Sonic hedgehog is required for cardiac outflow tract and neural crest cell development.

The Hedgehog signaling pathway is critical for a significant number of developmental patterning events. In this study, we focus on the defects in pharyngeal arch and cardiovascular patterning present in Sonic hedgehog (Shh) null mouse embryos. Our data indicate that, in the absence of Shh, there is general failure of the pharyngeal arch development leading to cardiac and craniofacial defects. The cardiac phenotype results from arch artery and outflow tract patterning defects, as well as abnormal development of migratory neural crest cells (NCCs). The constellation of cardiovascular defects resembles a severe form of the human birth defect syndrome tetralogy of Fallot with complete pulmonary artery atresia. Previous studies have demonstrated a role for Shh in NCC survival and proliferation at later stages of development. Our data suggest that SHH signaling does not act directly on NCCs as a survival factor, but rather acts to restrict the domains that NCCs can populate during early stages (e8.5-10.5) of cardiovascular and craniofacial development.

Two-generation reproductive study in mink fed diisopropyl methylphosphonate (DIMP).

Two generations of "Ranch Wild" mink (Mustela vison) were fed the organophosphate diisopropyl methylphosphonate (DIMP) at 0, 150, 450, or 1250ppm, to determine potential toxicity to the dams. Chemical, hematologic, necropsy, and microscopic examinations were performed on all parental animals and representative kits. The F0 and F1 dams had 3.4 and 4.6% mortality, respectively, distributed among all groups and not attributed to DIMP exposure. Adverse effects were mild and limited to the highest dose group. Plasma cholinesterase was reduced 40% (F0) and 31% (F1), as was whole blood cholinesterase (16 and 8.5%). Heinz bodies were present in 2.8% (F0) and 1.3% (F1) of erythrocytes. The erythrocyte count was reduced 6.3% in the F0. Reproductive efficiency was not affected. The mink were not uniquely susceptible to DIMP, relative to the literature on other species. The no observed adverse effect level (NOAEL), based on the 450ppm group of F1 females, was 56.5mg DIMP/kgBW per day; the lowest observed adverse effect level (LOAEL) was 329.5mg DIMP/kgBW per day.

Lumen loss in transplant coronary artery disease is a biphasic process involving early intimal thickening and late constrictive remodeling: results from a 5-year serial intravascular ultrasound study.

BACKGROUND: Coronary artery disease is the major cause of late cardiac allograft failure. However, few data exist regarding the natural history of changes in intimal and external elastic membrane (EEM) areas after heart transplantation. METHODS AND RESULTS: In 38 transplant recipients, serial intravascular ultrasound examinations were performed 3.7+/-2.2 weeks after transplantation and annually thereafter for 5 years. In 59 coronary arteries, we compared 135 matched segments among serial studies. In each segment, intravascular ultrasound images were digitized at 1-mm intervals, and mean values of EEM and lumen and intimal areas were analyzed. In the first year after transplantation, the intimal area increased significantly from 1.8+/-1.6 to 3.0+/-2.1 mm(2) (P<0.001). Subsequently, the annual increase in intimal area decreased. EEM area did not change during the first year; however, between years 1 and 3, significant expansion of EEM area occurred (15.4+/-4.6 to 17.2+/-5.4 mm(2), P<0.001). Thereafter, EEM area decreased significantly from 17.2+/-5.4 mm(2) (year 3) to 15.1+/-4.9 mm(2) (year 5, P=0.01). Different mechanisms of lumen loss were observed during 2 phases after transplantation: early lumen loss primarily caused by intimal thickening and late lumen loss caused by EEM area constriction. CONCLUSIONS: This serial ultrasound study revealed that most of the intimal thickening occurred during the first year after heart transplantation. Changes in the EEM area showed a biphasic response, consisting of early expansion and late constriction. Thus, different mechanisms of lumen loss were observed during the early and late phases after transplantation.

The BMP antagonists Chordin and Noggin have essential but redundant roles in mouse mandibular outgrowth.

