Epicardially derived fibroblasts preferentially contribute to the parietal leaflets of the atrioventricular valves in the murine heart.

The importance of the epicardium for myocardial and valvuloseptal development has been well established; perturbation of epicardial development results in cardiac abnormalities, including thinning of the ventricular myocardial wall and malformations of the atrioventricular valvuloseptal complex. To determine the spatiotemporal contribution of epicardially derived cells to the developing fibroblast population in the heart, we have used a mWt1/IRES/GFP-Cre mouse to trace the fate of EPDCs from embryonic day (ED)10 until birth. EPDCs begin to populate the compact ventricular myocardium around ED12. The migration of epicardially derived fibroblasts toward the interface between compact and trabecular myocardium is completed around ED14. Remarkably, epicardially derived fibroblasts do not migrate into the trabecular myocardium until after ED17. Migration of EPDCs into the atrioventricular cushion mesenchyme commences around ED12. As development progresses, the number of EPDCs increases significantly, specifically in the leaflets which derive from the lateral atrioventricular cushions. In these developing leaflets the epicardially derived fibroblasts eventually largely replace the endocardially derived cells. Importantly, the contribution of EPDCs to the leaflets derived from the major AV cushions is very limited. The differential contribution of EPDCs to the various leaflets of the atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the pathogenesis of abnormalities that preferentially affect individual components of this region of the heart. The notion that there is a significant difference in the contribution of epicardially and endocardially derived cells to the individual leaflets of the atrioventricular valves has also important pragmatic consequences for the use of endocardial and epicardial cre-mouse models in studies of heart development.

Differential Notch signaling in the epicardium is required for cardiac inflow development and coronary vessel morphogenesis.

RATIONALE: The proepicardium is a transient structure comprising epicardial progenitor cells located at the posterior limit of the embryonic cardiac inflow. A network of signals regulates proepicardial cell fate and defines myocardial and nonmyocardial domains at the venous pole of the heart. During cardiac development, epicardial-derived cells also contribute to coronary vessel morphogenesis. OBJECTIVE: To study Notch function during proepicardium development and coronary vessel formation in the mouse. METHODS AND RESULTS: Using in situ hybridization, RT-PCR, and immunohistochemistry, we find that Notch pathway elements are differentially activated throughout the proepicardial-epicardial-coronary transition. Analysis of RBPJk-targeted embryos indicates that Notch ablation causes ectopic procardiogenic signaling in the proepicardium that in turn promotes myocardial differentiation in adjacent mesodermal progenitors, resulting in a premature muscularization of the sinus venosus horns. Epicardium-specific Notch1 ablation using a Wt1-Cre driver line disrupts coronary artery differentiation, reduces myocardium wall thickness and myocyte proliferation, and reduces Raldh2 expression. Ectopic Notch1 activation disrupts epicardium development and causes thinning of ventricular walls. CONCLUSIONS: Epicardial Notch modulates cell differentiation in the proepicardium and adjacent pericardial mesoderm. Notch1 is later required for arterial endothelium commitment and differentiation and for vessel wall maturation during coronary vessel development and myocardium growth.

Defective ciliogenesis, embryonic lethality and severe impairment of the Sonic Hedgehog pathway caused by inactivation of the mouse complex A intraflagellar transport gene Ift122/Wdr10, partially overlapping with the DNA repair gene Med1/Mbd4.

Primary cilia are assembled and maintained by evolutionarily conserved intraflagellar transport (IFT) proteins that are involved in the coordinated movement of macromolecular cargo from the basal body to the cilium tip and back. The IFT machinery is organized in two structural complexes named complex A and complex B. Recently, inactivation in the mouse germline of Ift genes belonging to complex B revealed a requirement of ciliogenesis, or proteins involved in ciliogenesis, for Sonic Hedgehog (Shh) signaling in mammals. Here we report on a complex A mutant mouse, defective for the Ift122 gene. Ift122-null embryos show multiple developmental defects (exencephaly, situs viscerum inversus, delay in turning, hemorrhage and defects in limb development) that result in lethality. In the node, primary cilia were absent or malformed in homozygous mutant and heterozygous embryos, respectively. Impairment of the Shh pathway was apparent in both neural tube patterning (expansion of motoneurons and rostro-caudal level-dependent contraction or expansion of the dorso-lateral interneurons), and limb patterning (ectrosyndactyly). These phenotypes are distinct from both complex B IFT mutant embryos and embryos defective for the ciliary protein hennin/Arl13b, and suggest reduced levels of both Gli2/Gli3 activator and Gli3 repressor functions. We conclude that complex A and complex B factors play similar but distinct roles in ciliogenesis and Shh/Gli3 signaling.

