A unique feature of Weinstein Conferences is that all session speakers are chosen from submitted abstracts, and laboratory heads are encouraged to yield the podium to graduate students or postdocs. This provides exposure for young scientists and presentations that focus primarily on unpublished results. Meeting organizers arranged the following seven sessions from the abstract pool: Environment and Gene–Environment Interactions in Cardiovascular Defects; Imaging in the Developing Heart; Early Heart Development; Heart Valve Development; Proepicardium, Epicardium, Coronary Vessels, and Mouse Models of Coronary Heart disease (CHD); Myocardium Regulation, Differentiation, and Structure; and Neural Crest, Outflow Tract, and The Anterior Heart Field. The following paragraphs present highlights from each of these sessions.
Environment and Gene-Environmental Interactions
The first Thursday afternoon platform session sought to integrate the increasingly detailed understanding of regulatory pathways in cardiovascular biology with environmental factors that impact on development and disease. Karen Kuehl led off the session with an intriguing talk reporting on spatial associations in Maryland between ecologic exposures to solvent and the prevalence of hypoplastic left heart syndrome (HLH) and coarctation of the aorta (CoAo). Detailed analysis of data showed highly significant spatial clustering of malformations of heart defects, with significant clustering of male cases of HLH and AS, and of cases of CoAo in males and females. In regions where solvents and dioxin were released into the air, AS showed spatial clustering of male cases in a distinct spatial region of agricultural pesticide use. These ecologic associations provide testable hypotheses for gene environment interactions in the etiology of these heart malformations.
The second presentation by Janee Gelineau-van Waes from the University of Nebraska Medical Center investigated the reversal by folic acid administration of heart malformations in folate receptor (Folbp1) knockout mice. Results showed that, whereas Folbp1−/− embryos die in utero by E10.5, low-dose (12.5 mg/kg) daily oral maternal folinic acid supplementation is capable of extending survival until term. The “rescued” Folbp1−/− fetuses on low-dose folinic acid present with significant cardiac outflow tract malformations that can be further prevented when the maternal folinic acid supplementation dose is increased to 25 mg/kg per day. On low-dose folinic acid supplementation, increased apoptosis in the cardiac neural crest, dorsal neural tube, and surrounding ectoderm was observed, along with decreased Pax3 and Wnt1 expression in the dorsal neural tube, aberrant patterns of neural crest cell migration, and an overall reduction in the number of migrating neural crest cells reaching the outflow tract. Ornella Selmin concluded the session with a talk reporting on her work concerning the disparate effects on cardiac gene expression of TCE versus its metabolite TCAA. Vimentin transcript levels were reduced in embryonic rat hearts after maternal exposure to TCE, whereas maternal exposure to TCAA had the opposite effect. To further characterize the molecular mechanisms used by TCE and TCAA to disrupt normal heart development, the effects on vimentin levels of these environmental contaminants was investigated in the H9c2 heart muscle cell line. Similar changes in vimentin levels were observed in culture, indicating a direct effect on muscle cells. It was suggested that vimentin is a good marker for exposure to TCE and TCAA.
Imaging the Developing Heart
The second session of the afternoon brought together a group of speakers to highlight the increasingly sophisticated imaging methods that are being applied to visualize cardiovascular development and function. The first talk by Mesud Yelbuz reported on the direct, time-lapse visualization of cardiovascular development in chicken embryos in shell-less culture. Movies focused on vasculogenesis between 2 and 9 days of development, and particularly the complex process of extraembryonic vasculogenesis. Some embryos moved to shell-less culture died, and movies revealed that they displayed a pattern of cardiac failure that was similar to humans in end-stage heart failure.
Mary Dickinson reported on her work imaging movements of primitive blood cells in mice and zebrafish. Using vital, high-speed confocal microscopy, it was shown in mouse embryos that entry of primitive erythroblasts into the bloodstream results in a period of peak shear stress that correlates with remodeling of the yolk sac vasculature. Analysis of myosin light chain 2–deficient zebrafish embryos showed that mutant fish lacked atrial contractions and also had secondary defects in yolk sac remodeling that was attributed to the lack of laminar flow and erythroblast circulation. Finally, to gain a better understanding of how flow is established, three-dimensional models of young beating hearts were also generated.
