A multifunctional Wnt regulator underlies the evolution of rodent stripe patterns.

Animal pigment patterns are excellent models to elucidate mechanisms of biological organization. Although theoretical simulations, such as Turing reaction-diffusion systems, recapitulate many animal patterns, they are insufficient to account for those showing a high degree of spatial organization and reproducibility. Here, we study the coat of the African striped mouse (Rhabdomys pumilio) to uncover how periodic stripes form.

5'-Modifications improve potency and efficacy of DNA donors for precision genome editing.

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA-repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy.

CK2-Dependent Phosphorylation of the Brg1 Chromatin Remodeling Enzyme Occurs during Mitosis.

Brg1 (Brahma-related gene 1) is one of two mutually exclusive ATPases that can act as the catalytic subunit of mammalian SWI/SNF (mSWI/SfigureNF) chromatin remodeling enzymes that facilitate utilization of the DNA in eukaryotic cells. Brg1 is a phospho-protein, and its activity is regulated by specific kinases and phosphatases. Previously, we showed that Brg1 interacts with and is phosphorylated by casein kinase 2 (CK2) in a manner that regulates myoblast proliferation.

A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing.

CRISPR-Cas9 genome editing has transformed biotechnology and therapeutics. However, in vivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors), off-target editing, or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here, we exploited natural variation in the PAM-interacting domains (PIDs) of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis-Nme2Cas9-that recognizes a simple dinucleotide PAM (NCC) that provides for high target site density.

MED20 is essential for early embryogenesis and regulates NANOG expression.

Mediator is an evolutionarily conserved multi-subunit complex, bridging transcriptional activators and repressors to the general RNA polymerase II (Pol II) initiation machinery. Though the Mediator complex is crucial for the transcription of almost all Pol II promoters in eukaryotic organisms, the phenotypes of individual Mediator subunit mutants are each distinct. Here, we report for the first time, the essential role of subunit MED20 in early mammalian embryo development.

TIP55, a splice isoform of the KAT5 acetyltransferase, is essential for developmental gene regulation and organogenesis.

Regulation of chromatin structure is critical for cell type-specific gene expression. Many chromatin regulatory complexes exist in several different forms, due to alternative splicing and differential incorporation of accessory subunits. However, in vivo studies often utilize mutations that eliminate multiple forms of complexes, preventing assessment of the specific roles of each.

Small RNAs Gained during Epididymal Transit of Sperm Are Essential for Embryonic Development in Mice.

The small RNA payload of mammalian sperm undergoes dramatic remodeling during development, as several waves of microRNAs and tRNA fragments are shipped to sperm during post-testicular maturation in the epididymis. Here, we take advantage of this developmental process to probe the function of the sperm RNA payload in preimplantation development. We generated zygotes via intracytoplasmic sperm injection (ICSI) using sperm obtained from the proximal (caput) versus distal (cauda) epididymis and then characterized the development of the resulting embryos.

Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses.

Recent advances using CRISPR-Cas9 approaches have dramatically enhanced the ease for genetic manipulation in rodents. Notwithstanding, the methods to deliver nucleic acids into pre-implantation embryos have hardly changed since the original description of mouse transgenesis more than 30 years ago. Here we report a novel strategy to generate genetically modified mice by transduction of CRISPR-Cas9 components into pre-implantation mouse embryos via recombinant adeno-associated viruses (rAAVs).

KAT-Independent Gene Regulation by Tip60 Promotes ESC Self-Renewal but Not Pluripotency.

Although histone-modifying enzymes are generally assumed to function in a manner dependent on their enzymatic activities, this assumption remains untested for many factors. Here, we show that the Tip60 (Kat5) lysine acetyltransferase (KAT), which is essential for embryonic stem cell (ESC) self-renewal and pre-implantation development, performs these functions independently of its KAT activity. Unlike ESCs depleted of Tip60, KAT-deficient ESCs exhibited minimal alterations in gene expression, chromatin accessibility at Tip60 binding sites, and self-renewal, thus demonstrating a critical KAT-independent role of Tip60 in ESC maintenance.

Maternal expression and early induction of histone gene transcription factor Hinfp sustains development in pre-implantation embryos.

Fidelity of histone gene expression is important for normal cell growth and differentiation that is stringently controlled during development but is compromised during tumorigenesis. Efficient production of histones for packaging newly replicated DNA is particularly important for proper cell division and epigenetic control during the initial pre-implantation stages of embryonic development. Here, we addressed the unresolved question of when the machinery for histone gene transcription is activated in the developing zygote to accommodate temporal demands for histone gene expression.

Extra-embryonic Wnt3 regulates the establishment of the primitive streak in mice.

The establishment of the head to tail axis at early stages of development is a fundamental aspect of vertebrate embryogenesis. In mice, experimental embryology, genetics and expression studies have suggested that the visceral endoderm, an extra-embryonic tissue, plays an important role in anteroposterior axial development. Here we show that absence of Wnt3 in the posterior visceral endoderm leads to delayed formation of the primitive streak and that interplay between anterior and posterior visceral endoderm restricts the position of the primitive streak.

