A (true stemness marker containing unique exon 1), OCT4B (non-pluripotent variant) and OCT4B1 (putative stemness marker). In order to understand the mechanisms of hES cells pluripotency, it thus remains essential to concentrate upon the OCT4A isoform . Present study is focused on OCT-4A positive ES cells since we used primers specific to OCT4A. Moreover, in the ChIP sequencing studies, the occupancy of H3K27me3 mark on the promoter driving the expression of exon 1 that proves the involvement of ‘pluripotent OCT4 isoform’. Looking at the binding of EZH2 onto the OCT4 promoters, the primer pair specific to exon 1 which amplifies the distinct OCT4A gene further validates our findings, supporting the mechanism of binding of EZH2 and NR2F2 with OCT4A and repressing its expression for leading the hES cells to cardiac differentiation.OCT4 gene is reported to generate multiple transcripts that are translated into multiple isoforms of OCT4in vitro and in vivo44. Epigenetic remodelers found to be expressed were BRG1, MTA family and HDACs known to promote cardiac cell differentiation by regulating the levels of crucial cardiac transcripts like NKX2.5, TBX5 and GATA448. Similarly, the expression of other components of epigenetic machinery noted in our study is well studied in cardiac differentiation context; though their specific role is still unknown.Epigenetics adds another layer of gene expression control by modulating the transcriptional activities in a trimly and gene specific manner. The extensively studied epigenetic feature for stem cells is the simultaneous appearance of H3K27me3, H3K4me3 termed as bivalency that holds the equal potential to be expressed or repressed and that are resolved as per the signals and MLL2 enzymes specific for H3K27me3 and H3K4me marks respectively in our undifferentiated hES cells that were later subjected to cardiac lineage formation. However, on examining the cardiac progenitors and cardiomyocytes, EZH2 had a lowered expression in cardiac progenitors and much lower in cardiomyocytes, holding the evidence in support of reported studies that EZH2 is crucial for cardiac development 50. Additionally, it has been identified as a direct regulator of vascular endothelial cells by activating the NF-ĸB pathway51. With respect to cardiac development, LTB is identified as a risk factor altered for cardiovascular diseases and thus the TNF antagonist potentially acts as a likely candidate to reduce the CVD risk in rheumatoid arthritis patients55. However, though there exists a dearth in studies reporting molecular mechanisms associated with LTB in cardiac development and pathogenesis, present study reports reduced levels of LTB during differentiation of hES cells into the cardiac lineage.The sole down regulated gene LTB represents a TNF family ligand that majorly acts as the inducer of inflammatory response and is involved in the organization/development of lymphoid tissue57 or the endothelial cells58 at various stages of differentiation and the data was also compared with the transcriptional pattern in fetal and adult heart in vitro. However, majority of these have employed spontaneous embryoid body formation technique to obtain beating clusters that led to achieving the mixed population comprising of all the lineages. Secondly, the purifying techniques like percol gradient resulted in purified cardiac population of as low as 40%56. Third employed way was studying the cardiomyocytes differentiated from ES cells that were co-cultured onto the layer of END2 cells or inactivated mouse fibroblast feeders59. Despite the extensive characterization data and their comparison with the in vivo counterparts is available, the fact that the resulting population being studied contains the non-mesodermal population still remains. Our study on the other hand employs the directed differentiation approach that ensured the cardiac cell fate of majority of the population. Moreover, the protocol also allows studying the step wise formation of cardiomyocytes involving early nascent mesoderm, cardiac mesoderm formation followed by cardiac progenitors and beating cardiomyocytes. The protocol from the very beginning restricts the formation of ectodermal and endodermal population excluding the endodermal genes reported to be essential for mesodermal direction.Number of studies till date have reported the transcriptional profiling of ES derived beating clustersin vivo and in vitro that would provide deeper insights. Despite this, our study for the first time provides the mechanism of OCT4 repression by the combined actions of NR2F2 and EZH2 that would direct the future efforts to undertake more specific experiments towards understanding cardiac development. Analysing various histone modifications and their dynamics along with the lineage specific gene expression patterns could uncover the regulatory genes involved during early human cardiac development in vitro.To conclude, detailed transcriptomic expression of genetic and epigenetic factors in pluripotent hES cells and during their conversion into cardiac progenitors and cardiomyocytes is reported. With a view to identify a role of NR2F2 during this differentiation, we also show the expression pattern of H3K27me3 mark on NR2F2 and OCT4 genes. Limitations of the study include use of unsorted cardiac population which otherwise would bring much clearer picture of cardiac differentiation mechanisms. Secondly, the deficiency of more confirmations like loss of function tests with respect to NR2F2 and OCT4 both 60 were transitioned to feeder-free condition17 and used to differentiate into tripotent cardiac progenitors. Similar protocols (described in details in supplementary section) were used in the present study to obtain undifferentiated hES cells (D0), cardiovascular progenitors (D12) and beating cardiomyocytes (D20) for various studies.In-house derived human embryonic stem cells (KIND1) derived on human feedershttp://david.abcc.ncifcrf.gov/home.jsp). All data analysis and visualization of differentially expressed genes was conducted using R 3.1.2 (www.r-project.org). Genes with fold values between −2 and +2 were omitted from the analysis. Three combinations of data were analysed, namely – Day 0 Vs Day 12, Day 0 Vs Day 20 and Day 12 Vs Day 20. QRT-PCR analysis for pluripotent , cardiac specific markers  including LTB and other epigenetic machinery  validated microarray data. Human fetal and adult cardiac total RNA  were used to compare the maturity of the differentiated cells and GAPDH was used as an internal control. All the primers used in the study are mentioned in Table Microarray was performed using two biological replicates of each time point using Illumina, HT-12 v4.0 platform commercially. hES cells on days 0, 12 and 20 were harvested by mechanical dislodging using a cell lifter in TRIzol reagent  for RNA extraction as per manufacturer’s instructions. Genome-wide gene expression analysis was performed commercially using Illumina, HT-12 v4.0 platform . Gene-Enrichment and Functional Annotation analysis for significant probe list was performed using DAVID Bioinformatics Resources  were cross linked with 1% formaldehyde for 10 mins at room temperature (RT). Formaldehyde was quenched with 1.25 M glycine and cells were rinsed with ice cold 1X PBS, collected and centrifuged at 2500 rpm for 5 mins. Supernatant was aspirated leaving behind the pellet of cross linked cells. Cells were lysed with cell lysis buffer containing protease inhibitor (PI) . Lysed cells were subjected to sonication for about 25 cycles using pulse ON for 30 sec and pulse OFF for 50 sec on  to obtain fragments appropriate for performing qPCR (200–500 bp) or DNA sequencing (100–300 bp) analysis. Sonicated samples were then incubated with 10ug of anti H3K27me3 antibody  for ChIP-sequencing or anti EZH2 antibody  for ChIP-qPCR in blocking solution (0.5% BSA in 1X PBS). The beads with antibody - DNA complexes were washed 3 times with each of lysis buffer, IP1 buffer (lysis buffer with 500 mM NaCl), IP2 buffer  and Tris EDTA (TE) buffer . For eluting the bound complexes, the bead – complex solution was incubated with 200ul elution buffer (TE buffer with 1% SDS) at 65 °C for 50 mins. The eluted product was treated with RNase A and proteinase K at 60 °C for 60 mins. Following this the immuno-precipitated DNA was isolated Phenol:Chloroform:Isoamyl alcohol method.Sequential ChIP was performed using standard protocols. Briefly, the cross linked chromatin sample of undifferentiated and differentiated cells was immuno-precipitated using EZH2 antibody. Following this the complex was eluted using elution buffer with higher salt concentration and DTT  in order to remove traces of EZH2 antibody. The resulting chromatin sample was then immuno-precipitated with anti OCT4 antibody  and continued with standard ChIP protocol described above. The genomic DNA isolated after both ChIP and sequential ChIP experiments was quantified by qPCR  calculated as % of total input DNA. The genes included in study are listed in Table For ChIP sequencing step, the DNA isolated after the standard ChIP protocol immuno-precipitated with anti H3K27me3 antibody was quantified using Nanodrop  and then Illumina sequencing was done at sequencing facility of Genome Institute of Singapore (GIS). The data analysis was performed by Sandor Life Sciences Pvt Ltd, Hyderabad, India. The reads obtained were mapped with the Human hg19 and aligned using Bowtie . MACS  were employed for peak calling. The resulting peaks were visualized using Integrative Genome Viewer.Microarray data raw files are available on GEO NCBI data base accession number GSE97268. ChIP sequencing raw files are available on SRA NCBI data base accession number SRP115340.Supplementary Information


