Biology subjects

Wikramanayake, A.

Publications and source records attributed to Wikramanayake, A..

8 recordsLinked to original sources

bioRxiv2025

Alizarin red perturbs skeletal patterning and biomineralization via Catalase inhibition

Alizarin Red S (AZ) is an anthraquinone dye that is commonly used in histological studies and textiles. Exposure to AZ results in morphological perturbations in several species, including rats, frogs, and dogs; however, the mechanisms by which AZ has these effects is largely unexplored, and little is known about its effect on development. We have previously shown that AZ is teratogenic to sea urchin larvae, and that AZ was the only calcium-binding mineralization marker among five tested that perturbed skeletal patterning. Here, we further characterize these defects and demonstrate that embryos exposed to AZ have abnormal skeletal element rotation, branching, and bending. Immunostains and polychrome labeling reveal delayed migration of primary mesenchyme cells and initiation of biomineralization. Although gross ectodermal dorsal-ventral specification, ciliary band restriction, and neuronal specification occur normally in the majority of AZ-treated embryos, we find abnormal patterning and connectivity of the serotonergic neurons. Temporal transcriptomics comparisons confirm delayed development and implicate changes in neuron-related GO terms with AZ treatment. Particle imaging velocimetry experiments show that ciliary beating normally directs fluid flow into the larval mouth, while AZ treatment perturbs the normal pattern of vortices and redirects flow away from the mouth. These changes in fluid flow have functional consequences for larval feeding behaviors in AZ-treated embryos. Finally, we show that the effects of AZ on skeletal patterning are largely due to its inhibition of catalase and subsequent elevation of reactive oxygen species. Specifically, catalase knockdown and transient hydrogen peroxide treatment are each sufficient to phenocopy the hallmark AZ-mediated skeletal patterning defects. This study is the first to define the teratogenic consequences of AZ exposure and show how its effects on catalase activity impact development, skeletal patterning, and biomineralization. Summary StatementAlizarin Red exposure produces teratogenic effects on skeletal and nervous systems primarily via catalase inhibition.

developmental biology↗

bioRxiv2024

PFAS Compounds PFOA and Gen X are Teratogenic to Sea Urchin Embryos

Per-and polyfluorinated substances (PFAS) are synthetic chemicals that are used to make fluoropolymer coatings found in many products, such as non-stick pans, clothing, cosmetics, and food packaging. These highly persistent molecules are known as "forever chemicals" since they neither degrade environmentally nor break down enzymatically within biological systems. PFAS compounds readily contaminate water sources, and as a result, certain PFAS molecules have bioaccumulated in exposed species including humans. The purpose of this study was to define the effect of two PFAS molecules, the ostensibly more toxic perfluorooctanoic acid (PFOA) and the more recent, reportedly safer chemical hexafluoropropylene oxide dimer acid (Gen X), on the development of Lytechinus variegatus sea urchin embryos. We examined the effects of PFOA and Gen X on development and patterning using morphological analysis, immunostaining, HCR-FISH, and Particle Image Velocimetry (PIV). The results show that both PFAS compounds are teratogenic to sea urchin embryos. PFOA and Gen X each function at different intervals during development and provoke distinct phenotypic and gene expression outcomes. Despite beliefs that Gen X would be a safer alternative, our findings indicate that Gen X has earlier and more severe effects on endomesoderm and dorsal-ventral axis specification, neural development and function, and pattern formation compared to PFOA. These results illustrate the dangerous teratogenic potential of environmentally accumulating PFAS like Gen X, underscoring the negative ecological implications that accompany continuing commercial and industrial use of PFAS in the absence of remediation strategies. HIGHLIGHTSO_LIGen X is more teratogenic in sea urchins than PFOA C_LIO_LIGen X acts earlier than PFOA and perturbs axial and germ layer specification C_LIO_LIEach PFAS reduces neural numbers and perturbs ciliary behavior and swimming C_LI

developmental biology↗

bioRxiv2024

The Nematostella Wnt/β-catenin destruction complex provides insight into the evolution of the regulation of a major metazoan signal transduction pathway

