Jeffrey D. Brown

Mechanism and function of signal transduction by the Wnt/beta-catenin and Wnt/Ca2+ pathways.

Communication between cells is often mediated by secreted signaling molecules that bind cell surface receptors and modulate the activity of specific intracellular effectors. The Wnt family of secreted glycoproteins is one group of signaling molecules that has been shown to control a variety of developmental processes including cell fate specification, cell proliferation, cell polarity and cell migration. In addition, mis-regulation of Wnt signaling can cause developmental defects and is implicated in the genesis of several human cancers. The importance of Wnt signaling in development and in clinical pathologies is underscored by the large number of primary research papers examining various aspects of Wnt signaling that have been published in the past several years. In this review, we will present a synopsis of current research with particular attention paid to molecular mechanism of Wnt signal transduction and how the mis-regulation of Wnt signaling leads to cancer.

WNTs modulate cell fate and behavior during vertebrate development

Wnt genes encode a family of secreted glycoproteins that modulate cell fate and behavior in embryos through activation of receptor-mediated signaling pathways. Wnt sequences, patterns of expression and activities are highly conserved in evolution, so it has been possible to gain insights into the functions, and mechanisms of action, of the Wnt genes through a synthesis of genetic and cell biological approaches in different organisms. These studies suggest that there are functionally distinct WNT proteins as assayed by the ability to transform cells and by differences in embryonic responses to ectopic WNT signals. Moreover, gain-of-function and loss-of-function studies both support the involvement of Wnt proteins in modulating cell fate and cell behavior during vertebrate development, often through combinatorial interactions with other signaling pathways to regulate gene expression.

A frizzled homolog functions in a vertebrate Wnt signaling pathway

BACKGROUND Wnts are secreted proteins implicated in cell-cell interactions during embryogenesis and tumorigenesis, but receptors involved in transducing Wnt signals have not yet been definitively identified. Members of a large family of putative transmembrane receptors homologous to the frizzled protein in Drosophila have been identified recently in both vertebrates and invertebrates, raising the question of whether they are involved in transducing signals for any known signaling factors. RESULTS To test the potential involvement of frizzled homologs in Wnt signaling, we examined the effects of overexpressing rat frizzled-1 (Rfz-1) on the subcellular distribution of Wnts and of dishevelled, a cytoplasmic component of the Wnt signalling pathway. We demonstrate that ectopic expression of Rfz-1 recruits the dishevelled proten-as well as Xenopus Wnt-8 (Xwnt-8), but not the functionally distinct Xwnt-5A-to the plasma membrane. Moreover, Rfz-1 is sufficient to induce the expression of two Xwnt-8-responsive genes, siamois and Xnr-3, in Xenopus explants in a manner which is antagonized by glycogen synthase kinase-3, which also antagonizes Wnt signaling. When Rfz-1 and Xwnt-8 are expressed together in this assay, we observe greater induction of these genes, indicating that Rfz-1 can synergize with a Wnt. CONCLUSIONS The results demonstrate that a vertebrate frizzled homolog is involved in Wnt signaling in a manner which discriminates between functionally distinct Wnts, which involves translocation of the dishevelled protein to the plasma membrane, and which works in a synergistic manner with Wnts to induce gene expression. These data support the likely function of frizzled homologs as Wnt receptors, or as components of a receptor complex.

Generalized approach for modeling minimally invasive surgery as a stochastic process using a discrete Markov model

Minimally invasive surgery (MIS) involves a multidimensional series of tasks requiring a synthesis between visual information and the kinematics and dynamics of the surgical tools. Analysis of these sources of information is a key step in defining objective criteria for characterizing surgical performance. The Blue DRAGON is a new system for acquiring the kinematics and the dynamics of two endoscopic tools synchronized with the endoscopic view of the surgical scene. Modeling the process of MIS using a finite state model [Markov model (MM)] reveals the internal structure of the surgical task and is utilized as one of the key steps in objectively assessing surgical performance. The experimental protocol includes tying an intracorporeal knot in a MIS setup performed on an animal model (pig) by 30 surgeons at different levels of training including expert surgeons. An objective learning curve was defined based on measuring quantitative statistical distance (similarity) between MM of experts and MM of residents at different levels of training. The objective learning curve was similar to that of the subjective performance analysis. The MM proved to be a powerful and compact mathematical model for decomposing a complex task such as laparoscopic suturing. Systems like surgical robots or virtual reality simulators in which the kinematics and the dynamics of the surgical tool are inherently measured may benefit from incorporation of the proposed methodology.

