Development Function Research Articles

Kv2.1 voltage-gated potassium channels in developmental perspective.

Kv2.1 voltage-gated potassium channels consist of two types of α-subunits: (a) electrically-active Kcnb1 α-subunits and (b) silent or modulatory α-subunits plus β-subunits that, similar to silent α-subunits, also regulate electrically-active subunits. Voltage-gated potassium channels were traditionally viewed, mainly by electrophysiologists, as regulators of the electrical activity of the plasma membrane in excitable cells, a role that is performed by transmembrane protein domains of α-subunits that form the electric pore. Genetic studies revealed a role for this region of α-subunits of voltage-gated potassium channels in human neurodevelopmental disorders, such as epileptic encephalopathy. The N- and C-terminal domains of α-subunits interact to form the cytoplasmic subunit of heterotetrameric potassium channels that regulate electric pores. Subsequent animal studies revealed the developmental functions of Kcnb1-containing voltage-gated potassium channels and illustrated their role during brain development and reproduction. These functions of potassium channels are discussed in this review in the context of regulatory interactions between electrically-active and regulatory subunits.

P4999Cardiomyocyte ErbB4 receptors are essential for developmental cardiac hypertrophy and cardiac function in adult hearts

Abstract Background/Introduction Activation of ErbB4 by neuregulin 1 (NRG1) promotes cardiomyocyte hypertrophy and proliferation in both adult and neonatal mice, while treatment of patients with NRG1 following myocardial infarction reduces scar size and improves function. In mice, deletion of ErbB4 from cardiomyocytes mid-gestation results in development of dilated cardiomyopathy and reduced survival, pointing to a critical role for ErbB4 in the heart. Purpose We sought to evaluate the role of ErbB4 in the adult heart. Methods and results We deleted ErbB4 in αMHC-MerCreMer (cCre Tg+/)/ErbB4 homozygote floxed (ErbB4 fl/fl) mice at ∼2 months of age with 10 injections of Tamoxifen (20 mg/kg/day). Contractile function was reduced in vivo (echocardiography, 16%) and ex vivo (isolated-perfused, 33%) 3 months after gene deletion, while survival in mice up to 8 months after tamoxifen treatment was mot modified by cardiomyocyte ErbB4 deletion. We next evaluated the role of ErbB4 in response to physiological and pathological hypertrophic stressors. ErbB4 deletion did not modify cardiac enlargement in response to Angiotensin II (1000ng/kg/min, 14 days) or exercise (twice daily swimming, 20 min/session increasing 10 min/day to 90 min followed by 7 days at 90 min/session). Taken together, this indicated that ErbB4 is not essential for survival and adaptation in the adult heart, pointing instead towards a critical window for ErbB4 in neonatal heart development. To test this hypothesis, ErbB4ff and ErbB4ww neonates were injected at P1 with AAV9-cTNT-eGFP-iCre (2.16x1011viral particles, temporal vein) and culled at P6. We confirmed the presence of iCre mRNA, and suppression of ErbB4 in ErbB4 ff/ff mice, coincident with increased NRG1a, and reduced body and ventricular weights. By day 28, a number of hearts showed evidence of dilated cardiomyopathy. Conclusion Thus, ErbB4 is critical to cardiac hypertrophy and growth in neonatal mice, and maintains adult heart function. Acknowledgement/Funding National Health and Medical Research Council

Galactosemia.

Galactosemia.

Tracing bone marrow-derived microglia in brain metastatic tumors.

