The addition of MRI to mammography and ultrasound for breast cancer screening has been shown to improve screening sensitivity for high risk women, but there is little data to date for women at average or intermediate risk.CHEK2 mutation, 370 women had a first-degree relative with breast cancer (and no CHEK2 mutation) and 2269 women had neither risk factor. Subjects were followed for breast cancer for three years from the second screening examination.Two thousand nine hundred and ninety-five women, aged 40 to 65 years with no previous history of breast cancer were enrolled in a screening program, which consisted of two rounds of MRI, ultrasound and mammography, one year apart. Three hundred and fifty-six women had a n = 156) was greater than the number incurred by mammography (n = 35) or ultrasound (n = 57). Of the 19 invasive cancers detected by MRI, 17 (89%) were also detected by ultrasound or mammography.Twenty-seven invasive epithelial cancers, one angiosarcoma and six cases of DCIS were identified over the four-year period. Of the 27 invasive cancers, 20 were screen-detected, 2 were interval cancers, and five cancers were identified in the second or third follow-up year . For invasive cancer, the sensitivity of MRI was 86%, the sensitivity of ultrasound was 59% and the sensitivity of mammography was 50%. The number of biopsies incurred by MRI (In terms of sensitivity, MRI is slightly better than the combination of mammography and ultrasound for screening of women at average or intermediate risk of breast cancer. However, because of additional costs incurred by MRI screening, and the small gain in sensitivity, MRI screening is probably not warranted outside of high-risk populations. BRCA1 or BRCA2 mutation, or at moderate risk due to a family history. It is not clear if MRI should be used to screen women at average or intermediate risk. It is possible that MRI performs differently in women with and without BRCA mutations because of the differences in cancer risk and also potentially because of the difference in the natural history of cancers in the two groups. Most cancers in non-carriers or in CHEK2 carriers are ER-positive [BRCA1 carriers are usually triple-negative [Several studies have demonstrated that annual magnetic resonance imaging (MRI) is a more sensitive means of screening for breast cancer than annual mammography for women at high risk of cancer –10. Thespositive  whereas negative  and thesnegative . Women wnegative . Missensnegative .There are many relevant issues relating to expanding MRI screening to average - and moderate-risk populations, including the costs of screening, technical considerations including the availability of MRI-guided biopsy and the possibility of over-diagnosis. It is important to establish whether or not the gain in sensitivity obtained with MRI screening in terms of cancers detected over and above mammography (or a combination of mammography and ultrasound) justifies the expense. Ultimately the value of MRI screening should be determined by demonstrating an improvement in breast cancer mortality among women undergoing regular MRI screening.We conducted a multicenter observational study in Poland wherein we followed 2995 women (2269 at average risk and 726 at moderately elevated risk) who were screened with three screening modalities  on two occasions one year apart and then were followed for an additional three years. We estimate the risks of prevalent cancers, incident cancers and interval cancers in the cohort.CHEK2 mutation or a first-degree relative with breast cancer. To identify CHEK2-positive patients, the records of the hereditary cancer clinic of the Pomeranian Medical University were reviewed. Women who were between the ages of 40 and 65 and who had no previous history of breast cancer or of another cancer and were known to carry a CHEK2 mutation (truncating or missense) were invited to participate. Through this process, 496 CHEK2 carriers were invited, of whom 356 (72%) agreed to participate. The remaining study participants were recruited through a newspaper campaign conducted in different regions of Poland. Women with and without a family history of breast cancer were eligible to participate. Through this means, the study cohort was expanded to include an additional 2639 women at average risk, including 370 with a family history of breast cancer and 2269 with no family history of breast cancer. The distribution of patients was as follows . A blood sample was taken from each woman for DNA extraction. Genetic testing was done for four CHEK2 mutations  and for three BRCA1 mutations that are founder mutations in Poland  [BRCA1 mutation were excluded.The study group consisted of women at average risk of cancer and women with a moderately increased risk of cancer due to an inherited Women were screened twice, one year apart and were followed for three more years for incident breast cancers. Women were included in the study if they had all three examinations (MRI and ultrasound and mammography) at the first round. The ultrasound examination and mammography either took place the day before the MRI examination or on the same day. In some cases the radiologist had access to the MRI results at the time the ultrasound was conducted, but this was uncommon. Clinical breast examination was performed by the radiologist on a routine basis, but was not evaluated formally in this analysis. Ethics approval was obtained by the institutional review board of the Pomeranian Medical University and by all participating institutions. All participants provided written informed consent.prevalent cancers. Cancers detected on the second screening round were categorized as incident cancers. Cancers detected between screening rounds or within one year of the last screen were categorized as interval cancers. Cancers detected in the second or third year post-screening were categorized as post-screening cancers. These cancers were not considered in the evaluation of screening parameters (e. g. sensitivity) but were included in order to estimate the incidence rate of cancers in the cohort.The imaging studies were performed in nine different institutions (see above). MRI screening in each center was performed using General Electric Signa HDXT 1.5 Tesla scanner with gadolinium enhancement. The images generated by the ultrasound and mammography examinations were interpreted locally and the images generated by the MRI were interpreted centrally in Szczecin by two reference radiologists . All imaging studies were read by a radiologist affiliated with the study and were classified using the American College of Radiology Breast Imaging Reporting and Data System (BI-RADS) as follows: 0-further information or workup required; 1-negative; 2-benign finding; 3-probable benign finding ; 4-suspicious abnormality; 5-highly suggestive of malignancy . If any Women were contacted by telephone or by mail annually to inquire if they had been diagnosed with breast cancer in the three-year period following the second screening round. For 80% of the women who entered the study, follow-up was at least two years and for 76% of the women follow-up was three years after the second screening round. No screening MRI was done in the follow-up period. In some cases, the woman may have had screening mammography in the follow-up period outside of the study, but these details were not collected. Further, details on clinical breast examinations were not collected. For those women who developed cancer, details of the cancer were retrieved from medical records.Women with a BI-RADS 4 abnormality detected on MRI were followed for four to five years from the time of the abnormal MRI (until July 2015) in order to interpret the clinical impact of having a BI-RADS 4 abnormality without a biopsy on subsequent cancer risk. Follow-up was by telephone interview to the patients herself and inquiring about new diagnoses of breast cancer.We collected information on both invasive cancers and in situ cancers. Sensitivity was calculated for invasive epithelial cancers only. The sensitivity of a given modality was defined as the number of biopsy-proven cancers detected by that modality, divided by the total number of cases detected by all modalities, plus the interval cancers. The positive predictive value was the ratio of the number of biopsy-proven cancers detected by a given modality  divided by the number of biopsies performed that were occasioned by a positive screening test by the same modality. In the cost analysis, the costs used were the actual prices which were negotiated between the Pomeranian Medical University, with the Polish Ministry of Science and Euromedic. The cost analysis is restricted to the costs of screening only .CHEK2 mutation (49 truncationg and 307 missense). 51 women had both a first-degree relative with breast cancer and a CHEK2 mutation. Over the four year study period, breast cancer was diagnosed in 34 women, including six cases of ductal carcinoma in situ, one angiosarcoma and 27 invasive breast cancers. The following analyses are limited to the 27 invasive breast cancers.A total of 2995 women completed the first round of screening Table . The meaIn the first round of screening, 13 invasive cancers were detected  Table . Six of Of the 2565 women who participated in the second screening round, 2335 women were followed for an additional two years and 2273 women were followed for an additional three years through telephone interview and a mailed questionnaire. In the first year after the screening period (year three) there was only one invasive cancer diagnosed, in a 52 year old woman. It was 3.0 cm in size and node negative. She had been screened with MRI/ultrasound/mammography eight months prior to diagnosis. In the second year post screening, four additional cancers were diagnosed; on average these were 1.7 cm in size and one was node positive. In the third year after screening there was only one breast cancer diagnosed in the left breast of a 53-year old woman. She also had a breast cancer detected in the first round of screening in the right breast. The second  cancer was detected by the woman herself and mammography was negative. The cancer was 2.2 cm in size and was node-positive. In total, after the second screen, six cancers detected among 2335 women contributing 6643 person-years of follow-up .Of the 22 invasive cancers detected during the screening period (first screen to one year after second screen) 20 were detected by one of the screening modalities  and two were interval cancers. The highest sensitivity was seen with MRI (86%), followed by ultrasound (59%) and mammography (50%). Eighteen of the 22 