CTCF is essential for proper mitotic spindle structure and anaphase segregation.

Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis and has a role in localizing CENP-E, but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild-type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time-lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Long-term inhibition of CNEP-E via GSK923295 recapitulates CTCF knockdown abnormal mitotic spindles with polar chromosomes and increased nuclear sizes. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild-type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting that population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus likely through its known role in recruiting CENP-E.

Zebrafish crocc2 mutants exhibit divergent craniofacial shape, misregulated variability, and aberrant cartilage morphogenesis.

BACKGROUND: Phenotypic variation is of paramount importance in development, evolution, and human health; however, the molecular mechanisms that influence organ shape and shape variability are not well understood. During craniofacial development, the behavior of skeletal precursors is regulated by both biochemical and environmental inputs, and the primary cilia play critical roles in transducing both types of signals. Here, we examine a gene that encodes a key constituent of the ciliary rootlets, crocc2, and its role in cartilage morphogenesis in larval zebrafish. RESULTS: Geometric morphometric analysis of crocc2 mutants revealed altered craniofacial shapes and expanded variation. At the cellular level, we observed altered chondrocyte shapes and planar cell polarity across multiple stages in crocc2 mutants. Notably, cellular defects were specific to areas that experience direct mechanical input. Cartilage cell number, apoptosis, and bone patterning were not affected in crocc2 mutants. CONCLUSIONS: Whereas "regulatory" genes are widely implicated in patterning the craniofacial skeleton, genes that encode "structural" aspects of the cell are increasingly implicated in shaping the face. Our results add crocc2 to this list, and demonstrate that it affects craniofacial geometry and canalizes phenotypic variation. We propose that it does so via mechanosensing, possibly through the ciliary rootlet. If true, this would implicate a new organelle in skeletal development and evolution.

Ciliary Rootlet Coiled-Coil 2 (crocc2) Is Associated with Evolutionary Divergence and Plasticity of Cichlid Jaw Shape.

Cichlid fishes exhibit rapid, extensive, and replicative adaptive radiation in feeding morphology. Plasticity of the cichlid jaw has also been well documented, and this combination of iterative evolution and developmental plasticity has led to the proposition that the cichlid feeding apparatus represents a morphological "flexible stem." Under this scenario, the fixation of environmentally sensitive genetic variation drives evolutionary divergence along a phenotypic axis established by the initial plastic response. Thus, if plasticity is predictable then so too should be the evolutionary response. We set out to explore these ideas at the molecular level by identifying genes that underlie both the evolution and plasticity of the cichlid jaw. As a first step, we fine-mapped an environment-specific quantitative trait loci for lower jaw shape in cichlids, and identified a nonsynonymous mutation in the ciliary rootlet coiled-coil 2 (crocc2), which encodes a major structural component of the primary cilium. Given that primary cilia play key roles in skeletal mechanosensing, we reasoned that this gene may confer its effects by regulating the sensitivity of bone to respond to mechanical input. Using both cichlids and zebrafish, we confirmed this prediction through a series of experiments targeting multiple levels of biological organization. Taken together, our results implicate crocc2 as a novel mediator of bone formation, plasticity, and evolution.

Divergent Mechanisms of the Banert Cascade with Propargyl Azides.

Triazoles are privileged heterocycles for a variety of applications. The synthesis of 1H-triazoles can be accomplished by the Banert cascade from propargylic azides. Depending on the substrate and conditions, the Banert cascade can proceed by either a sigmatropic or prototropic mechanism. This report describes the first detailed kinetic analysis of the Banert cascade proceeding by both pathways including substituent effects and KIE. The analysis identified the inflection point in the divergent pathways, allowing future work to predict which Banert products are accessible.

Evidence for a Sigmatropic and an Ionic Pathway in the Winstein Rearrangement.