Here we investigate the roles of the Bone Morphogenetic Protein (BMP) antagonists Chordin and Noggin in development of the mandible, which is derived from the first branchial arch (BA1). Both genes are expressed in the pharynx during early mandibular outgrowth and later in the mandibular process. Mice mutant for either Nog or Chd have only mild mandibular defects; however, pups of the genotype Chd(-/-);Nog(+/-) exhibit a range of mandibular truncation phenotypes, from normal to agnathia. A few embryos homozygous null for both genes survive to late gestation; many are agnathic, though a few have significant mandibular outgrowth. In mandibular explants, ectopic BMP4 rapidly induces expression of both Chd and Nog, consistent with results obtained in vivo with mutant embryos. Previous work has shown that FGF8 is a survival factor for cells populating the mandibular bud. We find that excess BMP4 represses Fgf8 transcription in mandibular explants. Embryos lacking these BMP antagonists often show a strong reduction in Fgf8 expression in the pharyngeal ectoderm, and increased cell death in the mandibular bud. We suggest that the variable mandibular hypoplasia in double mutants involves increased BMP activity downregulating Fgf8 expression in the pharynx, decreasing cell survival during mandibular outgrowth.

Evaluation of three-dimensional segmentation algorithms for the identification of luminal and medial-adventitial borders in intravascular ultrasound images.

Intravascular ultrasound (IVUS) provides direct depiction of coronary artery anatomy, including plaque and vessel area, which is important in quantitative studies on the progression or regression of coronary artery disease. Traditionally, these studies have relied on manual evaluation, which is laborious, time consuming, and subject to large interobserver and intraobserver variability. A new technique, called active surface segmentation, alleviates these limitations and makes strides toward routine analyses. However, for three-dimensional (3-D) plaque assessment or 3-D reconstruction to become a clinical reality, methods must be developed which can analyze many images quickly. Presented is a comparison between two active surface techniques for three-dimensional segmentation of luminal and medial-adventitial borders. The force-acceleration technique and the neighborhood-search technique accurately detected both borders in vivo (r2 = 0.95 and 0.99, Williams' index = 0.67 and 0.65, and r2 = 0.95 and 0.99, WI = 0.67 and 0.70, respectively). However, the neighborhood-search technique was significantly faster and required less computation. Volume calculations for both techniques (r2 = 0.99 and r2 = 0.99) also agreed with a known-volume phantom. Active surface segmentation allows 3-D assessment of coronary morphology and further developments with this technology will provide clinical analysis tools.

Assessment of coronary compensatory enlargement by three-dimensional intravascular ultrasound.

Several techniques have been used to demonstrate that human arteries respond to atherosclerosis by increasing their total arterial area to prevent a decrease in blood flow. Three-dimensional reconstructions of coronary arteries can document this compensatory response accurately and specifically. Seven human coronary arteries were reconstructed using intravascular ultrasound and biplane angiography, and vessel geometries were quantified. In all seven vessels, as plaque area increased, overall vessel area increased (R = 0.986, 0.933, 0.984, 0.678, 0.763, 0.963, and 0.830), but luminal cross-sectional area did not significantly decrease. Focal compensatory enlargement was identified in each vessel, and in some cases this response appeared to occur until the vessel was 65% occluded. Luminal enlargement near the proximal ends was attributed to the natural taper of the vessel. The semi-automated, three-dimensional segmentation technique used in this study allows reproducible quantification, as there is no subjective manual tracing involved. Following the intravascular ultrasound transducer in time and space with biplane angiography allows for accurate reconstruction with or without automated pullback devices. Information on the rate of change of vessel measurements is also presented, which, when combined with visualization of accurate 3D geometry, provides a unique assessment of coronary compensatory enlargement. This reconstruction technique can be applied in a clinical environment with no major modification.

Vascular mechanics of the coronary artery.