Biomechanical Properties of Abdominal Organs In Vivo and Postmortem Under Compression Loads

Accurate knowledge of biomechanical characteristics of tissues is essential for developing realistic computer-based surgical simulators incorporating haptic feedback, as well as for the design of surgical robots and tools. As simulation technologies continue to be capable of modeling more complex behavior, an in vivo tissue property database is needed. Most past and current biomechanical research is focused on soft and hard anatomical structures that are subject to physiological loading, testing the organs in situ. Internal organs are different in that respect since they are not subject to extensive loads as part of their regular physiological function. However, during surgery, a different set of loading conditions are imposed on these organs as a result of the interaction with the surgical tools. Following previous research studying the kinematics and dynamics of tool/tissue interaction in real surgical procedures, the focus of the current study was to obtain the structural biomechanical properties (engineering stress-strain and stress relaxation) of seven abdominal organs, including bladder, gallbladder, large and small intestines, liver, spleen, and stomach, using a porcine animal model. The organs were tested in vivo, in situ, and ex corpus (the latter two conditions being postmortem) under cyclical and step strain compressions using a motorized endoscopic grasper and a universal-testing machine. The tissues were tested with the same loading conditions commonly applied by surgeons during minimally invasive surgical procedures. Phenomenological models were developed for the various organs, testing conditions, and experimental devices. A property database-unique to the literature-has been created that contains the average elastic and relaxation model parameters measured for these tissues in vivo and postmortem. The results quantitatively indicate the significant differences between tissue properties measured in vivo and postmortem. A quantitative understanding of how the unconditioned tissue properties and model parameters are influenced by time postmortem and loading condition has been obtained. The results provide the material property foundations for developing science-based haptic surgical simulators, as well as surgical tools for manual and robotic systems.

