Energy metabolism modulates the regulatory impact of activators on gene expression.

Gene expression is a regulated process fueled by ATP consumption. Therefore, regulation must be coupled to constraints imposed by the level of energy metabolism. Here, we explore this relationship both theoretically and experimentally. A stylized mathematical model predicts that activators of gene expression have variable impact depending on metabolic rate. Activators become less essential when metabolic rate is reduced and more essential when metabolic rate is enhanced. We find that, in the Drosophila eye, expression dynamics of the yan gene are less affected by loss of EGFR-mediated activation when metabolism is reduced, and the opposite effect is seen when metabolism is enhanced. The effects are also seen at the level of pattern regularity in the adult eye, where loss of EGFR-mediated activation is mitigated by lower metabolism. We propose that gene activation is tuned by energy metabolism to allow for faithful expression dynamics in the face of variable metabolic conditions.

An in vivo platform to identify pathogenic loci.

Rare genetic disease discovery efforts typically lead to the identification of new disease genes. PreMIER ( Pre cision M edicine Integrated E xperimental R esources) is a collaborative platform designed to facilitate functional evaluation of human genetic variants in model systems, and to date the PreMIER Consortium has evaluated over 50 variants in patients with genetic disorders. To understand if Drosophila could be used to identify pathogenic disease loci as part of the PreMIER Consortium, we used tissue-specific gene knockdown in the fly as a proof of principle experiment. Tissue-specific knockdown of seven conserved disease genes caused significant changes in viability, longevity, behavior, motor function, and neuronal survival arguing a set of defined assays can be used to determine if a gene of uncertain significance (GUS) regulates specific physiological processes. This study highlights the utility of a tissue-specific knockdown platform in Drosophila to characterize GUS, which may provide the first genephenotype correlations for patients with idiopathic genetic disorders.

Energy metabolism modulates the regulatory impact of activators on gene expression.

Gene expression is a regulated process fueled by ATP consumption. Therefore, regulation must be coupled to constraints imposed by the level of energy metabolism. Here, we explore this relationship both theoretically and experimentally. A stylized mathematical model predicts that activators of gene expression have variable impact depending on metabolic rate. Activators become less essential when metabolic rate is reduced and more essential when metabolic rate is enhanced. We find that in the Drosophila eye, expression dynamics of the yan gene are less affected by loss of EGFR-mediated activation when metabolism is reduced, and the opposite effect is seen when metabolism is enhanced. The effects are also seen at the level of pattern regularity in the adult eye, where loss of EGFR-mediated activation is mitigated by lower metabolism. We propose that gene activation is tuned by energy metabolism to allow for faithful expression dynamics in the face of variable metabolic conditions.

Instillation of Ophthalmic Formulation Containing Nilvadipine Nanocrystals Attenuates Lens Opacification in Shumiya Cataract Rats.

We developed ophthalmic formulations based on nilvadipine (NIL) nanocrystals (NIL-NP dispersions; mean particle size: 98 nm) by using bead mill treatment and investigated whether the instillation of NIL-NP dispersions delivers NIL to the lens and prevents lens opacification in hereditary cataractous Shumiya cataract rats (SCRs). Serious corneal stimulation was not detected in either human corneal epithelial cells or rats treated with NIL-NP dispersions. The NIL was directly delivered to the lens by the instillation of NIL-NP dispersions, and NIL content in the lenses of rats instilled with NIL-NP dispersions was significantly higher than that in the ophthalmic formulations based on NIL microcrystals (NIL-MP dispersions; mean particle size: 21 µm). Moreover, the supply of NIL prevented increases in Ca2+ content and calpain activity in the lenses of SCRs and delayed the onset of cataracts. In addition, the anti-cataract effect in the lens of rats instilled with NIL-NP dispersions was also significantly higher than that in NIL-MP dispersions. NIL-NPs could be used to prevent lens opacification.

Sub-millimeter endoscope demonstrates feasibility of in vivo reflectance imaging, fluorescence imaging, and cell collection in the fallopian tubes.

SIGNIFICANCE: Most cases of high-grade serous ovarian carcinoma originate as serous tubal intraepithelial carcinoma (STIC) lesions in the fallopian tube epithelium (FTE), enabling early endoscopic detection. AIM: The cell-acquiring fallopian endoscope (CAFE) was built to meet requirements for locating potentially pathological tissue indicated by an alteration in autofluorescence or presence of a targeted fluorophore. A channel was included for directed scrape biopsy of cells from regions of interest. APPROACH: Imaging resolution and fluorescence sensitivity were measured using a standard resolution target and fluorescence standards, respectively. A prototype was tested in ex vivo tissue, and collected cells were counted and processed. RESULTS: Measured imaging resolution was 88 µm at a 5-mm distance, and full field of view was ∼45 deg in air. Reflectance and fluorescence images in ex vivo porcine reproductive tracts were captured, and fit through human tracts was verified. Hemocytometry counts showed that on the order of 105 cells per scrape biopsy could be collected from ex vivo porcine tissue. CONCLUSIONS: All requirements for viewing STIC in the FTE were met, and collected cell counts exceeded input requirements for relevant analyses. Our benchtop findings suggest the potential utility of the CAFE device for in vivo imaging and cell collection in future clinical trials.

