A degron-based approach to manipulate Eomes functions in the context of the developing mouse embryo.

The T-box transcription factor Eomesodermin (Eomes), also known as Tbr2, plays essential roles in the early mouse embryo. Loss-of-function mutant embryos arrest at implantation due to Eomes requirements in the trophectoderm cell lineage. Slightly later, expression in the visceral endoderm promotes anterior visceral endoderm formation and anterior-posterior axis specification. Early induction in the epiblast beginning at day 6 is necessary for nascent mesoderm to undergo epithelial to mesenchymal transition (EMT). Eomes acts in a temporally and spatially restricted manner to sequentially specify the yolk sac haemogenic endothelium, cardiac mesoderm, definitive endoderm, and axial mesoderm progenitors during gastrulation. Little is known about the underlying molecular mechanisms governing Eomes actions during the formation of these distinct progenitor cell populations. Here, we introduced a degron-tag and mCherry reporter sequence into the Eomes locus. Our experiments analyzing homozygously tagged embryonic stem cells and embryos demonstrate that the degron-tagged Eomes protein is fully functional. dTAG (degradation fusion tag) treatment in vitro results in rapid protein degradation and recapitulates the Eomes-null phenotype. However in utero administration of dTAG resulted in variable and lineage-specific degradation, likely reflecting diverse cell type-specific Eomes expression dynamics. Finally, we demonstrate that Eomes protein rapidly recovers following dTAG wash-out in vitro. The ability to temporally manipulate Eomes protein expression in combination with cell marking by the mCherry-reporter offers a powerful tool for dissecting Eomes-dependent functional roles in these diverse cell types in the early embryo.

Lhx1 functions together with Otx2, Foxa2, and Ldb1 to govern anterior mesendoderm, node, and midline development.

Gene regulatory networks controlling functional activities of spatially and temporally distinct endodermal cell populations in the early mouse embryo remain ill defined. The T-box transcription factor Eomes, acting downstream from Nodal/Smad signals, directly activates the LIM domain homeobox transcription factor Lhx1 in the visceral endoderm. Here we demonstrate Smad4/Eomes-dependent Lhx1 expression in the epiblast marks the entire definitive endoderm lineage, the anterior mesendoderm, and midline progenitors. Conditional inactivation of Lhx1 disrupts anterior definitive endoderm development and impedes node and midline morphogenesis in part due to severe disturbances in visceral endoderm displacement. Transcriptional profiling and ChIP-seq (chromatin immunoprecipitation [ChIP] followed by high-throughput sequencing) experiments identified Lhx1 target genes, including numerous anterior definitive endoderm markers and components of the Wnt signaling pathway. Interestingly, Lhx1-binding sites were enriched at enhancers, including the Nodal-proximal epiblast enhancer element and enhancer regions controlling Otx2 and Foxa2 expression. Moreover, in proteomic experiments, we characterized a complex comprised of Lhx1, Otx2, and Foxa2 as well as the chromatin-looping protein Ldb1. These partnerships cooperatively regulate development of the anterior mesendoderm, node, and midline cell populations responsible for establishment of the left-right body axis and head formation.

Functional characterisation of cis-regulatory elements governing dynamic Eomes expression in the early mouse embryo.

The T-box transcription factor (TF) Eomes is a key regulator of cell fate decisions during early mouse development. The cis-acting regulatory elements that direct expression in the anterior visceral endoderm (AVE), primitive streak (PS) and definitive endoderm (DE) have yet to be defined. Here, we identified three gene-proximal enhancer-like sequences (PSE_a, PSE_b and VPE) that faithfully activate tissue-specific expression in transgenic embryos. However, targeted deletion experiments demonstrate that PSE_a and PSE_b are dispensable, and only VPE is required for optimal Eomes expression in vivo Embryos lacking this enhancer display variably penetrant defects in anterior-posterior axis orientation and DE formation. Chromosome conformation capture experiments reveal VPE-promoter interactions in embryonic stem cells (ESCs), prior to gene activation. The locus resides in a large (500 kb) pre-formed compartment in ESCs and activation during DE differentiation occurs in the absence of 3D structural changes. ATAC-seq analysis reveals that VPE, PSE_a and four additional putative enhancers display increased chromatin accessibility in DE that is associated with Smad2/3 binding coincident with transcriptional activation. By contrast, activation of the Eomes target genes Foxa2 and Lhx1 is associated with higher order chromatin reorganisation. Thus, diverse regulatory mechanisms govern activation of lineage specifying TFs during early development.

