Activation of the cGAS/STING Axis in Genome-Damaged Hematopoietic Cells Does Not Impact Blood Cell Formation or Leukemogenesis.

Genome damage is a main driver of malignant transformation, but it also induces aberrant inflammation via the cGAS/STING DNA-sensing pathway. Activation of cGAS/STING can trigger cell death and senescence, thereby potentially eliminating genome-damaged cells and preventing against malignant transformation. Here, we report that defective ribonucleotide excision repair (RER) in the hematopoietic system caused genome instability with concomitant activation of the cGAS/STING axis and compromised hematopoietic stem cell function, ultimately resulting in leukemogenesis. Additional inactivation of cGAS, STING, or type I IFN signaling, however, had no detectable effect on blood cell generation and leukemia development in RER-deficient hematopoietic cells. In wild-type mice, hematopoiesis under steady-state conditions and in response to genome damage was not affected by loss of cGAS. Together, these data challenge a role of the cGAS/STING pathway in protecting the hematopoietic system against DNA damage and leukemic transformation. SIGNIFICANCE: Loss of cGAS/STING signaling does not impact DNA damage-driven leukemogenesis or alter steady-state, perturbed or malignant hematopoiesis, indicating that the cGAS/STING axis is not a crucial antioncogenic mechanism in the hematopoietic system. See related commentary by Zierhut, p. 2807.

Laser Micromachining of Bone as a Tool for Studying Bone Marrow Biology.

The bone marrow (BM) has traditionally been a difficult tissue to access because it is embedded deep within the bone matrix. It is home to the hematopoietic stem cells (HSCs) that give rise to all blood cells in the body. It is also the site of origin for malignant blood cells such as leukemia and multiple myeloma, as well as a frequent site of metastasis for many solid tumors including prostate and breast cancer. The following chapter describes how laser micromachining of bone can be used to improve both optical and physical access to the BM. For example, laser thinning of the overlying bone can improve optical access, enabling deeper imaging into the BM as well as enhancing optical resolution by reducing scattering and aberration. Laser micromachining can also be used to provide physical access into the BM by creating access ports for micropipette insertion and delivery of cells to precise locations in the BM, as well as for the extraction of BM cells and interstitial fluid, all under image guidance. This chapter provides a detailed protocol for installing a laser-micromachining capability for users with an existing multiphoton microscope. Additionally, we briefly outline how such a system improves the optical resolution during imaging as well as its potential use to study injury response.

Image-seq: spatially resolved single-cell sequencing guided by in situ and in vivo imaging.

Tissue function depends on cellular organization. While the properties of individual cells are increasingly being deciphered using powerful single-cell sequencing technologies, understanding their spatial organization and temporal evolution remains a major challenge. Here, we present Image-seq, a technology that provides single-cell transcriptional data on cells that are isolated from specific spatial locations under image guidance, thus preserving the spatial information of the target cells. It is compatible with in situ and in vivo imaging and can document the temporal and dynamic history of the cells being analyzed. Cell samples are isolated from intact tissue and processed with state-of-the-art library preparation protocols. The technique therefore combines spatial information with highly sensitive RNA sequencing readouts from individual, intact cells. We have used both high-throughput, droplet-based sequencing as well as SMARTseq-v4 library preparation to demonstrate its application to bone marrow and leukemia biology. We discovered that DPP4 is a highly upregulated gene during early progression of acute myeloid leukemia and that it marks a more proliferative subpopulation that is confined to specific bone marrow microenvironments. Furthermore, the ability of Image-seq to isolate viable, intact cells should make it compatible with a range of downstream single-cell analysis tools including multi-omics protocols.

Unimpaired Responses to Vaccination With Protein Antigen Plus Adjuvant in Mice With Kit-Independent Mast Cell Deficiency.

