An interview with Christopher Wylie and Janet Heasman.

The 2014 Society for Developmental Biology (SDB) Lifetime Achievement Award was jointly awarded to Christopher Wylie and Janet Heasman in recognition of their outstanding and sustained contributions to the field. At the 73rd Annual SDB meeting, where they were presented with the award, we asked Chris and Janet about their careers and their advice for young researchers.

The roles of maternal Vangl2 and aPKC in Xenopus oocyte and embryo patterning.

The Xenopus oocyte contains components of both the planar cell polarity and apical-basal polarity pathways, but their roles are not known. Here, we examine the distribution, interactions and functions of the maternal planar cell polarity core protein Vangl2 and the apical-basal complex component aPKC. We show that Vangl2 is distributed in animally enriched islands in the subcortical cytoplasm in full-grown oocytes, where it interacts with a post-Golgi v-SNARE protein, VAMP1, and acetylated microtubules. We find that Vangl2 is required for the stability of VAMP1 as well as for the maintenance of the stable microtubule architecture of the oocyte. We show that Vangl2 interacts with atypical PKC, and that both the acetylated microtubule cytoskeleton and the Vangl2-VAMP1 distribution are dependent on the presence of aPKC. We also demonstrate that aPKC and Vangl2 are required for the cell membrane asymmetry that is established during oocyte maturation, and for the asymmetrical distribution of maternal transcripts for the germ layer and dorsal/ventral determinants VegT and Wnt11. This study demonstrates the interaction and interdependence of Vangl2, VAMP1, aPKC and the stable microtubule cytoskeleton in the oocyte, shows that maternal Vangl2 and aPKC are required for specific oocyte asymmetries and vertebrate embryonic patterning, and points to the usefulness of the oocyte as a model to study the polarity problem.

Nectin-2 and N-cadherin interact through extracellular domains and induce apical accumulation of F-actin in apical constriction of Xenopus neural tube morphogenesis.

Neural tube formation is one of the most dynamic morphogenetic processes of vertebrate development. However, the molecules regulating its initiation are mostly unknown. Here, we demonstrated that nectin-2, an immunoglobulin-like cell adhesion molecule, is involved in the neurulation of Xenopus embryos in cooperation with N-cadherin. First, we found that, at the beginning of neurulation, nectin-2 was strongly expressed in the superficial cells of neuroepithelium. The knockdown of nectin-2 impaired neural fold formation by attenuating F-actin accumulation and apical constriction, a cell-shape change that is required for neural tube folding. Conversely, the overexpression of nectin-2 in non-neural ectoderm induced ectopic apical constrictions with accumulated F-actin. However, experiments with domain-deleted nectin-2 revealed that the intracellular afadin-binding motif, which links nectin-2 and F-actin, was not required for the generation of the ectopic apical constriction. Furthermore, we found that nectin-2 physically interacts with N-cadherin through extracellular domains, and they cooperatively enhanced apical constriction by driving the accumulation of F-actin at the apical cell surface. Interestingly, the accumulation of N-cadherin at the apical surface of neuroepithelium was dependent on the presence of nectin-2, but that of nectin-2 was not affected by depletion of N-cadherin. We propose a novel mechanism of neural tube morphogenesis regulated by the two types of cell adhesion molecules.

Evolving strategies in mechanobiology to more effectively treat damaged musculoskeletal tissues.

In this paper, we had four primary objectives. (1) We reviewed a brief history of the Lissner award and the individual for whom it is named, H.R. Lissner. We examined the type (musculoskeletal, cardiovascular, and other) and scale (organism to molecular) of research performed by prior Lissner awardees using a hierarchical paradigm adopted at the 2007 Biomechanics Summit of the US National Committee on Biomechanics. (2) We compared the research conducted by the Lissner award winners working in the musculoskeletal (MS) field with the evolution of our MS research and showed similar trends in scale over the past 35 years. (3) We discussed our evolving mechanobiology strategies for treating musculoskeletal injuries by accounting for clinical, biomechanical, and biological considerations. These strategies included studies to determine the function of the anterior cruciate ligament and its graft replacements as well as novel methods to enhance soft tissue healing using tissue engineering, functional tissue engineering, and, more recently, fundamental tissue engineering approaches. (4) We concluded with thoughts about future directions, suggesting grand challenges still facing bioengineers as well as the immense opportunities for young investigators working in musculoskeletal research. Hopefully, these retrospective and prospective analyses will be useful as the ASME Bioengineering Division charts future research directions.