Blimp1/Prdm1 governs terminal differentiation of endovascular trophoblast giant cells and defines multipotent progenitors in the developing placenta.

Developmental arrest of Blimp1/Prdm1 mutant embryos at around embryonic day 10.5 (E10.5) has been attributed to placental disturbances. Here we investigate Blimp1/Prdm1 requirements in the trophoblast cell lineage. Loss of function disrupts specification of the invasive spiral artery-associated trophoblast giant cells (SpA-TGCs) surrounding maternal blood vessels and severely compromises the ability of the spongiotrophoblast layer to expand appropriately, secondarily causing collapse of the underlying labyrinth layer. Additionally, we identify a population of proliferating Blimp1(+) diploid cells present within the spongiotrophoblast layer. Lineage tracing experiments exploiting a novel Prdm1.Cre-LacZ allele demonstrate that these Blimp1(+) cells give rise to the mature SpA-TGCs, canal TGCs, and glycogen trophoblasts. In sum, the transcriptional repressor Blimp1/Prdm1 is required for terminal differentiation of SpA-TGCs and defines a lineage-restricted progenitor cell population contributing to placental growth and morphogenesis.

The transcriptional repressor Blimp1 is expressed in rare luminal progenitors and is essential for mammary gland development.

Mammary gland morphogenesis depends on a tight balance between cell proliferation, differentiation and apoptosis, to create a defined functional hierarchy within the epithelia. The limited availability of stem cell/progenitor markers has made it challenging to decipher lineage relationships. Here, we identify a rare subset of luminal progenitors that express the zinc finger transcriptional repressor Blimp1, and demonstrate that this subset of highly clonogenic luminal progenitors is required for mammary gland development. Conditional inactivation experiments using K14-Cre and WAPi-Cre deleter strains revealed essential functions at multiple developmental stages. Thus, Blimp1 regulates proliferation, apoptosis and alveolar cell maturation during puberty and pregnancy. Loss of Blimp1 disrupts epithelial architecture and lumen formation both in vivo and in three-dimensional (3D) primary cell cultures. Collectively, these results demonstrate that Blimp1 is required to maintain a highly proliferative luminal subset necessary for mammary gland development and homeostasis.

Blimp1/Prdm1 Functions in Opposition to Irf1 to Maintain Neonatal Tolerance during Postnatal Intestinal Maturation.

The neonatal intestine is a very complex and dynamic organ that must rapidly adapt and remodel in response to a barrage of environmental stimuli during the first few postnatal weeks. Recent studies demonstrate that the zinc finger transcriptional repressor Blimp1/Prdm1 plays an essential role governing postnatal reprogramming of intestinal enterocytes during this period. Functional loss results in global changes in gene expression patterns, particularly in genes associated with metabolic function. Here we engineered a knock-in allele expressing an eGFP-tagged fusion protein under control of the endogenous regulatory elements and performed genome wide ChIP-seq analysis to identify direct Blimp1 targets and further elucidate the function of Blimp1 in intestinal development. Comparison with published human and mouse datasets revealed a highly conserved core set of genes including interferon-inducible promoters. Here we show that the interferon-inducible transcriptional activator Irf1 is constitutively expressed throughout fetal and postnatal intestinal epithelium development. ChIP-seq demonstrates closely overlapping Blimp1 and Irf1 peaks at key components of the MHC class I pathway in fetal enterocytes. The onset of MHC class I expression coincides with down-regulated Blimp1 expression during the suckling to weaning transition. Collectively, these experiments strongly suggest that in addition to regulating the enterocyte metabolic switch, Blimp1 functions as a gatekeeper in opposition to Irf1 to prevent premature activation of the MHC class I pathway in villus epithelium to maintain tolerance in the neonatal intestine.

The transcriptional repressor Blimp1/Prdm1 regulates postnatal reprogramming of intestinal enterocytes.

Female mammals produce milk to feed their newborn offspring before teeth develop and permit the consumption of solid food. Intestinal enterocytes dramatically alter their biochemical signature during the suckling-to-weaning transition. The transcriptional repressor Blimp1 is strongly expressed in immature enterocytes in utero, but these are gradually replaced by Blimp1(-) crypt-derived adult enterocytes. Here we used a conditional inactivation strategy to eliminate Blimp1 function in the developing intestinal epithelium. There was no noticeable effect on gross morphology or formation of mature cell types before birth. However, survival of mutant neonates was severely compromised. Transcriptional profiling experiments reveal global changes in gene expression patterns. Key components of the adult enterocyte biochemical signature were substantially and prematurely activated. In contrast, those required for processing maternal milk were markedly reduced. Thus, we conclude Blimp1 governs the developmental switch responsible for postnatal intestinal maturation.