Innate inflammatory responses are crucial for induction and regulation of T cell and antibody responses. Mast cell (MC)-deficient Kit mutant mice showed impaired adaptive immunity, suggesting that MCs provide essential adjuvant activities, and pharmacological MC activation was proposed as a new adjuvant principle. However, the Kit mutations result in complex alterations of the immune system in addition to MC deficiency. We revisited the role of MCs in vaccination responses using Mcpt5-Cre R26DTA/DTA and Cpa3Cre/+ mice that lack connective tissue MCs or all MCs, respectively, but feature an otherwise normal immune system. These animals showed no impairment of T and B cell responses to intradermal vaccination with protein antigen plus complete Freund's adjuvant. Moreover, we demonstrate that the adjuvant effects of the MC secretagogue c48/80 in intradermal or mucosal immunization are independent of the presence of MCs. We hence find no evidence for a regulation by MCs of adaptive immune responses to protein antigens. The finding that immunological MC functions differ from those suggested by experiments in Kit mutants, emphasizes the importance of rigorous tests in Kit-independent MC-deficiency models.

Ribonucleotide Excision Repair Is Essential to Prevent Squamous Cell Carcinoma of the Skin.

Because of imperfect discrimination against ribonucleoside triphosphates by the replicative DNA polymerases, large numbers of ribonucleotides are incorporated into the eukaryotic nuclear genome during S-phase. Ribonucleotides, by far the most common DNA lesion in replicating cells, destabilize the DNA, and an evolutionarily conserved DNA repair machinery, ribonucleotide excision repair (RER), ensures ribonucleotide removal. Whereas complete lack of RER is embryonically lethal, partial loss-of-function mutations in the genes encoding subunits of RNase H2, the enzyme essential for initiation of RER, cause the SLE-related type I interferonopathy Aicardi-Goutières syndrome. Here, we demonstrate that selective inactivation of RER in mouse epidermis results in spontaneous DNA damage and epidermal hyperproliferation associated with loss of hair follicle stem cells and hair follicle function. The animals developed keratinocyte intraepithelial neoplasia and invasive squamous cell carcinoma with complete penetrance, despite potent type I interferon production and skin inflammation. These results suggest that compromises to RER-mediated genome maintenance might represent an important tumor-promoting principle in human cancer.Significance: Selective inactivation of ribonucleotide excision repair by loss of RNase H2 in the murine epidermis results in spontaneous DNA damage, type I interferon response, skin inflammation, and development of squamous cell carcinoma. Cancer Res; 78(20); 5917-26. ©2018 AACR.

Intravital imaging of osteocytes in mouse calvaria using third harmonic generation microscopy.

Osteocytes are the most abundant cell in the bone, and have multiple functions including mechanosensing and regulation of bone remodeling activities. Since osteocytes are embedded in the bone matrix, their inaccessibility makes in vivo studies problematic. Therefore, a non-invasive technique with high spatial resolution is desired. The purpose of this study is to investigate the use of third harmonic generation (THG) microscopy as a noninvasive technique for high-resolution imaging of the lacunar-canalicular network (LCN) in live mice. By performing THG imaging in combination with two- and three-photon fluorescence microscopy, we show that THG signal is produced from the bone-interstitial fluid boundary of the lacuna, while the interstitial fluid-osteocyte cell boundary shows a weaker THG signal. Canaliculi are also readily visualized by THG imaging, with canaliculi oriented at small angles relative to the optical axis exhibiting stronger signal intensity compared to those oriented perpendicular to the optical axis (parallel to the image plane). By measuring forward- versus epi-detected THG signals in thinned versus thick bone samples ex vivo, we found that the epi-collected THG from the LCN of intact bone contains a superposition of backward-directed and backscattered forward-THG. As an example of a biological application, THG was used as a label-free imaging technique to study structural variations in the LCN of live mice deficient in both histone deacetylase 4 and 5 (HDAC4, HDAC5). Three-dimensional analyses were performed and revealed statistically significant differences between the HDAC4/5 double knockout and wild type mice in the number of osteocytes per volume and the number of canaliculi per lacunar surface area. These changes in osteocyte density and dendritic projections occurred without differences in lacunar size. This study demonstrates that THG microscopy imaging of the LCN in live mice enables quantitative analysis of osteocytes in animal models without the use of dyes or physical sectioning.

Wnt signaling controls pro-regenerative Collagen XII in functional spinal cord regeneration in zebrafish.