Microglia are the resident macrophages in the central nervous system (CNS), and they constitute 15-20% of the total glial populations. They have wide developmental and protective functions during brain injury, infection and tumorigenesis. Originally thought to derive from postnatal hematopoietic progenitors, it has recently been demonstrated that microglia originate from primitive myeloid progenitor cells that arise during early development from the embryonic yolk sac. Circulating monocytes infiltrate the CNS upon inflammatory conditions, such as cancer, primarily differentiating into macrophages and dendritic cells. Both resident and recruited microglia respond to environmental cues and actively participate in pathogenic processes, albeit their transcriptomic profiles contain significant differences suggesting distinctive roles. Metastatic brain tumors are the most common intracranial neoplasm in adults, with an estimate incidence 10 times higher than all primary brain neoplasms combined, and with dismal prognosis. Microglia is a major immune population associated with brain metastatic tumors in patients. They are proposed to play multiple, and sometimes opposing roles, in tumor progression. However, our ability to evaluate individual contribution of resident and recruited populations is hindered by the fact that they express overlapping sets of surface markers. Tracking and interrogating tissue-resident vs recruited microglia in the brain tumor microenvironment becomes critical to dissect their respective roles and gain a better understanding of the mechanism governing their interaction. In this chapter, we describe the utilization of genetic reporter mice to identify recruited brain microglia, offer a comparison between the genetic method and the most widely used flow cytometric approach, and discuss potential downstream applications to interrogate BMDM function in brain metastatic disease.

Transcriptomic profiling of developing melanocortin neurons reveals a role for Prdm12 in energy balance

Abstract The hypothalamus is a critical regulator of many physiological processes essential for life. In the adult brain, each anatomically-defined hypothalamic nucleus consists of functionally heterogeneous neuronal subpopulations that dictate distinct survival behaviors. Nevertheless, how rich neuronal identities are established in the developing hypothalamus remains poorly understood. Despite their opposing actions on food intake, POMC and NPY/AgRP neurons in the arcuate nucleus of the hypothalamus (ARH) are derived from the same progenitors that give rise to ARH neurons. However, the mechanism whereby common neuronal precursors subsequently adopt either the anorexigenic (POMC) or the orexigenic (NPY/AgRP) identity remains elusive. We hypothesize that POMC and NPY/AgRP cell fates are specified and maintained by distinct intrinsic factors. In search of them, we profiled the transcriptomes of developing POMC and NPY/AgRP neurons with whole-genome RNA sequencing (RNA-seq). Moreover, cell-type-specific transcriptomic analyses revealed transcription regulators that are selectively enriched in either population, but whose developmental functions are unknown in these neurons. Among them, we found the expression of the PR domain-containing factor 12 (Prdm12) was enriched in POMC neurons but was absent in NPY/AgRP neurons. Selective ablation of Prdm12 in postmitotic POMC neurons led to early-onset obesity, accelerated linear growth, as well as impaired glucose tolerance, which recapitulates symptoms of POMC/MC4R deficiency in humans. These findings, therefore, establish a previously-unrecognized role for Prdm12 in energy balance. Furthermore, the combination of cell-type-specific genomic and genetic analyses provides a means to dissect cellular and functional diversity in the developing hypothalamus as well as the developmental origins of diverse survival behaviors.

The effectiveness of family therapy based on bowen's emotional treatment on developmental functions of family and maladaptive emotional schemes among women with major depressive disorder

The effectiveness of family therapy based on bowen's emotional treatment on developmental functions of family and maladaptive emotional schemes among women with major depressive disorder

Chloroplast Research Methods: Probing The Targeting, Localization And Interactions Of Chloroplast Proteins.

Chloroplast Research Methods: Probing The Targeting, Localization And Interactions Of Chloroplast Proteins.

Political Economy of Connectivity: China’s Belt and Road Initiative

There are ongoing academic and public debates on the nature of China’s Belt and Road Initiative (BRI). While some observers hold that the BRI is primarily about economic development, others see it as a grand strategy of a great power with hegemonic aspirations. Is the BRI about development or geopolitics? This article adopts a political economy approach to bridge the developmental and geopolitical perspectives. The main argument is that the BRI signifies an attempt by the Chinese state to manage internal problems of capital accumulation by externalizing development on a trans-regional scale. The problems of capital accumulation under China’s export-oriented growth model indicates a particular form of spatial fix via the BRI, which goes beyond the exportation of excess industrial/financial capacity and seeks to transform external productive spaces through inter-regional connectivity. The process of constructing new nodes and infrastructures of capital creates space for new forms of asymmetric interdependencies, rendering it prohibitively costly for most BRI partners to exit China-centered networks. To the extent that such asymmetric dependencies can be leveraged for strategic purposes, the BRI serves a geopolitical as well as a developmental function.

