Biology subjects

Papasaikas, P.

Publications and source records attributed to Papasaikas, P..

11 recordsLinked to original sources

bioRxiv2026

Optogenetic perturbations reveal temporal integration of Wnt signaling during pattern formation

The Wnt signaling pathway is a conserved regulator of tissue patterning and regeneration, yet how cells interpret dynamic Wnt inputs to generate robust developmental outcomes remains poorly understood. Here, we establish the first optogenetically activatable Hydra line, enabling precise temporal control of canonical Wnt signaling in vivo. Optogenetic stimulation induced dose-dependent patterning phenotypes whose rate of progression scaled with stimulation intensity. Transcriptomic analysis revealed that distinct combinations of signal intensity and duration converged onto shared transcriptional trajectories and could be described by an effective exposure metric that represents the cumulative signaling input. Functionally, Wnt activation rescued head regeneration under conditions that normally prevent organizer formation, and equivalent regenerative outcomes could be achieved through either strong, short-lived stimulation or weaker, prolonged activation. Together, our results indicate that Hydra tissues decode Wnt signaling through temporal integration of cumulative pathway activity, progressively accumulating transcriptional responses until patterning thresholds are reached. These findings establish a quantitative framework for understanding how dynamic morphogen signaling is translated into stable developmental decisions during regeneration.

developmental biology↗

bioRxiv2026

Large transgene arrays cause aberrant transcription and synthetic molting defects with myrf-1 mutation

Multicopy transgene arrays remain widely used in C. elegans research. It is usually assumed that they behave neutrally, not impacting the phenotype under investigation. Here, we reveal that a previously reported heterochronic extra-molt phenotype associated with myrf-1(mg412) depends on the presence of the integrated molting reporter mgIs49. Nanopore long-read sequencing shows that mgIs49 is a massive 8.8-Mb insertion - around 50% of the size of its host chromosome - which disrupts the prmt-9 gene. Both mgIs49 and another array, maIs105, cause dysregulation of the transcriptome and accumulation of reads mapping to the promoter sequences used as components of the array. We identify additional arrays exceeding 4 Mb and show that variable molting defects occur across different transgenic lines when combined with myrf-1(mg412), implicating array size or composition in the synthetic phenotype. Our results underscore the necessity of replacing multicopy reporters in developmental studies with single-copy insertions or endogenous tagging whenever possible.

genetics↗

bioRxiv2024

A transcription factor toggle switch determines differentiated epidermal cell identities in Hydra

In Hydra, a simple cnidarian model, epithelio-muscular cells play a crucial role in shaping and maintaining the body architecture. These cells are continuously renewed as undifferentiated cells from the bodys mid-region get displaced toward the extremities, replacing shed, differentiated cells and adopting specific identities. This ongoing differentiation, coupled with the maintenance of distinct anatomical regions, provides an ideal system to explore the relationship between cell type specification and axial patterning. However, the molecular mechanisms governing epithelial cell identity in Hydra remain largely unknown. In this study, we describe a double-negative feedback loop between the transcription factors Zic4 and Gata3 that functions as a toggle switch to control epidermal cell fate. Zic4 is activated by Wnt signaling from the mouth organizer and triggers battery cell specification in tentacles. In contrast, Gata3 promotes basal disk cell identity at the aboral end. Functional analyses demonstrate that Zic4 and Gata3 are mutually antagonistic; suppression of one leads to the dominance of the other, and vice versa, resulting in ectopic cell specification. Notably, simultaneous knockdown of both factors rescues the phenotype, indicating that it is the balance between these transcription factors, rather than their absolute levels, that dictates cell identity. This study highlights the mechanisms by which distinct cellular identities are established at Hydra body termini and reveals how cell fate decisions are coordinated with axial patterning.

