Detection of gelatinolytic activity in developing basement membranes of the mouse embryo head by combining sensitive in situ zymography with immunolabeling

Genetic evidence indicates that the major gelatinases MMP-2 and MMP-9 are involved in mammalian craniofacial development. Since these matrix metalloproteinases are secreted as proenzymes that require activation, their tissue distribution does not necessarily reflect the sites of enzymatic activity. Information regarding the spatial and temporal expression of gelatinolytic activity in the head of the mammalian embryo is sparse. Sensitive in situ zymography with dye-quenched gelatin (DQ-gelatin) has been introduced recently; gelatinolytic activity results in a local increase in fluorescence. Using frontal sections of wild-type mouse embryo heads from embryonic day 14.5-15.5, we optimized and validated a simple double-labeling in situ technique for combining DQ-gelatin zymography with immunofluorescence staining. MMP inhibitors were tested to confirm the specificity of the reaction in situ, and results were compared to standard SDS-gel zymography of tissue extracts. Double-labeling was used to show the spatial relationship in situ between gelatinolytic activity and immunostaining for gelatinases MMP-2 and MMP-9, collagenase 3 (MMP-13) and MT1-MMP (MMP-14), a major activator of pro-gelatinases. Strong gelatinolytic activity, which partially overlapped with MMP proteins, was confirmed for Meckel's cartilage and developing mandibular bone. In addition, we combined in situ zymography with immunostaining for extracellular matrix proteins that are potential gelatinase substrates. Interestingly, gelatinolytic activity colocalized precisely with laminin-positive basement membranes at specific sites around growing epithelia in the developing mouse head, such as the ducts of salivary glands or the epithelial fold between tongue and lower jaw region. Thus, this sensitive method allows to associate, with high spatial resolution, gelatinolytic activity with epithelial morphogenesis in the embryo.

Role for ephrinB2 in postnatal lung alveolar development and elastic matrix integrity

Alveoli are formed in the lung by the insertion of secondary tissue folds, termed septa, which are subsequently remodeled to form the mature alveolar wall. Secondary septation requires interplay between three cell types: endothelial cells forming capillaries, contractile interstitial myofibroblasts, and epithelial cells. Here, we report that postnatal lung alveolization critically requires ephrinB2, a ligand for Eph receptor tyrosine kinases expressed by the microvasculature. Mice homozygous for the hypomorphic knockin allele ephrinB2DeltaV/DeltaV, encoding mutant ephrinB2 with a disrupted C-terminal PDZ interaction motif, show severe postnatal lung defects including an almost complete absence of lung alveoli and abnormal and disorganized elastic matrix. Lung alveolar formation is not sensitive to loss of ephrinB2 cytoplasmic tyrosine phosphorylation sites. Postnatal day 1 mutant lungs show extracellular matrix alterations without differences in proportions of major distal cell populations. We conclude that lung alveolar formation relies on endothelial ephrinB2 function.

Development and simulation of DSP filters for transformer differential protection / by Oskar Reynisson.

Transformer protection is one of the most challenging applications within the power system protective relay field. Transformers with a capacity rating exceeding 10 MVA are usually protected using differential current relays. Transformers are an aging and vulnerable bottleneck in the present power grid; therefore, quick fault detection and corresponding transformer de-energization is the key element in minimizing transformer damage. Present differential current relays are based on digital signal processing (DSP). They combine DSP phasor estimation and protective-logic-based decision making. The limitations of existing DSP-based differential current relays must be identified to determine the best protection options for sensitive and quick fault detection. The development, implementation, and evaluation of a DSP differential current relay is detailed. The overall goal is to make fault detection faster without compromising secure and safe transformer operation. A detailed background on the DSP differential current relay is provided. Then different DSP phasor estimation filters are implemented and evaluated based on their ability to extract desired frequency components from the measured current signal quickly and accurately. The main focus of the phasor estimation evaluation is to identify the difference between using non-recursive and recursive filtering methods. Then the protective logic of the DSP differential current relay is implemented and required settings made in accordance with transformer application. Finally, the DSP differential current relay will be evaluated using available transformer models within the ATP simulation environment. Recursive filtering methods were found to have significant advantage over non-recursive filtering methods when evaluated individually and when applied in the DSP differential relay. Recursive filtering methods can be up to 50% faster than non-recursive methods, but can cause false trip due to overshoot if the only objective is speed. The relay sensitivity is however independent of filtering method and depends on the settings of the relay’s differential characteristics (pickup threshold and percent slope).