Tahitian tree snail mitochondrial clades survived recent mass extirpation.

Oceanic islands frequently support endemic faunal radiations that are highly vulnerable to introduced predators [1]. This vulnerability is epitomized by the rapid extinction in the wild of all but five of 61 described Society Islands partulid tree snails [2], following the deliberate introduction of an alien biological control agent: the carnivorous snail Euglandina rosea[3]. Tahiti's tree snail populations have been almost completely extirpated and three of the island's eight endemic Partula species are officially extinct, a fourth persisting only in captivity [2]. We report a molecular phylogenetic estimate of Tahitian Partula mitochondrial lineage survival calibrated with a 1970 reference museum collection that pre-dates the predator's 1974 introduction to the island [4]. Although severe winnowing of lineage diversity has occurred, none of the five primary Tahitian Partula clades present in the museum samples is extinct. Targeted conservation measures, especially of montane refuge populations, may yet preserve a representative sub-sample of Tahiti's endemic tree snail genetic diversity in the wild.

Moorean tree snail survival revisited: a multi-island genealogical perspective.

BACKGROUND: The mass extirpation of the island of Moorea's endemic partulid tree snail fauna, following the deliberate introduction of the alien predator Euglandina rosea, represents one of the highest profile conservation crises of the past thirty years. All of the island's partulids were thought to be extirpated by 1987, with five species persisting in zoos, but intensive field surveys have recently detected a number of surviving wild populations. We report here a mitochondrial (mt) phylogenetic estimate of Moorean partulid wild and captive lineage survival calibrated with a reference museum collection that pre-dates the predator's introduction and that also includes a parallel dataset from the neighboring island of Tahiti. RESULTS: Although severe winnowing of Moorea's mt lineage diversity has occurred, seven of eight (six Partula; two Samoana) partulid tip clades remain extant. The extinct mt clade occurred predominantly in the P. suturalis species complex and it represented a major component of Moorea's endemic partulid treespace. Extant Moorean mt clades exhibited a complex spectrum of persistence on Moorea, in captivity, and (in the form of five phylogenetically distinct sister lineages) on Tahiti. Most notably, three Partula taxa, bearing two multi-island mt lineages, have survived decades of E. rosea predation on Moorea (P. taeniata) and in the valleys of Tahiti (P. hyalina and P. clara). Their differential persistence was correlated with intrinsic attributes, such as taxonomy and mt lineages, rather than with their respective within-island distribution patterns. CONCLUSION: Conservation efforts directed toward Moorean and Tahitian partulids have typically operated within a single island frame of reference, but our discovery of robust genealogical ties among survivors on both islands implies that a multi-island perspective is required. Understanding what genetic and/or ecological factors have enabled Partula taeniata, P. hyalina and P. clara to differentially survive long-term direct exposure to the predator may provide important clues toward developing a viable long term conservation plan for Society Island partulid tree snails.

High-content imaging characterization of cell cycle therapeutics through in vitro and in vivo subpopulation analysis.