The spontaneous rearrangement of allylic azides is thought to be a sigmatropic reaction. Presented herein is a detailed investigation into the rearrangement of several allylic azides. A combination of experiments including equilibrium studies, kinetic analysis, density functional theory calculations, and selective 15N-isotopic labeling are included. We conclude that the Winstein rearrangement occurs by the assumed sigmatropic pathway under most conditions. However, racemization was observed for some cyclic allylic azides. A kinetic analysis of this process is provided, which supports a previously undescribed ionic pathway.

myFX: a turn-key software for laboratory desktops to analyze spatial patterns of gene expression in Drosophila embryos.

Spatial patterns of gene expression are of key importance in understanding developmental networks. Using in situ hybridization, many laboratories are generating images to describe these spatial patterns and to test biological hypotheses. To facilitate such analyses, we have developed biologist-centric software (myFX) that contains computational methods to automatically process and analyze images depicting embryonic gene expression in the fruit fly Drosophila melanogaster. It facilitates creating digital descriptions of spatial patterns in images and enables measurements of pattern similarity and visualization of expression across genes and developmental stages. myFX interacts directly with the online FlyExpress database, which allows users to search thousands of existing patterns to find co-expressed genes by image comparison.

DNA density is a better indicator of a nuclear bleb than lamin B loss.

Nuclear blebs are herniations of the nucleus that occur in diseased nuclei that cause nuclear rupture leading to cellular dysfunction. Chromatin and lamins are two of the major structural components of the nucleus that maintain its shape and function, but their relative roles in nuclear blebbing remain elusive. Lamin B is reported to be lost in blebs by qualitative data while quantitative studies reveal a spectrum of lamin B levels in nuclear blebs dependent on perturbation and cell type. Chromatin has been reported to be decreased or de-compacted in nuclear blebs, but again the data are not conclusive. To determine the composition of nuclear blebs, we compared the immunofluorescence intensity of lamin B and DNA in the main nucleus body and nuclear bleb across cell types and perturbations. Lamin B nuclear bleb levels varied drastically across MEF wild type and chromatin or lamins perturbations, HCT116 lamin B1-GFP imaging, and human disease model cells of progeria and prostate cancer. However, DNA concentration was consistently decreased to about half that of the main nucleus body across all measured conditions. Using Partial Wave Spectroscopic (PWS) microscopy to measure chromatin density in the nuclear bleb vs body we find similar results that DNA is consistently less dense in nuclear blebs. Thus, our data spanning many different cell types and perturbations supports that decreased DNA is a better marker of a nuclear bleb than lamin B levels that vary widely.

CTCF is essential for proper mitotic spindle structure and anaphase segregation.

Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus.

Recruitment of scribble to the synaptic scaffolding complex requires GUK-holder, a novel DLG binding protein.

BACKGROUND: Membrane-associated guanylate kinases (MAGUKs), such as Discs-Large (DLG), play critical roles in synapse maturation by regulating the assembly of synaptic multiprotein complexes. Previous studies have revealed a genetic interaction between DLG and another PDZ scaffolding protein, SCRIBBLE (SCRIB), during the establishment of cell polarity in developing epithelia. A possible interaction between DLG and SCRIB at synaptic junctions has not yet been addressed. Likewise, the biochemical nature of this interaction remains elusive, raising questions regarding the mechanisms by which the actions of both proteins are coordinated. RESULTS: Here we report the isolation of a new DLG-interacting protein, GUK-holder, that interacts with the GUK domain of DLG and which is dynamically expressed during synaptic bouton budding. We also show that at Drosophila synapses DLG colocalizes with SCRIB and that this colocalization is likely to be mediated by direct interactions between GUKH and the PDZ2 domain of SCRIB. We show that DLG, GUKH, and SCRIB form a tripartite complex at synapses, in which DLG and GUKH are required for the proper synaptic localization of SCRIB. CONCLUSIONS: Our results provide a mechanism by which developmentally important PDZ-mediated complexes are associated at the synapse.

Fasciclin II signals new synapse formation through amyloid precursor protein and the scaffolding protein dX11/Mint.