cancers were detected by either mammography or ultrasound and the sensitivity of mammography and ultrasound in combination (77%) was only slightly inferior to that of MRI (86%). This combination identified one cancer that was missed by the MRI. There were two cancers that were identified by the MRI that were not identified by any other screening modality. These were diagnosed in a 51-year old and a 66-year old woman and were 0.8 cm and 1.1 cm respectively. Both cancers were node-negative, non-palpable, ER-positive and HER2-negative.In the entire study, 571 women  had an abnormal examination by at least one screening modality . Two hundred and four women (36%) underwent a total of 216 biopsies, including all women with a BI-RADS 5 anomaly, 139 women with a BI-RADS 4 anomaly and 65 women with a BI-RADS 0 anomaly. These 204 women represent 6.8% of the entire study population. On 26 occasions (12.0%) the biopsy revealed cancer  and on 190 occasions (88.0%) the biopsy revealed either normal tissue or benign disease. Overall, the positive predictive value was 12.1% for MRI, was 14.2% for ultrasound and was 31.4% for mammography. There were 216 biopsies performed. There were 156 biopsies performed in women with an abnormal MRI, followed by 57 with an abnormal ultrasound and 35 women with an abnormal mammogram.CHEK2 mutation was 5.4 (95% CI 1.1 to 2.8) and that associated with a family history of breast cancer (first-degree relative) was 1.7 (95%CI 0.2 to 12). The detection rate was 0.43% at the first screening round  and was 0.27% at the second screening round (incident cancers).Overall, 22 invasive breast cancers were detected during the screening period . Two of the women had a positive family history of breast cancer and five women had a CHEK2 mutation (one case had both) Table . AssuminThe mean size of the 22 invasive cancers was 1.6 cm . The mean sizes of prevalent and incident invasive tumors were 1.7 cm and 1.2 cm respectively. Three out of 13 prevalent cancers were node-positive and two of seven incident cancers were node-positive Table .Table 4CIn our study, a biopsy was performed on all patients with an MRI BI-RADS 5 lesion, but not on all patients with MRI BI-RADS 4 lesion. This was either for a technical reason or due to patient preference or because of the radiologist’s interpretation of the other two imaging studies done concurrently. Of the 192 women who had a BI-RADS 4 (suspicious) lesion detected on MRI, 87 women had a biopsy and 11 breast cancers were found (12.6%). The 76 women who had a negative biopsy were followed for 4.0 years on average from the date of the abnormal MRI (BI-RADS 4) and two cases of invasive cancer were reported. There were also 105 women who had an abnormal MRI and who did not have a biopsy; these women were followed for an average of 4.3 years and two women were diagnosed with breast cancer. One was diagnosed with an invasive 4 cm node-positive cancer 55 months from the abnormal MRI. The other was diagnosed with a breast cancer in the breast contralateral the MRI abnormality, eight months after the initial abnormal MRI. The tumor was 5.0 cm and the patients was node-positive. In summary, of 192 women in the study who had a BI-RADS 4 abnormality, 11 breast cancers were detected at the time of screening and three cancers were diagnosed in the four year period following the abnormal MRI (one of these was diagnosed within the four year study period). Of the 151 women with an MRI BIRADS 4 abnormality and no other screening abnormality, four breast cancers were found, two at the time of the MRI and two thereafter. Of these, two were less than two centimeters and three were node negative.In this study of 2995 women at average or intermediate risk of breast cancer, we found the combination of mammography and ultrasound to be nearly equivalent to annual MRI. Of the 22 women who were diagnosed with invasive breast cancer over the two-year screening period, 19 were visualized with MRI and 17 were visualized with ultrasound and/or mammography. We report a high overall sensitivity (91%) and a very low proportion of interval cancers (9%) – both are indicators of an effective screening program. The average size of the breast cancers was small (1.6 cm) and the incident cancers were smaller, on average (1.2 cm) than were the prevalent cancers (1.7 cm). Fourteen of the 22 invasive cancers were detected in the cohort at a non-palpable stage and no cancer was detected solely by physical examination. This was an observational study and the management of the patients found to have an abnormal lesion was at the discretion of the treating physicians.We also identified six cases of DCIS. There is no consensus regarding the value of identifying Stage 0 breast cancers in the context of screening and in order to guard against over-interpretation, we have separated these from the invasive cancers.n = 2) might be a reflection of the reluctance of the radiologist or surgeon to biopsy a patient with an abnormal lesion detected on MRI (BI-RADS 4) when there was no corresponding lesion detected on mammography or ultrasound. However, among the 105 women with a BI-RADS 4 abnormality who did not have a biopsy, only one cancer was detected clinically in the three year period following the positive MRI test and this was detected in the contralateral breast. The other cancer was diagnosed 55 months post-MRI and the length of time elapsed suggests that this was an unrelated (new) event. These observations suggest that the reluctance to biopsy these women initially did not result in the failure to diagnosis many cancers that would have been clinically apparent in the three year interval following the second MRI examination. The lack of incident cancers in this group of 105 women in the three years following the abnormal MRI abnormal result suggests that there were some false positive MRI tests or that some cancers may be have been due to over-diagnosis. Over-diagnosis is defined as a cancer that presents on screening but would not otherwise become apparent in a patient’s lifetime. Miller et al estimate in the Canadian NBSS study [In this study, there is little evidence to support the position that annual MRI screening should be recommended for women from the average/moderate risk population who are being screened with annual mammography. Two cancers were identified through MRI screening that were missed by the other screening modalities. To some extent, the small number of cancers detected through MRI alone (SS study  that appBRCA mutation carriers) in general are more supportive of intensive screening than are studies of women at average or intermediate risk. In BRCA1 and BRCA2 carriers, the cancer prevalence (and incidence) is much higher than in the cohort studied here (approximately 6-fold higher) and the use of MRI as a screening tool can be justified. For example, in the recent paper by the Toronto group of 496 BRCA1 and BRCA2 carriers [BRCA mutation carriers, compared to 34 cancers among 1558 women at familial risk, but without a mutation. The detection rates for invasive cancers were 26.5 and 4.8 cancers per 1,000 woman-years in carriers and non-carriers respectively . They reported sensitivities of 71% and 41% for MRI and mammography respectively. We excluded women with a Polish founder mutation in BRCA1 from this cohort; three founder mutations comprise the majority of BRCA mutations in Poland. It is possible that there are other mutation carriers in this cohort but given the rarity of non-founder mutations in the Polish population and the fact that study subjects were unaffected, we expect the number of these to be very low.It is important that the results of screening studies be interpreted in the context of the level of risk of the participating subjects. Studies of women at high risk  were found through 216 biopsies (positive fraction 11.6%). In an early study of women with a BRCA1 or BRCA2 mutation, 33 biopsies revealed seven cancers (21.2%) . This diIn a recent report from the UK, (the FH01 study), the sensitivity of mammography alone was reported to be 77% for screening of 6710 young women at intermediate risk . They esIn the ACRIN study, 2662 women at high risk underwent 7443 mammogram and ultrasound screening tests over two years . Thirty three cancers were detected by mammography alone, 32 were detected by ultrasound alone, 26 were detected by both modalities and nine were not detected. The overall detection rate was 17 per 1000 woman-years. At the end of screening 612 women had an MRI and nine more cancers were detected (1.5%). The authors concluded that the routine use of MRI was not justified in this intermediate risk population.The data presented here do not support adding MRI screening to routine mammography and ultrasound for women at average risk at this time. The addition of MRI to the combination of ultrasound and mammography in this cohort of 2995 women led to the identification of two additional cancers. The resources expended to identify these two cancers were 5319 MRI examinations and 64 biopsies. This is equivalent to 3.7 cancers detected per 10,000 MRI examinations. Assuming a cost of 155 dollars per MRI , this is equivalent to 400,000 dollars per additional cancer detected in Poland, but is expected to be much higher than this in North America or western Europe. In this study, the cost of the ultrasound and mammography were 40 dollars per examination. Based on these prices, the total costs of screening 10,000 women would be 400,000 dollars for a combination of mammography and ultrasound and 1,550,000 using MRI alone. Our study is based on a relatively small number of detected cancers, and we recommend that further research be done in this area. It is important that the value of screening MRI ultimately be judged on studies with cancer mortality as the endpoint, but to date, there are no published studies with mortaity as the endpoint. It is also important to consider that there are potential harms associated with screening, particularly in low and average risk populations, such as increased numbers of confirmatory tests, anxiety, surgical morbidity and (in the case of over-diagnosis) unnecessary sugeries. Given the very low risk of cancer we observed in the follow-up period of women with BIRADS 4 lesion who did not undergo a biopsy, futher studies which consider the potential harms of overdiagnosis associated with MRI screening are warranted. Given the high cost of MRI examinations, until supporting evidence is available, we concur with other investigators  that the