DESIGN AND DEVELOPMENT OF BODIPY-BASED FLUORESCENT PROBES FOR SENSING AND IMAGING OF CYANIDE, Zn (II) IONS, LYSOSOMAL pH AND CANCER CELLS

BODIPY (4,4-Difluoro-3a,4a-diaza-s-indacene) dyes have gained lots of attention in application of fluorescence sensing and imaging in recent years because they possess many distinctive and desirable properties such as high extinction coefficient, narrow absorption and emission bands, high quantum yield and low photobleaching effect. However, most of BODIPY-based fluorescent probes have very poor solubilities in aqueous solution, emit less than 650 nm fluorescence that can cause cell and tissue photodamages compared with bio-desirable near infrared (650-900 nm) light. These undesirable properties extremely limit the applications of BODIPY-based fluorescent probes in sensing and imaging applications. In order to overcome these drawbacks, we have developed a very effective strategy to prepare a series of neutral highly water- soluble BODIPY dyes by enhancing the water solubilities of BODIPY dyes via incorporation of tri(ethylene glycol)methyl ether (TEG) and branched oligo(ethylene glycol)methyl ether (BEG) residues onto BODIPY dyes at 1,7-, 2,6-, 3,5-, 4- and meso- positions. We also have effectively tuned absorptions and emissions of BOIDPY dyes to red, deep red and near infrared regions via significant extension of π-conjugation of BODIPY dyes by condensation reactions of aromatic aldehydes with 2,6-diformyl BODIPY dyes at 1,3,5,7-positions. Based on the foundation that we built for enhancing water solubility and tuning wavelength, we have designed and developed a series of water-soluble, BODIPY-based fluorescent probes for sensitive and selective sensing and imaging of cyanide, Zn (II) ions, lysosomal pH and cancer cells. We have developed three BODIPY-based fluorescent probes for sensing of cyanide ions by incorporating indolium moieties onto the 6-position of TEG- or BEG-modified BOIDPY dyes. Two of them are highly water-soluble. These fluorescent probes showed selective and fast ratiometric fluorescent responses to cyanide ions with a dramatic fluorescence color change from red to green accompanying a significant increase in fluorescent intensity. The detection limit was measured as 0.5 mM of cyanide ions. We also have prepared three highly water-soluble fluorescent probes for sensing of Zn (II) ions by introducing dipicoylamine (DPA, Zn ion chelator) onto 2- and/or 6-positions of BEG-modified BODIPY dyes. These probes showed selective and sensitive responses to Zn (II) ion in the range from 0.5 mM to 24 mM in aqueous solution at pH 7.0. Particularly, one of the probes displayed ratiometric responses to Zn (II) ions with fluorescence quenching at 661 nm and fluorescence enhancement at 521 nm. This probe has been successfully applied to the detection of intracellular Zn (II) ions inside the living cells. Then, we have further developed three acidotropic, near infrared emissive BODIPY- based fluorescent probes for detection of lysosomal pH by incorporating piperazine moiety at 3,5-positions of TEG- or BEG-modified BODIPY dyes as parts of conjugation. The probes have low auto-fluorescence at physiological neutral condition while their fluorescence intensities will significant increase at 715 nm when pH shift to acidic condition. These three probes have been successfully applied to the in vitro imaging of lysosomes inside two types of living cells. At the end, we have synthesized one water- soluble, near infrared emissive cancer cell targetable BODIPY-based fluorescent polymer bearing cancer homing peptide (cRGD) residues for cancer cell imaging applications. This polymer exhibited excellent water-solubility, near infrared emission (712 nm), good biocompatibility. It also showed low nonspecific interactions to normal endothelial cells and can effectively detect breast tumor cells.

