Publications by authors named "Scott Holley"

38 Publications

Integrin intra-heterodimer affinity inversely correlates with integrin activatability.

Cell Rep 2021 Jun;35(10):109230

Department of Molecular, Cellular and Developmental Biology, Yale University, 260 Whitney Avenue, New Haven, CT 06520, USA. Electronic address:

Integrins are heterodimeric cell surface receptors composed of an α and β subunit that mediate cell adhesion to extracellular matrix proteins such as fibronectin. We previously studied integrin α5β1 activation during zebrafish somitogenesis, and in the present study, we characterize the integrin αV fibronectin receptors. Integrins are activated via a conformational change, and we perform single-molecule biophysical measurements of both integrin activation via fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) and integrin intra-heterodimer stability via fluorescence cross-correlation spectroscopy (FCCS) in living embryos. We find that integrin heterodimers that exhibit robust cell surface expression, including αVβ3, αVβ5, and αVβ6, are never activated in this in vivo context, even in the presence of fibronectin matrix. In contrast, activatable integrins, such as integrin αVβ1, and alleles of αVβ3, αVβ5, αVβ6 that are biased to the active conformation exhibit poor cell surface expression and have a higher intra-heterodimer dissociation constant (K). These observations suggest that a weak integrin intra-heterodimer affinity decreases integrin cell surface stability and increases integrin activatability.
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http://dx.doi.org/10.1016/j.celrep.2021.109230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8227800PMC
June 2021

Targeted degradation of transcription factors by TRAFTACs: TRAnscription Factor TArgeting Chimeras.

Cell Chem Biol 2021 05 8;28(5):648-661.e5. Epub 2021 Apr 8.

Department of Molecular, Cellular & Developmental Biology, Yale University, New Haven, CT 06511, USA; Department of Chemistry, Yale University, New Haven, CT 06511, USA; Department of Pharmacology, Yale University, New Haven, CT 06511, USA. Electronic address:

Many diseases, including cancer, stem from aberrant activation or overexpression of oncoproteins that are associated with multiple signaling pathways. Although proteins with catalytic activity can be successfully drugged, the majority of other protein families, such as transcription factors, remain intractable due to their lack of ligandable sites. In this study, we report the development of TRAnscription Factor TArgeting Chimeras (TRAFTACs) as a generalizable strategy for targeted transcription factor degradation. We show that TRAFTACs, which consist of a chimeric oligonucleotide that simultaneously binds to the transcription factor of interest (TOI) and to HaloTag-fused dCas9 protein, can induce degradation of the former via the proteasomal pathway. Application of TRAFTACs to two oncogenic TOIs, NF-κB and brachyury, suggests that TRAFTACs can be successfully employed for the targeted degradation of other DNA-binding proteins. Thus, TRAFTAC technology is potentially a generalizable strategy to induce degradation of other transcription factors both in vitro and in vivo.
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http://dx.doi.org/10.1016/j.chembiol.2021.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8524358PMC
May 2021

Mechanics as a Means of Information Propagation in Development.

Bioessays 2020 11 3;42(11):e2000121. Epub 2020 Sep 3.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA.

New research demonstrates that mechanics can serve as a means of information propagation in developing embryos. Historically, the study of embryonic development has had a dichotomy between morphogens and pattern formation on the one hand and morphogenesis and mechanics on the other. Secreted signals are the preeminent means of information propagation between cells and used to control cell fate, while physical forces act downstream or in parallel to shape tissue morphogenesis. However, recent work has blurred this division of function by demonstrating that mechanics can serve as a means of information propagation. Adhesive or repulsive interactions can propagate through a tissue as a wave. These waves are rapid and directional and can be used to control the flux of cells through a developmental trajectory. Here, two examples are reviewed in which mechanics both guides and mediates morphogenesis and two examples in which mechanics intertwines with morphogens to regulate cell fate.
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http://dx.doi.org/10.1002/bies.202000121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7722802PMC
November 2020

Fibronectin is a smart adhesive that both influences and responds to the mechanics of early spinal column development.

Elife 2020 03 31;9. Epub 2020 Mar 31.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, United States.