The last talk by Linda Leatherbury from Cecelia Lo's laboratory focused on a novel X-linked mutation that causes hypertrophic cardiomyopathy. To characterize the adult phenotype, echocardiograms were performed on mutant and age matched control mice. Of 23 females and 9 males analyzed, 10 females and 1 male had characteristics typical of hypertrophic cardiomyopathy, including increased ventricular wall thickness, reduced chamber size, and increased ejection fractions.
The early evening opening reception on the rooftop pavilion directly above the conference ballroom featured live mariachi music, a regional menu, and desert mountain vistas. As the sun descended beyond the Tucson Mountains, participants moved downstairs for the keynote address by Dr. Christine Seidman, Professor of Genetics and Medicine at Harvard Medical School and Director of the Cardiovascular Genetics Service at Brigham and Women's Hospital. Dr. Seidman provided an illuminating overview of her research program on the integration of functional studies in the mouse with human genetic studies and functional studies in the mouse of mutations in Tbx5, Nkx 2.5, and Id2.
Early Heart Development
Jun Takeuchi opened the first Friday morning session with his work on the role of Smarcd3, a gene encoding Baf60c of the mammalian BAF complex, in heart development and left–right asymmetry initiation. Using murine embryonic stem cell-directed RNAi to knock down Smarcd3 expression, Takeuchi described a range of heart defects, including randomized heart looping, irregular alignment of the node and morphology of nodal cilia, and loss of expression of left–right asymmetry markers such as lefty, pix2, and nodal. Smarcd3 displayed a similar expression pattern to notch1, and was suggested to act as an activator of Notch signaling, specifically functioning as an enhancer of the three-way association of the Notch Intracellular Domain, its DNA binding partner RBPj, and Brg1 (catalytic BAF complex subunit) to potentiate downstream nodal signaling. These data indicate that Baf60c is an important regulator of heart development and embryo patterning.
Brad Davidson discussed the role of the basic helix–loop–helix transcription factor Mesp in heart cell migration. Using Ciona intestinalis as a model, he described lineage tracing experiments with Mesp enhancer and repressor constructs. Expression of a Mesp enhancer construct resulted in the failure of cells to migrate to the head region, resulting to heart formation in the tail. In contrast, expression of a Mesp repressor construct did not inhibit cell migration, but cells were unable to fuse at the midline or form a heart. Mesp appears to perform two independent functions: to repress inhibitors of cell migration and to facilitate heart specification.
Insight into the function of Tbx5 in heart development was introduced by Frank Conlon, who presented evidence in Xenopus that Tbx5 expression in the endoderm at the onset of gastrulation is required to suppress the premature expression of early heart markers. Tbx5 expression was determined to be dependent upon VegT, and Tbx5 misexpression was capable of inducing endodermal gene expression but not markers of terminal cardiac differentiation. Morpholino inhibition of Tbx5 expression resulted in premature entry of cells into the cardiac lineages. These results suggest that Tbx5 is required for the proper timing and order of heart differentiation.
Deborah Yelon used time-lapse microscopy of a GFP-expressing transgene under the control of a cardiac myosin light chain promoter (cmlc2: GFP) to describe endocardium-directed cell movements driving heart tube assembly in zebrafish. Imaging identified two phases of cell movement: medial movement toward the midline and angular movement to a central point in a subset of cells. Using two mutant embryo lines (cloche, lacking endocardium and angular cell movement, and miles apart, in which medial cell movement fails), the two phases of movement were determined to occur independently of one another. Heart tube assembly appeared to be dependent upon molecular interactions with the endocardium.
Heart Valve Development
Tom Doetschman began the session with an interesting look at the regulation of epithelial–mesenchymal transformation (EMT) in endocardial cushion formation by transforming growth factor (TGF) β2. TGFβ2−/− mice showed evidence of endothelial activation, persistent EMT, undifferentiated cushion mesenchyme, up-regulation and phosphorylation of TGFβ1 signaling pathway components, and persistence of the E-cadherin repressor snail. An angiogenic switch model was proposed whereby TGFβ2 signaling controls the balance between activation and quiescence of endothelium. TGFβ2 may function to maintain endothelial homeostasis by modulating TGFβ1 signaling and by balancing ALK1/ALK5 and snail/GSK3β signaling.
