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Genomic architecture appears to play crucial roles in health and a variety of diseases. How nuclear structures reorganize over different timescales is elusive, partly because the tools needed to probe and perturb them are not as advanced as needed by the field. To fill this gap, the National Institutes of Health Common Fund started a program in 2015, called the 4D Nucleome (4DN), with the goal of developing and ultimately applying technologies to interrogate the structure and function of nuclear organization in space and time., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Large-scale generation of protein capture reagents remains a technical challenge, but their generation is just the beginning. Validation is a critical, iterative process that yields different results for different uses. Independent, community-based validation offers the possibility of transparent data sharing, with use case–specific results made broadly available. This type of resource, which can grow as new validation data are obtained for an expanding group of reagents, provides a community resource that should accompany future reagent-generating efforts. To address a pressing need for antibodies or other reagents that recognize human proteins, the National Institutes of Health Common Fund launched the Protein Capture Reagents Program in 2010 as a pilot to target human transcription factors. Here, we describe lessons learned from this program concerning generation and validation of research reagents, which we believe are generally applicable for future research endeavors working in a similar space.

Given the heterogeneity of senescent cells, our knowledge of both the drivers and consequences of cellular senescence in tissues and organs remains limited, as is our understanding of how this process could be harnessed for human health. Here we identified five broad areas that would help propel the field forward., (Published by Elsevier Inc.)

The NIH Roadmap Epigenomics Program was launched to deliver reference epigenomic data from human tissues and cells, develop tools and methods for analyzing the epigenome, discover novel epigenetic marks, develop methods to manipulate the epigenome, and determine epigenetic contributions to diverse human diseases. Here, we comment on the outcomes from this program: the scientific contributions made possible by a consortium approach and the challenges, benefits, and lessons learned from this group science effort.

It has become exceedingly important to understand the precise molecular profiles of the nearly 40 trillion cells in an adult human because of their role in determining health, disease, and therapeutic outcome. The National Institutes of Health (NIH) Common Fund-supported Single Cell Analysis Program (SCAP) was designed to address this challenge. In this review, we outline the original program goals and provide a perspective on the impact of the program as a catalyst for exploration of heterogeneity of human tissues at the cellular level. We believe that the technological advances in single-cell RNA sequencing and multiplexed imaging combined with computational methods made by this program will undoubtedly have an impact on broad and robust applications of single-cell analyses in both health and disease research.

In late 2004, an International Consortium of research groups were charged with the task of producing a high-quality molecular anatomy of the developing mammalian urogenital tract (UGT). Given the importance of these organ systems for human health and reproduction, the need for a systematic molecular and cellular description of their developmental programs was deemed a high priority. The information obtained through this initiative is anticipated to enable the highest level of basic and clinical research grounded on a 21st-century view of the developing anatomy. There are three components to the Genitourinary Developmental Molecular Anatomy Project GUDMAP; all of these are intended to provide resources that support research on the kidney and UGT. The first provides ontology of the cell types during UGT development and the molecular hallmarks of those cells as discerned by a variety of procedures, including in situ hybridization, transcriptional profiling, and immunostaining. The second generates novel mouse strains. In these strains, cell types of particular interest within an organ are labeled through the introduction of a specific marker into the context of a gene that exhibits appropriate cell type or structure-specific expression. In addition, the targeting construct enables genetic manipulation within the cell of interest in many of the strains. Finally, the information is annotated, collated, and promptly released at regular intervals, before publication, through a database that is accessed through a Web portal. Presented here is a brief overview of the Genitourinary Developmental Molecular Anatomy Project effort.