The genesis of signaling pathways likely drove metazoan evolution, but the origins of these pathways and how they acquired signaling activity is poorly understood. Here, we studied functional evolution of the Wnt/{beta}-catenin (cWnt) pathway destruction-complex (DC) that regulates {beta}-catenin signaling. Bilaterian DC function requires {beta}-catenin binding to Axin and APC proteins, and Axin-APC heterodimerization. However, bioinformatic analyses predicted that Axin and APC-like homologs in early-branching non-bilaterians lack important previously defined bilaterian {beta}-catenin binding domains, questioning if they have functional cWnt-DCs. We demonstrate that both Axin and APC proteins in the cnidarian Nematostella vectensis (a representative of the sister taxon to bilaterians) can regulate cWnt signaling indicating an active cWnt-DC. Using in vitro analyses, we show that NvAxin binds Nv{beta}-catenin weakly despite lacking the conserved bilaterian Axin {beta}-catenin-binding motif ({beta}catBM). Using AlphaFold3, we identified two predicted {beta}catBM-like sequences in NvAxin, one within the Axin-RGS domain and another towards the C-terminus. Similar analysis of placozoan, poriferan, and ctenophore Axin identified single {beta}catBM-like sequences located within Axin-RGS. We show that ctenophore Axin and {beta}-catenin do not interact and changing a conserved leucine on NvAxin-{beta}catBM-like motifs to resemble the ctenophore sequence abolished NvAxin-Nv{beta}-catenin interactions. We propose that an ancestral Axin-RGS sequence acquired low-affinity {beta}-catenin binding early in metazoan evolution, followed by motif duplication in the cnidarian-bilaterian ancestor. In bilaterians, the duplicated {beta}catBM evolved higher-affinity for {beta}-catenin, while the ancestral sequence was lost. Our results demonstrate how phylogenetic insights, AI tools and functional assays can be used to reconstruct the evolution of complex signaling pathways.

developmental biology↗

bioRxiv2023

Evolution of promoter-proximal pausing enabled a new layer of transcription control

Promoter-proximal pausing of RNA polymerase II (Pol II) is a key regulatory step during transcription. Despite the central role of pausing in gene regulation, we do not understand the evolutionary processes that led to the emergence of Pol II pausing or its transition to a rate-limiting step actively controlled by transcription factors. Here we analyzed transcription in species across the tree of life. Unicellular eukaryotes display a slow acceleration of Pol II near transcription start sites that transitioned to a longer-lived, focused pause in metazoans. This event coincided with the evolution of new subunits in the NELF and 7SK complexes. Depletion of NELF in mammals shifted the promoter-proximal buildup of Pol II from the pause site into the early gene body and compromised transcriptional activation for a set of heat shock genes. Our work details the evolutionary history of Pol II pausing and sheds light on how new transcriptional regulatory mechanisms evolve.

evolutionary biology↗

bioRxiv2021

Polarized Dishevelled dissolution and condensation drives embryonic axis specification in oocytes

The organismal body axes that are formed during embryogenesis are intimately linked to intrinsic asymmetries established at the cellular scale in oocytes [1]. Here, we report an axis-defining event in meiotic oocytes of the sea star Patiria miniata. Dishevelled is a cytoplasmic Wnt pathway effector required for axis development in diverse species [2-4], but the mechanisms governing its function and distribution remain poorly defined. Using time-lapse imaging, we find that Dishevelled localizes uniformly to puncta throughout the cell cortex in Prophase I-arrested oocytes, but becomes enriched at the vegetal pole following meiotic resumption through a dissolution-condensation mechanism. This process is driven by an initial disassembly phase of Dvl puncta, followed by selective reformation of Dvl assemblies at the vegetal pole. Rather than being driven by Wnt signaling, this localization behavior is coupled to meiotic cell cycle progression and influenced by Lamp1+ endosome association and Frizzled receptors pre-localized within the oocyte cortex. Our results reveal a cell cycle-linked mechanism by which maternal cellular polarity is transduced to the embryo through spatially-regulated Dishevelled dynamics.

cell biology↗

bioRxiv2021

The nanoscale organization of the Wnt signaling integrator Dishevelled in the development-essential vegetal cortex domain of an egg and early embryo

Wnt/{beta}-catenin (cWnt) signaling is a crucial regulator of development and Dishevelled (Dsh/Dvl) functions as an integral part of this pathway by linking Wnt binding to the frizzled:LRP5/6 receptor complex with {beta}-catenin-stimulated gene expression. In many cell types Dsh has been localized to ill-defined cytoplasmic puncta, however in sea urchin eggs and embryos confocal fluorescence microscopy has shown that Dsh is localized to puncta present in a novel and development-essential vegetal cortex domain (VCD). In the present study, we used super-resolution light microscopy and platinum replica TEM to provide the first views of the ultrastructural organization of Dsh within the sea urchin VCD. 3D-SIM imaging of isolated egg cortices demonstrated the concentration gradient-like distribution of Dsh in the VCD, whereas higher resolution STED imaging revealed that some individual Dsh puncta consisted of more than one fluorescent source. Platinum replica immuno-TEM localization showed that Dsh puncta on the cytoplasmic face of the plasma membrane consisted of aggregates of pedestal-like structures each individually labeled with the C-terminus specific Dsh antibody. These aggregates were resistant to detergent extraction and treatment with drugs that disrupt actin filaments or inhibit myosin II contraction, and coexisted with the first division actomyosin contractile ring. These results confirm and extend previous studies and reveal, for the first time in any cell type, the nanoscale organization of plasma membrane tethered Dsh. Our current working hypothesis is that these Dsh pedestals represent a prepositioned scaffold organization that is important for canonical Wnt pathway activation at the sea urchin vegetal organization and may also be relevant to the submembranous Dsh puncta present in other eggs and embryos.