The T-box transcription factor Eomesodermin governs haemogenic competence of yolk sac mesodermal progenitors.

Extra-embryonic mesoderm (ExM)-composed of the earliest cells that traverse the primitive streak-gives rise to the endothelium as well as haematopoietic progenitors in the developing yolk sac. How a specific subset of ExM becomes committed to a haematopoietic fate remains unclear. Here we demonstrate using an embryonic stem cell model that transient expression of the T-box transcription factor Eomesodermin (Eomes) governs haemogenic competency of ExM. Eomes regulates the accessibility of enhancers that the transcription factor stem cell leukaemia (SCL) normally utilizes to specify primitive erythrocytes and is essential for the normal development of Runx1+ haemogenic endothelium. Single-cell RNA sequencing suggests that Eomes loss of function profoundly blocks the formation of blood progenitors but not specification of Flk-1+ haematoendothelial progenitors. Our findings place Eomes at the top of the transcriptional hierarchy regulating early blood formation and suggest that haemogenic competence is endowed earlier during embryonic development than was previously appreciated.

The transcriptional repressor Blimp1/PRDM1 regulates the maternal decidual response in mice.

The transcriptional repressor Blimp1 controls cell fate decisions in the developing embryo and adult tissues. Here we describe Blimp1 expression and functional requirements within maternal uterine tissues during pregnancy. Expression is robustly up-regulated at early post-implantation stages in the primary decidual zone (PDZ) surrounding the embryo. Conditional inactivation results in defective formation of the PDZ barrier and abnormal trophectoderm invasion. RNA-Seq analysis demonstrates down-regulated expression of genes involved in cell adhesion and markers of decidualisation. In contrast, genes controlling immune responses including IFNγ are up-regulated. ChIP-Seq experiments identify candidate targets unique to the decidua as well as those shared across diverse cell types including a highly conserved peak at the Csf-1 gene promoter. Interestingly Blimp1 inactivation results in up-regulated Csf1 expression and macrophage recruitment into maternal decidual tissues. These results identify Blimp1 as a critical regulator of tissue remodelling and maternal tolerance during early stages of pregnancy.

Ventral closure, headfold fusion and definitive endoderm migration defects in mouse embryos lacking the fibronectin leucine-rich transmembrane protein FLRT3.

The three fibronectin leucine-rich repeat transmembrane (FLRT) proteins contain 10 leucine-rich repeats (LRR), a type III fibronectin (FN) domain, followed by the transmembrane region, and a short cytoplasmic tail. XFLRT3, a Nodal/TGFbeta target, regulates cell adhesion and modulates FGF signalling during Xenopus gastrulation. The present study describes the onset and pattern of FLRT1-3 expression in the early mouse embryo. FLRT3 expression is activated in the anterior visceral endoderm (AVE), and during gastrulation appears in anterior streak derivatives namely the node, notochord and the emerging definitive endoderm. To explore FLRT3 function we generated a null allele via gene targeting. Early Nodal activities required for anterior-posterior (A-P) patterning, primitive streak formation and left-right (L-R) axis determination were unperturbed. However, FLRT3 mutant embryos display defects in headfold fusion, definitive endoderm migration and a failure of the lateral edges of the ventral body wall to fuse, leading to cardia bifida. Surprisingly, the mutation has no effect on FGF signalling. Collectively these experiments demonstrate that FLRT3 plays a key role in controlling cell adhesion and tissue morphogenesis in the developing mouse embryo.

Smad4-dependent pathways control basement membrane deposition and endodermal cell migration at early stages of mouse development.

