Transcriptional remodeling by OTX2 directs specification and patterning of mammalian definitive endoderm.

The molecular mechanisms that drive essential developmental patterning events in the mammalian embryo remain poorly understood. To generate a conceptual framework for gene regulatory processes during germ layer specification, we analyzed transcription factor (TF) expression kinetics around gastrulation and during in vitro differentiation. This approach identified Otx2 as a candidate regulator of definitive endoderm (DE), the precursor of all gut- derived tissues. Analysis of multipurpose degron alleles in gastruloid and directed differentiation models revealed that loss of OTX2 before or after DE specification alters the expression of core components and targets of specific cellular signaling pathways, perturbs adhesion and migration programs as well as de-represses regulators of other lineages, resulting in impaired foregut specification. Key targets of OTX2 are conserved in human DE. Mechanistically, OTX2 is required to establish chromatin accessibility at candidate enhancers, which regulate genes critical to establishing an anterior cell identity in the developing gut. Our results provide a working model for the progressive establishment of spatiotemporal cell identity by developmental TFs across germ layers and species, which may facilitate the generation of gut cell types for regenerative medicine applications.

Integration of spatial and single-cell transcriptomic data elucidates mouse organogenesis.

Molecular profiling of single cells has advanced our knowledge of the molecular basis of development. However, current approaches mostly rely on dissociating cells from tissues, thereby losing the crucial spatial context of regulatory processes. Here, we apply an image-based single-cell transcriptomics method, sequential fluorescence in situ hybridization (seqFISH), to detect mRNAs for 387 target genes in tissue sections of mouse embryos at the 8-12 somite stage. By integrating spatial context and multiplexed transcriptional measurements with two single-cell transcriptome atlases, we characterize cell types across the embryo and demonstrate that spatially resolved expression of genes not profiled by seqFISH can be imputed. We use this high-resolution spatial map to characterize fundamental steps in the patterning of the midbrain-hindbrain boundary (MHB) and the developing gut tube. We uncover axes of cell differentiation that are not apparent from single-cell RNA-sequencing (scRNA-seq) data, such as early dorsal-ventral separation of esophageal and tracheal progenitor populations in the gut tube. Our method provides an approach for studying cell fate decisions in complex tissues and development.

Capturing Identity and Fate Ex Vivo: Stem Cells from the Mouse Blastocyst.

During mouse preimplantation development, three molecularly, morphologically, and spatially distinct lineages are formed, the embryonic epiblast, the extraembryonic primitive endoderm, and the trophectoderm. Stem cell lines representing each of these lineages have now been derived and can be indefinitely maintained and expanded in culture, providing an unlimited source of material to study the interplay of tissue-specific transcription factors and signaling pathways involved in these fundamental cell fate decisions. Here we outline our current understanding of the derivation, maintenance, and properties of these in vitro stem cell models representing the preimplantation embryonic lineages.

Calcium/NFAT signalling promotes early nephrogenesis.

A number of Wnt genes are expressed during, and are known to be essential for, early kidney development. It is typically assumed that their products will act through the canonical ß-catenin signalling pathway. We have found evidence that suggests canonical Wnt signalling is not active in the early nephrogenic metanephric mesenchyme, but instead provide expressional and functional evidence that implicates the non-canonical Calcium/NFAT Wnt signalling pathway in nephrogenesis. Members of the NFAT (Nuclear Factor Activated in T cells) transcription factor gene family are expressed throughout murine kidney morphogenesis and NFATc3 is localised to the developing nephrons. Treatment of kidney rudiments with Cyclosporin A (CSA), an inhibitor of Calcium/NFAT signalling, decreases nephron formation--a phenotype similar to that in Wnt4(-/-) embryos. Treatment of Wnt4(-/-) kidneys with Ionomycin, an activator of the pathway, partially rescues the phenotype. We propose that the non-canonical Calcium/NFAT Wnt signalling pathway plays an important role in early mammalian renal development and is required for complete MET during nephrogenesis, potentially acting downstream of Wnt4.

An X-linked GFP transgene reveals unexpected paternal X-chromosome activity in trophoblastic giant cells of the mouse placenta.

