Biomedical subjects

Cecilia W Lo

Publications and source records attributed to Cecilia W Lo.

17 recordsLinked to original sources

PubMed2006

Connexin 43-mediated modulation of polarized cell movement and the directional migration of cardiac neural crest cells.

Connexin 43 knockout (Cx43alpha1KO) mice have conotruncal heart defects that are associated with a reduction in the abundance of cardiac neural crest cells (CNCs) targeted to the heart. In this study, we show CNCs can respond to changing fibronectin matrix density by adjusting their migratory behavior, with directionality increasing and speed decreasing with increasing fibronectin density. However, compared with wild-type CNCs, Cx43alpha1KO CNCs show reduced directionality and speed, while CNCs overexpressing Cx43alpha1 from the CMV43 transgenic mice show increased directionality and speed. Altered integrin signaling was indicated by changes in the distribution of vinculin containing focal contacts, and altered temporal response of Cx43alpha1KO and CMV43 CNCs to beta1 integrin function blocking antibody treatment. High resolution motion analysis showed Cx43alpha1KO CNCs have increased cell protrusive activity accompanied by the loss of polarized cell movement. They exhibited an unusual polygonal arrangement of actin stress fibers that indicated a profound change in cytoskeletal organization. Semaphorin 3A, a chemorepellent known to inhibit integrin activation, was found to inhibit CNC motility, but in the Cx43alpha1KO and CMV43 CNCs, cell processes failed to retract with semaphorin 3A treatment. Immunohistochemical and biochemical analyses suggested close interactions between Cx43alpha1, vinculin and other actin-binding proteins. However, dye coupling analysis showed no correlation between gap junction communication level and fibronectin plating density. Overall, these findings indicate Cx43alpha1 may have a novel function in mediating crosstalk with cell signaling pathways that regulate polarized cell movement essential for the directional migration of CNCs.

Actins↗

PubMed2006

Primordial germ cell deficiency in the connexin 43 knockout mouse arises from apoptosis associated with abnormal p53 activation.

Connexin 43 knockout (Cx43alpha1KO) mice exhibit germ cell deficiency, but the underlying cause for the germ cell defect was unknown. Using an Oct4-GFP reporter transgene, we tracked the distribution and migration of primordial germ cells (PGCs) in the Cx43alpha1KO mouse embryo. Analysis with dye injections showed PGCs are gap-junction-communication competent, with dye coupling being markedly reduced in Cx43alpha1-deficient PGCs. Time-lapse videomicroscopy and motion analysis showed that the directionality and speed of cell motility were reduced in the Cx43alpha1KO PGCs. This was observed both in E8.5 and E11.5 embryos. By contrast, PGC abundance did not differ between wild-type and heterozygous/homozygous Cx43alpha1KO embryos until E11.5, when a marked reduction in PGC abundance was detected in the homozygous Cx43alpha1KO embryos. This was accompanied by increased PGC apoptosis and increased expression of activated p53. Injection of alpha-pifithrin, a p53 antagonist, inhibited PGC apoptosis and prevented the loss of PGC. Analysis using a cell adhesion assay indicated a reduction in beta1-integrin function in the Cx43alpha1KO PGCs. Together with the abnormal activation of p53, these findings suggest the possibility of anoikis-mediated apoptosis. Overall, these findings show Cx43alpha1 is essential for PGC survival, with abnormal p53 activation playing a crucial role in the apoptotic loss of PGCs in the Cx43alpha1KO mouse embryos.

Allantois↗

PubMed2006

Patterning of coronary arteries in wildtype and connexin43 knockout mice.