Although the cycling of eukaryotic cells has long been a primary focus for cancer therapeutics, recent advances in imaging and data analysis allow even further definition of cellular events as they occur in individual cells and cellular subpopulations in response to treatment. High-content imaging (HCI) has been an effective tool to elucidate cellular responses to a variety of agents; however, these data were most frequently observed as averages of the entire captured population, unnecessarily decreasing the resolution of each assay. Here, we dissect the eukaryotic cell cycle into individual cellular subpopulations using HCI in conjunction with unsupervised K-means clustering. We generate distinct phenotypic fingerprints for each major cell cycle and mitotic compartment and use those fingerprints to screen a library of 310 commercially available chemotherapeutic agents. We determine that the cell cycle arrest phenotypes caused by these agents are similar to, although distinct from, those found in untreated cells and that these distinctions frequently suggest the mechanism of action. We then show via subpopulation analysis that these arrest phenotypes are similar in both mouse models and in culture. HCI analysis of cell cycle using data obtained from individual cells under a broad range of research conditions and grouped into cellular subpopulations represents a powerful method to discern both cellular events and treatment effects. In particular, this technique allows for a more accurate means of assessing compound selectivity and leads to more meaningful comparisons between so-called targeted therapeutics.

Abnormal conduction and morphology in the atrioventricular node of mice with atrioventricular canal targeted deletion of Alk3/Bmpr1a receptor.

BACKGROUND: The atrioventricular (AV) node is essential for the sequential excitation and optimized contraction of the adult multichambered heart; however, relatively little is known about its formation from the embryonic AV canal. A recent study demonstrated that signaling by Alk3, the type 1a receptor for bone morphogenetic proteins, in the myocardium of the AV canal was required for the development of both the AV valves and annulus fibrosus. To test the hypothesis that bone morphogenetic protein signaling also plays a role in AV node formation, we investigated conduction system function and AV node morphology in adult mice with conditional deletion of Alk3 in the AV canal. METHODS AND RESULTS: High-resolution optical mapping with correlative histological analysis of 28 mutant hearts revealed 4 basic phenotypic classes based on electrical activation patterns and volume-conducted ECGs. The frequency of AV node conduction and morphological abnormalities increased from no detectable anomalies (class I) to severe defects (class IV), which included the presence of bypass tracts, abnormal ventricular activation patterns, fibrosis of the AV node, and twin AV nodes. CONCLUSIONS: The present findings demonstrate that bone morphogenetic protein signaling is required in the myocardium of the AV canal for proper AV junction development, including the AV node.

Prehistoric inter-archipelago trading of Polynesian tree snails leaves a conservation legacy.

Inter-archipelago exchange networks were an important aspect of prehistoric Polynesian societies. We report here a novel genetic characterization of a prehistoric exchange network involving an endemic Pacific island tree snail, Partula hyalina. It occurs in the Society (Tahiti only), Austral and Southern Cook Islands. Our genetic data, based on museum, captive and wild-caught samples, establish Tahiti as the source island. The source lineage is polymorphic in shell coloration and contains a second nominal species, the dark-shelled Partula clara, in addition to the white-shelled P. hyalina. Prehistoric inter-island introductions were non-random: they involved white-shelled snails only and were exclusively inter-archipelago in scope. Partulid shells were commonly used in regional Polynesian jewellery, and we propose that the white-shelled P. hyalina, originally restricted to Tahiti, had aesthetic value throughout these archipelagoes. Demand within the Society Islands could be best met by trading dead shells, but a low rate of inter-archipelago exchange may have prompted the establishment of multiple founder populations in the Australs and Southern Cooks. The alien carnivorous land snail Euglandina rosea has recently devastated populations of all 61 endemic species of Society Island partulid snails. Southern Cooks and Australs P. hyalina now represent the only unscathed wild populations remaining of this once spectacular land snail radiation.

Alk3/Bmpr1a receptor is required for development of the atrioventricular canal into valves and annulus fibrosus.

Endocardial cushions are precursors of mature atrioventricular (AV) valves. Their formation is induced by signaling molecules originating from the AV myocardium, including bone morphogenetic proteins (BMPs). Here, we hypothesized that BMP signaling plays an important role in the AV myocardium during the maturation of AV valves from the cushions. To test our hypothesis, we used a unique Cre/lox system to target the deletion of a floxed Alk3 allele, the type IA receptor for BMPs, to cardiac myocytes of the AV canal (AVC). Lineage analysis indicated that cardiac myocytes of the AVC contributed to the tricuspid mural and posterior leaflets, the mitral septal leaflet, and the atrial border of the annulus fibrosus. When Alk3 was deleted in these cells, defects were seen in the same leaflets, ie, the tricuspid mural leaflet and mitral septal leaflet were longer, the tricuspid posterior leaflet was displaced and adherent to the ventricular wall, and the annulus fibrosus was disrupted resulting in ventricular preexcitation. The defects seen in mice with AVC-targeted deletion of Alk3 provide strong support for a role of Alk3 in human congenital heart diseases, such as Ebstein's anomaly. In conclusion, our mouse model demonstrated critical roles for Alk3 signaling in the AV myocardium during the development of AV valves and the annulus fibrosus.