Cell adhesion molecules (CAMs) have been universally recognized for their essential roles during synapse remodeling. However, the downstream pathways activated by CAMs have remained mostly unknown. Here, we used the Drosophila larval neuromuscular junction to investigate the pathways activated by Fasciclin II (FasII), a transmembrane CAM of the Ig superfamily, during synapse remodeling. We show that the ability of FasII to stimulate or to prevent synapse formation depends on the symmetry of transmembrane FasII levels in the presynaptic and postsynaptic cell and requires the presence of the fly homolog of amyloid precursor protein (APPL). In turn, APPL is regulated by direct interactions with the PDZ (postsynaptic density-95/Discs large/zona occludens-1)-containing protein dX11/Mint/Lin-10, which also regulates synapse expansion downstream of FasII. These results provide a novel mechanism by which cell adhesion molecules are regulated and provide fresh insights into the normal operation of APP during synapse development.

FASt remodeling of synapses in Drosophila.

Cell adhesion molecules of the immunoglobulin superfamily (IgCAMS) play pivotal roles during synapse development and remodeling, being required in both activity-dependent and activity independent aspects of these processes. Recent advances using the fruit fly neuromuscular junction, as well as the mollusk Aplysia californica, have provided evidence for at least three mechanisms by which levels of IgCAMs are regulated during synapse remodeling--activity-dependent regulation of IgCAM clustering by PSD-95-type molecules, IgCAM internalization by MAP kinase pathway activation, and postsynaptic IgCAM exocytosis. These studies offer convincing evidence that synaptic cell adhesion brings about both negative and positive forces that stabilize synapses, while maintaining their ability to change in an activity-dependent manner.

Influence of the nest environment on bone mineral content in hatchling painted turtles (Chrysemys picta).

We performed an experiment at a field site in north-central Nebraska to assess the role of the nest environment in inducing variation in bone mineral content in hatchling painted turtles Chrysemys picta (Schneider 1783). The contents of several newly constructed nests were manipulated by reciprocal transplant, after which the eggs were allowed to incubate for 8 wk under natural conditions. The nests were then excavated, and the eggs were brought into the laboratory to complete incubation and hatch under standard conditions of temperature and moisture. The hatchlings were killed, and their carcasses and residual yolks were analyzed separately for calcium and phosphorus. More of the random variation in carcass calcium and phosphorus was related to the nest in which eggs incubated (37% and 42%, respectively) than was associated with the clutch of origin (21% and 37%). Moreover, hatchlings from some nests contained substantially more calcium and phosphorus than did hatchlings from other nests, both in terms of the absolute amounts of the elements in their carcasses (pointing to variation in body size) and in terms of the concentrations of those elements (pointing to variation in bone density). The amounts of calcium and phosphorus in carcasses of hatchlings were positively correlated with changes in mass of their eggs during the 8 wk that the eggs incubated in nests in the field, thereby indicating that the influence of the nest environment on developing embryos probably was mediated by water exchanges experienced by the eggs. These findings indicate that developmental plasticity underlies a major fraction of the variation in mineral content of hatchling painted turtles emerging from nests in the field. Phenotypic variation attributable to plasticity consequently needs to be addressed in models for life-history evolution of painted turtles and other chelonians producing eggs with soft, flexible shells.

Performance of African American preschool and kindergarten students on the expressive vocabulary test.

PURPOSE: To examine the validity of the Expressive Vocabulary Test (EVT; K. Williams, 1997) for assessing the expressive vocabulary skills of African American students. METHOD/RESULTS: One hundred sixty-five African American preschool and kindergarten students were administered the EVT. The mean EVT score for these African American students was 96.44 (SD = 11.42), which is not appreciably lower than the standardized mean of 100 (SD=15). CLINICAL IMPLICATIONS: Scores were normally distributed, indicating that the EVT is culturally fair and appropriate for use with some African American preschool and kindergarten children as part of an early screening battery. The importance of culturally fair vocabulary measures is discussed relative to this population.

Regulation of synaptic plasticity and synaptic vesicle dynamics by the PDZ protein Scribble.