The functions of maternal Dishevelled 2 and 3 in the early Xenopus embryo.

Of the three Dishevelled (Dvl) genes, only Dvl2 and Dvl3 are maternally encoded in the frog, Xenopus laevis. We show here by loss of function analysis that single depletion of either Dvl2 or Dvl3 from the oocyte causes the same embryonic phenotype. We find that the effects of loss of function of Dvl2 and 3 together are additive, and that the proteins physically interact, suggesting that both are required in the same complex. We show that maternal Dvl2 and 3 are required for convergence extension movements downstream of the dorsally localized signaling pathway activated by Xnr3, but not downstream of the pathway activated by activin. Also, depletion of maternal Dvl2 and 3 mRNAs causes the up-regulation of a subset of zygotic ectodermal genes, including Foxi1e, with surprisingly no significant effect on the canonical Wnt direct target genes Siamois and Xnr3. We suggest that the likely reason for continued expression of the Wnt target genes in Dvl2/3-depleted embryos is that maternal Dvl mRNA depletion is insufficient to deplete stored punctae of Dvl protein in the oocyte cortex, which may transduce dorsal signaling after fertilization.

IFITM1-mediated cell repulsion controls the initial steps of germ cell migration in the mouse.

In this issue of Developmental Cell, a novel mechanism for the initiation of germ cell migration in the mouse has been identified, based upon differential expression of interferon-inducible transmembrane proteins in the gastrula (Tanaka et al., 2005). Germ cells are displaced by a repulsion mechanism from the posterior mesoderm into the endoderm.

Predicting the viability of beta-lactamase: How folding and binding free energies correlate with beta-lactamase fitness.

One of the long-standing holy grails of molecular evolution has been the ability to predict an organism's fitness directly from its genotype. With such predictive abilities in hand, researchers would be able to more accurately forecast how organisms will evolve and how proteins with novel functions could be engineered, leading to revolutionary advances in medicine and biotechnology. In this work, we assemble the largest reported set of experimental TEM-1 ß-lactamase folding free energies and use this data in conjunction with previously acquired fitness data and computational free energy predictions to determine how much of the fitness of ß-lactamase can be directly predicted by thermodynamic folding and binding free energies. We focus upon ß-lactamase because of its long history as a model enzyme and its central role in antibiotic resistance. Based upon a set of 21 ß-lactamase single and double mutants expressly designed to influence protein folding, we first demonstrate that modeling software designed to compute folding free energies such as FoldX and PyRosetta can meaningfully, although not perfectly, predict the experimental folding free energies of single mutants. Interestingly, while these techniques also yield sensible double mutant free energies, we show that they do so for the wrong physical reasons. We then go on to assess how well both experimental and computational folding free energies explain single mutant fitness. We find that folding free energies account for, at most, 24% of the variance in ß-lactamase fitness values according to linear models and, somewhat surprisingly, complementing folding free energies with computationally-predicted binding free energies of residues near the active site only increases the folding-only figure by a few percent. This strongly suggests that the majority of ß-lactamase's fitness is controlled by factors other than free energies. Overall, our results shed a bright light on to what extent the community is justified in using thermodynamic measures to infer protein fitness as well as how applicable modern computational techniques for predicting free energies will be to the large data sets of multiply-mutated proteins forthcoming.

Plakoglobin is required for maintenance of the cortical actin skeleton in early Xenopus embryos and for cdc42-mediated wound healing.