The Drosophila Pioneer Factor Zelda Modulates the Nuclear Microenvironment of a Dorsal Target Enhancer to Potentiate Transcriptional Output.

Connecting the developmental patterning of tissues to the mechanistic control of RNA polymerase II remains a long-term goal of developmental biology. Many key elements have been identified in the establishment of spatial-temporal control of transcription in the early Drosophila embryo, a model system for transcriptional regulation. The dorsal-ventral axis of the Drosophila embryo is determined by the graded distribution of Dorsal (Dl), a homolog of the nuclear factor κB (NF-κB) family of transcriptional activators found in humans [1, 2]. A second maternally deposited factor, Zelda (Zld), is uniformly distributed in the embryo and is thought to act as a pioneer factor, increasing enhancer accessibility for transcription factors, such as Dl [3-9]. Here, we utilized the MS2 live imaging system to evaluate the expression of the Dl target gene short gastrulation (sog) to better understand how a pioneer factor affects the kinetic parameters of transcription. Our experiments indicate that Zld modifies probability of activation, the timing of this activation, and the rate at which transcription occurs. Our results further show that this effective rate increase is due to an increased accumulation of Dl at the site of transcription, suggesting that transcription factor "hubs" induced by Zld [10] functionally regulate transcription.

Metabolic Regulation of Developmental Cell Cycles and Zygotic Transcription.

The thirteen nuclear cleavages that give rise to the Drosophila blastoderm are some of the fastest known cell cycles [1]. Surprisingly, the fertilized egg is provided with at most one-third of the dNTPs needed to complete the thirteen rounds of DNA replication [2]. The rest must be synthesized by the embryo, concurrent with cleavage divisions. What is the reason for the limited supply of DNA building blocks? We propose that frugal control of dNTP synthesis contributes to the well-characterized deceleration of the cleavage cycles and is needed for robust accumulation of zygotic gene products. In support of this model, we demonstrate that when the levels of dNTPs are abnormally high, nuclear cleavages fail to sufficiently decelerate, the levels of zygotic transcription are dramatically reduced, and the embryo catastrophically fails early in gastrulation. Our work reveals a direct connection between metabolism, the cell cycle, and zygotic transcription.

Ciona intestinalis Noto4 contains a phosphotyrosine interaction domain and is involved in the midline intercalation of notochord cells.

Brachyury plays a pivotal role in the notochord formation in ascidian embryos. Ciona intestinalis Noto4 (Ci-Noto4) was isolated as a gene downstream of Ci-Bra. This gene encodes a 307 amino-acid protein with a C-terminal phosphotyrosine interaction domain (PTB/PID). Expression of Ci-Noto4 commences at the neural plate stage and is specific to notochord cells. Suppression of Ci-Noto4 levels with specific antisense morpholino oligonucleotides resulted in the formation of two rows of notochord cells owing to a lack of midline intercalation between the bilateral populations of progenitor cells. In contrast, overexpression of Ci-Noto4 by injection of a Ci-Bra(promoter):Ci-Noto4-EGFP construct into fertilized eggs disrupted the localization of notochord cells. Ci-Noto4 overexpression did not affect cellular differentiation in the notochord, muscle, mesenchyme, or nervous system. Analysis of Ci-Noto4 regions that are responsible for its function suggested significant roles for the PTB/PID and a central region, an area with no obvious sequence similarity to other known proteins. These results suggested that PTB/PID-containing Ci-Noto4 is essential for midline intercalation of notochord cells in chordate embryos.

Interaction of notochord-derived fibrinogen-like protein with Notch regulates the patterning of the central nervous system of Ciona intestinalis embryos.

The midline organ the notochord and its overlying dorsal neural tube are the most prominent features of the chordate body plan. Although the molecular mechanisms involved in the formation of the central nervous system (CNS) have been studied extensively in vertebrate embryos, none of the genes that are expressed exclusively in notochord cells has been shown to function in this process. Here, we report a gene in the urochordate Ciona intestinalis encoding a fibrinogen-like protein that plays a pivotal role in the notochord-dependent positioning of neuronal cells. While this gene (Ci-fibrn) is expressed exclusively in notochord cells, its protein product is not confined to these cells but is distributed underneath the CNS as fibril-like protrusions. We demonstrated that Ci-fibrn interacts physically and functionally with Ci-Notch that is expressed in the central nervous system, and that the correct distribution of Ci-fibrn protein is dependent on Notch signaling. Disturbance of the Ci-fibrn distribution caused an abnormal positioning of neuronal cells and an abnormal track of axon extension. Therefore, it is highly likely that the interaction between the notochord-based fibrinogen-like protein and the neural tube-based Notch signaling plays an essential role in the proper patterning of CNS.

