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Summary Gonadal sexual fate in mammals is determined during embryonic development and must be actively maintained in adulthood. Therefore, gonadal sex-specific transcription factors are required to prevent transdifferentiation of gonadal somatic cells to the other sexual fate. Mouse genetic experiments have shown that oestrogen receptor signalling and the transcription factor FOXL2 protect ovarian granulosa cells from transdifferentiation into Sertoli cells, their testicular counterpart. However, the mechanism underlying this protective mechanism is unknown. Here, we show that one post-translational modification (i.e. SUMOylation catalysed by TRIM28) is sufficient to prevent female-to-male sex reversal of the mouse ovary after birth. We found that upon loss of TRIM28 SUMO-E3 ligase activity, ovarian granulosa cells transdifferentiate to Sertoli cells through an intermediate cell type different from gonadal embryonic progenitors. TRIM28 binds to chromatin close to the critical transcription factor FOXL2 to maintain the female pathway through SUMOylation of specific chromatin regions. Therefore, FOXL2 signalling might maintain the adult ovary cell fate via TRIM28-dependent SUMOylation. Improper SUMOylation of chromatin regions in granulosa cells might lead to female reproductive disorders and infertility, the incidence of which is currently increasing.

Despite the importance of germ cell (GC) differentiation for sexual reproduction, the gene networks underlying their fate remain unclear. Here, we comprehensively characterize the gene expression dynamics during sex determination based on single-cell RNA sequencing of 14 914 XX and XY mouse GCs between embryonic days (E) 9.0 and 16.5. We found that XX and XY GCs diverge transcriptionally as early as E11.5 with upregulation of genes downstream of the bone morphogenic protein (BMP) and nodal/Activin pathways in XY and XX GCs, respectively. We also identified a sex-specific upregulation of genes associated with negative regulation of mRNA processing and an increase in intron retention consistent with a reduction in mRNA splicing in XY testicular GCs by E13.5. Using computational gene regulation network inference analysis, we identified sex-specific, sequential waves of putative key regulator genes during GC differentiation and revealed that the meiotic genes are regulated by positive and negative master modules acting in an antagonistic fashion. Finally, we found that rare adrenal GCs enter meiosis similarly to ovarian GCs but display altered expression of master genes controlling the female and male genetic programs, indicating that the somatic environment is important for GC function. Our data are available on a web platform and provide a molecular roadmap of GC sex determination at single-cell resolution, which will serve as a valuable resource for future studies of gonad development, function, and disease.

Leydig cells (LCs) are the major androgen-producing cells in the testis. They arise from steroidogenic progenitors (SPs), whose origins, maintenance, and differentiation dynamics remain largely unknown. Single-cell transcriptomics reveal that the mouse steroidogenic lineage is specified as early as embryonic day 12.5 (E12.5) and has a dual mesonephric and coelomic origin. SPs specifically express the Wnt5a gene and evolve rapidly. At E12.5 and E13.5, they give rise first to an intermediate population of pre-LCs, and finally to fetal LCs. At E16.5, SPs possess the characteristics of the dormant progenitors at the origin of adult LCs and are also transcriptionally closely related to peritubular myoid cells (PMCs). In agreement with our in silico analysis, in vivo lineage tracing indicates that Wnt5a-expressing cells are bona fide progenitors of PMCs as well as fetal and adult LCs, contributing to most of the LCs present in the fetal and adult testis.

SummaryLeydig cells (LCs) are the major androgen-producing cells in the testes. They arise from steroidogenic progenitors, whose origins, maintenance and differentiation dynamics remain largely unknown. Here, we identifiedWnt5aas a specific marker of steroidogenic progenitors, whose expression begins at around E11.5-E12.5 in interstitial cells of the fetal mouse testis.In vivolineage tracing indicates thatWnt5a-expressing progenitors are initially present in large numbers in the fetal testis and then progressively decrease as development progresses. We provide evidence thatWnt5a-expressing cells arebona fideprogenitors of peritubular myoid cells as well as fetal and adult LCs, contributing to most of the LCs present in the fetal and adult testis. Additionally, we show in the adult testis thatWnt5aexpression is restricted to a subset of LCs exhibiting a slow but noticeable clonal expansion, revealing hitherto unappreciated proliferation of fully differentiated LCs as a contribution to the adult LC pool.

