Tissue-Specific In Vivo Biotin Chromatin Immunoprecipitation with Sequencing in Zebrafish and Chicken.

Chromatin immunoprecipitation with sequencing (ChIP-seq) has been instrumental in understanding transcription factor (TF) binding during gene regulation. ChIP-seq requires specific antibodies against desired TFs, which are not available for numerous species. Here, we describe a tissue-specific biotin ChIP-seq protocol for zebrafish and chicken embryos which utilizes AVI tagging of TFs, permitting their biotinylation by a co-expressed nuclear biotin ligase. Subsequently, biotinylated factors can be precipitated with streptavidin beads, enabling the user to construct TF genome-wide binding landscapes like conventional ChIP-seq methods. For complete details on the use and execution of this protocol, please see Lukoseviciute et al. (2018) and Ling and Sauka-Spengler (2019).

Early chromatin shaping predetermines multipotent vagal neural crest into neural, neuronal and mesenchymal lineages.

The enteric nervous system (ENS) predominantly originates from vagal neural crest (VNC) cells that emerge from the caudal hindbrain, invade the foregut and populate the gastrointestinal tract. However, the gene regulatory network (GRN) orchestrating the early specification of VNC remains unknown. Using an EdnrB enhancer, we generated a comprehensive temporal map of the chromatin and transcriptional landscape of VNC in the avian model, revealing three VNC cell clusters (neural, neurogenic and mesenchymal), each predetermined epigenetically prior to neural tube delamination. We identify and functionally validate regulatory cores (Sox10/Tfap2B/SoxB/Hbox) mediating each programme and elucidate their combinatorial activities with other spatiotemporally specific transcription factors (bHLH/NR). Our global deconstruction of the VNC-GRN in vivo sheds light on critical early regulatory mechanisms that may influence the divergent neural phenotypes in enteric neuropathies.

A genome-wide assessment of the ancestral neural crest gene regulatory network.

The neural crest (NC) is an embryonic cell population that contributes to key vertebrate-specific features including the craniofacial skeleton and peripheral nervous system. Here we examine the transcriptional and epigenomic profiles of NC cells in the sea lamprey, in order to gain insight into the ancestral state of the NC gene regulatory network (GRN). Transcriptome analyses identify clusters of co-regulated genes during NC specification and migration that show high conservation across vertebrates but also identify transcription factors (TFs) and cell-adhesion molecules not previously implicated in NC migration. ATAC-seq analysis uncovers an ensemble of cis-regulatory elements, including enhancers of Tfap2B, SoxE1 and Hox-α2 validated in the embryo. Cross-species deployment of lamprey elements identifies the deep conservation of lamprey SoxE1 enhancer activity, mediating homologous expression in jawed vertebrates. Our data provide insight into the core GRN elements conserved to the base of the vertebrates and expose others that are unique to lampreys.

Reconstruction of the Global Neural Crest Gene Regulatory Network In Vivo.

Precise control of developmental processes is encoded in the genome in the form of gene regulatory networks (GRNs). Such multi-factorial systems are difficult to decode in vertebrates owing to their complex gene hierarchies and dynamic molecular interactions. Here we present a genome-wide in vivo reconstruction of the GRN underlying development of the multipotent neural crest (NC) embryonic cell population. By coupling NC-specific epigenomic and transcriptional profiling at population and single-cell levels with genome/epigenome engineering in vivo, we identify multiple regulatory layers governing NC ontogeny, including NC-specific enhancers and super-enhancers, novel trans-factors, and cis-signatures allowing reverse engineering of the NC-GRN at unprecedented resolution. Furthermore, identification and dissection of divergent upstream combinatorial regulatory codes has afforded new insights into opposing gene circuits that define canonical and neural NC fates early during NC ontogeny. Our integrated approach, allowing dissection of cell-type-specific regulatory circuits in vivo, has broad implications for GRN discovery and investigation.

Roles of Wnt pathway genes wls, wnt9a, wnt5b, frzb and gpc4 in regulating convergent-extension during zebrafish palate morphogenesis.

The Wnt signaling pathway is crucial for tissue morphogenesis, participating in cellular behavior changes, notably during the process of convergent-extension. Interactions between Wnt-secreting and receiving cells during convergent-extension remain elusive. We investigated the role and genetic interactions of Wnt ligands and their trafficking factors Wls, Gpc4 and Frzb in the context of palate morphogenesis in zebrafish. We describe that the chaperon Wls and its ligands Wnt9a and Wnt5b are expressed in the ectoderm, whereas juxtaposed chondrocytes express Frzb and Gpc4. Using wls, gpc4, frzb, wnt9a and wnt5b mutants, we genetically dissected the Wnt signals operating between secreting ectoderm and receiving chondrocytes. Our analysis delineates that non-canonical Wnt signaling is required for cell intercalation, and that wnt5b and wnt9a are required for palate extension in the anteroposterior and transverse axes, respectively.