The Tim17 family of proteins plays a fundamental role in the biogenesis of mitochondria. Three Tim17 family proteins, Tim17, Tim22, and Tim23, are the central components of the widely conserved multi-subunit protein translocases, TIM23 and TIM22, which mediate protein transport across and into the inner mitochondrial membrane, respectively. In addition, several Tim17 family proteins occupy the inner and outer membranes of plastids.We have performed comprehensive sequence analyses on 5631 proteomes from all domains of life deposited in the Uniprot database. The analyses showed that the Tim17 family of proteins is much more diverse than previously thought and involves at least ten functionally and phylogenetically distinct groups of proteins. As previously shown, mitochondrial inner membrane accommodates prototypical Tim17, Tim22 and Tim23 and two Tim17 proteins, TIMMDC1 and NDUFA11, which participate in the assembly of complex I of the respiratory chain. In addition, we have identified Romo1/Mgr2 as Tim17 family member. The protein has been shown to control lateral release of substrates fromTIM23 complex in yeast and to participate in the production of reactive oxygen species in mammalian cells. Two peroxisomal proteins, Pmp24 and Tmem135, of so far unknown function also belong to Tim17 protein family. Additionally, a new group of Tim17 family proteins carrying a C-terminal coiled-coil domain has been identified predominantly in fungi.We have mapped the distribution of Tim17 family members in the eukaryotic supergroups and found that the mitochondrial Tim17, Tim22 and Tim23 proteins, as well as the peroxisomal Tim17 family proteins, were all likely to be present in the last eukaryotic common ancestor (LECA). Thus, kinetoplastid mitochondria previously identified as carrying a single Tim17protein family homologue are likely to be the outcome of a secondary reduction. The eukaryotic cell has modified mitochondrial Tim17 family proteins to mediate different functions in multiple cellular compartments including mitochondria, plastids and peroxisomes.Concerning the origin of Tim17 protein family, our analyses do not support the affiliation of the protein family and the component of bacterial amino acid permease. Thus, it is likely that Tim17 protein family is exclusive to eukaryotes.The article was reviewed by Michael Gray, Martijn Huynen and Kira Makarova.The online version of this article (doi:10.1186/s13062-016-0157-y) contains supplementary material, which is available to authorized users. Mitochondria use several molecular machines to deliver proteins to the correct subcompartment of the organelle . All nucDespite being very different in their overall composition both of these molecular machines are built around core proteins from Tim17 family. Members of this protein family share four transmembrane helices, which constitute the translocation channel of so far unknown molecular structure . While tIn addition to these mitochondrial proteins, three further Tim17 family members  have been identified in chloroplasts, where they participate in the plastidial protein import system , 5. The Moreover, following the discovery that some organisms carry a single mitochondrial Tim17 family protein  it has bIn this paper, we provide a comprehensive analysis of Tim17 family of proteins in eukaryotes. We identify hitherto unknown Tim17 family members in mitochondria and peroxisomes, and demonstrate the presence of multiple Tim17 family proteins in the last common ancestor of eukaryotes. Our data also suggest that proteins of the Tim17 family lack evolutionary links to the bacterial LivH-type proteins and very likely represent exclusive eukaryotic protein family.Available genomic sequence data deposited in the Uniprot database of reference proteomes representing the major eukaryotic supergroups  were searched by HMMs (hidden Markov models) specific to the Tim17 family see . The retInspection of Tim22 subgroup showed that in addition to the mitochondrial proteins, two plastidial Tim17 family proteins, HP20 and HP30, represent Tim22 orthologues   and also stramenopiles, green and red algae  X10RX3DX6(G/A/S)GX3G number of protein sequences returned with no obvious link to Tim17 family of proteins XThus, we propose that Tim17 proteins represent unique eukaryotic protein family, which has first enabled the mitochondrion to import its proteins, while later provided additional roles in the peroxisome biology, plastidial protein import, the assembly of the mitochondrial complex I and the mitochondrial apoptotic pathway.In order to conduct an exhausting search of Tim17-like homologs, we employed an iterative search of the Uniprot database of reference proteomes which contains 5631 proteomes from all domains of life. Manually selected Tim17 homologs from the Swissprot database were used as query for the search. In each iteration, the discovered homologs were divided into groups by a 40 % identity cutoff using CD-HIT. Each group was then aligned and searched against the Uniprot database of reference proteomes using Hmmer . Hits wehttp://www.ch.embnet.org/software/TMPRED_form.html).The amino acid sequences of the identified proteins were analysed by BLAST and Hhpred in order to detect possible homology to other protein families. The occurrence of the transmembrane domains was predicted using Phobius , TMHMM  and TMprEutreptiella gymnastica. A dataset of known homologues of Tim17, Tim22 and Tim23 , mitochondrial Tim17-like proteins of kinetoplastids  and E. gymnastica sequences was aligned using Mafft  [The MMETSP database of transcriptomic data was searched using Hmmer  and homooptions) . The alioptions) . A phylooptions)  and MrBaoptions)  with thehttp://www.genome.jp/).The prokaryotic protein sequences were searched by a PRAT motif [GA]-x-x-[FY]-x-x-x-x-x-x-x-x-x-x-R-x-x-x-D-x-x-x-x-x-x-[GAS]-G-x-x-x-G using Motif search at Kyoto University Bioinformatics Centre  had all three proteins, arguing that the single Tim17 family member in kinetoplastid protozoa represents a simplification, rather than the ancestral state. The authors further identify Tim17 family members involved in different functions and located in different subcellular organelles (peroxisome as well as mitochondrion). Finally, the authors fail to find any convincing evidence indicating an evolutionary connection between the Tim17 family and bacterial LivH (amino acid permease), concluding that the Tim17 family is exclusive to eukaryotes.This paper is an interesting and generally well-executed contribution to our understanding of the evolution of the mitochondrial proteome. The authors have compiled a valuable data set and used it to make novel phylogenetic and functional inferences about the Tim17 protein family. This study nicely illustrates the utility of comprehensive comparative analyses in robustly supporting evolutionary conclusions.”Reviewer recommendations to authors“I have some reservations about how effectively the automated procedure used by the authors has resulted in a truly comprehensive retrieval of relevant sequences. For instance:• Inspection of Fig. Thanks. Unfortunately, this was caused by incomplete set of analyzed sequences, which we collected. We have re-done the analyses using the whole Uniprot database. In order to avoid false negatives we thus analyzed the whole Uniprot database of the 5631 reference proteomes from all domains of life. Doing so we have largely enriched the classification of Tim17 family members and their distribution in different groups of eukaryotes. This time the returned sequences contain the missed hits as noticed by the reviewer.Author’s response: • Figure In this case we wrongly labeled the absence of NDUFA11 in Fig.. The overall analysis was re-done and this mistake was corrected.Author’s response: • A. castellanii Trem125 was identified and annotated as an “uncharacterized conserved protein” (N375) in the study of Gawryluk et al. (2014) cited above. The complete Trem125 sequence (as well as a corrected sequence for A. castellanii Tim22) can be found in Gawryluk et al. (2014) Data in Brief 1, 12–14.Thanks. We have corrected that.Author’s response: • Romo1 and Oep16 are indicated as being present in A. castellanii in Fig. Admittedly, these omissions/inconsistencies do not affect the authors’ overall functional and evolutionary conclusions. However, some judicious manual searches to fill in gaps coupled with reference to previously published mitochondrial proteome analyses, as well as careful checking for consistency between Fig. Thanks. We have corrected these inconsistencies. Moreover, by using only Uniprot database, all sequences can now be more easily identified. Also, the taxonomic classification has been done to better detail.Author’s response: Minor issues“line 138: “indentified” should be “identified”doneAuthor’s response: Figure doneAuthor’s response: Reviewer summary“The manuscript handles an interesting subject, but I do find rather superficial in its analyses, in its comparisons with the literature  and in its discussion of the results. Often the logic the authors suggest in their language does not make sense to this referee. I do suggest the authors go back to the “drawing board”, do there analysis more thoroughly and spend some time on interpreting their results.”Reviewer recommendations to authors1) “There is no explanation of the choice of species, some model species  while three species of the Dictyostelium genus are present. Are these genomes particularly well sequences/annotated?There should be reciprocal checking of homologs, specifically for the “borderline” cases. In general E-values are used find homologs, not a “probability cutoff”.Thanks. Our intention was to cover all major eukaryotic supergroups. However, after the review we decided to run more thorough analyses on the whole Uniprot database of the reference 5631proteomes instead of selected proteomes. The detailed results of the analyses are submitted as Additional files, some of which are shown as Fig.  of the main text. We have also performed manual checks for the borderline sequences to eliminate false positives.Author’s response: 2) At least one reference is incomplete (Mokranjac and Neupert).The reference was corectedAuthor’s response: 3) The paper could have done with a more thorough analysis of the literature. The homology between NDUFA11, TIMMDC1 and the TIM17 family was already known in 2013. The authors actually cite a paper where that is mentioned but somehow forgot to read it? . There are actually multiple papers mentioning this homology. Similarly PMP24 has already been part of this family in PFAM, and this should be mentioned.Our mistake not referring to the previously published homology between NDUFA11 and TIMMDC1 and Tim17 is unfortunate. We