Development and characterization of fluorescent pH sensors based on porous silica and hydrogel support matrices

The hydrogen ion activity (pH) is a very important parameter in environment monitoring, biomedical research and other applications. Optical pH sensors have several advantages over traditional potentiometric pH measurement, such as high sensitivity, no need of constant calibration, easy for miniaturization and possibility for remote sensing. Several pH indicators has been successfully immobilized in three different solid porous materials to use as pH sensing probes. The fluorescent pH indicator fluorescein-5-isothiocyanate (FITC) was covalently bound onto the internal surface of porous silica (pore size ~10 nm) and retained its pH sensitivity. The excited state pK* a of FITC in porous silica (5.58) was slightly smaller than in solution (5.68) due to the free silanol groups (Si-OH) on the silica surface. The pH sensitive range for this probe is pH 4.5 - 7.0 with an error less than 0.1 pH units. The probe response was reproducible and stable for at least four month, stored in DI water, but exhibit a long equilibrium of up to 100 minutes. Sol-gel based pH sensors were developed with immobilization of two fluorescent pH indicators fluorescein-5-(and-6)-sulfonic acid, trisodium salt (FS) and 8-hydroxypyrene- 1,3,6-trisulfonic acid (HPTS) through physical entrapment. Prior to immobilization, the indicators were ion-paired with a common surfactant hexadecyltrimethylammonium bromide (CTAB) in order to prevent leaching. The sol-gel films were synthesized through the hydrolysis of two different precursors, ethyltriethoxysilane (ETEOS) and 3- glycidoxypropyltrimethoxysilane (GPTMS) and deposited on a quartz slide through spin coating. The pK a of the indicators immobilized in sol-gel films was much smaller than in solutions due to silanol groups on the inner surface of the sol-gel films and ammonium groups from the surrounding surfactants. Unlike in solution, the apparent pK a of the indicators in sol-gel films increased with increasing ionic strength. The equilibrium time for these sensors was within 5 minutes (with film thickness of ~470 nm). Polyethylene glycol (PEG) hydrogel was of interest for optical pH sensor development because it is highly proton permeable, transparent and easy to synthesize. pH indicators can be immobilized in hydrogel through physical entrapment and copolymerization. FS and HPTS ion-pairs were physically entrapped in hydrogel matrix synthesized via free radical initiation. For covalent immobilization, three indicators, 6,8-dihydroxypyrene-1,3- disulfonic acid (DHPDS), 2,7-dihydroxynaphthalene-3,6-disulfonic acid (DHNDS) and cresol red were first reacted with methacrylic anhydride (MA) to form methacryloylanalogs for copolymerization. These hydrogels were synthesized in aqueous solution with a redox initiation system. The thickness of the hydrogel film is controlled as ~ 0.5 cm and the porosity can be adjusted with the percentage of polyethylene glycol in the precursor solutions. The pK a of the indicators immobilized in the hydrogel both physically and covalently were higher than in solution due to the medium effect. The sensors are stable and reproducible with a short equilibrium time (less than 4 minutes). In addition, the color change of cresol red immobilized hydrogel is vivid from yellow (acidic condition) to purple (basic condition). Due to covalently binding, cresol red was not leaching out from the hydrogel, making it a good candidate of reusable "pH paper".

Monitoring thrombin generation by electrochemistry: development of an amperometric biosensor screening test for plasma and whole blood

BACKGROUND: Complete investigation of thrombophilic or hemorrhagic clinical presentations is a time-, apparatus-, and cost-intensive process. Sensitive screening tests for characterizing the overall function of the hemostatic system, or defined parts of it, would be very useful. For this purpose, we are developing an electrochemical biosensor system that allows measurement of thrombin generation in whole blood as well as in plasma. METHODS: The measuring system consists of a single-use electrochemical sensor in the shape of a strip and a measuring unit connected to a personal computer, recording the electrical signal. Blood is added to a specific reagent mixture immobilized in dry form on the strip, including a coagulation activator (e.g., tissue factor or silica) and an electrogenic substrate specific to thrombin. RESULTS: Increasing thrombin concentrations gave standard curves with progressively increasing maximal current and decreasing time to reach the peak. Because the measurement was unaffected by color or turbidity, any type of blood sample could be analyzed: platelet-poor plasma, platelet-rich plasma, and whole blood. The test strips with the predried reagents were stable when stored for several months before testing. Analysis of the combined results obtained with different activators allowed discrimination between defects of the extrinsic, intrinsic, and common coagulation pathways. Activated protein C (APC) predried on the strips allowed identification of APC-resistance in plasma and whole blood samples. CONCLUSIONS: The biosensor system provides a new method for assessing thrombin generation in plasma or whole blood samples as small as 10 microL. The assay is easy to use, thus allowing it to be performed in a point-of-care setting.