BACKGROUND: Smad4 mutant embryos arrest shortly after implantation and display a characteristic shortened proximodistal axis, a significantly reduced epiblast, as well as a thickened visceral endoderm layer. Conditional rescue experiments demonstrate that bypassing the primary requirement for Smad4 in the extra-embryonic endoderm allows the epiblast to gastrulate. Smad4-independent TGF-beta signals are thus sufficient to promote mesoderm formation and patterning. To further analyse essential Smad4 activities contributed by the extra-embryonic tissues, and characterise Smad4 dependent pathways in the early embryo, here we performed transcriptional profiling of Smad4 null embryonic stem (ES) cells and day 4 embryoid bodies (EBs). RESULTS: Transcripts from wild-type versus Smad4 null ES cells and day 4 EBs were analysed using Illumina arrays. In addition to several known TGF-beta/BMP target genes, we identified numerous Smad4-dependent transcripts that are mis-expressed in the mutants. As expected, mesodermal cell markers were dramatically down-regulated. We also observed an increase in non-canonical potency markers (Pramel7, Tbx3, Zscan4), germ cell markers (Aire, Tuba3a, Dnmt3l) as well as early endoderm markers (Dpp4, H19, Dcn). Additionally, expression of the extracellular matrix (ECM) remodelling enzymes Mmp14 and Mmp9 was decreased in Smad4 mutant ES and EB populations. These changes, in combination with increased levels of laminin alpha1, cause excessive basement membrane deposition. Similarly, in the context of the Smad4 null E6.5 embryos we observed an expanded basement membrane (BM) associated with the thickened endoderm layer. CONCLUSION: Smad4 functional loss results in a dramatic shift in gene expression patterns and in the endodermal cell lineage causes an excess deposition of, or an inability to breakdown and remodel, the underlying BM layer. These structural abnormalities probably disrupt reciprocal signalling between the epiblast and overlying visceral endoderm required for gastrulation.

Genetic dissection of Nodal and Bmp signalling requirements during primordial germ cell development in mouse.

The essential roles played by Nodal and Bmp signalling during early mouse development have been extensively documented. Here we use conditional deletion strategies to investigate functional contributions made by Nodal, Bmp and Smad downstream effectors during primordial germ cell (PGC) development. We demonstrate that Nodal and its target gene Eomes provide early instructions during formation of the PGC lineage. We discover that Smad2 inactivation in the visceral endoderm results in increased numbers of PGCs due to an expansion of the PGC niche. Smad1 is required for specification, whereas in contrast Smad4 controls the maintenance and migration of PGCs. Additionally we find that beside Blimp1, down-regulated phospho-Smad159 levels also distinguishes PGCs from their somatic neighbours so that emerging PGCs become refractory to Bmp signalling that otherwise promotes mesodermal development in the posterior epiblast. Thus balanced Nodal/Bmp signalling cues regulate germ cell versus somatic cell fate decisions in the early posterior epiblast.

Mice develop normally in the absence of Smad4 nucleocytoplasmic shuttling.

Smad4 in partnership with R-Smads (receptor-regulated Smads) activates TGF-beta (transforming growth factor-beta)-dependent signalling pathways essential for early mouse development. Smad4 null embryos die shortly after implantation due to severe defects in cell proliferation and visceral endoderm differentiation. In the basal state, Smad4 undergoes continuous shuttling between the cytoplasm and the nucleus due to the combined activities of an N-terminal NLS (nuclear localization signal) and an NES (nuclear export signal) located in its linker region. Cell culture experiments suggest that Smad4 nucleocytoplasmic shuttling plays an important role in TGF-beta signalling. In the present study we have investigated the role of Smad4 shuttling in vivo using gene targeting to engineer two independent mutations designed to eliminate Smad4 nuclear export. As predicted this results in increased levels of Smad4 in the nucleus of homozygous ES cells (embryonic stem cells) and primary keratinocytes, in the presence or absence of ligand. Neither mutation affects Smad4 expression levels nor its ability to mediate transcriptional activation in homozygous cell lines. Remarkably mouse mutants lacking the Smad4 NES develop normally. Smad4 NES mutants carrying one copy of a Smad4 null allele also fail to display developmental defects. The present study clearly demonstrates that Smad4 nucleocytoplasmic shuttling is not required for embryonic development or tissue homoeostasis in normal, healthy adult mice.

Publisher Correction: Single-cell RNA-seq reveals cell type-specific transcriptional signatures at the maternal-foetal interface during pregnancy.

This corrects the article DOI: 10.1038/ncomms11414.

Blimp-1/PRDM1 is a critical regulator of Type III Interferon responses in mammary epithelial cells.

The transcriptional repressor Blimp-1 originally cloned as a silencer of type I interferon (IFN)-ß gene expression controls cell fate decisions in multiple tissue contexts. Conditional inactivation in the mammary gland was recently shown to disrupt epithelial cell architecture. Here we report that Blimp-1 regulates expression of viral defense, IFN signaling and MHC class I pathways, and directly targets the transcriptional activator Stat1. Blimp-1 functional loss in 3D cultures of mammary epithelial cells (MECs) results in accumulation of dsRNA and expression of type III IFN-λ. Cultures treated with IFN lambda similarly display defective lumen formation. These results demonstrate that type III IFN-λ profoundly influences the behavior of MECs and identify Blimp-1 as a critical regulator of IFN signaling cascades.