Occult Histopathology and Its Predictors in Contralateral and Bilateral Prophylactic Mastectomies.

BACKGROUND: The last decade has seen an increasing prevalence of prophylactic mastectomies with decreasing age of patients treated for breast cancer. Data are limited on the prevalence of histopathologic abnormalities in this population. This study aimed to measure the prevalence of histopathologic findings in contralateral prophylactic mastectomy (CPM) and bilateral prophylactic mastectomy (BPM) patients and identify predictors of findings. METHODS: Our institution's prophylactic mastectomies from 2004 to 2011 were reviewed. Breast specimens with prior malignancies were excluded. Patient factors and pathology reports were collected. Independent predictive factors were identified with univariate and multivariate logistic analysis. RESULTS: A total of 524 specimens in 454 patients were identified. Malignancy was found in 7.0% of CPM and 5.7% of BPM specimens. In CPM patients, ipsilateral lobular carcinoma-in situ [odds ratio (OR) 4.0] and mammogram risk group (OR 2.0) were predictive of malignancy. Age group (OR 1.5), ipsilateral lobular carcinoma-in situ (OR 2.3), and prior bilateral salpingo-oophorectomy (OR 0.3) were predictive of moderate- to high-risk histopathology. Only increasing age group was predictive of increased moderate- to high-risk histopathology in BPM patients (OR 2.3). There were no independent predictors of malignancy in BPM. BRCA status was not predictive in either CPM or BPM. CONCLUSIONS: Patients with lobular carcinoma-in situ in the index breast or high-risk mammograms have a higher prevalence of malignancies. Although BRCA patients may benefit from prophylactic mastectomy, the genetic diagnosis does not increase the prevalence of detecting occult pathology. BPM patients can be counseled about relative risk, where occult pathology increases with age.

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel's Cartilage.

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel's cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel's cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel's cartilage. Multispectral cell labeling enables scientists to demonstrate extension of the Meckel's chondrocytes. During extension phase of the Meckel's cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

X-ray micro-diffraction studies on biological samples at the BioCAT Beamline 18-ID at the Advanced Photon Source.

The small source sizes of third-generation synchrotron sources are ideal for the production of microbeams for diffraction studies of crystalline and non-crystalline materials. While several such facilities have been available around the world for some time now, few have been optimized for the handling of delicate soft-tissue specimens under cryogenic conditions. Here the development of a new X-ray micro-diffraction instrument at the Biophysics Collaborative Access Team beamline 18-ID at the Advanced Photon Source, and its use with newly developed cryo-diffraction techniques for soft-tissue studies, are described. The combination of the small beam sizes delivered by this instrument, the high delivered flux and successful cryo-freezing of rat-tail tendon has enabled us to record data to better than 4 Šresolution. The ability to quickly raster scan samples in the beam allows selection of ordered regions in fibrous samples for markedly improved data quality. Examples of results of experiments obtainable using this instrument are presented.

The cross-bridge spring: can cool muscles store elastic energy?

Muscles not only generate force. They may act as springs, providing energy storage to drive locomotion. Although extensible myofilaments are implicated as sites of energy storage, we show that intramuscular temperature gradients may enable molecular motors (cross-bridges) to store elastic strain energy. By using time-resolved small-angle x-ray diffraction paired with in situ measurements of mechanical energy exchange in flight muscles of Manduca sexta, we produced high-speed movies of x-ray equatorial reflections, indicating cross-bridge association with myofilaments. A temperature gradient within the flight muscle leads to lower cross-bridge cycling in the cooler regions. Those cross-bridges could elastically return energy at the extrema of muscle lengthening and shortening, helping drive cyclic wing motions. These results suggest that cross-bridges can perform functions other than contraction, acting as molecular links for elastic energy storage.

Fast-scanning high-flux microprobe for biological X-ray fluorescence microscopy and microXAS.