An extracellular matrix of Fibronectin adheres the neural tube to the two flanking columns of paraxial mesoderm and is required for normal vertebrate development. Here, we find that the bilaterally symmetric interfaces between the zebrafish neural tube and paraxial mesoderm function as optimally engineered adhesive lap joints with rounded edges, graded Fibronectin 'adhesive' and an arced adhesive spew filet. Fibronectin is a 'smart adhesive' that remodels to the lateral edges of the neural tube-paraxial mesoderm interfaces where shear stress is highest. Fibronectin remodeling is mechanically responsive to contralateral variation morphogenesis, and Fibronectin-mediated inter-tissue adhesion is required for bilaterally symmetric morphogenesis of the paraxial mesoderm. Strikingly, however, perturbation of the Fibronectin matrix rescues the neural tube convergence defect of mutants. Therefore, Fibronectin-mediated inter-tissue adhesion dynamically coordinates bilaterally symmetric morphogenesis of the vertebrate trunk but predisposes the neural tube to convergence defects that lead to spina bifida.
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http://dx.doi.org/10.7554/eLife.48964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7108867PMC
March 2020

Organization of Embryonic Morphogenesis via Mechanical Information.

Dev Cell 2019 06 6;49(6):829-839.e5. Epub 2019 Jun 6.

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520, USA. Electronic address:

Embryonic organizers establish gradients of diffusible signaling molecules to pattern the surrounding cells. Here, we elucidate an additional mechanism of embryonic organizers that is a secondary consequence of morphogen signaling. Using pharmacological and localized transgenic perturbations, 4D imaging of the zebrafish embryo, systematic analysis of cell motion, and computational modeling, we find that the vertebrate tail organizer orchestrates morphogenesis over distances beyond the range of morphogen signaling. The organizer regulates the rate and coherence of cell motion in the elongating embryo using mechanical information that is transmitted via relay between neighboring cells. This mechanism is similar to a pressure front in granular media and other jammed systems, but in the embryo the mechanical information emerges from self-propelled cell movement and not force transfer between cells. The propagation likely relies upon local biochemical signaling that affects cell contractility, cell adhesion, and/or cell polarity but is independent of transcription and translation.
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http://dx.doi.org/10.1016/j.devcel.2019.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590525PMC
June 2019

Patterned Disordered Cell Motion Ensures Vertebral Column Symmetry.

Dev Cell 2017 07;42(2):170-180.e5

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA. Electronic address:

The biomechanics of posterior embryonic growth must be dynamically regulated to ensure bilateral symmetry of the spinal column. Throughout vertebrate trunk elongation, motile mesodermal progenitors undergo an order-to-disorder transition via an epithelial-to-mesenchymal transition and sort symmetrically into the left and right paraxial mesoderm. We combine theoretical modeling of cell migration in a tail-bud-like geometry with experimental data analysis to assess the importance of ordered and disordered cell motion. We find that increasing order in cell motion causes a phase transition from symmetric to asymmetric body elongation. In silico and in vivo, overly ordered cell motion converts normal anisotropic fluxes into stable vortices near the posterior tail bud, contributing to asymmetric cell sorting. Thus, disorder is a physical mechanism that ensures the bilateral symmetry of the spinal column. These physical properties of the tissue connect across scales such that patterned disorder at the cellular level leads to the emergence of organism-level order.
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http://dx.doi.org/10.1016/j.devcel.2017.06.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568629PMC
July 2017

A Sawtooth Pattern of Cadherin 2 Stability Mechanically Regulates Somite Morphogenesis.

Curr Biol 2016 Feb 4;26(4):542-9. Epub 2016 Feb 4.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA. Electronic address:

Differential cadherin (Cdh) expression is a classical mechanism for in vitro cell sorting. Studies have explored the roles of differential Cdh levels in cell aggregates and during vertebrate gastrulation, but the role of differential Cdh activity in forming in vivo tissue boundaries and boundary extracellular matrix (ECM) is unclear. Here, we examine the interactions between cell-cell and cell-ECM adhesion during somitogenesis, the formation of the segmented embryonic precursors of the vertebral column and musculature. We identify a sawtooth pattern of stable Cdh2 adhesions in which there is a posterior-to-anterior gradient of stable Cdh2 within each somite, while there is a step-like drop in stable Cdh2 along the somite boundary. Moreover, we find that the posterior somite boundary cells with high levels of stable Cdh2 have the most columnar morphology. Cdh2 is required for maximal cell aspect ratio and thus full epithelialization of the posterior somite. Loss-of-function analysis also indicates that Cdh2 acts with the fibronectin (FN) receptor integrin α5 (Itgα5) to promote somite boundary formation. Using genetic mosaics, we demonstrate that differential Cdh2 levels are sufficient to induce boundary formation, Itgα5 activation, and FN matrix assembly in the paraxial mesoderm. Elevated cytoskeletal contractility is sufficient to replace differential Cdh2 levels in genetic mosaics, suggesting that Cdh2 promotes ECM assembly by increasing cytoskeletal and tissue stiffness along the posterior somite boundary. Throughout somitogenesis, Cdh2 promotes ECM assembly along tissue boundaries and inhibits ECM assembly in the tissue mesenchyme.
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http://dx.doi.org/10.1016/j.cub.2015.12.055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822709PMC
February 2016

Integration of cell-cell and cell-ECM adhesion in vertebrate morphogenesis.

