Sherry G. Clendenon

A Multi-cell, Multi-scale Model of Vertebrate Segmentation and Somite Formation

Somitogenesis, the formation of the bodys primary segmental structure common to all vertebrate development, requires coordination between biological mechanisms at several scales. Explaining how these mechanisms interact across scales and how events are coordinated in space and time is necessary for a complete understanding of somitogenesis and its evolutionary flexibility. So far, mechanisms of somitogenesis have been studied independently. To test the consistency, integrability and combined explanatory power of current prevailing hypotheses, we built an integrated clock-and-wavefront model including submodels of the intracellular segmentation clock, intercellular segmentation-clock coupling via Delta/Notch signaling, an FGF8 determination front, delayed differentiation, clock-wavefront readout, and differential-cell-cell-adhesion-driven cell sorting. We identify inconsistencies between existing submodels and gaps in the current understanding of somitogenesis mechanisms, and propose novel submodels and extensions of existing submodels where necessary. For reasonable initial conditions, 2D simulations of our model robustly generate spatially and temporally regular somites, realistic dynamic morphologies and spontaneous emergence of anterior-traveling stripes of Lfng. We show that these traveling stripes are pseudo-waves rather than true propagating waves. Our model is flexible enough to generate interspecies-like variation in somite size in response to changes in the PSM growth rate and segmentation-clock period, and in the number and width of Lfng stripes in response to changes in the PSM growth rate, segmentation-clock period and PSM length.

Zebrafish fetal alcohol syndrome model: effects of ethanol are rescued by retinoic acid supplement

This study was designed to develop a zebrafish experimental model to examine defects in retinoic acid (RA) signaling caused by embryonic ethanol exposure. RA deficiency may be a causative factor leading to a spectrum of birth defects classified as fetal alcohol spectrum disorder (FASD). Experimental support for this hypothesis using Xenopus showed that effects of treatment with ethanol could be partially rescued by adding retinoids during ethanol treatment. Previous studies show that treating zebrafish embryos during gastrulation and somitogenesis stages with a pathophysiological concentration of ethanol (100mM) produces effects that are characteristic features of FASD. We found that treating zebrafish embryos with RA at a low concentration (10(-9)M) and 100mM ethanol during gastrulation and somitogenesis stages significantly rescued a spectrum of defects produced by treating embryos with 100mM ethanol alone. The rescued phenotype that we observed was quantitatively more similar to embryos treated with 10(-9)M RA alone (RA toxicity) than to untreated or 100mM ethanol-treated embryos. RA rescued defects caused by 100mM ethanol treatment during gastrulation and somitogenesis stages that include early gastrulation cell movements (anterior-posterior axis), craniofacial cartilage formation, and ear development. Morphological evidence also suggests that other characteristic features of FASD (e.g., neural axis patterning) are rescued by RA supplement.

Gα12/13 regulate epiboly by inhibitingE-cadherin activity and modulating the actin cytoskeleton

Epiboly spreads and thins the blastoderm over the yolk cell during zebrafish gastrulation, and involves coordinated movements of several cell layers. Although recent studies have begun to elucidate the processes that underlie these epibolic movements, the cellular and molecular mechanisms involved remain to be fully defined. Here, we show that gastrulae with altered Gα12/13 signaling display delayed epibolic movement of the deep cells, abnormal movement of dorsal forerunner cells, and dissociation of cells from the blastoderm, phenocopying e-cadherin mutants. Biochemical and genetic studies indicate that Gα12/13 regulate epiboly, in part by associating with the cytoplasmic terminus of E-cadherin, and thereby inhibiting E-cadherin activity and cell adhesion. Furthermore, we demonstrate that Gα12/13 modulate epibolic movements of the enveloping layer by regulating actin cytoskeleton organization through a RhoGEF/Rho-dependent pathway. These results provide the first in vivo evidence that Gα12/13 regulate epiboly through two distinct mechanisms: limiting E-cadherin activity and modulating the organization of the actin cytoskeleton.

The effects of spherical aberration on multiphoton fluorescence excitation microscopy

Multiphoton fluorescence excitation microscopy is almost invariably conducted with samples whose refractive index differ from that of the objective immersion medium, conditions that cause spherical aberration. Due to the quadratic nature of multiphoton fluorescence excitation, spherical aberration is expected to profoundly affect the depth dependence of fluorescence excitation. In order to determine the effect of refractive index mismatch in multiphoton fluorescence excitation microscopy, we measured signal attenuation, photobleaching rates and resolution degradation with depth in homogeneous samples with minimal light scattering and absorption over a range of refractive indices. These studies demonstrate that signal levels and resolution both rapidly decline with depth into refractive index mismatched samples. Analyses of photobleaching rates indicate that the preponderance of signal attenuation with depth results from decreased rates of fluorescence excitation, even in a system with a descanned emission collection pathway. Similar results were obtained in analyses of fluorescence microspheres embedded in rat kidney tissue, demonstrating that spherical aberration is an important limiting factor in multiphoton fluorescence excitation microscopy of biological samples.

Cadherin-11 controls otolith assembly: Evidence for extracellular cadherin activity

Cadherin‐11/Cdh11 is expressed through early development and strongly during inner ear development (otic placode and vesicle). Here we show that antisense knockdown of Cdh11 during early zebrafish development interferes with otolith formation. Immunofluorescence labeling showed that Cdh11 expression was concentrated on and within the otolith. Cdh11 was faintly detected at the lateral surface (sites of cell–cell contact) of otic epithelial cells and in the cytoplasm. Strongly labeled Cdh11 containing puncta were detected within the otolymph (the fluid within the otic vesicle) and associated with the otolith surface. BODIPY‐ceramine‐labeled vesicular structures detected in the otolymph were larger and more numerous in Cdh11 knockdown embryos. We present evidence supporting a working model that vesicular structures containing Cdh11 (perhaps containing biomineralization components) are exported from the otic epithelium into the otolymph, adhere to one another and to the surface of the growing otolith, facilitating otolith growth. Developmental Dynamics, 2009.

