Retinoic acid temporally orchestrates colonization of the gut by vagal neural crest cells

The enteric nervous system arises from neural crest cells that migrate as chains into and along the primitive gut, subsequently differentiating into enteric neurons and glia. Little is known about the mechanisms governing neural crest migration en route to and along the gut in vivo. Here, we report that Retinoic Acid (RA) temporally controls zebrafish enteric neural crest cell chain migration. In vivo imaging reveals that RA loss severely compromises the integrity and migration of the chain of neural crest cells during the window of time window when they are moving along the foregut. After loss of RA, enteric progenitors accumulate in the foregut and differentiate into enteric neurons, but subsequently undergo apoptosis resulting in a striking neuronal deficit. Moreover, ectopic expression of the transcription factor meis3 and/or the receptor ret, partially rescues enteric neuron colonization after RA attenuation. Collectively, our findings suggest that retinoic acid plays a critical temporal role in promoting enteric neural crest chain migration and neuronal survival upstream of Meis3 and RET in vivo

Zebrafish Stem/Progenitor Factor msi2b Exhibits Two Phases of Activity Mediated by Different Splice Variants

The Musashi (Msi) family of RNA-binding proteins is important in stem and differentiating cells in many species. Here, we present a zebrafish gene/protein trap line gt(msi2b-citrine)(ct) (57) (a) that expresses a Citrine fusion protein with endogenous Msi2b. Our results reveal two phases of Msi2b expression: ubiquitous expression in progenitor cells in the early embryo and later, tissue-specific expression in differentiating cells in the olfactory organ, pineal gland, and subpopulations of neurons in the central nervous system (CNS). Interestingly, this division between early and late phases is paralleled by differential expression of msi2b alternative splicing products. Whereas the full-length and long variant v3 Msi2b predominate at early stages, the later expression of variants in differentiating tissues appears to be tissue specific. Using the gt(msi2b-citrine)(ct) (57) (a), we characterized tissue-specific expression of Msi2b with cellular resolution in subsets of differentiating cells in the olfactory organ, pineal gland, CNS, and ventral neural tube. By performing transcription activator-like effectors nuclease-mediated biallelic genome editing or morpholino knockdown of Msi2b in zebrafish, our results show that early inactivation of Msi2b results in severe embryonic defects including hypertrophy of the ventricles and shortening of the body, consistent with an important role in cell proliferation and survival. Moreover, specific inactivation of Msi2b full-length indicates that this species is essential for the early role of Msi2b. This line provides a valuable tool both for live imaging of the endogenous Msi2b at subcellular resolution and manipulation of Msi2b-expressing cells

Tracking the Care of Patients with Severe Chronic Illness - The Dartmouth Atlas of Health Care 2008

In 2001 the Institute of Medicine (IOM) issued Crossing the Quality Chasm, a report that sent a wake-up call to patients, providers, and policy makers about the poor quality of American health care. The IOM argued that one of the central drivers of poor quality has been the unsystematic and fragmentary nature of our health care delivery system. Nowhere are the system’s failings more apparent than in the care of the chronically ill. More than 90 million Americans live with at least one chronic illness, and seven out of ten Americans die from chronic disease. Among the Medicare population, the toll is even greater: about nine out of ten deaths are associated with just nine chronic illnesses, including congestive heart failure, chronic lung disease, cancer, coronary artery disease, renal failure, peripheral vascular disease, diabetes, chronic liver disease, and dementia

Laminin β1a controls distinct steps during the establishment of digestive organ laterality

Visceral organs, including the liver and pancreas, adopt asymmetric positions to ensure proper function. Yet the molecular and cellular mechanisms controlling organ laterality are not well understood. We identified a mutation affecting zebrafish laminin β1a (lamb1a) that disrupts left-right asymmetry of the liver and pancreas. In these mutants, the liver spans the midline and the ventral pancreatic bud remains split into bilateral structures. We show that lamb1a regulates asymmetric left-right gene expression in the lateral plate mesoderm (LPM). In particular, lamb1a functions in Kupffer’s vesicle (KV), a ciliated organ analogous to the mouse node, to control the length and function of the KV cilia. Later during gut-looping stages, dynamic expression of Lamb1a is required for the bilayered organization and asymmetric migration of the LPM. Loss of Lamb1a function also results in aberrant protrusion of LPM cells into the gut. Collectively, our results provide cellular and molecular mechanisms by which extracellular matrix proteins regulate left-right organ morphogenesis

SOXE neofunctionalization and elaboration of the neural crest during chordate evolution