Abnormal patterning of coronary arteries (CAs) is a clinically significant problem, and as yet, few animal models have been systematically investigated for coronary patterning defects. Here we characterized coronary artery (CA) insertion and branching patterns of the proximal coronary stems in the hearts of wildtype and heterozygous connexin43 knockout (Cx43alpha1 KO) mice. This study entailed the use of high-resolution micro CT imaging for three-dimensional coronary reconstructions. MicroCT of 17 wildtype mice showed a remarkably consistent pattern of CA deployment in the normal mouse heart. Two main CA stems are inserted from the left and right into the aorta. The right coronary artery then branches immediately into the right main and the septal-conal branch, while the left coronary artery branches further distally into the circumflex and the anterior descending CA. This patterning of CA anatomy was confirmed by histology, and by using a vascular smooth muscle or endothelial cell specific lacZ reporter gene to delineate the CAs. A parallel analysis of 25 heterozygous Cx43alpha1 KO mouse hearts showed 22 had defects in patterning of the CAs. They exhibited a wide variation in CA anatomy, including abnormal origin and course of the main CA stems, multiple accessories, and dual septal-conal branches. Overall, these studies show loss of one Cx43alpha1 allele (haploinsufficiency) leads to a high incidence of coronary patterning defects. These findings suggest CA patterning is sensitive to Cx43alpha1 gene dosage.

Animals↗

PubMed2006

Klf2 is an essential regulator of vascular hemodynamic forces in vivo.

Hemodynamic responses that control blood pressure and the distribution of blood flow to different organs are essential for survival. Shear forces generated by blood flow regulate hemodynamic responses, but the molecular and genetic basis for such regulation is not known. The transcription factor KLF2 is activated by fluid shear stress in cultured endothelial cells, where it regulates a large number of vasoactive endothelial genes. Here, we show that Klf2 expression during development mirrors the rise of fluid shear forces, and that endothelial loss of Klf2 results in lethal embryonic heart failure due to a high-cardiac-output state. Klf2 deficiency does not result in anemia or structural vascular defects, and it can be rescued by administration of phenylephrine, a catecholamine that raises vessel tone. These findings identify Klf2 as an essential hemodynamic regulator in vivo and suggest that hemodynamic regulation in response to fluid shear stress is required for cardiovascular development and function.

Anemia↗

PubMed2006

Fetal mouse imaging using echocardiography: a review of current technology.

Advances in genetic research have led to the need for phenotypic analysis of small animal models. However, often these genetic alterations, especially when affecting the cardiovascular system, can result in fetal or perinatal death. Noninvasive ultrasound imaging is an ideal method for detecting and studying such congenital malformations, as it allows early recognition of abnormalities in the living fetus and the progression of disease can be followed in utero with longitudinal studies. Two platforms for fetal mouse echocardiography exist, the clinical systems with 15-MHz phased array transducers and research systems with 20-55-MHz mechanical transducers. The clinical ultrasound system has limited two-dimensional (2D) resolution (axial resolution of 440 microm), but the availability of color and spectral Doppler allows quick interrogations of blood flows, facilitating the detection of structural abnormalities. M-mode imaging further provides important functional data, although, the proper imaging planes are often difficult to obtain. In comparison, the research biomicroscope system has significantly improved 2D resolution (axial resolution of 28 microm). Spectral Doppler imaging is also available, but in the absence of color Doppler, imaging times are increased and the detection of flow abnormalities is more difficult. M-mode imaging is available and equivalent to the clinical ultrasound system. Overall, the research system, given its higher 2D resolution, is best suited for in-depth analysis of mouse fetal cardiovascular structure and function, while the clinical ultrasound systems, equipped with phase array transducers and color Doppler imaging, are ideal for high-throughput fetal cardiovascular screens.

Animals↗

PubMed2005

Connexin43 deficiency causes dysregulation of coronary vasculogenesis.