Contrasting roles for MyoD in organizing myogenic promoter structures during embryonic skeletal muscle development.

Background: Among the complexities of skeletal muscle differentiation is a temporal distinction in the onset of expression of different lineage-specific genes. The lineage-determining factor MyoD is bound to myogenic genes at the onset of differentiation whether gene activation is immediate or delayed. How temporal regulation of differentiation-specific genes is established remains unclear.

The Dynamics of Morphogenesis in the Early Mouse Embryo.

SUMMARYOver the past two decades, our understanding of mouse development from implantation to gastrulation has grown exponentially with an upsurge of genetic, molecular, cellular, and morphogenetic information. New discoveries have exalted the role of extraembryonic tissues in orchestrating embryonic patterning and axial specification. At the same time, the identification of unexpected morphogenetic processes occurring during mouse gastrulation has challenged established dogmas and brought new insights into the mechanisms driving germ layer formation.

Senescence-associated β-galactosidase activity marks the visceral endoderm of mouse embryos but is not indicative of senescence.

Senescence-associated β-galactosidase (SA-β-gal) activity is widely used as a marker of cellular senescence and as an indicator of organismal aging. Here, we report that SA-β-gal activity is present in the visceral endoderm layer of early postimplantation mouse embryos in predictable patterns that vary as the embryo progresses in development. However, determination of the mitotic index and analysis of the expression of Cdkn1a (p21), a marker of senescent cells, do not indicate cellular senescence.

Arf4 is required for Mammalian development but dispensable for ciliary assembly.

The primary cilium is a sensory organelle, defects in which cause a wide range of human diseases including retinal degeneration, polycystic kidney disease and birth defects. The sensory functions of cilia require specific receptors to be targeted to the ciliary subdomain of the plasma membrane. Arf4 has been proposed to sort cargo destined for the cilium at the Golgi complex and deemed a key regulator of ciliary protein trafficking.

A fast and sensitive alternative for β-galactosidase detection in mouse embryos.

The bacterial lacZ gene is widely used as a reporter in a myriad of mouse transgenic experiments. β-Galactosidase, encoded by lacZ, is usually detected using X-gal in combination with ferric and ferrous ions. This assay produces a blue indole precipitate that is easy to detect visually.

Wnt3 function in the epiblast is required for the maintenance but not the initiation of gastrulation in mice.

The formation of the anteroposterior axis in mice requires a Wnt3-dependent symmetry-breaking event that leads to the formation of the primitive streak and gastrulation. Wnt3 is expressed sequentially in two distinct areas of the mouse embryo before the appearance of the primitive streak; first in the posterior visceral endoderm and soon after in the adjacent posterior epiblast. Hence, although an axial requirement for Wnt3 is well established, its temporal and tissue specific requirements remain an open question.

Conditional Aurora A deficiency differentially affects early mouse embryo patterning.

Aurora A is a mitotic kinase essential for cell proliferation. In mice, ablation of Aurora A results in mitotic arrest and pre-implantation lethality, preventing studies at later stages of development. Here we report the effects of Aurora A ablation on embryo patterning at early post-implantation stages.

A non-specific effect associated with conditional transgene expression based on Cre-loxP strategy in mice.

Transgenes flanked by loxP sites have been widely used to generate transgenic mice where the transgene expression can be controlled spatially and temporally by Cre recombinase. Data from this approach has led to important conclusions in cancer, neurodevelopment and neurodegeneration. Using this approach to conditionally express micro RNAs (miRNAs) in mice, we found that Cre-mediated recombination in neural progenitor cells caused microcephaly in five of our ten independent transgenic lines.

Chromatin immunoprecipitation assay for tissue-specific genes using early-stage mouse embryos.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo.

Culture of whole mouse embryos at early postimplantation to organogenesis stages: developmental staging and methods.

In vitro culture of whole mouse embryos enables the maintenance of growth and morphogenesis of postimplantation embryos outside the uterine environment. This technological advent facilitates the observation of the development of embryos in real time whereby cell lineage and tissue morphogenesis can be traced with appropriate vital cell labels and molecular markers. Embryos in culture are also amenable to direct experimental manipulations for elucidating developmental mechanisms of embryogenesis, germ layer formation, and embryonic patterning.

The histone gene activator HINFP is a nonredundant cyclin E/CDK2 effector during early embryonic cell cycles.

Competency for DNA replication is functionally coupled to the activation of histone gene expression at the onset of S phase to form chromatin. Human histone nuclear factor P (HiNF-P; gene symbol HINFP) bound to its cyclin E/cyclin-dependent kinase 2 (CDK2) responsive coactivator p220(NPAT) is a key regulator of multiple human histone H4 genes that encode a major subunit of the nucleosome. Induction of the histone H4 transcription factor (HINFP)/p220(NPAT) coactivation complex occurs in parallel with the CDK-dependent release of pRB from E2F at the restriction point.