Long non-coding RNAs: Fine-tuning the developmental responses in plants

Plant developmental biology is associated with various gene regulatory pathways involved in different phases of their life cycle. In course of development, growth and differentiation of different organs in plants are regulated by specific sets of gene expression. With the advances in genomic and bioinformatic techniques, particularly high-throughput sequencing technology, many transcriptional units with no protein-coding potential have been discovered. Previously thought to be the dark matters of genome, long non-coding RNAs (lncRNAs) are gradually gaining importance as crucial players in gene regulation during different developmental phases. Some lncRNAs, showing complementarity to microRNAs (miRNAs), are used as endogenous target mimics of specific miRNA family. A number of lncRNAs can also act as natural antisense transcripts to attenuate the expression of coding genes. Although lncRNA-mediated regulations have extensively been studied in animals, plant lncRNA research is still in its initial phase. The present review highlights the regulatory mechanism and different physiological aspects of lncRNAs in plant development. In plants, lncRNAs are found to be associated with a number of plant developmental functions such as lateral root development, vernalization, photomorphogenesis, pollen development, fiber development and nodulation. Understanding these potent roles of lncRNAs in plant development can further provide novel tools for crop improvement programs in future.

Noncoding RNAs in development and teratology, with focus on effects of cannabis, cocaine, nicotine, and ethanol.

Completion of the Human Genome Project has led to the identification of a large number of transcription start sites that are not paired with protein-coding genes, supporting the growing recognition of the abundance of encoded nonprotein-coding RNAs (ncRNAs) and their importance for speciation and species-specific development. Present in both plants and animals, ncRNAs vary in size, function, primary sequence, and secondary structure. While microRNAs (miRNAs) are the best known, there are a number of other ncRNAs (long[er] nonprotein-coding RNA, pseudogenes, circular RNAs, and so on) that have been shown to play an important role in the development either directly or via networks of proteins and other ncRNAs, including modulating the impact of miRNAs. Furthermore, these ncRNAs and their developmental regulatory networks are sensitive to teratogens such as ethanol, cannabis, cocaine, and nicotine. A better understanding of the developmental role of ncRNAs and their capacity to mediate teratogenesis is a necessary step in efforts to minimize the long-term consequences of developmental exposures to drugs-of-abuse. Moreover, with increasing awareness of the prevalence of polydrug use, experimental models will need to incorporate more complex drug exposure paradigms into meaningful assessments of developmental ncRNA function.

EMB-7L is required for embryogenesis and plant development in maize involved in RNA splicing of multiple chloroplast genes

Embryo and endosperm originate from the double fertilization, but they have different developmental fates and biological functions. We identified a previously undescribed maize seed mutant, wherein the embryo appears to be more severely affected than the endosperm (embryo-specific, emb). In the W22 background, the emb embryo arrests at the transition stage whereas its endosperm appears nearly normal in size. At maturity, the embryo in W22-emb is apparently small or even invisible. In contrast, the emb endosperm develops into a relative normal size. We cloned the mutant gene on the Chromosome 7L and designated it emb-7L. This gene is generally expressed, but it has a relatively higher expression level in leaves. Emb-7L encodes a chloroplast-localized P-type pentatricopeptide repeat (PPR) protein, consistent with the severe chloroplast deficiency in emb-7L albino seedling leaves. Full transcriptome analysis of the leaves of WT and emb-7L seedlings reveals that transcription of chloroplast protein-encoding genes are dramatically variable with pre-mRNA intron splicing apparently affected in a tissue-dependent pattern and the chloroplast structure and activity were dramatically affected including chloroplast membrane and photosynthesis machinery component and synthesis of metabolic products (e.g., fatty acids, amino acids, starch).

