Mendelian genetics posits equal transmission of alleles, but selfish alleles can bias the transmission of large genomic regions or entire chromosomes. One long-standing question is how transmission bias evolves to encompass large genomic regions. Mus musculus (house mouse) t-haplotypes exhibit up to 99% transmission bias from heterozygous males and harbor selfish alleles genetically linked to large inversions spanning the proximal half of chromosome 17. Here, by generating a high-quality, single-haplotype assembly of a t-haplotype, we reveal the evolution of eight large amplicons with known and candidate selfish alleles as a distinct genetic feature. Three amplicons are conserved in closely related Mus species, and two have known selfish alleles in the oldest inversion, implicating amplicons and an inversion drove the origins of a selfish chromosome 17 ~3MYA. The remaining t-haplotype amplicons harbor gene families expressed predominantly in haploid spermatids, newly acquired retrogenes, and the most differentially expressed genes in wild-type/t-haplotype spermatids. Targeted deletion of a ~1.8 Mb amplicon with candidate selfish alleles on the t-haplotype reduces selfish transmission in heterozygous males by ~3%. Notably, the evolution of selfish allele-containing amplicons and inversions on the t-haplotype parallels mammalian sex chromosome evolution as signatures of selfish transmission. We propose amplicon acquisition and large inversions initiate evolutionary arms races between selfish haplotypes and serve as genome-wide signatures of selfish transmission.

In animal breeding, female or male progeny is preferred depending on the use of the animals. Especially in livestock, offspring with undesired sex is of low economic value and often culled. Here we achieve targeted sex ratio distortion in the mouse, leveraging components of a selfish supergene, the t -haplotype. Utilizing its central element, the t -responder, we generate a highly “selfless” genetic element disabling transgenic sperm. When integrated on the X or Y chromosome it results in a high prevalence of non-transgenic offspring of the desired sex. Our strategy overcomes key limitations of existing strategies, including compromised animal health, reduced fertility, and fully transgenic breeding stocks, which might foster acceptance of its application to livestock. The efficacy of our approach marks a groundbreaking advancement in animal breeding. ![Figure][1] Graphical abstract Transgene integration on X or Y chromosome affects the motility of transgenic sperm resulting in a strong sex-bias towards non-transgenic/wildtype offspring. Application to livestock can reduce unwanted progeny and transform farm animal breeding. ### Competing Interest Statement BGH, HB and FK have submitted a patent application on the described technology to the European Patent Office. Max Planck Society, https://ror.org/01hhn8329 [1]: pending:yes

Despite a distinct developmental origin, extraembryonic cells in mice contribute to gut endoderm and converge to transcriptionally resemble their embryonic counterparts. Notably, all extraembryonic progenitors share a non-canonical epigenome, raising several pertinent questions, including whether this landscape is reset to match the embryonic regulation and if extraembryonic cells persist into later development. Here we developed a two-colour lineage-tracing strategy to track and isolate extraembryonic cells over time. We find that extraembryonic gut cells display substantial memory of their developmental origin including retention of the original DNA methylation landscape and resulting transcriptional signatures. Furthermore, we show that extraembryonic gut cells undergo programmed cell death and neighbouring embryonic cells clear their remnants via non-professional phagocytosis. By midgestation, we no longer detect extraembryonic cells in the wild-type gut, whereas they persist and differentiate further in p53-mutant embryos. Our study provides key insights into the molecular and developmental fate of extraembryonic cells inside the embryo. Batki, Hetzel et al. report a lineage-tracing strategy to track extraembryonic gut endoderm cells over development. They find that these cells are eventually eliminated in a p53-dependent manner and neighbouring embryonic cells clear their remnants.

Serum response factor (SRF) is a transcription factor essential for cell proliferation, differentiation, and migration and is required for primitive streak and mesoderm formation in the embryo. The canonical roles of SRF are mediated by a diverse set of context-dependent cofactors. Here, we show that SRF physically interacts with CTCF and cohesin subunits at topologically associating domain (TAD) boundaries and loop anchors. SRF promotes long-range chromatin loop formation and contributes to TAD insulation. In embryonic stem cells (ESCs), SRF associates with SOX2 and NANOG and contributes to the formation of three-dimensional (3D) pluripotency hubs. Our findings reveal additional roles of SRF in higher-order chromatin organization.