have corrected that and also toned down the title of the section of the manuscript accordingly. Reference to Pfam database was also included.Author’s response: 4) What does appear new, is that they find ROMO1/MGR2 to be homologous to the TIM17 family. Given the novelty of that, and given that MGR2 is part of a complex with the TIM17 homolog TIM23, and given the recent interest in its function, this does deserve more attention, including an alignment and interpretation of conserved residues in light of what is known about these proteins (their interactions in the membrane).We have added the alignment of Romo1/Mgr2, Tim17 and Tim23 sequences as Additional file: Figure S2. However, so far there is limited data available on the role of particular amino acid residues of Romo1/Mgr2 or its interaction with Tim23 or Tim17.Author’s response: In the repeated analyses using much larger dataset we have also uncovered additional group of proteins carrying a C-terminal coiled coil domain, referred to as T17lp1 (Tim17-like protein 1).5) The lack of homology between LivH and TIM17 has been noted before, even in this journal .Yes, we and others have been aware of the lack of homology between LivH and Tim17, which was originally described by Rassow et al., 1999. However, the preposition made against the two proteins relationship were usually based on unsuccessful pair-wise sequence analyses such as blast . Therefore, we have felt that more sensitive analysis had to done. We have added following paragraph to the text: “[6]. In order to test the bacterial link to the eukaryotic Tim17 family proteins we searched all available bacterial protein sequences deposited in the UniProt Knowledgebase with Tim17 specific HMMs built from our complete dataset of all identified Tim17 homologues or Pfam seed alignment. The returned bacterial proteins contained a glycine-zipper motif (GxxxGxxxG) (Pfam clan CL0500) and interestingly, the highest ranking hits were from the alphaproteobacterial group of Rhizobiales. However, except the the glycine- zipper motif, these proteins did not share other Tim17 characteristics. In support of that, when the glycine-zipper motif was masked, the similarity score dropped dramatically (data not shown).”Author’s response: 6) The table with the phylogenetic distribution of the protein subfamilies appears inconsistent with the results: e.g. the authors say that they find a NDUFA11 protein in plants, and it appears in their phylogeny, but then do not show an NDUFA11 (which appears under many names in the manuscript & supplement) ortholog in the table. In general I do find the many “holes” in the table suspiscious. I suggest they do a better job of their homology searches, and also search against the DNA/RNA sequences of the species they are analyzing to search for genes that were not predicted as such.In order to minimize the false negatives in our analyses, we have completely re-done the analyses using the entire Uniprot database of reference proteomes as a sequence source. However, still some “holes” remain. The detailed table of Tim17 proteins can be found in Additional file : Table S1 and Additional file: Table S2.Author’s response: 7) In Fig. The entire Fig.has been changed on the base of the different and larger dataset.Author’s response: 8) The following two sentences do not make much sense:“Analogously to mitochondrial Tim22, both HP20 and HP30, participate in the import of the inner membrane plastidial proteins. The presence of the conserved cysteine residues suggests that these also participate in the stabilization of the translocase complex in the plastid as the redox-regulated protein import analogous to MIA pathway does not occur here.”Why does the absence of the MIA complex support that the cysteines are involved in stabilization?Given that the cysteine residues in Tim22 have been show to serve for two purposes – the complex stabilization and/or the Mia40 dependent import, we propose that, when the MIA pathway is missing, the function of the cysteine residues is rather to stabilize the mature protein complex.Author’s response: 9) A sentence like “The peroxisomal localization of Tim17 family protein is reminiscent to the peroxisomal ADP/ATP carrier PMP34, a member of mitochondrial carrier protein family .” also carries more meaning than it actually has.Yes, the expression is not precise and it was rephrased to the following:Author’s response: “That a member of the originally described mitochondrial protein family was found in the peroxisomes has a precedent in the case of the peroxisomal ADP/ATP carrier PMP34, a member of mitochondrial carrier protein family [30]. Considering that a Tim17 protein (Tim22) assembles the mitochondrial carriers into the inner mitochondrial membrane, it is tempting to speculate that PMP24 mediates the insertion and the assembly of PMP34 into the peroxisomal membrane.”10) “This fact is accentuated by the presence of one or both invariant cysteine residues . Analogously to mitochondrial Tim22, both HP20 and HP30, take part in the import of the inner membrane plastidial proteins [5]. The relationship is further supported by the presence of one or both invariant cysteine residues . In Tim22, the residues were shown to participate during the Mia40- dependent protein import into mitochondria [10] and/or contribute to the TIM22 complex stability via the formation of an intramolecular disulphide bond [11]. The lack of MIA pathway in the plastids suggests that plastidial Tim17 proteins do not use disulfide relay system during the import and the cysteine residues may, at least in case of HP20, also participate in the stabilization of the translocase complex in the plastid”.11) I do find Fig. Now the support for this claim can be found in the Additional file: Figure S2, reference to it was added to the text.Author’s response: “The manuscript describes a relatively straightforward but accurate sequence and comparative genomic analysis of Tim17 family. The significance, originality and novelty of presented results are not very high, but the analysis is done carefully on a large genome set and the paper still could be useful for those who are interested in evolution of the components of mitochondrial translocases.”Reviewer recommendations to authors“The manuscript describes a relatively straightforward but accurate sequence and comparative genomic analysis of Tim17 family. These proteins were first characterized as components of protein translocase of inner mitochondrial membrane and are essential for mitochondria functioning. As it is the case for many membrane protein families, they are very poorly annotated and this analysis could be helpful to improve annotation of these important proteins. In addition the family members are found in perxisomes and plastids. I don’t have much criticism for the methods applied and I find that conclusions are generally justified, pending some answers to the questions below. I however have a number of suggestions and questions to the authors.1. The title of the paper is misleading since all the different functions of the superfamily members have been characterized before elsewhere. As far as I can see there is no additional groups found in this paper which function has not been known before. This paper simply presents comparative genomic analysis of the family but does not analyze their diverse roles. Thus the title should better reflect the work which has been done here.Yes, we agree and we have change the title to better described the content of the paper to:Author’s response: “Evolution of the Tim17 protein family”2. Furthermore I suggest that authors should rephrase the abstract considerably, since as mentioned above they have not studied function of these proteins. Also the novelty of the presented results is not obvious from the abstract. It should be clarified if authors detected any new subfamilies or showed similarity between the families that was not detected before or showed relationships between subfamilies that have not been shown before. See also the next suggestion.Yes, the abstract has been modified accordinglyAuthor’s response: 3. As far as I can tell there are two pfam profiles that include Tim17 family members (Tim17 Tim17/Tim22/Tim23/Pmp24 family) and pfam10247 (Romo1). It should be clearly stated if authors have identified any subfamilies that are not present in the respective pfam alignments. Note that the protein sequence (such as NDUFA11) could be included in the pfam alignments but have not identified by this ID. Authors should check that. This is also important to understand if additional pfam alignments should be made to cover the whole family better or if the ones that exist are sufficient.Our inspection of the pfam alignments confirmed that NDUFA11, TIMMDC1, Oep16 sequences except the newly define T17lp1 proteins are already included in the dataset of Tim17 Tim17/Tim22/Tim23/Pmp24 family - we have included that information in the text.Author’s response: 4. Alignment of a few representatives of each subfamily should be shown as a main figure and it should be discussed. In particular if still there is a motif/consensus characteristic for the whole family (discuss if the new motif (if any) is different from the old one called PRAT family motif), which region of the sequences is the most conserved, etc.The aligned HMM logos of different Tim17 family members were added to the paper as Fig. Author’s response: 5. Authors made a considerable effort trying to provide some supporting evidence for the previously published hypothesis that Tim17 family originated from LivH amino acid transporter component. That hypothesis was very weak so not surprising that they could not find any support for it. But the conclusion that Tim17 family is specific to eukaryotes is also dangerous, since membrane proteins evolve fast and it is difficult to identify there distant homologs. Authors mentioned that HHpred search identifies a number of bacterial families with G zipper motif . I would recommend authors to analyze this similarity very carefully with respect of additional potential shared motifs, number of transmembrane domains, relevance to electron transport complexes or any other relevant sequence features. The paper would be much more interesting if authors could present a better hypothesis on the origin of Tim17 family.During our searches we found number of hits among bacterial proteins that contain a glycine-zipper motif (Pfam clan CL0500), interestingly the highest ranking hits were from the alphaproteobacterial group Rhizobiales. In addition to the glycine- zipper motif these proteins, however, they did not share other Tim17 characteristics. In