UNLABELLED: This paper describes our research into the vascular mechanics of the coronary artery and plaque. The three sections describe the determination of arterial mechanical properties using intravascular ultrasound (IVUS), a constitutive relation for the arterial wall, and finite element method (FEM) models of the arterial wall and atheroma. METHODS: Inflation testing of porcine left anterior descending coronary arteries was conducted. The changes in the vessel geometry were monitored using IVUS, and intracoronary pressure was recorded using a pressure transducer. The creep and quasistatic stress/strain responses were determined. A Standard Linear Solid (SLS) was modified to reproduce the non-linear elastic behavior of the arterial wall. This Standard Non-linear Solid (SNS) was implemented into an axisymetric thick-walled cylinder numerical model. Finite element analysis models were created for five age groups and four levels of stenosis using the Pathobiological Determinants of Atherosclerosis Youth (PDAY) database. RESULTS: The arteries exhibited non-linear elastic behavior. The total tissue creep strain was epsilon creep = 0.082 +/- 0.018 mm/mm. The numerical model could reproduce both the non-linearity of the porcine data and time dependent behavior of the arterial wall found in the literature with a correlation coefficient of 0.985. Increasing age had a strong positive correlation with the shoulder stress level, (r = 0.95). The 30% stenosis had the highest shoulder stress due to the combination of a fully formed lipid pool and a thin cap. CONCLUSIONS: Studying the solid mechanics of the arterial wall and the atheroma provide important insights into the mechanisms involved in plaque rupture.

The organizer factors Chordin and Noggin are required for mouse forebrain development.

In mice, there is evidence suggesting that the development of head and trunk structures is organized by distinctly separated cell populations. The head organizer is located in the anterior visceral endoderm (AVE) and the trunk organizer in the node and anterior primitive streak. In amphibians, Spemann's organizer, which is homologous to the node, partially overlaps with anterior endoderm cells expressing homologues of the AVE markers cerberus, Hex and Hesx1. For mice, this raises the question of whether the AVE and node are independent of each other, as suggested by their anatomical separation, or functionally interdependent as is the case in amphibians. Chordin and Noggin are secreted bone morphogenetic protein (BMP) antagonists expressed in the mouse node, but not in the AVE. Here we show that mice double-homozygous mutants that are for chordin and noggin display severe defects in the development of the prosencephalon. The results show that BMP antagonists in the node and its derivatives are required for head development.

Neural induction and patterning in the mouse in the absence of the node and its derivatives.

The signals which induce vertebrate neural tissue and pattern it along the anterior-posterior (A-P) axis have been proposed to emanate from Spemann's organizer, which in mammals is a structure termed the node. However, mouse embryos mutant for HNF3 beta lack a morphological node and node derivatives yet undergo neural induction. Gene expression domains occur at their normal A-P axial positions along the mutant neural tubes in an apparently normal temporal manner, including the most anterior and posterior markers. This neural patterning occurs in the absence of expression of known organizer genes, including the neural inducers chordin and noggin. Other potential signaling centers in gastrulating mutant embryos appear to express their normal constellation of putative secreted factors, consistent with the possibility that neural-inducing and -patterning signals emanate from elsewhere or at an earlier time. Nevertheless, we find that the node and the anterior primitive streak, from which the node derives, are direct sources of neural-inducing signals, as judged by expression of the early midbrain marker Engrailed, in explant-recombination experiments. Similar experiments showed the neural-inducing activity in HNF3 beta mutants to be diffusely distributed. Our results indicate that the mammalian organizer is capable of neural induction and patterning of the neural plate, but that maintenance of an organizer-like signaling center is not necessary for either process.

The segment polarity gene porcupine encodes a putative multitransmembrane protein involved in Wingless processing.

The Wnt protein Wingless (Wg) functions as a signal in patterning of both the Drosophila embryo and imaginal discs. Lack of porcupine (porc) activity is associated with mutant phenotypes similar to those of wg mutations. In porc mutant embryos, Wg protein is confined to the cells that produce it, suggesting that Porc plays a role in processing or secretion of Wg. porc encodes a novel transmembrane protein that appears to be concentrated at the endoplasmic reticulum. We present both genetic and in vitro evidence demonstrating that porc is involved specifically in the processing of Wg. We identified a human sequence related to Porc suggesting the existence of a family of proteins involved in processing of Wnts.

Regulation of flt-1 expression during mouse embryogenesis suggests a role in the establishment of vascular endothelium.

