The formation of a complex multicellular organism from a single cell is one of the most amazing processes of biology. Embryonic development is characterized by the careful regulation of cellular behaviors such that cells proliferate, migrate, differentiate, and form tissues at the correct place and time. These processes are genetically controlled and depend both on the history of cells, their lineage, and on the activities of signaling pathways, which coordinate the cell interactions leading to organogenesis.A limited number of key signaling pathways—Fgf, Hedgehog, Wnt, TGFß, Notch among the most important—operate during development, acting repeatedly at different times and in different regions in the embryo and eliciting diverse cellular responses. This raises the question of how cells integrate all the information they receive and can respond in cell type-specific ways to the same signals. Classical concepts in embryology such as organizers  and competence (ability of cells to respond) can now be analyzed in molecular terms. In recent years many advances have been made in identifying the signals acting during embryo development and understanding their properties and functions, which is equally of relevance for human pathology and evolution. An important discovery is the conservation of signals and mechanisms, not only in evolutionary terms , but also in the repeated use of the same signaling pathways at different times and places in the embryos. Moreover, many of those mechanisms are involved in adult tissue homeostasis and regeneration.Understanding developmental signaling pathways is important for several reasons. It gives us information about basic mechanisms of cell function and interactions needed for morphogenesis and organogenesis. It uncovers the basis of congenital malformations, since errors at any step of cell signaling during development are a major cause of defects. Fundamental insight also gives us clues to understand the mechanisms operating in evolution that explain diversity in form and function. And finally, it allows the identification of possible causes of disease in the adult organism (such as cancer or degenerative diseases) pinpointing possible targets for therapeutic approaches.In this context, the aim of the Frontiers research topic “Signaling pathways in embryonic development” has been to provide a forum for experts in cell and developmental biology to share recent advances in the field of signaling during embryonic development. Sixteen articles in a variety of formats are united in this Topic, offering a valuable collection for researchers looking for an update in the knowledge of signaling pathways operating during embryogenesis. The works, focused mainly on vertebrates, explore different aspects of this theme from cell communication to organ formation and have implications for areas as distant as evolution or pathology.2+ pathways], illustrating how the same signal can elicit diverse cellular responses depending on the cell type, context, and developmental timing. Fujimura reviews the role of canonical Wnt signaling in eye development, highlighting the important roles it plays in patterning of ocular tissue, differentiation of retinal pigment epithelium, and morphogenesis of the optic cup. Importantly, mis-regulation of the signaling cascade can lead to eye malformations and disease. Gentzel and Schambony review a group of core intracellular effectors of the Wnt pathway, disheveled (DVL) proteins, which comprise three members in vertebrates. Although all DVLs share a common basic function in Wnt signaling, the expression patterns, and functions of the different isoforms are not totally redundant and have also diverged between different species, suggesting they play specific roles depending on the tissue distribution and specific interactions. Again, mutations in DVL genes can cause human congenital disease, highlighting their important role in development. Additionally, Berger et al. review the role of PTK7  in the fine-tuning of the Wnt signaling network. Its functions in establishing cell polarity, regulation of cell movements, and migration are also essential for development and disease, particularly in cancer and metastasis.Among the signaling pathways with important and widespread roles in development is the Wnt pathway, comprising a family of ligands with homology to wingless in Drosophila. Wnts can bind to multiple receptor complexes and trigger several downstream signaling cascades [including the so-called canonical WNT/β-catenin dependent signaling pathway, the non-canonical WNT/planar cell polarity (PCP), and the WNT/CaCarrieri and Dale review the particularly well-studied function of Notch in somitogenesis and also present recent data on the role of FBXW7 protein in regulating the turnover of Notch intracellular domain , in development and cancer. This relates to an often-overlooked essential point in signaling, which is the termination of activation and resetting of the components, allowing the cells to become competent again. Multiple mechanisms of regulation exist (positive and negative feedback loops) that allow a fine control of signaling pathways at different steps of the intracellular cascades.Another important signaling pathway is Notch, a transmembrane protein that mediates juxtacrine cell-cell communication. Notch has many functions in organ formation and adult homeostasis, including cell determination and stem cell maintenance. Bernatik et al. reporting on the role of the BMP antagonist Noggin in sensitizing cells and potentiating the activation of non-canonical Wnt signaling in skeletal development. They also provide evidence for a genetic interaction between these two pathways, which are involved in human congenital malformations.Crosstalk between the limited numbers of signaling pathways is a mechanism that allows cells to respond differently to the same signal, producing the diverse cellular behaviors that are needed to build tissues and organs. A new example of this is provided by Díez del Corral and Morales review the multiple roles of Fgf signaling in the developing spinal cord. This important structure of the nervous system arises from neural derivatives of an early neuromesodermal population located at the caudal part of the embryo. Extension of this region is coupled to spinal cord formation and several essential processes such as neurogenesis, ventral patterning or neural crest specification are controlled by Fgf signaling. These embryonic functions of Fgfs could be related to its ability to promote regeneration in the injured spinal cord of adults.The role of specific signaling pathways in the formation of particular organs is discussed in other articles. Gálvez et al. which also highlight that these same mechanisms are involved in hair cell regeneration, opening avenues for possible therapeutic approaches in hearing impairment. Ear development is also the topic reviewed by Magariños et al. They present evidence for a crucial role of autophagy, the regulated process of degradation, and recycling of cellular components, in vertebrate inner ear formation.Signalling pathways often converge on controlling the expression of transcription factors, which regulate cell fate specification. The integration of Notch signaling and bHLH transcription factors during inner ear development is analyzed by Tickle and Towers review the role of Shh in this process, a paradigm of how signals control and integrate tissue pattern and growth. They also discuss the implications of this important pathway for congenital malformations in humans and for the generation of limb morphological diversity during evolution. Montero et al. also treat this evolutionary aspect in their perspective article. They present a detailed analysis of Sox9 expression in developing digits of several species. This transcription factor, regulated by signaling pathways such as BMPs, Tgfßs, or Fgfs is involved in formation of the chondrogenic template of the skeleton. Differences in Sox9 expression patterns among species that have specific morphologies may reflect differences in signaling pathways controlling its expression. Also related to skeletal development, Amara et al. show that the effects of Calcium/Calmodulin dependent kinase II (CAMKII), an effector for Ca+2 -dependent signal transduction, in promoting chondrogenic differentiation seems to be specific for chicken embryos. This function is not observed in the mouse, thus highlighting the existence of differences in signaling functions and regulation among different species.The limb is a classic model in embryology and some of the most important discoveries related to the roles of signaling pathways in pattern formation, growth, and differentiation have been made studying limb development. Dueñas et al. review the role of signals, transcription factors and cellular processes in the formation of the epicardium. This is the external-most layer of the heart that serves not only as the outer cover for this organ, but also seems to play a role in regeneration. Thus, understanding the basis of its development may have important therapeutic implications. Two articles deal with muscle development. Hernandez-Torres et al. review the role of Pitx2 in embryonic and adult myogenesis. A hierarchy of transcription factors controls skeletal muscle differentiation and Pitx2 plays an important role in the regulation of this process. Importantly, it also seems to be involved in the establishment and function of satellite cells, the stem cells resident in adult muscle, thus opening new avenues for development of regenerative therapies. Additionally, Nassari et al. review the role of connective tissues in muscle development. Apart from the intrinsic molecular signals mentioned above, the interaction of muscle cells with surrounding tissues  is critical for the correct assembly of the musculoskeletal system during development and for maintaining adult homeostasis.Integration of extrinsic and intrinsic regulatory cues is essential for organ formation. Valdivia et al. review the mechanical control of myotendinous junction formation and tendon differentiation, highlighting again the importance of the interplay between chemical and mechanical signaling during embryogenesis. In the same line, Stricker et al. provide a timely discussion reminding us that cells in embryos and adult organisms are not present in isolation, but embedded in extracellular matrices into complex tissues. Cells attach to the ECM and sense its mechanical properties. Typically, experimental in vitro conditions do not fully reproduce this environment, which is however critical for the physiological cellular responses to signaling cascades. The challenge for the future is to try and integrate as many interactions as possible when designing experiments.An emerging theme in developmental biology is the control of tissue morphogenesis by physical forces (mechanotransduction). We hope that the articles in this topic will be of interest to researchers working in development and cell biology, fuelling discussion on this area and opening new avenues for thinking and investigation.JS was the Guest editor of this Research Topic, inviting co-editors AM and SS and working with them to define the subjects to be treated. They identified and invited leaders in specific research fields to contribute their work to the Research Topic. They acted as handling editors of manuscripts in the topic. JS wrote the Editorial with input from the other co-editors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Synechococcus spp. at the genomic level, despite being recognised of considerable ecological importance in aquatic ecosystems. So far, it has not been shown whether these unicellular picocyanobacteria have the potential for nitrogen fixation. Here, we present