The Drosophila tumor suppressor Scribble (Scrib) is a PDZ-containing protein required for maintaining epithelial cell polarity. At the larval neuromuscular junction, Scrib colocalizes and indirectly interacts with another tumor suppressor and PDZ protein, Discs-Large (Dlg). Previous studies demonstrate that Dlg is critical for development of normal synapse structure and function, as well as for normal synaptic Scrib localization. Here we show that Scrib is also an important regulator of synaptic architecture and physiology. The most notable ultrastructural defect in scrib mutants is an increase in the number of synaptic vesicles in an area of the synaptic bouton thought to contain the reserve vesicle pool. Additionally, the number of active zones is reduced in scrib mutants. Functionally, the scrib synapse behaves relatively normally at low-frequency stimulation. However, several forms of plasticity at this synapse are drastically altered in the mutants. Specifically, scrib mutants exhibit loss of facilitation and post-tetanic potentiation, and faster synaptic depression. In addition, FM1-43 imaging of recycling synaptic vesicles shows that vesicle dynamics are impaired in scrib mutants. These results identify Scrib as an essential regulator of short-term synaptic plasticity. Taken together, our results are consistent with a model in which Scrib is required to sustain synaptic vesicle concentrations at their sites of release.

Nucleus to Synapse Nesprin1 Railroad Tracks Direct Synapse Maturation through RNA Localization.

An important mechanism underlying synapse development and plasticity is the localization of mRNAs that travel from the nucleus to synaptic sites. Here we demonstrate that the giant nuclear-associated Nesprin1 (dNesp1) forms striated F-actin-based filaments, which we dubbed "railroad tracks," that span from muscle nuclei to postsynaptic sites at the neuromuscular junction in Drosophila. These railroad tracks specifically wrap around immature boutons formed during development and in response to electrical activity. In the absence of dNesp1, mRNAs normally localized at postsynaptic sites are lacking and synaptic maturation is inhibited. This dNesp1 function does not depend on direct association of dNesp1 isoforms with the nuclear envelope. We also show that dNesp1 functions with an unconventional myosin, Myo1D, and that both dNesp1 and Myo1D are mutually required for their localization to immature boutons. These studies unravel a novel pathway directing the transport of mRNAs from the nucleus to postsynaptic sites during synaptic maturation. VIDEO ABSTRACT.

Environmentally induced variation in body size of turtles hatching in natural nests.

Eggs from three snapping turtles (Chelydra serpentina) were divided between two natural nests in a factorial experiment assessing the role of the nest environment as a cause for variation in body size and energy reserves of hatchlings at our study site in northcentral Nebraska. Nest # 1 was located in an unshaded area on the south side of a high sandhill, whereas nest #2 was located in an unshaded area on level ground. Eggs in nest #1 increased in mass over the course of incubation, with eggs at the bottom of the nest gaining more mass than eggs nearer to the surface. In constrast, eggs in nest #2 lost mass during incubation, with eggs at the bottom declining less in mass than eggs at the top of the cavity. Hatchlings from nest #1 were much larger (but contained smaller masses of unused yolk) than hatchlings from nest #2. Additionally, eggs from the lower layers in both nests tended to produce larger hatchings (but with smaller masses of unused yolk) than eggs from the upper layers. Thus, ecologically important variation in body size and nutrient reserves of hatchling snapping turtles results from variation in the environment among and within nests.

Accumulation of lactate by frozen painted turtles (Chrysemys picta) and its relationship to freeze tolerance.

Hatchling painted turtles (Chrysemys picta) survived freezing at -2 degrees C for 4 d, few recovered from freezing lasting 6 d, and none survived being frozen for 8 d. Whole-body glucose and lactate were low in animals that had not been subjected to cold and ice but increased precipitously in animals that were frozen for 2 d. Both metabolites continued to increase, but at a somewhat lower rate, in animals frozen for 4, 6, or 8 d. The increase in whole-body lactate reflects a reliance by frozen hatchlings on anaerobiosis, whereas the increase in glucose presumably results from mobilization of glycogen reserves to support anaerobic metabolism. Mortality of frozen hatchlings is correlated with the increase in whole-body lactate. Factors that may contribute to the observed correlation include a compromised capacity for individual organs to cope with the lactic acidosis that accompanies anaerobic metabolism and organ-specific depletion of energy reserves. Individual organs must rely on buffering and glucose reserves available in situ because blood of frozen hatchlings does not circulate. Thus, buffer from the shell cannot be transported to other organs, lactate cannot be sequestered in the shell, and glucose mobilized from liver glycogen is not available to supplement glucose reserves of other tissues. This integrated suite of physiological disruptions may limit tolerance of freezing to conditions with little or no ecological relevance.