Cell motility is regulated by extracellular cues and by intracellular factors that accumulate at sites of contact between cells and the extracellular matrix. One of these factors, focal adhesion kinase (FAK), regulates the cycle of focal adhesion formation and disassembly that is required for cell movement to occur. Recently, Wnt signaling has also been implicated in the control of cell movement in vertebrates, but the mechanism through which Wnt proteins influence motility is unclear. We demonstrate that Drosphila Wnt4 is required for cell movement and FAK regulation during ovarian morphogenesis. Dfrizzled2, Disheveled, and protein kinase C are also required. The DWnt4 cell motility pathway is distinct from both the canonical Wnt pathway and the planar polarity pathway. Our data suggest that DWnt4 facilitates motility through regulation of focal adhesions.

beta-Catenin plays an important role in signal transduction pathways that regulate cellular differentiation and proliferation. The increased concentration of this protein in the cytoplasm favors its binding to the T-cell factor (TCF) family of DNA-binding proteins, and it subsequently translocates to the nucleus, where it induces transcription of specific genes. We explored mechanisms that lead to activation of beta-catenin/TCF-dependent transcription in esophageal squamous cell carcinoma (ESCC) independent of adenomatous polyposis coli and beta-catenin mutation. Electrophoresis mobility shift assay demonstrated that TCF4 and beta-catenin form a complex and have DNA binding activity. However, there was no constitutive activation of beta-catenin/TCF-dependent transcription. Coculture experiments demonstrated that Wnt-1, but not Wnt-5A and Wnt-7A, activated the TCF reporter gene. Additionally, when cultured with Wnt-1-conditioned media, ESCC cell lines showed an accumulation of beta-catenin in the cytoplasm. Although both Wnt and epidermal growth factor inactivate glycogen synthase kinase 3beta, activation of epidermal growth factor receptor did not stabilize beta-catenin. A comparison of extracellular stimuli suggests that specific Wnt family members stabilize beta-catenin with resulting activation of TCF-dependent transcription in ESCC.

Members of the Wnt gene family encode secreted proteins that signal through the Frizzled family of receptors to function in many aspects of development. In this study, we have analyzed the expression of two Wnt genes and one Frizzled family member that were recently identified through the Drosophila genome sequencing project. We show that DWnt6 is only weakly expressed in developing embryos, with transcripts faintly detected in the gut. By late third instar however, this gene is expressed in a pattern that is identical to that of wingless (wg) in the imaginal discs. DWnt10 is expressed in the embryonic mesoderm, central nervous system and gut, whereas its expression is below the levels of our detection in third instar imaginal discs. We find that DFz4 is also expressed in a dynamic pattern in the mesoderm, gut, and central nervous system.

Wnt genes are often expressed in overlapping patterns, where they affect a wide array of developmental processes. To address the way in which various Wnt signals elicit distinct effects we compared the activities of two Wnt genes in Drosophila, DWnt-4, and wingless. We show that these Wnt signals produce distinct responses in cells of the dorsal embryonic epidermis. Whereas wingless acts independently of hedgehog signaling in these cells, we show that DWnt-4 requires Hh to elicit its effects. We also show that expression of Wg signal transduction components does not mimic expression of DWnt-4, suggesting that DWnt-4 signaling proceeds through a distinct pathway. The dorsal epidermis may therefore be useful in the identification of novel Wnt signaling components.

Organismal size is determined by a tightly regulated mechanism that coordinates cell growth, cell proliferation and cell death. The Drosophila insulin receptor/Chico/Dp110 pathway regulates cell and organismal size. Here we show that genetic manipulation of the phosphoinositide-3-OH-kinase-dependent serine/threonine protein kinase Akt (protein kinase B) during development of the Drosophila imaginal disc affects cell and organ size in an autonomous manner. Ectopic expression of Akt does not affect cell-fate determination, apoptosis or proliferation rates in imaginal discs. Thus, Akt appears to stimulate intracellular pathways that specifically regulate cell and compartment size independently of cell proliferation in vivo.

Wnt genes encode secreted signalling molecules involved in a number of basic developmental processes. In Drosophila, wingless and DWnt-4 are two physically clustered Wnt genes, which are transcribed in overlapping patterns during embryogenesis and, in several instances, are controlled by the same regulatory molecules. To address the question of the functional relationship of wingless and DWnt-4, we analysed how embryonic cells respond when they are exposed, simultaneously or not, to the encoded Wnt signals. We show that DWnt-4 has the capacity to antagonise Wingless signalling both in the Drosophila ventral epidermis and in a heterologous system, the Xenopus embryo. We provide evidence that DWnt-4 inhibits the Wingless/Wnt-1 signalling pathway upstream of the activation of transcriptional targets. This is the first report that antagonising Wnt signals exist in Drosophila.