support of that, when the glycine-zipper motif was masked, the score dropped dramatically. We thus conclude that there are similar domains in bacterial proteins, these can not be used to unambiguously pinpoint the prokaryotic ancestor of the Tim17-like protein family. We have added this information to the manuscript.Author’s response: 6. Are there any similarity  of the Tom40 channel and Tim17 based complexes? It would be interesting to read some discussion to this extent. Also in the HHpred searches there is a weak similarity to Mitochondrial uncoupling protein 2 (PDB: 2lck) and Tom5. Could any of those be real based on additional considerations mentioned above?So far, there has been no similarity found in any aspects of the TIM and TOM complexes and our analyses support that. Tom40 is a beta-barrel protein, which can be found only in the outer membranes of gram-negative bacteria, plastids and mitochondria. Also the receptor subunits and small Toms are exclusive to the TOM complex. The relationship of Tim17 protein to mitochondrial uncoupling protein 2  rather reflects a similarity among the transmembrane segments of different mitochondrial inner membrane proteins than the true affiliation of the proteins.Author’s response: 1. Mention .Author’s response: 2. Figure Yes, we agree, figure was meant as a graphical summary of the Tim17 evolution. We have merged Fig.to Fig.Author’s response: 3. Additional file We have used only Uniprot IDs in the revision.Author’s response: 1) I do not have much to add to my previous comments. Small remarks: “The lack of MIA pathway in the plastids suggests that the cysteine residues may, at least in case of HP20, also participate in the stabilization of the translocase complex in the plastid.” How does the lack of the MIA pathway support this? Please clarify this in the manuscript (not just in the answer to me). The MIA dependent import pathways works via the oxidation of the cysteines. In its absence you actually have the problem that it is not clear how these cysteines are oxidized.We have rewritten the paragraph to the following:” The lack of MIA pathway in the plastids suggests that plastidial Tim17 proteins do not use disulfide relay system during the import and the cysteine residues may, at least in case of HP20, also participate in the stabilization of the translocase complex in the plastid.Author’s response: 2) Regarding the lack of homology between LivH and TIM17. Please be so kind to cite the previous discussion about this, as I have asked before and show what you did differently.Concerning the homology between LivH and TIM17. We have added following paragraph to the text: “[6]. In order to test the bacterial link to the eukaryotic Tim17 family proteins we searched all available bacterial protein sequences deposited in the UniProt Knowledgebase with Tim17 specific HMMs built from our complete dataset of all identified Tim17 homologues or Pfam seed alignment. The returned bacterial proteins contained a glycine-zipper motif (GxxxGxxxG) (Pfam clan CL0500) and interestingly, the highest ranking hits were from the alphaproteobacterial group of Rhizobiales. However, except the the glycine- zipper motif, these proteins did not share other Tim17 characteristics. In support of that, when the glycine-zipper motif was masked, the similarity score dropped dramatically (data not shown).”Author’s response: 3) “Inspection of Tim22 subgroup revealed that in addition to the mitochondrial proteins, two plastidial Tim17 family proteins, HP20 and HP30, represent Tim22 orthologues [5] .Author’s response: 4) The affinity of TIMMDC1 and NDUFA11, to Tim17 protein family was already suggested“ This was more than just ’suggested” in that paper. They showed an actual alignment and mentioned the PFAM family.We have corrected that.Author’s response: The manuscript describes a relatively straightforward but fairly accurate sequence and comparative genomic analysis of Tim17 family. These proteins are components of protein translocase of inner mitochondrial membrane and are essential for mitochondria functioning. As it is the case for many membrane protein families, they are very poorly annotated and this analysis could be helpful to improve annotation of these important proteins in eukaryotic genomes. I don’t have a lot of criticism for the methodology and I find that conclusions are generally justified. However authors’ answers to a few questions below could clarify some additional details regarding results and conclusions mentioned in the main text.1) Abstract still needs another round of refinement and restructuring. Results and novelty should be better clarified. “Two further Tim17 family proteins are present in the peroxisomal membranes” – this is not a result of this work. Inference about LECA should go to conclusions.We have changed the abstract accordingly.Author’s response: 2) Could authors provide here more technical details of their attempts to identify homologs in prokaryotes ?We have added the information into the manuscript.Author’s response: 3) Any speculation on the origin of Tim17 membrane proteins? It would be interesting to read more discussion on the comparison  of the Tom complex and Tim complexes.So far, there has been no similarity found in any aspects of the TIM and TOM complexes and our analyses support that. Tom40 is a beta-barrel protein, which can be found only in the outer membranes of gram-negative bacteria, plastids and mitochondria. Also the receptor subunits and small Toms are exclusive to the TOM complex. The relationship of Tim17 protein to mitochondrial uncoupling protein 2  rather reflects a similarity among the transmembrane segments of different mitochondrial inner membrane proteins than the true affiliation of the proteins.Author’s response:


Tek either ubiquitously or specifically in embryonic ECs. Postnatal Tie2 attenuation resulted in the degeneration of newly formed veins followed by the formation of haemangioma-like vascular tufts in retina and venous tortuosity. Mechanistically, Tie2 insufficiency compromised venous EC identity, as indicated by a significant decrease of COUP-TFII protein level, a key regulator in venogenesis. Consistently, angiopoietin-1 stimulation increased COUP-TFII in cultured ECs, while Tie2 knockdown or blockade of Tie2 downstream PI3K/Akt pathway reduced COUP-TFII which could be reverted by the proteasome inhibition. Together, our results imply that Tie2 is essential for venous specification and maintenance via Akt mediated stabilization of COUP-TFII.Mechanisms underlying the vein development remain largely unknown. Tie2 signaling mediates endothelial cell (EC) survival and vascular maturation and its activating mutations are linked to venous malformations. Here we show that vein formation are disrupted in mouse skin and mesentery when Tie2 signals are diminished by targeted deletion of DOI:http://dx.doi.org/10.7554/eLife.21032.001 Mechanisms underlying arteriovenous specification have been under intensive investigation during the past years, and this has led to the identification of several signaling pathways involved in the coordination of this process. The VEGF-A/VEGFR-2 pathway mediates activation of RAF1 and ERK1/2 kinases to induce the expression of genes required for arterial development , includiTie2 is a receptor tyrosine kinase that mediates angiopoietin signaling for EC survival, vascular remodeling and integrity . Tie2 deTek gene . Flox/+/Ubc-CreERT2Tek or Flox/+/Cdh5-CreERT2Tek littermate mice were used as controls. Tie2 deletion efficiency was examined by immunostaining with skin and also by real-time RT-PCR with lung of Tek mutant mice . Analysis of veins in skin at E15.5 showed that venogenesis was disrupted in both types of mutant embryos, in which Tek deletion was induced by intraperitoneal injections of tamoxifen into pregnant mice at E10.5–12.5  and analyzed at P7 . The partial Tie2 deletion was also indicated by the mosaic GFP expression when the mTmG allele was crossed into the Tek mutant mice to generate the compound genetic mouse model   . No obviav-iCre) .10.7554/−/iUCKO mutants compared with controls at P7 , but an increase of Erk1/2 phosphorylation  in the lungs of Tek−/iUCKO mice compared with controls  resulted in a similar but milder vascular phenotype . Interestingly, the venous tortuosity was also observed in retinas of Tek−/iUCKO mice at the adult stage (2.5 month-old) while the increased angiogenesis along the retinal veins regressed . Interestingly, there was also a trend of reduction in the arterial gene expression in Tek−/iUCKO mice , suggesting that Tie2 retardation may also affect arteries at later stages.Mechanistically, we found that Tie2 attenuation led to the alteration of venous EC identity as shown by the change of EC marker expression . Transcrrols P7; . Furtherrols P7;   for gene deletion were generated by the National Resource Center for Mutant Mice, Nanjing University, as previously described  (Vav1-iCre) (ERT2Ubc-Cre or EIIa-Cre) (IMSR_JAX:000664) with at least five backcrosses. In all the phenotype analysis, littermates were used as control.Conditional mice with escribed . All aniCreERT2) , hematopv1-iCre) , or ubiqIIa-Cre) . The floInduction of gene deletion was performed by tamoxifen treatment as previously described . BrieflyTek mutant and control mice were collected and homogenized in TRIzol (Ambion). RNA extraction and reverse transcription were performed by standard procedures . Quantitative real-time RT–PCR was carried out using the SYBR premix Ex Taq kit (TaKaRa). Briefly, for each reaction, 50 ng of total RNA was transcribed for 2 min at 50°C with a denaturing step at 95°C for 30 s followed by 40 cycles of 5 s at 95°C and 34 s at 60°C. Fluorescence signal was analyzed by using ABI PRISM 7500. The primers used were as follows: GAPDH: 5'-GGTGAAGGTCGGTGTGAACG-3', 5'-CTCGCTCCTGGAAGATGGTG-3'; Tie2: 5'-GATTTTGGATTGTCCCGAGGTCAAG-3', 5'-CACCAATATCTGGGCAAATGATGG-3'; APJ: 5’-CAGTCTGAATGCGACTACGC-3', 5'-CCATGACAGGCACAGCTAGA-3'; Ephb4: 5'-CTGGATGGAGAACCCCTACA-3', 5'-CCAGGTAGAAGCCAGCTTTG-3'; COUP-TFII: 5'-GCAAGTGGAGAAGCTCAAGG-3', 5'-TTCCAAAGCACACTGGGACT-3'; NRP1: 5’-CCGGAACCCTACCAGAGAAT-3', 5'-AAGGTGCAATCTTCCCACAG-3'; EphrinB2: 5'-TGTTGGGGACTTTTGATGGT-3', 5'-GTCCACTTTGGGGCAAATAA-3'; NOTCH1: 5'-TGTTGTGCTCCTGAAGAACG-3', 5'-TCCATGTGATCCGTGATGTC-3'; Dll4: 5'-TGCCTGGGAAGTATCCTCAC-3', 5'-GTGGCAATCACACACTCGTT-3'. The transcripts of venous and arterial markers were normalized against GAPDH, and the relative expression level of every gene in the Tek mutants (Tek−/iUCKO) was normalized against that of littermate control mice.Tissues from Tek mutant and control mice were homogenized following