NKG2D is a critical activatory molecule expressed on cytotoxic cells such as NK cells, γδ T cells and subsets of αβ T cells . LigatioULBP6 is the most polymorphic gene within the ULBP family and in recent work our team contrasted the molecular and functional correlates of the two most common protein variants, ULBP601 and ULBP602 Figure  3]. Str. Str3]. The mechanisms that underlie the association of NKG2DL alleles with clinical disorders such as auto-immunity  or tumorin vivo murine studies have shown that ectopic expression of NKG2D ligands on tumor cells can be sufficient to mediate control of tumor growth[The critical role of NKG2DL in tumour-specific immune responses is highlighted by the fact that tumours frequently evolve a range of mechanisms to evade NKG2D-mediated responses. These include NKG2DL cleavage from the surface, and NKG2D downregulation which is frequently observed on cytotoxic cells in patients with cancer . Interesor growth. In ordeThe central importance of NKG2D interactions within immune homeostasis has been relatively under investigated but an emerging hope is that detailed interrogation of the significance of natural alleleic variation within the ULBPL family members  will ser


TP53 mutations in about 50%, however, only little is known about alternative pathogenic mechanisms within the p53 regulatory network. Particularly, data on alterations of the oncogenes MDM2 and CDK4 located in the chromosomal region 12q13-15 are limited in SDC, while genomic rearrangements of the adjacent HMGA2 gene locus are well documented in subsets of SDCs. We here analyzed the mutational status of the TP53 gene, genomic amplification of MDM2, CDK4 and HMGA2 rearrangement/amplification as well as protein expression of TP53 (p53), MDM2 and CDK4 in 51 de novo and ex pleomorphic adenoma SDCs.Salivary duct carcinoma (SDC) is an aggressive adenocarcinoma of the salivary glands associated with poor clinical outcome. SDCs are known to carry TP53 mutations, associated with extreme positive immunohistochemical p53 staining levels in 13 cases. Three out of 51 tumors had an MDM2 amplification, one of them coinciding with a CDK4 amplification and two with a HMGA2 rearrangement/amplification. Two of the MDM2 amplifications occurred in the setting of a TP53 mutation. Two out of 51 cases showed a CDK4 amplification, one synchronously being MDM2 amplified and the other one displaying concurrent low copy number increases of both, MDM2 and HMGA2.25 of 51 cases were found to carry MDM2 and/or CDK4, partly in association with TP53 mutations and rearrangement/amplification of HMGA2. Further research is necessary to clarify the role of chromosomal region 12q13-15 alterations in SDC tumorigenesis and their potential prognostic and therapeutic relevance.In summary, we here show that subgroups of SDCs display genomic amplifications of  TP53 mutations were detected, involving around 50% of cases of de novo and ex pleomorphic adenoma SDCs [TP53 wildtype (WT) tumors which might harbor alternative alterations in the p53 regulatory network.Salivary duct carcinoma (SDC) is an aggressive adenocarcinoma of the salivary glands, most commonly involving the parotid gland. It is one of the most aggressive salivary gland malignancies, most frequently associated with the occurrence of early distant metastasis and poor prognosis . In the oma SDCs , 3. HoweMDM2-amplified well-differentiated or dedifferentiated liposarcomas (LS), which are consistently characterized by a high-level genomic amplification (frequently >15-30 copies as clusters) of sequences derived from chromosomal region 12q13-15 comprising the oncogenes MDM2 and CDK4, showed that treatment with the MDM2 antagonist RG7112 activates the p53 pathway and decreases cell proliferation [TP53, but MDM2 alterations have also been described in subsets of TP53 mutated tumors [MDM2 transgenic mice developing mammary gland tumors suggest a crucial role for MDM2 in epithelial tumors of glandular differentiation [For decades, p53 has been a well-known tumor suppressor that is mutated or functionally inactivated in large subsets of human cancers . Physiolferation . Most frd tumors . It has ntiation .CDK4 together with MDM2 [MDM2+/CDK4- LS shows favorable prognostic features compared to MDM2+/CDK4+ LS [CDK4 amplification status therefore provides genomic information on the structural characteristics of the amplicon with MDM2 being located at 12q15 and CDK4 at 12q13.3-12 and it might add further information on an independent oncogenic mechanism apart from p53 dysfunction. Since CDK4 is the key regulator of the G1-S cell-cycle transition and drives cell-cycle progression, CDK4 inhibitors might offer new strategies for a targeted cancer therapy [In LS, the 12q13-15 amplicon usually, but not always, shows a co-amplification of the cell cycle regulator ith MDM2 . It has CDK4+ LS . Knowled therapy , 12.HMGA2, encoding a high-mobility group protein. Chromosomal breaks of the HMGA2 locus have been described in several benign mesenchymal tumors including lipomas and uterine leiomyomas [HMGA2 amplification was shown in several soft tissue malignancies including liposarcomas where it is almost always co-amplified with MDM2 [HMGA2 are well known in subsets of pleomorphic adenomas (PA) [ex PA have been reported to generally retain HMGA2 rearrangements along with further gene alterations in tumor progression making HMGA2 a potential marker for SDCs arising in PA [Another gene in chromosomal region 12q13-15 frequently subject to structural alterations is iomyomas , and HMGith MDM2 , 15. In mas (PA) , 16. CarMDM2 and CDK4 alterations in SDC and to put them in context with HMGA2 alterations known in SDC. We here report on the rare occurrence of MDM2 and CDK4 amplifications in a large collection of these aggressive salivary neoplasms showing a heterogeneous distribution among TP53 wildtype tumors and those carrying a TP53 mutation.Only very little is known about the role of MDM2 or CDK4 in SDC tumorigenesis. The major aim of this study therefore was to systematically evaluate the involvement of TP53 mutational status, MDM2, CDK4 and HMGA2 genomic amplification as well as HMGA2 rearrangement and p53, MDM2 and CDK4 protein expression. The clinicopathological characteristics of these 51 patients are summarized in Table 51 SDC cases were analyzed for TP53 mutations were detected in 25 of these 51 cases  staining result in 13 cases, all of these cases displaying a TP53 missense point mutation except for one case in which the TP53 mutational status could not be evaluated due to minor DNA quality. In 8 cases with various TP53 mutations, an extreme negative (EN) p53 immunohistochemical staining was detected and 5 cases with TP53 mutations showed a non-extreme (NE) staining level for p53. An extreme negative (EN) p53 staining was detected in 8 cases without TP53 mutation and in 5 further cases in which TP53 mutational status could not be evaluated.MDM2 amplification, one case showed an MDM2 grey-zone amplification in FISH analyses. Two cases with MDM2 amplification strongly stained for MDM2, one MDM2 amplified case and the grey-zone amplified case showed low MDM2 immunohistochemical staining levels. Two of the MDM2 amplified cases synchronously carried TP53 mutations. All MDM2 amplifications and the MDM2 grey-zone amplification occurred in de novo SDCs, none was detected in an SDC ex pleomorphic adenoma.Three out of the total 51 cases displayed an CDK4 amplification, one coinciding with an MDM2 amplification and one in the setting of an MDM2 grey-zone amplification. Nine cases showed a CDK4 grey-zone amplification, one of these cases in the setting of an MDM2 amplification. However, the immunohistochemical CDK4 staining was negative in all cases.In two cases, we detected a HMGA2 FISH could be analyzed in 42 out of 51 cases. 