The inhibitory extracellular matrix in a spinal lesion site is a major impediment to axonal regeneration in mammals. In contrast, the extracellular matrix in zebrafish allows substantial axon re-growth, leading to recovery of movement. However, little is known about regulation and composition of the growth-promoting extracellular matrix. Here we demonstrate that activity of the Wnt/ß-catenin pathway in fibroblast-like cells in the lesion site is pivotal for axon re-growth and functional recovery. Wnt/ß-catenin signaling induces expression of col12a1a/b and deposition of Collagen XII, which is necessary for axons to actively navigate the non-neural lesion site environment. Overexpression of col12a1a rescues the effects of Wnt/ß-catenin pathway inhibition and is sufficient to accelerate regeneration. We demonstrate that in a vertebrate of high regenerative capacity, Wnt/ß-catenin signaling controls the composition of the lesion site extracellular matrix and we identify Collagen XII as a promoter of axonal regeneration. These findings imply that the Wnt/ß-catenin pathway and Collagen XII may be targets for extracellular matrix manipulations in non-regenerating species.Following spinal injury in zebrafish, non-neural cells establish an extracellular matrix to promote axon re-growth but how this is regulated is unclear. Here, the authors show that Wnt/ß-catenin signaling in fibroblast-like cells at a lesion activates axon re-growth via deposition of Collagen XII.

The fundamental rotational interval of para-H2 (+) by MQDT-assisted Rydberg spectroscopy of H2.

Transitions from selected nd Rydberg states of H2 to n'p/f Rydberg series converging on the lowest two (N(+) = 0 and 2) rotational levels of the X(+) (2)Σg (+) (v(+) = 0) ground state of para-H2 (+) have been measured in the range 1-7.4 THz using a laser-based, pulsed, narrow-band source of submillimeter-wave radiation. The analysis of the spectra by multichannel quantum-defect theory (MQDT) has allowed a complete interpretation of the fine structures of the Rydberg series and their dependence on the principal quantum number. The extrapolation of the series to their limits with MQDT has enabled the determination of the first rotational interval of para-H2 (+), which is 174.236 71(7) cm(-1) (5 223 485.1(2.3) MHz).

Wnt/ß-catenin signaling defines organizing centers that orchestrate growth and differentiation of the regenerating zebrafish caudal fin.

Zebrafish regenerate their fins via the formation of a population of progenitor cells, the blastema. Wnt/ß-catenin signaling is essential for blastemal cell proliferation and patterning of the overlying epidermis. Yet, we find that ß-catenin signaling is neither active in the epidermis nor the majority of the proliferative blastemal cells. Rather, tissue-specific pathway interference indicates that Wnt signaling in the nonproliferative distal blastema is required for cell proliferation in the proximal blastema, and signaling in cells lining the osteoblasts directs osteoblast differentiation. Thus, Wnt signaling regulates epidermal patterning, blastemal cell proliferation, and osteoblast maturation indirectly via secondary signals. Gene expression profiling, chromatin immunoprecipitation, and functional rescue experiments suggest that Wnt/ß-catenin signaling acts through Fgf and Bmp signaling to control epidermal patterning, whereas retinoic acid and Hedgehog signals mediate its effects on blastemal cell proliferation. We propose that Wnt signaling orchestrates fin regeneration by defining organizing centers that instruct cellular behaviors of adjacent tissues.

High-resolution laser spectroscopy between 0.9 and 14.3 THz in a supersonic beam: Rydberg-Rydberg transitions of atomic Xe at intermediate n values.

A laser-based, pulsed, narrow-band source of submillimeter-wave radiation has been developed that is continuously tunable from 0.1 THz to 14.3 THz. The source is based on difference-frequency mixing in the nonlinear crystal trans-4(')-(dimethylamino)-N-methyl-4-stilbazolium tosylate. By varying the pulse length, the bandwidth of the submillimeter-wave radiation can be adjusted between 85 MHz and 2.8 MHz. This new radiation source has been integrated in a vacuum-ultraviolet-submillimeter-ware double-resonance spectrometer, with which low-frequency transitions of atoms and molecules in supersonic beams can be detected mass-selectively by photoionization and time-of-flight mass spectrometry. The properties of the radiation source and spectrometer are demonstrated in a study of 33f ← nd Rydberg-Rydberg transitions in Xe with n in the range 16-31. The frequency calibration of the submillimeter-wave radiation was performed with an accuracy of 2.8 MHz. The narrowest lines observed experimentally have a full-width at half-maximum of ∼3 MHz, which is sufficient to fully resolve the hyperfine structure of the Rydberg-Rydberg transitions of (129)Xe and (131)Xe. A total of 72 transitions were measured in the range between 0.937 THz and 14.245 THz and their frequencies are compared with frequencies calculated by multichannel quantum defect theory.