standard procedures. Antibodies used included rabbit polyclonal anti-Tie2 , rabbit polyclonal anti-Akt , rabbit monoclonal anti-phospho-Akt473 , rabbit polyclonal anti-phospho-p56Dok-2 , rabbit polyclonal anti-p56Dok-2 , rabbit polyclonal anti-Erk1/2 and phospho-Erk1/2 , mouse monoclonal anti-COUP-TFII , and mouse monoclonal to beta–actin antibody .To analyze Tie2 mediated signaling pathway, lung tissues from TM siRNA duplex oligoribonucleotides targeting human Tie2  using Lipofectamine RNAiMax ; Stealth RNAi negative control duplexes (medium GC) were used as a control. COMP-Ang1  or Ang1  was used to stimulate Tie2/Akt pathway. To inhibit PI3K and downstream Akt signaling, HUVECs were treated with LY294002  or MK2206  and analyzed at 3 hr, 6 hr or 12 hr after treatment. For the experiment with proteasome inhibition, HUVECs were treated with MG132 . The cells were washed with ice-cold PBS and lysed in the lysis buffer , 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 10% glycerol, 50 mM NaF, 10 mM β-glycerolphosphate, 5 mM sodium pyrophosphate, 5 mM EDTA, 0.5 mM EGTA and 1% NP-40). The lysates were incubated on ice for 0.5 hr with rotation and centrifuged. Protein concentration was determined using the BCA protein assay kit (PIERCE), and equal amounts of protein were used for analysis.HUVECs  were cultured in endothelial cell basal medium plus supplements . To knock down Tie2 expressoin in HUVECs, cells were transfected with Stealth RNAiAB_394816), goat anti-mouse Tie2 , goat anti-mouse Dll4 , goat anti-mouse EphB4 , Cy3-conjugated mouse anti-mouse αSMA . Alexa488, Alexa594 (Invitrogen), Cy5- or Cy3- (Jackson) conjugated secondary antibodies were used for staining. Slides were mounted with Vectashield (VectorLabs), and analyzed with the Olympus FluoView 1000 confocal microscope or Olympus BX51 fluorescent dissection microscope. For staining of frozen sections, retinas were collected and fixed in 4% PFA for 1 hr at 4°C, incubated in 20% sucrose overnight and then embedded in OCT. Consecutive sections (10 µm in thickness) were incubated with antibodies against PECAM-1  and NG2 , followed by staining with the appropriate fluorochrome-conjugated secondary antibodies and mounted as described above.For whole-mount immunostaining with embryonic skin, mesentery and retina, tissues were harvested and processed as previously described . The tisRetinal vascularization index was quantified as the ratio of vascularized area to total retinal area. For the quantification of blood vessel parameters in the retina, fluorescent images were taken from similar regions in all samples. Blood vessel density was measured and analyzed by using Image Pro Plus (MediaCybernetics), as previously described .t test. All statistical tests were two-sided. Data are presented as mean ± S.D.Statistical analysis was performed with the unpaired  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Janet Rossant as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Hellmut Augustin (Reviewer #2); Lena Claesson-Welsh (Reviewer #3).Thank you for submitting your work entitled "Angiopoietin receptor Tie2 is required for vein specification and maintenance via regulating COUP-TFII" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in All the reviewers agree that the paper contains interesting and rather novel observations. However they feel that the paper needs additional experiments to make the results stronger and to get a more mechanistic view of the effect of Tie2 in venous differentiation. Both reviewers 2 and 3 list major points to be considered and specific experiments to add to improve the manuscript. Since these revisions require considerable additional work, we are not able to consider a revised manuscript.eLife in the future.We encourage the authors to complete the recommended revision of their work and consider resubmitting to Reviewer #1:The paper by Man Chu et al. describes the effect of inactivation of Tie-2 expression on artero-venous differentiation in the mouse embryo, pups and cultured endothelial cells.As previously reported, Tie 2 null embryos die early during gestation for vascular and heart developmental problems. Here the authors show that Tie-2 deficient embryos and pups present a defective venous organization when Tie-2 expression is missing. In some organs and tissues, veins are absent along the arteries. In the retina there is a reduced horizontal growth of the vasculature and the presence of malformations along the length of the veins. In the skin, veins look tortuous. Arterial and venous markers are partially modified. In HUVEC silenced for Tie2 or lung and liver tissues Coup-TFII is reduced.Mechanistically, data suggest that Tie-2 may act through phosphorylation of Akt that in turn would increase COUP-TFII and venous differentiation.Although the paper contains some interesting observations it is essentially descriptive. The major weakness is the lack of a detailed mechanistic analysis of how Tie-2 acts in inducing venous differentiation. Data on the role of Akt are suggestive but a marked reduction of COUP-TFII is detectable only in HUVEC KD for Tie-2 and is much less apparent in the tissues. Other published studies showed an increase in cultured cells of Akt and STAT 1 upon infection with Tie-2 gain of function mutant. In addition, inherited gain of function mutations of Tie-2 induce a strong venous endothelial cell proliferation and formation of large hemangiomas inhibited by Rapamycin . To my view the authors should extend their studies on the mechanism of action of Tie-2 also adding novel observations as compared to previously published literature.Reviewer #2:The authors of the manuscript, "Angiopoietin receptor Tie2 is required for vein specification and maintenance via regulating COUP-TFII" have used global and endothelial cell-specific conditional Tie2 knockout mouse models to demonstrate the critical role of Tie2 during vein development. The study is novel and addresses a fundamental question regarding the pathways  involved in forming venous endothelial identity and function. Although there is already a lot of circumstantial evidence in the published literature on vein-specific functions of Ang/Tie signaling, the manuscript makes an important point in showing this conclusively using definite genetic models. The study will be of interest for the broad readership of the journal. In further advancing their work, the authors should consider the following:ERT2UBC-Cre) to knockout Tie2. Even though the authors have also used an endothelial specific Cre line (ERT2Cad5-Cre) to demonstrate the phenotypes in embryos and early postnatal retina, all the mechanistic studies are only performed using the ERT2UBC-Cre line. This is a major concern especially because -/iUCKOTie2 seems to show better Tie2 deletion as well as stronger phenotypes compared to -/iECKOTie2 line. Is this solely due to different recombination efficacies of the different drivers or would this difference raise the question whether endothelial cell autonomous effects may not be the only cause of defective vein development in Tie2-deficient mice? Why did the authors opt to study different time points in 1) The major limitation in the study is the use of a ubiquitously expressed Cre deletor line . In -/iECKOTie2 mice which is clearly not corresponding to the extremely weak or absent Tie2 staining shown in 2) The immunofluorescence images show essentially black boxes in -/iUCKOTie2 mice showing vascular tuft formation is interesting. The same analysis should be included using the -/iECKOTie2 mice.3) The retina vasculature analysis at P11, P15 and P21 in -/iUCKOTie2 mice at P21, the adult retina did not show any such malformations, whereas ear skin showed tortuous veins in adult mice. These data have been discussed rather insufficiently. The authors should comparatively analyze COUP-TFII transcript levels and other downstream players such as pAkt and pDok-2 in these two vascular beds in order to dissect the mechanisms, which help the retinal veins to "recover" but not the cutaneous veins. Likewise, partial recombination will lead to a mosaic vasculature of WT and KO cells. If such mosaic mice are traced over time, there is a good chance that the KO cells will be competed out by WT cells. As such, it is critical, particularly in the longer term experiments to carefully trace the percentage of cells with Tie2 deletion at the later time points of analysis.4) Although hemangioma-like vascular tufts were observed in 5) The authors claim that Tie2 is required for the maintenance of venous EC identity. This is suggested by the observed phenotypes . A systematic gene expression analysis of adult retina similar to 6) The authors are encouraged to reconsider their discussion. They conclude at the end of the discussion that the findings put a cautionary note on long term Tie2 targeting therapies, e.g., during tumor angiogenesis. This may not be the most important point of the study. In fact, nobody tries to target Tie2. Ang2 neutralizing strategies primarily work cooperatively with VEGF pathway targeting drugs based on their vascular normalization Tie2 gain-of-function effect. The same holds true for VE-PTP inhibitors or the recently published ABTAA antibody. What this reviewer finds much more exciting is the role of Tie2 on venogenesis. Venogenesis has mostly been considered as the default pathway with transcriptional programs driving lymphatic and arterial differentiation. The identification of Tie2 signaling as upstream regulator of COUP-TFII is a major finding that should likely be discussed in more detail. The fact that Tie2 KO leads in part to the acquisition of an arterial phenotype is noteworthy and suggests something of an arteriovenous identity balance mechanism. Likewise, angiogenesis is an arterializing process. As such, the physiological downregulation of Tie2 in the angiogenic tip cell vasculature should/could likely be discussed in the context of the findings of this manuscript. Obviously, it is at the discretion of the authors to prioritize the discussion, but it is felt that the authors may in the present version of the manuscript not have discussed the most obvious and most exciting implications of their work.Reviewer #3:The study by Chu et al. characterizes the effect of Tie2 gene inactivation either using a ubiquitous or endothelial promoter to drive Cre expression during different stages of embryonic and postnatal stages. Interestingly, Tie2 deficiency leads to loss in vein formation or in stage-dependent maintenance of veins in different organs. The analyses are thorough and neatly presented. The study is ambitious, novel and of general