Antibiotic resistance is increasing among pathogens, and the human microbiome contains a reservoir of antibiotic resistance genes. Acidaminococcus intestini is the first Negativicute bacterium (Gram-negative Firmicute) shown to be resistant to beta-lactam antibiotics. Resistance is conferred by the aci1 gene, but its evolutionary history and prevalence remain obscure. We discovered that ACI-1 proteins are phylogenetically distinct from beta-lactamases of Gram-positive Firmicutes and that aci1 occurs in bacteria scattered across the Negativicute clade, suggesting lateral gene transfer. In the reference A. intestini RyC-MR95 genome, we found transposons residing within a tailed prophage context are likely vehicles for aci1's mobility. We found aci1 in 56 (4.4%) of 1,267 human gut metagenomes, mostly hosted within A. intestini, and, where could be determined, mostly within a consistent mobile element constellation. These samples are from Europe, China and the USA, showing that aci1 is distributed globally. We found that for most Negativicute assemblies with aci1, the prophage observed in A. instestini is absent, but in all cases aci1 is flanked by varying transposons. The chimeric mobile elements we identify here likely have a complex evolutionary history and potentially provide multiple complementary mechanisms for antibiotic resistance gene transfer both within and between cells.

SummaryDespite the importance of germ cell (GC) differentiation for sexual reproduction, the gene networks underlying their fate remain unclear. Here, we comprehensively characterize the gene expression dynamics during sex determination based on single-cell RNA sequencing of 14,914 XX and XY mouse GCs between embryonic days (E) 9.0 and 16.5. We found that XX and XY GCs diverge transcriptionally as early as E11.5 with upregulation of genes downstream of the Bone morphogenic protein (BMP) and Nodal/Activin pathways in XY and XX GCs, respectively. We also identified a sex-specific upregulation of genes associated with negative regulation of mRNA processing and an increase in intron retention consistent with a reduction in mRNA splicing in XY testicular GCs by E13.5. Using computational gene regulation network inference analysis, we identified sex-specific, sequential waves of putative key regulator genes during GC differentiation and revealed that the meiotic genes are regulated by positive and negative master modules acting in an antagonistic fashion. Finally, we found that rare adrenal GCs enter meiosis similarly to ovarian GCs but display altered expression of master genes controlling the female and male genetic programs, indicating that the somatic environment is important for GC function. Our data is available on a web platform and provides a molecular roadmap of GC sex determination at single-cell resolution, which will serve as a valuable resource for future studies of gonad development, function and disease.

Negativicutes are gram-negative bacteria characterized by two cell membranes, but they are phylogenetically a side-branch of gram-positive Firmicutes that contain only a single membrane. We asked whether viruses (phages) infecting Negativicutes were horizontally acquired from gram-negative Proteobacteria, given the shared outer cell structure of their bacterial hosts, or if Negativicute phages co-evolved vertically with their hosts and thus resemble gram-positive Firmicute prophages. We predicted and characterized 485 prophages (mostly Caudovirales) from gram-negative Firmicute genomes plus 2977 prophages from other bacterial clades, and we used virome sequence data from 183 human stool samples to support our predictions. The majority of identified Negativicute prophages were lambdoids closer related to prophages from other Firmicutes than Proteobacteria by sequence relationship and genome organization (position of the lysis module). Only a single Mu-like candidate prophage and no clear P2-like prophages were identified in Negativicutes, both common in Proteobacteria. Given this collective evidence, it is unlikely that Negativicute phages were acquired from Proteobacteria. Sequence-related prophages, which occasionally harboured antibiotic resistance genes, were identified in two distinct Negativicute orders (Veillonellales and Acidaminococcales), possibly suggesting horizontal cross-order phage infection between human gut commensals. Our results reveal ancient genomic signatures of phage and bacteria co-evolution despite horizontal phage mobilization.