developmental biology↗

bioRxiv2024

Preventing CpG island hypermethylation in oocytes safeguards mouse development

In mammalian somatic and male germline cells, genomes are extensively DNA methylated (DNAme). In oocytes, however, DNAme is largely limited to transcribed regions only. Regulatory CpG-island (CGI) sequences are also devoid of repressive DNAme in somatic and germ cells of both sexes. The mechanisms restricting de novo DNAme acquisition in developing oocytes, at CGIs and globally, and the relevance thereof for regulating zygotic gene expression and embryo development after fertilization are largely unknown. Here we show that the histone H3 lysine 36 dimethyl (H3K36me2) demethylases KDM2A and KDM2B prevent genome-wide accumulation of H3K36me2, thereby impeding global DNMT3A-catalyzed de novo DNAme, including at CGI gene promoters. By recruiting variant Polycomb Repressive Complex 1 (vPRC1), they further control H2A mono-ubiquitin deposition and vPRC1-dependent gene repression. Through genetic perturbations, we demonstrate that aberrant Dnmt3a-dependent DNAme established in Kdm2a/Kdm2b double mutant oocytes represses transcription from maternal loci in two-cell embryos. The lethality of Kdm2a/Kdm2b maternally deficient pre-implantation embryos is suppressed by Dnmt3a deficiency during oogenesis. Hence, KDM2A/KDM2B are essential for confining the oocyte DNA methylome, conferring competence for early embryonic development. Our research implies that the reprogramming capacity eminent to early embryos is insufficient to erase aberrant DNAme from maternal chromatin, and that early development is vulnerable to gene dosage haplo-insufficiency effects. HIGHLIGHTSDemethylation of H3K36me2 by KDM2A and KDM2B prevents aberrant de novo DNA methylation in mouse oocytes. Sequence composition and H3K4me3 modulate the probability for aberrant H3K36me2 and DNA methylation at CpG islands. Aberrant oocyte DNA methylation is not reprogrammed in early embryos and suppresses maternal gene transcription. Aberrant oocyte DNA methylation causes embryonic lethality during pre-implantation development. GRAPHICAL SUMMARY O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/595726v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@198608org.highwire.dtl.DTLVardef@1aea04dorg.highwire.dtl.DTLVardef@19e3dd7org.highwire.dtl.DTLVardef@1ee11f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

Progressive cell fate specification in morphallactic regeneration

Through regeneration various species replace lost parts of their body. This is achieved either by growth of new structures at the amputation side (epimorphosis), as is the case of axolotl limb regeneration, or through remodeling of the remaining tissue (morphallaxis), as happens in Hydra. Whereas work on epimorphic regeneration support a gradual proximal to distal establishment of cell identities, morphallactic regeneration is believed to rely on initial establishment of boundary conditions that organize the re-adjustment of the pattern. Performing single cell RNA sequencing during regeneration in Hydra, we revealed the sequence of cells transdifferentiation into the missing identities. We provide evidence that morphallaxis proceeds with progressive specification of cell fates, unifying its mechanism with the one found for epimorphosis.

developmental biology↗

bioRxiv2023

Distinct roles of LARP1 and 4EBP1/2 in regulating translation and stability of 5'TOP mRNAs

A central mechanism of mTOR complex 1 (mTORC1) signaling is the coordinated translation of ribosomal protein and translation factor mRNAs mediated by the 5'-terminal oligopyrimidine motif (5'TOP). Recently, La-related protein 1 (LARP1) has been proposed to be the specific regulator of 5'TOP mRNA translation downstream of mTORC1, while eIF4E-binding proteins (4EBP1/2) were suggested to have a general role in repression. Here, we employ single-molecule translation site imaging of 5'TOP and canonical mRNAs to study the translational dynamics of single mRNAs in living cells. Our data reveals that 4EBP1/2 has a dominant role in translation repression of both 5'TOP and canonical mRNAs during pharmacological inhibition of mTOR. In contrast, we find that LARP1 selectively protects 5'TOP mRNAs from degradation in a transcriptome-wide analysis of mRNA half-lives. Our results clarify the roles of 4EBP1/2 and LARP1 in regulating 5'TOP mRNAs and provides a framework to further study how these factors control cell growth during development and disease.

molecular biology↗

bioRxiv2021

The transcription factor Zic4 acts as a transdifferentiation switch

The molecular mechanisms that maintain cell identities and prevent transdifferentiation remain mysterious. Interestingly, both dedifferentiation and transdifferentiation are transiently reshuffled during regeneration. Therefore, organisms that regenerate readily offer a fruitful paradigm to investigate the regulation of cell fate stability. Here, we used Hydra as a model system and show that Zic4 silencing is sufficient to induce transdifferentiation of tentacle into foot cells. We identified a Wnt-controlled Gene Regulatory Network that controls a transcriptional switch of cell identity. Furthermore, we show that this switch also controls the re-entry into the cell cycle. Our data indicate that maintenance of cell fate by a Wnt-controlled GRN is a key mechanism during both homeostasis and regeneration. One-Sentence SummaryA Wnt-controlled GRN controls fate maintenance in Hydra.