Curr Opin Cell Biol 2015 Oct 17;36:48-53. Epub 2015 Jul 17.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, United States. Electronic address:

In this review, we highlight recent re-evaluations of the classical cell sorting models and their application to understanding embryonic morphogenesis. Modern genetic and biophysical techniques reveal that tissue self-assembly is not solely a result of differential adhesion, but rather incorporates dynamic cytoskeletal tension and extracellular matrix assembly. There is growing evidence that these biomechanical modules cooperate to organize developing tissues. We describe the contributions of Cadherins and Integrins to tissue assembly and propose a model in which these very different adhesive regimes affect the same outcome through separate but convergent mechanisms.
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http://dx.doi.org/10.1016/j.ceb.2015.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4639458PMC
October 2015

Cross-Scale Integrin Regulation Organizes ECM and Tissue Topology.

Dev Cell 2015 Jul 18;34(1):33-44. Epub 2015 Jun 18.

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06511, USA. Electronic address:

The diverse morphologies of animal tissues are underlain by different configurations of adherent cells and extracellular matrix (ECM). Here, we elucidate a cross-scale mechanism for tissue assembly and ECM remodeling involving Cadherin 2, the ECM protein Fibronectin, and its receptor Integrin α5. Fluorescence cross-correlation spectroscopy within the zebrafish paraxial mesoderm mesenchyme reveals a physical association between Integrin α5 on adjacent cell membranes. This Integrin-Integrin complex correlates with conformationally inactive Integrin. Cadherin 2 stabilizes both the Integrin association and inactive Integrin conformation. Thus, Integrin repression within the adherent mesenchymal interior of the tissue biases Fibronectin fibrillogenesis to the tissue surface lacking cell-cell adhesions. Along nascent somite boundaries, Cadherin 2 levels decrease, becoming anti-correlated with levels of Integrin α5. Simultaneously, Integrin α5 clusters and adopts the active conformation and then commences ECM assembly. This cross-scale regulation of Integrin activation organizes a stereotypic pattern of ECM necessary for vertebrate body elongation and segmentation.
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http://dx.doi.org/10.1016/j.devcel.2015.05.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496283PMC
July 2015

Fusaric acid induces a notochord malformation in zebrafish via copper chelation.

Biometals 2015 Aug 26;28(4):783-9. Epub 2015 Apr 26.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.

Over a thousand extracts were tested for phenotypic effects in developing zebrafish embryos to identify bioactive molecules produced by endophytic fungi. One extract isolated from Fusarium sp., a widely distributed fungal genus found in soil and often associated with plants, induced an undulated notochord in developing zebrafish embryos. The active compound was isolated and identified as fusaric acid. Previous literature has shown this phenotype to be associated with copper chelation from the active site of lysyl oxidase, but the ability of fusaric acid to bind copper ions has not been well described. Isothermal titration calorimetry revealed that fusaric acid is a modest copper chelator with a binding constant of 4.4 × 10(5) M(-1). These results shed light on the toxicity of fusaric acid and the potential teratogenic effects of consuming plants infected with Fusarium sp.
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http://dx.doi.org/10.1007/s10534-015-9855-7DOI Listing
August 2015

The tissue mechanics of vertebrate body elongation and segmentation.

Curr Opin Genet Dev 2015 Jun 19;32:106-11. Epub 2015 Mar 19.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, United States. Electronic address:

England's King Richard III, whose skeleton was recently discovered lying ignobly beneath a parking lot, suffered from a lateral curvature of his spinal column called scoliosis. We now know that his scoliosis was not caused by 'imbalanced bodily humors', rather vertebral defects arise from defects in embryonic elongation and segmentation. This review highlights recent advances in our understanding of post-gastrulation biomechanics of the posteriorly advancing tailbud and somite morphogenesis. These processes are beginning to be deciphered from the level of gene networks to a cross-scale physical model incorporating cellular mechanics, the extracellular matrix, and tissue fluidity.
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http://dx.doi.org/10.1016/j.gde.2015.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4470730PMC
June 2015

Modeling the zebrafish segmentation clock's gene regulatory network constrained by expression data suggests evolutionary transitions between oscillating and nonoscillating transcription.

Genetics 2014 Jun 24;197(2):725-38. Epub 2014 Mar 24.