A GFP transgene has been integrated on the proximal part of the mouse X chromosome just distal of Timp and Syn1. During development, this X-linked GFP transgene exhibits widespread green fluorescence throughout the embryonic and adult life of male mice but displays mosaic expression in tissues as a result of X-inactivation in females. In living female embryos, inactivation of the transgene is imprinted in extraembryonic regions and random in the embryo proper, demonstrating that this reporter is behaving in a similar fashion to the majority of X-linked loci, and so provides a vital readout of X chromosome activity. This is observation is further supported in T16H/X female mice harboring the GFP transgene on the normal X chromosome where reporter inactivation is observed in somatic cells. The differential expression of GFP activity facilitates fluorescence activated cell sorting for the purification of GFP+ vs. GFP- cells from female embryonic tissues, thereby allowing access to populations of cells that have kept active a particular X chromosome. By tracking the activity of this X-linked GFP transgene, we discovered that the primary and secondary giant cells of the X/X placenta maintain an active paternal copy of this transgene on the presumed silenced paternal X-chromosome. This finding implies that the imprint on the paternal X chromosome may be relaxed in these trophectodermal derivatives.

The color of mice: in the light of GFP-variant reporters.

The mouse currently represents the premier model organism for mammalian genetic studies. Over the past decade the production of targeted and transgenic lines of mice has become commonplace, with current technology allowing the creation of mutations at base pair resolution. Such genome modifications are becoming increasingly elaborate and often incorporate gene-based reporters for tagging different cellular populations. Until recently, lacZ, the bacterial beta-galactosidase gene has been the marker of choice for most studies in the mouse. However, over the past 3 years another valuable reporter has emerged, and its attractiveness is reflected by an explosion in its use in mice. Green fluorescent protein (GFP), a novel autofluorescent genetic reporter derived from the bioluminescent jellyfish Aequorea victoria, currently represents a unique alternative to other gene-based reporters in that its visualization is non-invasive and so can be monitored in real-time in vitro or in vivo. It has the added advantage that it can be quantified by, for example, flow cytometry, confocal microscopy, and fluorometric assays. Several mutants of the original wild-type GFP gene that improve thermostability and fluorescence have been engineered. Enhanced GFP is one such variant, which has gained popularity for use in transgenic or targeted mice. Moreover, various GFP spectral variants have also been developed, and two of these novel color variants, enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP), can also be used in mice. Since the spectral profiles of the ECFP and EYFP color variants are distinct and non-overlapping, these two reporters can be co-visualized, and are therefore ideal for in vivo double-labeling or fluorescent energy transfer analyses. The use of GFP and its color variants as reporters provides an unprecedented level of sophistication and represents the next step in mouse genome engineering technology by opening up the possibility of combinatorial non-invasive reporter usage within a single animal.

The stem cells of early embryos.

Cells resident in an organism that possess the dual capacity for self-renewal and differentiation into a spectrum of subtypes are referred to as stem cells. In the past decade, basic research performed on stem cells has shed light on the molecular pathways operating in vivo which can be harnessed in vitro for the establishment of cell lines mirroring the stem cells in the organism. The attractiveness of stem cells as in vitro models of organotypic differentiation and their potential application in a clinical context holds great promise and is only beginning to be exploited. Stem cells can be broadly grouped into two categories based on their origin from either the embryonic or the adult. Only the early embryo possesses truly pluripotent cells that can give rise to all the cell types present in the embryo proper and adult. The adult, on the other hand, possesses specialized, tissue- or organ-specific stem cell types, which can give rise to the differentiated cell types of that specific organ and have in some instances been shown to transdifferentiate. However, no stem cell obtained from an adult organism has yet been shown to exhibit developmental potential matching the breadth of that of stem cells obtained from embryos. This review focuses on the different types of stem cells that are resident in early stage mammalian embryos, detailing their derivation and propagation in addition to highlighting their developmental potential and opportunities for future applications.

FACS for the isolation of individual cells from transgenic mice harboring a fluorescent protein reporter.

Flow cytometry is extensively used for the isolation of discreet populations of cells from complex pools. The advent of autofluorescent (AFP) reporters such as wild type Green Fluorescent Protein (wtGFP) (Chalfie et al., 1994) and its variants, including enhanced green fluorescent protein (EGFP) and enhanced yellow fluorescent protein (EYFP) (Cormack et al., 1996), as vital reporters opens up the possibility of sorting live reporter-expressing cells. Moreover the use of these reporters in transgenics (Okabe et al., 1997) or mice carrying homologously targeted loci (Godwin et al., 1998) should enable the direct isolation of reporter-expressing cells from any desired lineage. Here we have assessed this approach in transgenic mice. ES cell-mediated transgenesis was used for generating a line of mice that express an autofluorescent EYFP reporter in the heart and part of the neural tube at midgestation. Pools of fluorescent cells harboring and expressing the EYFP reporter were isolated from defined regions of embryos and their origin confirmed by assaying the expression of domain-defined marker genes. Such a tool should prove useful for gaining access to any given lineage that can be fluorescent protein reporter tagged.