Vesa Kaartinen discussed the phenotype of a mouse line carrying a conditional, endothelial-specific deletion of the Alk2 gene (a bone morphogenetic protein [BMP] type 1 receptor). Cardiac defects in these animals included small cardiac cushions and atrioventricular valve and septal defects, resulting from a failure of endothelial cells to contribute to the atrioventricular (AV) canal and valve cushion mesenchyme. Molecular studies indicated a decrease in msx1 and snail. Endothelial cells explanted from knockout mice failed to migrate and retained lower (or absent) phosphorylation content of multiple smad proteins. It was concluded that ALK2 is required for the initiation of TGFβ2/BMP-induced endocardial cushion formation in mouse AV canals.
The roles of BMP and FGF signaling in heart valve formation were discussed by Joy Lincoln, who described the regulation of cartilage and tendon cell lineage markers in valve leaflets and chordae tendineae, respectively. BMP2 treatment of prefused endocardial cushion cultures and whole cushion explants implicated BMP signaling in activation of the cartilage cell lineage markers sox9 and aggrecan, whereas FGF4 treatment activated the tendon cell lineage markers scleraxis and tenascin. Interestingly, antagonism of BMP signaling with noggin inhibited cartilage cell lineage markers but promoted expression of tendon markers. Contrastingly, treatment with the FGF inhibitor SU5402 resulted in the activation of cartilage cell lineage markers and suppression of markers of the tendon cell lineage. It was proposed that BMP and FGF signaling pathways direct cell-fate specification of multipotent valve progenitor cells.
Using Wnt1Cre:R26R and P0Cre:GFP transgenic mouse lines, Tomoki Nakamura observed neural crest cell derivatives in valve leaflets, the outflow tract, and atrioventricular valves that persisted in the adult heart. Neural crest cells failed to express endothelial, fibroblast, or chondrocyte markers, but surprisingly retained neurogenic potential, evidenced by expression of the neural marker tuj1.
Proepicardium, Epicardium, Coronary Vessels, and Mouse Models of CHD
Yasuo Ishii opened the session by proposing that the liver serves as an inducer of the proepicardial organ. In quail–chick chimera experiments, the liver primordium was found to up-regulate expression of the proepicardial organ markers capsulin and WT-1 in mesothelium but not in other tissues. Results suggested that the liver bud is capable of inducing competent mesothelium to form the proepicardial organ.
Ravi Misra discussed identification of a DNA enhancer region in the serum response factor (SRF) gene that directs expression in the proepicardial organ and in proepicardial organ-derived endothelial cells. Regulation of heart-specific expression of SRF was confined to a −1,500-bp promoter region able to drive expression of a reporter gene in the outflow tract (OFT)/AV junction and proepicardial organ. Mutational analysis of this promoter region identified a 270-bp enhancer that when fused to a 322-bp proximal promoter sequence directed proepicardial organ-specific expression. OFT expression, however, appeared to be dependent upon some region in the 322-bp proximal SRF promoter. It was suggested that SRF is expressed in a subset of cells in the proepicardial organ that can form subepicardial mesenchymal cells in a nonepicardial manner.
The presentation by Thomas Brand also focused on gene regulation by BMP signaling, with specific reference to proepicardial organ gene expression. Based upon the expression patterns of BMP2 and BMP4, a functional role for BMP signaling was assessed by implanting BMP or noggin soaked beads into whole embryos, and treatment of proepicardial explants with BMP or noggin in culture. Surprisingly, high concentrations of both molecules interfered with proepicardial marker gene expression and induced cardiac muscle marker genes. Titration studies led to the conclusion that expression of proepicardial marker genes requires a distinct level of BMP signaling.
Monica Zamora from the Riuz-Lozano lab presented studies suggesting that Wnt signaling is important for epicardium formation. The generation of a transgenic mouse line with an epicardial-specific β-catenin mutation proved informative, as animals displayed cardiac defects, including reduced heart size, thin and uncompacted myocardium, disrupted epicardium, and diminished epicardial EMT. Treatment with lithium chloride to activate signaling downstream of Wnt/β-catenin activated epicardial EMT; similar results were obtained using the EMC epicardial cell line. It appears that β-catenin expression in the epicardium is important for normal development of neighboring tissues such as the myocardium.
