Extraembryonic gut endoderm cells undergo programmed cell death during development.

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.

Early neural specification of stem cells is mediated by a set of SOX2-dependent neural-associated enhancers.

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.

The lncRNA Sweetheart regulates compensatory cardiac hypertrophy after myocardial injury in murine males.

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.

Genome-wide identification of notochord enhancers comprising the regulatory landscape of the brachyury locus in mouse.

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.

Fendrr synergizes with Wnt signalling to regulate fibrosis related genes during lung development via its RNA:dsDNA triplex element.

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 an RNA:dsDNA triplex formation. We genetically deleted the triplex forming sequence (FendrrBox) from the lncRNA Fendrr in mice and found that this FendrrBox is partially required for Fendrr function in vivo. We found 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 lung fibroblasts. We biophysically confirmed the formation of an RNA:dsDNA triplex with target promoters in vitro. We found that Fendrr with the Wnt signalling pathway regulates these genes, implicating that Fendrr synergizes with Wnt signalling in lung fibrosis.

BMP4 triggers regulatory circuits specifying the cardiac mesoderm lineage.

Cardiac lineage specification in the mouse is controlled by TGFß 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.

First genome-wide association study of esophageal atresia identifies three genetic risk loci at CTNNA3, FOXF1/FOXC2/FOXL1, and HNF1B.

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 × 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 × 10-10; OR = 1.47; 95% CI, 1.38-1.55) and on chromosome 17q12 next to the gene HNF1B (p = 3.35 × 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.

A 37†kb region upstream of brachyury comprising a notochord enhancer is essential for notochord and tail development.

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 T expression in NMPs and nascent mesoderm has been characterized in detail; however, control elements for T expression 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.

Generation of Mouse Pluripotent Stem Cell-derived Trunk-like Structures: An in vitro Model of Post-implantation Embryogenesis.

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.

RAC1 controls progressive movement and competitiveness of mammalian spermatozoa.

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.

Modeling mammalian trunk development in a dish.

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.

PWD/Ph-Encoded Genetic Variants Modulate the Cellular Wnt/ß-Catenin Response to Suppress Apc Min-Triggered Intestinal Tumor Formation.

Genetic predisposition affects the penetrance of tumor-initiating mutations, such as APC mutations that stabilize ß-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 Apc Min/+ mice on highly variant C57BL/6J (B6) and PWD/Ph (PWD) genetic backgrounds. (B6 × PWD) F1 APC Min 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 ß-catenin-driven and stem cell-specific gene expression in the presence of Apc Min 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.

Mouse embryonic stem cells self-organize into trunk-like structures with neural tube and somites.

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.

Cell type-dependent differential activation of ERK by oncogenic KRAS in colon cancer and intestinal epithelium.

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 ß-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.

Two isoforms of the RAC-specific guanine nucleotide exchange factor TIAM2 act oppositely on transmission ratio distortion by the mouse t-haplotype.

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.

Exome sequencing in syndromic brain malformations identifies novel mutations in ACTB, and SLC9A6, and suggests BAZ1A as a new candidate gene.

BACKGROUND: Syndromic brain malformations comprise a large group of anomalies with a birth prevalence of about 1 in 1,000 live births. Their etiological factors remain largely unknown. To identify causative mutations, we used whole-exome sequencing (WES) in aborted fetuses and children with syndromic brain malformations in which chromosomal microarray analysis was previously unremarkable. METHODS: WES analysis was applied in eight case-parent trios, six aborted fetuses, and two children. RESULTS: WES identified a novel de novo mutation (p.Gly268Arg) in ACTB (Baraitser-Winter syndrome-1), a homozygous stop mutation (p.R2442*) in ASPM (primary microcephaly type 5), and a novel hemizygous X-chromosomal mutation (p.I250V) in SLC9A6 (X-linked syndromic mentaly retardation, Christianson type). Furthermore, WES identified a de novo mutation (p.Arg1093Gln) in BAZ1A. This mutation was previously reported in only one allele in 121.362 alleles tested (dbSNP build 147). BAZ1A has been associated with neurodevelopmental impairment and dysregulation of several pathways including vitamin D metabolism. Here, serum vitamin-D (25-(OH)D) levels were insufficient and gene expression comparison between the child and her parents identified 27 differentially expressed genes. Of note, 10 out of these 27 genes are associated to cytoskeleton, integrin and synaptic related pathways, pinpointing to the relevance of BAZ1A in neural development. In situ hybridization in mouse embryos between E10.5 and E13.5 detected Baz1a expression in the central and peripheral nervous system. CONCLUSION: In syndromic brain malformations, WES is likely to identify causative mutations when chromosomal microarray analysis is unremarkable. Our findings suggest BAZ1A as a possible new candidate gene.