There is a growing interest in the biomedical community in obtaining information concerning the distribution and local chemical environment of metals in tissues and cells. Recently, biological X-ray fluorescence microscopy (XFM) has emerged as the tool of choice to address these questions. A fast-scanning high-flux X-ray microprobe, built around a recently commissioned pair of 200 mm-long Rh-coated silicon Kirkpatrick-Baez mirrors, has been constructed at BioCAT beamline 18ID at the Advanced Photon Source. The new optical system delivers a flux of 1.3 x 10(12) photons s(-1) into a minimum focal spot size of approximately 3-5 microm FWHM. A set of Si drift detectors and bent Laue crystal analyzers may be used in combination with standard ionization chambers for X-ray fluorescence measurements. BioCAT's scanning software allows fast continuous scans to be performed while acquiring and storing full multichannel analyzer spectra per pixel on-the-fly with minimal overhead time (<20 ms per pixel). Together, the high-flux X-ray microbeam and the rapid-scanning capabilities of the BioCAT beamline allow the collection of XFM and micro X-ray absorption spectroscopy (microXAS) measurements from as many as 48 tissue sections per day. This paper reports the commissioning results of the new instrument with representative XFM and microXAS results from tissue samples.

Experimental approaches for solution X-ray scattering and fiber diffraction.

X-ray scattering and diffraction from non-crystalline systems have gained renewed interest in recent years, as focus shifts from the structural chemistry information gained by high-resolution studies to the context of structural physiology at larger length scales. Such techniques permit the study of isolated macromolecules as well as highly organized macromolecular assemblies as a whole under near-physiological conditions. Time-resolved approaches, made possible by advanced synchrotron instrumentation, add a crucial dimension to many of these investigations. This article reviews experimental approaches in non-crystalline X-ray scattering and diffraction that may be used to illuminate important scientific questions such as protein/nucleic acid folding and structure-function relationships in large macromolecular assemblies.

Reverse actin sliding triggers strong myosin binding that moves tropomyosin.

Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the "steric blocking" mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca(2+) with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca(2+)], and stretch activation, at lower [Ca(2+)], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored "actin target zones." Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca(2+)] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca(2+)], Vi-"paralyzed" fibers produce force substantially above passive response at pCa approximately 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding "brakes" by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

The BioCAT undulator beamline 18ID: a facility for biological non-crystalline diffraction and X-ray absorption spectroscopy at the Advanced Photon Source.

The 18ID undulator beamline of the Biophysics Collaborative Access Team at the Advanced Photon Source, Argonne, IL, USA, is a high-performance instrument designed for, and dedicated to, the study of partially ordered and disordered biological materials using the techniques of small-angle X-ray scattering, fiber diffraction, and X-ray absorption spectroscopy. The beamline and associated instrumentation are described in detail and examples of the representative experimental results are presented.

X-ray diffraction of the molecular substructure of human articular cartilage.

The molecular substructure of human articular cartilage has been difficult to study because of its complex composition and high degree of hydration. Using newly available small-angle X-ray diffraction (SAX) instrumentation that allows very short exposure times (0.1 to 10 sec), we have obtained spatially resolved information concerning the disposition of collagen fibers in the matrix of cartilage from the normal and osteoarthritic ankle and knee joints of human cadavers. Surprisingly, in zones of cartilage damage, such as in preosteoarthritic lesions or in the severely degenerated cartilage of osteoarthritic joints, collagen fibers of the deeper layers tended to be reoriented from the vertical. The SAX technique represents a nondestructive method of analyzing the collagen network in cartilage. Taken together, the data suggest a rigid control mechanism for the fiber network and an extensive passive reorganization of the collagen fiber orientation in diseased joint cartilage.

The in situ supermolecular structure of type I collagen.

BACKGROUND: The proteins belonging to the collagen family are ubiquitous throughout the animal kingdom. The most abundant collagen, type I, readily forms fibrils that convey the principal mechanical support and structural organization in the extracellular matrix of connective tissues such as bone, skin, tendon, and vasculature. An understanding of the molecular arrangement of collagen in fibrils is essential since it relates molecular interactions to the mechanical strength of fibrous tissues and may reveal the underlying molecular pathology of numerous connective tissue diseases. RESULTS: Using synchrotron radiation, we have conducted a study of the native fibril structure at anisotropic resolution (5.4 A axial and 10 A lateral). The intensities of the tendon X-ray diffraction pattern that arise from the lateral packing (three-dimensional arrangement) of collagen molecules were measured by using a method analogous to Rietveld methods in powder crystallography and to the separation of closely spaced peaks in Laue diffraction patterns. These were then used to determine the packing structure of collagen by MIR. CONCLUSIONS: Our electron density map is the first obtained from a natural fiber using these techniques (more commonly applied to single crystal crystallography). It reveals the three-dimensional molecular packing arrangement of type I collagen and conclusively proves that the molecules are arranged on a quasihexagonal lattice. The molecular segments that contain the telopeptides (central to the function of collagen fibrils in health and disease) have been identified, revealing that they form a corrugated arrangement of crosslinked molecules that strengthen and stabilize the native fibril.