Early Xenopus embryos are large, and during the egg to gastrula stages, when there is little extracellular matrix, the cytoskeletons of the individual blastomeres are thought to maintain their spherical architecture and provide scaffolding for the cellular movements of gastrulation. We showed previously that depletion of plakoglobin protein during the egg to gastrula stages caused collapse of embryonic architecture. Here, we show that this is due to loss of the cortical actin skeleton after depletion of plakoglobin, whereas the microtubule and cytokeratin skeletons are still present. As a functional assay for the actin skeleton, we show that wound healing, an actin-based behavior in embryos, is also abrogated by plakoglobin depletion. Both wound healing and the amount of cortical actin are enhanced by overexpression of plakoglobin. To begin to identify links between plakoglobin and the cortical actin polymerization machinery, we show here that the Rho family GTPase cdc42, is required for wound healing in the Xenopus blastula. Myc-tagged cdc42 colocalizes with actin in purse-strings surrounding wounds. Overexpression of cdc42 dramatically enhances wound healing, whereas depletion of maternal cdc42 mRNA blocks it. In combinatorial experiments we show that cdc42 cannot rescue the effects of plakoglobin depletion, showing that plakoglobin is required for cdc42-mediated cortical actin assembly during wound healing. However, plakoglobin does rescue the effect of cdc42 depletion, suggesting that cdc42 somehow mediates the distribution or function of plakoglobin. Depletion of alpha-catenin does not remove the cortical actin skeleton, showing that plakoglobin does not mediate its effect by its known linkage through alpha-catenin to the actin skeleton. We conclude that in Xenopus, the actin skeleton is a major determinant of cell shape and overall architecture in the early embryo, and that plakoglobin plays an essential role in the assembly, maintenance, or organization of this cortical actin.

The Xenopus laevis isoform of G protein-coupled receptor 3 (GPR3) is a constitutively active cell surface receptor that participates in maintaining meiotic arrest in X. laevis oocytes.

Oocytes are held in meiotic arrest in prophase I until ovulation, when gonadotropins trigger a subpopulation of oocytes to resume meiosis in a process termed "maturation." Meiotic arrest is maintained through a mechanism whereby constitutive cAMP production exceeds phosphodiesterase-mediated degradation, leading to elevated intracellular cAMP. Studies have implicated a constitutively activated Galpha(s)-coupled receptor, G protein-coupled receptor 3 (GPR3), as one of the molecules responsible for maintaining meiotic arrest in mouse oocytes. Here we characterized the signaling and functional properties of GPR3 using the more amenable model system of Xenopus laevis oocytes. We cloned the X. laevis isoform of GPR3 (XGPR3) from oocytes and showed that overexpressed XGPR3 elevated intraoocyte cAMP, in large part via Gbetagamma signaling. Overexpressed XGPR3 suppressed steroid-triggered kinase activation and maturation of isolated oocytes, as well as gonadotropin-induced maturation of follicle-enclosed oocytes. In contrast, depletion of XGPR3 using antisense oligodeoxynucleotides reduced intracellular cAMP levels and enhanced steroid- and gonadotropin-mediated oocyte maturation. Interestingly, collagenase treatment of Xenopus oocytes cleaved and inactivated cell surface XGPR3, which enhanced steroid-triggered oocyte maturation and activation of MAPK. In addition, human chorionic gonadotropin-treatment of follicle-enclosed oocytes triggered metalloproteinase-mediated cleavage of XGPR3 at the oocyte cell surface. Together, these results suggest that GPR3 moderates the oocyte response to maturation-promoting signals, and that gonadotropin-mediated activation of metalloproteinases may play a partial role in sensitizing oocytes for maturation by inactivating constitutive GPR3 signaling.

The distribution and behavior of extragonadal primordial germ cells in Bax mutant mice suggest a novel origin for sacrococcygeal germ cell tumors.