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520

During segmentation of vertebrate embryos, somites form in accordance with a periodic pattern established by the segmentation clock. In the zebrafish (Danio rerio), the segmentation clock includes six hairy/enhancer of split-related (her/hes) genes, five of which oscillate due to negative autofeedback. The nonoscillating gene hes6 forms the hub of a network of 10 Her/Hes protein dimers, which includes 7 DNA-binding dimers and 4 weak or non-DNA-binding dimers. The balance of dimer species is critical for segmentation clock function, and loss-of-function studies suggest that the her genes have both unique and redundant functions within the clock. However, the precise regulatory interactions underlying the negative feedback loop are unknown. Here, we combine quantitative experimental data, in silico modeling, and a global optimization algorithm to identify a gene regulatory network (GRN) designed to fit measured transcriptional responses to gene knockdown. Surprisingly, we find that hes6, the clock gene that does not oscillate, responds to negative feedback. Consistent with prior in silico analyses, we find that variation in transcription, translation, and degradation rates can mediate the gain and loss of oscillatory behavior for genes regulated by negative feedback. Extending our study, we found that transcription of the nonoscillating Fgf pathway gene sef responds to her/hes perturbation similarly to oscillating her genes. These observations suggest a more extensive underlying regulatory similarity between the zebrafish segmentation clock and the mouse and chick segmentation clocks, which exhibit oscillations of her/hes genes as well as numerous other Notch, Fgf, and Wnt pathway genes.
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http://dx.doi.org/10.1534/genetics.114.163642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4063927PMC
June 2014

Cell-fibronectin interactions propel vertebrate trunk elongation via tissue mechanics.

Curr Biol 2013 Jul 27;23(14):1335-41. Epub 2013 Jun 27.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

During embryonic development and tissue homeostasis, cells produce and remodel the extracellular matrix (ECM). The ECM maintains tissue integrity and can serve as a substrate for cell migration. Integrin α5 (Itgα5) and αV (ItgαV) are the α subunits of the integrins most responsible for both cell adhesion to the ECM protein fibronectin (FN) and FN matrix fibrillogenesis. We perform a systems-level analysis of cell motion in the zebrafish tail bud during trunk elongation in the presence and absence of normal cell-FN interactions. Itgα5 and ItgαV have well-described roles in cell migration in vitro. However, we find that concomitant loss of itgα5 and itgαV leads to a trunk elongation defect without substantive alteration of cell migration. Tissue-specific transgenic rescue experiments suggest that the FN matrix on the surface of the paraxial mesoderm is required for body elongation via its role in defining tissue mechanics and intertissue adhesion.
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http://dx.doi.org/10.1016/j.cub.2013.05.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3725194PMC
July 2013

Regulated tissue fluidity steers zebrafish body elongation.

Development 2013 Feb;140(3):573-82

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

The tailbud is the posterior leading edge of the growing vertebrate embryo and consists of motile progenitors of the axial skeleton, musculature and spinal cord. We measure the 3D cell flow field of the zebrafish tailbud and identify changes in tissue fluidity revealed by reductions in the coherence of cell motion without alteration of cell velocities. We find a directed posterior flow wherein the polarization between individual cell motion is high, reflecting ordered collective migration. At the posterior tip of the tailbud, this flow makes sharp bilateral turns facilitated by extensive cell mixing due to increased directional variability of individual cell motions. Inhibition of Wnt or Fgf signaling or cadherin 2 function reduces the coherence of the flow but has different consequences for trunk and tail extension. Modeling and additional data analyses suggest that the balance between the coherence and rate of cell flow determines whether body elongation is linear or whether congestion forms within the flow and the body axis becomes contorted.
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http://dx.doi.org/10.1242/dev.090381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561786PMC
February 2013

Segmental assembly of fibronectin matrix requires rap1b and integrin α5.

Dev Dyn 2013 Feb 7;242(2):122-31. Epub 2013 Jan 7.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06511, USA.

Background: During segmentation of the zebrafish embryo, inside-out signaling activates Integrin α5, which is necessary for somite border morphogenesis. The direct activator of Integrin α5 during this process is unknown. One candidate is Rap1b, a small monomeric GTPase implicated in Integrin activation in the immune system.

Results: Knockdown of rap1b, or overexpression of a dominant negative rap1b, causes a mild axis elongation defect in zebrafish. However, disruption of rap1b function in integrin α5(-/-) mutants results in a strong reduction in Fibronectin (FN) matrix assembly in the paraxial mesoderm and a failure in somite border morphogenesis along the entire anterior-posterior axis. Somite patterning appears unaffected, as her1 oscillations are maintained in single and double morphants/mutants, but somite polarity is gradually lost in itgα5(-/-) ; rap1b MO embryos.

