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Biomedical subjects

Richard P Harvey

Publications and source records attributed to Richard P Harvey.

At least 19 recordsLinked to original sources
PubMed2006

The Hlx homeobox transcription factor is required early in enteric nervous system development.

BACKGROUND: Development of the enteric nervous system (ENS) requires interactions between migrating neural crest cells and the nascent gastrointestinal tract that are dependent upon genes expressed by both cell compartments. Hlx, a homeobox transcription factor gene that is expressed in mouse intestinal and hepatic mesenchyme, is required for normal embryonic growth of intestine and liver, and the Hlx-/- genotype is embryonic lethal. We hypothesized that Hlx is required for ENS development. RESULTS: Enteric neurons were identified in Hlx+/+ and Hlx-/- mouse embryos by immunostaining of embryo sections for the neural markers PGP9.5 and Phox2b, or by staining for beta-galactosidase in whole-mount embryos containing the dopamine beta-hydroxylase-nLacZ transgene. In Hlx+/+ embryos, neural crest cells/enteric neurons have moved from the stomach into the intestine by E10.5. By contrast, neural crest cells/enteric neurons remain largely restricted to the lateral stomach mesenchyme of Hlx-/- embryos, with only a few scattered neural crest cells/enteric neurons in the intestine between E10.5-16.5. CONCLUSION: The Hlx homeobox transcription factor is required for early aspects of ENS development.

Animals↗
PubMed2006

Formation of the venous pole of the heart from an Nkx2-5-negative precursor population requires Tbx18.

The venous pole of the mammalian heart is a structurally and electrically complex region, yet the lineage and molecular mechanisms underlying its formation have remained largely unexplored. In contrast to classical studies that attribute the origin of the myocardial sinus horns to the embryonic venous pole, we find that the sinus horns form only after heart looping by differentiation of mesenchymal cells of the septum transversum region into myocardium. The myocardial sinus horns and their mesenchymal precursor cells never express Nkx2-5, a transcription factor critical for heart development. In addition, lineage studies show that the sinus horns do not derive from cells previously positive for Nkx2-5. In contrast, the sinus horns express the T-box transcription factor gene Tbx18. Mice deficient for Tbx18 fail to form sinus horns from the pericardial mesenchyme and have defective caval veins, whereas the pulmonary vein and atrial structures are unaffected. Our studies define a novel heart precursor population that contributes exclusively to the myocardium surrounding the sinus horns or systemic venous tributaries of the developing heart, which are a source of congenital malformation and cardiac arrhythmias.

Animals↗
PubMed2006

A tyrosine-rich domain within homeodomain transcription factor Nkx2-5 is an essential element in the early cardiac transcriptional regulatory machinery.

Homeodomain factor Nkx2-5 is a central component of the transcription factor network that guides cardiac development; in humans, mutations in NKX2.5 lead to congenital heart disease (CHD). We have genetically defined a novel conserved tyrosine-rich domain (YRD) within Nkx2-5 that has co-evolved with its homeodomain. Mutation of the YRD did not affect DNA binding and only slightly diminished transcriptional activity of Nkx2-5 in a context-specific manner in vitro. However, the YRD was absolutely essential for the function of Nkx2-5 in cardiogenesis during ES cell differentiation and in the developing embryo. Furthermore, heterozygous mutation of all nine tyrosines to alanine created an allele with a strong dominant-negative-like activity in vivo: ES cell<-->embryo chimaeras bearing the heterozygous mutation died before term with cardiac malformations similar to the more severe anomalies seen in NKX2.5 mutant families. These studies suggest a functional interdependence between the NK2 class homeodomain and YRD in cardiac development and evolution, and establish a new model for analysis of Nkx2-5 function in CHD.

Amino Acid Sequence↗
PubMed2006

Quantitative trait loci modifying cardiac atrial septal morphology and risk of patent foramen ovale in the mouse.