A Secreted Ig-Domain Protein Required in Both Astrocytes and Neurons for Regulation of Drosophila Night Sleep

Endogenous rhythmic behaviors are evolutionarily conserved and essential for life. In mammalian and invertebrate models, well-characterized neuronal circuits and evolutionarily conserved mechanisms regulate circadian behavior and sleep [1-4]. In Drosophila, neuronal populations located in multiple brain regions mediate arousal, sleep drive, and homeostasis (reviewed in [3, 5-7]). Similar to mammals [8], there is also evidence that fly glial cells modulate the neuronal circuits controlling rhythmic behaviors, including sleep [1]. Here, we describe a novel gene (CG14141; aka Nkt) that is required for normal sleep. NKT is a 162-amino-acid protein with a single IgC2 immunoglobulin (Ig) domain and a high-quality signal peptide [9], and we show evidence that it is secreted, similar to its C.elegans ortholog (OIG-4) [10]. We demonstrate that Nkt-null flies or those with selective knockdown in either neurons or glia have decreased and fragmented night sleep, indicative of a non-redundant requirement in both cell types. We show that Nkt is required in fly astrocytes and in a specific set of wake-promoting neurons-the mushroom body (MB) α'β' cells that link sleep to memory consolidation [11]. Importantly, Nkt gene expression is required in the adult nervous system for normal sleep, consistent with a physiological rather than developmental function for the Ig-domain protein.

Multilevel regulation of the glass locus during Drosophila eye development.

Development of eye tissue is initiated by a conserved set of transcription factors termed retinal determination network (RDN). In the fruit fly Drosophila melanogaster, the zinc-finger transcription factor Glass acts directly downstream of the RDN to control identity of photoreceptor as well as non-photoreceptor cells. Tight control of spatial and temporal gene expression is a critical feature during development, cell-fate determination as well as maintenance of differentiated tissues. The molecular mechanisms that control expression of glass, however, remain largely unknown. We here identify complex regulatory mechanisms controlling expression of the glass locus. All information to recapitulate glass expression are contained in a compact 5.2 kb cis-acting genomic element by combining different cell-type specific and general enhancers with repressor elements. Moreover, the immature RNA of the locus contains an alternative small open reading frame (smORF) upstream of the actual glass translation start, resulting in a small peptide instead of the three possible Glass protein isoforms. CRISPR/Cas9-based mutagenesis shows that the smORF is not required for the formation of functioning photoreceptors, but is able to attenuate effects of glass misexpression. Furthermore, editing the genome to generate glass loci eliminating either one or two isoforms shows that only one of the three proteins is critical for formation of functioning photoreceptors, while removing the two other isoforms did not cause defects in developmental or photoreceptor function. Our results show that eye development and function is largely unaffected by targeted manipulations of critical features of the glass transcript, suggesting a strong selection pressure to allow the formation of a functioning eye.

The Kinetics of Nucleotide Binding to Isolated Chlamydomonas Axonemes Using UV-TIRF Microscopy

Cilia and flagella are long, slender organelles found in many eukaryotic cells, where they have sensory, developmental, and motile functions. All cilia and flagella contain a microtubule-based structure called the axoneme. In motile cilia and flagella, which drive cell locomotion and fluid transport, the axoneme contains, along most of its length, motor proteins from the axonemal dynein family. These motor proteins drive motility by using energy derived from the hydrolysis of ATP to generate a bending wave, which travels down the axoneme. As a first step toward visualizing the ATPase activity of the axonemal dyneins during bending, we have investigated the kinetics of nucleotide binding to axonemes. Using a specially built ultraviolet total internal reflection fluorescence microscope, we found that the fluorescent ATP analog methylanthraniloyl ATP (mantATP), which has been shown to support axonemal motility, binds all along isolated, immobilized axonemes. By studying the recovery of fluorescence after photobleaching, we found that there are three mantATP binding sites: one that bleaches rapidly (time constant ≈ 1.7 s) and recovers slowly (time constant ≈ 44 s), one that bleaches with the same time constant but does not recover, and one that does not bleach. By reducing the dynein content in the axoneme using mutants and salt extraction, we provide evidence that the slow-recovering component, but not the other components, corresponds to axonemal dyneins. The recovery rate of this component, however, is too slow to be consistent with the activation of beating observed at higher mantATP concentrations; this indicates that the dyneins may be inhibited due to their immobilization at the surface. The development of this method is a first step toward direct observation of the traveling wave of dynein activity.