SummaryPhigaro is a standalone command-line application that is able to detect prophage regions taking raw genome and metagenome assemblies as an input. It also produces dynamic annotated “prophage genome maps” and marks possible transposon insertion spots inside prophages. It provides putative taxonomic annotations that can distinguish tailed from non-tailed phages. It is applicable for mining prophage regions from large metagenomic datasets.AvailabilitySource code for Phigaro is freely available for download at https://github.com/bobeobibo/phigaro along with test data. The code is written in Python.

BackgroundImmuno-oncology therapies are now part of the standard of care for cancer in many indications. However, durable objective responses remain limited to a subset of patients. As such, there is a critical need to identify biomarkers that can predict or enrich for treatment response. So far, the majority of putative biomarkers consist of features of the tumor microenvironment (TME). However, in preclinical mouse models, the collection of tumor tissue for this type of analysis is a terminal procedure, obviating the ability to directly link potential biomarkers to long-term treatment outcomes.MethodsTo address this, we developed and validated a novel non-terminal tumor sampling method to enable biopsy of the TME in mouse models based on fine needle aspiration.ResultsWe show that this technique enables repeated in-life sampling of subcutaneous flank tumors and yields sufficient material to support downstream analyses of tumor-infiltrating immune cells using methods such as flow cytometry and single-cell transcriptomics. Moreover, using this technique we demonstrate that we can link TME biomarkers to treatment response outcomes, which is not possible using the current method of terminal tumor sampling.ConclusionThus, this minimally invasive technique is an important refinement for the pharmacodynamic analysis of the TME facilitating paired evaluation of treatment response biomarkers with outcomes and reducing the number of animals used in preclinical research.

Antibiotic resistance is increasing among pathogens at unprecedented rates and the human body contains a large pool of antibiotic resistance genes that can be spread among bacteria by mobile genetic elements. Acidaminococcus intestini, a bacterium found in the human gut that belongs to the class of Negativicutes, is the first gram-negative coccus shown to be resistant to beta-lactam antibiotics. Resistance is conferred by aci1, a gene encoding the ACI-1 class A beta-lactamase, but the evolutionary history of aci1 and its distribution across other Negativicutes and in the human gut microbiota remains obscure. We discovered that ACI-1 proteins are phylogenetically distinct from class A beta-lactamases of gram-positive Firmicutes and that the aci1 gene occurs in bacteria scattered across the Negativicutes clade, suggesting possible mobilization. In the reference A. intestini RyC-MR95 strain, we found that aci1 is surrounded by mobile DNA, transposon derived sequences directly flank aci1 and are likely the vehicle for its mobility. These transposon sequences reside within a prophage context consisting of two likely degraded tailed prophages, the first prophages to be characterised in A. intestini. We found aci1 in at least 56 (4.4%) out of 1,267 human gut metagenome samples, mostly hosted within A. intestini, and, where could be determined, mostly within a similar constellation of mobile elements to that found in the reference A. intestini genome. These human samples are from individuals in Europe, China and the USA, showing that aci1 is widely distributed globally. Additionally, we examined the nine different Negativicute genome assemblies that contain aci1, and found that only two of these strains show a similar mobile element context around aci1 to the reference A. intestini with transposons adjacent to a tailed prophage. However, in all nine cases aci1 is flanked by transposon derived sequences, and these sequences are diverse, suggesting the activity and degradation of multiple transposons. Overall, we show that ACI-1 proteins form a distinct class A beta lactamase family, and that the aci1 gene is present in human guts worldwide within Negativicute bacterial hosts, due to transposons, sometimes inserted into tailed prophages.