SOX2 is a transcription factor involved in the regulatory network maintaining the pluripotency of embryonic stem cells in culture as well as in early embryos. In addition, SOX2 plays a pivotal role in neural stem cell formation and neurogenesis. How SOX2 can serve both processes has remained elusive. Here, we identified a set of SOX2-dependent neural-associated enhancers required for neural lineage priming. They form a distinct subgroup (1,898) among 8,531 OCT4/SOX2/NANOG-bound enhancers characterized by enhanced SOX2 binding and chromatin accessibility. Activation of these enhancers is triggered by neural induction of wild-type cells or by default in Smad4-ablated cells resistant to mesoderm induction and is antagonized by mesodermal transcription factors via Sox2 repression. Our data provide mechanistic insight into the transition from the pluripotency state to the early neural fate and into the regulation of early neural versus mesodermal specification in embryonic stem cells and embryos.

Supplementary Figures 1-7. Supplementary Figure 1: Assembly of the PWD genome and alignment to the C57BL/6 genome. Supplementary Figure 2: RNA-seq read assignment. Supplementary Figure 3: MeDIP-seq quality controls. Supplementary Figure 4: Histology and distribution of tumors in B6 x PWD consomic mice. Supplementary Figure 5: Transcriptome analyses identify modifier candidate genes on other chromosomes than chromosome 5. Supplementary Figure 6: Strain-specific DNA methylation on chromosome 5 is correlated to CpG availability. Supplementary Figure 7: Assessment of cell types in the intestinal crypt.

After myocardial infarction in the adult heart the remaining, non-infarcted tissue adapts to compensate the loss of functional tissue. This adaptation requires changes in gene expression networks, which are mostly controlled by transcription regulating proteins. Long non-coding transcripts (lncRNAs) are taking part in fine-tuning such gene programs. We describe and characterize the cardiomyocyte specific lncRNA Sweetheart RNA ( Swhtr ), an approximately 10 kb long transcript divergently expressed from the cardiac core transcription factor coding gene Nkx2-5 . We show that Swhtr is dispensable for normal heart development and function but becomes essential for the tissue adaptation process after myocardial infarction in murine males. Re-expressing Swhtr from an exogenous locus rescues the Swhtr null phenotype. Genes that depend on Swhtr after cardiac stress are significantly occupied and therefore most likely regulated by NKX2-5. The Swhtr transcript interacts with NKX2-5 and disperses upon hypoxic stress in cardiomyocytes, indicating an auxiliary role of Swhtr for NKX2-5 function in tissue adaptation after myocardial injury.

The node and notochord are important signaling centers organizing the dorso-ventral patterning of cells arising from neuro-mesodermal progenitors forming the embryonic body anlage. Owing to the scarcity of notochord progenitors and notochord cells, a comprehensive identification of regulatory elements driving notochord-specific gene expression has been lacking. Here, we have used ATAC-seq analysis of FACS-purified notochord cells from Theiler stage 12-13 mouse embryos to identify 8921 putative notochord enhancers. In addition, we established a new model for generating notochord-like cells in culture, and found 3728 of these enhancers occupied by the essential notochord control factors brachyury (T) and/or Foxa2. We describe the regulatory landscape of the T locus, comprising ten putative enhancers occupied by these factors, and confirmed the regulatory activity of three of these elements. Moreover, we characterized seven new elements by knockout analysis in embryos and identified one new notochord enhancer, termed TNE2. TNE2 cooperates with TNE in the trunk notochord, and is essential for notochord differentiation in the tail. Our data reveal an essential role of Foxa2 in directing T-expressing cells towards the notochord lineage.

Long non-coding RNAs are a very versatile class of molecules that can have important roles in regulating a cells function, including regulating other genes on the transcriptional level. One of these mechanisms is that RNA can directly interact with DNA thereby recruiting additional components such as proteins to these sites via a RNA:dsDNA triplex formation. We genetically deleted the triplex forming sequence (FendrrBox) from the lncRNA Fendrr in mice and find that this FendrrBox is partially required for Fendrr function in vivo. We find that the loss of the triplex forming site in developing lungs causes a dysregulation of gene programs, associated with lung fibrosis. A set of these genes contain a triplex site directly at their promoter and are expressed in fibroblasts. We confirm the formation of RNA:dsDNA formation with target promoters. We find that Fendrr with the Wnt signalling pathway regulates these genes, implicating that Fendrr synergizes with Wnt signalling in lung fibrosis.