Atrial septal defect (ASD) is a common congenital heart disease (CHD) occurring in 5 to 7 per 10,000 live births. Mutations in 5 human genes (NKX2.5, TBX5, GATA4, MYHC, ACTC) are known to cause dominant ASD, but these account for a minority of cases. Human and mouse data suggest that ASD exists in an anatomical continuum with milder septal variants patent foramen ovale (PFO) and atrial septal aneurysm, strongly associated with ischemic stroke and migraine. We have previously shown in inbred mice that the incidence of PFO strongly correlates with length of the interatrial septum primum, defining a quantitative trait underlying PFO risk. To better understand genetic causation of atrial septal abnormalities, we mapped quantitative trait loci (QTL) influencing septal morphology using mouse strains (QSi5 and 129T2/SvEms) maximally informative for PFO incidence and 3 quantitative septal anatomical traits including septum primum length. [QSi5x129T2/SvEms]F2 intercross animals (n=1437) were phenotyped and a whole genome scan performed at an average 17-cM interval. Statistical methodology scoring PFO as a binary phenotype was developed as a confirmatory mapping technique. We mapped 7 significant and 6 suggestive QTL modifying quantitative phenotypes, with 4 supported by binary analysis. Quantitative traits, although strongly associated with PFO (P<0.001), correlated poorly with each other and in all but 1 case QTL for different traits were nonoverlapping. Thus, multiple anatomical processes under separate genetic control contribute to risk of PFO. Our findings demonstrate the feasibility of modeling the genetic basis of common CHD using animal genetic and genomic technologies.

Animals↗
PubMed2006

Sexually dimorphic changes in the exploratory and habituation profiles of heterozygous neuregulin-1 knockout mice.

The neuregulin-1 gene is widely expressed in the central nervous system and is associated with increased risk for schizophrenia. Using an ethologically based approach, the phenotype of neuregulin-1 heterozygous knockout mice was examined by revealing the individual elements of behaviour in the murine repertoire over the prolonged course of interaction with the environment. During initial exploration, neuregulin-1 mutants displayed a phenotype characterized by increases in locomotion and rearing free, with sex-specific alterations in sifting and grooming. Over subsequent habituation, certain initial effects endured while new phenotypic effects emerged, some of which were again sex-specific. These studies elaborate a pleiotropic role of neuregulin-1 in development, plasticity and function, including sexual dimorphism, by defining the elemental, temporal and sex-specific characteristics of the neuregulin-1 mutant ethogram.

Animals↗
PubMed2006

Generation of conditional Cited2 null alleles.

Cited2 is a transcriptional co-factor that is widely expressed in both embryonic and extraembryonic cells during early development. It is essential for embryonic development with Cited2 null embryos showing abnormal development of organs including heart, neural tube, adrenal glands, and placenta (both in trophoblast derivatives and invading fetal vasculature), as well as having defects in the establishment of the left-right body axis. We report the generation of two conditional null alleles allowing Cre-recombinase-mediated somatic cell gene inactivation. Mice heterozygous or homozygous for these alleles are viable and fertile. Crossing conditional mutants with CMV-Cre transgenic mice produces an embryonic-lethal phenotype in the offspring indistinguishable from germline null mutants. We also demonstrate that conditional deletion results in lacZ expression under the control of the Cited2 promoter. These alleles are therefore useful genetic tools for dissecting the functions of Cited2 in the formation of different organs and patterning of the developing embryo. genesis

Alleles↗
PubMed2006

Conditional (loxP-flanked) allele for the gene encoding the retinoic acid-synthesizing enzyme retinaldehyde dehydrogenase 2 (RALDH2).

Retinoic acid, the active vitamin A derivative, has pleiotropic functions during vertebrate development and postnatal life. Retinaldehyde dehydrogenase 2 (RALDH2) acts as the main retinoic acid-synthesizing enzyme during development. Mouse Raldh2 germline null mutants are early embryonic lethal and exhibit complex abnormalities that include defective heart looping morphogenesis. To investigate later functions of this enzyme, we have engineered a "floxed" (loxP-flanked) allele allowing Cre-mediated somatic gene inactivations. Mice heterozygous or homozygous for the floxed Raldh2 allele are viable and fertile. We tested whether the novel Raldh2 allele behaves as a null mutation after Cre-mediated in vivo excision by crossing the conditional mutants with CMV-Cre transgenic mice. An embryonic lethal phenotype indistinguishable from that of germline mutants was obtained. The conditional allele described herein is a genetic tool for studying tissue-specific, RALDH2-dependent functions of retinoic acid during development and in adult life.