The connexin43 knockout (Cx43alpha1 KO) mouse dies at birth from outflow obstruction associated with infundibular pouches. To elucidate the origin of the infundibular pouches, we used microarray analysis to investigate gene expression changes in the pouch tissue. We found elevated expression of many genes encoding markers for vascular smooth muscle (VSM), endothelial cells, and fibroblasts, cell types that are epicardially derived and essential for coronary vasculogenesis. This was accompanied by increased expression of VEGF and genes in the TGFbeta and VEGF/Notch/Eph cell-signaling pathways known to regulate vasculogenesis/angiogenesis. Using immunohistochemistry and a VSM lacZ reporter gene, we confirmed an abundance of ectopic VSM and endothelial cells in the infundibular pouch and in some regions of the right ventricle forming secondary pouches. This was associated with distinct thinning of the compact myocardium. TUNEL labeling showed increased apoptosis in the pouch tissue, in agreement with the finding of altered expression of many apoptotic genes. Defects in vascular remodeling were indicated by a marked reduction in the branching complexity of the distal coronary arteries. In the near term KO mouse, we also observed a profusion of large coronary vascular plexuses subepicardially. This was associated with elevated epicardial expression of VEGF and abnormal epicardial cell morphology. Together, these observations indicate that dysregulated coronary vasculogenesis plays a pivotal role in formation of the infundibular pouches and suggests an essential role for Cx43alpha1 gap junctions in coronary vasculogenesis and vascular remodeling.

Animals↗

PubMed2005

Connexin43 associated with an N-cadherin-containing multiprotein complex is required for gap junction formation in NIH3T3 cells.

Previous studies have indicated an intimate linkage between gap junction and adherens junction formation. It was suggested this could reflect the close membrane-membrane apposition required for junction formation. In NIH3T3 cells, we observed the colocalization of connexin43 (Cx43alpha1) gap junction protein with N-cadherin, p120, and other N-cadherin-associated proteins at regions of cell-cell contact. We also found that Cx43alpha1, N-cadherin, and N-cadherin-associated proteins were coimmunoprecipitated by antibodies to either Cx43alpha1, N-cadherin, or various N-cadherin-associated proteins. These findings suggest that Cx43alpha1 and N-cadherin are coassembled in a multiprotein complex containing various N-cadherin-associated proteins. Studies using siRNA knockdown indicated that cell surface expression of Cx43alpha1 required N-cadherin, and conversely, N-cadherin cell surface expression required Cx43alpha1. Pulse-chase labeling and cell surface biotinylation experiments indicated that in the absence of N-cadherin, Cx43alpha1 cell surface trafficking is blocked. Surprisingly, siRNA knockdown of p120, an N-cadherin-associated protein known to modulate cell surface turnover of N-cadherin, reduced N-cadherin cell surface expression without altering Cx43alpha1 expression. These observations suggest that in contrast to the coregulated cell surface trafficking of Cx43alpha1 and N-cadherin, N-cadherin turnover at the cell surface may be regulated independently of Cx43alpha1. Functional studies showed gap junctional communication is reduced and cell motility inhibited with N-cadherin or Cx43alpha1 knockdown, consistent with the observed loss of both gap junction and cadherin contacts with either knockdown. Overall, these studies indicate that the intracellular coassembly of connexin and cadherin is required for gap junction and adherens junction formation, a process that likely underlies the intimate association between gap junction and adherens junction formation.

Adherens Junctions↗

PubMed2005

Overexpression of connexin43 alters the mutant phenotype of midgestational wnt-1 null mice resulting in recovery of the midbrain and cerebellum.

The midbrain-hindbrain (MHB) junction plays a key role in the patterning of the embryonic neural tube and the formation of brain structures such as the cerebellum. The mitogen wnt-1 is critical for cerebellar development, as evidenced by the lack of MHB region and cerebellar formation in the wnt-1 null embryo. We have generated wnt-1 null embryos overexpressing the gap junction gene connexin43 by crossing wnt-1 null heterozygotes into the CMV43 mouse line. We have confirmed that these mice show an increase in gap junctional communication by dye coupling analysis. Two-thirds of wnt-1 null CMV43(+) mouse embryos at E18.5 have a cerebellum. In addition, changes in the wnt-1 null phenotype in mouse embryos overexpressing connexin43 are observed as early as E9.5. At this stage, one-quarter of wnt-1 null CMV43(+) embryos display extra or expanded tissue present at the MHB boundary (a wnt-1 null enlarged phenotype). In situ hybridization studies conducted on these embryos have indicated no changes in the expression of embryonic brain positional markers in this region. We conclude from these studies that overexpression of the connexin43 gap junction restores cerebellar formation by compensating for the loss of wnt-1.