Conclusions: In itgα5(-) (/) (-) mutants, rap1b is required for proper somite border morphogenesis in zebrafish. The loss of somite borders is not a result of aberrant segmental patterning. Rather, somite boundary formation initiates but is not completed, due to the failure to assemble FN matrix along the nascent boundary. We propose a model in which Rap1b activates Integrin/Fibronectin receptors as part of an "inside-out" signaling pathway that promotes Integrin binding to FN, FN matrix assembly, and subsequent stabilization of morphological somite boundaries.
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http://dx.doi.org/10.1002/dvdy.23909DOI Listing
February 2013

Crosstalk between Fgf and Wnt signaling in the zebrafish tailbud.

Dev Biol 2012 Sep 14;369(2):298-307. Epub 2012 Jul 14.

Department of Molecular, Cellular, and Developmental Biology, New Haven, CT, USA.

Fibroblast growth factor (Fgf) and Wnt signaling are necessary for the intertwined processes of tail elongation, mesodermal development and somitogenesis. Here, we use pharmacological modifiers and time-resolved quantitative analysis of both nascent transcription and protein phosphorylation in the tailbud, to distinguish early effects of signal perturbation from later consequences related to cell fate changes. We demonstrate that Fgf activity elevates Wnt signaling by inhibiting transcription of the Wnt antagonists dkk1 and notum1a. PI3 kinase signaling also increases Wnt signaling via phosphorylation of Gsk3β. Conversely, Wnt can increase signaling within the Mapk branch of the Fgf pathway as Gsk3β phosphorylation elevates phosphorylation levels of Erk. Despite the reciprocal positive regulation between Fgf and Wnt, the two pathways generally have opposing effects on the transcription of co-regulated genes. This opposing regulation of target genes may represent a rudimentary relationship that manifests as out-of-phase oscillation of Fgf and Wnt target genes in the mouse and chick tailbud. In summary, these data suggest that Fgf and Wnt signaling are tightly integrated to maintain proportional levels of activity in the zebrafish tailbud, and this balance is important for axis elongation, cell fate specification and somitogenesis.
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http://dx.doi.org/10.1016/j.ydbio.2012.07.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3423502PMC
September 2012

The Her7 node modulates the network topology of the zebrafish segmentation clock via sequestration of the Hes6 hub.

Development 2012 Mar 25;139(5):940-7. Epub 2012 Jan 25.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

Using in vitro and in vivo assays, we define a network of Her/Hes dimers underlying transcriptional negative feedback within the zebrafish segmentation clock. Some of the dimers do not appear to be DNA-binding, whereas those dimers that do interact with DNA have distinct preferences for cis regulatory sequences. Dimerization is specific, with Hes6 serving as the hub of the network. Her1 binds DNA only as a homodimer but will also dimerize with Hes6. Her12 and Her15 bind DNA both as homodimers and as heterodimers with Hes6. Her7 dimerizes strongly with Hes6 and weakly with Her15. This network structure engenders specific network dynamics and imparts greater influence to the Her7 node. Computational analysis supports the hypothesis that Her7 disproportionately influences the availability of Hes6 to heterodimerize with other Her proteins. Genetic experiments suggest that this regulation is important for operation of the network. Her7 therefore has two functions within the zebrafish segmentation clock. Her7 acts directly within the delayed negative feedback as a DNA-binding heterodimer with Hes6. Her7 also has an emergent function, independent of DNA binding, in which it modulates network topology via sequestration of the network hub.
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http://dx.doi.org/10.1242/dev.073544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3274355PMC
March 2012

Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins.

Nat Chem Biol 2011 Jul 3;7(8):538-43. Epub 2011 Jul 3.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA.

The ability to regulate any protein of interest in living systems with small molecules remains a challenge. We hypothesized that appending a hydrophobic moiety to the surface of a protein would mimic the partially denatured state of the protein, thus engaging the cellular quality control machinery to induce its proteasomal degradation. We designed and synthesized bifunctional small molecules to bind a bacterial dehalogenase (the HaloTag protein) and present a hydrophobic group on its surface. Hydrophobic tagging of the HaloTag protein with an adamantyl moiety induced the degradation of cytosolic, isoprenylated and transmembrane HaloTag fusion proteins in cell culture. We demonstrated the in vivo utility of hydrophobic tagging by degrading proteins expressed in zebrafish embryos and by inhibiting Hras1(G12V)-driven tumor progression in mice. Therefore, hydrophobic tagging of HaloTag fusion proteins affords small-molecule control over any protein of interest, making it an ideal system for validating potential drug targets in disease models.
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http://dx.doi.org/10.1038/nchembio.597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3139752PMC
July 2011

Essential roles of fibronectin in the development of the left-right embryonic body plan.