The Drosophila segment polarity gene wingless (wg) is required in the regulation of engrailed (en) expression and the determination of cell fates in neighboring cells. This paracrine wg activity also regulates transcription of wg itself, through a positive feedback loop including en activity. In addition, wg has a second, more direct autoregulatory requirement that is distinct from the en-dependent feedback loop. Four gene products, encoded by armadillo (arm), dishevelled (dsh), porcupine (porc) and zeste-white 3 (zw3), have been previously implicated as components of wg paracrine signaling. Here we have used three different assays to assess the requirements of these genes in the more direct wg autoregulatory pathway. While the activities of dsh, zw3 and arm appear to be specific to the paracrine feedback pathway, the more direct autoregulatory pathway requires porc.

We have characterized a Drosophila gene that is a highly conserved homolog of the mammalian cyclic AMP (cAMP)-responsive transcription factors CREB and CREM. Uniquely among Drosophila genes characterized to date, it codes for a cAMP-responsive transcriptional activator. An alternatively spliced product of the same gene is a specific antagonist of cAMP-inducible transcription. Analysis of the splicing pattern of the gene suggests that the gene may be the predecessor of the mammalian CREB and CREM genes.

Proper spatial expression of the wingless (wg) gene in the Drosophila embryonic epidermis is crucial to intrasegmental patterning. Single cell wide wg expression is initiated at the blastoderm stage in response to combinatorial regulation by the pair rule genes. Later, during gastrulation, when the epidermal expression of the pair rule genes has disappeared, wg becomes regulated by the activity of the segment polarity genes. The segment polarity gene engrailed (en) is expressed in cells adjacent to the wg-expressing cells and is required to maintain wg transcription. Since wg is in turn required to maintain en expression, wg appears to autoregulate its own expression through an endependent paracrine feedback loop. In this paper, we demonstrate that wild-type wg expression requires wg activity during stage 9, prior to its requirement for en maintenance, indicating that wg has an autoregulatory role that is distinct from its paracrine feedback loop through en. In addition, by misexpressing Wg and En in distinct spatial patterns in the epidermis, we find that En is capable of inducing expression from the endogenous wg gene only in immediate adjacent cells which have been exposed to Wg. Furthermore, exogenous Wg expression enables maintenance of endogenous wg transcription in both wg and en mutant embryos. Our results support the model that in the wild-type embryo, wg has an autoregulatory function which is distinct and separable from paracrine regulation via en. We also provide evidence that late, localized Wg expression is crucial for the asymmetric patterning of epidermal cell types as reflected in the larval cuticle.

The Drosophila gene wingless is a member of the Wnt gene family, a group of genes that are involved in embryonic development and the regulation of cell proliferation. wingless encodes a secreted glycoprotein that plays a role in embryogenesis as well as in the development of adult structures. In the primordia of the adult limbs, the imaginal discs, wingless is expressed in an anterior ventral sector and is required for specification of ventral fate. Ectopic expression of low levels of Wingless in the leg discs leads to partial ventralization and outgrowths of the proximodistal axis. Wingless has thus been proposed to specify ventral fate in a concentration dependent manner (i.e., as a morphogen) and to organize the proximodistal axis. We have extended the analysis of Wingless function in the leg primordium through targeted ectopic expression. We find that Wingless has two functions in the leg disc. In the specification of ventral fate, our data indicate that Wingless does not function as a morphogen but instead appears to collaborate with other factors. In addition to its role in ventral fate specification, Wingless inhibits the commitment of dorsal cells toward a determined state and influences the regulation of proliferation. We propose a model in which Wingless achieves separate functions via spatially regulated mechanisms and discuss the significance of these functions during axial patterning and organization.