Titin-based modulation of calcium sensitivity of active tension in mouse skinned cardiac myocytes.

We studied the effect of titin-based passive force on the length dependence of activation of cardiac myocytes to explore whether titin may play a role in the generation of systolic force. Force-pCa relations were measured at sarcomere lengths (SLs) of 2.0 and 2.3 microm. Passive tension at 2.3 microm SL was varied from approximately 1 to approximately 10 mN/mm(2) by adjusting the characteristics of the stretch imposed on the passive cell before activation. Relative to 2.0 microm SL, the force-pCa curve at 2.3 microm SL and low passive tension showed a leftward shift (pCa(50) [change in pCa at half-maximal activation]) of 0.09+/-0.02 pCa units while at 2.3 microm SL and high passive tension the shift was increased to 0.25+/-0.03 pCa units. Passive tension also increased pCa(50) at reduced interfilament lattice spacing achieved with dextran. We tested whether titin-based passive tension influences the interfilament lattice spacing by measuring the width of the myocyte and by using small-angle x-ray diffraction of mouse left ventricular wall muscle. Cell width and interfilament lattice spacing varied inversely with passive tension, in the presence and absence of dextran. The passive tension effect on length-dependent activation may therefore result from a radial titin-based force that modulates the interfilament lattice spacing.

Myofilament lattice spacing as a function of sarcomere length in isolated rat myocardium.

The Frank-Starling relationship of the heart has, as its molecular basis, an increase in the activation of myofibrils by calcium as the sarcomere length increases. It has been suggested that this phenomenon may be due to myofilaments moving closer together at longer lengths, thereby enhancing the probability of favorable acto-myosin interaction, resulting in increased calcium sensitivity. Accordingly, we have developed an apparatus so as to obtain accurate measurements of myocardial interfilament spacing (by synchrotron X-ray diffraction) as a function of sarcomere length (by video microscopy) over the working range of the heart, using skinned as well as intact rat trabeculas as model systems. In both these systems, lattice spacing decreased significantly as sarcomere length was increased. Furthermore, lattice spacing in the intact muscle was significantly smaller than that in the skinned muscle at all sarcomere lengths studied. These observations are consistent with the hypothesis that lattice spacing underlies length-dependent activation in the myocardium.

In vivo x-ray diffraction of indirect flight muscle from Drosophila melanogaster.

Small-angle x-ray diffraction from isolated muscle preparations is commonly used to obtain time-resolved structural information during contraction. We extended this technique to the thoracic flight muscles of living fruit flies, at rest and during tethered flight. Precise measurements at 1-ms time resolution indicate that the myofilament lattice spacing does not change significantly during oscillatory contraction. This result is consistent with the notion that a net radial force maintains the thick filaments at an equilibrium interfilament spacing of approximately 56 nm throughout the contractile cycle. Transgenic flies with amino-acid substitutions in the conserved phosphorylation site of the myosin regulatory light chain (RLC) exhibit structural abnormalities that can explain their flight impairment. The I(20)/I(10) equatorial intensity ratio of the mutant fly is 35% less than that of wild type, supporting the hypothesis that myosin heads that lack phosphorylated RLC remain close to the thick filament backbone. This new experimental system facilitates investigation of the relation between molecular structure and muscle function in living organisms.

Z/I and A-band lattice spacings in frog skeletal muscle: effects of contraction and osmolarity.

A-band and Z-line/I-band lattice spacings were measured by small-angle X-ray diffraction from relaxed and isometrically-contracting whole frog sartorius muscles with lattice spacings reduced or swollen by changing the osmolarity of the bathing solution. A-band spacing increased by approximately 3% upon isometric contraction at reduced lattice spacings (245-356 mOsm) and decreased by approximately 1% at swollen spacings (172 mOsm), similarly to the behaviour of skinned muscles upon changing from the relaxed state to rigor. The Z/I lattice underwent a significant lattice expansion (3-8%) upon isometric contraction at all osmolarities, in qualitative agreement (but quantitative disagreement) with results from electron microscopy on mammalian skeletal muscle. Lattice areas calculated for the Z/I and A-band lattices indicate a barrel-shaped sarcomere in the resting state, which may provide a partial explanation for how longitudinal forces produced in the A-band can produce a radial expansive force in the Z-line during contraction. The radial component of cross-bridge stiffness was calculated from the A-band data for contracting muscle, using a lattice stability model incorporating structural, osmotic and electrostatic forces. The calculations gave a radial cross-bridge stiffness during contraction of about 9 x 10(5) N m-2, and outward radial force per thick filament in normal Ringer's solution of 6 x 10(-9) N, corresponding to a radial force per cross-bridge of 10(-11) N.