Aldehyde Oxidoreductases↗
PubMed2005

Muscle costameric protein, Chisel/Smpx, associates with focal adhesion complexes and modulates cell spreading in vitro via a Rac1/p38 pathway.

The murine X-linked gene Chisel (Csl/Smpx) encodes a 9-kDa protein that associates in heart and skeletal muscle cells with the costameric cytoskeleton, implicated in maintaining muscle integrity and responses to biomechanical stress. After expression in C2C12 myoblasts, MYC epitope-tagged Csl co-localized with actin networks at peripheral membranes, and with focal adhesion proteins vinculin, paxillin, integrin beta1, and the small GTPase Rac1. Csl could be co-immunoprecipitated with vinculin from extracts of C2C12 cells and native muscle. MYC-Csl induced cell spreading and lamellipodia formation in C2C12 cells at the expense of filopodia, suggestive of modulation of Rac1 activity. Lamellipodia formation was indeed Rac1-dependent, and in MYC-Csl cells replated on fibronectin, Rac1 activity was increased relative to controls. Expression of MYC-Csl led to an increased association between vinculin and p34, a subunit of the Arp2/3 actin nucleation complex, a Rac1-dependent event. Induced cell spreading was also dependent upon p38 kinases that act downstream of Rac1 to control the actin capping activity of heat shock protein 27. Our data suggest that Csl localizes to the costameric cytoskeleton of muscle cells through an association with focal adhesion proteins, where it may participate in regulation of cytoskeletal dynamics through the Rac1-p38 pathway.

Actin-Related Protein 2↗
PubMed2005

Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation.

The genetic hierarchies guiding lineage specification and morphogenesis of the mammalian embryonic heart are poorly understood. We now show by gene targeting that murine T-box transcription factor Tbx20 plays a central role in these pathways, and has important activities in both cardiac development and adult function. Loss of Tbx20 results in death of embryos at mid-gestation with grossly abnormal heart morphogenesis. Underlying these disturbances was a severely compromised cardiac transcriptional program, defects in the molecular pre-pattern, reduced expansion of cardiac progenitors and a block to chamber differentiation. Notably, Tbx20-null embryos showed ectopic activation of Tbx2 across the whole heart myogenic field. Tbx2 encodes a transcriptional repressor normally expressed in non-chamber myocardium, and in the atrioventricular canal it has been proposed to inhibit chamber-specific gene expression through competition with positive factor Tbx5. Our data demonstrate a repressive activity for Tbx20 and place it upstream of Tbx2 in the cardiac genetic program. Thus, hierarchical, repressive interactions between Tbx20 and other T-box genes and factors underlie the primary lineage split into chamber and non-chamber myocardium in the forming heart, an early event upon which all subsequent morphogenesis depends. Additional roles for Tbx20 in adult heart integrity and contractile function were revealed by in-vivo cardiac functional analysis of Tbx20 heterozygous mutant mice. These data suggest that mutations in human cardiac transcription factor genes, possibly including TBX20, underlie both congenital heart disease and adult cardiomyopathies.

Animals↗
PubMed2005

T-box transcription factors and their roles in regulatory hierarchies in the developing heart.

T-box transcription factors are important players in the molecular circuitry that generates lineage diversity and form in the developing embryo. At least seven family members are expressed in the developing mammalian heart, and the human T-box genes TBX1 and TBX5 are mutated in cardiac congenital anomaly syndromes. Here, we review T-box gene function during mammalian heart development in the light of new insights into heart morphogenesis. We see for the first time how hierarchies of transcriptional activation and repression involving multiple T-box factors play out in three-dimensional space to establish the cardiac progenitors fields, to define their subservient lineages, and to generate heart form and function.

Animals↗
PubMed2004

Foxh1 is essential for development of the anterior heart field.

The anterior heart field (AHF) mediates formation of the outflow tract (OFT) and right ventricle (RV) during looping morphogenesis of the heart. Foxh1 is a forkhead DNA binding transcription factor in the TGFbeta-Smad pathway. Here we demonstrate that Foxh1-/- mutant mouse embryos form a primitive heart tube, but fail to form OFT and RV and display loss of outer curvature markers of the future working myocardium, similar to the phenotype of Mef2c-/- mutant hearts. Further, we show that Mef2c is a direct target of Foxh1, which physically and functionally interacts with Nkx2-5 to mediate strong Smad-dependent activation of a TGFbeta response element in the Mef2c gene. This element directs transgene expression to the presumptive AHF, as well as the RV and OFT, a pattern that closely parallels endogenous Mef2c expression in the heart. Thus, Foxh1 and Nkx2-5 functionally interact and are essential for development of the AHF and its derivatives, the RV and OFT, in response to TGFbeta-like signals.

