Multi-labeling Live Imaging to Quantify Gene Expression Dynamics During Drosophila Embryonic Development.

Transcription in developing metazoans is inherently stochastic, involving transient and dynamic interactions among transcriptional machinery. A fundamental challenge with traditional techniques, including fixed-tissue protein and RNA staining, is the lack of temporal resolution. Quantifying kinetic changes in transcription can elucidate underlying mechanisms of interaction among regulatory modules. In this protocol, we describe the successful implementation of a combination of MS2/MCP and PP7/PCP systems in living Drosophila embryos to further our understanding of transcriptional dynamics during development. Our technique can be extended to visualize transcriptional activities of multiple genes or alleles simultaneously, characterize allele-specific expression of a target gene, and quantitatively analyze RNA polymerase II activity in a single-cell resolution.

Modulation of protein-DNA binding reveals mechanisms of spatiotemporal gene control in early Drosophila embryos.

It is well known that enhancers regulate the spatiotemporal expression of their target genes by recruiting transcription factors (TFs) to the cognate binding sites in the region. However, the role of multiple binding sites for the same TFs and their specific spatial arrangement in determining the overall competency of the enhancer has yet to be fully understood. In this study, we utilized the MS2-MCP live imaging technique to quantitatively analyze the regulatory logic of the snail distal enhancer in early Drosophila embryos. Through systematic modulation of Dorsal and Twist binding motifs in this enhancer, we found that a mutation in any one of these binding sites causes a drastic reduction in transcriptional amplitude, resulting in a reduction in mRNA production of the target gene. We provide evidence of synergy, such that multiple binding sites with moderate affinities cooperatively recruit more TFs to drive stronger transcriptional activity than a single site. Moreover, a Hidden Markov-based stochastic model of transcription reveals that embryos with mutated binding sites have a higher probability of returning to the inactive promoter state. We propose that TF-DNA binding regulates spatial and temporal gene expression and drives robust pattern formation by modulating transcriptional kinetics and tuning bursting rates.

Modulation of protein-DNA binding reveals mechanisms of spatiotemporal gene control in early Drosophila embryos.

It is well known that enhancers regulate the spatiotemporal expression of their target genes by recruiting transcription factors (TFs) to the cognate binding sites in the region. However, the role of multiple binding sites for the same TFs and their specific spatial arrangement in determining the overall competency of the enhancer has yet to be fully understood. In this study, we utilized the MS2-MCP live imaging technique to quantitatively analyze the regulatory logic of the snail distal enhancer in early Drosophila embryos. Through systematic modulation of Dorsal and Twist binding motifs in this enhancer, we found that a mutation in any one of these binding sites causes a drastic reduction in transcriptional amplitude, resulting in a reduction in total mRNA production of the target gene. We provide evidence of synergy, such that multiple binding sites with moderate affinities cooperatively recruit more TFs to drive stronger transcriptional activity than a single site. Moreover, a Hidden Markov-based stochastic model of transcription reveals that embryos with mutated binding sites have a higher probability of returning to the inactive promoter state. We propose that TF-DNA binding regulates spatial and temporal gene expression and drives robust pattern formation by modulating transcriptional kinetics and tuning bursting rates.

The nuclear to cytoplasmic ratio directly regulates zygotic transcription in Drosophila through multiple modalities.

Early embryos must rapidly generate large numbers of cells to form an organism. Many species accomplish this through a series of rapid, reductive, and transcriptionally silent cleavage divisions. Previous work has demonstrated that the number of divisions before both cell cycle elongation and zygotic genome activation (ZGA) is regulated by the ratio of nuclear content to cytoplasm (N/C). To understand how the N/C ratio affects the timing of ZGA, we directly assayed the behavior of several previously identified N/C ratio-dependent genes using the MS2-MCP reporter system in living Drosophila embryos with altered ploidy and cell cycle durations. For every gene that we examined, we found that nascent RNA output per cycle is delayed in haploid embryos. Moreover, we found that the N/C ratio influences transcription through three overlapping modes of action. For some genes (knirps, fushi tarazu, and snail), the effect of ploidy can be primarily attributed to changes in cell cycle duration. However, additional N/C ratio-mediated mechanisms contribute significantly to transcription delays for other genes. For giant and bottleneck, the kinetics of transcription activation are significantly disrupted in haploids, while for frühstart and Krüppel, the N/C ratio controls the probability of transcription initiation. Our data demonstrate that the regulatory elements of N/C ratio-dependent genes respond directly to the N/C ratio through multiple modes of regulation.

Bioactive hydrogels for bone regeneration.

Bone self-healing is limited and generally requires external intervention to augment bone repair and regeneration. While traditional methods for repairing bone defects such as autografts, allografts, and xenografts have been widely used, they all have corresponding disadvantages, thus limiting their clinical use. Despite the development of a variety of biomaterials, including metal implants, calcium phosphate cements (CPC), hydroxyapatite, etc., the desired therapeutic effect is not fully achieved. Currently, polymeric scaffolds, particularly hydrogels, are of interest and their unique configurations and tunable physicochemical properties have been extensively studied. This review will focus on the applications of various cutting-edge bioactive hydrogels systems in bone regeneration, as well as their advantages and limitations. We will examine the composition and defects of the bone, discuss the current biomaterials for bone regeneration, and classify recently developed polymeric materials for hydrogel synthesis. We will also elaborate on the properties of desirable hydrogels as well as the fabrication techniques and different delivery strategies. Finally, the existing challenges, considerations, and the future prospective of hydrogels in bone regeneration will be outlined.

Drug Delivery to the Brain across the Blood-Brain Barrier Using Nanomaterials.

A major obstacle facing brain diseases such as Alzheimer's disease, multiple sclerosis, brain tumors, and strokes is the blood-brain barrier (BBB). The BBB prevents the passage of certain molecules and pathogens from the circulatory system into the brain. Therefore, it is nearly impossible for therapeutic drugs to target the diseased cells without the assistance of carriers. Nanotechnology is an area of growing public interest; nanocarriers, such as polymer-based, lipid-based, and inorganic-based nanoparticles can be engineered in different sizes, shapes, and surface charges, and they can be modified with functional groups to enhance their penetration and targeting capabilities. Hence, understanding the interaction between nanomaterials and the BBB is crucial. In this Review, the components and properties of the BBB are revisited and the types of nanocarriers that are most commonly used for brain drug delivery are discussed. The properties of the nanocarriers and the factors that affect drug delivery across the BBB are elaborated upon in this review. Additionally, the most recent developments of nanoformulations and nonconventional drug delivery strategies are highlighted. Finally, challenges and considerations for the development of brain targeting nanomedicines are discussed. The overall objective is to broaden the understanding of the design and to develop nanomedicines for the treatment of brain diseases.