Dev Biol 2011 Jun 3;354(2):208-20. Epub 2011 Apr 3.

Weill Cornell Medical College, Department of Medicine, Division of Cardiology, New York, NY, USA.

Studies in Xenopus laevis suggested that cell-extracellular matrix (ECM) interactions regulate the development of the left-right axis of asymmetry; however, the identities of ECM components and their receptors important for this process have remained unknown. We discovered that FN is required for the establishment of the asymmetric gene expression pattern in early mouse embryos by regulating morphogenesis of the node, while cellular fates of the nodal cells, canonical Wnt and Shh signaling within the node were not perturbed by the absence of FN. FN is also required for the expression of Lefty 1/2 and activation of SMADs 2 and 3 at the floor plate, while cell fate specification of the notochord and the floor plate, as well as signaling within and between these two embryonic organizing centers remained intact in FN-null mutants. Furthermore, our experiments indicate that a major cell surface receptor for FN, integrin α5β1, is also required for the development of the left-right asymmetry, and that this requirement is evolutionarily conserved in fish and mice. Taken together, our studies demonstrate the requisite role for a structural ECM protein and its integrin receptor in the development of the left-right axis of asymmetry in vertebrates.
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http://dx.doi.org/10.1016/j.ydbio.2011.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3225965PMC
June 2011

Expression of the oscillating gene her1 is directly regulated by Hairy/Enhancer of Split, T-box, and Suppressor of Hairless proteins in the zebrafish segmentation clock.

Dev Dyn 2009 Nov;238(11):2745-59

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA.

Somites are segmental units of the mesoderm in vertebrate embryos that give rise to the axial skeleton, muscle, and dermis. Somitogenesis occurs in a periodic manner and is governed by a segmentation clock that causes cells to undergo repeated oscillations of gene expression. Here, we present a detailed analysis of cis-regulatory elements that control oscillating expression of the zebrafish her1 gene in the anterior presomitic mesoderm. We identify binding sites for Her proteins and demonstrate that they are necessary for transcriptional repression. This result confirms that direct negative autoregulation of her gene expression constitutes part of the oscillator mechanism. We also characterize binding sites for fused somites/Tbx24 and Suppressor of Hairless proteins and show that they are required for activation of her1 expression. These data provide the foundation for a precise description of the regulatory grammar that defines oscillating gene expression in the zebrafish segmentation clock.
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http://dx.doi.org/10.1002/dvdy.22100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943421PMC
November 2009

Control of extracellular matrix assembly along tissue boundaries via Integrin and Eph/Ephrin signaling.

Development 2009 Sep 29;136(17):2913-21. Epub 2009 Jul 29.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

Extracellular matrixes (ECMs) coat and subdivide animal tissues, but it is unclear how ECM formation is restricted to tissue surfaces and specific cell interfaces. During zebrafish somite morphogenesis, segmental assembly of an ECM composed of Fibronectin (FN) depends on the FN receptor Integrin alpha5beta1. Using in vivo imaging and genetic mosaics, our studies suggest that incipient Itgalpha5 clustering along the nascent border precedes matrix formation and is independent of FN binding. Integrin clustering can be initiated by Eph/Ephrin signaling, with Ephrin reverse signaling being sufficient for clustering. Prior to activation, Itgalpha5 expressed on adjacent cells reciprocally and non-cell-autonomously inhibits spontaneous Integrin clustering and assembly of an ECM. Surface derepression of this inhibition provides a self-organizing mechanism for the formation and maintenance of ECM along the tissue surface. Within the tissue, interplay between Eph/Ephrin signaling, ligand-independent Integrin clustering and reciprocal Integrin inhibition restricts de novo ECM production to somite boundaries.
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http://dx.doi.org/10.1242/dev.038935DOI Listing
September 2009

Zebrafish whole mount high-resolution double fluorescent in situ hybridization.

J Vis Exp 2009 Mar 25(25). Epub 2009 Mar 25.

Department of Molecular, Cellular and Developmental Biology, Yale University.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red (1,2). This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) (3). The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy (3,4). One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein (5). When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.
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http://dx.doi.org/10.3791/1229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2789764PMC
March 2009

Balancing segmentation and laterality during vertebrate development.

Semin Cell Dev Biol 2009 Jun 25;20(4):472-8. Epub 2008 Nov 25.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8103, USA.