BRACHYURY directs histone acetylation to target loci during mesoderm development.

T-box transcription factors play essential roles in multiple aspects of vertebrate development. Here, we show that cooperative function of BRACHYURY (T) with histone-modifying enzymes is essential for mouse embryogenesis. A single point mutation (TY88A) results in decreased histone 3 lysine 27 acetylation (H3K27ac) at T target sites, including the T locus, suggesting that T autoregulates the maintenance of its expression and functions by recruiting permissive chromatin modifications to putative enhancers during mesoderm specification. Our data indicate that T mediates H3K27ac recruitment through a physical interaction with p300. In addition, we determine that T plays a prominent role in the specification of hematopoietic and endothelial cell types. Hematopoietic and endothelial gene expression programs are disrupted in TY88A mutant embryos, leading to a defect in the differentiation of hematopoietic progenitors. We show that this role of T is mediated, at least in part, through activation of a distal Lmo2 enhancer.

Antagonistic Activities of Sox2 and Brachyury Control the Fate Choice of Neuro-Mesodermal Progenitors.

The spinal cord and mesodermal tissues of the trunk such as the vertebral column and skeletal musculature derive from neuro-mesodermal progenitors (NMPs). Sox2, Brachyury (T), and Tbx6 have been correlated with NMP potency and lineage choice; however, their exact role and interaction in these processes have not yet been revealed. Here we present a global analysis of NMPs and their descending lineages performed on purified cells from embryonic day 8.5 wild-type and mutant embryos. We show that T, cooperatively with WNT signaling, controls the progenitor state and the switch toward the mesodermal fate. Sox2 acts antagonistically and promotes neural development. T is also involved in remodeling the chromatin for mesodermal development. Tbx6 reinforces the mesodermal fate choice, represses the progenitor state, and confers paraxial fate commitment. Our findings refine previous models and establish molecular principles underlying mammalian trunk development, comprising NMP maintenance, lineage choice, and mesoderm formation.

Patterning and gastrulation defects caused by the tw18 lethal are due to loss of Ppp2r1a.

The mouse t haplotype, a variant 20 cM genomic region on Chromosome 17, harbors 16 embryonic control genes identified by recessive lethal mutations isolated from wild mouse populations. Due to technical constraints so far only one of these, the tw5 lethal, has been cloned and molecularly characterized. Here we report the molecular isolation of the tw18 lethal. Embryos carrying the tw18 lethal die from major gastrulation defects commencing with primitive streak formation at E6.5. We have used transcriptome and marker gene analyses to describe the molecular etiology of the tw18 phenotype. We show that both WNT and Nodal signal transduction are impaired in the mutant epiblast, causing embryonic patterning defects and failure of primitive streak and mesoderm formation. By using a candidate gene approach, gene knockout by homologous recombination and genetic rescue, we have identified the gene causing the tw18 phenotype as Ppp2r1a, encoding the PP2A scaffolding subunit PR65alpha. Our work highlights the importance of phosphatase 2A in embryonic patterning, primitive streak formation, gastrulation, and mesoderm formation downstream of WNT and Nodal signaling.

Oncogenic ß-catenin and PIK3CA instruct network states and cancer phenotypes in intestinal organoids.

Colorectal cancer is driven by cooperating oncogenic mutations. In this study, we use organotypic cultures derived from transgenic mice inducibly expressing oncogenic ß-catenin and/or PIK3CAH1047R to follow sequential changes in cancer-related signaling networks, intestinal cell metabolism, and physiology in a three-dimensional environment mimicking tissue architecture. Activation of ß-catenin alone results in the formation of highly clonogenic cells that are nonmotile and prone to undergo apoptosis. In contrast, coexpression of stabilized ß-catenin and PIK3CAH1047R gives rise to intestinal cells that are apoptosis-resistant, proliferative, stem cell-like, and motile. Systematic inhibitor treatments of organoids followed by quantitative phenotyping and phosphoprotein analyses uncover key changes in the signaling network topology of intestinal cells after induction of stabilized ß-catenin and PIK3CAH1047R We find that survival and motility of organoid cells are associated with 4EBP1 and AKT phosphorylation, respectively. Our work defines phenotypes, signaling network states, and vulnerabilities of transgenic intestinal organoids as a novel approach to understanding oncogene activities and guiding the development of targeted therapies.