Architectural plan for the heart: early patterning and delineation of the chambers and the nodes.

During folding of the embryo, lateroanterior visceral mesoderm forms the embryonic tubular heart at the midline, just ventral to the foregut. In mice, this nascent tube contains the future left ventricle and atrioventricular canal. Mesenchymal cells subsequently recruited to the cardiac lineage at the intake and the outflow of the tube will form the atria and the right ventricle and outflow tract, respectively. Shortly after its emergence, the embryonic heart tube starts to loop, and the first signs of left ventricular chamber differentiation become visible on the outer curvature of the middle portion of the tube. Subsequently, the right ventricle differentiates cranially, and the atria caudally, while the inflow tract, atrioventricular canal, inner curvatures, and outflow tract form recognizable components flanking the chambers. The latter, nonchamber regions in turn provide signals for the formation of the cushion mesenchyme, are involved in remodeling of the heart, and form the nodes of the conduction system. This review discusses how the patterning of the heart tube relates to the localized differentiation of atrial and ventricular chambers, why some parts of the heart do not form chambers, and how this relates to the formation of the conduction system.

Transcriptional regulation in the mouse atrioventricular conduction system.

We identified a GATA6 gene enhancer that selectively marks the developing atrioventricular conduction system (AVCS) in transgenic mice. This enhancer reads anterior/posterior and medial/lateral positional information early in the cardiogenic programme and remains active in progressively more restricted subsets of heart cells leading up to AVCS formation. Additional experiments will be required to determine if the potential to be recruited into the AVCS is similarly restricted to a subset of myocardial cells early in the cardiogenic programme or if this enhancer can also be activated de novo in cells that initially reside outside this field. We are using several strategies to identify factors that regulate this and other AVCS enhancers and hence govern AVCS function. We are also using this enhancer to make transgenic mice that express Cre, or an inducible form of Cre, to track lineages and to delete floxed genes in the developing or mature AVCS. This Cre/lox approach provides a means to deconstruct complex congenital heart phenotypes that involve the conduction system and to test whether genes are required to form the AVCS or to maintain AVCS function. Lastly, we are exploring strategies to isolate and analyse AVCS cells from normal and affected hearts.

Mouse models for cardiac conduction system development.

The mouse is the animal of choice for the study of molecular mechanisms involved in the regulation of cardiovascular morphogenesis and function. Recently, a series of genetically engineered mouse models have been reported (e.g. cGATA6/lacZ, MinK/lacZ knock-in/knock-out, engrailed2/lacZ, Cardiac troponin I/lacZ) that provide new and exciting information on the development of the atrioventricular conduction system (AVCS). On the basis of these and ongoing studies, concepts for the formation of the AVCS are continuously being adjusted. A proper understanding of the normal developmental mechanisms underlying the cardiac remodelling leading to the formation of the AVCS is imperative for the interpretation of cardiac abnormalities, including conduction disturbances, as observed in some genetically perturbed (knockout) mice. In this paper information on murine AVCS development will be integrated with published and unpublished results from studies in other vertebrates, including human and rabbit. We will illustrate that although many pieces of the puzzle still remain to be gathered, the outline of a very complex and critical event in cardiac morphogenesis is slowly emerging. Specifically, we will re-evaluate the concept of the 'primary ring' in the context of the new insights in the development of the AV junction as provided by the respective mouse models described above.

A cohort study of thyroid cancer and other thyroid diseases after the Chornobyl accident: objectives, design and methods.