5 cases showed rearrangement and/or amplification of HMGA2, among these 3 de novo and 2 ex PA SDCs. Two of the 5 cases displayed HMGA2 rearrangement and amplification, both occurring in association with an MDM2 amplification; the third MDM2 (and CDK4) amplified case displayed no HMGA2 alteration. One of the HMGA2 rearrangements occurred in the setting of a CDK4 grey-zone amplification, the two further HMGA2 rearrangements were detected in cases without MDM2 and/or CDK4 amplification. One CDK4 amplified tumor with grey-zone MDM2 amplification showed only a low HMGA2 copy number increase.TP53 mutated cases showed a tendency to a worse 5-year OS (20.5% with TP53 mutation vs. 53.3% without TP53 mutation) but statistical significance was not reached (p=0.267)  in our collection of SDCs was 39.6% Figure . TP53 mu) Figure . No statTP53 mutations in de novo and ex PA SDCs [TP53, the major aim of this study was to elucidate the role of another key player within the p53 regulatory network, i.e. the ubiquitin ligase MDM2, in SDC. Due to the co-localization of MDM2 with the cell cycle regulator CDK4 in the chromosomal region 12q13-15 and a documented proneness of this region to genomic alterations, these regulators of cell growth and fate are frequently co-amplified in some human malignancies including a subset of soft tissue tumors [CDK4 amplification status might add further information on an independent oncogenic mechanism apart from p53 dysfunction, opening therapeutic options. Involvement of the chromosomal region 12q13-15 in salivary gland tumorigenesis has been documented before with the HMGA2 gene showing rearrangements and genomic amplification in subsets of PA and SDCs ex PA [HMGA2 furthermore represents an almost constant partner of MDM2 in the 12q13-15 amplicon known in other tumors, we complemented our analysis with the analysis of HMGA2 to get further insight into the structure of the amplicon [Recent genomic profiling studies confirmed a high frequency of  PA SDCs , 3, affee tumors . Apart fCs ex PA . Since Hamplicon .MDM2 amplifications in 3 cases, interestingly all classified as de novo SDCs. Two of these MDM2 amplified cases showed a concurrent rearrangement/co-amplification of HMGA2. According to Bahrami et al. [HMGA2 as a marker for SDCs ex PA, it could be hypothesized if these two cases might also have arisen in totally obscured PA, however, there was no evidence for a pre-existing PA, neither by histopathology nor with respect to clinical history. One of the MDM2 amplified SDCs harbored no HMGA2 alteration while 3 SDCs with HMGA2 rearrangement did not show an MDM2 amplification, implying that alterations of these two genes are obviously not strictly connected in SDCs. Thus, MDM2 amplification appears to be a genomic alteration at least partly independent from HMGA2 status and particularly from pathogenesis (de novo vs. ex PA) in SDC. Since only two of the cases harboring an MDM2 amplification revealed a strong immunohistochemical staining for MDM2, immunohistochemical staining alone appears not to be a reliable marker of MDM2 amplification in SDC. Interestingly, two of the MDM2 amplified cases synchronously carried TP53 mutations, while one MDM2 amplification occurred in a TP53-wildtype setting. In another TP53-wildtype tumor a grey-zone MDM2 amplification was detected. A very recent study on 37 SDCs revealed one MDM2 amplified case, also in the setting of a TP53 mutation [TP53 cannot be expected to substantially gain in oncogenic potential due to a concomitant amplification of MDM2 since it usually acts as an oncogenic factor by downregulation of wildtype p53 protein. In contrast, in tumors harboring particular TP53 mutations and retaining their wildtype TP53 allele, MDM2 amplification might contribute to oncogenesis by additionally disrupting the remaining functional p53 levels. Additionally, several reports argue in favor of an oncogenic role for MDM2 independently from the p53 status [MDM2 amplification obviously may serve as an alternative mechanism leading to dysregulation of the p53 network, thereby offering an option for a targeted MDM2-directed therapy [MDM2 amplification might represent an alternative or additional mechanism in deregulating the p53 network besides well-known and pathogenetically relevant genetic alterations of the PI3K/AKT signaling pathway in SDC [We detected i et al. , who pro3 status , 17, 18. therapy . With re therapy , 20, MDMTP53 mutational status: With 48% of TP53 mutated cases and none of the TP53 wildtype cases showing an extreme positive (EP) staining for p53 protein expression, EP p53 staining reliably went along with a missense TP53 mutation. 32% of TP53 mutated cases showed an extreme negative (EN) p53 staining and 20% of TP53 mutated cases a non-extreme (NE) p53 staining. In contrast, cases without TP53 mutation displayed an EN p53 staining result in 44% and a NE staining in 56%. So, in summary, in case of an EN or a NE p53 staining, a reliable prediction of TP53 mutational status is not possible in SDCs whereas an EP p53 expression is strongly suggestive of an underlying TP53 missense mutation.The immunohistochemical staining for p53 partly correlated with the TP53 mutated cases showed a tendency to a worse 5-year OS , pointing to a CDK4-mediated cell-cycle dysregulation in SDC tumorigenesis. CDK4 inhibitors might therefore offer new strategies for targeted therapeutic approaches in SDCs as recently discussed for several entities [CDK4 amplified or grey-zone amplified cases showed a positive immunohistochemical staining for CDK4 (with solid staining result of the positive control), so immunohistochemical CDK4 staining obviously is not reliably suitable for the detection of CDK4 amplified SDC. Anyway, lack of immunohistochemical CDK4 protein detection does not exclude a pathogenic role of the CDK4 oncogene and may be due to tight protein regulation beyond the threshold of staining sensitivity. Contrasting with our immunohistochemical results and potentially pointing to different characteristics of diagnostically employed antibodies, a previously reported study on five SDC cases demonstrated CDK4 protein expression in four of five cases, accompanied by MDM2 protein expression in two cases; however, CDK4 and MDM2 amplification status was not determined in that study [Out of the entities , 12. Intat study .MDM2 and/or CDK4 amplified/grey-zone amplified subgroups, which is probably due to the limited case numbers contained in the smaller subgroups. Larger cohorts of these aggressive tumors should be analyzed for a better understanding of a potential prognostic impact of MDM2 and/or CDK4 alterations.As described above, in a subset of liposarcomas the small subgroup of MDM2+/CDK4- tumors shows favorable prognostic features compared to MDM2+/CDK4+ tumors . In our TP53 mutational status, MDM2 and CDK4 amplification and HMGA2 rearrangement/amplification as well as protein expression of p53, MDM2 and CDK4. We showed that in subgroups of SDCs, MDM2 and/or CDK4 amplification might play a pathogenic role, in part apparently in association with other genetic alterations. Further work is mandatory to clarify the role of MDM2 and CDK4 alterations in the tumorigenesis of SDCs , the potential prognostic relevance of these alterations in SDCs and the feasibility of MDM2- and/or CDK4-directed therapeutic strategies in SDCs.In this study, we investigated for the first time systematically the involvement of MDM2 and CDK4 in the carcinogenesis of SDCs by analyzing the th edition of the UICC TNM classification for carcinomas of the salivary glands. Patients were followed up at the outpatients department of Cologne or Muenster, respectively, at periodic visits in 3 to 6 months. At the time of analysis, 22 patients had died and 11 patients had developed a histologically confirmed relapse. Mean follow-up time was 22.2 months (range 0 to 161). The study was approved by the local Ethics Committees.The investigation was conducted according to the Declaration of Helsinki on biomedical research involving human subjects. The retrospective study included 51 patients with newly diagnosed salivary duct carcinoma. Among these, 30 cases were derived from the University Hospital of Muenster and 21 cases from the University Hospital of Cologne  material of the patients was obtained from the archives of the Departments of Pathology at the University Hospitals of Cologne and Muenster, respectively. All tumors were re-evaluated microscopically and by means of immunohistochemistry by two experienced pathologists with regard to histopathological diagnosis in accordance with WHO 2005 classification of tumors of salivary glands.From FFPE material of all included cases, two core biopsies out of the tumor area were taken to assemble tissue microarrays (TMA).For statistical analysis, the IBM SPSS Statistics 22 software  was applied and Kaplan-Meier survival analysis and Log rank test were performed. The significance level was set at p<0.05.in-situ hybridization (FISH) analyses and immunohistochemical stainings were conducted on slides from TMAs. FISH analyses were performed as described previously [® SPEC MDM2/CEN 12 Dual Color Probe for assessment of MDM2 amplification and the ZytoLight® SPEC CDK4/CEN 12 Dual Color Probe for assessment of CDK4 amplification . HMGA2 FISH analysis was performed according to a previously published assay [MDM2 and CDK4 was defined as an MDM2/centromer 12 (CEN12) or CDK4/centromer 12 (CEN12) ratio ≥2.0 or an average number of MDM2 or CDK4 signals per tumor cell nucleus ≥6 or large clusters of MDM2 or CDK4 signals ≥10%, respectively. Grey-zone amplification was defined as an MDM2/centromer 12 (CEN12) or CDK4/centromer 12 (CEN12) ratio ≥1.8 or as microclusters of ≥5 MDM2 or CDK4 signals in ≥15% of tumor cell nuclei, respectively, based on modified scoring algorithms for HER2 and FGFR1 as published before [HMGA2 rearrangement and amplification were evaluated as published before [Fluorescence eviously , 23 usined assay , 24 usind before . For HMGd before .et al. [Immunohistochemical staining was conducted on a Dako Autostainer  following the manufacturer's instructions. Three μm sections were cut from the TMAs, followed by heat induced antigen retrieval in low (for p53) or high (for MDM2 and CDK4) pH buffer. For visualization LSAB method with AP/RED was used. Following antibodies and concentrations were used: p53 , MDM2  and CDK4 . Sections were counterstained with hematoxylin and tiled with Cytoseal (Thermo Fisher Scientific Inc.). For scoring of the immunohistochemical