the draft-genome of the new pink-pigmented Synechococcus-like strain Vulcanococcus limneticus.Many cyanobacteria are capable of fixing atmospheric nitrogen, playing a crucial role in biogeochemical cycling. Little is known about freshwater unicellular cyanobacteria sp. nov., isolated from the volcanic Lake Albano .Vulcanococcus limneticus sp. nov. falls inside the sub-cluster 5.2, close to the estuarine/marine strains in a maximum-likelihood phylogenetic tree generated with 259 marker genes with representatives from marine, brackish, euryhaline and freshwater habitats. V.limneticus sp. nov. possesses a complete nitrogenase and nif operon. In an experimental setup under nitrogen limiting and non-limiting conditions, growth was observed in both cases. However, the nitrogenase genes (nifHDK) were not transcribed, i.e., V.limneticus sp. nov. did not fix nitrogen, but instead degraded the phycobilisomes to produce sufficient amounts of ammonia. Moreover, the strain encoded many other pathways to incorporate ammonia, nitrate and sulphate, which are energetically less expensive for the cell than fixing nitrogen. The association of the nif operon to a genomic island, the relatively high amount of mobile genetic elements (52 transposases) and the lower observed GC content of V.limneticus sp. nov. nif operon (60.54%) compared to the average of the strain (68.35%) support the theory that this planktonic strain may have obtained, at some point of its evolution, the nif operon by horizontal gene transfer (HGT) from a filamentous or heterocystous cyanobacterium.The novel species Vulcanococcus limneticus sp. nov., which possesses a complete nif operon for nitrogen fixation. The finding that in our experimental conditions V.limneticus sp. nov. did not express the nifHDK genes led us to reconsider the actual ecological meaning of these accessory genes located in genomic island that have possibly been acquired via HGT.In this study, we describe the novel species The online version of this article (10.1186/s12864-018-4648-3) contains supplementary material, which is available to authorized users. Three code for structural and catalytic components (nifHDK) and three (nifENB) for components involved in the biosynthesis of FeMoco enzymes, which are involved in dinitrogenase activation [nifHDK can be used as a proxy for nitrogen fixation levels [The reduction of atmospheric dinitrogen gas , which lack heterocysts, segregate nitrogen fixation and photosynthesis both spatially and temporally [Cyanothece, Synechococcus) evolved a temporal separation strategy, fixing nitrogen during the night and performing photosynthesis during the day, thus separating oxygenic PSII activity from the nitrogenase complex [Diazotrophs are microorganisms capable of converting dinitrogen into ammonia . Cyanobad nifVZT . The nitmporally , 9. Non- complex .nif operon in the cyanobacterial ancestor is controversial: on the one hand it could be acquired by horizontal gene transfer (HGT) after the origin of this clade, probably as consequence of the “fixed nitrogen crisis” between the Archean and Proterozoic era, which led to a lack of available nitrogen in the upper ocean promoting acquisition of nif genes from a heterotrophic prokaryote via HGT [nif genes were present in the cyanobacterial ancestor, repeatedly lost and gained again within the cyanobacterial phylum.These last kind of unicellular cyanobacteria have been recognized to be important in marine systems and are potentially abundant enough to significantly contribute to oceanic nitrogen fixation , 12. The via HGT . Alterna via HGT  proposednif operon in terms of presence, phylogenetic relationships and functionality in strains within the cyanobacterial clades. The ubiquitous unicellular cyanobacteria, Synechococcus, is a polyphyletic genus [Synechococcus from Yellowstone hot spring mat [nif operon and were described as nitrogen fixing. Certain strains showed nitrogenase activity only under anaerobic conditions [Synechococcus SF1 isolated from the blades of Sargassum fluitans which protects the nitrogenase complex by consuming excess oxygen through Uptake Hydrogenase activity [Synechococcus RF-1, isolated from a rice field, capable of rhythmic nitrogen-fixing activity [Synechococcus strains Miami BG43511 and BG43522 developed a temporal regulation [Synechococcus-like species with the potential for nitrogen fixation, nor for the activity of nif genes by RNA transcript quantification.In the light of the central role of nitrogen fixation in biogeochemical cycles, it is crucial to study the ic genus  currentlic genus  and is ric genus . Yet it ring mat  were repnditions , while oactivity , 21. Anoactivity . The margulation . NeverthVulcanococcus limneticus sp. nov. (formerly Synechococcus LL) isolated from a volcanic lake in central Italy, previously sequenced at draft genome level, finding it positive for the nif operon. Thus: i) we analyzed the sequences of the genes within the nif operon and the phylogenetic relationships between this cluster and others from Synechococcus and other cyanobacteria strains, ii) we measured nifHDK transcripts under nitrogen limitation conditions and iii) we analyzed the genome to find other ways for this strain to use nitrogen.In order to better understand the possible contribution of freshwater picocyanobacteria to nitrogen fixation we studied V.limneticus sp. nov., a novel planktonic strain isolated from the volcanic Lake Albano in central Italy   , the molybdenum transporter modAB and the ferredoxins fdxB and fdxH. The latter was described as important for the maximum nitrogenase activity in heterocystous cyanobacteria as its inactivation delays nitrogen fixation [nifP was absent from this strain. We compared the structure of the nif operon with some representatives of cyanobacteria and provided a phylogeny of the nifHDK concatamer among different molybdenum, vanadium and iron nitrogenases from different bacterial phyla representatives as previously described [Anabaena variabilis and Nostoc display a completely different organization of the nif operon with some subunits located elsewhere in their genomes. V.limneticus sp. nov. contains a slightly different nif structure compared with the rest of the other strains, although the closest gene organization is observed in the Yellowstone Hot Springs Synechococcus JA-3-3Ab and JA-2-3Ba [nifBSUHDKVZ is maintained except for the insertion of the ferredoxin fdxB in these strains between nifB and nifS subunits. The gene organization compared to anaerobic cyanobacteria also displays different arrangements  of the nif operon for Synechococcus JA-3-3Ab and JA-2-3Ba, we observed low levels of genetic identity (from 50 to 80%) despite the well conserved synteny of the different subunits between the different cyanobacteria tested, confirming an enormous genetic diversity of nitrogenases in the cyanobacterial phylum.Here, for the first time, we show the presence of nitrogenase operon in the new planktonic freshwater picocyanobacteria fixation , but it fixation . Only niescribed –38. CompJA-2-3Ba , where tPCC 7425 . The novMicrocoleus chthonoplastes showed acquisition of its nif operon by HGT [nif operon. Following metagenomic fragment recruitment methods described in other publications for freshwater Synechococcus [V.limneticus sp. nov. in two Amazon lakes from which metagenomic studies were carried out [V.limneticus sp. nov. genome is fully covered both above the species level (95%) and between 80 and 95% of identity values, which confirms that V.limneticus sp. nov. clones from the same species (> 95%) are present in these tropical Amazon lakes together with closely and distantly related species (80–95%). The regions of low coverage in these metagenomes, also called genomic islands, were associated to LPS biosynthesis, genetic mobile elements or other phage defense systems, as previously described in marine Synechococcus [. However, we noted that nif operon was also associated with a genomic island, being absent from the majority of the Synechococcus population and apparently rare in the tropical Lake Ananá and Mancapuru Great lake. The GC content of the contig containing the nif operon  also contrasts with the GC content of the strain (68.35%). The genes flanking the nif operon contain an average GC content of 65–67%, whilst the majority of nif genes present lower values from 56 to 61% and give top BLAST hits to heterocystous, filamentous and other Cyanobacteria as Leptolyngbya, Lyngbya, Phormidium, Oscillatoria, Microcoleus, Kamptonema, Calothrix, Pseudoanabaena or Tolypothrix species. Moreover, we noted a relatively high amount of transposases and mobile elements in V.limneticus sp. nov. (52), mostly at the beginning and at the end of the different contigs, which confirms that this strain is genetically prepared for gene transfer events. These features may reinforce the theory that this planktonic strain may have obtained, at some point of its evolution, the nif operon by HGT from a filamentous or heterocystous cyanobacterium.A previous study carried out in the cyanobacterium n by HGT . We founhococcus  we notedried out . This sthococcus . HowevernifHDK genes of V.limneticus sp. nov.we measured RNA transcripts using real time PCR in two experimental conditions: in the presence and absence of nitrogen. V.limneticus sp. nov.was able to grow in both conditions  . We acknowledge the need to test the transcript quantification of the nifHDK genes in other laboratory conditions  to make our results more comprehensive. We recognized some similarities with the filamentous, non-heterocystous cyanobacterium Microcoleus chthonoplastes that possesses nif genes but is unable to express nitrogenase in culture [To estimate the expression level of the  culture .V.limneticus sp. nov. is under nutrient limitation, it accumulates glycogen  producing sufficient amounts of ammonia [V.limneticus sp. nov. genome pointing to other mechanisms of nitrogen acquisition. The V.limneticus sp. nov. genome harbored multiple mechanisms to incorporate ammonia into the cell. For instance, we detected the ubiquitous and widespread ammonia assimilatory pathway via glutamine synthetase, ferredoxin-dependent glutamate synthetase, glutamine amidotransferase, nitrogen regulatory protein pII and amt transporters. Cyanate hydrolysis has been reported in marine Synechococcus and Prochlorococcus under nitrogen depleted conditions [2, which could provide an evolutionary advantage of V.limneticus sp. nov. under nitrogen limitation in Lake Albano. We also observed the nitrate and nitrite ammonification pathway as an alternative source to get ammonia; we detected a nitrate assimilatory reductase  and an ammonia ferredoxin oxidoreductase or nitrite reductase . Together with these enzymes, we also found two nitrate ABC transporters and four nitrate/sulfonate ABC transporters that could also be used for the uptake of sulfonates and alkanesulfonates.When  ammonia . This genditions . Interes2 consumption and is present in almost all nitrogen-fixing strains [Synechocystis PCC 6803, the bidirectional HoxEFUYH hydrogenase functions for H2 uptake or production [V.limneticus sp. nov. we detected only this bidirectional hydrogenase (HoxEFUYH). This is surprising since almost all nitrogen-fixing cyanobacteria possess also the uptake hydrogenase (HupSL) except for Synechococcus BG 043511 [Synechococcus there is an absence of the uptake hydrogenase. The recycling of hydrogen by these type of enzymes generates an anoxic environment necessary for nitrogenase activity [2 production could be assumed both by nitrogenases and bidirectional hydrogenases [It was reported that in nitrogen-fixing cyanobacteria hydrogen is synthesized as a by-product of nitrogenase activity and is further oxidized by a hydrogenase , 8. Ther strains  and a bi strains . In Syneoduction . In V.liG 043511 . Hence, activity  and the ogenases .V.limneticus sp. nov. Two alkanesulfonate monooxygenases and six sulfonate ABC transporters (TauABCD and ssuEADCB) were detected in the genome. These organosulfur compounds were also reported to be degraded by freshwater Actinobacteria [Achromobacter or Rhodococcus strains [Cyanobium and Synechococcus [nagA, nagB and the NAG kinase nagK) were also detected in the genome, which suggests that V.limneticus sp. nov. may also be capable of chitin degradation. All these features enhance the ecological relevance of the novel V.limneticus sp. nov.Regarding sulfur metabolism, we observed mechanisms for uptake and utilization of alkanesulfonates in bacteria  and Achr strains . The uti strains  and mari strains  and some strains  or herbi strains  are conshococcus . A chitiVulcanococcus limneticus sp. nov. possess a complete nitrogenase and nif operon, possibly acquired by HGT and located in a genomic island. However, in our experimental conditions, V.limneticus sp. nov. did not express the nifHDK genes. We cannot eliminate the possibility that in other conditions the nifHDK genes might still be expressed. Nevertheless, the presence of genes coding for different enzymes in the V.limneticus sp. nov. genome like alanine dehydrogenase or hydrogenase points to other pathways to incorporate ammonia, like direct ammonia assimilation from phycobilisome degradation, nitrate/nitrite ammonification or cyanate hydrolysis, which are energetically less expensive for the cell.This study shows that Vulcanococcus ; limneticus limnetic .3). It can form microcolonies of 10–20 cells. Vulcanococcus limneticus sp. nov. presents a G + C content of 68.35 mol%. The genome has a total size of 3,548,882 bp. The strain grows optimally in BG11 media for freshwater cyanobacteria at neutral pH and between 19 and 25 °C at low light (10–15 μmol photons m− 2 s− 1). The strain has a maximum absorbance at 573 nm typical of phycoerythrin and develops a type IIB intense pink-red pigmentation.The isolation source was the freshwater volcanic Lake Albano located in central Italy. It is composed by aerobic gram-negative non-motile cells approximately 0.97 ± 0.21 μm long and 0.76 ± 0.12 μm wide (volume: 0.36 ± 0.19 μm− 1) to eliminate the picoeukaryotes. Then the culture was purified by sorting  one single cell each into wells of 96-well plates containing 100 μl of BG11 substrate and kept in a thermostat at 18–20 °C at low light (10–15 μmol photons m− 2 s− 1) [Synechococcus LL based on 16S rDNA. The cells with phycoerythrin are well recognized by the flow cytometer, forming a defined cloud in the cytograms with signal collected in orange and red channels [, submitted) and following the recent Cyanobacteria classification scheme [Vulcanococcus limneticus sp. nov.The strain used for this experiment was a phycoerythrin-rich (PE) picocyanobacteria . This pi 2 s− 1) . In this 2 s− 1) ). This s 2 s− 1) , 55 and − 10) and visualising using Bandage v0.07 [DNA was extracted using AXG  gravity flow columns as per the manufacturer’s protocol. Library prep was performed with the Illumina TruSeq Nano DNA Library Preparation Kit  and sequenced on an Illumina Hi-Seq 2500. Reads were trimmed using Trimmomatic v0.32  before bge v0.07  as previge v0.07 . The finSynechococcus and Cyanobium genomes originating from multiple habitats. Nine Prochlorococcus marinus were also used to complete the phylogeny.A reference protein-concatenate-based tree with a total of 259 universal markers based on PhyloPlAn tool  was creaA NifHDK protein concatamer tree was constructed with NifHDK concatamers from a wide range of bacteria as previously described –38. SequVulcanococcus limneticus sp. nov., was assessed by tBLASTX [The structure and similarity of the nitrogenase subunits for the different compared cyanobacteria, including the novel planktonic  tBLASTX  with > 5V.limneticus sp. nov.We performed BLAST , BATCH wV.limneticus sp. nov. and its low covered genomic islands in the tested metagenomes [We performed recruitment plots following methods from other publications . We usedagenomes .Vulcanococcus limneticus sp. nov., in conditions in which it grows very well, by simply using two treatments, with (+N) and without (-N) nitrogen in the culture media, to measure nifHDK transcripts under nitrogen limitation conditions. In this way, we were able to use a non-axenic culture to recognize the activation of the nifHDK gene for the nitrogenase activity specific for V.limneticus sp. nov. The culture media was specific for cyanobacteria with nitrogen present as NaNO3 at a final concentration of 8 mmol L− 1 of N in the medium and N: P of 20 [V.limneticus sp. nov. around 8 × 105 cells ml− 1. The volume of the inoculum was low, so that the nitrogen added in the –N treatment was considered negligible. Each treatment was performed in triplicate, in semi-continuous cultures, kept in the same condition of maintenance in a culture room at 20 °C, with a 12 h light: 12 h dark diel-cycle using cool white fluorescent tubes at an intensity of 20 μmol photons m− 2 s− 1. Each day at the end of the light cycle and at the beginning of the dark cycle , equipped with a 50 mW laser emitting at a fixed excitation wavelength of 488 nm. The instrument provides light scattering signals , green fluorescence (FL1 channel = 533/30 nm), orange fluorescence (FL2 channel = 585/40 nm) and red fluorescence (FL3 channel > 670 nm and FL4 channel 675/25). For counting we used FL2-H vs FL3-H which better singled out the phycoerythrin-rich White polycarbonate filters  were used for microscopic evaluation of the cells at the beginning of the experiment, at the sampling for RNA analyses and at the end of the experiment. We used a Zeiss Axioplan microscope equipped with an HBO 100 W lamp, a Neofluar 100 x objective 1.25 x additional magnification and filter sets for blue  and green light excitation .V.limneticus sp. nov. cultivated in the presence and absence of nitrogen. Aliquots of the all replicates were analyzed for the abundance of V.limneticus sp. nov. by flow cytometry as described above. A volume of 20 ml of culture correspondent to 108 cells ml− 1 was centrifuged for 5 min at 13000 g and the pellets were processed for the RNA extraction using a commercial kit following the manufacturer’s instructions  with some modifications. The chemical and mechanical lysis were conducted together . Moreover, a DNase  treatment was carried out before the RNA purification. The RNA concentration was evaluated by fluorometric approach using Qubit assay following the manufacturer’s instructions . Comparable amounts of RNA (around 50 ng) of each sample were retro-transcribed following the manufacturer’s protocol . NORT for each sample was used as non-template control for the estimation of genomic DNA amplification signal. All the samples of cDNA and NORT were tested for the presence/abundance of the nitrogenase genes (nifHDK) and of 16S rDNA of V.limneticus sp. nov. by real time PCR. The primers for all the tested genes were designed using the genome sequence of V.limneticus sp. nov. as template using NetPrimer software (http://www.premierbiosoft.com/netprimer/index.html), the specificity of each primer was verified by blasting all the primer sequences against that of the V.limneticus sp. nov. full genome. The primer sequences are reported in supplementary material  and by the electrophoresis gel run. The abundance of each gene for all the samples was expressed as CT value.The RNA extraction was carried out from the broth cultures of the strain of Additional file 1:Table S1. Main characteristics of Lake Albano  from where V.limneticus sp. nov. was isolated. A more detailed study on water chemistry is published in Ellwood et al. 2009. This volcanic lake has trace level oxygen concentration in deep water (below 30 m), increase of NH4-N concentration below 70 m and calcite precipitation events in spring. (PDF 182 kb)Additional file 2:Fig. S2. Growth experiment of V.limneticus sp. nov. kept in two culture conditions: with and without nitrogen in the culture medium. The dark periods are reported as grey area. The number of cells are reported as mean of 3 replicates ± standard deviation. (PDF 337 kb)Additional file 3:Table S2. ANI , AAI  and GGDH  between Vulcanococcus limneticus sp. nov. and phylogenetically closest species. (PDF 88 kb)Additional file 4:Fig. S1. Metagenomic fragment recruitment of V.limneticus sp. nov. concatenated genome on Amazon lake metagenomic datasets. A) Recruitment plot on Lake Ananá. B) Recruitment plot on Mancapuru Great lake. LPS biosynthesis and nif operon genomic islands are highlighted in red. (PDF 1778 kb)Additional file 5:Table S3. Abundance of the genes tested by real time PCR expressed as threshold cycle value. (PDF 83 kb)Additional file 6:Fig. S3. Microphotography at epifluorescence microscopy of V.limneticus sp. nov. cultures in the two treatments with (+N) and whithout (-N) nitrogen. Glycogen granules are clearly visible in the N limited culture. . V.limneticus sp. nov. monoclonal culture was isolated from a volcanic freshwater mesotrophic lake in central Italy, Lake Albano. Dimension of cells: 0.97 ± 0.21 × 0.76 ± 0.12 μm, Volume: 0.36 ± 0.19 μm3. (PDF 644 kb)Additional file 7:Table S4. Primer pairs used to quantify 16S rDNA and nif genes of V.limneticus sp. nov. (PDF 163 kb)


Due to substantial improvement in the diagnosis and treatment of gynaecologic cancers, a better understanding of patient care needs to be revised. We reviewed the literature related to fertility preservation strategies in gynaecological cancer and discussed current general management approaches. New technical modalities and patients’ own desire for motherhood should be integral and paramount in the clinical evaluation to significantly contribute to preserving fertility in those women diagnosed with gynaecologic cancers during the reproductive years. Gynaecologic malignancies are relatively frequent in the female population, with a global estimated incidence of 222,700 new cases in Europe .In the last decade, the percentage of patients who had a first pregnancy after 40 years was approximately 20% . Thus, aHowever, compared to other types of tumours, the gonadotoxicity derived from medical and/or radiation treatment did not represent the main cause of infertility.In fact, the diagnosis of a gynaecologic malignancy is able to affect the fertility capability from an anatomical point of view, to the extent that a definitive damage is caused by the surgical approach.In this scenario, the consideration of fertility preservation represents an important issue –6.For sure, treatment strategy must be optimised to guarantee tumour eradication and excellent clinical outcomes. However, patient’s survivorship wishes to preserve fertility should be taken into consideration and nowadays a personalised care plan should become paramount. Identification of patients with early stage disease, as well as new and improved therapeutic strategies, is key to the safe selection of patients for fertility-preserving treatment.Fertility-sparing surgical strategies are feasible in early stage tumours, but advanced diseases usually require a radical approach leading to irreversible infertility, even if some authors hypothesised the futuristic use of uterus transplantation after an eventual previous oocyte cryopreservation –9.Thus, most of the scientific evidence regarding fertility preservation in gynaecologic malignancies is mainly focused on surgical modulation.In this context, we reviewed the literature related to onco-fertility in cervical, ovarian and endometrial cancers with particular attention to both radiation therapy (RT) and chemotheraphy (CHT) gonadotoxic effects. Moreover, we hereby discussed current general management approaches and related