Helen J. McBride

Surface imaging microscopy, an automated method for visualizing whole embryo samples in three dimensions at high resolution

Modern biology is faced with the challenge of understanding the specification, generation, and maintenance of structures ranging from cells and tissues to organs and organisms. By acquiring images directly from the block face of an embedded sample, surface imaging microscopy (SIM) generates high‐resolution volumetric images of biological specimens across all of these scales. Surface imaging microscopy expands our range of imaging tools by generating three‐dimensional reconstructions of embryo samples at high resolution and high contrast. SIM image quality is not limited by depth or the optical properties of overlying tissue, and intrinsic or extrinsic alignment markers are not required for volume reconstruction. These volumes are highly isotropic, enabling them to be virtually sectioned in any direction without loss of image quality. Surface imaging microscopy provided a more accurate three‐dimensional representation of a chick embryo than confocal microscopy of the same sample. SIM offers excellent imaging of embryos from three major vertebrate systems in developmental biology: mouse, chicken, and frog. Immediate applications of this technology are in visualizing and understanding complex morphogenetic events and in making detailed comparisons between normal and genetically modified embryos.

Distinct regions of the Swi5 and Ace2 transcription factors are required for specific gene activation

Swi5 and Ace2 are cell cycle-regulated transcription factors that activate expression of early G1-specific genes in Saccharomyces cerevisiae. Swi5 and Ace2 have zinc finger DNA-binding domains that are highly conserved, and the two proteins bind to the same DNA sequences in vitro. Despite this similarity in DNA binding, Swi5 and Ace2 activate different genes in vivo, with Swi5 activating the HO gene and Ace2 activating CTS1expression. In this report we have used chimeric fusions between Swi5 and Ace2 to determine what regions of these proteins are necessary for promoter-specific activation of HO and CTS1. We have identified specific regions of Swi5 and Ace2 that are required for activation of HO and CTS1, respectively. The Swi5 protein bindsHO promoter DNA cooperatively with the Pho2 homeodomain protein, and the HO specificity region of Swi5 identified in the chimeric analysis coincides with the region of Swi5 previously identified that interacts with Pho2 in vitro. Swi5 and Ace2 also activate expression of a number of other genes expressed in G1 phase of the cell cycle, including ASH1,CDC6, EGT2, PCL2, PCL9,RME1, and SIC1. Analysis of the Swi5/Ace2 chimeras shows that distinct regions of Swi5 and Ace2 contribute to the transcriptional activation of some of these other G1-regulated genes.

Store-Operated CRAC Channels Regulate Gene Expression and Proliferation in Neural Progenitor Cells

Calcium signals regulate many critical processes during vertebrate brain development including neurogenesis, neurotransmitter specification, and axonal outgrowth. However, the identity of the ion channels mediating Ca2+ signaling in the developing nervous system is not well defined. Here, we report that embryonic and adult mouse neural stem/progenitor cells (NSCs/NPCs) exhibit store-operated Ca2+ entry (SOCE) mediated by Ca2+ release-activated Ca2+ (CRAC) channels. SOCE in NPCs was blocked by the CRAC channel inhibitors La3+, BTP2, and 2-APB and Western blots revealed the presence of the canonical CRAC channel proteins STIM1 and Orai1. Knock down of STIM1 or Orai1 significantly diminished SOCE in NPCs, and SOCE was lost in NPCs from transgenic mice lacking Orai1 or STIM1 and in knock-in mice expressing the loss-of-function Orai1 mutant, R93W. Therefore, STIM1 and Orai1 make essential contributions to SOCE in NPCs. SOCE in NPCs was activated by epidermal growth factor and acetylcholine, the latter occurring through muscarinic receptors. Activation of SOCE stimulated gene transcription through calcineurin/NFAT (nuclear factor of activated T cells) signaling through a mechanism consistent with local Ca2+ signaling by Ca2+ microdomains near CRAC channels. Importantly, suppression or deletion of STIM1 and Orai1 expression significantly attenuated proliferation of embryonic and adult NPCs cultured as neurospheres and, in vivo, in the subventricular zone of adult mice. These findings show that CRAC channels serve as a major route of Ca2+ entry in NPCs and regulate key effector functions including gene expression and proliferation, indicating that CRAC channels are important regulators of mammalian neurogenesis.