Abstract 2456: NetZen: A comprehensive network-based pathway and target discovery platform

Abstract BACKGROUND: Success in precision medicine depends on the ability to accurately and rapidly identify master regulators of critical gene subnetworks. To achieve this goal requires a seamless computational target discovery platform that integrates global network visualization with identification of master regulatory subnetworks (MRS) and their key influencers. METHODS: We have created NetZen - a computer cluster-capable computational suite allowing for 1) robust network generation with million edges; 2) network-based MRS identification; 3) hierarchical analysis with automated annotations; and 4) whole-network 3D visualization of all 20,000 genes. To identify cancer MRS, we applied NetZen to expression datasets of tumors and normal tissues, tumor-initiating cells (TICs), non-TIC cancer cells, and normal stem and non-stem cells from TCGA, GEO and internal sources. For validation, we introduced these subnetworks in normal cells to recapitulate TIC phenotypes in vivo or disrupted them in patients-derived TICs and mouse xenografts (PDX). To identify MRS of mortality, we applied NetZen to survival data in TCGA. RESULTS: Networks of 28 human cancers were generated, integrating transcriptomics, microRNAs, proteomics, and clinical data. For cancers with TIC datasets, we identified TIC-specific MRS with clear-cut hierarchy controlling immune surveillance, EMT and developmental functions. Interestingly, in some cancers, the immune subnetwork including immune checkpoints was entirely encompassed in the developmental subnetwork. We present representative validation on glioblastoma TIC (GIC) MRS with features common in other cancers’ TICs. Exogenous introduction of this subnetwork in normal astrocytes forced them to become GICs and form tumors in mice. Disrupting this subnetwork in 10 independent PDXs profoundly blunted tumor growth and prolonged survival. Lastly, we present detailed MRS of mortality analysis of multiple cancer types and identify common pathways directly influencing outcomes. DISCUSSION: NetZen provides a complete solution to big data analysis by effortlessly processing million-edge hierarchical networks with biological insights through automated annotations. The findings that the immune and developmental subnetworks were intimately linked suggest that immune escape by TICs is intrinsic of cellular transformation rather than a product of selection. Lastly, detailed network hierarchy provides a logical roadmap for cancer therapeutic development. Citation Format: Son Le, Alberto Riva, Changwang Deng, David D. Tran. NetZen: A comprehensive network-based pathway and target discovery platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2456.

Abstract LB-285: SAGA functions in development and disease

Abstract Our research is focused on defining the functions of histone modifying enzymes in normal cells, so that we can understand how alterations in these activities contribute to tumor development or progression. Our recent studies on the SAGA complex, which houses both histone acetyltransferase (HAT) and deubiquitylase (DUB) activities, revealed that the Gcn5 HAT, is an important co-activator for Myc in regulating self-renewal of mouse ES cells. Gcn5 is also important for Myc functions as a Yamanaka factor in stimulating conversion of differentiated cells towards an induced pluripotent state. These developmental functions predict that Gcn5 will also be important during Myc functions as an oncogene. In support of this idea, recent studies reveal that deletion of Gcn5 delays lymphoma onset and more than doubles survival of Eμ-Myc mice. The SAGA DUB module is also implicated in cancer, as the USP22 catalytic subunit is overexpressed in several highly aggressive human tumors. We have created USP22 knock out mice to probe the functions of this DUB. Our data indicate that USP22 drives TGF-beta and receptor tyrosine kinase signaling pathways that are critical both to normal developmental processes and to oncogenesis. Altogether, our studies indicate that both Gcn5 and USP22 may provide unique targets for development of new cancer therapies. Note: This abstract was not presented at the meeting. Citation Format: Sharon Y. Dent, Aimee Farria, Evangelia Koutelou. SAGA functions in development and disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-285.