fertility preservation techniques, emphasising the need for multidisciplinary team involvement. Finally, we mentioned the emerging topic of fertility management of women with BRCA germline mutations and the psychological aspect of women with gynaecological cancer diagnosis. Indeed, despite many studies showing the connection between adequate counseling regarding fertility preservation treatments and the quality of life of women, there is no adequate information about this type of treatment .RT to the abdomen–pelvis is associated with a high risk of permanent amenorrhoea and can damage the uterus and uterine vessels, affecting the ability of a woman to carry a pregnancy to term .The risk of radiation sequelae increases with an advanced stage disease, extensive residual tumour size and abdominal carcinomatosis. Improvement in radiation techniques, including intensity modulated RT (IMRT) and proton therapy, may allow the radiotherapist to spare the ovaries and uterus from significant radiation dose, minimizsing the potential adverse effects on fertility. Amenorrhoea and infertility can also be subsequent to cranial irradiation.Ovarian dysfunction: Abdomino-pelvic irradiation may directly affect the gonads, leading to infertility , follicle-stimulating hormone (FSH), luteinising hormone (LH), estradiol, progesterone and prolactin, the hypothalamic–pituitary–ovarian axis is responsible for fine regulation of menstruation and fertility. However, this possible effect is of minimal concern to the vast majority of gynaecologic patients who do not present brain metastases at diagnosis. This issue is increasingly important in pediatric cancer survivors, but it is outside the scope of this review.The effect of chemotherapeutic drugs on ovarian function varies extensively according to age, as demonstrated for other tumours, and to CHT schedule . The main common CHT agents used in gynaecological cancer are alkylating agents (cyclophosphamide and ifosfamide), platinum compounds (cisplatin and carboplatin), taxanes (paclitaxel), anthracyclines (doxorubicin) and antimetabolites (gemcitabine and 5-fluorouracil) . Details2) of alkylating agents have a high risk (more than 70%) of developing permanent amenorrhoea [Alkylating agents are gonadotoxic chemotherapeutic agents and have most consistently been associated with ovarian failure in a dose-dependent manner. The American Society of Clinical Oncology (ASCO) Clinical Practice Guideline Committee stated that women treated with high doses  and tumour characteristics . Furthermore, oncofertility counseling represents a crucial step to inform patients with cancer on the risk of developing treatment-induced premature ovarian failure and infertility with the proposed anticancer therapies and to present the different available options to preserve ovarian function and fertility by discussing the pros and cons of each option. A summary of the main fertility preservation techniques is provided in Cervical cancer: The appropriate strategy is based on each stage of the disease. Generally, conization and trachelectomy are performed in early cervical cancer (stage IA-IB1), whereas in the advanced stage (stage > IB1) the current standard recommendation is RT with or without CHT, although neoadjuvant CHT followed by radical surgery represents a valid alternative.Regarding fertility preservation strategies, most of the international guidelines suggest that the first diagnostic and curative step for microscopic tumours consists of conization, a procedure consisting of the en bloc removal of the ectocervix and endocervical canals.In the presence of negative margins and the absence of clinical contraindications to surgery, the cone biopsy may represent a definitive treatment.Moreover, due to its extremely low incidence of lymphatic metastasis (<1%), conization alone without lymphadenectomy can be safely proposed in stage IA1 disease without the presence of lymphovascular space invasion (LVSI) .In the case of LVSI, radical trachelectomy with laparoscopic pelvic lymphadenectomy is preferable, when fertility preservation is desired.This technique includes surgical removal of the cervix to the level of the internal os, with en bloc resection of the vaginal fornix and supporting ligaments. The vagina is then sutured to the lower uterine body, and a cerclage suture is placed at the level of the internal os to prevent miscarriage during a future pregnancy . In geneOf note, some authors tested the use of conization eventually preceded by a neoadjuvant chemotherapy approach also in stage IA2, IB1 and even IB2 tumours with interesting results , 21.In the case of primary definitive RT, ovarian transposition out of the radiation field is a treatment option to preserve ovarian function with a success rate of 70–100% . At presOvarian cancer : Most patients with ovarian cancer present with advanced disease that is not eligible for fertility preservation. However, borderline and early stage tumours, including stage I epithelial ovarian cancers, malignant ovarian germ cell tumours and sex cord stromal tumours, are potentially suitable for fertility-sparing surgical procedures. Since scientific evidence has showed that the rate of microscopic tumour in the contralateral ovary is very low, ranging from 0% to 2.5%, the contralateral ovary such as the uterus are retained. However, since the recurrence after conservative treatment, also in IA stage, is not an excluded hypothesis, some authors suggest the use of adjuvant platinum-based chemotherapy in order to limit this risk [his risk .Endometrial cancer: The majority of endometrial cancer occurs in postmenopausal women. Endometrial cancer in women of reproductive age is often associated with a clinical history of polycystic ovarian syndrome – —which significantly reduces the chance of becoming pregnant and thus should be considered when proposing fertility-sparing treatment. In patients with stage IA disease who desire to preserve their fertility, a fertility-sparing treatment can be proposed [proposed . Continuproposed . Most ofproposed . Fertiliproposed .Embryo and oocyte cryopreservation are the standard assisted fertility preservation strategies. To offer the opportunity to conserve fertility, cryopreservation should be performed before the start of cancer therapy.Embryo cryopreservation: Embryo cryopreservation is the most established fertility preservation strategy if the patient has no urgency to start cancer therapy. A male partner or donor sperm is needed. Embryo cryopreservation is a safe technique. Both protocols, including randomly ovarian stimulation with subcutaneously injected gonadotropins for 8–14 days or within 3 days of the start of the menstrual cycle, are effective. After retrieval, oocytes are examined for maturity and then cryopreserved [reserved .Oocyte cryopreservation: Unfertilised oocyte cryopreservation requires ovarian stimulation with daily gonadotropin injections and ultrasound-guided oocyte retrieval under sedation. This technique is particularly important for patients who do not have a male partner or prefer not to use donor sperm [or sperm . Compareor sperm , 24.Ovarian cryopreservation: Ovarian tissue cryopreservation is currently considered an experimental technique to preserve reproductive potential [in vitro maturation of eggs [otential . Ovarianotential . Technic of eggs .Gonadotropin-releasing hormone agonists: The use of gonadotropin-releasing hormone agonists (GnRHa) is an investigational strategy, based on the use of GnRHa to suppress ovarian function during CHT. Theoretically, the use of GnRHa reduces the gonadotropins level, placing the ovaries in an artificial prepubertal state [al state . Moreoveal state . Howeveral state . HoweverTheir application has been largely investigated in fertility preservation of patients with breast cancer, but few data are available about their use in gynaecologic malignancies.However, a potential role for fertility preservation of gynaecologic malignancies might be hypothesised for the preservation of ovarian failure after a conservative surgical treatment of ovarian cancer or for the use of neoadjuvant approaches before a conservative surgical procedure for early stage cervical tumours.Actual and future perspectives for hysterectomised women (beyond the uterus transplantation):All the strategies reported until now may be considered in early stage tumours not requiring the removal of reproductive organs. The only motherhood options for women who undergo hysterectomy are adoption  or pregnancy in a gestational surrogate carrier after oocyte or embryo preservation, if applicable, in order to acquire genetic motherhood, followed by adoption to achieve legal motherhood.et al [Despite the interesting results reported by Brännström et al  about thHowever, it could represent a revolutionary approach for the management of gynaecologic cancer patients with fertility preservation purposes.It is well established that women with germline mutation BRCA genes have an increased lifetime risk of developing ovarian and breast cancer. To minimise cancer risk and maximise survival, risk-reduction surgeries should be performed at 25 years old . AlthougIn addition, controversy exists about fertility potential in this setting of patients. In fact, BRCA germline mutations seem to be related to a lower ovarian reserve – and thus a shorter reproductive lifespan – compared to women without BRCA mutations. The loss of BRCA function predisposes to DNA damage and subsequent accelerated follicular atresia and apoptotic oocytes . At presCancer is one of the diseases most commonly accompanied by psychosocial problems, and cancer patients may suffer, over the long term, from psychosocial disorders like fatigue, sleep disorders, cognitive and functional limitations, sexual dysfunctions, infertility, psychological problems, and psychiatric disorders , 35. In et al [Depression may have a negative impact on patients’ daily life and communication with their environment. McCorkle et al  showed iet al .It’s important to identify patients with particular risk of developing sexual and psychological problems. It would always be advisable to carry out an interview, using the Brief Index of Sexual Functioning for Women  or the FThe real effectiveness of different strategies for fertility preservation of gynaecologic malignancies needs to be determined. Since the main cause of infertility consists of surgical removal of the reproductive organs, a modulation of the surgical approach is the most desirable procedure.Embryo and oocyte cryopreservation are considered standard practice and are widely available in most other tumours; these procedures can also be used in gynaecologic cancers, for example in cases of uterus sparing or in cases of uterus-surrogate carriers. Other fertility preservation strategies should be considered experimental and should be reserved for patients who are treated in specialised centres with the necessary expertise.Although patients and physicians should be focused initially on cancer diagnosis, the patient and her family should be extensively counseled for fertility preservation. The risk of conservative therapy balanced against the disadvantages of more radical treatment should be explained. The patient should realize that by choosing fertility-sparing treatment, she is assuming a less but not well-defined risk of recurrent disease. Moreover, patients and physicians should evaluate the realistic probabilities of achieving conception based on the patient’s age, as well as her family history and fertility evaluation.A multidisciplinary team  evaluation should be the standard. Therefore, the presence of a psychologist is important to convey the correct information to patients with gynaecological cancer who wish to preserve their reproductive capacity , 45.