Animals↗

PubMed2005

Developmental regulation and expression of the zebrafish connexin43 gene.

We cloned and sequenced the zebrafish (Danio rerio) connexin43 (Cx43alpha1) gene. The predicted protein sequence shows a high degree of sequence conservation. Transcript analyses revealed multiple transcription start sites and a potential alternative transcript encoding a N-terminally truncated Cx43alpha1 protein. Maternal Cx43alpha1 transcripts were detected, with zygotic expression initiated before gastrulation. In situ hybridization revealed many Cx43alpha1 expression domains, including the notochord and brain, heart and vasculature, many resembling patterns seen in mammalian embryos. Of interest, a reporter construct under control of the mouse Cx43alpha1 promoter was observed to drive green fluorescent protein expression in zebrafish embryos in domains mimicking the native Cx43alpha1 expression pattern in fish and mice. Sequence comparison between the mouse and zebrafish Cx43alpha1 promoter sequences showed the conservation of several transcription factor motifs, which otherwise shared little overall sequence homology. The conservation of protein sequence and developmental gene regulation would suggest that Cx43alpha1 gap junctions are likely to have conserved roles in vertebrate embryonic development.

Amino Acid Sequence↗

PubMed2005

High-throughput mouse genotyping using robotics automation.

The use of mouse models is rapidly expanding in biomedical research. This has dictated the need for the rapid genotyping of mutant mouse colonies for more efficient utilization of animal holding space. We have established a high-throughput protocol for mouse genotyping using two robotics workstations: a liquid-handling robot to assemble PCR and a microfluidics electrophoresis robot for PCR product analysis. This dual-robotics setup incurs lower start-up costs than a fully automated system while still minimizing human intervention. Essential to this automation scheme is the construction of a database containing customized scripts for programming the robotics workstations. Using these scripts and the robotics systems, multiple combinations of genotyping reactions can be assembled simultaneously, allowing even complex genotyping data to be generated rapidly with consistency and accuracy. A detailed protocol, database, scripts, and additional background information are available at http://dir.nhlbi.nih.gov/labs/ldb-chd/autogene/.

Algorithms↗

PubMed2004

ENU induced mutations causing congenital cardiovascular anomalies.

We used non-invasive high frequency ultrasound to screen N-ethyl-N-nitrosourea mutagenized mouse fetuses for congenital cardiovascular anomalies. We ultrasound scanned 7546 mouse fetuses from 262 mutagenized families, and identified 124 families with cardiovascular defects. Represented were most of the major congenital cardiovascular anomalies seen clinically. The ENU-induced mutations in several families were mapped using polymorphic microsatellite DNA markers. One family with forelimb anomalies and ventricular septal defects, phenotypes similar to Holt-Oram syndrome, and one family with transposition of the great arteries and heart situs anomalies were mapped to different regions of mouse chromosome 4. A third mutation causing persistent truncus arteriosus and craniofacial defects, phenotypes reminiscent of DiGeorge syndrome, was mapped to mouse chromosome 2. We note that mouse chromosomes 4 and 2 do not contain Tbx5 or Tbx1, genes previously linked to Holt-Oram and DiGeorge syndromes, respectively. In two other families, the ENU-induced mutation was identified--Sema3CL605P was associated with persistent truncus arteriosus with interrupted aortic arch, and the Gja1W45X connexin43 mutation caused conotruncal malformation and coronary aneurysms. Although our screen was designed as a recessive screen, a number of the mutations showed cardiovascular phenotypes in both heterozygote and homozygote animals. These studies show the efficacy of ENU mutagenesis and high-throughput ultrasound phenotyping in recovering mutations causing a wide spectrum of congenital heart defects. These ENU-induced mutations hold promise in yielding new insights into the genetic basis for human congenital heart disease.