Somites are the mesodermal segments of vertebrate embryos that become the vertebral column, skeletal muscle and dermis. Somites arise within the paraxial mesoderm by the periodic, bilaterally symmetric process of somitogenesis. However, specification of left-right asymmetry occurs in close spatial and temporal proximity to somitogenesis and involves some of the same cell signaling pathways that govern segmentation. Here, we review recent evidence that identifies cross-talk between these processes and that demonstrates a role for retinoic acid in maintaining symmetrical somitogenesis by preventing impingement of left-right patterning signals upon the paraxial mesoderm.
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http://dx.doi.org/10.1016/j.semcdb.2008.11.009DOI Listing
June 2009

Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function.

Development 2008 Jun 14;135(12):2065-70. Epub 2008 May 14.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

Cell division, differentiation and morphogenesis are coordinated during embryonic development, and frequently are in disarray in pathologies such as cancer. Here, we present a zebrafish mutant that ceases mitosis at the beginning of gastrulation, but that undergoes axis elongation and develops blood, muscle and a beating heart. We identify the mutation as being in early mitotic inhibitor 1 (emi1), a negative regulator of the Anaphase Promoting Complex, and use the mutant to examine the role of the cell cycle in somitogenesis. The mutant phenotype indicates that axis elongation during the segmentation period is driven substantially by cell migration. We find that the segmentation clock, which regulates somitogenesis, functions normally in the absence of cell cycle progression, and observe that mitosis is a modest source of noise for the clock. Somite morphogenesis involves the epithelialization of the somite border cells around a core of mesenchyme. As in wild-type embryos, somite boundary cells are polarized along a Fibronectin matrix in emi1(-/-). The mutants also display evidence of segment polarity. However, in the absence of a normal cell cycle, somites appear to hyper-epithelialize, as the internal mesenchymal cells exit the core of the somite after initial boundary formation. Thus, cell cycle progression is not required during the segmentation period for segmentation clock function but is necessary for the normal segmental arrangement of epithelial borders and internal mesenchymal cells.
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http://dx.doi.org/10.1242/dev.022673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2923836PMC
June 2008

Two deltaC splice-variants have distinct signaling abilities during somitogenesis and midline patterning.

Dev Biol 2008 Jun 20;318(1):126-32. Epub 2008 Mar 20.

Department of Molecular, Cellular and Developmental Biology, Yale University, PO Box 208103, New Haven, CT 06520, USA.

Notch signaling is required for many developmental processes, yet differences in the signaling abilities of various Notch ligands are poorly understood. Here, we have isolated a splice variant of the zebrafish Notch ligand deltaC in which the inclusion of the last intron leads to a truncation of the C-terminal 39 amino acids (deltaC(tv2)). We show that, unlike deltaC(tv1), deltaC(tv2) cannot function effectively in somitogenesis but has an enhanced ability to signal during midline development. Additionally, over-expression of deltaC(tv2) preferentially affects anterior midline development, while another Notch ligand, deltaD, shows a posterior bias. Using chimeric Deltas we show that the intracellular domain is responsible for the strength of signal in midline development, while the extracellular domain influences the anterior-posterior bias of the effect. Together our data show that different deltas can signal in biologically distinct ways in both midline formation and somitogenesis. Moreover, it illustrates the importance of cell-type-dependent modifiers of Notch signaling in providing ligand specificity.
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http://dx.doi.org/10.1016/j.ydbio.2008.03.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2442715PMC
June 2008

Oscillators and the emergence of tissue organization during zebrafish somitogenesis.

Trends Cell Biol 2007 Dec 7;17(12):593-9. Epub 2007 Nov 7.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

Genetic networks that include positive and negative feedback can exhibit oscillations. These oscillations are a form of emergence, which is when novel patterns or properties arise during self organization of complex systems. Within the extending trunk and tail of the developing vertebrate embryo, the somitogenesis oscillator governs the periodic formation of segments that ultimately become the vertebral column and musculature. These oscillations occur within the context of noise created by cell movement, mitosis, and stochastic gene expression. Here, we review recent progress in our understanding of the role of the Notch signaling pathway in the zebrafish segmentation oscillator and our appreciation of how the oscillator interfaces with different sources of noise.
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http://dx.doi.org/10.1016/j.tcb.2007.09.005DOI Listing
December 2007

3D cell nuclei segmentation based on gradient flow tracking.

BMC Cell Biol 2007 Sep 4;8:40. Epub 2007 Sep 4.

Center for Bioinformatics, Harvard Center for Neurodegeneration and Repair, Harvard Medical School, Boston, MA, USA.

Background: Reliable segmentation of cell nuclei from three dimensional (3D) microscopic images is an important task in many biological studies. We present a novel, fully automated method for the segmentation of cell nuclei from 3D microscopic images. It was designed specifically to segment nuclei in images where the nuclei are closely juxtaposed or touching each other. The segmentation approach has three stages: 1) a gradient diffusion procedure, 2) gradient flow tracking and grouping, and 3) local adaptive thresholding.