Evaluating Integration. Research contributions for a gender sensitive migration policy: Research contributions for a gender sensitive migration policy

The integration of academic and non-academic knowledge is a key concern for researchers who aim at bridging the gap between research and policy. Researchers involved in the sustainability-oriented NCCR North-South programme have made the experience that linking different types of knowledge requires time and effort, and that methodologies are still lacking. One programme component was created at the inception of this transdisciplinary research programme to support exchange between researchers, development practitioners and policymakers. After 8 years of research, the programme is assessing whether research has indeed enabled a continuous communication across and beyond academic boundaries and has effected changes in the public policies of poor countries. In a first review of the data, we selected two case studies explicitly addressing the lives of women. In both cases – one in Pakistan, the other in Nepal – the dialogue between researchers and development practitioners contributed to important policy changes for female migration. In both countries, outmigration has become an increasingly important livelihood strategy. National migration policies are gendered, limiting the international migration of women. In Nepal, women were not allowed to migrate to specific countries such as the Gulf States or Malaysia. This was done in the name of positive discrimination, to protect women from potential exploitation and harassment in domestic work. However, women continued to migrate in many other and often illegal and more risky ways, increasing their vulnerability. In Pakistan, female labour migration was not allowed at all and male migration increased the vulnerability of the families remaining back home. Researchers and development practitioners in Nepal and Pakistan brought women’s shared experience of and exposure to the mechanisms of male domination into the public debate, and addressed the discriminating laws. Now, for the first time in Pakistan, the new draft policy currently under discussion would enable broadly-based female labour migration. What can we learn from the two case studies with regard to ways of relating experience- and research-based knowledge? The paper offers insights into the sequence of interactions between researchers, local people, development practitioners, and policy-makers, which eventually contributed to the formulation of a rights-based migration policy. The reflection aims at exploring the gendered dimension of ways to co-produce and share knowledge for development across boundaries. Above all, it should help researchers to better tighten the links between the spheres of research and policy in future.

Teachers' professional development in a community

The goal of this article was to study teachers' professional development related to web-based learning in the context of the teacher community. The object was to learn in what kind of networks teachers share the knowledge of web-based learning and what are the factors in the community that support or challenge teachers professional development of web-based learning. The findings of the study revealed that there are teachers who are especially active, called the central actors in this study, in the teacher community who collaborate and share knowledge of web-based learning. These central actors share both technical and pedagogical knowledge of web-based learning in networks that include both internal and external relations in the community and involve people, artefacts and a variety of media. Furthermore, the central actors appear to bridge different fields of teaching expertise in their community. According to the central actors' experiences the important factors that support teachers' professional development of web-based learning in the community are; the possibility to learn from colleagues and from everyday working practices, an emotionally safe atmosphere, the leader's personal support and community-level commitment. Also, the flexibility in work planning, challenging pupils, shared lessons with colleagues, training events in an authentic work environment and colleagues' professionalism are considered meaningful for professional development. As challenges, the knowledge sharing of web-based learning in the community needs mutual interests, transactive memory, time and facilities, peer support, a safe atmosphere and meaningful pedagogical practices. On the basis of the findings of the study it is suggested that by intensive collaboration related to web-based learning it may be possible to break the boundaries of individual teachership and create such sociocultural activities which support collaborative professional development in the teacher community. Teachers' in-service training programs should be more sensitive to the culture of teacher communities and teachers' reciprocal relations. Further, teacher trainers should design teachers' in-service training of web-based learning in co-evolution with supporting networks which include the media and artefacts as well as people.

Teachers' professional development in a community

The goal of this article was to study teachers' professional development related to web-based learning in the context of the teacher community. The object was to learn in what kind of networks teachers share the knowledge of web-based learning and what are the factors in the community that support or challenge teachers professional development of web-based learning. The findings of the study revealed that there are teachers who are especially active, called the central actors in this study, in the teacher community who collaborate and share knowledge of web-based learning. These central actors share both technical and pedagogical knowledge of web-based learning in networks that include both internal and external relations in the community and involve people, artefacts and a variety of media. Furthermore, the central actors appear to bridge different fields of teaching expertise in their community. According to the central actors' experiences the important factors that support teachers' professional development of web-based learning in the community are; the possibility to learn from colleagues and from everyday working practices, an emotionally safe atmosphere, the leader's personal support and community-level commitment. Also, the flexibility in work planning, challenging pupils, shared lessons with colleagues, training events in an authentic work environment and colleagues' professionalism are considered meaningful for professional development. As challenges, the knowledge sharing of web-based learning in the community needs mutual interests, transactive memory, time and facilities, peer support, a safe atmosphere and meaningful pedagogical practices. On the basis of the findings of the study it is suggested that by intensive collaboration related to web-based learning it may be possible to break the boundaries of individual teachership and create such sociocultural activities which support collaborative professional development in the teacher community. Teachers' in-service training programs should be more sensitive to the culture of teacher communities and teachers' reciprocal relations. Further, teacher trainers should design teachers' in-service training of web-based learning in co-evolution with supporting networks which include the media and artefacts as well as people.