Wnt signaling regulates atrioventricular canal formation upstream of BMP and Tbx2.

In the developing heart, the atrioventricular canal (AVC) is essential for separation and alignment of the cardiac chambers, for valve formation, and serves to delay the electrical impulse from the atria to the ventricles. Defects in various aspects of its formation are the most common form of congenital heart defects. Using mutant and transgenic approaches in zebrafish, this study demonstrates that Wnt/ß-catenin signaling is both sufficient and required for the induction of BMP4 and Tbx2b expression in the AVC and consequently the proper patterning of the myocardium. Furthermore, genetic analysis shows that Wnt/ß-catenin signaling is upstream and in a linear pathway with BMP and Tbx2 during AVC specification.

Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish.

Systems for spatial and temporal control of gene expression are essential for developmental studies and are of particular importance for research in adult model organisms. We present two modified dually inducible TetON systems for tissue-specific conditional control of gene expression in zebrafish based on (i) a tetracycline inducible transcriptional activator (TetActivator) fused to the ligand binding domain of a mutated glucocorticoid receptor (TetA-GBD) and (ii) a TetActivator fused with a domain of the Ecdysone receptor (TetA-EcR). Both systems showed strong induction of tetracycline-responsive promoters upon administration of the appropriate ligands (doxycycline and dexamethasone for TetA-GBD, and doxycycline and tebufenozide for TetA-EcR), and undetectable leakiness when compared with classical TetActivators. Combinations of transgenic lines expressing TetA-GBD specifically in the heart or the CNS with different Tet-responsive transgenic lines allows conditional and tissue-specific control of gene expression in embryos and adults. Importantly, induction is fully reversible and tunable by the doses of drugs used. The TetA-EcR system avoids the possible side effects of dexamethasone and displays improved sensitivity both in zebrafish and in mammalian cells. These results show that dually inducible TetON systems are convenient tools for reversible and very tightly controlled conditional gene expression in zebrafish.

High-precision optical measurements of 13C/12C isotope ratios in organic compounds at natural abundance.

A continuous-flow cavity ring-down spectroscopy (CRDS) system integrating a chromatographic separation technique, a catalytic combustor, and an isotopic (13)C/(12)C optical analyzer is described for the isotopic analysis of a mixture of organic compounds. A demonstration of its potential is made for the geochemically important class of short-chain hydrocarbons. The system proved to be linear over a 3-fold injection volume dynamic range with an average precision of 0.95 per thousand and 0.67 per thousand for ethane and propane, respectively. The calibrated accuracy for methane, ethane, and propane is within 3 per thousand of the values determined using isotope ratio mass spectrometry (IRMS), which is the current method of choice for compound-specific isotope analysis. With anticipated improvements, the low-cost, portable, and easy-to-use CRDS-based instrumental setup is poised to evolve into a credible challenge to the high-cost and complex IRMS-based technique.

Activity of nitrogen-containing and non-nitrogen-containing bisphosphonates on tumor cell lines.

We synthesized and tested three series of bisphosphonates for their activity in inhibiting the growth of three human tumor cell lines: MCF-7 (breast), NCI-H460 (lung), and SF-268 (CNS). The first series of compounds consisted of 49 nitrogen-containing bisphosphonates, the most active species being a tetrakispivaloyloxymethyl (POM) ester, having an (average) IC(50) of 6.8 microM. The second series of compounds consisted of nine terphenylbisphosphonates, the most active species also being a POM ester, having an IC(50) of 2.2 microM. The third series of compounds consisted of seven halogen or cyanophenylbisphosphonates, the most active species again being a POM ester, having an IC(50) of 500 nM. Taken together, these results are of interest because they show that bisphosphonate esters can have potent activity against a variety of tumor cell lines, with the most active terphenyl- and halophenyl-containing species having IC(50) values approximately 10-40x lower than the most potent commercially available bisphosphonates.