Emergent Stratification in Solid Tumors Selects for Reduced Cohesion of Tumor Cells: A Multi-Cell, Virtual-Tissue Model of Tumor Evolution Using CompuCell3D.

Tumor cells and structure both evolve due to heritable variation of cell behaviors and selection over periods of weeks to years (somatic evolution). Micro-environmental factors exert selection pressures on tumor-cell behaviors, which influence both the rate and direction of evolution of specific behaviors, especially the development of tumor-cell aggression and resistance to chemotherapies. In this paper, we present, step-by-step, the development of a multi-cell, virtual-tissue model of tumor somatic evolution, simulated using the open-source CompuCell3D modeling environment. Our model includes essential cell behaviors, microenvironmental components and their interactions. Our model provides a platform for exploring selection pressures leading to the evolution of tumor-cell aggression, showing that emergent stratification into regions with different cell survival rates drives the evolution of less cohesive cells with lower levels of cadherins and higher levels of integrins. Such reduced cohesivity is a key hallmark in the progression of many types of solid tumors.

A Liver-Centric Multiscale Modeling Framework for Xenobiotics.

We describe a multi-scale, liver-centric in silico modeling framework for acetaminophen pharmacology and metabolism. We focus on a computational model to characterize whole body uptake and clearance, liver transport and phase I and phase II metabolism. We do this by incorporating sub-models that span three scales; Physiologically Based Pharmacokinetic (PBPK) modeling of acetaminophen uptake and distribution at the whole body level, cell and blood flow modeling at the tissue/organ level and metabolism at the sub-cellular level. We have used standard modeling modalities at each of the three scales. In particular, we have used the Systems Biology Markup Language (SBML) to create both the whole-body and sub-cellular scales. Our modeling approach allows us to run the individual sub-models separately and allows us to easily exchange models at a particular scale without the need to extensively rework the sub-models at other scales. In addition, the use of SBML greatly facilitates the inclusion of biological annotations directly in the model code. The model was calibrated using human in vivo data for acetaminophen and its sulfate and glucuronate metabolites. We then carried out extensive parameter sensitivity studies including the pairwise interaction of parameters. We also simulated population variation of exposure and sensitivity to acetaminophen. Our modeling framework can be extended to the prediction of liver toxicity following acetaminophen overdose, or used as a general purpose pharmacokinetic model for xenobiotics.

3D quantitative analyses of angiogenic sprout growth dynamics

Background: Zebrafish intersegmental vessel (ISV) growth is widely used to study angiogenesis and to screen drugs and toxins that perturb angiogenesis. Most current ISV growth assays observe the presence or absence of ISVs or perturbation of ISV morphology but do not measure growth dynamics. We have developed a four‐dimensional (4D, space plus time) quantitative analysis of angiogenic sprout growth dynamics for characterization of both normal and perturbed growth. Results: We tracked the positions of the ISV base and tip for each ISV sprout in 4D. Despite immobilization, zebrafish embryos translocated globally and non‐uniformly during development. We used displacement of the ISV base and the angle between the ISV and the dorsal aorta to correct for displacement and rotation during development. From corrected tip cell coordinates, we computed average ISV trajectories. We fitted a quadratic curve to the average ISV trajectories to produce a canonical ISV trajectory for each experimental group, arsenic treated and untreated. From the canonical ISV trajectories, we computed curvature, average directed migration speed and directionality. Canonical trajectories from treated (arsenic exposed) and untreated groups differed in curvature, average directed migration speed and angle between the ISV and dorsal aorta. Conclusions: 4D analysis of angiogenic sprout growth dynamics: (1) Allows quantitative assessment of ISV growth dynamics and perturbation, and (2) provides critical inputs for computational models of angiogenesis. Developmental Dynamics 242:508–516, 2013.

Zebrafish cadherin‐11 participates in retinal differentiation and retinotectal axon projection during visual system development

Background: Cadherins orchestrate tissue morphogenesis by controlling cell adhesion, migration and differentiation. Various cadherin family members are expressed in the retina and other neural tissues during embryogenesis, regulating development of these tissues. Cadherin‐11 (Cdh11) is expressed in mesenchymal, bone, epithelial, neural and other tissues, and this cadherin was shown to control cell migration and differentiation in neural crest, tumor and bone cells. Our previous studies characterized Cdh11 expression and function in zebrafish. Results: Here, we report effects of Cdh11 loss‐of‐function on visual system development using morpholino oligonucleotide knockdown methods. Cdh11 is expressed in the retina and lens during retinal differentiation. Cdh11 loss‐of‐function produced defects in retinal differentiation and lens development. Cdh11 loss‐of‐function also reduced retinotectal axon projection and organization, consistent with known Cdh11 function in cell migration. Conclusion: Cdh11 expression in the developing visual system and Cdh11 loss‐of‐function phenotype illustrates the critical role for differential cadherin activity in visual system differentiation and organization. Developmental Dynamics 241:442–454, 2012.

Deep tissue fluorescent imaging in scattering specimens using confocal microscopy.

In scattering specimens, multiphoton excitation and nondescanned detection improve imaging depth by a factor of 2 or more over confocal microscopy; however, imaging depth is still limited by scattering. We applied the concept of clearing to deep tissue imaging of highly scattering specimens. Clearing is a remarkably effective approach to improving image quality at depth using either confocal or multiphoton microscopy. Tissue clearing appears to eliminate the need for multiphoton excitation for deep tissue imaging.