Ching-Pin Chang described cardiac defects in Pbx mutant mouse lines generated by his group. Pbx1 is a homeobox transcription factor that forms DNA-binding complexes with Hox and Meis genes, and mice lacking Pbx1 displayed persistent truncus arteriosus and brachial arch defects and presented with aberrant subclavian and carotid arteries. Deficiency of Pbx1 coincided with a severe reduction of Pax3 expression in the heart, particularly in the outflow tract, suggesting that Pbx1 regulates Pax3. Interestingly, Pbx and Meis1 were found to bind cooperatively to the Pax3 promoter; a relationship necessary for normal conotruncal development.
The role of neurofibromin (NF-1) was discussed by Fraz Ismat who suggested that NF-1 might regulate ras signaling in cardiovascular development. Increased ras activity was observed in endocardial cushions derived from Nf1 knockout mice and also in endocardial cushions that coincidentally displayed overgrowth resulting from enhanced EMT. Contrastingly, mice expressing a cre-recombinase dependent Nf1 GAP-related domain (GRD) exhibited down-regulated ras activity, and ubiquitous expression of this Nf1 GRD rescued cardiac defects and embryonic lethality in Nf1 null animals.
Myocardium Regulation, Differentiation, and Structure
With a forecast of record temperatures near 108°F, the dry heat hypothesis was slated for testing as the first Saturday morning session got under way. Da-Zhi Wang led off by proposing that the transcription factor myocardin interacts synergistically with Smad1 and Smad4 to regulate cardiac gene expression. The CarG box sequence was determined to be both necessary and sufficient for the synergistic activation of cardiac genes by myocardin and Smad1 but not to depend upon binding of Smad1 to DNA. A direct interaction was mapped to the SAP and Q domains of myocardin. It was concluded that the myocardin:Smad1/4 complex binds SRF to drive expression of cardiac genes.
Joshua Wythe introduced the function of HADP1, a novel pleckstrin homology (PH) domain protein, in cardiovascular development. HADP1 is conserved across species, and in zebrafish is expressed in the heart in a pattern that becomes restricted to the ventricle as development progresses. Morpholino knockdown of HADP1 led to decreased blood circulation and contractility defects, whereas some animals also displayed an abnormal atrium–ventricle boundary angle. It was suggested that HADP1 might play a role in proper assembly of the Z-disk in cardiac muscle sarcomeres.
Youngsook Lee suggested that Jumonji (JMJ) is required for normal cardiac development by regulating cell proliferation by means of an interaction with the retinoblastoma gene product Rb. Using reporter assays, JMJ was determined to indirectly repress the transcriptional activity of ANF by binding to and inhibiting the ANF activators Nkx2.5 and GATA4. Interestingly, cell proliferation was increased in the trabecular layer of transgenic mouse hearts lacking JMJ, and reporter assays suggested this was due to the inability of Rb, in the absence of JMJ, to sequester E2F, which can bind the E2F-dependent promoter and promote cell growth. Youngsook concluded that JMJ plays an important role in cell cycle regulation during cardiovascular development.
Joseph Ruiz reported that SNF1LK, an AMPK family member, might function through its kinase and ubiquitin binding domains as a cell cycle regulator during early cardiogenesis. Expression of a truncated SNF1LK (kinase and ubiquitin binding domains only) in CHO cells induced endoreduplication, a G2/M defect in which multiple rounds of DNA replication occur in the absence of cell division. Expression of the truncated SNF1LK in the C2C12 skeletal muscle cell line caused cells to stall in G2/M and fail to undergo terminal differentiation and form myotubes. Interestingly, in transgenic mice with specific cardiac expression of SNF1LK under the control of the ANF promoter, hypoplastic ventricles and atrial septal defects were observed, suggesting that SNF1LK functions as a cell cycle checkpoint regulator.
Stephan Lange introduced us to the structure and function of the sarcomeric M-band Myomesin and to sarcomeric protein signaling downstream of titin kinase. The presented crystal structure of Myomesin demonstrated how this structural M-band component is able to form antiparallel dimers and subsequently crosslinks the myosin filaments in the M-band. Signaling through the proposed “molecular ruler” titin was suggested to propagate by means of its kinase domain and a cascade of interacting muscle proteins (NBR1, p62, and MURF2), resulting in gene expression regulation by means of SRF. This signaling cascade was verified by transfection studies in cultured cardiomyocytes, and by stimulating mechanical arrest in these cells, it was suggested that the signaling cascade may be regulated mechanically, with titin acting as a mechanical stretch sensor.