Animals↗
PubMed2003

Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4, and GATA5 in regulation of gene expression in the developing heart.

Tbx20 is a member of the T-box transcription factor family expressed in the forming hearts of vertebrate and invertebrate embryos. We report here analysis of Tbx20 expression during murine cardiac development and assessment of DNA-binding and transcriptional properties of Tbx20 isoforms. Tbx20 was expressed in myocardium and endocardium, including high levels in endocardial cushions. cDNAs generated by alternative splicing encode at least four Tbx20 isoforms, and Tbx20a uniquely carried strong transactivation and transrepression domains in its C terminus. Isoforms with an intact T-box bound specifically to DNA sites resembling the consensus brachyury half site, although with less avidity compared with the related factor, Tbx5. Tbx20 physically interacted with cardiac transcription factors Nkx2-5, GATA4, and GATA5, collaborating to synergistically activate cardiac gene expression. Among cardiac GATA factors, there was preferential synergy with GATA5, implicated in endocardial differentiation. In Xenopus embryos, enforced expression of Tbx20a, but not Tbx20b, led to induction of mesodermal and endodermal lineage markers as well as cell migration, indicating that the long Tbx20a isoform uniquely bears functional domains that can alter gene expression and developmental behaviour in an in vivo context. We propose that Tbx20 plays an integrated role in the ancient myogenic program of the heart, and has been additionally coopted during evolution of vertebrates for endocardial cushion development.

Alternative Splicing↗
PubMed2003

Cardiac homeobox gene NKX2-5 mutations and congenital heart disease: associations with atrial septal defect and hypoplastic left heart syndrome.

OBJECTIVES: We sought to examine the importance of mutations in the cardiac transcription factor gene NKX2-5 in patients with an atrial septal defect (ASD), patent foramen ovale (PFO), or hypoplastic left heart syndrome (HLHS). BACKGROUND: Mutations in NKX2-5 have been found in families showing secundum ASD and atrioventricular (AV) conduction block and in some individuals with tetralogy of Fallot. The prevalence of NKX2-5 mutations in sporadic cases of ASD/PFO and other forms of congenital heart disease is unknown. METHODS: A cohort of 146 individuals with secundum ASD, PFO complicated by paradoxical embolism, or HLHS were evaluated. Patients with ASD or PFO were ascertained irrespective of family history or associated cardiac abnormalities. The coding region of the NKX2-5 locus was amplified by polymerase chain reaction and sequenced. RESULTS: Among 102 ASD and 25 PFO patients screened, 13 patients (10%) had a positive family history and 5 patients (4%) had AV conduction block. We found one previously documented NKX2-5 missense mutation, T178M, in members of a family with ASD without AV conduction block. One NKX2-5 mutation-positive child from this family had HLHS, although no mutations were subsequently found in 18 patients with sporadic or familial HLHS. In a second ASD family without AV conduction block, we found a missense change, E21Q, previously reported as pathogenic. Because this change did not segregate with disease status, we propose that it is a non-disease-causing polymorphism. CONCLUSIONS: Our findings suggest that NKX2-5 mutations are a relatively infrequent cause of sporadic ASD and HLHS. Screening for NKX2-5 mutations may be warranted in individuals with ASD and a positive family history, irrespective of the presence or absence of AV conduction block.

Adolescent↗
PubMed2003

Molecular pathways in myocardial development: a stem cell perspective.

The heart has long been considered to adapt to increased work or pathology through the cellular growth process of hypertrophy. However, recent evidence for the existence of endogenous stem cells and regenerative capacity in the adult heart has given new impetus to the quest for cell therapies for heart failure, which remains the number one killer in Western cultures. The molecular cues driving cardiac development are now being explored in detail and will come into sharp focus as regimes for stem cell differentiation and efforts to augment endogenous regeneration are trialed. This review briefly outlines the current state of knowledge on the molecular basis of the four modalities of myogenesis that have been identified in the developing vertebrate heart. Stem cell-mediated myogenic repair in the heart represents a fifth modality, and an exciting frontier with basic and practical implications that remain to be explored.