In the mouse, germ cells that do not reach the genital ridges rapidly die by a wave of apoptosis that requires the pro-apoptotic protein Bax. In Bax-null embryos, large numbers of ectopic (extragonadal) germ cells fail to die. We have studied the fates of these, in an effort to understand the etiology of human extragonadal germ cell tumors, which are thought to arise from ectopic germ cells. We find that ectopic germ cells in which apoptosis is blocked form a heterogeneous population, which partially differentiates along the gonocyte pathway to different extents in different regions of the embryo, and in the two genders. In particular, a previously undescribed population of ectopic germ cells was identified in the tail. These germ cells retained primitive markers for longer than ectopic germ cells in other regions, and represent a possible origin for sacrococcygeal type I extragonadal germ cell tumors found in neonates and infants. This hypothesis is supported, but not proved, by the finding of cells expressing the germ cell marker Oct4 associated with a coccygeal germ cell tumor in a human infant.

Corrosion of nickel-based dental casting alloys.

OBJECTIVES: To study the microstructure, corrosion behaviour and cell culture response of two nickel-based dental casting alloys before and after a heat treatment to simulate porcelain firing. METHODS: The microstructure was studied using scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX). Corrosion behaviour was evaluated by electrochemical measurements in artificial saliva at different values of pH in the presence of a crevice. 3T3 mouse fibroblasts were exposed indirectly to alloy specimens and the number of viable cells counted after 3 and 6 days compared to a control culture. RESULTS: Small changes in microstructure were observed after heat treatment but had a negligible effect on the corrosion properties in the conditions tested. The alloy with a lower bulk level of Cr (12.6 wt.%) showed lower corrosion resistance, indicated by an increased passive current density and this stability was greatly reduced at pH 2.5, where crevice corrosion was observed. Selective dissolution occurred at regions within the microstructure containing lower levels of Cr and Mo. Furthermore, the proliferation of 3T3 mouse fibroblasts was reduced (p<0.05) when exposed indirectly to this alloy. The alloy containing a higher level of Cr (25 wt.%) showed superior corrosion resistance, which was associated with a more uniform distribution of Cr in the alloy microstructure. SIGNIFICANCE: The presence of crevices combined with an inhomogeneous distribution of Cr in the microstructure can lead to accelerated corrosion of Ni-based alloys with lower Cr contents. This effect can be avoided by increasing the Cr content of the alloy.

Nodal signalling in Xenopus: the role of Xnr5 in left/right asymmetry and heart development.

Nodal class TGF-ß signalling molecules play essential roles in establishing the vertebrate body plan. In all vertebrates, nodal family members have specific waves of expression required for tissue specification and axis formation. In Xenopus laevis, six nodal genes are expressed before gastrulation, raising the question of whether they have specific roles or act redundantly with each other. Here, we examine the role of Xnr5. We find it acts at the late blastula stage as a mesoderm inducer and repressor of ectodermal gene expression, a role it shares with Vg1. However, unlike Vg1, Xnr5 depletion reduces the expression of the nodal family member xnr1 at the gastrula stage. It is also required for left/right laterality by controlling the expression of the laterality genes xnr1, antivin (lefty) and pitx2 at the tailbud stage. In Xnr5-depleted embryos, the heart field is established normally, but symmetrical reduction in Xnr5 levels causes a severely stunted midline heart, first evidenced by a reduction in cardiac troponin mRNA levels, while left-sided reduction leads to randomization of the left/right axis. This work identifies Xnr5 as the earliest step in the signalling pathway establishing normal heart laterality in Xenopus.

Primordial germ cell migration.

Mutational and antisense screens in Drosophila and zebrafish, and transcriptional profiling and time-lapse analysis in the mouse, have contributed greatly to our understanding of PGC development. In all three systems, the behavior of PGCs is controlled by growth factors which signal through G-protein coupled receptors and/or tyrosine kinase receptors. Additionally, regulated cell-cell and cell-substrate adhesion is important for PGC motility. Finally, localized growth factors may control PGC survival and consequently PGC position. Chemotaxis, regulated adhesion and cell survival are important for multiple migration processes which occur during development and disease. PGC migration shares these features.