Gregor Andelfinger presented data suggesting that the Kruppel-like family member KLF13 regulates transcription of cardiac-specific genes during Xenopus heart development. In cell culture studies, KLF13 was shown to bind a CACCC site in the BNP promoter and to activate transcription cooperatively with GATA4. Morpholino knockdown of KLF13 in Xenopus embryos induced a range of heart defects including ventricular hypoplasia, hypotrabeculation, and atrial septal defects. The early heart markers GATA5, TBX5 and NKX2.5 showed reduced or delayed initiation, whereas GATA6 is normally initiated at stage 15. In later stages, all markers later showed decreased maintenance during cardiac morphogenesis. Time-lapse analysis showed that heart function was disrupted in these embryos, with smaller ventricles exhibiting slower and less vigorous contractions, a phenotype that could be rescued in a dose-dependent manner by coinjection with a mouse KLF13 capped mRNA, which is not blocked by the morpholino.
Xianghu Qu reported the characterization of NDRG2 and NDRG4, and suggested a role for these molecules in early cardiac development. NDRG4 is expressed robustly, and NDRG2 weakly, throughout the embryonic myocardium of Zebrafish and mice. Morpholino knockdown of NDRG4 induced a range of malformations, including pericardial edema, weakened heart beat, and insufficient circulation. Similarly, overexpression of NDRG2 mRNA resulted in defects including pericardial edema and dilated atrium. Coinjection of morpholinos to NDRG4 along with NDRG2 mRNA did not rescue these heart defects, arguing against functional redundancy between these family members.
David Sedmera described a model of chick–quail parabiosis and presented exciting work indicating that circulating bone marrow-derived stem cells could contribute to cardiac lineages during normal cardiac development. Culture of chick and quail embryos together facilitates a connection of blood vessels that permits cells to pass between embryos. Assessment of chick embryos for the presence of quail-derived cells showed that circulating quail cells could incorporate into multiple chick organ systems, including cell populations in the myocardium, valve leaflets, and developing coronary vessels. These results suggested a potential therapeutic role for bone marrow–derived stem cells, and this strategy is being pursued in a chick model of hypoplastic left heart syndrome.
Neural Crest, Outflow Tract, and the Anterior Heart Field
Yi-Hui Chen discussed the function of Msx1 and Msx2 in cardiac neural crest cell regulation. Msx1 and Msx2 have overlapping expression patterns and redundant functions and are expressed in neural crest cells. Mice deficient in both Msx1 and Msx2 displayed incomplete aorticopulmonary septation and inappropriate alignment of the aortic and pulmonary trunk. Using a Wnt1cre/R26R mouse line to follow a subset of neural crest cells, migration defects were not detected in Msx1/Msx2 double-knockout embryos, although cardiac neural crest cell derivatives were more abundant. Hearts displayed decreased p27 expression, implicating loss of cell cycle regulation, showed an up-regulation of BMP signaling (by means of phospho-smad1), and exhibited decreased expression in the ectoderm and pharyngeal mesoderm, and decreased AP-2α and FGF10 in the anterior heart field. It was hypothesized that, in the absence of Msx1 and Msx2, down-regulation of FGF10 and AP-2α expression led to defective postmigratory neural crest cell regulation.
Brian Black described the differential functions of MEF2C in the anterior versus primary heart fields. Analysis of potential upstream regulators of MEF2C implicated multiple modular transcriptional elements, and in particular a regulatory element that was sufficient to drive expression in the secondary heart field throughout development. Activity of this enhancer is dependent upon binding of GATA4 and Islet-1. MEF2C enhancer-expressing cells from the region of the anterior heart field contributed to the right ventricle, outflow tract, and ventricular septum. Mice with a conditional anterior heart field-specific MEF2C gene inactivation exhibited a range of cardiac defects, including outflow tract and ventricular septal defects, suggesting that MEF2C functions in the anterior heart field to direct normal cardiovascular development.
The role of FGFR2-IIIb signaling in outflow tract septation was presented by Stephane Zaffran, who described previously unreported cardiac defects in transgenic mice deficient in either fibroblast growth factor receptor (FGFR) 2-IIIb or FGF10. FGFR2-IIIb mice exhibited ventricular septal defects, overriding aorta, outflow tract defects, and abnormal trabeculation. Interestingly, FGF10 mutants did not display outflow tract defects but demonstrated aberrant heart positioning, suggesting compensation by other FGF family members. Both mutants, however, lacked pulmonary veins and arteries.