stainings for p53, MDM2 and CDK4 only nuclear staining was rated. The staining intensity was evaluated semiquantitavely into 0, 1, 2 or 3 by comparing within the different tumor samples. To determine percentage labelling indices, all tumor cells within the cores were analyzed using high-power (400x) magnification. A sum score was calculated out of the staining intensity and the percentage labelling index, and for MDM2 and CDK4 value ranges for staining level were defined as follows: 0: no staining; >0 to 50: low staining level; >50 to 100: intermediate staining level; >100: strong staining. Sum score of p53 staining was interpreted modified according to Boyle et al.  as folloTP53 mutational status as previously described for a smaller subset of samples [TP53 mutational status was carried out as follows:To complete the data on 2 tumor area corresponding to the tumor area of H&E-stained section was scraped off with a scalpel and collected into plastic tubes. Subsequently, the DNA was automatically extracted using the Maxwell DNA FFPE isolation kit on a Maxwell platform . Fluorometric DNA quantification was performed according to the Qubit dsDNA HS assay .Sections were prepared from FFPE material and stained with hematoxylin & eosin (H&E). Six additional sections of 6 μm thickness were cut, mounted onto glass slides and used for macrodissection. In total, 1 cmTP53 . Target enrichment was processed by means of the GeneRead DNAseq Panel PCR V2 Kit (Qiagen), following the manufacturer's instructions. All purification and size selection steps were performed utilizing Agencourt AMPure XP magnetic beads . End repair, A-addition and ligation to NEXTflex-96 DNA barcodes  was carried out using the GeneRead DNA Library I Core Kit (Qiagen). Amplification of adapter-ligated DNA was conducted using NEXTflex primers (Bioo Scientific) and the HiFi PCR Master Mix . Next generation sequencing was performed applying 12.5 pM library pools (2% PhiX V3 control) and the MiSeq Reagent v2 chemistry .Pre-verified multiplex PCR primer sets  and all variants below 4% allelic frequency were filtered, ending up with variants listed in Supporting Information TP53 mutations is provided in Supporting Information Fastq files were generated by the MiSeq Reporter software (Illumina) and further analyzed by means of the CLC Biomedical Genomics Workbench software . The total batch of identified TP53 primer sets  and various


Scientific Reports7:9384; doi:10.1038/s41598-017-09296-w; Article published online 24 August 2017This Article contains an error in the legend of Figure 5.“Multiple sequence alignment of the prepropeptides of AtCLE18 and LjCLE34. CLE domains are highlighted with a red box and the CLEL domain is underlined in blue. Conservation between amino acid residues of the two sequences is represented by grey  and black (100%) shading”.should read:LjCLE5 and MtCLE12. The LjCLE5 translation start site corresponding to that of MtCLE12 results in a truncated prepropeptide (denoted by the asterisk shaded in black). The blue box represents an alternative start codon that results in the CLE domain region of LjCLE5 (underlined in red) being translated in frame. Black highlighted nucleic acid bases are 100% conserved”.“Sequence alignment of


Rint1) influences cellular homeostasis through maintenance of endoplasmic reticulum, Golgi and centrosome integrity and regulation of vesicle transport, autophagy and the G2/M checkpoint. Rint1 has been postulated to function as a tumor suppressor as well as an oncogene, with its role depending perhaps upon the precise cellular and/or experimental context. In humans, heterozygosity for germline missense variants in RINT1 have, in some studies, been associated with increased risk of both breast and Lynch syndrome type cancers. However, it is not known if these germline variants represent loss of function alleles or gain of function alleles. Based upon these findings, as well as our initial consideration of Rint1 as a potential candidate for Mom5, a genetic modifier of intestinal tumorigenesis in ApcMin/+ mice, we sought to explicitly examine the impact of Rint1 on tumorigenesis in ApcMin/+ mice. However, heterozygosity for a knockout of Rint1 had no impact on tumorigenesis in Rint1+/-; ApcMin/+ mice. Likewise, we found no evidence to suggest that the remaining Rint1 allele was lost somatically in intestinal tumors in ApcMin/+ mice. Interestingly, in contrast to what has been observed in Rint1+/- mice on a mixed genetic background, Rint1+/- mice on a pure C57BL/6J background did not show spontaneous tumor development. We also evaluated colorectal cancer data available in the COSMIC and ONCOMINE databases and found that RINT1 overexpression, as well as the presence of somatic missense mutations in RINT1 were associated with colorectal cancer development. In vitro evaluation of two missense variants in RINT1 suggested that such variants do have the potential to impact RINT1 function.The Rad50 Interacting Protein 1 ( Th. ThRint1Rint1 had no discernible impact on tumorigenesis in ApcMin/+ mice. Mean tumor multiplicity in the Rint1+/-; Mom5129/B6;ApcMin/+ mice was 49.6±4, which was essentially identical to the mean of 49.8±7.4 in Rint1+/+; Mom5129/B6;ApcMin/+ mice  and Rint1 expression is not reduced in colon tumors in ApcMin/+ mice , this phenotype was not enhanced in Rint1+/-; ApcMin/+ mice (data not shown).Our analysis indicated that heterozygosity for n/+ mice . Since Rn/+ mice . AlthougRint1 knockout did not support the hypothesis that heterozygosity for the Rint1 knockout allele promotes tumorigenesis in ApcMin/+ mice. These observations were consistent with data extracted from the COSMIC database, which revealed no evidence for loss of heterozygosity or deletion of RINT1 in colorectal cancers, suggesting that somatic loss of RINT1 was rare during colorectal tumorigenesis. Furthermore, we note that just 0.16% (1 of 607) colorectal tumors in the COSMIC database showed loss of RINT1 expression. By contrast, 6.1% (37 of 607) of colorectal tumors in the COSMIC database overexpressed RINT1. Likewise, comparison of colorectal cancers and normal mucosa in the Oncomine database showed that RINT1 transcript levels were significantly greater in many colon adenocarcinoma subtypes (RINT1were detected in 2% (30 of 1493) of cancers of the large intestine, and ~57% (17/30) of these somatic mutations were missense mutations  developed lymphomas [Rint1+/- mice were on a highly mixed 129 x B6 genetic background, which is known to have a high incidence of spontaneous lymphoma development[Rint1+/- mice observed in Lin et al is attributable to genetic background rather than the Rint1 knockout allele. This possibility cannot be excluded because this original description of the tumor phenotype in Rint1+/- mice did not include a parallel analysis of tumorigenesis in Rint1+/+ mice on this same mixed genetic background. Because of the possibility that genetic background might impact the tumor phenotype in Rint1+/- mice, we sought to determine if genetic background might also impact the embryonic lethality associated with homozygosity for the Rint1 knockout allele. However, as was observed on the mixed 129 x B6 genetic background, homozygosity for the Rint1 knockout allele on a pure B6 genetic background also resulted in embryonic lethality.It has been reported previously that mice heterozygous for the velopment, we cannRint1 on tumorigenesis in ApcMin/+ mice. However, this analysis revealed that heterozygosity for Rint1 did not impact intestinal tumor number or size. We also found no evidence for somatic loss of Rint1 in colon tumors in ApcMin/+ mice. Altogether, our studies with Rint1+/- mice did not support the idea that the Rint1 knockout allele functioned as either a modifier allele of tumorigenesis in ApcMin/+ mice or a cancer predisposing allele on the B6 background. Thus, these studies did not provide robust evidence to support the hypothesis that Rint1 functions as a tumor suppressor. Likewise, analysis of data available in the COSMIC and ONCOMINE databases failed to yield strong evidence that loss of RINT1 was associated with intestinal carcinogenesis in humans. Rather, analyses of available colorectal cancer data in these databases suggested that overexpression of RINT1 as well as somatic missense mutations in RINT1 may be associated with colorectal carcinogenesis. It has been reported previously that germline missense variants in RINT1 are associated with increased risk of breast and Lynch Syndrome type cancers [RINT1 mutations, most of which are missense mutations, on the function of RINT1 is not known.The original, primary purpose of this study was to evaluate the impact of heterozygosity for a knockout of  cancers . HoweverRINT1 variants in vitro and examined the impact of these variants on the interaction of RINT1 with some of its binding partners. Compared to wild type, some of the RINT1 variants we generated showed a more robust association with UVRAG, a known RINT1 interacting protein that is involved in retrograde transport and autophagy [in vitro are similar to some of those associated with Lynch Syndrome type cancers in that they are missense variants in the coil-coil domain, which is thought to mediate the interaction of RINT1 with many of its binding partners. Thus, our results raise the possibility that missense mutations in RINT1 may represent dominant, gain of function alleles that may be functionally equivalent, at least in a certain respect, to RINT1 overexpression. Altogether, these data are more consistent with the hypothesis that RINT1 functions as an oncogene rather than a tumor suppressor gene in the context of colorectal cancer.To explore the possible impact of RINT1 missense variants on RINT1 function, we expressed several missense utophagy . The RIN