Cloning and expression throughout mouse development of mfat1, a homologue of the Drosophila tumour suppressor gene fat.

We present the entire sequence of the mouse Fat orthologue (mFat1), a protein of 4,588 amino acids with 34 cadherin repeats, 27 potential N-glycosylation sites, five EGF repeats and a laminin A G-motif in its extracellular domain. A single transmembrane region is followed by a cytoplasmic domain containing putative catenin-binding sequences. mFat1 shows high homology to human FAT and lesser homology to Drosophila Fat. The sequence of this giant cadherin suggests that it is unlikely to have a homophilic adhesive function, but may mediate heterophilic adhesion or play a signalling role. Expression analysis shows that the mfat1 gene is expressed early in pre-implantation mouse development, at the compact eight cell stage. Whole-mount and section in situ analyses show that transcripts are widely expressed throughout post-implantation development, most notably in the limb buds, branchial arches, forming somites, and in particular in the proliferating ventricular zones in the brain, being down-regulated as cells cease dividing. RT-PCR detects widespread expression in the adult suggesting a role in proliferation and differentiation of many tissues and cell types.

Mice lacking both presenilin genes exhibit early embryonic patterning defects.

Genetic studies in worms, flies, and humans have implicated the presenilins in the regulation of the Notch signaling pathway and in the pathogenesis of Alzheimer's Disease. There are two highly homologous presenilin genes in mammals, presenilin 1 (PS1) and presenilin 2 (PS2). In mice, inactivation of PS1 leads to developmental defects that culminate in a perinatal lethality. To test the possibility that the late lethality of PS1-null mice reflects genetic redundancy of the presenilins, we have generated PS2-null mice by gene targeting, and subsequently, PS1/PS2 double-null mice. Mice homozygous for a targeted null mutation in PS2 exhibit no obvious defects; however, loss of PS2 on a PS1-null background leads to embryonic lethality at embryonic day 9.5. Embryos lacking both presenilins, and surprisingly, those carrying only a single copy of PS2 on a PS1-null background, exhibit multiple early patterning defects, including lack of somite segmentation, disorganization of the trunk ventral neural tube, midbrain mesenchyme cell loss, anterior neuropore closure delays, and abnormal heart and second branchial arch development. In addition, Delta like-1 (Dll1) and Hes-5, two genes that lie downstream in the Notch pathway, were misexpressed in presenilin double-null embryos: Hes-5 expression was undetectable in these mice, whereas Dll1 was expressed ectopically in the neural tube and brain of double-null embryos. We conclude that the presenilins play a widespread role in embryogenesis, that there is a functional redundancy between PS1 and PS2, and that both vertebrate presenilins, like their invertebrate homologs, are essential for Notch signaling.

The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo.

The prospective fate of cells in the primitive streak was examined at early, mid and late stages of mouse gastrula development to determine the order of allocation of primitive streak cells to the mesoderm of the extraembryonic membranes and to the fetal tissues. At the early-streak stage, primitive streak cells contribute predominantly to tissues of the extraembryonic mesoderm as previously found. However, a surprising observation is that the erythropoietic precursors of the yolk sac emerge earlier than the bulk of the vitelline endothelium, which is formed continuously throughout gastrula development. This may suggest that the erythropoietic and the endothelial cell lineages may arise independently of one another. Furthermore, the extraembryonic mesoderm that is localized to the anterior and chorionic side of the yolk sac is recruited ahead of that destined for the posterior and amnionic side. For the mesodermal derivatives in the embryo, those destined for the rostral structures such as heart and forebrain mesoderm ingress through the primitive streak early during a narrow window of development. They are then followed by those for the rest of the cranial mesoderm and lastly the paraxial and lateral mesoderm of the trunk. Results of this study, which represent snapshots of the types of precursor cells in the primitive streak, have provided a better delineation of the timing of allocation of the various mesodermal lineages to specific compartments in the extraembryonic membranes and different locations in the embryonic anteroposterior axis.

Promotion of trophoblast stem cell proliferation by FGF4.

The trophoblast cell lineage is essential for the survival of the mammalian embryo in utero. This lineage is specified before implantation into the uterus and is restricted to form the fetal portion of the placenta. A culture of mouse blastocysts or early postimplantation trophoblasts in the presence of fibroblast growth factor 4 (FGF4) permitted the isolation of permanent trophoblast stem cell lines. These cell lines differentiated to other trophoblast subtypes in vitro in the absence of FGF4 and exclusively contributed to the trophoblast lineage in vivo in chimeras.

mCelsr1 is an evolutionarily conserved seven-pass transmembrane receptor and is expressed during mouse embryonic development.