Amino Acid Sequence↗

PubMed2004

The complex of ciliary neurotrophic factor-ciliary neurotrophic factor receptor alpha up-regulates connexin43 and intercellular coupling in astrocytes via the Janus tyrosine kinase/signal transducer and activator of transcription pathway.

Cytokines regulate numerous cell processes, including connexin expression and gap junctional coupling. In this study, we examined the effect of ciliary neurotrophic factor (CNTF) on connexin43 (Cx43) expression and intercellular coupling in astrocytes. Murine cortical astrocytes matured in vitro were treated with CNTF (20 ng/ml), soluble ciliary neurotrophic factor receptor alpha (CNTFRalpha) (200 ng/ml), or CNTF-CNTFRalpha. Although CNTF and CNTFRalpha alone had no effect on Cx43 expression, the heterodimer CNTF-CNTFRalpha significantly increased both Cx43 mRNA and protein levels. Cx43 immunostaining correlated with increased intercellular coupling as determined by dye transfer analysis. By using the pharmacological inhibitor alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG490), the increase in Cx43 was found to be dependent on the Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Immunocytochemical analysis revealed that CNTF-CNTFRalpha treatment produced nuclear localization of phosphorylated STAT3, whereas CNTF treatment alone did not. Transient transfection of constructs containing various sequences of the Cx43 promoter tagged to a LacZ reporter into ROS 17/2.8 cells confirmed that the promoter region between -838 to -1693 was deemed necessary for CNTF-CNTFRalpha to induce heightened expression. CNTF-CNTFRalpha did not alter Cx30 mRNA levels, suggesting selectivity of CNTF-CNTFRalpha for connexin signaling. Together in the presence of soluble receptor, CNTF activates the JAK/STAT pathway leading to enhanced Cx43 expression and intercellular coupling.

Animals↗

PubMed2004

Rapid high resolution three dimensional reconstruction of embryos with episcopic fluorescence image capture.

One of the overarching goals in developmental biology is the elucidation of mechanisms that elaborate form and function. To this end, an accurate morphological description of embryonic development is essential. However, visualizing dynamic changes in the three-dimensional (3D) structure of the developing embryo has been a "holy grail" in the field of developmental biology. The fundamental difficulties that have hindered all efforts in 3D reconstruction using two-dimensional (2D) image stacks revolve around the seemingly intractable problems of section registration and distortion. A remarkably simple solution has come about with the development of a new technique referred to as episcopic fluorescence image capture (EFIC). With EFIC imaging, tissue autofluorescence is used to image the block face prior to cutting each section. The 2D resolution obtained is close to that achieved by histology, and such 2D image stacks can be readily reconstructed in 3D. The 3D models generated provide fine structural details with resolution unmatched by 3D reconstructions obtained with any other imaging modalities. Given the perfect registration of EFIC image stacks, another important capability provided by EFIC is digital resectioning in any plane. This provides complete flexibility in the selection of optimal virtual sectioning planes for viewing different features in a specimen, and is invaluable for analyzing dynamic changes in tissue structure in the developing embryo. The capabilities provided by EFIC for rapid high resolution 3D reconstruction together with digital resectioning make this an unparalleled tool for characterizing morphogenetic events in the developing embryo. Although our review is focused on using EFIC for studying embryonic development, it is important to note that there is no intrinsic limitation on the size of the specimen that can be analyzed by EFIC imaging. Overall, EFIC should serve as an important imaging technique that will complement other 3D imaging modalities such as MRI and optical tomography. Given the feasibility of generating EFIC image stacks using cryoembedded or polyethylene glycol (PEG)-embedded specimens, there is the possibility that EFIC may be combined with 3D RNA or protein expression profiling. Together, such studies may help further elucidate the relationship between form and function.

Animals↗

PubMed2004

High-frequency ultrasound database profiling growth, development, and cardiovascular function in C57BL/6J mouse fetuses.