CRLF2 gene (hiCRLF2) have poor prognosis. CRLF2 protein overexpression leads to activated JAK/STAT signaling and trials are underway using JAK inhibitors to overcome treatment failure. Pre-clinical studies indicated limited efficacy of single JAK inhibitors, thus additional pathways must be targeted in hiCRLF2 cells. To identify additional activated networks, we used single-cell mass cytometry to examine 15 BCP-ALL primary patient samples. We uncovered a coordinated signaling network downstream of CRLF2 characterized by co-activation of JAK/STAT, PI3K, and CREB pathways. This CRLF2-driven network could be more effectively disrupted by SRC/ABL inhibition than single-agent JAK or PI3K inhibition, and this could be demonstrated even in primary minimal residual disease (MRD) cells. Our study suggests SCR/ABL inhibition as effective in disrupting the cooperative functional networks present in hiCRLF2 BCP-ALL patients, supporting further investigation of this strategy in pre-clinical studies.Children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) overexpressing the  CRLF2 (Cytokine Receptor Like Factor 2) gene are frequently present in high-risk BCP-ALL patients [B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a heterogeneous disease resulting from the accumulation of genetic alterations in B-lymphoid precursor cells and represents the most common malignant disease in childhood , 2. Althpatients  as well patients  and resupatients .CRLF2 is present in up to 15% of high risk BCP-ALL patients [JAK, IL7R and NRAS [IKZF1 gene [Overexpression of patients -10. Subsand NRAS , as wellZF1 gene , 13, whiZF1 gene . Since tJAK2 and IL7R alterations in TSLPR downstream signaling of murine pro-B Ba/F3 has been widely investigated by several groups [CRLF2 and/or JAK2 are responsible for increased TSLP-dependent activation of JAK2, STAT5, and rpS6 phospho-species, suggesting that targeting these molecules may be a valid therapeutic option for these patients [CRLF2 alterations . However, Weigert et al. and Scheartzman et al. demonstrated limited in vitro efficacy of ruxolitinib in human BCP-ALL CRLF2 rearranged (r)/JAK2 mutated cell lines [hiCRLF2 patients, as also recently described by Tasian et al. [The important role played by l groups , 16. As patients , 18. Thell lines -21, suggn et al. .hiCRLF2 BCP-ALL bone marrow samples. CyTOF enabled examination of multiple signaling pathways simultaneously and we identified a network involving JAK/STAT, PI3K and CREB pathways activated in hiCRLF2 patients. Perturbation of cells with inhibitors of the downstream TSLPR pathways, including a monoclonal antibody against the CRLF2 subunit, revealed the dual SRC/ABL inhibitor, dasatinib, to be effective in disrupting this network ex vivo and in inducing cell death to a similar degree as with the combination of JAK and PI3K inhibition. To determine if this network was relevant in drug resistance in hiCRLF2 patients, we examined minimal residual disease (MRD) samples and observed the same network present at the time of diagnosis in these patients. Further, in two of three patients classified as poor responders, cells harboring this network phenotype were enriched at Day 8 and Day 15 time-points, suggesting that this network may be important in the early persistence of leukemic cells. Thanks to this single-cell analysis, we uncovered distinct and clinically-relevant signaling nodes that can be successfully targeted by using a dual SRC/ABLi both in diagnostic and MRD cells, suggesting new therapeutic perspectives for patients with BCP-ALL bearing CRLF2 alterations.To better understand the complexity and impact of CRLF2 overexpression, we utilized single-cell mass cytometry (CyTOF)  to examihiCRLF2 and 6 loCRLF2, were analyzed by CyTOF using a 39-antibody panel  consistent with previous data [hiCRLF2 cohort contained two patients bearing mutations in JAK2 .ous data , althougin vitro stimulation with TSLP increased the phosphorylation levels of both STAT5 and rpS6 in hiCRLF2 compared to loCRLF2 cells   supporting the hypothesis that multiple pathways are involved in CRLF2-driven signaling.Yet, ) Figure , as prevTo understand if the TSLP-activated molecules were activated as part of a network and to quantify the strengths of these relationships, we used conditional-Density Resampled Estimate of Mutual Information (DREMI) to reveal pairwise signaling interactions between molecules and conditional-Density Rescaled Visualization (DREVI) to visually render this function as a rescaled heatmap, as previously described .hiCRLF2 patients  targeting JAK/STAT (ruxolitinib), SRC/ABL (dasatinib), or PI3K/mTOR (NVP-BEZ235) pathways  and in Ba/F3-hTSLPR expressing cells. Although all clones could specifically bind the CRLF2 subunit in both cell lines  for their binding strength and signaling inhibition in ll lines , only thll lines , thus fohiCRLF2 or loCRLF2) to these treatments (hiCRLF2 samples but the response was heterogeneous (p=0.3338) as patients #1, #2  and patient #6 demonstrated a strong pSTAT5 inhibition with ruxolitinib. However, pSTAT5 was more efficiently and consistently inhibited by the SRC/ABLi, dasatinib, (p=0.0254) or by the A10 anti-CRLF2 mAb (p=0.0357) , but activated CREB was only inhibited by the JAKi (p=0.0338) supporting previous evidence of active crosstalk within the pathways.TSLP-mediated activation of pERK and pCREB in hiCRLF2 patients, 4 were Ph-like  based on gene expression signature  we found that, although all the agents were able to affect single phosphoproteins, the only treatment disrupting all their interactions was the SRC/ABLi, dasatinib, as shown in Figure hiCRLF2 BCP-ALL cases, it is clear, as shown in our data and others [hiCRLF2 primary samples . SRC/ABL inhibition was still most effective at inhibiting downstream TSLP signaling in the STAT5 NR cells (p<0.0001). The A10 anti-CRLF2 mAb also showed a strong inhibitory effect (p=0.0081), as compared to JAKi or PI3K/mTORi (p=0.0227 for both agents) .Although our previous data demonstrated the effects of single agent kinase inhibition to inhibit activated TSLP signaling, it was not known if this translated into cell death. To assess how the signaling inhibition affects cellular proliferation and to evaluate if combination treatment might be more effective than single agents, we tested different cellular settings hiCRLF2 cells tested  at Day 8 and Day 15 after initiation of therapy.Although there is a correlation between hiCRLF2 samples  . CRLF2 ps Figure . Analyzis Figure , demonstTo determine if the TSLPR is functional and can be similarly inhibited by the tested TKIs, MRD cells of patient #13 (based on cell availability) were treated with either TSLP alone or in combination with JAKi or A10 or SRC/ABLi . Consistent with the diagnostic samples, the SRC/ABLi was the most effective treatment, inhibiting the TSLP-activated signaling, activated prpS6 and pCREB, in the MRD cells at Day 8 Figure , red boxhiCRLF2 cells at diagnosis is enriched in treatment-resistant cells. This network can be disabled by JAK inhibition but a more effective strategy may be inhibition of SRC/ABL in treatment-resistant cells.Together, we assessed the feasibility of using CyTOF to detect MRD cells but we were further able to interrogate their signaling network, demonstrating that the network profile present in the CRLF2 rearrangements and other Ph-like-related kinase alterations are associated with a greater risk of relapse and inferior outcomes in ALL patients [CRLF2 alterations and their related signaling in leukemic transformation of B-cells remains only partially understood.patients , 28. In patients , anti-CRpatients , 31 or, patients . HoweverHere, we have further investigated CRLF2 signaling in order to identify signaling nodes downstream of TSLPR that confer a survival advantage to these cells and could be targeted by using kinase inhibitors already used clinically for other subtypes of patients.hiCRLF2 primary samples using mass cytometry, which allows the simultaneous measurement of both surface and signaling molecules in a large number of cells [in vitro effects on signaling and cell death induction of a selected monoclonal antibody directed against the CRLF2 subunit, and three different KIs  acting at different levels of TSLPR-mediated signaling.To this end, we performed an extensive single-cell profiling of activated phosphoproteins in BCP-ALL leukemic cells in two series of of cells , 33. FurhiCRLF2 patients and this could be explained by the activity of dasatinib to act against SRC downstream targets which are also part of the TSLP-driven signaling pathways as demonstrated by Zhong et al [in vitro activity of the A10 anti-CRLF2 mAb, which could inhibit the TSLP-induced pSTAT5, prpS6 and pERK activation in some hiCRLF2 patients but was not generally as effective as the SRC/ABLi.Our findings confirmed and further extended previous data , 18, 24 ng et al . ConvershiCRLF2 patients, which did not phosphorylate STAT5 upon TSLP stimulation. These cells displayed enhanced TSLP-mediated phosphorylation of rpS6 and a strong signaling connection between prpS6 and pCREB, which was completely inhibited by the SRC/ABLi suggesting that SRC family members, already shown by Zhong et al. to be involved in the TSLPR pathway, may be a crucial signaling node that needs to be targeted to affect the coordinated TSLPR signaling.Taking advantage of the single-cell nature of these data and applying bioinformatics tools  to inveshiCRLF2 cells. Nonetheless, combining dasatinib with a PI3K/mTORi was even more effective in cells bearing not only the CRLF2 overexpression but also the activating mutation R683G in the JAK2 gene.We next asked how the inhibition of the CRLF2 network affected cell survival. In accordance with the short-term signaling data, we found that the SRC/ABLi dasatinib was most efficient as a single-agent in inducing cell death of in vivo persistence of these cells. The emergence of a stronger dependency between prpS6 and pCREB in the MRD cells compared to that in the total leukemia population at diagnosis is similar to that observed in the STAT5-non-responsive TSLP cells  and 6 were CRLF2 low (loCRLF2) (assessed by FCM and RQ-PCR). Genetic characterization of patients for JAK2 and IL7R alterations as well as for other BCP-ALL associated aberrations were performed based on samples availability  were first analyzed by CyTOF to study the TSLP-related pathways. In addition, 3 further hiCRLF2 patients were studied during early phases of remission induction therapy  and 1 hiCRLF2 patient out of 9 was used for the in vitro agents’ combinations. Further patient’s information is reported as Twelve patients , 1% L-glutamine and 1% penicillin/streptomycin at 37°C in humidified air with 5% CO2. The murine pro-B cell line Ba/F3-hTSLPR, provided by Prof. Shai Izraeli, is derived from Ba/F3 cells transduced with a retrovirus containing human CRLF2 and IL-7Rɑ genes to stably express on their surface a functional human TSLP complex, as previously described [2 incubator. Assessment of cell death assays was carried out by FACS analysis  and used for escribed . Ba/F3-hysis see .