Brachyury-downstream notochord genes and convergent extension in Ciona intestinalis embryos.

Formation of the chordate body is accomplished by a complex set of morphogenetic movements including convergent extension of notochord cells. In the ascidian Ciona intestinalis, Brachyury plays a key role in the formation of the notochord, and more than 30 Bra-downstream notochord genes have been identified. In the present study, we examined the effects of functional suppression of nine Bra-downstream notochord genes, which include Ci-PTP, Ci-ACL, Ci-prickle, Ci-netrin, Ci-trop, Ci-Noto3, Ci-ASAK, Ci-ERM and Ci-pellino. When the function of the first two genes (Ci-PTP and Ci-ACL) was suppressed with specific morpholinos, the notochord cells failed to converge, while functional suppression of Ci-prickle resulted in a failure of intercalation, and therefore the cells in these three types of embryo remained in the mid-dorsal region of the embryo. Functional suppression of the next four genes (Ci-netrin, Ci-trop, Ci-Noto3 and Ci-ASAK) resulted in the partial defect of intercalation, and the notochord did not consist of a single row. In addition, when the function of the last two genes (Ci-ERM and Ci-pellino) was suppressed, notochord cells failed to elongate in the embryo, even though convergence/extension took place normally. These results indicate that many Bra-downstream notochord genes are involved in convergence/extension of the embryo.

Accumulation of maize response regulator proteins in mesophyll cells after cytokinin treatment.

The maize response regulator genes ZmRR1 and ZmRR2 respond to cytokinin, and the translated products seem to be involved in nitrogen signal transduction mediated by cytokinin through the His-Asp phosphorelay. To elucidate the physiological function of the proteins, we examined the temporal and spatial distribution in maize leaves by immunochemical analysis and use of transgenic plants. ZmRR1 and ZmRR2 polypeptides could be distinctively detected by western blotting. The polypeptides accumulated in leaves within 5 h of the supply of nitrate to nitrogen-depleted maize, and the accumulation was transient. The extent of induction was larger in the leaf tip, which is rich in photosynthetically matured cells, than elsewhere. In leaves, the polypeptides accumulated mostly in mesophyll cells. Histochemical analyses of transgenic maize harboring a ZmRR1 promoter-beta-glucuronidase fusion gene also showed most of the expression to be in these cells. These results suggest that ZmRR1 and ZmRR2 are induced in mesophyll cells and function in nitrogen signal transduction mediated by cytokinin.

Growth and development of Mesembryanthemum crystallinum (Aizoaceae).

This review describes the life cycle of Mesembryantheum crystallinum L. (the common ice plant, Aizoaceae, Caryophyllales), a halophyte with a developmentally programmed switch from C3 photosynthesis to Crassulacean acid metabolism (CAM) which is accelerated by salinity and drought. Since there has been controversy regarding the interplay between genes and environmental stimuli during the development of M. crystallinum, it is timely to summarize the life cycle for a defined set of conditions. We seek to establish the framework whereby five stages of development can be described in terms of morphology, physiology, and molecular biology. Stages 1 and 2, representing germination and growth of a juvenile form, show a determinate pattern of growth. Although specific genes for salt tolerance can be induced at these stages, stress early in development prevents progression to the mature form (stages 3-5) in which the plants advance to mature growth, flowering, and seed development. Growth in stage 3 is indeterminate in the absence of stress, but development and flowering are accelerated by environmental stresses, and CAM is constitutively expressed. Depending on the severity of the stress, plants start to flower (stage 4) and then die from the roots, ultimately with only seed capsules remaining viable, with salt sequestered into large epidermal bladder cells (stage 5). We highlight responses to salinity leading to compartmentation of ions and compatible solutes, turgor maintenance, and CAM. Finally, the molecular genetics of the ice plant are characterized, emphasizing selected genes and their products. We conclude with an analysis of the multiple stages of growth as an ecological adaptation to progressive stress. The initial determinate and inflexible juvenile phase provides a critical mass of plant material which supports the indeterminate, mature phase. Depending on the degree of stress, the mature form is then propelled towards flowering and seedset. CONTENTS Summary 171 I. Introduction 172 II. Standardizing methodology 173 III. Growth and development 175 IV. Effects of salt stress on developmental physiology 176 V. Ionic composition of cells during development 179 VI. Water transport within the plant 179 VII. The switch from C3 to Crassulacean Acid Metabolism 181 VIII. Stress and plant-growth regulators 182 IX. Molecular biology 183 X. Genetics, ploidy and mutants 184 XI. Conclusions and future directions 185 Acknowledgements 187 References 187.