interest.1) The study shows that loss of Tie2 is accompanied by vascular abnormalities and a deficiency in vein formation. It is not entirely clear from the images that veins are missing entirely or if they are malformed. Please complement images with aSMA stainings .2) The author have previously described the iUCKO-/- mice in their paper in ATVB Jun;34(6):1221-30. Here, they have examined lymphatic vessel formation in the skin and show that deletion of Tie2 in neonate mice did not affect lymphatic vessel growth and maturation . I'm surprised that the lack of veins, as shown in the current study, has had no consequence for lymphatic vessel formation. The authors need to clarify and confirm that lymphatics are unaffected although veins are lacking.3) In 4) In [Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor.Thank you for submitting your work entitled "Angiopoietin receptor Tie2 is required for vein specification and maintenance via regulating COUP-TFII" for consideration by eLife only allows for a single round of reviews. Given the importance of your work and the fact that all three reviewers have urged me to give you another opportunity to revise the paper, we are willing to give you a second chance to make the original required major revisions. I would like to stress that the reviewers are your advocates and trying to help you improve the paper. The reviewers’ comments from the second round of review are listed below, but I urge you to make the revisions requested in both the first and second round of review.While the reviewers feel that your work is potentially very important, they were disappointed that you chose to argue points rather than making the necessary revisions elaborated in the original set of reviews. You will see from their comments, that none of the reviewers felt you adequately addressed their original concerns. Normally, This will be your final opportunity to provide acceptable additional experimental evidence as required by the reviewers.Reviewer #1:Overall my feeling is that the authors could have a very nice story but they do not want to make an effort to make it more complete. Data on the mechanism of action remain the same, as poor as before.I do not think that they can conclude that since COUP-TFII is reduced when Akt activation is inhibited (in HUVEC) Akt activation is the mechanism of action of Tie-2 in maintaining a venous identity. Although there is a partial inhibition of Akt (24% reduction) in Tie-2 deficient mice other pathways are likely implicated.pDok- 2, for instance, seems more dramatically affected. It is sad that they do not want to make a relatively small effort to improve their paper.The same applies to the relevant points raised such as the use of a model of ubiquitous inactivation of Tie-2 versus endothelial specific inactivation, or the lack of studies on the possible alterations of the lymphatics in absence of a correct venous development.Reviewer #2:The manuscript by Man Chu and coworkers is resubmitted after revision. That global deletion of Tie2 casus venous defects is convincingly demonstrated. I have the following comments:1) A major concern with this study is the weak phenotype of the endothelial deletion compared to the ubiquitous deletion. The strong effects in the global deletion could be primary as well as secondary.2) The question on how lack of veins affect lymphatics remains unanswered. The authors published in ATVB in 2014, that lymphatic vessel development is unaffected in neonate pups with the same global Tie2 deletion as described here. It is unexpected that the lack of veins would not affect lymphatic development.3) Mechanistic in vivo data remain scarce.Reviewer #3:This reviewer is rather disappointed on how the authors have addressed the specific critique to the original submission of the manuscript. The idea of the peer review process is to initiate an iterative process aimed at improving the eventually published manuscript. As such, this reviewer had constructively raised a number of issues that he felt would help to improve the manuscript. Generally speaking, the authors have not accepted this invitation, but essentially argued away all of the reviewer's critique and suggestions. They thereby missed the opportunity to improve their manuscript (at least in response to the comments and suggestions made by this reviewer). For example, they did not even opt to replace the black IF images for the supposed Tie2 ECKO analyses. In the rebuttal, they argue at length about different recombination efficacies using the two different driver lines. Clearly, incomplete recombination and the resulting competition of WT and KO cells in long term experiments is key to the interpretation of conditional KO data. If the authors' recombination analyses are correct, then this reviewer would insist that the corresponding IF images should show mosaic Tie2 expression and not black images.Despite this disappointment about the revised manuscript, this reviewer considers the manuscript of high quality and in principle deserving of publication. As such, if the authors are either not able or not willing to adequately address this reviewer's concern and suggestions, the manuscript should be published as is. [Editors’ note: the author responses to the first round of peer review follow.][…] Reviewer #1:The paper by Man Chu et al. describes the effect of inactivation of Tie-2 expression on artero-venous differentiation in the mouse embryo, pups and cultured endothelial cells.As previously reported, Tie 2 null embryos die early during gestation for vascular and heart developmental problems. Here the authors show that Tie-2 deficient embryos and pups present a defective venous organization when Tie-2 expression is missing. In some organs and tissues, veins are absent along the arteries. In the retina there is a reduced horizontal growth of the vasculature and the presence of malformations along the length of the veins. In the skin, veins look tortuous. Arterial and venous markers are partially modified. In HUVEC silenced for Tie2 or lung and liver tissues Coup-TFII is reduced.Mechanistically, data suggest that Tie-2 may act through phosphorylation of Akt that in turn would increase COUP-TFII and venous differentiation.Although the paper contains some interesting observations it is essentially descriptive. The major weakness is the lack of a detailed mechanistic analysis of how Tie-2 acts in inducing venous differentiation. Data on the role of Akt are suggestive but a marked reduction of COUP-TFII is detectable only in HUVEC KD for Tie-2 and is much less apparent in the tissues. Other published studies showed an increase in cultured cells of Akt and STAT 1 upon infection with Tie-2 gain of function mutant. In addition, inherited gain of function mutations of Tie-2 induce a strong venous endothelial cell proliferation and formation of large hemangiomas inhibited by Rapamycin . To my view the authors should extend their studies on the mechanism of action of Tie-2 also adding novel observations as compared to previously published literature.Tie2, and this is confirmed by the siRNA mediated knockdown of Tie2 in HUVECs  involved in forming venous endothelial identity and function. Although there is already a lot of circumstantial evidence in the published literature on vein-specific functions of Ang/Tie signaling, the manuscript makes an important point in showing this conclusively using definite genetic models. The study will be of interest for the broad readership of the journal. In further advancing their work, the authors should consider the following:ERT2) to knockout Tie2. Even though the authors have also used an endothelial specific Cre line (Cad5-CreERT2) to demonstrate the phenotypes in embryos and early postnatal retina, all the mechanistic studies are only performed using the UBC-CreERT2 line. This is a major concern especially because Tie2-/iUCKO seems to show better Tie2 deletion as well as stronger phenotypes compared to Tie2-/iECKO line. Is this solely due to different recombination efficacies of the different drivers or would this difference raise the question whether endothelial cell autonomous effects may not be the only cause of defective vein development in Tie2-deficient mice? Why did the authors opt to study different time points in 1) The major limitation in the study is the use of a ubiquitously expressed Cre deletor line , suggesting that it is an endothelial cell autonomous effect. Postnatally, the milder vascular effect observed in retina of -/iECKOTie2 mice may be mainly due to the lower deletion efficiency of Tie2. The remaining Tie2 mRNA level is approximately 10-20% in -/iUCKOTie2 mice .The abnormal vein development was observed in skin of -/iUCKOTie2 mice at the same stage . In -/iECKO mice which is clearly not corresponding to the extremely weak or absent Tie2 staining shown in 2) The immunofluorescence images show essentially black boxes in -/iUCKOTie2 mice were shown in -/iECKOTie2 mice is now added as discussed above.The deletion efficiency of Tie2 was also examined by western blot analysis and real-time RT-PCR, in addition to the immunostaining. The remaining levels of Tie2 transcripts in lung and retina of -/iUCKO mice showing vascular tuft formation is interesting. The same analysis should be included using the Tie2-/iECKO mice.3) The retina vasculature analysis at P11, P15 and P21 in Tie2-/iECKOTie2 mice at P21, and this may be due to the lower efficiency of Tie2 gene deletion as discussed above. According to the recent work in the lab of Dr. Gou Young Koh , similar vascular defects were observed in retinas of -/iECKOTie2 mice when a higher efficiency of Tie2 deletion was achieved using a different VE-ERT2Cadherin-Cre mouse line.We did not observe the vascular tufts in retina of -/iUCKO mice at P21, the adult retina did not show any such malformations, whereas ear skin showed tortuous veins in adult mice. These data have been discussed rather insufficiently. The authors should comparatively analyze COUP-TFII transcript levels and other downstream players such as pAkt and pDok-2 in these two vascular beds in order to dissect the mechanisms, which help the retinal veins to "recover" but not the cutaneous veins. Likewise, partial recombination will lead to a mosaic vasculature of WT and KO cells. If such mosaic mice are traced over time, there is a good chance that the KO cells will be competed out by WT cells. As such, it is critical, particularly in the longer term experiments to carefully trace the percentage of cells with Tie2 deletion at the later time points of analysis.4) Although hemangioma-like vascular tufts were observed in Tie2-/iUCKOTie2 