Structurally Related Receptors and Antagonists Compete for Secreted Wnt Ligands

Since Frzb can block the signaling activity of ectopic Xwnt-8, this raises the question of whether Frzb and Wnts are ever expressed in a manner consistent with their normally functioning in an antagonistic manner. In mouse embryos, Frzb is expressed in the primitive streak during gastrulation, and in mouse and human adults Frzb is expressed in a variety of adult organs, including the heart, brain, and skeletal muscle, pancreas, and kidney (Leyns et al. 1997xLeyns, L., Bouwmeester, T., Kim, S.-H., Piccolo, S., and DeRobertis, E.M. Cell. 1997; 88Abstract | Full Text | Full Text PDF | PubMed | Scopus (505)See all ReferencesLeyns et al. 1997). In Xenopus embryos, Frzb is also expressed during gastrulation, in a remarkable region of the embryo known as the gastrula organizer, or Spemanns organizer (Figure 1Figure 1) (8xLeyns, L., Bouwmeester, T., Kim, S.-H., Piccolo, S., and DeRobertis, E.M. Cell. 1997; 88Abstract | Full Text | Full Text PDF | PubMed | Scopus (505)See all References, 16xWang, S., Krinks, M., Lin, K., Luyten, F.P., and Moos, M. Jr. Cell. 1997; 88Abstract | Full Text | Full Text PDF | Scopus (371)See all References). This dorsal region, when transplanted to the ventral side of a host embryo, is able to induce the formation of a new body axis, complete with notochord, neural structures, and muscle (Spemann 1938xSee all ReferencesSpemann 1938; reviewed byLemaire and Kodjabachian 1996xLemaire, P. and Kodjabachian, L. Trends Genet. 1996; 12: 525–531Abstract | Full Text PDF | PubMed | Scopus (138)See all ReferencesLemaire and Kodjabachian 1996). Like Frzb, Wnts are also expressed in vertebrate embryos and adult tissues, including some of the tissues that express Frzb (9xMcMahon, A.P. Trends Genet. 1992; 8: 236–242Abstract | Full Text PDF | Scopus (89)See all References, 10xNusse, R. and Varmus, H.E. Cell. 1992; 69: 1073–1087Abstract | Full Text PDF | PubMed | Scopus (656)See all References). While careful mapping of the spatial patterns of expression of Frzb, Frizzled homologs, and Wnts has not yet been conducted, the complementary patterns of Frzb and Xwnt-8 in Xenopus (Figure 1Figure 1) are certainly consistent with these secreted factors working in an antagonistic manner in the future mesoderm.The ability of ectopic Frzb to block embryonic responses to ectopic Xwnt-8 does not establish that Frzb normally works to counteract the functions of endogenous Xwnt-8; however, further experiments by Leyns et al. 1997xLeyns, L., Bouwmeester, T., Kim, S.-H., Piccolo, S., and DeRobertis, E.M. Cell. 1997; 88Abstract | Full Text | Full Text PDF | PubMed | Scopus (505)See all ReferencesLeyns et al. 1997 and Wang et al. 1997xWang, S., Krinks, M., Lin, K., Luyten, F.P., and Moos, M. Jr. Cell. 1997; 88Abstract | Full Text | Full Text PDF | Scopus (371)See all ReferencesWang et al. 1997 support this likelihood. As noted above, endogenous Xwnt-8 is expressed in prospective ventro-lateral mesoderm of the gastrula, and its overexpression in regions of the embryo in which it is not normally expressed can divert the fate of those cells to a ventro-lateral mesodermal fate (Christian and Moon 1993xChristian, J.L. and Moon, R.T. Genes Dev. 1993; 7: 13–28CrossRef | PubMedSee all ReferencesChristian and Moon 1993). If a normal function of Xwnt-8 were to participate in forming ventro-lateral mesoderm, then its loss-of-function should perturb the somites and skeletal muscle, which are derived from this region (Figure 1Figure 1). Supporting this role, a dominant negative Xwnt-8 blocks induction of MyoD in the embryo, and inhibits formation of skeletal muscle (Hoppler et al. 1996xHoppler, S., Brown, J.D., and Moon, R.T. Genes Dev. 1996; 10: 2805–2817CrossRef | PubMedSee all ReferencesHoppler et al. 1996). Consistent with Frzb functioning to antagonize endogenous Xwnt-8, ectopic expression of Frzb throughout early Xenopus embryos also blocks induction of MyoD, and inhibits formation of skeletal muscle (8xLeyns, L., Bouwmeester, T., Kim, S.-H., Piccolo, S., and DeRobertis, E.M. Cell. 1997; 88Abstract | Full Text | Full Text PDF | PubMed | Scopus (505)See all References, 16xWang, S., Krinks, M., Lin, K., Luyten, F.P., and Moos, M. Jr. Cell. 1997; 88Abstract | Full Text | Full Text PDF | Scopus (371)See all References). Local ectopic expression of Frzb in ventral blastomeres at the 4-cell stage results in partial dorsalized phenotypes, also likely by antagonizing the ventral signal of endogenous Xwnt-8 (Wang et al. 1997xWang, S., Krinks, M., Lin, K., Luyten, F.P., and Moos, M. Jr. Cell. 1997; 88Abstract | Full Text | Full Text PDF | Scopus (371)See all ReferencesWang et al. 1997). These experiments support the conclusion that endogenous Frzb is a novel antagonist of the functions of some endogenous Wnts, including Xwnt-8.The presence of Frzb in the gastrula organizer may serve several functions, depending upon the largely unknown extent of diffusion of both Frzb and Xwnts. Within the extracellular space of the gastrula organizer, Frzb may locally block the instructive signal of Xwnt-8 which promotes ventro-lateral mesoderm formation, thereby allowing the development of dorsal cell fates. Second, there may be a region at the boundary of expression of Xwnt-8 and Frzb where the extracellular environment contains both proteins, which could yield a graded response to Xwnt-8 in the prospective lateral mesoderm. Third, since signals from the organizer induce and pattern the nervous system (Hemmati-Brivanlou and Melton 1997xHemmati-Brivanlou, A. and Melton, D. Cell. 1997; 88: 13–17Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesHemmati-Brivanlou and Melton 1997), it is possible that Frzb is involved in modulating neural cell fate, or endodermal cell fate, in addition to its likely roles in the mesoderm. Further testing of the functions of Frzb requires both a better understanding of the range of action of Frzb and Wnt signals, as well as genetic loss-of-function data. The loss-of-function studies would presumably need to come from another organism, since methods for interfering with gene expression in Xenopus embryos are not very precise, and the approach of interfering with protein function through design of a dominant negative protein is probably not suitable when the protein in question is essentially a dominant negative protein to begin with.While showing that the gastrula organizer is the source of a Wnt antagonist was completely unexpected, the new work on Frzb also adds to the growing evidence that the gastrula organizer is the surprising source of multiple secreted antagonists. Previously, the gastrula organizer has been shown to be a source of noggin, follistatin, and chordin that can bind to bone morphogenetic proteins (BMPs), thereby preventing their activating receptor-mediated signaling pathways (reviewed by7xLemaire, P. and Kodjabachian, L. Trends Genet. 1996; 12: 525–531Abstract | Full Text PDF | PubMed | Scopus (138)See all References, 3xHemmati-Brivanlou, A. and Melton, D. Cell. 1997; 88: 13–17Abstract | Full Text | Full Text PDF | PubMedSee all References). The consequence of antagonizing BMPs is that some of the dorsal ectodermal tissue then follows a default developmental pathway and becomes the nervous system. These examples underscore the important role of inhibitory signals, along with receptor-activating signals, in establishing regions of distinct cell fate and function in the embryo.In developmental biology one is constantly reminded that modern cell and molecular studies are often filling the gaps in processes identified or suspected many years ago by the embryologists and philosophers who asked how eggs exploit adults to make more eggs. The idea that different regions of embryos can be the sources of different signals that work in gradients, or in synergistic or antagonistic combinations, was well developed from the work of Boveri, Child, Holtfreter, and others (reviewed bySpemann 1938xSee all ReferencesSpemann 1938). Indeed, “the inducing effect of the organizer was, from the beginning, characterized as a releasing one” (Spemann 1938xSee all ReferencesSpemann 1938). One suspects that these embryologists would have been satisfied to learn of the complementary patterns of expression of Xwnt-8 and Frzb and to learn of their antagonistic functions in the Xenopus embryo.sc