Flt-1 is a high affinity binding receptor for the vascular endothelial cell growth factor (VEGF) and is primarily expressed in endothelial cells. In this study we have investigated the temporal and spatial regulation of its expression by establishing mouse lines containing the lacZ gene targeted into the flt-1 locus through homologous recombination in embryonic stem (ES) cells. In the yolk sac as well as in the embryo proper, lacZ expression faithfully reflected the endogenous expression pattern of the flt-1 gene. LacZ staining of heterozygous embryos led to the following observations: (1) the onset of flt-1 expression is detected at the early primitive streak stage in the extraembryonic mesoderm, and is strongly up-regulated thereafter, reaching a maximum by early to midsomite stages and declining subsequently; (2) while flt-1 is widely expressed within the developing vascular endothelium, its expression level is differentially regulated both spatially and temporally. The pattern of flt-1 expression suggests that it may play an important role in the initiation of endothelium development; and (3) flt-1 is expressed in essentially all the cells in early blood islands, but later its expression is gradually restricted to the endothelial lineage. Our results indicate that flt-1 is a marker for hemangioblasts, the presumed progenitor for both hematopoietic and angioblastic lineage. The flt-1 expression pattern also suggests that it may play important roles in both vasculogenesis and angiogenesis.

Conservation of dishevelled structure and function between flies and mice: isolation and characterization of Dvl2.

The segment polarity gene dishevelled (dsh) of Drosophila is required for pattern formation of the embryonic segments and the adult imaginal discs. dsh encodes the earliest-acting and most specific known component of the signal transduction pathway of Wingless, an extracellular signal homologous to Wnt1 in mice. We have previously described the isolation and characterization of the Dvl1 mouse dsh homolog. We report here the isolation of a second mouse dsh homolog, Dvl2, which maps to chromosome 11. The Dvl2 amino acid sequence is equally related to the dsh sequence as is that of Dvl1, but Dvl2 is most similar to the Xenopus homolog Xdsh. However, unlike the other vertebrate dsh homologs. Like the other genes, Dvl2 is ubiquitously expressed throughout most of embryogenesis and is expressed in many adult organs. We have developed an assay for dsh function in fly embryos, and show that Dvl2 can partially rescue the segmentation defects of embryos devoid of dsh. Thus, Dvl2 encodes a mammalian homolog of dsh which can transduce the Wingless signal.

Alternative tertiary care pathways for a rural Department of Veterans Affairs Medical Center.

This study addresses the cost of rural health care delivery where veterans do not have ready access to tertiary Department of Veterans Affairs Medical Centers (VAMCs) but where local community health care is available. The study sample was 209 patients referred for tertiary care to a VAMC 50 miles distant from the referring rural VAMC. The cost of tertiary referral VAMC care was retrospectively compared with the cost had the patients received the tertiary care in the local community hospital located in the immediate vicinity. In addition, the cost of travel resulting from the remote access was also computed. Findings indicate that a savings of +309,293 could have been obtained had a local community hospital provided the tertiary care utilizing the Health Care Financing Administration Medicare rate. Data generated by the methodology of this study are expected to provide a baseline for policy decisions relating to alternative pathways for tertiary care in the Department of Veterans Affairs.

A Drosophila CREB/CREM homolog encodes multiple isoforms, including a cyclic AMP-dependent protein kinase-responsive transcriptional activator and antagonist.

We have characterized a Drosophila gene that is a highly conserved homolog of the mammalian cyclic AMP (cAMP)-responsive transcription factors CREB and CREM. Uniquely among Drosophila genes characterized to date, it codes for a cAMP-responsive transcriptional activator. An alternatively spliced product of the same gene is a specific antagonist of cAMP-inducible transcription. Analysis of the splicing pattern of the gene suggests that the gene may be the predecessor of the mammalian CREB and CREM genes.

Dorsalizing and neuralizing properties of Xdsh, a maternally expressed Xenopus homolog of dishevelled.

Signaling factors of the Wnt proto-oncogene family are implicated in dorsal axis formation during vertebrate development, but the molecular mechanism of this process is not known. Studies in Drosophila have indicated that the dishevelled gene product is required for wingless (Wnt1 homolog) signal transduction. We demonstrate that injection of mRNA encoding a Xenopus homolog of dishevelled (Xdsh) into prospective ventral mesodermal cells triggers a complete dorsal axis formation in Xenopus embryos. Lineage tracing experiments show that cells derived from the injected blastomere contribute to anterior and dorsal structures of the induced axis. In contrast to its effect on mesoderm, overexpression of Xdsh mRNA in prospective ectodermal cells triggers anterior neural tissue differentiation. These studies suggest that Wnt signal transduction pathway is conserved between Drosophila and vertebrates and point to a role for maternal Xdsh product in dorsal axis formation and in neural induction.