The thyroid gland in children is one of the organs that is most sensitive to external exposure to X and gamma rays. However, data on the risk of thyroid cancer in children after exposure to radioactive iodines are sparse. The Chornobyl accident in Ukraine in 1986 led to the exposure of large populations to radioactive iodines, particularly (131)I. This paper describes an ongoing cohort study being conducted in Belarus and Ukraine that includes 25,161 subjects under the age of 18 years in 1986 who are being screened for thyroid diseases every 2 years. Individual thyroid doses are being estimated for all study subjects based on measurement of the radioactivity of the thyroid gland made in 1986 together with a radioecological model and interview data. Approximately 100 histologically confirmed thyroid cancers were detected as a consequence of the first round of screening. The data will enable fitting appropriate dose-response models, which are important in both radiation epidemiology and public health for prediction of risks from exposure to radioactive iodines from medical sources and any future nuclear accidents. Plans are to continue to follow-up the cohort for at least three screening cycles, which will lead to more precise estimates of risk.

GATA-6 gene enhancer contains nested regulatory modules for primary myocardium and the embedded nascent atrioventricular conduction system.

The cGATA-6 gene is flanked by an enhancer that selectively marks the atrioventricular conduction system (AVCS) in transgenic mice. This enhancer reads anterior/posterior and medial/lateral positional information very early in the cardiogenic program and remains active in progressively more restricted regions of primary myocardium leading up to the emergence of a histologically distinct AVCS. We undertook to parse this enhancer to resolve how the respective AVCS-specific transcription program is regulated at the molecular level. We determined that this AVCS enhancer includes a 102 bp module that is sufficient to restrict expression to primary nonchamber myocardium. This offers a novel tool to analyze the early molecular delineation of primary and chamber myocardium, which subsequently give rise to components of the central and peripheral conduction system, respectively. Furthermore, we show that this 102 bp module in turn contains a nested 47 bp core module that has the potential to direct expression specifically to the AVCS domain of primary myocardium, albeit with low efficiency. Accordingly, we show that a GATA site and a GC-rich site in the 102 bp region bolster the activity of the nested 47 bp AVCS core region even within the context of the parental 1,478 bp enhancer. These are the first functional elements to be reported for a cardiac conduction system-specific control region.

Advances in geroscience: impact on healthspan and chronic disease.

Population aging is unprecedented, without parallel in human history, and the 21st century will witness even more rapid aging than did the century just past. Improvements in public health and medicine are having a profound effect on population demographics worldwide. By 2017, there will be more people over the age of 65 than under age 5, and by 2050, two billion of the estimated nine billion people on Earth will be older than 60 (http://unfpa.org/ageingreport/). Although we can reasonably expect to live longer today than past generations did, the age-related disease burden we will have to confront has not changed. With the proportion of older people among the global population being now higher than at any time in history and still expanding, maintaining health into old age (or healthspan) has become a new and urgent frontier for modern medicine. Geroscience is a cross-disciplinary field focused on understanding the relationships between the processes of aging and age-related chronic diseases. On October 30-31, 2013, the trans-National Institutes of Health GeroScience Interest Group hosted a Summit to promote collaborations between the aging and chronic disease research communities with the goal of developing innovative strategies to improve healthspan and reduce the burden of chronic disease.

Conservation genetics of a critically endangered limpet genus and rediscovery of an extinct species.

BACKGROUND: A third of all known freshwater mollusk extinctions worldwide have occurred within a single medium-sized American drainage. The Mobile River Basin (MRB) of Alabama, a global hotspot of temperate freshwater biodiversity, was intensively industrialized during the 20(th) century, driving 47 of its 139 endemic mollusk species to extinction. These include the ancylinid limpet Rhodacmea filosa, currently classified as extinct (IUCN Red List), a member of a critically endangered southeastern North American genus reduced to a single known extant population (of R. elatior) in the MRB. METHODOLOGY/PRINCIPAL FINDINGS: We document here the tripling of known extant populations of this North American limpet genus with the rediscovery of enduring Rhodacmea filosa in a MRB tributary and of R. elatior in its type locality: the Green River, Kentucky, an Ohio River Basin (ORB) tributary. Rhodacmea species are diagnosed using untested conchological traits and we reassessed their systematic and conservation status across both basins using morphometric and genetic characters. Our data corroborated the taxonomic validity of Rhodacmea filosa and we inferred a within-MRB cladogenic origin from a common ancestor bearing the R. elatior shell phenotype. The geographically-isolated MRB and ORB R. elatior populations formed a cryptic species complex: although overlapping morphometrically, they exhibited a pronounced phylogenetic disjunction that greatly exceeded that of within-MRB R. elatior and R. filosa sister species. CONCLUSIONS/SIGNIFICANCE: Rhodacmea filosa, the type species of the genus, is not extinct. It persists in a Coosa River tributary and morphometric and phylogenetic analyses confirm its taxonomic validity. All three surviving populations of the genus Rhodacmea merit specific status. They collectively contain all known survivors of a phylogenetically highly distinctive North American endemic genus and therefore represent a concentrated fraction of continental freshwater gastropod biodiversity. We recommend the establishment of a proactive targeted conservation program that may include their captive propagation and reintroduction.

