Biology subjects

Prabhakara, C.

Publications and source records attributed to Prabhakara, C..

5 recordsLinked to original sources

bioRxiv2026

Integration of in situ hybridization and scRNA-seq data provides a 2D topographical map of the developing retina across species

Precise regional patterning is fundamental to tissue organization, yet the spatial logic that governs it remains poorly defined for many tissues. In the vertebrate retina, molecular domains along the dorsoventral and nasotemporal axes provide positional cues for regional specializations such as the high-acuity area (HAA). We combined multiplexed in situ hybridization data with single-cell transcriptomic data to create quantitative two-dimensional maps of developing retinal cells. In the developing chicken retina, this approach resolved sharp expression boundaries of genes involved in patterning, and revealed novel candidates enriched in the anlagen of the HAA. Comparative analysis of chicken, mouse, and human data demonstrated conserved axis-based programs, but distinct fine-scale organization consistent with presence/absence of an HAA. Here, we show that spatial reconstruction from scRNA-seq data, anchored by experimental benchmarks, enables comparative 2D topographic mapping of gene expression across species and provides a generalizable strategy to investigate the spatial logic of molecular organization in developing tissues. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/697548v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@e9b84aorg.highwire.dtl.DTLVardef@4207deorg.highwire.dtl.DTLVardef@112bc79org.highwire.dtl.DTLVardef@113e1d3_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIQuantification of multiplexed RNA-FISH signals generated reproducible spatial expression maps in the developing chicken retina. C_LIO_LIResolution of relative boundaries for early patterning genes revealed nested expression domains marking the boundaries of the developing HAA. C_LIO_LIRNA-FISH experimental data using genes with axis-specific expression allowed the generation of DV and NT scores which enabled the localization of each cell in a scRNA-seq dataset onto a 2D topographical map of the developing retina. C_LIO_LI2D topographic maps derived from scRNA-seq data revealed novel HAA-enriched genes in the developing chicken retina. C_LIO_LICross-species comparisons showed conserved DV/NT programs but divergent fine-scale organization in retinal development. C_LIO_LICyp26c1 expression pattern was validated in human retina correlating with the location of the future HAA. C_LI

developmental biology↗

bioRxiv2024

Integration of sexual dimorphism and left-right asymmetry in the development of the duck syrinx

Embryonic morphogenesis is regulated across multiple dimensions. In ducks, the syrinx, the avian vocal organ, undergoes morphogenic processes that result in both left-right asymmetric and sexually dimorphic development. Although these properties are thought to be controlled by the NODAL-PITX2 left-right signaling cascade and the sex steroid pathways, how these mechanisms work together to produce an asymmetric structure in a sexually dimorphic manner remains unclear. Here, we first establish evidence for sexual selection driving the evolution of the duck syrinx. During its development, we observe that PITX2 is expressed on the left side in both male and female ducks, although not in other birds. Asymmetric activation of PITX2 in this domain is triggered by bilateral BMP signaling but is limited to the left side by left-specific stably accessible chromatin established during an earlier asymmetric wave of PITX2 expression. Ultimately, there is an induction of left-specific WNT and BMP signaling in the syrinx primordium, which synergistically elevates cell proliferation on the left, leading to asymmetric growth. Estrogen receptor expression is also shown to be induced on the left side of the forming syrinx. This has no effect in males, but in females where the hormone is present, estrogen signaling reduces left-sided cell proliferation, thus promoting bilaterally symmetric growth. These data demonstrate how sexually dimorphic left-right asymmetry can be integrated to produce an adaptive trait.

developmental biology↗

bioRxiv2022

Quantitative analysis of three-dimensional cell organisation and concentration profiles within curved epithelial tissues

Organogenesis involves folding of flat epithelial tissues into three-dimensional (3D) shapes. Quantitative analysis in 3D of the concentration gradients of biochemical and mechano-chemical cues that shape the tissue has remained a challenge, due to the complex tissue geometries. Towards addressing this, we present a methodology that transforms the cartesian images acquired by high-resolution confocal microscopy of curved tissues into a laminar organisation from the outer-most tissue surface. We further detail a data-based intensity correction method to account for the intensity variations that arise as a consequence of the global geometry. Applying our approach to the dome-shaped Drosophila wing disc, we quantitatively estimate the concentration profiles of different biochemical signals, including the Wingless morphogen gradient. The laminar data-organisation method also enabled visualisation of apical and basal layers facilitating an accurate 3D reconstruction of cell shapes within the pseudostratified epithelium. We find that the columnar disc proper cells have irregular 3D shapes and undergo frequent apico-basal cell intercalations, concomitantly leading to cell neighbour exchanges along the apico-basal axis. The workflow of image processing described here may be employed to quantify 3D concentration gradients and 3D cellular organisation in any layered curved tissue, provided a marker describing the reference surface manifold is available.

developmental biology↗

bioRxiv2022

Cellular compartmentalisation and receptor promiscuity as a strategy for accurate and robust inference of position during morphogenesis

Precise spatial patterning of cell fate during morphogenesis requires accurate inference of cellular position. In making such inferences from morphogen profiles, cells must contend with inherent stochasticity in morphogen production, transport, sensing and signalling. Motivated by the multitude of signalling mechanisms in various developmental contexts, we show how cells may utilise multiple tiers of processing (compartmentalisation) and parallel branches (multiple receptor types), together with feedback control, to bring about fidelity in morphogenetic decoding of their positions within a developing tissue. By simultaneously deploying specific and nonspecific receptors, cells achieve a more accurate and robust inference. We explore these ideas in the patterning of Drosophila melanogaster wing imaginal disc by Wingless morphogen signalling, where multiple endocytic pathways participate in decoding the morphogen gradient. The geometry of the inference landscape in the high dimensional space of parameters provides a measure for robustness and delineates stiff and sloppy directions. This distributed information processing at the scale of the cell highlights how local cell autonomous control facilitates global tissue scale design.

biophysics↗

bioRxiv2020

Niclosamide inhibits SARS-CoV2 entry by blocking internalization through pH-dependent CLIC/GEEC endocytic pathway

Many viruses utilize the host endo-lysosomal network to infect cells. Tracing the endocytic itinerary of SARS-CoV2 can provide insights into viral trafficking and aid in designing new therapeutic targets. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV2 is internalized via the clathrin and dynamin-independent, pH-dependent CLIC/GEEC (CG) endocytic pathway. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, strongly block the uptake of RBD. Using transduction assays with SARS-CoV2 Spike-pseudovirus, we confirmed that these acidification inhibitors also impede viral infection. By contrast, Chloroquine neither affects RBD uptake nor extensively alters the endosomal pH, yet attenuates Spike-pseudovirus entry, indicating a pH-independent mechanism of intervention. We screened a subset of FDA-approved acidification inhibitors and found Niclosamide to be a potential SARS-CoV2 entry inhibitor. Niclosamide, thus, could provide broader applicability in subverting infection of similar category viruses entering host cells via this pH-dependent endocytic pathway.

cell biology↗