® (www.LCLabs.com). The three anti-CRLF2 monoclonal antibodies  were provided by MRCT Centre of Therapeutic Discovery of Leicester under MTA (see SRC/ABLi (dasatinib), JAKi (ruxolitinib) and PI3K/mTORi (NVP-BEZ235) were purchased from LC Laboratoriest-test was used to analyze the statistically significant differences between the hiCRLF2 and loCRLF2 phosphoprotein groups. Statistical analyses of phosphoprotein expression levels after treatment, calculated as arcsinh ratio in treated (treatment + TSLP) versus untreated condition , were performed with one-way ANOVA test followed by Dunn’s test for multiple comparisons with α=0.05.Unpaired two-sided Student’s in vitro cell death assay to evaluate the cell viability in treated vs non-treated condition, or in the combined vs single treatment. A p value ≤ 0.05 was considered statistically significant. All statistical analyses were performed with GraphPad Prism v 6.0 software . * p<0.5, ** p<0.01, *** p<0.001, **** p<0.0001.One-way ANOVA test, followed by Tukey’s test for multiple comparisons, was also applied to the


Allograft bone is a widely used as a convenient tool for reconstructing massive bone defects in orthopedic surgery. However, allografts are associated with the risk of viral disease transmission. One of the viruses transmitted in this manner is human T-lymphotropic virus type 1 (HTLV-1), which is found worldwide but is unevenly distributed. The southwestern parts of Japan are a highly endemic for HTLV-1. We investigated the HTLV-1 seroprevalence in candidate allograft donors at the regional bone bank in Kagoshima, Japan during its first 5 years of service. Between 2008 and 2012, we collected 282 femoral heads at the Kagoshima regional bone bank from living donors with osteoarthritis of the hip joint. Among the 282 candidate donors, 32 donors (11.3 %) were seropositive for anti-HTLV-1 antibody; notably, this prevalence is higher than that reported for blood donors in this area. Additionally, to determine if HTLV-1 genes are detectable after processing, we examined the bone marrow of the femoral heads from seropositive donors by conducting PCR assays. Our results confirm the existence of viral genes following the heat treatment processing of the femoral heads. Therefore, it is important to inactivate a virus completely by heat-treatment. Together, our findings highlight the importance of HTLV-1 screening at bone banks, particularly in HTLV-1-endemic areas such as southwest Japan. Allograft bone is a widely used as a convenient tool for reconstructing massive bone defects in orthopedic surgery (Engh and Ammeen HTLV-1 is a retrovirus associated with adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis . Furthermore, we performed postoperative screening of the donors by performing serologic tests for human immunodeficiency virus and HTLV-1 as well as PCR tests to detect hepatitis B virus and HCV genes Fig. . The heaAfter the heat treatment, the resulting solutions were cultured to confirm the efficacy of the thermal disinfection. We finally reconfirmed the results of the screening tests and medical history; based on these findings, we discarded 70 femoral heads.The femoral heads from 32 donors were not used for transplantation because the donors were found to be seropositive for HTLV-1. In order to verify the existence of HTLV-1 provirus in the femoral heads, DNA was extracted from the bone marrow tissue from these femoral necks, which included many blood cells, by using a DNeasy tissue kit according to the manufacturer’s instructions . The bone marrow tissue, including the bone itself, was crushed into pieces before the DNA extractions were performed . HTLV-1 tax primers and TaqMan probes were used for the real-time PCR using β-actin as an internal control. /(mean of β-actin copy number) × 2 × 10Among the 282 procured femoral heads, 212 were used for allografts in orthopedic surgery. However, 70 femoral heads (24.8 %) were discarded for various reasons  were positive for HTLV-1-specific antibody. All donors less than 50 years old were anti-HTLV-1 antibody-negative, whereas 14.2 % of the donors who were at least 50 years old were HTLV-1-positive . Alternatively, the PCR demonstration of proviral DNA may just indicate DNA from dead cells. Our detection of HTLV-1 genes in all three post-processing samples demonstrates that viral genes can remain in bone marrow lymphocytes in spite of processing. The heat treatment reduced the risk of virus transmission by inactivating the infected cells (Nowak et al. These findings emphasize the importance of screening donors for HTLV-1 prior to the use of their donations in transplantation (Schreiber et al. HTLV-1 screening in endemic areas such as Kagoshima is especially important. Because HTLV-1 seroprevalence is high in Kagoshima, we have performed serologic tests for HTLV-1 in candidate donors as part of their preoperative screening since 2013. Preoperative screening for HTLV1 eliminates the need for later screening tests, improving the cost-effectiveness of bone allografts.We detected HTLV-1 genes in samples from bone tissue that had been through sterilization processing. Therefor, it is important that the virus was completely disinfected by heat treatment. Additionally, our data reconfirm the importance of HTLV-1 screening at bone banks, particularly in endemic areas such as southwest Japan.


Reverse signaling is a signaling mechanism where transmembrane or membrane-bound ligands transduce signals and exert biological effects upon binding of their specific receptors, enabling a bidirectional signaling between ligand and receptor-expressing cells. In this study, we address the question of whether the transmembrane chemokine (C-X-C motif) ligand 16, CXCL16 is able to transduce reverse signaling and investigate the biological consequences. For this, we used human glioblastoma cell lines and a melanoma cell line as in vitro models to show that stimulation with recombinant C-X-C chemokine receptor 6 (CXCR6) or CXCR6-containing membrane preparations induces intracellular (reverse) signaling. Specificity was verified by RNAi experiments and by transfection with expression vectors for the intact CXCL16 and an intracellularly-truncated form of CXCL16. We showed that reverse signaling via CXCL16 promotes migration in CXCL16-expressing melanoma and glioblastoma cells, but does not affect proliferation or protection from chemically-induced apoptosis. Additionally, fast migrating cells isolated from freshly surgically-resected gliomas show a differential expression pattern for CXCL16 in comparison to slowly-migrating cells, enabling a possible functional role of the reverse signaling of the CXCL16/CXCR6 pair in human brain tumor progression in vivo. Cellular communication is frequently mediated by more or less specific binding of a ligand to its corresponding receptor, exerting intracellular signaling cascades and downstream effects in the receptor-expressing cell. However, transmembrane or membrane-bound ligands can also serve as signaling “receptors” and thus enable a bidirectional cellular communication. This signaling mode is termed “reverse signaling” and has so far been described for members of some (super)families of transmembrane ligands including the tumor necrosis factor-α (TNFα) superfamily, the ephrin ligand family and the semaphorins; for a review, see ,2,3. RevRecently, we were able to report another alternative signaling mode that is mediated via the transmembrane chemokines CXCL16 and CX3CL1 (chemokine (C-X3-C motif) ligand 1). In this process, upon shedding by matrix metalloproteinases  10 and ADAM17), the chemokine domain can be released from the transmembrane stack ,12, bindThus, regarding the facts that (1) reverse signaling via transmembrane ligands has been reported for a considerable number of ligand-receptor pairs and (2) we recently could show that transmembrane CXCL16 can transduce signals via its intracellular domain upon binding of its soluble form , we wondered if reverse signaling may also take place in the interaction between transmembrane CXCL16 and its known receptor CXCR6.To investigate intracellular signaling of CXCL16 upon stimulation with CXCR6, initially, we used human glioblastoma cell lines  and applied CXCR6 in different forms. The specificity of reverse signaling was proven by silencing experiments, as well as by transfection experiments using a CXCL16-negative, CXCR6-negative melanoma cell line to investigate intracellular signaling and biological effects upon stimulation with CXCR6.From our recent investigations, we know that CXCL16 is highly expressed in different human gliomas, while the corresponding receptor CXCR6 is restricted to a small subset of glioma cells with stem cell