Rapid Evolution of Gained Essential Developmental Functions of a Young Gene via Interactions with Other Essential Genes.

New genes are of recent origin and only present in a subset of species in a phylogeny. Accumulated evidence suggests that new genes, like old genes that are conserved across species, can also take on important functions and be essential for the survival and reproductive success of organisms. Although there are detailed analyses of the mechanisms underlying new genes' gaining fertility functions, how new genes rapidly become essential for viability remains unclear. We focused on a young retro-duplicated gene (CG7804, which we named Cocoon) in Drosophila that originated between 4 and 10 Ma. We found that, unlike its evolutionarily conserved parental gene, Cocoon has evolved under positive selection and accumulated many amino acid differences at functional sites from the parental gene. Despite its young age, Cocoon is essential for the survival of Drosophila melanogaster at multiple developmental stages, including the critical embryonic stage, and its expression is essential in different tissues from those of its parental gene. Functional genomic analyses found that Cocoon acquired unique DNA-binding sites and has a contrasting effect on gene expression to that of its parental gene. Importantly, Cocoon binding predominantly locates at genes that have other essential functions and/or have multiple gene-gene interactions, suggesting that Cocoon acquired novel essential function to survival through forming interactions that have large impacts on the gene interaction network. Our study is an important step toward deciphering the evolutionary trajectory by which new genes functionally diverge from parental genes and become essential.

Expression of the guanine nucleotide exchange factor, RAPGEF5, during mouse and human embryogenesis

Rap GTPases mediate fundamental cellular processes, including cell adhesion, migration and intracellular signal transduction. The subcellular activity of these GTPases is regulated by dedicated activators (guanine nucleotide exchange factors, GEFs) and deactivators (GTPase-activating proteins, GAPs). RAPGEF5 is a potent activator of Rap proteins and mutations in RAPGEF5 have been linked to both neurological disorders and congenital heart disease. In the frog model, Xenopus tropicalis, Rapgef5 is a critical regulator of the canonical Wnt signalling pathway and is required for normal gastrulation and correct establishment of the left-right body axis. However, requirements for RAPGEF5 in other developmental contexts, and in mammalian embryogenesis in particular, remain undefined. Here, we describe RAPGEF5 mRNA expression patterns during mouse (E9.5 – E16.5) and human (Carnegie stage 21) development, as an initial step towards better understanding its developmental functions.

Nutrition, epigenetics and health

Nutrition, epigenetics and health

Professional development activities for pre-service foreign language and culture teachers to build their methodological skills within test competency construct

A professional development activity is considered by the author as a means of educational and cognitive performance aimed at building pre-service foreign language and culture teachers’ methodological abilities to select test materials, analyse test items and design tests for implementing test assessment of their students’ foreign language communication competence performance outcomes. Due to the suggested professional development activities, methodological knowledge is activated and specific methodological skills of pre-service teachers to design, implement, and monitor language testing are developed. While methodological skills are formed and developed, professional development activities perform their educational, motivational, integration, developmental and assessment functions. The article substantiates and presents the classification of teacher professional development activities based on one principal criterion (the characteristics of pre-service foreign language and culture teachers’ methodological performance) and two additional criteria (place and mode of doing professional development activities). According to the criterion “the characteristics of pre-service foreign language and culture teachers’ methodological performance”, the receptive analytical, reproductive, and creative professional development activities are determined. These activities foster prospective teachers’ skills to observe, analyse, generalise, classify, reproduce, forecast, develop, present. According to the criterion “place of doing professional development activities”, classroom and homework assignments are distinguished. In accordance with the criterion “mode of doing professional development activities”, individual, paired, group (in small groups) activities are identified. The sub-types of professional development activities are determined according to the principal criterion: analysis, classification (receptive-analytical activities), addition, correction (reproductive activities), design (creative activities). The requirements for professional development activities are formulated: functionality, hands-on attitude, taking into account specific features of foreign language and culture teacher’s performance while designing, planning, and organising students’ language competence test assessment. Several examples of professional development activities are presented in the French language.