Mcelsr1 encodes a protein of 3034 amino acids predicted to contain seven membrane spanning domains having homology to a group of peptide hormone binding G-protein coupled receptors. Its extracellular domain comprises epidermal growth factor-like repeats, laminin A G-domains and cadherin repeats. Homologous genes have been identified in C. elegans and D. melanogaster suggesting that the Celsr gene family is ancient. mCelsr1 mRNA expression precedes gastrulation, is subsequently restricted primarily to ectodermal derivatives and is tightly regulated in the developing central nervous system (CNS). We observe segmentally-restricted gene expression in the developing hindbrain and in the spinal cord dynamic dorso-ventrally restricted 'stripes' of expression.

Cloning and characterization of Sel-1l, a murine homolog of the C. elegans sel-1 gene.

The Notch signaling pathway regulates specification and proliferation in a variety of cell lineages in invertebrates and vertebrates. We have cloned a murine homolog of SEL-1, a key negative regulator of the Notch pathway in Caenorhabditis elegans. Murine SEL-1L (mSEL-1L) protein exhibits a high degree of similarity to SEL-1, including a signal peptide and the C-terminal region required for SEL-1 function in C. elegans. This mammalian homolog of sel-1 is widely expressed in adult mouse and human tissues, with particularly high levels in the pancreas. RNA in situ analysis of developing mouse embryos indicates that mSEL-1L is moderately expressed throughout the neural tube and dorsal root ganglia, with particularly high levels in the floor plate of the neural tube beginning at E10.5 and increasing at E11.5. Expression is high at E14.5 and E17.5 in the acini of the pancreas, and moderate in the epithelial cells of the gut villi. We localized the SEL-1L protein to the cytosol, possibly in intracellular vesicles, in a beta-islet-derived tumor cell line (RinM).

Generating green fluorescent mice by germline transmission of green fluorescent ES cells.

Green fluorescent protein (GFP) and its variants currently represent the only non-invasive markers available for labeling mammalian cells in culture or in a multicellular organism through transgenesis. To date this marker gene has been widely used in the study of many organisms, but as yet has not found large-scale application in mammals due to problems encountered with weak fluorescence and instability of the wild-type protein at higher temperatures. Recently, though, several mutants have been made in the wild-type (wt) GFP so as to improve its thermostability and fluorescence. EGFP (enhanced GFP) is one such wtGFP variant. As a first step in assessing the use of EGFP in ES cell-mediated strategies, we have established a mouse embryonic stem (ES) cell lines expressing EGFP, which can be propagated in culture, reintroduced into mice. or induced to differentiate in vitro, while still maintaining ubiquitous EGFP expression. From the results presented we can suggest that: 1) possible improvements in the efficiency of transgenic regimes requiring the germline transmission of ES cells by aggregation chimeras can be made by the preselection chimeric embryos at the blastocyst stage: (2) the expression of a noninvasive marker, driven by a promoter that is active during early postimplantation development, allows access to embryos during a window of embryonic development that has previously been difficult to investigate (3) the behavior of mutant ES cells can be followed with simple microscopic observation of chimeric embryos or adult animals comprising green fluorescent cells/tissues. and (4) intercrosses of F1 mice and subsequent generations of animals show that progeny can be genotyped by UV light, such that mice homozygous for the transgene can be distinguished from hemizygotes due to their increased fluorescence.

Embryonic stem cells, creating transgenic animals.

Embryonic stem (ES) cells have afforded a means of directly modifying the mouse genome in vitro and then introducing such changes directly into animals. The advent of this technology has made the mouse the mammal of choice for mutagenesis approaches used in the study of embryonic development and disease conditions. This chapter deals with the maintenance and modification of these pluripotent cell lines and describes the routes that can be taken for their efficient introduction to the in vivo environment.

Dissecting the role of N-myc in development using a single targeting vector to generate a series of alleles.

The N-myc proto-oncogene is expressed in many organs of the mouse embryo, suggesting that it has multiple functions. A null mutation leads to mid-gestation lethality [1-4], obscuring the later roles of the gene in organogenesis. We have generated a multi-purpose gene alteration by combining the potential for homologous and site-specific recombination in a single targeting vector, and using the selectable marker for neomycin-resistance, neo, to downregulate gene activity. This allowed us to create a series of alleles that led to different levels of N-myc expression. The phenotypes revealed a spectrum of developmental problems. The hypomorphic allele produced can be repaired in situ by Cre-recombinase-mediated DNA excision. We show here for the first time the use of a single targeting vector to generate an allelic series. This, and the possibility of subsequent lineage-specific or conditional allele repair in situ, represent new genome modification strategies that can be used to investigate multiple functions of a single gene.