Matthew Goddeeris described his graduate studies of hedgehog (Hh) signaling in outflow tract development. Using cre-recombinase–mediated conditional ablation of Hh signaling components from the anterior heart field and cardiac neural crest cells, sonic hedgehog signaling was implicated in normal outflow tract formation and atrioventricular septation, and in normal cardiac neural crest cell migration. Studies demonstrated that a sonic hedgehog signal from the pharyngeal endoderm to the cardiac neural crest and the anterior heart field is required for normal outflow tract patterning. The signaling pathways appear to be complex, and it was suggested that the endoderm might require sonic hedgehog signaling in an autocrine manner to produce a secondary signal needed for proper anterior heart field development.
Jau-Nain Chen introduced the Zebrafish tremblor mutant that exhibits a fibrillation defect, and mapped the mutation to a cardiac-specific sodium calcium exchanger gene, NCX1h, that is expressed specifically in the zebrafish heart. Embryos injected with a morpholino to NCX1h exhibited uncoordinated, individually contracting cardiomyocytes. A calcium uptake assay determined that the tremblor mutant allele, encoding a truncated version of the NCX1h protein, lacked calcium transporter activity and caused mutant cardiomyocytes to become overloaded with calcium and fail to propagate calcium waves. Injection of wild-type NCX1h mRNA rescued the tremblor phenotype and restored normal heartbeat coordination. Furthermore, coinjection of SERCA2 (to facilitate calcium storage) and PMCA (to assist with calcium extrusion) mRNAs restored normal rhythmic contractions, suggesting a critical role for calcium homeostasis in maintaining cardiac rhythm and also that NCX1h is important in normal cardiac function.
The function of cardiac neural crest cells in compaction of the conduction system was presented by Robert Gourdie, who described optical mapping of activation in comparing control versus laser-mediated neural crest cell ablated embryos. When neural crest was ablated at or after Hamburger and Hamilton (HH) stage 34, embryos exhibited larger, less compact, and poorly organized His bundles compared with controls. Electrode stimulation indicated that abnormal His bundles were lacking normal insulation, suggesting that neural crest cells contribute to the insulation around bundles of His in the conduction system.
Eon Joo Park rounded out the session and the conference by discussing the function of FGF8 in normal cardiovascular development. Regional expression of FGF8 is observed in the primitive streak, precardiac mesoderm, and pharyngeal mesoderm, and multiple cardiac defects are apparent in FGF8 knockout mice. Ablation of FGF8 specifically in the early mesoderm resulted in embryonic lethality due to disrupted outflow tract formation and right ventricular hypoplasia. Mice deficient in FGF8 within the anterior heart field and foregut endoderm were viable but uniformly exhibited persistent truncus arteriosus, small pharyngeal arches, and abnormal heart looping. Expression of FGF8 in the pharyngeal ectoderm was implicated in vasculogenesis, as embryos deficient in FGF8 expression in this region exhibited an interrupted aortic arch phenotype. Results indicate that FGF8 has temporospatial-specific functions in normal cardiovascular development.
Sundown Barbecue and Awards
The meeting concluded with an evening barbecue as the thermometer plummeted below 100°F and the sky shaded to purple above the Tucson Mountains and Saguaro National Park. Awards were presented, and several student platform presentations received special mention. Brad Davidson (Berkeley), Joy Lincoln (Cincinnati), and Matthew Goddeeris (Duke) gave very strong platform presentations and the Goddeeris talk was selected for the Best Platform Award. Among the posters, Anne Foley (Burnham Institute) was selected for the Novartis Best Poster Award. Leigh Compton (Vanderbilt), Bin Zou (Vanderbilt), and Audrey Hettinger (Albert Einstein) also received Novartis Poster Awards for their contributions. Travel awards to pay for lodging were awarded to each of the students selected for platform presentation.
By all accounts, the meeting was a resounding success, and participants departed the Old Pueblo with memories of great science, cattle prods, and Skopopellis, plus newfound appreciation for the true meaning of “dry heat.” Special acknowledgment to the organizers: Raymond Runyan, Parker Antin, Carol Gregorio, Scott Klewer, Paul Krieg, Todd Camenisch, Ron Heimark, and Abigail McElhinny. The next meeting is scheduled for May 11–13, 2006 in St. Petersburg, Florida, and information is available at www.weinsteinmeeting.org. Happy Trails!