The BlueDRAGON - a system for measuring the kinematics and dynamics of minimally invasive surgical tools in-vivo

Minimally invasive surgery involves a multidimensional series of tasks requiring a synthesis between visual information and the kinematics and dynamics of the surgical tools. Analysis of these sources of information is a key step in mastering MIS, but may also be used to define objective criteria for characterizing surgical performance. The BlueDRAGON is a new system for acquiring the kinematics and dynamics of two endoscopic tools synchronized with the visual view of the surgical scene. It includes two four-bar passive mechanisms equipped with position and force torque sensors for measuring the positions and orientations of the two endoscopic tools along with the forces and torques (F/T) applied by the surgeons hands. The methodology of decomposing the surgical task is based on a fully connected, 28 finite-states Markov model where each states corresponded to a fundamental tool/tissue interaction based on the tool kinematics and associated with unique F/T signatures. The experimental protocol includes seven MIS tasks performed on an animal model by 30 surgeons at different levels of their residency training including expert surgeons. From the preliminary analysis of these data, the major differences between residents at different skill levels are discussed. Systems like surgical robots or virtual reality simulators that inherently measure the kinematics and dynamics of the surgical tool may benefit from inclusion of the proposed methodology for the analysis of efficacy and objective evaluation of surgical skills during training.