Generation and Characterization of Fully Human Monoclonal Antibodies Against Human Orai1 for Autoimmune Disease

Calcium entry into T cells following antigen stimulation is crucial for nuclear factor of activated T cells (NFAT)–mediated T cell activation. The movement of calcium is mediated by calcium release–activated calcium (CRAC) channels. There are two key components of this channel: Orai1 is the pore-forming subunit located in the plasma membrane, and stromal interaction molecule 1 (STIM1) functions as a Ca2+ sensor in the endoplasmic reticulum. A subset of human patients carry mutations in either STIM1 or Orai1 that affect protein function or expression, resulting in defective store-operated Ca2+ influx and CRAC channel function, and impaired T cell activation. These patients suffer from a hereditary form of severe combined immune deficiency syndrome, highlighting the importance of the CRAC channel for T lymphocyte function in humans. Since autoreactive T cells play an important role in the development of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and organ transplantation, Orai1 becomes an attractive therapeutic target for ameliorating autoimmune disease. We developed a novel approach to inhibiting CRAC function by generating high-affinity fully human monoclonal antibodies to human Orai1. These antibodies inhibited ICRAC current, store-operated Ca2+ influx, NFAT transcription, and cytokine release. These fully human antibodies to human Orai1 may represent a novel therapeutic approach for the treatment of autoimmunity.

Wnt signaling components in the chicken intestinal tract

Wnt signal transduction has emerged as an increasingly complex pathway due to the numerous ligands, receptors, and modulators identified in multiple developmental systems. Wnt signaling has been implicated in the renewal of the intestinal epithelium within adult animals and the progression of cancer in the colon. The Wnt family, however, has not been explored for function during embryonic gut development. Thus, to dissect the role of Wnt signaling in the developing gastrointestinal tract, it is necessary to first obtain a complete picture of the spatiotemporal expression of the Wnt signaling factors with respect to the different tissue layers of the gut. Here, we offer an in depth in situ gene expression study of Wnt ligands, frizzled receptors, and frizzled related modulators over several days of chicken gut development. These data show some expected locations of Wnt signaling as well as a surprising lack of expression of factors in the hindgut. This paper describes the first comprehensive characterization of the dynamic expression of Wnt signaling molecules during gut development. These data form the basis for future studies to determine the role of Wnt signaling in the developing gastrointestinal tract.

Interactions between Pho85 cyclin‐dependent kinase complexes and the Swi5 transcription factor in budding yeast

Pho85 is a cyclin‐dependent protein kinase (Cdk) in budding yeast with roles in cell metabolism and cell cycle progression. Activation of Pho85 occurs through association with Pho85 cyclins (Pcls), of which 10 are known. When complexed with the G1 cyclins, Pcl1 and Pcl2, Pho85 is required for cell cycle progression in the absence of the Cdc28‐dependent cyclins, Cln1 and Cln2. To identify potential targets of Pcl2‐Pho85, we performed a two‐hybrid screen using the Pcl2 cyclin as bait and recovered the transcription factor Swi5 as a Pcl2‐interacting protein. We performed both biochemical and genetic tests to discover the biological significance of the interaction between Pcl2 and Swi5 seen in the two‐hybrid assay. We found that Swi5 interacts in vitro with Pho85 cyclins and is phosphorylated in vitro by the Pho80‐Pho85 kinase. We discovered that a subset of genes that are controlled by Swi5 and a homologous transcription factor, Ace2, was misregulated in a pho85 deletion strain; expression of the ASH1 and CTS1 genes was reduced in an ace2 deletion strain, whereas expression of both genes was increased in an ace2Δ pho85Δ double mutant. We also found that overexpression of SWI5 caused cell lethality in a pho85 deletion strain. Our results are consistent with misregulation of Swi5 activity in vivo in the absence of Pho85 and implicate Swi5 as a potential substrate of Pho85 cyclin‐dependent kinase complexes.

Discovery and in Vivo Evaluation of Dual PI3Kβ/δ Inhibitors

Structure-based rational design led to the synthesis of a novel series of potent PI3K inhibitors. The optimized pyrrolopyridine analogue 63 was a potent and selective PI3Kβ/δ dual inhibitor that displayed suitable physicochemical properties and pharmacokinetic profile for animal studies. Analogue 63 was found to be efficacious in animal models of inflammation including a keyhole limpet hemocyanin (KLH) study and a collagen-induced arthritis (CIA) disease model of rheumatoid arthritis. These studies highlight the potential therapeutic value of inhibiting both the PI3Kβ and δ isoforms in the treatment of a number of inflammatory diseases.