Whole transcriptome expression profiling of mouse limb tendon development by using RNA-seq.

Tendons are fibrous connective tissues that transmit force between muscle and bone. Whereas the molecular and cellular mechanisms of bone and muscle development have been well studied, that of tendon development is poorly understood. Using the Scx-GFP transgenic mice, we isolated GFP(+) cells from the developing mouse limbs at E11.5, E13.5, and E15.5, respectively, and carried out whole transcriptome RNA-seq analysis. Comparing the gene expression profiles of GFP(+) and GFP(-) cells in the E13.5 limb isolated over 1,500 genes that exhibited enrichment of mRNA expression by at least 1.5-fold in the GFP(+) cells. Of these, 778 genes showed expression up-regulated by more than 1.5-fold from E11.5 to E13.5 and 516 genes showed expression up-regulated by more than 1.5-fold from E13.5 to E15.5 in the GFP(+) cell population. Interestingly, over 30 genes encoding transcription factors are among the early-activated genes in the GFP(+) cells. Whole mount and section in situ hybridization analyses showed that many of these transcription factor genes have distinct patterns of expression during limb development and identified Foxf2 expression as a specific marker for differentiated dorsal limb tendon cells. Together, these data provide a valuable resource for further investigation of the molecular mechanisms regulating tendon development.

Wnt signaling activates Shh signaling in early postnatal intervertebral discs, and re-activates Shh signaling in old discs in the mouse.

Intervertebral discs (IVDs) are strong fibrocartilaginous joints that connect adjacent vertebrae of the spine. As discs age they become prone to failure, with neurological consequences that are often severe. Surgical repair of discs treats the result of the disease, which affects as many as one in seven people, rather than its cause. An ideal solution would be to repair degenerating discs using the mechanisms of their normal differentiation. However, these mechanisms are poorly understood. Using the mouse as a model, we previously showed that Shh signaling produced by nucleus pulposus cells activates the expression of differentiation markers, and cell proliferation, in the postnatal IVD. In the present study, we show that canonical Wnt signaling is required for the expression of Shh signaling targets in the IVD. We also show that Shh and canonical Wnt signaling pathways are down-regulated in adult IVDs. Furthermore, this down-regulation is reversible, since re-activation of the Wnt or Shh pathways in older discs can re-activate molecular markers of the IVD that are lost with age. These data suggest that biological treatments targeting Wnt and Shh signaling pathways may be feasible as a therapeutic for degenerative disc disease.

A lifetime of migration.

Careers in any profession can take a curious path. One small choice can seemingly change a career path and chance encounters open doors to new opportunities that take a person in new, unforeseen directions. For Chris Wylie this has certainly been the case. This interview highlights how someone can build a successful career in science, how that career can be fulfilling and fun and at the same time, it is possible to have a family and a life outside of science. Chris has certainly had success in science, having built very successful labs at many institutions and helped found and grow world-renowned research centers. He gives great credit for his success to his longtime collaborator and wife, Janet Heasman. Although they have indeed made major contributions to their chosen fields of study, what is remarkable is the number of trainees that they have had pass through their labs. Ultimately for any scientist that might be their greatest legacy and it is obvious the impact that great mentors such as J.Z. Young and Ruth Bellairs had on how Chris ran his own lab. As Chris moves on to the next stage of his career, it seems likely that he will pursue it with as much vigor and passion as he pursued his love of scientific research and have a lot of fun. I can't wait for the next interview!

Par6b regulates the dynamics of apicobasal polarity during development of the stratified Xenopus epidermis.