Results: Both qualitative and quantitative results on synthesized and original 3D images are provided to demonstrate the performance and generality of the proposed method. Both the over-segmentation and under-segmentation percentages of the proposed method are around 5%. The volume overlap, compared to expert manual segmentation, is consistently over 90%.

Conclusion: The proposed algorithm is able to segment closely juxtaposed or touching cell nuclei obtained from 3D microscopy imaging with reasonable accuracy.
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http://dx.doi.org/10.1186/1471-2121-8-40DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064921PMC
September 2007

Detection of blob objects in microscopic zebrafish images based on gradient vector diffusion.

Cytometry A 2007 Oct;71(10):835-45

School of Automation, Northwestern Polytechnic University, Xi'an, China.

The zebrafish has become an important vertebrate animal model for the study of developmental biology, functional genomics, and disease mechanisms. It is also being used for drug discovery. Computerized detection of blob objects has been one of the important tasks in quantitative phenotyping of zebrafish. We present a new automated method that is able to detect blob objects, such as nuclei or cells in microscopic zebrafish images. This method is composed of three key steps. The first step is to produce a diffused gradient vector field by a physical elastic deformable model. In the second step, the flux image is computed on the diffused gradient vector field. The third step performs thresholding and nonmaximum suppression based on the flux image. We report the validation and experimental results of this method using zebrafish image datasets from three independent research labs. Both sensitivity and specificity of this method are over 90%. This method is able to differentiate closely juxtaposed or connected blob objects, with high sensitivity and specificity in different situations. It is characterized by a good, consistent performance in blob object detection.
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http://dx.doi.org/10.1002/cyto.a.20436DOI Listing
October 2007

The genetics and embryology of zebrafish metamerism.

Authors:
Scott A Holley

Dev Dyn 2007 Jun;236(6):1422-49

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA.

Somites are the most obvious metameric structures in the vertebrate embryo. They are mesodermal segments that form in bilateral pairs flanking the notochord and are created sequentially in an anterior to posterior sequence concomitant with the posterior growth of the trunk and tail. Zebrafish somitogenesis is regulated by a clock that causes cells in the presomitic mesoderm (PSM) to undergo cyclical activation and repression of several notch pathway genes. Coordinated oscillation among neighboring cells manifests as stripes of gene expression that pass through the cells of the PSM in a posterior to anterior direction. As axial growth continually adds new cells to the posterior tail bud, cells of the PSM become relatively less posterior. This gradual assumption of a more anterior position occurs over developmental time and constitutes part of a maturation process that governs morphological segmentation in conjunction with the clock. Segment morphogenesis involves a mesenchymal to epithelial transition as prospective border cells at the anterior end of the mesenchymal PSM adopt a polarized, columnar morphology and surround a mesenchymal core of cells. The segmental pattern influences the development of the somite derivatives such as the myotome, and the myotome reciprocates to affect the formation of segment boundaries. While somites appear to be serially homologous, there may be variation in the segmentation mechanism along the body axis. Moreover, whereas the genetic architecture of the zebrafish, mouse, and chick segmentation clocks shares many common elements, there is evidence that the gene networks have undergone independent modification during evolution.
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http://dx.doi.org/10.1002/dvdy.21162DOI Listing
June 2007

Priming, initiation and synchronization of the segmentation clock by deltaD and deltaC.

Nat Cell Biol 2007 May 8;9(5):523-30. Epub 2007 Apr 8.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520, USA.

Zebrafish somitogenesis is governed by a segmentation clock that generates oscillations in expression of several Notch pathway genes, including her1, her7 and deltaC. Using a combination of pharmacological inhibition and Mendelian genetics, we show that DeltaD and DeltaC, two Notch ligands, represent functionally distinct signals within the segmentation clock. Using high-resolution fluorescent in situ hybridization, the oscillations were divided into phases based on eight distinct subcellular patterns of mRNA localization for 140,000 cells. her1, her7 and deltaC expression was examined in wild-type, deltaD(-/-) and deltaC(-/-) embryos. We identified areas within the tailbud where the clock is set up in the progenitor cells (priming), where the clock starts running (initiation), and where the clocks of neighbouring cells are entrained (synchronization). We find that the clocks of motile cells are primed by deltaD in a progenitor zone in the posterior tailbud and that deltaD is required for cells to initiate oscillations on exiting this zone. Oscillations of adjacent cells are synchronized and amplified by deltaC in the posterior presomitic mesoderm as cell movement subsides and cells maintain stable neighbour relationships.
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http://dx.doi.org/10.1038/ncb1578DOI Listing
May 2007
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