Differential requirements for segment polarity genes in wingless signaling.

The segment polarity genes wingless and engrailed are required throughout development of Drosophila. During early embryogenesis, these two genes are expressed in adjacent domains, in an inter-dependent way. Later, their expression is regulated by different mechanisms and becomes maintained by auto-regulation. To dissect the genetic requirements for the initial signaling between wingless and engrailed expressing cells, we have previously used a transgenic Drosophila strain that expresses wingless under the control of the heat shock promoter (HS-wg). Focusing on the later phases of wingless and engrailed regulation, we have now extended these studies, using embryos carrying various combinations of segment polarity mutations and the HS-wg transgene. We confirm some of the existing models of regulation of the expression of wingless and engrailed. In addition, we find that HS-wg embryos require engrailed for induction of ectopic endogenous wingless expression. Signaling from engrailed cells to this novel wingless expression domain is dependent on hedgehog but also on porcupine. We further demonstrate a novel requirement for hedgehog in maintenance of expression of engrailed itself.

The dishevelled protein is modified by wingless signaling in Drosophila.

Wingless (Wg) is an important signaling molecule in the development of Drosophila, but little is known about its signal transduction pathway. Genetic evidence indicates that another segment polarity gene, dishevelled (dsh) is required for Wg signaling. We have recently developed a cell culture system for Wg protein activity, and using this in vitro system as well as intact Drosophila embryos, we have analyzed biochemical changes in the Dsh protein as a consequence of Wg signaling. We find that Dsh is a phosphoprotein, normally present in the cytoplasm. Wg signaling generates a hyperphosphorylated form of Dsh, which is associated with a membrane fraction. Overexpressed Dsh becomes hyperphosphorylated in the absence of extracellular Wg and increases levels of the Armadillo protein, thereby mimicking the Wg signal. A deletional analysis of Dsh identifies several conserved domains essential for activity, among which is a so-called GLGF/DHR motif. We conclude that dsh, a highly conserved gene, is not merely a permissive factor in Wg signaling but encodes a novel signal transduction molecule, which may function between the Wg receptor and more downstream signaling molecules.

Characterization of a gene trap insertion into a novel gene, cordon-bleu, expressed in axial structures of the gastrulating mouse embryo.

We have used a gene trap (GT) vector and embryonic stem (ES) cell chimeras to screen for insertions of the lacZ reporter gene into transcription units that are spatially and temporally regulated during early mouse embryogenesis. GT vectors which can act as both a reporter and a mutagen have been previously used to isolate new genes that are essential for mouse development. In this paper we describe a GT insertion which displays a very restricted pattern of expression in the gastrulating embryo. beta-Galactosidase activity was first detected at 7.5 days post-coitum (E7.5) in the node region of the embryo and extended to the midline structures at E8.0. At E9.5 expression was restricted to the floor plate, the notochord, the roof of the gut, and the liver anlage. Expression appeared in the somites at E10.0 and later became more widespread. We used rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) to clone a partial 360 base pair (bp) cDNA representing an endogenous sequence and containing an open reading frame (ORF) fused in frame to the lacZ reporter gene. The sequence showed no homology to any known protein or protein domain. An overlapping 1,200 bp fragment from a wild-type cDNA library was cloned and it detected the same pattern of expression as the reporter gene in E7.5, E8.5, and E9.5 wild-type embryos. It hybridized to a 5.4 kb lacZ fusion transcript and to an endogenous transcript of 6.5 kb. The gene was mapped to chromosome 11 and was named cordon-bleu (cobl). No phenotype was detected in mice homozygous for the insertion. However, the insertion may not cause a complete disruption of the gene function. The pattern of expression of cobl is very similar to that of hepatic nuclear factor 3 beta (HNF3 beta) and sonic hedgehog (Shh), both of which are involved in axial patterning. Therefore, the product of the cobl gene may also prove to be an important component of the genetic pathway regulating vertebrate axis formation.