Fog2 re-expression in the Fog2 null embryos is sufficient to extend their life span, identification of GATA4-FOG2 target genes in cardiomyocytes could shed light on the molecular mechanism of GATA4-FOG2 action in these cells. We report here that cardiac expression of slow skeletal troponin T (Tnnt1) strictly depends on the physical interaction between GATA4-FOG2 in the myocardium of both atria and ventricles.Previous work by us and others has shown that the loss of interaction between GATA4 and FOG2 protein partners is embryonic lethal due to heart failure at embryonic day (E) 13.5; however, the role of this important protein duo in various cardiac compartments  remains to be understood. Although a dual role (both as an activator and a repressor) for the GATA4-FOG2 transcriptional complex has been put forward, the specific genes under GATA4-FOG2 control in the developing heart have remained largely elusive. Since the myocardial-restricted


TNFAIP8 family. Members of the TNFAIP8 gene family have been linked to regulation of immune function and homeostasis and the development of multiple cancer types. Through a conserved gene synteny analysis, we identified zebrafish orthologs to human TNFAIP8L1 and TNFAIP8L3 genes and two co-orthologs to human TNFAIP8L2, but failed to identify an ortholog to human TNFAIP8. Through the application of the orthology bridge, we determined that teleost orthologs to human TNFAIP8 genes were likely lost in a genome inversion event after their divergence from their common ancestor with spotted gar. These findings demonstrate the value of this enhanced approach to gene history analysis and support the development of teleost models to study complex questions related to an array of biomedical issues, including immunity and cancer.Comparative functional genomic studies require the proper identification of gene orthologs to properly exploit animal biomedical research models. To identify gene orthologs, comprehensive, conserved gene synteny analyses are necessary to unwind gene histories that are convoluted by two rounds of early vertebrate genome duplication, and in the case of the teleosts, a third round, the teleost genome duplication (TGD). Recently, the genome of the spotted gar, a holostean outgroup to the teleosts that did not undergo this third genome duplication, was sequenced and applied as an orthology bridge to facilitate the identification of teleost orthologs to human genes and to enhance the power of teleosts as biomedical models. In this study, we apply the spotted gar orthology bridge to help describe the gene history of the vertebrate  Many organisms lack formalized gene and protein nomenclature conventions. We apply zebrafish nomenclature conventions to stickleback [Gasterosteus aculeatus], spotted gar [Lepisosteus oculatus], and coelacanth [Latimeria chalumnae] and human nomenclature conventions to Chinese softshell turtle (Pelodiscus sinensis) and anole lizard (Anolis carolinensis) genes and proteins.Nomenclature rules for vertebrate genes and proteins follow accepted conventions. This work presents gene and protein nomenclature for specific species according to their respective naming conventions  software version 3.6b  , TNFAIP8L1 , TNFAIP8L2 , and TNFAIP8L3  display 50–57% sequence identity , we determined that the zebrafish genome encodes four members of the vertebrate TNFAIP8 gene family, called tnfaip8 (ENSDARG00000086457), tnfaip8l2a (ENSDARG00000075592), tnfaip8l2b (ENSDARG00000046148), and tnfaip8l3 (ENSDARG00000088709). To investigate relationships of members of the zebrafish tnfaip8 gene family to their human orthologs, we compared conserved syntenies of chromosome segments containing the four zebrafish genes to the human genome. We found that the gene called ‘tnfaip8’ (ENSDARG00000086457) occupies a chromosome segment on Dre22 whose genes are mostly orthologous to the region of Hsa19 that contains TNFAIP8L1, not to the region of Hsa5 that contains TNFAIP8  but not with TNFAIP8 (Hsa5). The region of Hsa19 between 0 and 10Mb displays conserved synteny with both Dre 22 and Dre2 , the locus originally designated as tnfaip8 was changed to tnfaip8l1, which is in agreement with our earlier analysis (ENSDARG00000086457 22:4769086–4787454:-1). We carefully examined EST data that had been previously associated with the incorrectly annotated term “zebrafish tnfaip8” (located on UniGene [https://www.ncbi.nlm.nih.gov/unigene] at UGID: 2554148) and determined that there was no evidence of a zebrafish tnfaip8 ortholog in the expression data.In our initial investigation using Ensembl Zv9  and tnfaip8l2b. (ENSDARG00000046148). Conserved synteny analysis verified that the sections of the zebrafish genome on chromosomes Dre19 and Dre16 that contain these two genes are both orthologous to the section of Hsa1 that contains TNFAIP8L2, consistent with their gene nomenclature  and that a true ortholog of human TNFAIP8 is absent from the zebrafish genome.These data lead to the conclusion that zebrafish has one ortholog of TNFAIP8 lies at 118.6Mb on chromosome Hsa5 and is flanked by DMXL1 and HSD17B4. All three genes are transcribed in the same direction (DMXL1 (dmxl1[1of2] and dmxl1[2of2]) and an adjacent ortholog of HSD17B4 lie adjacent to one another, 3.2 kB apart at the left tip of Dre8 at location 0.3 Mb. The 3.2 Kb between zebrafish dmxl1 and hsd17b4 contains no sequence recognizable as a TNFAIP8 ortholog (or any other known genetic element)   as foundrections  rather tebrafish , suggestdmxl1, tnfaip8, and hsd17b4 genes in the same order and orientation as in the human genome , hsd17b4, [TNFAIP8 gene, however, resides in the region of Hsa5 between 117Mb and 125Mb, whose TGD paralog is less unclear. To investigate this point further, we looked for paralogs of genes occupying the left tip of Dre8 (note that hsd17b4 is the tenth gene from the left telomere). Results failed to reveal an obvious paralogon for this region, although Dre5 and Dre10 possess some more distantly related homologs  form a monophyletic group with 99.8% support , two zebrafish co-orthologs to mammalian Tnfaip8l2 (tnfaip8l2a and tnfaip8l2b), and one zebrafish ortholog to mammalian Tnfaip8l3 (tnfaip8l3). The phylogenetic tree suggests, although without strong support (59% for the TNFAIP8/TNFAIP8L1 clade and 48.4% for the TNFAIP8L2/TNFAIP8L3 clade), that VGD1 produced a TNFAIP8/8L1 ancestor gene and a TNFAIP8L2/8L3 ancestor gene, and then VGD2 produced the full group of four genes  that is represented in the current human genome  and percomorphs (including stickleback) teleosts. This loss could have resulted after the divergence with the spotted gar but before the VGD because no tnfaip8 orthologs have been identified in other representative teleost species, including stickleback and zebrafish. It is also possible that the tnfaip8-containing region duplicated with the TGD to produce, tnfaip8a and tnfaip8b ohnologs and that two independent events—one associated with an inversion break point and the other associated with the loss of several adjacent genes—led to the loss of both tnfaip8 TGD ohnologs. Although the two-loss scenario seems less likely by strict parsimony, biologically it seems to be more likely. To help answer this question, we attempted to identify tnfaip8 gene family members in the genomes of the Japanese and European eels , which are representatives of the Anguilliformes, a basally diverging lineage that arose immediately after the TGD [The vertebrate L3 genes . Our ana the TGD . We perfTNFAIP8L1 and TNFAIP8L3 genes  and TNFAIP8L3 (tnfaip8l3a and tnfaip8l3b) were present in the common teleost ancestor genome and that one of the co-orthologs for each gene was subsequently lost, leading to the current condition in teleost genomes. The loss of tnfaip8 may reflect a non-essential function and/or functional redundancy among tnfaip8 family members. In contrast, two co-orthologs to the human TNFAIP8L2 gene remain present in teleosts (tnfaip8l2a and tnfaip8l2b)  . Based ulization . It is afunction . Going ftnfaip8 gene from the teleost lineage and described a genome inversion mechanism by which it likely occurred. Our findings highlight the value of the spotted gar as an orthology bridge that can be used to not only identify gene orthologs shared between mammalian and teleost genomes but also to resolve discrepancies related to ohnolog loss. These sorts of studies have broad applicability and will serve to strengthen rationales for the application of teleost models to study problems in human health and disease.Our findings support the importance of characterizing vertebrate gene histories in developing teleost models for human biomedicine , 48. TelS1 Fig(A) Clustal Omega alignment of human TNFAIP8, TNFAIP8L1, TNFAIP8L2, and TNFAIP8L3 protein sequences. (B) Percent amino acid identity based on pairwise comparisons between each of the TNFAIP8 family members.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.