Animals↗
PubMed2003

Architectural defects in the spleens of Nkx2-3-deficient mice are intrinsic and associated with defects in both B cell maturation and T cell-dependent immune responses.

Mice lacking the homeodomain transcription factor Nkx2-3 are either asplenic or develop a spleen of significantly reduced size with poorly organized white pulp. In this report, we analyze the effect of this mutation on B lymphocyte development and differentiation. Follicular dendritic cells in spleen, but not lymph node, of Nkx2-3(-/-) mice fail to express a developmental Ag (follicular dendritic cell-M2) and show an abnormal association with B cells, despite essentially normal expression of several chemokine genes. Bone marrow reconstitution studies show the splenic disorganization and absence of marginal zone B cells to be of stromal rather than hemopoietic origin. Furthermore, Nkx2-3(-/-) mice show an excess of conventional B cells in mesenteric lymph node and peritoneal cavity, whereas transitional B cells are rare in spleen but overrepresented in bone marrow. Finally, immunization of Nkx2-3(-/-) mice with a T cell-dependent Ag elicits clusters of germinal center B cells, although these fail to develop to the same extent as in controls and there is no evidence of affinity maturation in serum Ab. Similarly, Ab-forming cells fail to aggregate into foci early in the response. Collectively, these data indicate a substantial role for Nkx2-3 in the correct association of lymphocytes and splenic stromal elements that is independent of chemokine expression.

Anemia↗
PubMed2003

The creation of an historical meteorological database for environmental dose assessment.

The focus of this study is to develop wind data for the Savannah River Site (SRS) between 1955 and 1961 to be used in an assessment of estimates of atmospheric dispersion and downwind risk at the Savannah River Site. In particular, a study of the uncertainties of radioiodine dosimetry from the late 1950s provides the underlying motivation for developing historical windroses at the Savannah River Site (SRS). Wind measurement towers did not exist at the SRS until the early 1970s. Three relatively simple methods were used to create a 1955-1961 meteorological database for the SRS for a dose reconstruction project. The winds were estimated from onsite measurements in the 1990s and National Weather Service (NWS) observations in the 1990s and 1950s using (1) a linear regression method, (2) a similarity theory approach, and (3) a simple statistical differences method. The criteria for determining success were based on (1) how well the mean values and standard deviations of the predicted wind speed agree with the known SRS values from the 1990s, (2) the shape of the predicted frequency distribution functions for wind speed, and (3) how closely the predicted windroses resembled the SRS windrose for the 1990s. The linear regression model's wind speed distribution function was broad, flat, and skewed too much toward higher wind speeds. The similarity theory approach produced a wind speed distribution function that contained excess predicted speeds in the range 0-1.54 m s(-1) (0-3 kts) and had 'excluded' bins caused by predictions being made from integer values of knots in the NWS data. The distribution function from the mean difference method was smooth with a shape like a Weibull distribution with a shape parameter of 2 and appeared to resemble closely the SRS 1992-1996 distribution. The wind directions for all three methods of approach were successfully based on the mean difference method. It was difficult to discern differences among the wind roses produced by the three methods so the wind speed distribution functions need to be examined in order to make an informed choice for dose reconstruction.

Databases, Factual↗
PubMed2002

Hop is an unusual homeobox gene that modulates cardiac development.

Hop is a small, divergent homeodomain protein that lacks certain conserved residues required for DNA binding. Hop gene expression initiates early in cardiogenesis and continues in cardiomyocytes throughout embryonic and postnatal development. Genetic and biochemical data indicate that Hop functions directly downstream of Nkx2-5. Inactivation of Hop in mice by homologous recombination results in a partially penetrant embryonic lethal phenotype with severe developmental cardiac defects involving the myocardium. Inhibition of Hop activity in zebrafish embryos likewise disrupts cardiac development and results in severely impaired cardiac function. Hop physically interacts with serum response factor (SRF) and inhibits activation of SRF-dependent transcription by inhibiting SRF binding to DNA. Hop encodes an unusual homeodomain protein that modulates SRF-dependent cardiac-specific gene expression and cardiac development.

3T3 Cells↗