ABSTRACT Cardiac lineage specification in the mouse is controlled by TGFβ and WNT signaling. From fly to fish, BMP has been identified as an indispensable heart inducer. A detailed analysis of the role of Bmp4 and its effectors Smad1/5, however, was still missing. We show that Bmp4 induces cardiac mesoderm formation in murine embryonic stem cells in vitro. Bmp4 first activates Wnt3 and upregulates Nodal. pSmad1/5 and the WNT effector Tcf3 form a complex, and together with pSmad2/3 activate mesoderm enhancers and Eomes. They then cooperate with Eomes to consolidate the expression of many mesoderm factors, including T. Eomes and T form a positive- feedback loop and open additional enhancers regulating early mesoderm genes, including the transcription factor Mesp1, establishing the cardiac mesoderm lineage. In parallel, the neural fate is suppressed. Our data confirm the pivotal role of Bmp4 in cardiac mesoderm formation in the mouse. We describe in detail the consecutive and cooperative actions of three signaling pathways, BMP, WNT and Nodal, and their effector transcription factors, during cardiac mesoderm specification.

Supplementary tables 1-7. Supplementary Table 1: GSEA signatures and references (Wnt target genes are taken from the HALLMARK Signature). Supplementary Table 2: 58 differentially expressed genes on chromosome 5, B6 vs C5 APC min normal intestine. Supplementary Table 3: 116 differentially expressed genes not located on chromosome 5, B6 vs C5 APC min normal intestine. Supplementary table 4: Differentially expressed genes on chromosome 5 regulated in cis, trans, or both. Supplementary table 5: Candidate modifier genes differentially expressed between B6 or PWD chromosome 5. Supplementary Table 6: Polymorphisms in human orthologs of mouse genes located on chromosome 5, polymorphic between B6 and PWD, and expressed in the intestine. Supplementary Table 7: PWD polymorphisms on chr5 in genes that are expressed in the intestine and/or in adenoma.

Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) is the most common congenital malformation of the upper digestive tract. This study represents the first genome-wide association study (GWAS) to identify risk loci for EA/TEF. We used a European case-control sample comprising 764 EA/TEF patients and 5,778 controls and observed genome-wide significant associations at three loci. On chromosome 10q21 within the gene CTNNA3 (p = 2.11 x 10(-8); odds ratio [OR] = 3.94; 95% confidence interval [CI], 3.10-5.00), on chromosome 16q24 next to the FOX gene cluster (p = 2.25 x 10(-10); OR = 1.47; 95% CI, 1.38-1.55) and on chromosome 17q12 next to the gene HNF1B (p = 3.35 x 10(-16); OR = 1.75; 95% CI, 1.64-1.87). We next carried out an esophageal/tracheal transcriptome profiling in rat embryos at four selected embryonic time points. Based on these data and on already published data, the implicated genes at all three GWAS loci are promising candidates for EA/TEF development. We also analyzed the genetic EA/TEF architecture beyond the single marker level, which revealed an estimated single-nucleotide polymorphism (SNP)-based heritability of around 37% +/- 14% standard deviation. In addition, we examined the polygenicity of EA/TEF and found that EA/TEF is less polygenic than other complex genetic diseases. In conclusion, the results of our study contribute to a better understanding on the underlying genetic architecture of ET/TEF with the identification of three risk loci and candidate genes.

Post-implantation mammalian embryogenesis involves profound molecular, cellular, and morphogenetic changes. The study of these highly dynamic processes is complicated by the limited accessibility of in utero development. In recent years, several complementary in vitro systems comprising self-organized assemblies of mouse embryonic stem cells, such as gastruloids, have been reported. We recently demonstrated that the morphogenetic potential of gastruloids can be further unlocked by the addition of a low percentage of Matrigel as an extracellular matrix surrogate. This resulted in the formation of highly organized trunk-like structures (TLSs) with a neural tube that is frequently flanked by bilateral somites. Notably, development at the molecular and morphogenetic levels is highly reminiscent of the natural embryo. To facilitate access to this powerful model, here we provide a detailed step-by-step protocol that should allow any lab with access to standard cell culture techniques to implement the culture system. This will provide the user with a means to investigate early mid-gestational mouse embryogenesis at an unprecedented spatiotemporal resolution.