Teachers' professional development in a community

The goal of this article was to study teachers' professional development related to web-based learning in the context of the teacher community. The object was to learn in what kind of networks teachers share the knowledge of web-based learning and what are the factors in the community that support or challenge teachers professional development of web-based learning. The findings of the study revealed that there are teachers who are especially active, called the central actors in this study, in the teacher community who collaborate and share knowledge of web-based learning. These central actors share both technical and pedagogical knowledge of web-based learning in networks that include both internal and external relations in the community and involve people, artefacts and a variety of media. Furthermore, the central actors appear to bridge different fields of teaching expertise in their community. According to the central actors' experiences the important factors that support teachers' professional development of web-based learning in the community are; the possibility to learn from colleagues and from everyday working practices, an emotionally safe atmosphere, the leader's personal support and community-level commitment. Also, the flexibility in work planning, challenging pupils, shared lessons with colleagues, training events in an authentic work environment and colleagues' professionalism are considered meaningful for professional development. As challenges, the knowledge sharing of web-based learning in the community needs mutual interests, transactive memory, time and facilities, peer support, a safe atmosphere and meaningful pedagogical practices. On the basis of the findings of the study it is suggested that by intensive collaboration related to web-based learning it may be possible to break the boundaries of individual teachership and create such sociocultural activities which support collaborative professional development in the teacher community. Teachers' in-service training programs should be more sensitive to the culture of teacher communities and teachers' reciprocal relations. Further, teacher trainers should design teachers' in-service training of web-based learning in co-evolution with supporting networks which include the media and artefacts as well as people.

Development and Validation of a Fast and Homogeneous Cell-Based Fluorescence Screening Assay for Divalent Metal Transporter 1 (DMT1/SLC11A2) Using the FLIPR Tetra.

Divalent metal ion transporter 1 (DMT1) is a proton-coupled Fe(2+) transporter that is essential for iron uptake in enterocytes and for transferrin-associated endosomal iron transport in many other cell types. DMT1 dysfunction is associated with several diseases such as iron overload disorders and neurodegenerative diseases. The main objective of the present work is to develop and validate a fluorescence-based screening assay for DMT1 modulators. We found that Fe(2+) or Cd(2+) influx could be reliably monitored in calcium 5-loaded DMT1-expressing HEK293 cells using the FLIPR Tetra fluorescence microplate reader. DMT1-mediated metal transport shows saturation kinetics depending on the extracellular substrate concentration, with a K0.5 value of 1.4 µM and 3.5 µM for Fe(2+) and Cd(2+), respectively. In addition, Cd(2+) was used as a substrate for DMT1, and we find a Ki value of 2.1 µM for a compound (2-(3-carbamimidoylsulfanylmethyl-benzyl)-isothiourea) belonging to the benzylisothioureas family, which has been identified as a DMT1 inhibitor. The optimized screening method using this compound as a reference demonstrated a Z' factor of 0.51. In summary, we developed and validated a sensitive and reproducible cell-based fluorescence assay suitable for the identification of compounds that specifically modulate DMT1 transport activity.