BACKGROUND: High-frequency ultrasound is effective for noninvasive phenotypic analysis of cardiovascular development and function in mutagenized fetal mice. However, lacking is a normative database of echocardiographic variables for monitoring growth and cardiovascular function. METHODS: C57BL/6J fetal mice were scanned in utero using an ultrasound system with a 15-MHz linear phased-array transducer. Pregnant mothers were anesthetized with 1% isoflurane mixed with 100% oxygen. Quantitative variables for monitoring fetal growth and cardiac function were obtained in several imaging planes and modalities. RESULTS: Fetal growth measurements increased linearly. Inflow velocities showed significant A-wave dominance. The E wave progressively increased during development. Cardiac function was best assessed through M-mode analysis, but short-axis images were difficult to obtain. Spectral Doppler was readily obtained and the myocardial performance index was calculated. CONCLUSIONS: These results provide an essential foundation for the evaluation of cardiovascular defects in mutagenized and transgenic mice.

Animals↗

PubMed2004

Connexins and cell signaling in development and disease.

Gap junctions contain hydrophilic membrane channels that allow direct communication between neighboring cells through the diffusion of ions, metabolites, and small cell signaling molecules. They are made up of a hexameric array of polypeptides encoded by the connexin multi-gene family. Cell-cell communication mediated by connexins is crucial to various cellular functions, including the regulation of cell growth, differentiation, and development. Mutations in connexin genes have been linked to a variety of human diseases, including cardiovascular anomalies, peripheral neuropathy, deafness, skin disorders, and cataracts. In addition to their coupling function, recent studies suggest that connexin proteins may also mediate signaling. This could involve interactions with other protein partners that may play a role not only in connexin assembly, trafficking, gating and turnover, but also in the coordinate regulation of cell-cell communication with cell adhesion and cell motility. The integration of these cell functions is likely to be important in the role of gap junctions in development and disease.

Animals↗

PubMed2003

Correlation of GDF5 and connexin 43 mRNA expression during embryonic development.

Growth/differentiation factor 5 (GDF5) regulates connexin expression and enhances embryonic chondrogenesis in a gap junction-dependent manner, suggesting that GDF5 action on developmental skeletogenesis is coordinated with gap junction activities. The results shown here demonstrate concordance between the mRNA expression profiles of GDF5 and the gap junction gene, Cx43, in the mouse embryonic limb, spine, and heart, consistent with coordinated functions for these gene products during developmental organogenesis.

Animals↗

PubMed2003

Noninvasive phenotypic analysis of cardiovascular structure and function in fetal mice using ultrasound.

METHODS: We established methods for noninvasive mouse fetal heart imaging using an Acuson/Siemens Sequoia ultrasound scanner equipped with a single-pulse CHIRP Coded Excitation program, and a highfrequency linear array transducer. Mouse fetuses spanning gestation day 12.5 to 18.5 (E12.5-E18.5) were studied. RESULTS: Controlled anesthetic and constant body temperature were found to be essential for hemodynamic stability of the mother and fetuses. Fetal heart rates increased from 160 to 220 beats/min as development progressed. These heart rates were lower than those of newborn mice, so that frame rates above 100 Hz adequately resolved structural details in 2D without misregistration. Analysis of 2D images showed a doubling in crown-torump length (8-19 mm), and rapid growth of the heart from 1 to 3 mm in diameter as fetuses developed from E12.5 to E18.5. A cumulative increase in scanning modalities was achieved with increasing developmental age, with the optimal stage for scanning being E16.5. At E16.5 right and left could be distinguished, and it was possible to obtain diagnostic 2D color flow Doppler in the four-chamber, apical long axis 3/5-chamber and short axis views. In addition, M-mode images of high quality were obtainable from E15.5 to E18.5, whereas spectral Doppler signals could be obtained readily from E12.5 onwards. CONCLUSIONS: These studies show that ultrasound imaging can be used for structural and functional analysis of the developing mammalian heart, even at early stages of development. Such noninvasive cardiovascular ultrasonic evaluation should be ideally suited for high throughput screening of mutagenized mice.

Algorithms↗