During early vertebrate development, epithelial cells establish and maintain apicobasal polarity, failure of which can cause developmental defects or cancer metastasis. This process has been mostly studied in simple epithelia that have only one layer of cells, but is poorly understood in stratified epithelia. In this paper we address the role of the polarity protein Partitioning defective-6 homolog beta (Par6b) in the developing stratified epidermis of Xenopus laevis. At the blastula stage, animal blastomeres divide perpendicularly to the apicobasal axis to generate partially polarized superficial cells and non-polarized deep cells. Both cell populations modify their apicobasal polarity during the gastrula stage, before differentiating into the superficial and deep layers of epidermis. Early differentiation of the epidermis is normal in Par6b-depleted embryos; however, epidermal cells dissociate and detach from embryos at the tailbud stage. Par6b-depleted epidermal cells exhibit a significant reduction in basolaterally localized E-cadherin. Examination of the apical marker Crumbs homolog 3 (Crb3) and the basolateral marker Lethal giant larvae 2 (Lgl2) after Par6b depletion reveals that Par6b cell-autonomously regulates the dynamics of apicobasal polarity in both superficial and deep epidermal layers. Par6b is required to maintain the "basolateral" state in both epidermal layers, which explains the reduction of basolateral adhesion complexes and epidermal cells shedding.

A role for hedgehog signaling in the differentiation of the insertion site of the patellar tendon in the mouse.

Tendons are typically composed of two histologically different regions: the midsubstance and insertion site. We previously showed that Gli1, a downstream effector of the hedgehog (Hh) signaling pathway, is expressed only in the insertion site of the mouse patellar tendon during its differentiation. To test for a functional role of Hh signaling, we targeted the Smoothened (Smo) gene in vivo using a Cre/Lox system. Constitutive activation of the Hh pathway in the mid-substance caused molecular markers of the insertion site, e.g. type II collagen, to be ectopically expressed or up-regulated in the midsubstance. This was confirmed using a novel organ culture method in vitro. Conversely, when Smo was excised in the scleraxis-positive cell population, the development of the fibrocartilaginous insertion site was affected. Whole transcriptome analysis revealed that the expression of genes involved in chondrogenesis and mineralization was down-regulated in the insertion site, and expression of insertion site markers was decreased. Biomechanical testing of murine adult patellar tendon, which developed in the absence of Hh signaling, showed impairment of tendon structural properties (lower linear stiffness and greater displacement) and material properties (greater strain), although the linear modulus of the mutant group was not significantly lower than controls. These studies provide new insights into the role of Hh signaling during tendon development.

The paratenon contributes to scleraxis-expressing cells during patellar tendon healing.

The origin of cells that contribute to tendon healing, specifically extrinsic epitenon/paratenon cells vs. internal tendon fibroblasts, is still debated. The purpose of this study is to determine the location and phenotype of cells that contribute to healing of a central patellar tendon defect injury in the mouse. Normal adult patellar tendon consists of scleraxis-expressing (Scx) tendon fibroblasts situated among aligned collagen fibrils. The tendon body is surrounded by paratenon, which consists of a thin layer of cells that do not express Scx and collagen fibers oriented circumferentially around the tendon. At 3 days following injury, the paratenon thickens as cells within the paratenon proliferate and begin producing tenascin-C and fibromodulin. These cells migrate toward the defect site and express scleraxis and smooth muscle actin alpha by day 7. The thickened paratenon tissue eventually bridges the tendon defect by day 14. Similarly, cells within the periphery of the adjacent tendon struts express these markers and become disorganized. Cells within the defect region show increased expression of fibrillar collagens (Col1a1 and Col3a1) but decreased expression of tenogenic transcription factors (scleraxis and mohawk homeobox) and collagen assembly genes (fibromodulin and decorin). By contrast, early growth response 1 and 2 are upregulated in these tissues along with tenascin-C. These results suggest that paratenon cells, which normally do not express Scx, respond to injury by turning on Scx and assembling matrix to bridge the defect. Future studies are needed to determine the signaling pathways that drive these cells and whether they are capable of producing a functional tendon matrix. Understanding this process may guide tissue engineering strategies in the future by stimulating these cells to improve tendon repair.