XENOESTROGEN-SPECIFIC MECHANISMS OF DEVELOPMENTAL REPROGRAMMING CORRELATE WITH GENE EXPRESSION AND TUMOR DEVELOPMENT

Environmental exposures during sensitive windows of development can reprogram normal physiological responses and alter disease susceptibility later in life in a process known as developmental reprogramming. We have shown that neonatal exposure to the xenoestrogen diethylstilbestrol (DES) can developmentally reprogram the reproductive tract in genetically susceptible Eker rats giving rise to complete penetrance of uterine leiomyoma. Based on this, we hypothesized that xenoestrogens, including genistein (GEN) and bisphenol A (BPA), reprogram estrogen-responsive gene expression in the myometrium and promote the development of uterine leiomyoma. We proposed the mechanism that is responsible for the developmental reprogramming of gene expression was through estrogen (E2)/ xenoestrogen inducedrapid ER signaling, which modifies the histone methyltransferase Enhancer of Zeste homolog 2 (EZH2) via activation of the PI3K/AKT pathway. We further hypothesized that there is a xenostrogen-specific effect on this pathway altering patterns of histone modification, DNA methylation and gene expression. In addition to our novel finding that E2/DES-induced phosphorylation of EZH2 by AKT reduces the levels of H3K27me3 in vitro and in vivo, this work demonstrates in vivo that a brief neonatal exposure to GEN, in contrast to BPA, activates the PI3K/AKT pathway to regulate EZH2 and decreases H3K27me3 levels in the neonatal uterus. Given that H3K27me3 is a repressive mark that has been shown to result in DNA methylation and gene silencing we investigated the methylation of developmentally reprogrammed genes. In support of this evidence, we show that neonatal DES exposure in comparison to VEH, leads to hypomethylation of the promoter of a developmentally reprogrammed gene, Gria2, that become hyper-responsive to estrogen in the adult myometrium indicating vi that DES exposure alter gene expression via chromatin remodeling and loss of DNA methylation. In the adult uterus, GEN and BPA exposure developmentally reprogrammed expression of estrogen-responsive genes in a manner opposite of one another, correlating with our previous data. Furthermore, the ability of GEN and BPA to developmental reprogram gene expression correlated with tumor incidence and multiplicity. These data show that xenoestrogens have unique effects on the activation of non-genomic signaling in the developing uterus that promotes epigenetic and genetic alterations, which are predictive of developmental reprogramming and correlate with their ability to modulate hormone-dependent tumor development.

Development of nuclear emulsions with 1 μm spatial resolution for the AEgIS experiment

The main goal of the AEgIS experiment at CERN is to test the weak equivalence principle for antimatter. We will measure the Earth ' s gravitational acceleration g with antihydrogen atoms being launched in a horizontal vacuum tube and traversing a moiré de fl ectometer. We intend to use a position sensitive device made of nuclear emulsions (combined with a time-of- fl ight detector such as silicon μ strips) to measure precisely their annihilation points at the end of the tube. The goal is to determine g with a 1% relative accuracy. In 2012 we tested emulsion fi lms in vacuum and at room temperature with low energy antiprotons from the CERN antiproton decelerator. First results on the expected performance for AEgIS are presented

Use of a hypomorphic allele of myogenin to analyze Myogenin-dependent processes in mouse development

Myogenin is a muscle-specific transcription factor essential for skeletal muscle differentiation. A severe reduction in the number of fused myotubes is seen in myogenin-null mice, and the expression of genes characteristic of differentiated skeletal muscle is reduced. Additionally, sternebrae defects are seen in myogenin-null mice, a secondary defect in the sternal cartilage precursors. Very little is known about the quantitative requirement for myogenin in muscle differentiation and thoracic skeletal development in vivo. In this thesis I describe experiments utilizing a mouse line harboring a hypomorphic allele of myogenin, generated by gene targeting techniques in embryonic stem cells. The nature of the hypomorphism was due to lowered levels of myogenin from this allele. In embryos homozygous for the hypomorphic allele, normal sternum formation and extensive muscle differentiation was observed. However, muscle hypoplasia and reduced muscle-specific gene expression were apparent in these embryos, and the mice were not viable after birth. These results suggest skeletal muscle differentiation is highly sensitive to the absolute amounts of myogenin, and reveal distinct threshold requirements for myogenin in skeletal muscle differentiation, sternum formation, and viability in vivo. The hypomorphic allele was utilized as a genetically sensitized background to identify other components of myogenin-mediated processes. Using a candidate gene approach I crossed null mutations in MEF2C and MRF4 into the hypomorphic background and examined whether these mutations affected muscle differentiation and skeleton formation in the myogenin hypomorph. Although MEF2C mutation did not affect any phenotypes seen in the hypomorphic background, MRF4 was observed to be an essential component of myogenin-mediated processes of thoracic skeletal development. Additionally, the hypomorphic allele was very sensitive to genetic effects, suggesting the existence of mappable genetic modifiers of the hypomorphic allele of myogenin. ^