characteristics . To inveTo prove specificity, we used stable transfected LOX melanoma cell clones. LOX melanoma cells do not endogenously express CXCL16, nor CXCR6, and so, we generated LOX clones expressing transmembrane CXCL16 (LOX-CXCL16) or a C–terminally truncated version of transmembrane CXCL16 (LOX-ΔCXCL16) and a clone from the empty expression vector (LOX-pcDNA) . To veriTo investigate a putative reverse signaling of transmembrane CXCL16 upon binding of its known receptor CXCR6, for the first approach, we used different CXCL16-positive, CXCR6-negative glioblastoma cell lines and stimulated them with 25 ng/mL recombinant CXCR6 for 10 or 15 min. As shown in In the next step, we stimulated stably CXCL16 expressing LOX melanoma cells (LOX-CXCL16) with recombinant CXCR6 and observed again an activation of ERK1/2 B. FurtheThese results indicate that signaling upon CXCR6 stimulation specifically depends on the expression of CXCL16 including its intracellular domain and may physiologically occur upon exposition of transmembrane CXCL16 to CXCR6-expressing membranes.To investigate which biological consequences might result from the reverse signaling of the CXCL16-CXCR6 axis, we first referred to the effects observed with inverse signaling in glioma cells  and testNext, we investigated the migratory potential of LOX-CXCL16 cells in comparison to LOX-pcDNA (control) and LOX-ΔCXCL16  and of T98G glioblastoma cells in a scratch assay with or without stimulation with recombinant CXCR6 C. Here, Interestingly, when we investigated the expression of CXCL16 in fast migrating in comparison to slowly-migrating cells isolated from freshly-dissected glioblastomas, as described previously , we coulSummarizing, our data show that transmembrane CXCL16 transduces signals upon stimulation with its known receptor CXCR6, activating intracellular ERK1/2 signaling. This reverse signaling depends on the intracellular domain of CXCL16 and promotes migration in CXCL16-expressing melanoma and glioblastoma cells in vitro. Additionally, we could show that fast migrating glioblastoma cells isolated from freshly-dissected glioblastomas express CXCL16 at higher levels in comparison to slowly-migrating cells, giving a first hint that reverse signaling might also contribute to glioblastoma migration processes in vivo.Physiologically, transmembrane CXCL16 is among others expressed by immune and endothelial cells and can be induced in inflammatory conditions ,15,16,27However, recently, we showed that CXCL16 can also induce signals independently from CXCR6 by a mechanism we termed inverse signaling. In this signaling mode, the chemokine domain of CXCL16 binds to the transmembrane form of CXCL16, induces intracellular ERK1/2 and Akt signaling and promotes proliferation and rescue from chemically-induced apoptosis ,14. In tRegarding effects via transmembrane ligands in a more general view, the reverse signaling is often involved in modulating the balance in dynamic changing systems and plasticity, like for example the Eph (erythropoietin-producing human hepatocellular receptors)/ephrin interactions in the formation and maintenance of synapses , in angiThus, reverse signaling contributes to tumor biology in a multifaceted way. We were able to show now that the transmembrane chemokine CXCL16 can also mediate reverse signaling and promotes migration in the tumor context. In this line, we observed that fast migrating glioblastoma cells show higher CXCL16 expression levels in comparison to slowly-migrating cells of the same tumors.Mycoplasma contamination. LOX melanoma cells were a gift from Udo Schumacher, Department of Anatomy, University of Hamburg, Germany [The human glioblastoma multiforme (GBM) cell lines A172 (ECACC 880624218), U251MG , T98G (ECACC 92090213) and LN229 ATCC-CRL-2611) were obtained from the European Collection of Cell Cultures  or the American Type Culture Collection  and cultured as described before 11 were o; compare Germany . Cell liStable transfected LOX-pcDNA, LOX-CXCL16 and intracellularly truncated LOX-ΔCXCL16 clones were generated as described previously .Expression vectors for CXCR6  and pCMV  were obtained from OriGene , and transfection of LOX melanoma cells  was performed with TurboFect  in serum-free Dulbecco’s Modified Eagle’s Medium (dulbecco’s modified eagle’s medium (DMEM); Invitrogen, Carlsbad, CA, USA) without antibiotics using 3 µg of the respective expression vectors and 3 µL TurboFect in a total volume of 1 mL. After 6 h, cells were rinsed, and normal growth medium ) was added. Successful transfection was controlled by immunocytochemistry and quantitative real-time PCR (qRT-PCR). Stable clones were generated by selection with 0.8 mg/mL G418 , and colonies were picked after 10–20 days, amplified and checked for expression by qRT-PCR and immunocytochemistry.Glioblastoma cell lines and different stably-transfected LOX melanoma cells grown on glass cover slips were prepared as described before . Cells w® Reagent  or with the ARCTURUS® PicoPure® RNA Isolation Kit  according to the manufacturer’s instructions. DNase digestion, cDNA synthesis and qRT-PCR were performed as described before [CT) were determined, and ∆CT values of each sample were calculated as CTgene of interest − CTGAPDH. A ∆CT of 3.33 corresponds to a 10-fold lower expression compared to GAPDH. For statistical analysis, the relative gene expression compared to GAPDH (2CT−∆) was employed. The induction of gene expression upon stimulation is displayed as relative gene expression; n-fold expression changes were calculated as ∆∆CT values = 2C∆TCcontrol−∆Tstimulus.RNA of the different cell types was isolated with the TRIzold before  using TaAfter cultivation of human glioblastoma cell lines in DMEM plus 10% FBS in 6-well dishes  for 24 h, cells were transfected with siCXCL16 RNA  dissolved in a mixture of Opti-MEM medium and lipofectamine (Life Technologies) for 5 h as described before . In paraFor isolation of cell membranes, stable transfected LOX-CXCR6 and LOX-pCMV clone cells were lysed in 5 mM HEPES buffer, then 200 mM HEPES buffer containing 1.4 mM sodium chloride was added, and the mixture was centrifuged at 800 rpm for 8 min at 4 °C. Supernatants were transferred into a new tube and centrifuged once again at 14,000 rpm for 60 min at 4 °C. The remaining pellets were resuspended with 50 µL of 20 mM HEPES buffer including 0.14 mM sodium chloride, and membranes were kept at 4 °C until usage.Western blotting was performed as described . Briefly5) and T98G glioblastoma cells (5 × 104) were grown for one day in 10% FBS-supplemented DMEM and stimulated in DMEM plus 0.5% FBS (LOX clones) or 2% FBS (T98G cells) with 50 ng/mL recombinant CXCR6 protein (BIOZOL) for 24 h up to 48 h. In parallel, control groups without stimulation were used. Then, 250 μL of CyQUANT GR dye/cell-lysis buffer and 2.5 μL RNase (CyQUANT® Cell Proliferation Assay Kit (C-7026); Thermo Fisher Scientific, Waltham, MA, USA) were added to the cells, and lysates were scraped off, briefly centrifuged and added 250 µL 2× CyQUANT GR. Sample fluorescence was measured using a fluorescence microplate reader  with filters appropriate for 480-nm excitation and 520-nm emission maxima. Results were calculated in ng DNA as the percentage of unstimulated controls.Proliferation assays were performed as described . Briefly2 flask, grown for 30 h and stimulated for 18 h) as described above using an antibody specifically detecting cleaved PARP . An antibody against GAPDH  served as the loading control.Apoptosis was induced in LOX-CXCL16, LOX-pcDNA and truncated LOX-ΔCXCL16 clones by the addition of 0.1 µg/mL camptothecin  applied in a stock solution in DMSO, in the presence or absence of 50 ng/mL recombinant CXCR6 . The final solvent concentration of 0.1% DMSO in camptothecin-treated cultures was also used in controls. After stimulation, cleavage of poly(ADP Ribose) polymerase (PARP) was measured by Western blot , apoptosis was induced with 400 µg/mL TMZ (solved in DMSO) for 48 h in the presence or absence of 50 ng/mL recombinant CXCR6 . Cells were lysed, and caspase 3/7 activity was measured as previously described [In T98G glioblastoma cells  with 50 ng/mL recombinant CXCR6 protein (BIOZOL). In each experiment, three scratch regions were photographed at 0 and 8 h. Scratch areas were measured, and differences between 8 and 0 h were determined (yielding the settled area). Stimuli were normalized to non-stimulated controls.Migration was analyzed in wound healing assays . Brieflt-test was used. Significance levels were p < 0.05 (indicated by *), p < 0.01 (indicated by **) and p < 0.001 (indicated by ***).For statistical analysis, a two-tailed Student’s In this study, we could show that the transmembrane chemokine CXCL16 can mediate intracellular signaling upon stimulation with its receptor CXCR6 in the ligand expressing cell. This signaling mechanism has previously been reported for other transmembrane ligands like ephrins, semaphorins and TNF family members and was termed reverse signaling. We now observed that reverse signaling via the transmembrane chemokine CXCL16 promotes migration in the tumor context, but does not affect proliferation or rescue from apoptosis in melanoma or glioblastoma cells. In this line, we could detect that fast migrating glioblastoma cells show higher CXCL16 expression levels in comparison to slowly-migrating cell fractions of the same tumor.Taken together, being produced as a transmembrane ligand, CXCL16 harbors a broad range of para- and autocrine communication options that may be regulated via expression levels of ligand and receptor  and via cleavage and release of the chemokine domain by ADAMs. Apart from the classical forward signaling via CXCR6, the transmembrane CXCL16 form may also mediate signaling on its own, either upon binding its soluble CXCL16  or upon binding of its receptor CXCR6 , inducing proliferation and survival, as well as migration in tumor cells.