The sinus venosus progenitors separate and diversify from the first and second heart fields early in development.

AIMS: During development, the heart tube grows by differentiation of Isl1(+)/Nkx2-5(+) progenitors to the arterial and venous pole and dorsal mesocardium. However, after the establishment of the heart tube, Tbx18(+) progenitors were proposed to form the Tbx18(+)/Nkx2-5(-) sinus venosus and proepicardium. To elucidate the relationship between these contributions, we investigated the origin of the Tbx18(+) sinus venosus progenitor population in the cardiogenic mesoderm and its spatial and temporal relation to the second heart field during murine heart development. METHODS AND RESULTS: Explant culture revealed that the Tbx18(+) cell population has the potential to form Nkx2-5(-) sinus venosus myocardium. Three-dimensional reconstruction of expression patterns showed that during heart tube elongation, the Tbx18(+) progenitors remained spatially and temporally separate from the Isl1(+) second heart field, only overlapping with the Isl1(+) domain at the right lateral side of the inflow tract, where the sinus node developed. Consistently, genetic lineage analysis revealed that the Tbx18(+) descendants formed the sinus venosus myocardium, but did not contribute to the pulmonary vein myocardium that developed in the Isl1(+) second heart field. By means of DiI labelling and expression analysis, the origin of the sinus venosus progenitor population was traced to the lateral rim of splanchnic mesoderm that down-regulated Nkx2-5 expression approximately 2 days before its differentiation into sinus venosus myocardium. CONCLUSION: Our data indicate that the cardiogenic mesoderm contains an additional progenitor subpopulation that contributes to the sinus venosus myocardium. After patterning of the cardiogenic mesoderm, this progenitor population remains spatially separated and genetically distinctive from the second heart field subpopulation.

Identification and characterization of a novel Schwann and outflow tract endocardial cushion lineage-restricted periostin enhancer.

Periostin is a fasciclin-containing adhesive glycoprotein that facilitates the migration and differentiation of cells that have undergone epithelial-mesenchymal transformation during embryogenesis and in pathological conditions. Despite the importance of post-transformational differentiation as a general developmental mechanism, little is known how periostin's embryonic expression is regulated. To help resolve this deficiency, a 3.9-kb periostin proximal promoter was isolated and shown to drive tissue-specific expression in the neural crest-derived Schwann cell lineage and in a subpopulation of periostin-expressing cells in the cardiac outflow tract endocardial cushions. In order to identify the enhancer and associated DNA binding factor(s) responsible, in vitro promoter dissection was undertaken in a Schwannoma line. Ultimately a 304-bp(peri) enhancer was identified and shown to be capable of recapitulating 3.9 kb(peri-lacZ)in vivo spatiotemporal patterns. Further mutational and EMSA analysis helped identify a minimal 37-bp region that is bound by the YY1 transcription factor. The 37-bp enhancer was subsequently shown to be essential for in vivo 3.9 kb(peri-lacZ) promoter activity. Taken together, these studies identify an evolutionary-conserved YY1-binding 37-bp region within a 304-bp periostin core enhancer that is capable of regulating simultaneous novel tissue-specific periostin expression in the cardiac outflow-tract cushion mesenchyme and Schwann cell lineages.