Consolidated memory after olfactory learning in Drosophila consists of two components, a cycloheximide-sensitive, long-term memory (LTM) and a cycloheximide-insensitive, anesthesia-resistant memory (ARM). Using an inducible transgene that expresses a dominant negative member of the fly CREB family, LTM was specifically and completely blocked only after induction, while ARM and learning were unaffected. These results suggest that LTM formation requires de novo gene expression probably mediated by CREB family genes.

The determination of specific cell fates and polarity within each segmental unit of the Drosophila embryo involves the products of the segment polarity genes. One of these, wingless (wg), encodes a secreted protein that is homologous to the mammalian proto-oncogene Wnt-1 (refs 4, 5). In the embryonic epidermis, wg is expressed in a single row of cells within each segmental unit, although its activity is required for the correct patterning of most of the epidermis. Initially Wg signals to adjacent posterior cells, maintaining engrailed (en) expression. Later during embryogenesis, wg specifies the differentiation of naked cuticle. Wg signalling functions by inactivating or antagonizing the activity of zestewhite 3 (zw3). We have investigated the requirement in the Wg signal transduction pathway for the three genes armadillo (arm), dishevelled (dsh) and porcupine (porc), all of which have embryonic mutant phenotypes similar to wg. Our results indicate that dsh and porc act upstream of zw3, and arm acts downstream of zw3.

Proliferin (PLF) is a secreted glycoprotein in the prolactin-growth hormone family in mice. PLF expression was detected in C3H 10T1/2 fibroblasts, but not in two 10T1/2-derived myogenic cell lines, and was restored in two nondifferentiating variants of one of these myogenic cell lines. Transient expression of one form of PLF (PLF1) inhibited expression from a muscle-specific gene promoter; a second form of PLF, which differed at three amino acid residues, displayed no activity in this transient assay. Introduction of a PLF1 expression construct into both muscle- and 10T1/2-derived myoblasts resulted in cell lines that were no longer myogenic or that differentiated only partially. Analysis of these cell lines revealed that differentiation could be obstructed at several steps and by one or more factors in addition to PLF. Although expected to function in vivo as an extracellular hormone, PLF did not appear to be acting through a cell surface receptor to inhibit differentiation in these cultured myoblasts.

The serum-inducible expression of proliferin genes in BALB/c 3T3 cells was found to be dependent on both protein synthesis and an extended presence of serum in the medium. Even though no mature proliferin mRNA was detected in serum-starved cells, transcription of the proliferin genes occurred in these resting-cell cultures, indicating that posttranscriptional events may be important for regulating proliferin mRNA levels. These results suggest that protein synthesis after serum stimulation of quiescent mouse fibroblasts is required for posttranscriptional processing or stabilization of proliferin RNA. Proliferin RNA levels were found to be heterogeneous among serum-stimulated cells analyzed by in situ hybridization. This heterogeneity is probably due to asynchrony in the population and may point to a correlation between the time of proliferin expression and the time of entry of a cell into S phase.

Several copies of the prolactin-related proliferin gene were detected in the mouse genome, encoding at least two distinct forms of proliferin. These forms correspond to proliferin cDNA clones derived from BALB/c 3T3 tissue culture and BALB/c placental RNA. Tissue culture and placental cells were each found to express both forms of proliferin.

Proliferin-related protein (mPRP) is a member of the PRL/GH family in the mouse. We have generated an antiserum against mPRP expressed as a bacterial fusion protein; this antiserum detects mPRP in the conditioned media of placental tissue cultures as a heterogeneous population of glycoproteins. We have also expressed mPRP in mammalian tissue culture cells and purified the secreted protein. N-terminal sequence analysis of the purified protein reveals that it is secreted as a 214 amino acid protein after removal of a 30 amino acid signal polypeptide. An antiserum raised against the purified protein detects high levels of mPRP in maternal serum during gestation. The site of synthesis of this protein has been localized by in situ hybridization to the basal zone of the day-10 mouse placenta, which is distinct from the site of synthesis of other placental proteins in this family.