During chordate evolution, two genome-wide duplications facilitated acquisition of vertebrate traits, including emergence of neural crest cells (NCCs), in which neofunctionalization of the duplicated genes are thought to have facilitated development of craniofacial structures and the peripheral nervous system. How these duplicated genes evolve and acquire the ability to specify NC and their derivatives are largely unknown. Vertebrate SoxE paralogues, most notably Sox9/10, are essential for NC induction, delamination and lineage specification. In contrast, the basal chordate, amphioxus, has a single SoxE gene and lacks NC-like cells. Here, we test the hypothesis that duplication and divergence of an ancestral SoxE gene may have facilitated elaboration of NC lineages. By using an in vivo expression assay to compare effects of AmphiSoxE and vertebrate Sox9 on NC development, we demonstrate that all SOXE proteins possess similar DNA binding and homodimerization properties and can induce NCCs. However, AmphiSOXE is less efficient than SOX9 in transactivation activity and in the ability to preferentially promote glial over neuronal fate, a difference that lies within the combined properties of amino terminal and transactivation domains. We propose that acquisition of AmphiSoxE expression in the neural plate border led to NCC emergence while duplication and divergence produced advantageous mutations in vertebrate homologues, promoting elaboration of NC traits

Rbms3 functions in craniofacial development by posttranscriptionally modulating TGF-β signaling

Cranial neural crest cells form much of the facial skeleton, and abnormalities in their development lead to severe birth defects. In a novel zebrafish protein trap screen, we identified an RNA-binding protein, Rbms3, that is transiently expressed in the cytoplasm of condensing neural crest cells within the pharyngeal arches. Morphants for rbms3 displayed reduced proliferation of prechondrogenic crest and significantly altered expression for chondrogenic/osteogenic lineage markers. This phenotype strongly resembles cartilage/crest defects observed in Tgf-βr2:Wnt1-Cre mutants, which suggests a possible link with TGF-β signaling. Consistent with this are the findings that: (a) Rbms3 stabilized a reporter transcript with smad2 3′ untranslated region, (b) RNA immunoprecipitation with full-length Rbms3 showed enrichment for smad2/3, and (c) pSmad2 levels were reduced in rbms3 morphants. Overall, these results suggest that Rbms3 posttranscriptionally regulates one of the major pathways that promotes chondrogenesis, the transforming growth factor β receptor (TGF-βr) pathway

Dynamic and Differential Regulation of Stem Cell Factor FoxD3 in the Neural Crest Is Encrypted in the Genome

The critical stem cell transcription factor FoxD3 is expressed by the premigratory and migrating neural crest, an embryonic stem cell population that forms diverse derivatives. Despite its important role in development and stem cell biology, little is known about what mediates FoxD3 activity in these cells. We have uncovered two FoxD3 enhancers, NC1 and NC2, that drive reporter expression in spatially and temporally distinct manners. Whereas NC1 activity recapitulates initial FoxD3 expression in the cranial neural crest, NC2 activity recapitulates initial FoxD3 expression at vagal/trunk levels while appearing only later in migrating cranial crest. Detailed mutational analysis, in vivo chromatin immunoprecipitation, and morpholino knock-downs reveal that transcription factors Pax7 and Msx1/2 cooperate with the neural crest specifier gene, Ets1, to bind to the cranial NC1 regulatory element. However, at vagal/trunk levels, they function together with the neural plate border gene, Zic1, which directly binds to the NC2 enhancer. These results reveal dynamic and differential regulation of FoxD3 in distinct neural crest subpopulations, suggesting that heterogeneity is encrypted at the regulatory level. Isolation of neural crest enhancers not only allows establishment of direct regulatory connections underlying neural crest formation, but also provides valuable tools for tissue specific manipulation and investigation of neural crest cell identity in amniotes

Tokamak Physics Experiment (TPX) power supply design and development

The Tokamak Physics Experiment (TPX) is an advanced tokamak project aimed at the production of quasi-steady state plasmas with advanced shape, heating, and particle control. TPX is to be built at the Princeton Plasma Physics Laboratory (PPPL) using many of the facilities from the Tokamak Fusion Test Reactor (TFTR). TPX will be the first tokamak to utilize superconducting (SC) magnets in both the toroidal field (TF) and poloidal field (PF) systems. This new feature requires a departure from the traditional tokamak power supply schemes. This paper describes the plan for the adaptation of the PPPL/FTR power system facilities to supply TPX. Five major areas are addressed, namely the AC power system, the TF, PF and Fast Plasma Position Control (FPPC) power supplies, and quench protection for the TF and PF systems. Special emphasis is placed on the development of new power supply and protection schemes