Bone marrow cells transdifferentiate to cardiomyocytes when introduced into the embryonic heart.

Since rates of cardiomyocyte generation in the embryo are much higher than within the adult, we explored whether the embryonic heart would serve as useful experimental system for examining the myocardial potential of adult stem cells. Previously, we reported that the long-term culturing of adult mouse bone marrow produced a cell population that was both highly enriched for macrophages and cardiac competent. In this study, the myocardial potential of this cell population was analyzed in greater detail using the embryonic chick heart as recipient tissue. Experiments involving the co-incubation of labeled bone marrow cells with embryonic heart tissue showed that bone marrow (BM) cells incorporated into the myocardium and immunostained for myocyte proteins. Reverse transcription-polymerase chain reaction analysis demonstrated that the heart tissue induced bone marrow cells to express the differentiated cardiomyocyte marker alpha-cardiac myosin heavy chain. The cardiomyocyte conversion of the bone marrow cells was verified by harvesting donor cells from mice that were genetically labeled with a myocardial-specific beta-galactosidase reporter. Embryonic hearts exposed to the transgenic bone marrow in culture exhibited significant numbers of beta-galactosidase-positive cells, indicating the presence of bone marrow-derived cells that had converted to a myocardial phenotype. Furthermore, when transgenic mouse BM cells were injected into living chick embryos, donor cells incorporated into the developing heart and exhibited a myocardial phenotype. Immunofluorescence analysis demonstrated that donor BM cells exhibiting myocyte markers contained only nuclei from mouse cells, indicating that differentiation and not cell fusion was the predominant mechanism for the acquisition of a myocyte phenotype. These data confirm that adult mouse bone marrow contain cells with the ability to form cardiomyocytes. In addition, the predominance of the macrophage phenotype within the donor bone marrow cell population suggests that transdifferentiation of immune response cells may play a role in cellular regeneration in the adult.

E pluribus unum: A phylogenetic and phylogeographic reassessment of Laevapex (Pulmonata: Ancylidae), a North American genus of freshwater limpets.

The North American freshwater limpet genus Laevapex (Walker, 1903) is a ubiquitous inhabitant of lentic and slow-moving lotic habitats east of the Rocky Mountains, but uncertainty clouds its systematic affinities, the phylogenetic validity of its constituent nominal species, and its degree of genetic connectivity among drainages. We addressed these issues by sampling the genus throughout much of its collective range and constructing representative nuclear and mitochondrial (mt) gene trees, in addition to performing morphometric analyses of shell shape variation. Our results identify neotropical Gundlachia and South American Uncancylus as sister lineages for Laevapex and reveal a pronounced sub-familial dichotomy within the Ancylidae, separating these three New World genera from a Holarctic (Ferrissia (Ancylus, Rhodacmea)) sister clade. Five nominal taxa (L. fuscus, L. diaphanus, L. peninsulae, L. sp., and "F."arkansasensis), indistinguishable in our morphometric analyses, were polyphyletic in the mt gene trees, exhibited modest levels (< 3.9%) of genetic divergence in the primary (103 of 109 individuals) mt clade and, with one minor exception, they appeared fixed for a single nuclear ITS-2 genotype. Although complicated by the presence of rare, highly divergent mt lineages (of either introgressive or persistent ancestral polymorphic origin) in some populations, the molecular data were consistent with a taxonomic conclusion that these five nominal taxa represent a single polymorphic lineage of the type species L. fuscus. AMOVA analyses indicated that 56% of the observed mt variation could be attributed to among population differences, only two of 36 haplotypes were detected in more than one sampling location, and estimates of among-population mt gene flow were generally low at both regional and continental scales. Unrooted network analyses revealed a number of mt tip clades, one restricted to the southwestern part of the range, the remainder having overlapping distributions in eastern North America. All of the eastern tip clades occurred in the Mid-Atlantic region, and these samples displayed by far the highest levels of collective mt diversity. However, directional gene flow estimates indicated that this region has been a recipient (especially from Alabama populations), rather than a source of haplotypic diversity, implying that it likely represents a center of overlap, not a primary ice age refugium, for this limpet species.