cell biology↗

bioRxiv2020

A dual role for Axin in establishing the anterior-posterior axis in the early sea urchin embryo

The activation of Wnt/{beta}-catenin (cWnt) signaling at the future posterior end of early embryos is a highly conserved mechanism for initiating pattern formation along the anterior-posterior (AP) axis in bilaterians. Moreover, in many bilaterian taxa, in addition, to activation of cWnt signaling at the posterior end, inhibition of cWnt signaling at the anterior end is required for normal development of anterior structures. In most cases, inhibition of cWnt signaling at the anterior end occurs around gastrulation and it is typically mediated by secreted factors that block signal transduction through the cWnt cell surface receptor-ligand complex. This phenomenon has been fairly well characterized, but the emerging role for intracellular inhibition of cWnt signaling in future anterior blastomeres--in cleavage stage embryos--to regulate correct AP patterning is less well understood. To investigate this process in an invertebrate deuterostome embryo we studied the function of Axin, a critical negative regulator of cWnt signaling, during early sea urchin embryogenesis. Sea urchin Axin is ubiquitously expressed in early embryos and by the blastula stage the expression of the gene becomes restricted to the posterior end of the embryo. Strikingly, knockdown of Axin protein levels using antisense Axin morpholinos (MO) led to ectopic nuclearization of {beta}-catenin and activation of endomesoderm gene expression in anterior blastomeres in early embryos. These embryos developed a severely posteriorized phenotype that could be fully rescued by co-injection of Axin MO with wild-type Axin mRNA. Axin is known to negatively regulate cWnt by its role in mediating {beta}-catenin stability within the destruction complex. Consistent with this function overexpression of Axin by mRNA injection led to the downregulation of nuclear {beta}-catenin, inhibition of endomesoderm specification and a strong anteriorization of embryos. Axin has several well-defined domains that regulate its interaction with {beta}-catenin and the key regulators of the destruction complex, Adenomatous Polyposis Coli (APC), Glycogen Synthase Kinase 3{beta}(GSK-3{beta}), and Dishevelled (Dvl). Using Axin constructs with single deletions of the binding sites for these proteins we showed that only the GSK-3{beta}binding site on Axin is required for its inhibition of cWnt in the sea urchin embryo. Strikingly, overexpression of the GSK-3{beta}-binding domain alone led to embryos with elevated levels of endomesoderm gene expression and a strongly posteriorized phenotype. These results indicated that Axin has a critical global role in inhibiting cWnt signaling in the early sea urchin embryo, and moreover, that the interaction of Axin with GSK-3{beta}is critical for this inhibition. These results also add to the growing body of evidence that Axin plays a global function in suppressing cWnt signaling in early embryos and indicates that modulation of Axin function may be a critical early step during patterning of the AP axis during bilaterian development

developmental biology↗

bioRxiv2019

β-catenin has an ancestral role in cell fate specification but not cell adhesion

The {beta}-catenin protein has two major known functions in animal cells. It keeps epithelial tissue homeostasis by its connection with Adherens Junctions (AJ), and it serves as a transcriptional cofactor along with Lef/Tcf to enter the nucleus and regulate target genes of the Wnt/{beta}-catenin (cWnt) signaling pathway. To assess the ancestral role of {beta}-catenin during development we examined its distribution and function in the ctenophore Mnemiopsis leidyi (one of the earliest branching animal phyla) by using ctenophore-specific antibodies and mRNA injection. We found that {beta}-catenin protein never localizes to cell-cell contacts during embryogenesis as it does in other metazoans, most likely because ctenophore-cadherins do not have the cytoplasmic domain required for interaction with the catenin proteins. Downregulation of zygotic Ml{beta}-catenin signaling led to the loss of endodermal and mesodermal tissues indicating that nuclear {beta}-catenin may have a deep role in germ-layer evolution. Our results indicate that the ancestral role for {beta}-catenin was in the cell-fate specification and not in cell adhesion and also further emphasizes the critical role of this protein in the evolution of tissue layers in metazoans.

developmental biology↗