The relationship between gut contents and supercooling capacity in hatchling painted turtles (Chrysemys picta).

Painted turtles (Chrysemys picta) typically spend their first winter of life in a shallow, subterranean hibernaculum (the natal nest) where they seemingly withstand exposure to ice and cold by resisting freezing and becoming supercooled. However, turtles ingest soil and fragments of eggshell as they are hatching from their eggs, and the ingestate usually contains efficient nucleating agents that cause water to freeze at high subzero temperatures. Consequently, neonatal painted turtles have only a modest ability to undergo supercooling in the period immediately after hatching. We studied the limit for supercooling (SCP) in hatchlings that were acclimating to different thermal regimes and then related SCPs of the turtles to the amount of particulate matter in their gastrointestinal (GI) tract. Turtles that were transferred directly from 26 degrees C (the incubation temperature) to 2 degrees C did not purge soil from their gut, and SCPs for these animals remained near -4 degrees C for the 60 days of the study. Animals that were held at 26 degrees C for the duration of the experiment usually cleared soil from their GI tract within 24 days, but SCPs for these turtles were only slightly lower after 60 days than they were at the outset of the experiment. Hatchlings that were acclimating slowly to 2 degrees C cleared soil from their gut within 24 days and realized a modest reduction in their SCP. However, the limit of supercooling in the slowly acclimating animals continued to decline even after all particulate material had been removed from their GI tract, thereby indicating that factors intrinsic to the nucleating agents themselves also may have been involved in the acclimation of hatchlings to low temperature. The lowest SCPs for turtles that were acclimating slowly to 2 degrees C were similar to SCPs recorded in an earlier study of animals taken from natural nests in late autumn, so the current findings affirm the importance of seasonally declining temperatures in preparing animals in the field to withstand conditions that they will encounter during winter.

Patterns of variation in glycogen, free glucose and lactate in organs of supercooled hatchling painted turtles (Chrysemys picta).

Hatchling painted turtles (Chrysemys picta) typically spend their first winter of life in a shallow, subterranean hibernaculum (the natal nest), where they may be exposed for extended periods to ice and cold. The key to their survival seems to be to avoid freezing and to sustain a state of supercooling. As temperature declines below 0 degrees C, however, the heart of an unfrozen turtle beats progressively slower, the diminished perfusion of peripheral tissues with blood induces a functional hypoxia, and anaerobic glycolysis assumes ever greater importance as a source of ATP. We hypothesized that diminished circulatory function in supercooled turtles also reduces the delivery of metabolic substrates to peripheral tissues from central stores in the liver, so that the tissues depend increasingly on endogenous stores to fuel their metabolism. We discovered in the current investigation that part of the glycogen reserve in hearts and brains of hatchlings is mobilized during the first 10 days of exposure to -6 degrees C but that glucose from hepatic glycogen supports metabolism of the organs thereafter. Hatchlings that were held at -6 degrees C for 10 days and then at +3 degrees C for another 10 days were able to reconstitute some of the reserve of glycogen in heart and liver but not the glycogen reserve in brain. Patterns of accumulation of lactate in individual organs were very similar to those reported for whole animals in a companion study, and point to a high degree of reliance on anaerobic metabolism at -6 degrees C and to a lesser degree of reliance on anaerobiosis at higher subzero temperatures. Lactate had returned to baseline levels in organs of animals that were held for 10 days at -6 degrees C and for another 10 days at +3 degrees C, but free glucose remained elevated. Indeed, carbohydrate metabolism probably does not return to the pre-exposure state in any of the major organs until well after the exposure to subzero temperatures has ended, circulatory sufficiency has been restored, and aerobic respiration has fully supplanted anaerobic respiration as a source of ATP.