Editorial):“Next, Duchemin-Pelletier et al. present data about the role of CK2β, the regulatory subunit of the ubiquitous protein kinase CK2 [16]. In MCF10A mammary epithelial cells where CK2β expression was downregulated, the authors provide evidence that CK2β downregulation can promote the acquisition of characteristics commonly associated with the cancer stem cell phenotype. They demonstrate that a CK2β level establishes a critical cell fate threshold in the control of epithelial cell plasticity. Since EMT supports the induction stem-cell phenotype, identifying the CK2 substrates will improve the discovery of new specific markers for breast cell stemness.”The author wishes to make the following correction to the paper . The folThe reference numbering was also corrected.The author would like to apologize for any inconvenience caused. The manuscript will be updated and the original will remain online on the article webpage.


A novel highly pathogenic avian influenza A(H5N6) virus affecting wild birds and commercial poultry was detected in the Netherlands in December 2017. Phylogenetic analysis demonstrated that the virus is a reassortant of H5N8 clade 2.3.4.4 viruses and not related to the Asian H5N6 viruses that caused human infections. The samples tested positive by real-time PCR on the matrix gene (Cygnus olor) and a tufted duck (Aythya fuligula) were found dead in this area , as described previously  that was detected in February 2017 but likely represents a separate introduction related to wild bird migration in fall 2017. The reassortment events may have occurred on breeding grounds in Siberia, where large numbers of wild birds congregate, and the virus may have spread by long-distance flights of infected migratory birds  can be infected by the novel reassortant HPAI H5N6 viruses detected in the Netherlands, Greece, Japan, and Taiwan. We recommend further studies in mammals (ferrets or mice) to provide experimental data on the virulence for mammals.Additional information about the emergence of HPAI H5N6 virus in the Netherlands in December 2017.


Cdca7 (Cell division cycle associated 7) expression by transcription factor Pax6 contributes to Pax6’s cellular actions during corticogenesis. The function of Cdca7 in mediating Pax6’s effects during corticogenesis has not been explored. Pax6 is expressed by radial glial progenitors in the ventricular zone of the embryonic cortical neuroepithelium, where it is required for the development of a normal complement of Tbr2-expressing intermediate progenitor cells in the subventricular zone. Pax6’s expression levels are graded across the ventricular zone, with highest levels laterally where Tbr2-expressing progenitors are generated in greatest numbers at early stages of corticogenesis.We studied whether regulation of Cdca7 and Pax6 expression in cortical tissue from wild-type and Pax6/−− embryos. In each genotype we compared the graded expression of the two genes quantitatively at several ages. To test whether defects in Cdca7 expression in lateral cortical cells might contribute to the cellular defects in this region caused by Pax6 loss, we electroporated a Cdca7 expression vector into wild-type lateral cortex and examined the effect on the production of Tbr2-expressing cells.We used in situ hybridization and immunohistochemistry to analyse patterns of Cdca7 is co-expressed with Pax6 in cortical progenitors, at levels opposite to those of Pax6. Lowest levels of Cdca7 are found in the radial glial progenitors of lateral cortex, where Pax6 levels are highest. Higher levels of Cdca7 are found in ventral telencephalon, where Pax6 levels are low. Loss of Pax6 causes Cdca7 expression to increase in the lateral cortex. Elevating Cdca7 in normal lateral cortical progenitors to levels close to those normally found in ventral telencephalon reduces their production of Tbr2-expressing cells early in lateral cortical formation.We found that Cdca7 expression in the lateral cortex and that repression of Cdca7 in cells of this region is required for their production of a normal complement of Tbr2-expressing intermediate progenitors.Our results suggest that Pax6 normally represses  In rodents, about two thirds of excitatory, pyramidal neurons in the cerebral cortex are generated from progenitor cells that divide beneath the apical surface of the embryonic cortical neuroepithelium in a region called the subventricular zone –3. TheseEarly in corticogenesis, Pax6 expression levels are graded across the ventricular zone of the developing cortical neuroepithelium. Levels increase with proximity to the boundary between the lateral cortex and the subpallium. Pax6 exerts regional control of cell proliferation, with its greatest effects in anterior and lateral regions where its levels are highest . It is rPax6 identified Cdca7 (Cell division cycle associated 7) as a gene whose expression is increased by loss of Pax6 function  to caudo-medial [low]. As corticogenesis continues, this gradient of Pax6 expression becomes progressively less steep [Cdca7 normally correlates with that of Pax6 and whether the expression of Cdca7 is affected in mutants that are null for Pax6.Pax6 is expressed at high levels in the ventricular zone of the developing cerebral cortex and at lower levels in the ventricular zone of the lateral ganglionic eminence (LGE). At the earliest stages of murine cortical neurogenesis, around embryonic days 12–13 (E12-13), ss steep . At all Pax6 and Cdca7 in adjacent sections from wild-type  and Pax6/−− (n = 4) embryos at E12.5 . In contrast, Cdca7 expression levels were lower in the lateral cortex than in the LGE , Pax6 expression declined more gradually than normal across the PSPB, as at E12.5  and a ribbon whose width corresponded to that of the gene expression domains was drawn through the telencephalon from dorsal to ventral as shown in Fig. Pax6 gene. Regression analysis gave values for the slopes on the two sides .We used a quantitative approach to confirm the breakdown in the normal expression of Pax6 and Cdca7, with significant interaction effects for both genes . In wild-types, the gradients of Pax6 and Cdca7 expression were opposite at E12.5  or reaching (in the case of Cdca7) zero by E14.5 . In Pax6/−− pallium, the gradient of Pax6 expression was much smaller than normal at E12.5  and then reversed  but one appeared, with a slope opposite to normal, over the subsequent 2 days .In the pallium . Pax6 and Cdca7 were expressed in opposing gradients from E12.5-14.5  sequence to generate an HA epitope-tagged version of Cdca7. This was cloned into the expression vector pCAGGS_GFP, which expresses green fluorescent protein (GFP) tagged with a nuclear localization signal (NLS)  Fig. B. The CdLS) Fig. C. The leLS) Fig. D. GFP+ cLS) Fig. E. ImmunoLS) Fig. F, G. Sinitiation .Pax6/−− mutants (Fig. We estimated the level of Cdca7 expression from the electroporated cells relative to surrounding non-electroporated cells by randomly selecting 30 GFP-expressing cells and 30 intermingled GFP-non-expressing cells within the lateral cortex, outlining each cell and measuring the intensity of the Cdca7 signal from each using Fiji software. We found that the average intensity of the Cdca7 signal was 4.4-fold higher in the GFP+ than in the GFP− cells. To put this difference into context, we then carried out the same analysis with 30 GFP-non-expressing cells from the ventral telencephalon, where Cdca7 is highly expressed by some cells; in this case we selected cells with high levels of expression, located in the ventral telencephalic subventricular zone (Fig. We examined all GFP+ electroporated cells 16 h after electroporation at either E12.5 or E14.5 in a series of sections stained for: GFP and Tbr2; GFP and Tbr1; GFP and BrdU. We found no evidence that the elevation of Cdca7 levels altered the distribution of affected cells through the cortical depth 16 h after electroporation Fig. .Fig. 6ThThere was a significant reduction by ~20–25% in the proportion of GFP+ cells that were Tbr2+ in embryos electroporated with Cdca7 at E12.5 (Fig. Analysis of the proportion of cells labelled by BrdU shortly before animals were sacrificed allowed us to assess whether elevated Cdca7 levels in the lateral cortex had a drastic effect on proliferation, for example bringing effected cells out of the cell cycle. We found that 20–30% of GFP+ cells took up BrdU in both control and experimental groups following electroporation at E12.5 or E14.5 Fig. . At neitOur findings indicate that the main effect of elevated Cdca7 expression in early embryonic lateral cortical progenitors is to reduce the production of cells expressing Tbr2, which is the hallmark of intermediate progenitors.Pax6/−− forebrain is a failure of the lateral cortex to expand ahead of more medial regions. This regional defect has been linked to a disproportionate underproduction of Tbr2+ cells in the lateral cortex of Pax6/−− embryos, a loss which equilibrates the numbers of Tbr2+ cells across all cortical regions [Based on new findings reported here, we suggest that one of the actions of Pax6 in the embryonic cerebral cortex is to suppress Cdca7 expression and thereby enhance the production of Tbr2+ intermediate progenitors. Interestingly, our experiments suggested that this suppressive effect is most prominent in the lateral cortex early in corticogenesis, where cortical levels of Pax6 expression are highest . Since T regions . It is pOn the ventral side of the PSPB in wild-type embryos, Pax6 levels are lower and Cdca7 levels are higher, particularly in the subventricular zone of the subpallium. As an extension of the present study, it would be interesting to test with gene knock-out or knock-down experiments whether the particularly high levels of Cdca7 that we observed in the subpallial subventricular zone contribute to the lack of Tbr2 expression in the cells of this region.CDCA7 was first identified as a MYC direct target and CDCA7 protein is often found to be overexpressed in human solid tumours [Cdca7 expression and whether the repressive effects of Pax6 on Cdca7 expression are direct, or an indirect consequence of effects on Notch signalling, or both.Relatively little is known about the mechanisms of action of Cdca7. Human and mouse CDCA7/Cdca7 are nuclear proteins which contain a zinc finger domain in the C-terminus , 15. In  tumours , 26–28.  tumours . The evi tumours . In the  tumours , 31. It Cdca7 overexpression early in corticogenesis reduced not only the production of Tbr2+ intermediate progenitors but also the production of Tbr1+ cells. The divisions of Tbr2+ cells generate Tbr1+ neuronal precursors early in corticogenesis. The length of cell cycles in the cerebral cortex is around 10 h at E12.5, which is a few hours shorter than the 16 h between electroporation and sacrifice in our experiments , 33. It Pax6 and Cdca7 mRNA cortical expression gradients following mutation of the Pax6 gene indicated that both become highly abnormal from E12.5 to E14.5. By E14.5, the slopes of both gradients had reversed from normal. This result highlights the complexity of the molecular interactions that establish graded expression patterns. It indicates that Pax6 is not the only factor that can affect differential Cdca7 expression levels across the developing cortex. Changes in the expression of Pax6 mRNA in Pax6/−− mice are likely to result from a loss of direct or indirect autoregulation of the Pax6 locus, which is known to be a prominent element in the control of expression of the Pax6 gene [Finally, our quantitative analysis of ax6 gene .In summary, this study provides evidence of counter-gradients of Pax6 and Cdca7 during embryonic ages E12.5 to E14.5 in mice and this relationship is disrupted when Pax6 is absent. In addition, elevated Cdca7 in the lateral cortex reduces the production of intermediate progenitors and postmitotic neurons, suggesting that repression of Cdca7 by Pax6 is required for the normal production of intermediate progenitors in this region.


Drosophila melanogaster, three Robo paralogs  each have specialized roles in regulating midline crossing and the formation of longitudinal axon pathways in the embryonic ventral nerve cord. The number of robo genes differs in other insects, and it is unknown whether the roles and/or signaling mechanisms of Drosophila Robos are shared in other insect species. To directly compare the axon guidance activities of Robo receptors in Drosophila and the flour beetle Tribolium castaneum, I have used a CRISPR/Cas9-based approach to replace Drosophila robo3 with Tribolium robo2/3.Axon guidance receptors of the Roundabout (Robo) family regulate a number of axon guidance outcomes in bilaterian animals in addition to their canonical role in Slit-dependent midline repulsion. In the fruit fly robo3 locus in Drosophila embryos, Tribolium Robo2/3 (TcRobo2/3) protein is properly translated and localized to axons, where it reproduces the normal expression pattern of Drosophila Robo3. In embryos expressing TcRobo2/3 in place of robo3, two distinct subsets of longitudinal axons are guided properly to their normal positions in the intermediate neuropile, indicating that TcRobo2/3 can promote Robo3-dependent axon guidance decisions in developing Drosophila neurons.I show that when expressed from the Drosophila Robo3 promotes longitudinal pathway formation is evolutionarily conserved in Tribolium, where it is performed by TcRobo2/3. The CRISPR/Cas9-based gene replacement approach described here can be applied to comparative evolutionary developmental studies of other Drosophila genes and their orthologs in other species.These observations suggest that the mechanism by which  Drosophila melanogaster, where the underlying genetics has been best studied, the medial–lateral and dorsal–ventral position of dendrites, axon terminals, and longitudinal axon pathways within each neuromere is specified by a combination of extracellular guidance cues including Slit and semaphorin ligands, and their neuronal receptors of the Roundabout (Robo) and Plexin families, respectively [During development of the nervous system, neural circuits form as axons and dendrites follow directional cues which guide them to specific regions of the central nervous system (CNS), where local contact-dependent cues promote the formation of synaptic connections. In the insect ventral nerve cord, a stereotypical three-dimensional arrangement of axons and dendrites forms within each segmentally repeated neuromere , 2. In tectively –5.Drosophila, the secreted Slit ligand and its Robo receptors regulate midline crossing of axons in the developing embryonic CNS. In addition, two of the three Robo family members in Drosophila (Robo2 and Robo3) specify the medial–lateral position of longitudinal axon pathways. The functional specialization of the three Drosophila Robos depends on both their differential expression in distinct neuronal subsets and distinct activities of the receptors themselves [Drosophila robo3 gene, along with all of the intervening introns, can be replaced with a non-native cDNA encoding robo3 without any detectable changes to its expression pattern or function [robo3 with robo1 or robo2 cDNAs further demonstrated that each of these paralogs can fully substitute for robo3 to promote axon pathway formation in intermediate regions of the neuropile in the Drosophila embryonic CNS. In contrast, neither robo1 nor robo3 can rescue robo2-dependent formation of lateral pathways, suggesting that unique structural features of Robo2 specify this role [Drosophila Robo receptors regulate lateral position in response to a gradient of Slit originating at the CNS midline, the precise mechanism(s) underlying this activity have not been fully characterized [In emselves –9. Previfunction . Equivalhis role . Althougcterized .robo2 and robo3 have distinct expression patterns and axon guidance roles in Drosophila, these two genes are a product of a recent gene duplication and do not exist as separate genes outside of dipteran insects. Instead, most insect groups have retained an ancestral robo2/3 gene [Tribolium castaneum, this gene (TcRobo2/3) appears to combine the activities of Drosophila robo2 and robo3 and is required for the formation of both intermediate and lateral axon pathways [Drosophila Robo2 and Robo3 throughout its extracellular portion, with the highest degree of sequence conservation in the Ig domains, especially the Slit-binding Ig1 domain . Despite this size difference, the two conserved cytoplasmic (CC) motifs present in Drosophila Robo3 are also present in TcRobo2/3. These two motifs, named CC0 and CC1, are conserved in most Robo family members, including Drosophila Robo1, Robo2, and Robo3, as well as Robo1 and Robo2 in vertebrates [While 2/3 gene , 12. In pathways . The TcRtebrates .Fig. 1Serobo2 and robo3 was required for both intermediate and lateral pathway formation, and that this dual activity is retained by the robo2/3 gene in most insect species (including Tribolium), while the activities of Drosophila robo2 and robo3 have diverged through subfunctionalization after gene duplication [TcRobo2/3 should be able to substitute for both Drosophila robo3 and robo2 to promote intermediate and lateral pathway formation, respectively. Here, I test this prediction by replacing the robo3 gene in Drosophila with the robo2/3 gene from Tribolium via CRISPR/Cas9-mediated gene replacement. I find that when expressed from the robo3 locus, TcRobo2/3 protein reproduces the endogenous Robo3 expression pattern in the Drosophila embryonic CNS, including proper localization to neuronal axons and distribution on longitudinal axon pathways within intermediate and lateral regions of the neuropile. Further, TcRobo2/3 is able to substitute for robo3 to direct two distinct subsets of longitudinal axons to intermediate regions of the neuropile. These results suggest that Drosophila robo3 and Tribolium robo2/3 regulate axon pathway formation through an evolutionarily conserved mechanism and