Abstract Genetic predisposition affects the penetrance of tumor-initiating mutations, such as APC mutations that stabilize β-catenin and cause intestinal tumors in mice and humans. However, the mechanisms involved in genetically predisposed penetrance are not well understood. Here, we analyzed tumor multiplicity and gene expression in tumor-prone ApcMin/+ mice on highly variant C57BL/6J (B6) and PWD/Ph (PWD) genetic backgrounds. (B6 × PWD) F1 APCMin offspring mice were largely free of intestinal adenoma, and several chromosome substitution (consomic) strains carrying single PWD chromosomes on the B6 genetic background displayed reduced adenoma numbers. Multiple dosage-dependent modifier loci on PWD chromosome 5 each contributed to tumor suppression. Activation of β-catenin–driven and stem cell–specific gene expression in the presence of ApcMin or following APC loss remained moderate in intestines carrying PWD chromosome 5, suggesting that PWD variants restrict adenoma initiation by controlling stem cell homeostasis. Gene expression of modifier candidates and DNA methylation on chromosome 5 were predominantly cis controlled and largely reflected parental patterns, providing a genetic basis for inheritance of tumor susceptibility. Human SNP variants of several modifier candidates were depleted in colorectal cancer genomes, suggesting that similar mechanisms may also affect the penetrance of cancer driver mutations in humans. Overall, our analysis highlights the strong impact that multiple genetic variants acting in networks can exert on tumor development. Significance: These findings in mice show that, in addition to accidental mutations, cancer risk is determined by networks of individual gene variants.

Mammalian spermatozoa employ calcium (Ca2+) and cyclic adenosine monophosphate (cAMP) signaling in generating flagellar beat. However, how sperm direct their movement towards the egg cells has remained elusive. Here we show that the Rho small G protein RAC1 plays an important role in controlling progressive motility, in particular average path velocity and linearity. Upon RAC1 inhibition of wild type sperm with the drug NSC23766, progressive movement is impaired. Moreover, sperm from mice homozygous for the genetically variant t-haplotype region (tw5/tw32), which are sterile, show strongly enhanced RAC1 activity in comparison to wild type (+/+) controls, and quickly become immotile in vitro. Sperm from heterozygous (t/+) males, on the other hand, display intermediate RAC1 activity, impaired progressive motility and transmission ratio distortion (TRD) in favor of t-sperm. We show that t/+-derived sperm consist of two subpopulations, highly progressive and less progressive. The majority of highly progressive sperm carry the t-haplotype, while most less progressive sperm contain the wild type (+) chromosome. Dosage-controlled RAC1 inhibition in t/+ sperm by NSC23766 rescues progressive movement of (+)-sperm in vitro, directly demonstrating that impairment of progressive motility in the latter is caused by enhanced RAC1 activity. The combined data show that RAC1 plays a pivotal role in controlling progressive motility in sperm, and that inappropriate, enhanced or reduced RAC1 activity interferes with sperm progressive movement. Differential RAC1 activity within a sperm population impairs the competitiveness of sperm cells expressing suboptimal RAC1 activity and thus their fertilization success, as demonstrated by t/+-derived sperm. In conjunction with t-haplotype triggered TRD, we propose that Rho GTPase signaling is essential for directing sperm towards the egg cells.

Mammalian post-implantation development comprises the coordination of complex lineage decisions and morphogenetic processes shaping the embryo. Despite technological advances, a comprehensive understanding of the dynamics of these processes and of the self-organization capabilities of stem cells and their descendants remains elusive. Building synthetic embryo-like structures from pluripotent embryonic stem cells in vitro promises to fill these knowledge gaps and thereby may prove transformative for developmental biology. Initial efforts to model the post-implantation embryo resulted in structures with compromised morphology (gastruloids). Recent approaches employing modified culture media, an extracellular matrix surrogate or extra-embryonic stem cells, however, succeeded in establishing embryo-like architecture. For example, embedding of gastruloids in Matrigel unlocked self-organization into trunk-like structures with bilateral somites and a neural tube-like structure, together with gut tissue and primordial germ cell-like cells. In this review, we describe the currently available models, discuss how these can be employed to acquire novel biological insights, and detail the imminent challenges for improving current models by in vitro engineering.