Discovery and evaluation of 7-alkyl-1,5-bis-aryl-pyrazolopyridinones as highly potent, selective, and orally efficacious inhibitors of p38alpha mitogen-activated protein kinase.

The p38alpha mitogen-activated protein (MAP) kinase is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1beta and tumor necrosis factor alpha. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, and Crohns disease, as well as other diseases where aberrant cytokine signaling is the driver of disease. In this communication, we describe a novel class of 7-alkyl-1,5-bis-aryl-pyrazolopyridinone-based p38alpha inhibitors. In particular, compound 3f is highly potent in the enzyme and cell-based assays, selective in an Ambit kinase screen, and efficacious (ED(50) < or = 0.01 mg/kg) in the rat collagen induced arthritis (CIA) model.

LONG-RANGE INTERACTIONS AT THE HO PROMOTER

The SWI5 gene encodes a zinc finger DNA-binding protein required for the transcriptional activation of the yeast HO gene. There are two Swi5p binding sites in the HO promoter, site A at -1800 and site B at -1300. Swi5p binding at site B has been investigated in some detail, and we have shown that Swi5p binds site B in a mutually cooperative fashion with Pho2p, a homeodomain protein. In this report, we demonstrate that Swi5p and Pho2p bind cooperatively to both sites A and B but that there are differences in binding to these two promoter sites. It has been shown previously that point mutations in either Swi5p binding site only modestly reduce HO expression in a PHO2 strain. We show that these mutant promoters are completely inactive in a pho2 mutant. We have created stronger point mutations at the two Swi5p binding sites within the HO promoter, and we show that the two binding sites, separated by 500 bp, are both absolutely required for HO expression, independent of PHO2. These results create an apparent dilemma, as the strong mutations at the Swi5p binding sites show that both binding sites are required for HO expression, but the earlier binding site mutations allow Swi5p to activate HO, but only in the presence of Pho2p. To explain these results, a model is proposed in which physical interaction between Swi5p proteins bound to these two sites separated by 500 bp is required for activation of the HO promoter. Experimental evidence is presented that supports the model. In addition, through deletion analysis we have identified a region near the amino terminus of Swi5p that is required for PHO2-independent activation of HO, suggesting that this region mediates the long-range interactions between Swi5p molecules bound at the distant sites.

Bone CLARITY: Clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow

Bone clearing and light-sheet microscopy enables visualization and quantification of fluorescent single cells in intact bone. Peeking in on osteoprogenitors The eyes may be the windows to the soul, but a window into the bone—specifically bone marrow—would be useful for studying bone development and disease. Greenbaum et al. developed a method of whole-bone optical clearing, using a series of reagents under continuous flow to delipidate and decalcify bone tissue. This process renders the entire bone transparent but does not affect endogenous fluorescence, making this compatible with reporter mice. Using light sheet fluorescence microscopy, the authors counted and mapped the number of fluorescently labeled osteoprogenitors within cleared mouse tibia, vertebral column, and femur bones treated with sclerostin antibody. With reduced variability compared to standard section analysis, this Bone CLARITY and computational analysis will be a useful tool for bone research. Bone tissue harbors unique and essential physiological processes, such as hematopoiesis, bone growth, and bone remodeling. To enable visualization of these processes at the cellular level in an intact environment, we developed “Bone CLARITY,” a bone tissue clearing method. We used Bone CLARITY and a custom-built light-sheet fluorescence microscope to detect the endogenous fluorescence of Sox9-tdTomato+ osteoprogenitor cells in the tibia, femur, and vertebral column of adult transgenic mice. To obtain a complete distribution map of these osteoprogenitor cells, we developed a computational pipeline that semiautomatically detects individual Sox9-tdTomato+ cells in their native three-dimensional environment. Our computational method counted all labeled osteoprogenitor cells without relying on sampling techniques and displayed increased precision when compared with traditional stereology techniques for estimating the total number of these rare cells. We demonstrate the value of the clearing-imaging pipeline by quantifying changes in the population of Sox9-tdTomato–labeled osteoprogenitor cells after sclerostin antibody treatment. Bone tissue clearing is able to provide fast and comprehensive visualization of biological processes in intact bone tissue.