demonstrate the utility of CRISPR/Cas9-mediated gene replacement for comparative studies in evolutionary developmental biology.We have previously hypothesized that the ancestor of lication , 11. A probo3 donor construct was assembled from four PCR fragments via Gibson assembly (New England Biolabs E2611). The four fragments were derived from pBluescript , the wild-type robo3 genomic locus , and the robo3 cDNA . This initial donor construct contained an untagged robo3 cDNA flanked by BglII sites. To make the HA-tagged robo3TcRobo2/3 donor, the robo3 coding sequence was excised with BglII and a 4xHA sequence flanked by BamHI (upstream) and BglII (downstream) sites was cloned into the BglII site, thus retaining a single BglII cloning site immediately downstream of the 4xHA tag. The TcRobo2/3 coding sequence was amplified by PCR using primers 279 and 280, then digested with BglII, and cloned into the 4× HA-containing donor backbone. The entire donor region including TcRobo2/3 coding sequence and robo3 flanking regions was sequenced prior to injection.The initial robo3 gRNA sequences were cloned into the tandem expression vector pCFD4 [or pCFD4  via PCR Drosophila strains, transgenes, and mutant alleles were used: Canton-S (wild type), robo31 [robo3robo3 [robo3TcRobo2/3 (this study), P{sema2b-TauMyc} [w1118; snaSco/CyO,P{en1}wgen11 (“Sco/CyOwg”). All crosses were carried out at 25 °C.The following  robo31 , robo3ro3robo3 , robo3T-TauMyc} , w1118robo3 gRNA and robo3TcRobo2/3 homologous donor plasmids were coinjected into nos-Cas9.P embryos [Sco/CyOwg. Founders  were identified by testing two pools of three F1 females per G0 cross by genomic PCR with primers 229 and 280, which produce a 0.8-kb product only when TcRobo2/3 sequences are present. From each identified founder, 5–10 F1 males were then crossed individually to Sco/CyOwg virgin females. After three days, the F1 males were removed from the crosses and tested by PCR with primers 229 and 280 to determine whether they carried the modified allele. F2 flies from positive F1 crosses were used to generate balanced stocks, and the modified alleles were fully sequenced by amplifying the entire modified locus (approx. 6 kb) from genomic DNA using primers 589 and 590, then sequencing the PCR product after cloning via CloneJET PCR cloning kit (Thermo Scientific). Details of G0 survival, fertility, and modified allele transmission rates are provided in Table The  embryos  by BestGDrosophila embryo collection, fixation, and antibody staining were carried out as previously described . The folTribolium Robo2/3 can substitute for Drosophila robo3 to promote axon guidance outcomes in the Drosophila embryonic CNS, I used a CRISPR/Cas9-based approach to replace the robo3 gene with TcRobo2/3. Two guide RNAs (gRNAs) targeting exons 2 and 12 were combined with a homologous donor plasmid containing 1-kb flanking regions to induce homology-directed repair, replacing robo3 exons 2-12 with an HA-tagged TcRobo2/3 cDNA  [robo3TcRobo2/3 donor plasmid into Drosophila embryos expressing Cas9 under the control of the germline-specific nanos promoter [TcRobo2/3 sequences. Additional PCR screening and DNA sequencing were used to identify correctly modified robo3TcRobo2/3 loci among the lines recovered from the positive F1 flies. Further details are provided in Methods and Table To test whether  (pCFD4)  was injepromoter , and F1 robo3TcRobo2/3 allele, I first compared its expression to that of the endogenous Robo3 protein in various wild-type and modified genetic strains , there are no detectable large-scale defects in the axon scaffold in robo31 mutants, as assayed by an antibody against horseradish peroxidase (anti-HRP), which labels all of the axons in the CNS . In robo3robo3 homozygous embryos, the Robo3 protein expressed from the modified locus precisely reproduces Robo3’s normal expression pattern  axon segments and longitudinal axons in the medial neuropile. No appreciable levels of TcRobo2/3 appear to accumulate within the cell bodies of normally robo3-expressing neurons, or on the cell body plasma membrane, consistent with proper subcellular localization of the Tribolium Robo2/3 protein in this heterologous context. These observations indicate that the TcRobo2/3 protein is properly translated and trafficked within Drosophila neurons, and suggest that axons that would normally select intermediate or lateral pathways under conditions of wild-type Robo3 expression make equivalent pathway choices when TcRobo2/3 is expressed in its place.In the robo3TcRobo2/3 embryos, I used an antibody against the cell adhesion molecule Fasciclin II (FasII) to label a subset of longitudinal axon pathways in these embryos  by which robo3 and TcRobo2/3 regulate axon guidance outcomes in the Drosophila and Tribolium embryonic CNS, respectively, are evolutionarily conserved. In addition, the gene replacement approach described here should be broadly applicable to evo-devo studies of other genes in Drosophila and other insects.In this paper, I have used a CRISPR/Cas9-based gene replacement approach to directly examine the evolutionary conservation of axon guidance activities within the Robo family of axon guidance receptors. I have replaced the Drosophila axons: (1) The subset of longitudinal axons that normally express Robo3 are still localized to the lateral two-thirds of the neuropile when they express TcRobo2/3 instead of Robo3; (2) FasII-positive axon pathways in the intermediate neuropile form in their proper location when robo3 is replaced by TcRobo2/3; and (3) sema2b-positive longitudinal axons also select their correct intermediate position when robo3 is replaced by TcRobo2/3. These observations, together with a previous report that RNAi-mediated knockdown of TcRobo2/3 disrupts intermediate pathway formation in the Tribolium embryonic CNS [Drosophila Robo3 promotes longitudinal pathway formation is evolutionarily conserved in Tribolium, where it is performed by TcRobo2/3. CRISPR approaches have recently been used successfully in Tribolium [TcRobo2/3 with Drosophila robo3. In this case, robo3 should be able to replace the function of TcRobo2/3 to promote intermediate pathway formation, but the lateral pathways (which presumably depend on a robo2-like activity of TcRobo2/3) may not form properly.Three lines of evidence suggest that TcRobo2/3 can act equivalently to Robo3 to guide onic CNS , suggestribolium , so it sDrosophila embryonic CNS suggests that any sequences that are necessary for this role in Robo3 are conserved in TcRobo2/3. Notably, the highest degree of sequence conservation between these two proteins occurs within the extracellular Ig1 and Ig3 domains  are conserved in Robo3 and Robo2/3, but there is little conservation in the cytodomain outside of these motifs . A similar approach should be applicable to other genes assuming that, like all three Drosophila robo genes, the introns that would be removed by the cDNA replacement are dispensable for proper expression of the gene to be modified.The gene replacement approach described here has several advantages for both structure–function studies and evo-devo functional comparisons of orthologs from different species. First, replacing multiple coding exons in the endogenous gene with a single cDNA sequence allows for epitope tagging of the modified locus in addition to replacing the entire coding region with a foreign sequence or a modified cDNA carrying engineered deletions, additions, or sequence swaps. For multi-exon genes, this eliminates the need to replace multiple exons individually. Second, the endogenous promoter and translational start site remain unmodified, minimizing effects on gene expression. Third, using two gRNA targets that flank the majority of the gene should allow recovery of a null deletion allele via non-homologous end joining (NHEJ) in addition to the HDR-mediated gene replacement allele. This would be of particular benefit for genes for which a null allele is not currently available. Finally, this approach allows the same gRNA plasmid and donor backbone to be used for multiple gene replacement experiments, minimizing troubleshooting and optimization for each subsequent experiment. We have used the same donor construct backbone to replace Drosophila and used this strategy to demonstrate the evolutionary conservation of axon guidance mechanism(s) between the Drosophila robo3 and Tribolium robo2/3 genes. When expressed from the robo3 locus, TcRobo2/3 protein is properly translated and localized to neuronal axons in the Drosophila embryonic CNS and can guide developing axons to pathways in the intermediate region of the neuropile in an equivalent manner to Drosophila Robo3. The approach described here should be generally applicable to other genes and developmental contexts and will be useful to interrogate the evolutionary conservation or divergence of additional axon guidance mechanisms in insects.Here I have described a strategy for CRISPR/Cas9-based trans-species gene replacement in