and control mice at the adult stage (2.5 month-old). Similar defects with venous tortuosity was also observed in the retina of -/iUCKOTie2 mice. Consistently, there was little vascular growth to the second layer and no blood vessel growth into the third layer of retina in -/iUCKOTie2 mice. Furthermore, Tie2 transcript level remained low in -/iUCKOTie2 mice as analysed by the immunostaining . The data is now included in the text.As shown in staining  and real5) The authors claim that Tie2 is required for the maintenance of venous EC identity. This is suggested by the observed phenotypes . A systematic gene expression analysis of adult retina similar to -/iUCKOTie2 mice at the adult stage, and retina was abnormally vascularized as shown in -/iUCKOTie2 mice compared with that of controls . This may be due to the fact that endothelial cell numbers are different between Tie2 knockout and littermate control mice due to the disruption of EC survival pathway.As discussed above, Tie2 transcript level remained low in 6) The authors are encouraged to reconsider their discussion. They conclude at the end of the discussion that the findings put a cautionary note on long term Tie2 targeting therapies, e.g., during tumor angiogenesis. This may not be the most important point of the study. In fact, nobody tries to target Tie2. Ang2 neutralizing strategies primarily work cooperatively with VEGF pathway targeting drugs based on their vascular normalization Tie2 gain-of-function effect. The same holds true for VE-PTP inhibitors or the recently published ABTAA antibody. What this reviewer finds much more exciting is the role of Tie2 on venogenesis. Venogenesis has mostly been considered as the default pathway with transcriptional programs driving lymphatic and arterial differentiation. The identification of Tie2 signaling as upstream regulator of COUP-TFII is a major finding that should likely be discussed in more detail. The fact that Tie2 KO leads in part to the acquisition of an arterial phenotype is noteworthy and suggests something of an arteriovenous identity balance mechanism. Likewise, angiogenesis is an arterializing process. As such, the physiological downregulation of Tie2 in the angiogenic tip cell vasculature should/could likely be discussed in the context of the findings of this manuscript. Obviously, it is at the discretion of the authors to prioritize the discussion, but it is felt that the authors may in the present version of the manuscript not have discussed the most obvious and most exciting implications of their work.The cautionary note in the Discussion is deleted. A new sentence has been added to the first paragraph of Discussion- “This implies that Tie2/Akt signaling may counterbalance VEGFR2/MAPK pathway in arteriovenous specification during the vascular development.”Reviewer #3:The study by Chu et al. characterizes the effect of Tie2 gene inactivation either using a ubiquitous or endothelial promoter to drive Cre expression during different stages of embryonic and postnatal stages. Interestingly, Tie2 deficiency leads to loss in vein formation or in stage-dependent maintenance of veins in different organs. The analyses are thorough and neatly presented. The study is ambitious, novel and of general interest.1) The study shows that loss of Tie2 is accompanied by vascular abnormalities and a deficiency in vein formation. It is not entirely clear from the images that veins are missing entirely or if they are malformed. Please complement images with aSMA stainings .In embryos, skin veins were completely missing when the induced deletion of Tie2 gene started from E10.5. The absence of veins in the skin were confirmed by co-staining of PECAM-1 and Dll4 as shown in 2) The author have previously described the iUCKO-/- mice in their paper in ATVB Jun;34(6):1221-30. Here, they have examined lymphatic vessel formation in the skin and show that deletion of Tie2 in neonate mice did not affect lymphatic vessel growth and maturation . I'm surprised that the lack of veins, as shown in the current study, has had no consequence for lymphatic vessel formation. The authors need to clarify and confirm that lymphatics are unaffected although veins are lacking.-/iUCKOTie2 and control mice during embryogenesis, and the data was not included in the paper. We found that there was abnormality about the lymphatic network in the skin analyzed at E17.5 (e.g. lymphatic vessel dilation). However, this could be secondary to the tissue edema resulting from the defective vein development. The detailed mechanism underlying the lymphatic defects in -/iUCKOTie2 mice during embryogenesis is still to be investigated.We have performed the analysis of lymphatic development with 3) In The images in 4) In As shown in -/iUCKOTie2 mice. However, as Tie2 is lowly expressed by lymphatic endothelium, it remains to be investigated whether it is a primary or secondary defects resulting from Tie2 insufficiency.As discussed above, lymphatic abnormalities were observed in [Editors’ note: the author responses to the re-review follow.][..] While the reviewers feel that your work is potentially very important, they were disappointed that you chose to argue points rather than making the necessary revisions elaborated in the original set of reviews. You will see from their comments, that none of the reviewers felt you adequately addressed their original concerns. Normally, eLife only allows for a single round of reviews. Given the importance of your work and the fact that all three reviewers have urged me to give you another opportunity to revise the paper, we are willing to give you a second chance to make the original required major revisions. I would like to stress that the reviewers are your advocates and trying to help you improve the paper. The reviewers’ comments from the second round of review are listed below, but I urge you to make the revisions requested in both the first and second round of review.This will be your final opportunity to provide acceptable additional experimental evidence as required by the reviewers.Reviewer #1:Overall my feeling is that the authors could have a very nice story but they do not want to make an effort to make it more complete. Data on the mechanism of action remain the same, as poor as before.I do not think that they can conclude that since COUPTFII is reduced when Akt activation is inhibited (in HUVEC) Akt activation is the mechanism of action of Tie-2 in maintaining a venous identity. Although there is a partial inhibition of Akt (24% reduction) in Tie-2 deficient mice other pathways are likely implicated.-/iUCKO mice with gene targeting efficiency approximately 87% in lung as shown in We agree with the reviewer that other downstream pathways, in addition to Akt mediated signaling, may also be involved in the venous abnormality when Tie2 mediated signaling was absent or insufficient. Considering that Akt is downstream of many signaling pathways and that it is also activated in many cell types, it is significant that we observed the decrease of pAkt in the total lung lysates after Tie2 deletion Tek as shown -/iUCKOFlox/-/Ubc-CreERT2. This may result from the lower Tie2 deletion efficiency in Tek-/iECKO mice than that of Tek-/iUCKO mice  A major concern with this study is the weak phenotype of the endothelial deletion compared to the ubiquitous deletion. The strong effects in the global deletion could be primary as well as secondary.-/iUCKO mutant mice, especially at postnatal stages. This requires further investigation. We have revised the Discussion section to point this out.We also thank the reviewer to point this out. As discussed above, based on the embryo studies, the disruption of vein development is likely to be a cell-autonomous effect resulting from the deletion of Tie2 in endothelial cells . However2) The question on how lack of veins affect lymphatics remains unanswered. The authors published in ATVB in 2014, that lymphatic vessel development is unaffected in neonate pups with the same global Tie2 deletion as described here. It is unexpected that the lack of veins would not affect lymphatic development.-/iECKO (Flox/-/Cdh5-CreERT2)Tek mice during embryogenesis. Lymphatic vessels were detected in Tek-/iECKO mice but became dilated  Mechanistic in vivo data remain scarce.We agree with the reviewer that further in vivo mechanistic studies are required for validating and fully elucidating Tie2 downstream mediators in the regulation of venous formation and maintenance. This may include the rescue experiments with Tie2 mutant mice as well as the generation of new genetic mouse models targeting the signal mediators downstream of Tie2 pathway (e.g. Dok-2). We have revised the Discussion section to reflect the reviewer’s valid point.Reviewer #3:This reviewer is rather disappointed on how the authors have addressed the specific critique to the original submission of the manuscript. The idea of the peer review process is to initiate an iterative process aimed at improving the eventually published manuscript. As such, this reviewer had constructively raised a number of issues that he felt would help to improve the manuscript. Generally speaking, the authors have not accepted this invitation, but essentially argued away all of the reviewer's critique and suggestions. They thereby missed the opportunity to improve their manuscript (at least in response to the comments and suggestions made by this reviewer). For example, they did not even opt to replace the black IF images for the supposed Tie2 ECKO analyses. In the rebuttal, they argue at length about different recombination efficacies using the two different driver lines. Clearly, incomplete recombination and the resulting competition of WT and KO cells in long term experiments is key to the interpretation of conditional KO data. If the authors' recombination analyses are correct, then this reviewer would insist that the corresponding IF images should show mosaic Tie2 expression and not black images.-/iECKO mice (-/iECKO mice, we have performed new experiments with the mTmG allele bred into the Tie2 mutant mice. The partial Tie2 deletion was indicated by the mosaic GFP expression in the compound genetic mouse model (Tek-/iECKO;mTmG; We now have replaced the IF images for TekCKO mice . In addiDespite this disappointment about the revised manuscript, this reviewer considers the manuscript of high quality and in principle deserving of publication. As such, if the authors are either not able or not willing to adequately address this reviewer's concern and suggestions, the manuscript should be published as is.We appreciate the highly positive comment and encouragement.