Combinatorial Smad2/3 Activities Downstream of Nodal Signaling Maintain Embryonic/Extra-Embryonic Cell Identities during Lineage Priming.

Epiblast cells in the early post-implantation stage mammalian embryo undergo a transition described as lineage priming before cell fate allocation, but signaling pathways acting upstream remain ill defined. Genetic studies demonstrate that Smad2/3 double-mutant mouse embryos die shortly after implantation. To learn more about the molecular disturbances underlying this abrupt failure, here we characterized Smad2/3-deficient embryonic stem cells (ESCs). We found that Smad2/3 double-knockout ESCs induced to form epiblast-like cells (EpiLCs) display changes in naive and primed pluripotency marker gene expression, associated with the disruption of Oct4-bound distal regulatory elements. In the absence of Smad2/3, we observed enhanced Bmp target gene expression and de-repression of extra-embryonic gene expression. Cell fate allocation into all three embryonic germ layers is disrupted. Collectively, these experiments demonstrate that combinatorial Smad2/3 functional activities are required to maintain distinct embryonic and/or extra-embryonic cell identity during lineage priming in the epiblast before gastrulation.

Mapping the chromatin landscape and Blimp1 transcriptional targets that regulate trophoblast differentiation.

Trophoblast stem cells (TSCs) give rise to specialized cell types within the placenta. However, the regulatory mechanisms that guide trophoblast cell fate decisions during placenta development remain ill defined. Here we exploited ATAC-seq and transcriptional profiling strategies to describe dynamic changes in gene expression and chromatin accessibility during TSC differentiation. We detect significantly increased chromatin accessibility at key genes upregulated as TSCs exit from the stem cell state. However, downregulated gene expression is not simply due to the loss of chromatin accessibility in proximal regions. Additionally, transcriptional targets recognized by the zinc finger transcriptional repressor Prdm1/Blimp1, an essential regulator of placenta development, were identified in ChIP-seq experiments. Comparisons with previously reported ChIP-seq datasets for primordial germ cell-like cells and E18.5 small intestine, combined with functional annotation analysis revealed that Blimp1 has broadly shared as well as cell type-specific functional activities unique to the trophoblast lineage. Importantly, Blimp1 not only silences TSC gene expression but also prevents aberrant activation of divergent developmental programmes. Overall the present study provides new insights into the chromatin landscape and Blimp1-dependent regulatory networks governing trophoblast gene expression.

Long-lived unipotent Blimp1-positive luminal stem cells drive mammary gland organogenesis throughout adult life.

The hierarchical relationships between various stem and progenitor cell subpopulations driving mammary gland morphogenesis and homoeostasis are poorly understood. Conditional inactivation experiments previously demonstrated that expression of the zinc finger transcriptional repressor Blimp1/PRDM1 is essential for the establishment of epithelial cell polarity and functional maturation of alveolar cells. Here we exploit a Prdm1.CreERT2-LacZ reporter allele for lineage tracing experiments. Blimp1 expression marks a rare subpopulation of unipotent luminal stem cells that initially appear in the embryonic mammary gland at around E17.5 coincident with the segregation of the luminal and basal compartments. Fate mapping at multiple time points in combination with whole-mount confocal imaging revealed these long-lived unipotent luminal stem cells survive consecutive involutions and retain their identity throughout adult life. Blimp1+ luminal stem cells give rise to Blimp1- progeny that are invariably Elf5+ERα-PR-. Thus, Blimp1 expression defines a mammary stem cell subpopulation with unique functional characteristics.

Single-cell RNA-seq reveals cell type-specific transcriptional signatures at the maternal-foetal interface during pregnancy.

Growth and survival of the mammalian embryo within the uterine environment depends on the placenta, a highly complex vascularized organ comprised of both maternal and foetal tissues. Recent experiments demonstrate that the zinc finger transcriptional repressor Prdm1/Blimp1 is essential for specification of spiral artery trophoblast giant cells (SpA-TGCs) that invade and remodel maternal blood vessels. To learn more about functional contributions made by Blimp1+ cell lineages here we perform the first single-cell RNA-seq analysis of the placenta. Cell types of both foetal and maternal origin are profiled. Comparisons with microarray datasets from mutant placenta and in vitro differentiated trophoblast stem cells allow us to identify Blimp1-dependent transcripts enriched in SpA-TGCs. Our experiments provide new insights into the functionally distinct cell types present at the maternal-foetal interface and advance our knowledge of dynamic gene expression patterns controlling placental morphogenesis and vascular mimicry.