developmental biology↗

bioRxiv2020

Wnt3 expression as a readout of tissue stretching during Hydra regeneration

Mechanical forces shape cell fate decisions during development and regeneration in many systems. Epithelial lumen volume changes, for example, generate mechanical forces that can be perceived by the surrounding tissue and integrated into cell fate decisions. Similar behavior occurs in regenerating Hydra tissue spheroids, where periodic osmotically driven inflation and deflation cycles generate mechanical stimuli in the form of tissue stretching. Using this model, we investigate how such mechanical input guides the de novo formation of differentiated body parts. We show that the expression of the organizer-defining factor Wnt3 functions as a quantitative readout of cellular stretching and, when supplied externally, enables successful regeneration without mechanical stimulation. This finding represents a previously undescribed cellular mechanism for converting mechanical stimuli to a biochemical signaling readout and guiding cell fate transitions. It also elucidates the role of mechanical oscillations in Hydra regeneration, which long remained unclear. The presence the Wnt/mechanics interplay in Hydra and its relatives underscores the ancient evolutionary history of this crosstalk, possibly extending back to the first metazoans. Since Wnt signaling crosstalks with cellular mechanics in various developmental and disease contexts, it can also represent a conserved feature of this signaling pathway.

developmental biology↗

bioRxiv2020

Dynamics of alternative splicing during somatic cell reprogramming reveals functions for RNA-binding proteins CPSF3, hnRNP UL1 and TIA1

In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for cell reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we have studied the dynamics of alternative splicing (AS) changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells. These changes, generally uncoupled from transcriptional regulation, significantly overlapped with splicing programs reported during reprogramming of mouse embryonic fibroblasts (MEFs). Correlation between gene expression of potential regulators and specific clusters of AS changes enabled the identification and subsequent validation of CPSF3 and hnRNP UL1 as facilitators, and TIA1 as repressor of MEFs reprogramming. These RNA-binding proteins control partially overlapping programs of splicing regulation affecting genes involved in developmental and morphogenetic processes. Our results reveal common programs of splicing regulation during reprogramming of different cell types and identify three novel regulators of this process.

molecular biology↗

bioRxiv2020

REPRESSION BY HDAC3 AND DAX1 MEDIATES LINEAGE RESTRICTION OF EMBRYONIC STEM CELLS

Mouse embryonic stem cells (mESCs) give rise to embryonic but not extraembryonic endoderm fates. Here, we identify the mechanism of this lineage barrier and report that the histone deacetylase Hdac3 and the corepressor Dax1 cooperatively restrict transdifferentiation of mESCs by silencing an enhancer of the extraembryonic endoderm-specifying transcription factor (TF) Gata6. This restriction is opposed by the pluripotency TFs Nr5a2 and Esrrb, which promote cell type conversion. Perturbation of the barrier extends mESC potency, and allows formation of 3D spheroids that mimic the spatial segregation of embryonic epiblast and extraembryonic endoderm in early embryos. Overall, this study shows that transcriptional repressors stabilize pluripotency by biasing the equilibrium between embryonic and extraembryonic lineages that is hardwired into the mESC TF network.

developmental biology↗

bioRxiv2019

Cell types of the human retina and its organoids at single-cell resolution: developmental convergence, transcriptomic identity, and disease map

How closely human organoids recapitulate cell-type diversity and cell-type maturation of their target organs is not well understood. We developed human retinal organoids with multiple nuclear and synaptic layers. We sequenced the RNA of 158,844 single cells from these organoids at six developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable developed state at a rate similar to human retina development in vivo and the transcriptomes of organoid cell types converged towards the transcriptomes of adult peripheral retinal cell types. The expression of disease-associated genes was significantly cell-type specific in adult retina and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in adult human retinas.

neuroscience↗