Biology subjects

Athilingam, T.

Publications and source records attributed to Athilingam, T..

5 recordsLinked to original sources

bioRxiv2025

Identification of optimal fluorophores for use in the Drosophila embryo

The use of fluorescent proteins has become ubiquitous throughout the life sciences as a key method for understanding molecular, cellular and tissue scale processes. Genetically encodable fluorophores have enabled stable genetic lines to be generated in a large array of organisms. There are now suites of fluorophores available, particularly in the green and red spectra. Yet, which fluorophore works best in vivo can depend on a range of factors, both extrinsic (e.g. pH, temperature) and intrinsic (e.g. photobleaching, brightness). While fluorophores have been well characterised in cell culture, such measures within in vivo systems are more limited. Here, we present a quantitative screen of nine green and eight red fluorophore lines in Drosophila, with the fluorescent protein expressed from the same genomic location and imaged under identical conditions. We analyse the expression of the fluorophores in both early and late Drosophila embryos. We provide a quantitative analysis of the bleaching and folding rates. We find amongst the green fluorophores that the suitable choice - e.g., mEGFP, mNeonGreen, mStayGold - depends on timing and imaging requirements. For the red fluorophores, mScarlet-I performed consistently well, though no particular fluorophore stood out as ideal under all conditions. These results provide a powerful database for selecting optimal fluorophores for imaging in the Drosophila embryo in green and red channels.

developmental biology↗

bioRxiv2024

Regulation of bicoid mRNA throughout oogenesis and early embryogenesis impacts protein gradient formation

The transcription factor Bicoid (Bcd) protein guides early Drosophila patterning and is the best-characterised morphogen. The source of the morphogen, bcd mRNA, is maternally deposited during oogenesis and localised to the anterior pole of the mature oocyte. While the spatiotemporal interpretation of the Bcd morphogen gradient has been intensely studied, when and where Bcd protein is produced and how this protein gradient is dynamically shaped remains contentious. Here, we use the SunTag reporter system to quantitatively examine the spatiotemporal profile of bcd mRNA translation in vivo. We show that association with Processing bodies (P bodies) in mature oocytes prevent premature bcd mRNA translation. Following egg activation, bcd mRNA dissociates from P bodies and translation is observed at the anterior pole. Translation remains restricted to the anterior domain throughout early development, even after nuclear migration in the syncytial blastoderm. At cellularisation, translation ceases and the remaining bcd mRNA associates with reformed P bodies, which appear to block any further translation. We use these observations to create a new modified source-diffusion-degradation model of Bcd gradient formation that has spatiotemporally varying production. Overall, our study reveals that bcd mRNA translation is tightly controlled in space and time during oogenesis and early embryogenesis.

developmental biology↗

bioRxiv2023

Deep learning reduces data requirements and allows real-time measurements in Imaging Fluorescence Correlation Spectroscopy

Imaging Fluorescence Correlation Spectroscopy (Imaging FCS) is a powerful tool to extract information on molecular mobilities, actions and interactions in live cells, tissues and organisms. Nevertheless, several limitations restrict its applicability. First, FCS is data hungry, requiring 50,000 frames at 1 ms time resolution to obtain accurate parameter estimates. Second, the data size makes evaluation slow. Thirdly, as FCS evaluation is model-dependent, data evaluation is significantly slowed unless analytic models are available. Here we introduce two convolutional neural networks (CNNs) - FCSNet and Im-FCSNet - for correlation and intensity trace analysis, respectively. FCSNet robustly predicts parameters in 2D and 3D live samples. ImFCSNet reduces the amount of data required for accurate parameter retrieval by at least one order of magnitude and makes correct estimates even in moderately defocused samples. Both CNNs are trained on simulated data, are model-agnostic, and allow autonomous, real-time evaluation of Imaging FCS measurements.

biophysics↗

bioRxiv2022

Long-ranged formation of the Bicoid gradient requires multiple dynamic modes that spatially vary across the embryo

Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution. Yet, how morphogen gradients form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. We utilise fluorescence correlation spectroscopy (FCS) and perturbations to Bcd, to dissect Bcd dynamics at multiple spatial and temporal locations. In both the cytoplasm and nucleus, we find two dynamic modes for Bicoid diffusion dynamics, consisting of fast and slow populations of Bcd. Surprisingly, there are spatial differences in Bcd diffusivity along the AP-axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in the Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. We subsequently investigated which binding elements of Bcd are playing a role in its dynamics. In the nucleus, we demonstrate that the slower mode of Bcd transport is due to Bcd DNA binding. Addition of the Bcd homeodomain to eGFP::NLS can qualitatively replicate the observed Bcd concentration profile, suggesting this domain is the primary region regulating Bcd dynamics. This study provides a detailed analysis of morphogen dynamics at different spatial and temporal locations, revealing multiple modes of transport. These results explain how a long-ranged gradient can form while retaining a steep profile through much of its range.

developmental biology↗

bioRxiv2020

Degenerating Drosophila Larval Epidermal Cells Drive Thorax Closure

Adult thorax formation in Drosophila begins during pre-pupal development by fusion of its two contralateral progenitor halves, the heminotal epithelia (HE). HEs migrate and replace an underlying cell layer of thoracic larval epidermal cells (LECs) during a morphogenetic process called thorax closure. The LEC layer has so far been proposed to be a passive substrate over which HEs migrate before their zipping. By contrast, here we show that the pull forces generated within the LEC layer drive HE migration. During thorax closure, the LECs display actomyosin-mediated contraction, via enrichment of non-muscle myosin-II and actin, besides squamous-to-pseudostratified columnar epithelial transition and tissue shrinkage. This shrinkage of the LEC layer is further accompanied by cell extrusion and death, that prevent overcrowding of LECs, thereby promoting further shrinkage. The pull forces thus generated by the shrinking LEC layer are then relayed to the HEs by their mutual adhesions via {beta}PS1 (Mys) and PS3 (Scb) integrins. Suppression of cell death in the LEC layer by a gain of p35 leads to cell overcrowding, which impedes HE migration and zipping. Further, knockdown of sqh, the light chain of non-muscle myosin II, in LECs or integrins (mys or scb) in either the LEC layer or in the HEs, or both abrogate thorax closure. Mathematical modeling also reveals the biophysical underpinnings of the forces that drive this tissue closure process wherein a degenerating LEC layer mediates its succession by the future adult primodia. These essential principles of thorax closure appear ancient in origin and recur in multiple morphogenetic contexts and tissue repair.

developmental biology↗