Biology subjects

Steward, R.

Publications and source records attributed to Steward, R..

3 recordsLinked to original sources

bioRxiv2023

Tet Controls Axon Guidance in Early Brain Development through Glutamatergic Signaling

Mutations in human TET proteins have been found in individuals with neurodevelopmental disorders. Here we report a new function of Tet in regulating Drosophila early brain development. We found that mutation in the Tet DNA-binding domain (TetAXXC) resulted in axon guidance defects in the mushroom body (MB). Tet is required in early brain development during the outgrowth of MB {beta} axons. Transcriptomic study shows that glutamine synthetase 2 (Gs2), a key enzyme in glutamatergic signaling, is significantly downregulated in the TetAXXC mutant brains. CRISPR/Cas9 mutagenesis or RNAi knockdown of Gs2 recapitulates the TetAXXC mutant phenotype. Surprisingly, Tet and Gs2 act in the insulin-producing cells (IPCs) to control MB axon guidance, and overexpression of Gs2 in these cells rescues the axon guidance defects of TetAXXC. Treating TetAXXC with the metabotropic glutamate receptor antagonist MPEP can rescue while treating with glutamate enhances the phenotype confirming Tet function in regulating glutamatergic signaling. TetAXXC and the Drosophila homolog of Fragile X Messenger Ribonucleoprotein protein mutant (Fmr13) have similar axon guidance defects and reduction in Gs2 mRNA levels. Interestingly, overexpression of Gs2 in the IPCs also rescues the Fmr13 phenotype, suggesting functional overlapping of the two genes. Our studies provide the first evidence that Tet can control the guidance of axons in the developing brain by modulating glutamatergic signaling and the function is mediated by its DNA-binding domain.

developmental biology↗

bioRxiv2023

A Machine Learning Approach to Predict Cellular Mechanical Stresses in Response to Chemical Perturbation

Mechanical stresses generated at the cell-cell level and cell-substrate level have been suggested to be important in a host of physiological and pathological processes. However, the influence various chemical compounds have on the mechanical stresses mentioned above is poorly understood, hindering the discovery of novel therapeutics and representing a barrier in the field. To overcome this barrier, we implemented two machine learning (ML) Models: Stepwise Linear Regression (SLR) and Quadratic Support Vector Machine (QSVM) to predict the dose-dependent response of tractions and intercellular stresses to chemical perturbation. We used traction and intercellular stress experimental data gathered from 0.2 g/mL and 2 g/mL drug concentrations along with cell morphological properties to train our model. To demonstrate the predictive capability of our ML model we predicted tractions and intercellular stresses in response to 0 g/ml & 1 g/ml drug concentrations. Results revealed the QSVM model to best predict intercellular stresses, while SLR best predicted tractions. Author SummaryThe ML framework we present here can be used to predict the mechanical response of any anchorage-dependent cell type to any chemical perturbation. The proposed ML can directly predict the intercellular stresses or tractions as a function of drug dosage and/or monolayer/cell coverage area which could potentially reduce the experimental time on studying the mechanics of cells to external chemicals or mechanical constraints. We believe our findings could be helpful in accelerating drug discovery and increase our understanding in the role of cellular stresses during disease progression.

biophysics↗

bioRxiv2023

Tet-dependent 5-hydroxymethyl-Cytosine modification of mRNA regulates the axon guidance genes robo2 and slit in Drosophila

Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases and catalyze the transition of 5mC to 5hmC in DNA. These enzymes have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila as Tet knock-out animals do not reach adulthood. We describe the identification of Tet-target genes in the embryo and larval brain by mapping one, Tet DNA-binding sites throughout the genome and two, the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC modifications are distributed along the entire transcript, while Tet DNA-binding sites are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are preferentially involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. Our results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs primarily in cells of the nervous system.

neuroscience↗