The blue DRAGON - A system for monitoring the kinematics and the dynamics of endoscopic tools in minimally invasive surgery for objective laparoscopic skill assessment

Minimally invasive surgeiy (MIS) involves a multi-dimensional series of tasks requiring a synthesis between visual information and the kinematics and dynamics of the surgical tools. Analysis of these sources of information is a key step in mastering MIS surgery but may also be used to define objective criteria for characterizing surgical performance. The BIueDRAGON is a new system for acquiring the kinematics and the dynamics of two endoscopic tools along with the visual view of the surgical scene. It includes two four-bar mechanisms equipped with position and force torque sensors for measuring the positions and the orientations (P/O) of two endoscopic tools along with the forces and torques applied by the surgeons hands. The methodology of decomposing the surgical task is based on a fully connected, finite-states (28 states) Markov model where each states corresponded to a fundamental tool/tissue interaction based on the tool kinematics and associated with unique F/T signatures. The experimental protocol included seven MIS tasks performed on an animal model (pig) by 30 surgeons at different levels of their residency training. Preliminary analysis of these data showed that major differences between residents at different skill levels were: (i) the types of tool/tissue interactions being used, (ii) the transitions between tool/tissue interactions being applied by each hand, (iii) time spent while perfonning each tool/tissue interaction, (iv) the overall completion time, and (v) the variable F/T magnitudes being applied by the subjects through the endoscopic tools. Systems like surgical robots or virtual reality simulators that inherently measure the kinematics and the dynamics of the surgical tool may benefit from inclusion of the proposed methodology for analysis of efficacy and objective evaluation of surgical skills during training.

Quantifying Surgeon Grasping Mechanics in Laparoscopy Using the Blue DRAGON System

Mechanical testing of abdominal organs has a profound impact on surgical simulation and surgical robotics development. Due to the nonlinear and viscoelastic nature of soft tissue it is crucial to test them in surgically relevant ranges of applied force, deformation, and duration for incorporating haptic realism into surgical simulators and for safe operation of surgical robots. In order to determine these ranges, a system known as the Blue DRAGON was used to track the motions and the forces applied to surgical tools during live procedures for quantifying how surgeons typically perform a minimally invasive surgical procedure. Thirty-one surgeons of varying skill were recorded performing three different surgical tasks. Grasping force (as applied to the tool handles) and handle angle for each tool were the signals of interest among 26 channels total acquired by the system in real time. These data were analyzed for their magnitudes and frequency content. Using the tool contact state, an algorithm selected tissue grasps to analyze measures during grasps only, as well as obtain grasp durations. The mean force applied to the tool handles during tissue grasps was 8.52 N +/- 2.77 N; maximum force was 68.17 N. Ninety-five percent of the handle angle frequency content was below 1.98 Hz +/- 0.98 Hz. Average grasp time was 2.29 s +/- 1.65 s, and 95% of all grasps were held for 8.86 s +/- 7.06 s or less. The average maximum grasp time during these tasks was 13.37 s +/- 11.42 s. These results form the basis for determining how abdominal tissues are to be mechanically tested in ranges and durations of force and deformation that are surgically realistic. Additionally, this information may serve as design specifications for new surgical robots or haptic simulators.

Computer-controlled motorized endoscopic grasper for in vivo measurement of soft tissue biomechanical characteristics.

Accurate biomechanical characteristics of tissues are essential for developing realistic virtual reality surgical simulators utilizing haptic devices. Surgical simulation technology has progressed rapidly but without a large database of soft tissue mechanical properties with which to incorporate. The device described here is a computer-controlled, motorized endoscopic grasper capable of applying surgically relevant levels of force to tissue in vivo and measuring the tissues force-deformation properties.