Twelve providers (eight institutions) participating in a Gender and Sex Diversity Fertility Working Group completed a survey assessing their hospital's transgender, disorders/differences of sex development (DSD), and fertility healthcare teams. Participants also prioritized the development of a: (1) Provider Assessment Tool (questionnaire assessing knowledge/feelings about future fertility), or (2) decision aid (DA). Healthcare team presence by institution: transgender , DSD (75%), and fertility preservation (75%). Two-thirds of providers reported that the DA was most needed. Respondents recommended the DA to: help manage complex information, have a take-home reference, and provide neutral information. Other identified resource needs included: fertility specialists in clinic and financial resources. In addition, many treatments  currently offered to young transgender and DSD patients can affect future fertility.2 Medical decisions impacting the future fertility of transgender and DSD patients are complex, and often must be made in childhood, yet no clinical tools or guidelines exist to guide discussions about future fertility and fertility preservation (FP) with the families of gender and sex diverse individuals.The term “gender and sex diversity” encompasses the following: (1) transgender individuals for whom gender identity is incongruent with phenotypic sex, and (2) individuals with disorders/differences of sex development (DSD), for whom chromosomal, gonadal, or anatomic sex development is atypical.4 and patient decision aids (DAs) are available to guide care.5Similar to the parents of gender and sex diverse children, parents of pediatric cancer patients also face decisions related to potentially preserving their children's future fertility. Challenges in this setting include the following: (1) Necessity of parental proxy decision-making; (2) uncertainty regarding future assisted reproductive technologies, particularly related to maturation of immature reproductive tissue into usable adult eggs or sperm; and (3) cost of long-term tissue storage. For pediatric cancer patients, clinical guidelines regarding FP,Parents contemplating the idea of preserving future fertility for their transgender child, or child with a DSD, face some distinct challenges compared with the pediatric cancer population. For example, families, patients, or providers may express hesitancy or unfamiliarity with utilizing gonads incongruent with gender identity to produce biological offspring. In addition, the fertility potential in many DSD conditions is still being defined. Despite the complexity of, and uncertainty about, pediatric FP options, and the unique issues facing gender and sex diverse individuals, no clinical tools exist to guide fertility-related care for these patients.Our aim was to conduct a clinical needs-assessment of transgender and DSD care providers participating in a working group focused on fertility-related care. The needs-assessment was conducted to understand the perspectives and priorities of clinicians who care for transgender and DSD patients and to guide next steps for clinical tool development. We hypothesized that providers would recommend creating a clinical Provider Assessment Tool (vs. a DA) as the first step in clinical tool development.® Meeting in Chicago.6 All Oncofertility Consortium Meeting attendees were invited to attend; participants were also invited based on previously expressed interest in the topic. Healthcare professionals from around North America with expertise in transgender, DSD, and fertility medicine met to share knowledge and experiences and to outline next steps for the field of fertility-related care for gender and sex diverse individuals. A needs-assessment survey was conducted to understand the current state of transgender, DSD, and FP teams at the institutions represented by working group members and to determine an initial priority for clinical tool development.The first multidisciplinary Gender and Sex Diversity Fertility Working Group was convened as a subgroup of the 2015 Oncofertility ConsortiumIdeas for the two potential clinical tools came from the Lurie Children's Gender and Sex Development Program (GSDP) Providers. GSDP providers who care for transgender and DSD patients have expressed difficulty with: (1) assessing families' knowledge, information needs, and desires about possible FP, and (2) counseling families about complex, often experimental, FP options. The GSDP clinic group expressed that a Provider Assessment Tool could potentially address the challenge with assessing knowledge and information needs and that a DA may assist with counseling about preservation options.Providers participating in the Gender and Sex Diversity Fertility Working Group completed a two-part self-administered survey . SectionDescriptive statistics were used to characterize survey responses. Reasons for recommending a Provider Assessment Tool or a DA were categorized and organized into themes. The Lurie Children's Institutional Review Board approved the study. Verbal consent was obtained from all participants.The Gender and Sex Diversity Fertility Working Group consisted of 14 participants , 12 of whom completed the survey (86% response rate). Freestanding children's hospitals, other pediatric hospitals, and adult institutions were all represented. Specialties and teams represented are detailed in Nine providers (75%) expressed a preference for developing either a Provider Assessment Tool or a DA. Three providers felt that both were equally important. Of providers with a preference, 6/9 (67%) prioritized the development of a patient DA and 3/9 (33%) preferred the Provider Assessment Tool. Reasons for preferring the DA included the following: (1) the DA could facilitate fertility discussions in the clinic, and (2) as an informational source, the DA could confer multiple benefits to patients and families. Specifically, respondents reported that the DA could help families manage a large volume of complex information, provide a take-home reference for later review, and serve as a neutral source of information about fertility options.N=3) believed that it could facilitate fertility-related discussions in clinic, similar to the DA. However, respondents also noted that the questionnaire-based assessment tool could help patients to clarify their values and help triage patients for referral to fertility specialists.Participants who preferred the Provider Assessment Tool (Other resource needs suggested included the following: (1) presence of a fertility specialist in transgender and DSD clinics, (2) a handout about what to expect if FP is desired, (3) connection to support groups to discuss fertility-related issues, (4) financial resources, (5) access to patient testimonials, and (6) development of visual aids to illustrate complex fertility-related concepts.A multidisciplinary group of healthcare providers is necessary to provide fertility-related care for gender and sex diverse individuals. A sample of such providers recommended development of a patient DA to facilitate fertility-related care for transgender and DSD patients. The recommendation to create a patient DA about fertility was contrary to our expectation that providers would prefer a Provider Assessment Tool. Survey participants cited potential patient and family unfamiliarity with fertility as a topic, as well as management of a large volume of fertility-related information as potential benefits of the DA. Despite the potential for a Provider Assessment Tool to help focus initial clinic visits, the working group noted that topic of fertility should be introduced in person before use of a clinical tool.7 but long-term effects are yet to be fully described. For DSD patients, fertility is more often impaired by the inherent gonadal developmental abnormalities or prophylactic gonadectomy performed due to malignancy risk. Fertility potential has been reported in the literature among individuals with selected DSD conditions, including Klinefelter8–10 and Turner Syndromes,12 but has been incompletely described for other DSD conditions. Relevant to both transgender and DSD individuals, pediatric FP options are still evolving. Many gender and sex diverse youth would require prepubertal gonadal cryopreservation to preserve future fertility. The technology to mature this tissue into adult forms of eggs or sperm is still in development, but fertility experts expect this option to be available in the future.13–15 Fertility-related decision-making for both DSD and transgender individuals occurs in the context of a high level of clinical uncertainty, highlighting the importance of developing clinical tools to guide care.Consideration of future fertility is relevant for both transgender and DSD patients, but the evidence base guiding fertility-related care for both groups is limited. For transgender individuals, medical interventions intended to alleviate gender dysphoria, including pubertal suppression and gender-affirming hormone therapy, may affect future fertility,5 but similar tools are not available for gender and sex diverse individuals. We aim to develop a modular DA that could be adapted based on each patient's clinical situation and updated with emerging information and technologies. Previous investigators have noted the benefits of building in patient-level flexibility to clinical DAs.16 DAs have also been shown to improve care for many conditions by reducing decisional conflict, improving patient-clinician communication, and increasing satisfaction with decisions.17 Specific to fertility-related care for gender and sex diverse individuals, a patient-specific modular DA could help clinicians focus and structure discussions about this complicated evolving topic. Such a DA could also help to ensure that the entire multidisciplinary team is delivering a consistent message.Developing a patient-centered DA about fertility is an important next step for our research group. DAs are now available for pediatric cancer patients,The institutions represented in our working group recognized the importance of a multidisciplinary team approach to transgender, DSD, and fertility-related care. However, the composition and organization of each of these teams varied by institution. In addition, the optimal model for interaction of these teams to provide fertility-related care to transgender and DSD patients is yet to be defined. It is encouraging that 63% of participating hospitals had either formal or informal teams covering all three care categories, suggesting that comprehensive fertility-related care for gender and sex diverse individuals could be accomplished at multiple sites nationwide.7 and the 2006 DSD consensus statement1 both advocate for coordinated multidisciplinary care; we suggest that fertility-related care may be incorporated within these existing frameworks. A recommended minimum team composition includes: endocrinology, urology, pediatric surgery, fertility medicine, genetic counseling, and mental health, with other disciplines involved based on specific identified needs of each patient. Given that fertility-related care for gender and sex diverse individuals is a small and emerging field, telemedicine may facilitate this aspect of care for patients who otherwise may not have access. While all aspects of fertility-related care  cannot be conducted remotely, telemedicine has an established role for certain aspects of care, including mental health,18 surgical consultation, and routine postoperative care.19 Thus, organizing and supporting teams to provide components of their clinical services remotely are recommended as the new healthcare field expands.The World Professional Association for Transgender Health Standards of CareThis study was limited by the sample size, and survey participants constituted a convenience sample with specialized interest and expertise in transgender, DSD, and fertility medicine. Given the small sample size, we were unable to stratify results by provider or hospital characteristics. However, we felt the survey was an important first step to query engaged providers about their needs, as there was no previous information available to guide our clinical tool development priorities. Another limitation was the format assessing preferred choice for clinical tool development—it was presented as a predefined dichotomous option. Although there was the opportunity for open-ended suggestions, there may be other clinical tools that would be more helpful that were not considered by the study team or survey participants. In addition, the survey did not request specific content recommendations for a Provider Assessment Tool or DA. To address this, we will be iteratively incorporating provider suggestions and feedback into the process of developing a DA about fertility for gender and sex diverse individuals. Finally, our investigation only represents the provider perspective, and we queried providers about both populations in aggregate. To mitigate this limitation, we are also currently obtaining and evaluating transgender and DSD patients and perspectives about how best to deliver fertility-related care.Fertility-related care for gender and sex diverse individuals is an emerging multidisciplinary field requiring multiple clinical tools. The first priority is to develop a patient DA, which will help families understand complex information about future fertility.