The node-streak border region comprising notochord progenitor cells (NPCs) at the posterior node and neuro-mesodermal progenitor cells (NMPs) in the adjacent epiblast is the prime organizing center for axial elongation in mouse embryos. The T-box transcription factor brachyury (T) is essential for both formation of the notochord and maintenance of NMPs, and thus is a key regulator of trunk and tail development. The T promoter controlling Texpression in NMPs and nascent mesoderm has been characterized in detail; however, control elements for Texpression in the notochord have not been identified yet. We have generated a series of deletion alleles by CRISPR/Cas9 genome editing in mESCs, and analyzed their effects in mutant mouse embryos. We identified a 37 kb region upstream of T that is essential for notochord function and tailbud outgrowth. Within that region, we discovered a T-binding enhancer required for notochord cell specification and differentiation. Our data reveal a complex regulatory landscape controlling cell type-specific expression and function of T in NMP/nascent mesoderm and node/notochord, allowing proper trunk and tail development.

Post-implantation embryogenesis is a highly dynamic process comprising multiple lineage decisions and morphogenetic changes that are inaccessible to deep analysis in vivo. We found that pluripotent mouse embryonic stem cells (mESCs) form aggregates that upon embedding in an extracellular matrix compound induce the formation of highly organized "trunk-like structures" (TLSs) comprising the neural tube and somites. Comparative single-cell RNA sequencing analysis confirmed that this process is highly analogous to mouse development and follows the same stepwise gene-regulatory program. Tbx6 knockout TLSs developed additional neural tubes mirroring the embryonic mutant phenotype, and chemical modulation could induce excess somite formation. TLSs thus reveal an advanced level of self-organization and provide a powerful platform for investigating post-implantation embryogenesis in a dish.

Oncogenic mutations in KRAS or BRAF are frequent in colorectal cancer and activate the ERK kinase. Here, we find graded ERK phosphorylation correlating with cell differentiation in patient-derived colorectal cancer organoids with and without KRAS mutations. Using reporters, single cell transcriptomics and mass cytometry, we observe cell type-specific phosphorylation of ERK in response to transgenic KRASG12V in mouse intestinal organoids, while transgenic BRAFV600E activates ERK in all cells. Quantitative network modelling from perturbation data reveals that activation of ERK is shaped by cell type-specific MEK to ERK feed forward and negative feedback signalling. We identify dual-specificity phosphatases as candidate modulators of ERK in the intestine. Furthermore, we find that oncogenic KRAS, together with β-Catenin, favours expansion of crypt cells with high ERK activity. Our experiments highlight key differences between oncogenic BRAF and KRAS in colorectal cancer and find unexpected heterogeneity in a signalling pathway with fundamental relevance for cancer therapy.

Transmission ratio distortion (TRD) by the mouse t-haplotype, a variant region on chromosome 17, is a well-studied model of non-Mendelian inheritance. It is characterized by the high transmission ratio (up to 99%) of the t-haplotype from t/+ males to their offspring. TRD is achieved by the exquisite ability of the responder (Tcr) to trigger non-Mendelian inheritance of homologous chromosomes. Several distorters (Tcd1-Tcd4), which act cumulatively, together promote the high transmission ratio of Tcr and the t-haplotype. Molecularly, TRD is brought about by deregulation of Rho signaling pathways via the distorter products, which impair sperm motility, and the t-sperm specific rescue of sperm motility by the responder. The t-sperm thus can reach the egg cells faster than +-sperm and fertilize them. Previously we have shown that the responder function is accomplished by a dominant negative form of sperm motility kinase (SMOKTCR), while the distorter functions are accomplished by the Rho G protein regulators TAGAP, FGD2 and NME3 proposed to function in two oppositely acting pathways. Here we identify the RAC1-specific guanine nucleotide exchange factor TIAM2 as modifier of t-haplotype TRD. Tiam2 is expressed in two isoforms, the full-length (Tiam2l) and a short transcript (Tiam2s). Tiam2s expression from the t-allele is strongly increased compared to the wild-type allele. By transgenic approaches we show that Tiam2s enhances t-haplotype transmission, while Tiam2l has the opposite effect. Our data show that a single modifier locus can encode different gene products exerting opposite effects on a trait. They also suggest that the expression ratio of the isoforms determines if the outcome is an enhancing or a suppressive effect on the trait.